STAPLED PEPTIDES AND METHODS THEREOF
20240376151 ยท 2024-11-14
Inventors
- Olena Tokareva (Andover, MA, US)
- Justin Robert Lawrie (Somerville, MA, US)
- Kunhua Li (Newton, MA, US)
- Tara Lynne Travaline (Arlington, MA, US)
- Jean-Marie Swiecicki (Somerville, MA, US)
- Gregory L. Verdine (Boston, MA)
- John Hanney McGee (Somerville, MA, US)
- Ty Matthew Thomson (Arlington, MA, US)
Cpc classification
C07K2319/90
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure provides powerful technologies for the development, production, characterization, and/or use of stapled peptide compositions. Among other things, the present disclosure provides strategies for defining amino acid sequences particularly amenable or useful for stapling, as well as technologies, reagents, and systems for developing, producing, characterizing, and/or using stapled peptides having such amino acid sequences. In some embodiments, the present disclosure provides stapled peptide agents and uses thereof including for treating various conditions, disorders or diseases.
Claims
1-2145. (canceled)
2146. An agent, comprising:
X.sup.86X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.5X.sup.16X.sup.17, wherein: each of X.sup.86, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.5 comprises a side chain comprising a polar group; X.sup.6 comprises a hydrophobic side chain; X.sup.9 comprises a side chain comprising a polar group; X.sup.10 comprises a hydrophobic side chain; X.sup.12 comprises a hydrophobic side chain; X.sup.13 comprises a hydrophobic side chain; and X.sup.14 comprises a side chain comprising an acidic group; or an agent, comprising:
X.sup.87X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.5X.sup.16X.sup.17, wherein: each of X.sup.87, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.0 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.1 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.9 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.88X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.5X.sup.16X.sup.17, wherein: each of X.sup.88, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.2 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.3 comprises a side chain comprising a polar group; X.sup.5 comprises a hydrophobic side chain; X.sup.6 comprises a side chain comprising an acidic group; X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.10 comprises a hydrophobic side chain; X.sup.12 comprises a side chain comprising an acidic group; X.sup.13 comprises a side chain comprising a polar group; X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.15 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.89X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein: each of X.sup.89, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.9 comprises a side chain comprising a polar group; X.sup.10 comprises a hydrophobic side chain; X.sup.12 comprises a hydrophobic side chain; X.sup.13 comprises a hydrophobic side chain; and X.sup.14 comprises a side chain comprising an acidic group; or an agent, comprising:
X.sup.90X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein: each of X.sup.90, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.1 comprises a hydrophobic side chain; X.sup.2 comprises a side chain comprising an acidic group; X.sup.3 comprises a hydrophobic side chain; X.sup.6 comprises a side chain comprising an aromatic group; X.sup.9 comprises a side chain comprising an acidic group; X.sup.10 comprises a hydrophobic side chain; X.sup.12 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.13 comprises a hydrophobic side chain; and X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.94X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein: each of X.sup.94, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.9 comprises a side chain comprising an acid group; X.sup.10 comprises a hydrophobic side chain; X.sup.12 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.13 comprises a hydrophobic side chain; X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.15 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.16 comprises a hydrophobic side chain; and X.sup.17 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.91X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein: each of X.sup.91, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.3 comprises a hydrophobic side chain; X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.6 comprises a side chain comprising a polar group; X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.12 comprises a hydrophobic side chain; X.sup.14 comprises a side chain comprising an acidic group; X.sup.15 comprises a side chain comprising an acidic group; and X.sup.16 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.92X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17X.sup.18X.sup.19X.sup.20, wherein: each of X.sup.92, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.3 comprises a side chain comprising an acidic group; X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.12 comprises a hydrophobic side chain; and X.sup.15 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.93X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein: each of X.sup.93, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.11 comprises a hydrophobic side chain; X.sup.16 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.17 comprises an acidic side chain.
2147. An agent, comprising:
X.sup.31X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.31, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.3 comprises a side chain comprising an acidic group; X.sup.5 comprises a hydrophobic side chain; X.sup.6 comprises a hydrophobic side chain; X.sup.9 comprises a side chain comprising an aromatic group or a basic group; and X.sup.10 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.32X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.32, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.2 comprises a hydrophobic side chain; X.sup.3 comprises a side chain comprising an acidic group; X.sup.5 comprises a side chain comprising an aromatic group; X.sup.6 comprises a hydrophobic side chain; and X.sup.10 comprises a side chain comprising an acidic group; an agent, comprising:
X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.5 comprises a side chain comprising an acidic group; X.sup.6 comprises a side chain comprising an aromatic group; X.sup.10 comprises a side chain comprising an aromatic group; X.sup.13comprises a hydrophobic side chain; and X.sup.14 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.34X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.34, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.2 comprises a side chain comprising an aromatic group; X.sup.5 comprises a side chain comprising an acidic side chain; X.sup.6 comprises a side chain comprising a basic group or an aromatic group; X.sup.9 comprises a hydrophobic side chain; and X.sup.10 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.35X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.35, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.5 comprises a side chain comprising an aromatic group; X.sup.6 comprises a side chain comprising an acidic group; X.sup.9 comprises a side chain comprising an aromatic group; and X.sup.13 comprises a side chain comprising an aromatic group; or an agent, comprising:
X.sup.41X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.41, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.6 comprises a side chain comprising an aromatic group; X.sup.9 comprises a hydrophobic side chain; X.sup.10 comprises a side chain comprising an aromatic group; and X.sup.12 comprises a side chain comprising an acidic group; or an agent, comprising:
X.sup.42X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.42, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.2 comprises a side chain comprising an aromatic group; X.sup.5 comprises a hydrophobic side chain; and X.sup.6 comprises a side chain comprising an aromatic group; or an agent, comprising:
X.sup.43X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.43, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.2 comprises a side chain comprising an acidic group; X.sup.3 comprises a side chain comprising an aromatic group; X.sup.5 comprises a hydrophobic side chain; X.sup.6 comprises a side chain comprising a polar group or an aromatic group; and X.sup.10 comprises a side chain comprising an acidic group; or an agent, comprising:
X.sup.44X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.44, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.10 comprises a hydrophobic side chain; X.sup.13 comprises a side chain comprising a polar group; and X.sup.14 comprises a side chain comprising s polar group or an aromatic group; or an agent, comprising: an agent, comprising:
X.sup.45X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.45, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.6 comprises a side chain comprising an acidic group; X.sup.9 comprises a hydrophobic side chain; X.sup.12 comprises a side chain comprising a polar group; and X.sup.13 comprises a side chain comprising a polar group or an aromatic group; or an agent, comprising:
X.sup.46X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.31, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.6 comprises a side chain comprising a polar group; X.sup.9 comprises a hydrophobic side chain X.sup.10 comprises a side chain comprising an aromatic group; or an agent, comprising:
X.sup.47X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.31, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.2 comprises a side chain comprising a polar group; X.sup.5 comprises a hydrophobic side chain; X.sup.6 comprises a side chain comprising a polar group or an aromatic group; and X.sup.9 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.48X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.48, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.3 comprises a side chain comprising a basic group or an aromatic group; X.sup.10 comprises a side chain comprising an aromatic group; X.sup.12 comprises a hydrophobic side chain; and X.sup.13 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.51X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.51, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.1 comprises a side chain comprising an aromatic group; X.sup.2 comprises a side chain comprising an aromatic group; X.sup.6 comprises a hydrophobic side chain; X.sup.9 comprises a hydrophobic side chain; and X.sup.10 comprises a side chain comprising an acidic group; or an agent, comprising
X.sup.52X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13, wherein: each of X.sup.52, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, and X.sup.13 is independently an amino acid residue; X.sup.5 comprises a hydrophobic side chain; X.sup.8 comprises a side chain comprising an aromatic group; and X.sup.9 comprises a side chain comprising an aromatic group; or an agent, comprising
X.sup.53X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.53, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.3 comprises a side chain comprising an acidic group; X.sup.5 comprises a side chain comprising a basic or aromatic group; X.sup.6 comprises a hydrophobic side chain; and X.sup.9 comprises a side chain comprising an aromatic group or a polar group; or an agent, comprising:
X.sup.54X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12, wherein: each of X.sup.54, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, and X.sup.12 is independently an amino acid residue; X.sup.5 comprises a side chain comprising an aromatic group or a basic group; X.sup.6 comprises a hydrophobic side chain; X.sup.8 comprises a hydrophobic side chain; and X.sup.9 comprises a side chain comprising an aromatic group; or an agent, comprising:
X.sup.61X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.61, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.2 comprises a side chain comprising an aromatic group; X.sup.6 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.9 comprises a side chain comprising a basic group; or an agent, comprising:
X.sup.62X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.62, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.3 comprises a side chain comprising a polar group or an acidic group; X.sup.6 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.9 comprises a side chain comprising an aromatic group; or
2148. A method for modulating a beta-catenin activity, reducing beta-catenin interaction with axin, reducing beta-catenin interaction with TCF, modulating a beta-catenin activity in a system, reducing beta-catenin interaction with axin in a system, or reducing beta-catenin interaction with TCF in a system, comprising contacting beta-catenin, or administering or delivering to the system, with an agent of claim 2147.
2149. A method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of claim 2147, optionally wherein the condition, disorder or disease is associated with beta-catenin, optionally wherein the condition, disorder or disease is cancer.
2150. A method for modulating a RNF31 activity or modulating a RNF31 activity in a system, comprising contacting RNF31, or administering or delivering to the system, with an agent claim 2147; or
2151. A method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of claim 2147, optionally wherein the condition, disorder or disease is associated with RNF3, optionally wherein the condition, disorder or disease is cancer.
2152. A method for modulating a CDK2 activity or modulating a CDK2 activity in a system, comprising contacting CDK2, or administering or delivering to the system, with an agent of claim 2147.
2153. A method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of claim 2147, optionally wherein the condition, disorder or disease is associated with CDK2, optionally wherein the condition, disorder or disease is cancer.
2154. A method for modulating a PPIA activity, inhibiting PPIA peptidyl-prolyl cis-trans isomerase activity, modulating a PPIA activity in a system, or inhibiting PPIA peptidyl-prolyl cis-trans isomerase activity in a system, comprising contacting PPIA with, or administering or delivering to the system, an agent of claim 2147.
2155. A method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of claim 2147, optionally wherein the condition, disorder or disease is associated with PPIA, optionally wherein the condition, disorder or disease is cancer.
2156. An agent, comprising:
X.sup.71X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.71, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, Xi, X.sup.12, X, and X.sup.14 is independently an amino acid residue; X.sup.10 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.72X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.72, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.9 comprises a side chain comprising a polar group; X.sup.13 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.73X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.73, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; and X.sup.2 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.74X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.74, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.10 comprises a hydrophobic side chain; and X.sup.12 comprises a side chain comprising a basic group or an aromatic group; or an agent, comprising:
X.sup.75X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.75, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.75 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.12 comprises a side chain comprising an acidic group; or an agent, comprising:
X.sup.76X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.76, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.5 comprises a hydrophobic side chain; and X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.77X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.77, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.6 comprises a hydrophobic side chain; and X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.78X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.78, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.5 comprises a side chain comprising an acidic group; and X.sup.13 comprises an aliphatic side chain; or an agent, comprising:
X.sup.79X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.79, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.13 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
[X.sup.80]p1[X.sup.2]p2[X.sup.3]p3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of p1, p2 and p3 is independently 0 or 1; each of X.sup.80, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.7 comprises a hydrophobic side chain; and X.sup.9 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.81X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.81, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.6 comprises a side chain comprising a polar group; X.sup.10 comprises a side chain comprising an acidic group; and X.sup.14 comprises hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.82X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.82, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.13 comprises a hydrophobic side chain or a side chain comprising an aromatic group; or an agent, comprising:
X.sup.83X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.83, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.83 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.5 comprises a side chain comprising a polar group; and X.sup.9 comprises a hydrophobic side chain; or an agent, comprising:
X.sup.84X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein: each of X.sup.84, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; X.sup.10 comprises a hydrophobic side chain; X.sup.13 comprises a hydrophobic side chain; and X.sup.14 comprises a side chain comprising an acidic group; or an agent, comprising:
X.sup.85X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.3X.sup.14, wherein: each of X.sup.85, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, Xi, X.sup.12, X, and X.sup.14 is independently an amino acid residue; X.sup.3 comprises a side chain comprising an acidic or polar group; X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and X.sup.12 comprises a hydrophobic side chain.
2157. A method, comprising: contacting a first collection of stapled peptides with a first target of interest so that a number of stapled peptides of the first collection binds to the target; determining amino acid sequences of stapled peptides of the first collection that bind to the first target; identifying one or more enriched amino acid residues shared by a cluster of stapled peptides of the first collection that bind to the first target independently at one or more enriched positions (first collection enriched amino acid residues); contacting a second collection of stapled peptides with a second target of interest so that one or more stapled peptides of the second collection binds to the second target, wherein the second collection is enriched for one or more first collection enriched amino acid residues independently at one or more enriched positions or positions corresponding thereto; and determining amino acid sequences of stapled peptides that bind to the second target.
2158. A method for recognizing or reprogramming a polypeptide surface, or for reprograming a polypeptide to cooperatively bind a target polypeptide, comprising contacting the polypeptide with an agent identified with the method of claim 2157.
2159. A method for modulating polypeptide interaction, comprising contacting two polypeptides with an agent of claim 2146.
2160. A method for modulating polypeptide interaction, comprising contacting two polypeptides with an agent of claim 2156.
2161. A method for modulating polypeptide interaction, comprising contacting two polypeptides with an agent identified with the method of claim 2157.
2162. A method for modulating level of a target polypeptide, or for degrading a target polypeptide, comprising contacting the target polypeptide with an agent of claim 2156.
2163. A method for modulating function or level of a target polypeptide, comprising contacting the target polypeptide with an agent of claim 2146.
2164. A method for modulating function or level of a target polypeptide, comprising contacting the target polypeptide with an agent of claim 2147.
2165. A method for modulating function or level of a target polypeptide, comprising contacting the target polypeptide with an agent identified with a method of claim 2157.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Definitions
[0070] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed. Additionally, general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, 5.sup.th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001.
[0071] Administration: As used herein, the term administration typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
[0072] Agent: In general, the term agent, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term agent may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term agent may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety. In some embodiments, an agent is a compound. In some embodiments, an agent is a stapled peptide.
[0073] Aliphatic: As used herein, aliphatic means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation, or combinations thereof. Unless otherwise specified, aliphatic groups contain 1-100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof.
[0074] Alkenyl: As used herein, the term alkenyl refers to an aliphatic group, as defined herein, having one or more double bonds.
[0075] Alkenylene: The term alkenylene refers to a bivalent alkenyl group.
[0076] Alkyl: As used herein, the term alkyl is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C.sub.1-C.sub.20 for straight chain, C.sub.2-C.sub.20 for branched chain), and alternatively, about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C.sub.1-C.sub.4 for straight chain lower alkyls).
[0077] Alkylene: The term alkylene refers to a bivalent alkyl group.
[0078] Amino acid: In its broadest sense, as used herein, refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid comprising an amino group and an a carboxylic acid group. In some embodiments, an amino acid has the structure of NH(R.sup.a1)-L.sup.a1-C(R.sup.a2)(R.sup.a3)-L.sup.a2-COOH, wherein each variable is independently as described in the present disclosure. In some embodiments, an amino acid has the general structure NH(R)C(R).sub.2COOH, wherein each R is independently as described in the present disclosure. In some embodiments, an amino acid has the general structure H.sub.2NC(R).sub.2COOH, wherein R is as described in the present disclosure. In some embodiments, an amino acid has the general structure H.sub.2NC(H)(R)COOH, wherein R is as described in the present disclosure. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, one or more hydrogens, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term amino acid may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
[0079] Analog: As used herein, the term analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an analog shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.
[0080] Animal: As used herein refers to any member of the animal kingdom. In some embodiments, animal refers to humans, of either sex and at any stage of development. In some embodiments, animal refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically engineered animal, and/or a clone.
[0081] Approximately: As used herein, the term approximately or about, as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term approximately or about refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%,8%,7%,6%,5%,4%,3%,2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0082] Aryl: The term aryl used alone or as part of a larger moiety as in aralkyl, aralkoxy, aryloxyalkyl, etc. refers to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. The term aryl may be used interchangeably with the term aryl ring. In certain embodiments of the present disclosure, aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. In some embodiments, also included within the scope of the term aryl, as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like, where a radical or point of attachment is on an aryl ring.
[0083] Associated with: Two events or entities are associated with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., nucleic acid (e.g., genomic DNA, transcripts, mRNA, etc.), polypeptide, genetic signature, metabolite, microbe, etc..) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population).
[0084] Carrier: as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.
[0085] Comparable: As used herein, the term comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.
[0086] Composition: Those skilled in the art will appreciate that the term composition may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any forme.g., gas, gel, liquid, solid, etc.
[0087] Comprising: A composition or method described herein as comprising one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as comprising (or which comprises) one or more named elements or steps also describes the corresponding, more limited composition or method consisting essentially of (or which consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as comprising or consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method consisting of (or consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
[0088] Cycloaliphatic: The term cycloaliphatic, as used herein, refers to saturated or partially unsaturated aliphatic monocyclic, bicyclic, or polycyclic ring systems having, e.g., from 3 to 30, members, wherein the aliphatic ring system is optionally substituted. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons. The terms cycloaliphatic may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where a radical or point of attachment is on an aliphatic ring. In some embodiments, a carbocyclic group is bicyclic. In some embodiments, a carbocyclic group is tricyclic. In some embodiments, a carbocyclic group is polycyclic. In some embodiments, cycloaliphatic (or carbocycle or cycloalkyl) refers to a monocyclic C.sub.3-C.sub.6 hydrocarbon, or a C.sub.8-C.sub.10 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, or a C.sub.9-C.sub.16 tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic.
[0089] Derivative: As used herein, the term derivative refers to a structural analogue of a reference substance. That is, a derivative is a substance that shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, a derivative is a substance that can be generated from the reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.
[0090] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term dosage form may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
[0091] Dosing regimen: Those skilled in the art will appreciate that the term dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
[0092] Halogen: The term halogen means F, Cl, Br, or I.
[0093] Heteroaliphatic: The term heteroaliphatic is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms are replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).
[0094] Heteroalkyl: The term heteroalkyl is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.
[0095] Heteroaryl: The terms heteroaryl and heteroar-, used alone or as part of a larger moiety, e.g., heteroaralkyl, or heteroaralkoxy, refer to monocyclic, bicyclic or polycyclic ring systems having, for example, a total of five to thirty, e.g., 5, 6, 9, 10, 14, etc., ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur. In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl group has 6, 10, or 14 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms heteroaryl and heteroar-, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where a radical or point of attachment is on a heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be monocyclic, bicyclic or polycyclic. The term heteroaryl may be used interchangeably with the terms heteroaryl ring, heteroaryl group, or heteroaromatic, any of which terms include rings that are optionally substituted. The term heteroaralkyl refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.
[0096] Heteroatom: The term heteroatom means an atom that is not carbon and is not hydrogen. In some embodiments, a heteroatom is oxygen, sulfur, nitrogen, phosphorus, boron or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR* (as in N-substituted pyrrolidinyl); etc.). In some embodiments, a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur.
[0097] Heterocyclyl: As used herein, the terms heterocycle, heterocyclyl, heterocyclic radical, and heterocyclic ring are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term nitrogen includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or .sup.+NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms heterocycle, heterocyclyl, heterocyclyl ring, heterocyclic group, heterocyclic moiety, and heterocyclic radical, are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where a radical or point of attachment is on a heteroaliphatic ring. A heterocyclyl group may be monocyclic, bicyclic or polycyclic. The term heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
[0098] Homology: As used herein, the term homology refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be homologous to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be homologous to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as hydrophobic or hydrophilic amino acids, and/or as having polar or non-polar side chains. Substitution of one amino acid for another of the same type may often be considered a homologous substitution. Typical amino acid categorizations are summarized below:
TABLE-US-00001 Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive 4.5 Asparagine Asn N polar neutral 3.5 Aspartic acid Asp D polar negative 3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu E polar negative 3.5 Glutamine Gln Q polar neutral 3.5 Glycine Gly G nonpolar neutral 0.4 Histidine His H polar positive 3.2 Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys K polar positive 3.9 Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral 1.6 Serine Ser S polar neutral 0.8 Threonine Thr T polar neutral 0.7 Tryptophan Trp W nonpolar neutral 0.9 Tyrosine Tyr Y polar neutral 1.3 Valine Val V nonpolar neutral 4.2
TABLE-US-00002 Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or aspartic acid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa X
[0099] As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues correspond to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
[0100] Improved, increased or reduced: As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be improved relative to that obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject or system of interest may be improved relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.
[0101] Partially unsaturated: As used herein, the term partially unsaturated refers to a moiety that includes at least one double or triple bond. The term partially unsaturated is intended to encompass groups having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties.
[0102] Peptide: The term peptide as used herein refers to a polypeptide that is typically relatively short, for example having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids.
[0103] Pharmaceutical composition: As used herein, the term pharmaceutical composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
[0104] Pharmaceutically acceptable: As used herein, the phrase pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0105] Pharmaceutically acceptable carrier: As used herein, the term pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; RingeR's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.
[0106] Pharmaceutically acceptable salt: The term pharmaceutically acceptable salt, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other known methods such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic base addition salts, such as those formed by acidic groups of provided compounds (e.g., phosphate linkage groups of oligonucleotides, phosphorothioate linkage groups of oligonucleotides, etc.) with bases. Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts are ammonium salts (e.g., N(R).sub.3/). In some embodiments, pharmaceutically acceptable salts are sodium salts. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
[0107] Polypeptide: As used herein refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term polypeptide may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
[0108] Prevent or prevention: as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
[0109] Protecting Group: The phrase protecting group, as used herein, refers to temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. A Si protecting group is a protecting group comprising a Si atom, such as Si-trialkyl (e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g., t-butyldiphenylsilyl), or Si-aryl-dialkyl (e.g., Si-phenyldialkyl). Generally, a Si protecting group is attached to an oxygen atom. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). Such protecting groups (and associated protected moieties) are described in detail below.
[0110] Protected hydroxyl groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitably protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, propionates, pentanoates, crotonates, and benzoates. Specific examples of suitable esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benzylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.
[0111] Protected amines are well known in the art and include those described in detail in Greene (1999). Suitable mono-protected amines further include, but are not limited to, aralkylamines, carbamates, allyl amines, amides, and the like. Examples of suitable mono-protected amino moieties include t-butyloxycarbonylamino (NHBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (NHAlloc), benzyloxocarbonylamino (NHCBZ), allylamino, benzylamino (NHBn), fluorenylmethylcarbonyl (NHFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like. Suitable di-protected amines include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides, such as phthalimide, maleimide, succinimide, and the like. Suitable di-protected amines also include pyrroles and the like, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.
[0112] Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Suitable protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes, semicarbazones, and derivatives thereof.
[0113] Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Suitable protected carboxylic acids further include, but are not limited to, optionally substituted C.sub.1-6 aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, and the like. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, wherein each group is optionally substituted. Additional suitable protected carboxylic acids include oxazolines and ortho esters.
[0114] Protected thiols are well known in the art and include those described in detail in Greene (1999). Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioester, to name but a few.
[0115] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
[0116] Substitution: As described herein, compounds of the disclosure may contain optionally substituted and/or substituted moieties. In general, the term substituted, whether preceded by the term optionally or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an optionally substituted group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term stable, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, example substituents are described below.
[0117] Suitable monovalent substituents include halogen; (CH.sub.2).sub.0-4R.sup.; (CH.sub.2).sub.0-4OR.sup.; O(CH.sub.2).sub.0-4R.sup., O(CH.sub.2).sub.0-4C(O)OR.sup.; (CH.sub.2).sub.0-4CH(OR.sup.).sub.2; (CH.sub.2).sub.0-4Ph, which may be substituted with R.sup.; (CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1Ph which may be substituted with R.sup.; CHCHPh, which may be substituted with R.sup.; (CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1-pyridyl which may be substituted with R.sup.; NO.sub.2; CN; N.sub.3; (CH.sub.2).sub.0-4N(R.sup.).sub.2; (CH.sub.2).sub.0-4N(R.sup.)C(O)R.sup.; N(R.sup.)C(S)R.sup.; (CH.sub.2).sub.0-4N(R.sup.)C(O)NR.sup..sub.2; N(R.sup.)C(S)NR.sup..sub.2; (CH.sub.2).sub.0-4N(R.sup.)C(O)OR.sup.; N(R.sup.)N(R.sup.)C(O)R.sup.; N(R.sup.)N(R.sup.)C(O)NR.sup..sub.2; N(R.sup.)N(R.sup.)C(O)OR.sup.; (CH.sub.2).sub.0-4C(O)R.sup.; C(S)R.sup.; (CH.sub.2).sub.0-4C(O)OR.sup.; (CH.sub.2).sub.0-4C(O)SR.sup.; (CH.sub.2).sub.0-4C(O)OSiR.sup..sub.3; (CH.sub.2).sub.0-4OC(O)R.sup.; OC(O)(CH.sub.2).sub.0-4SR, SC(S)SR.sup.; (CH.sub.2).sub.0-4SC(O)R.sup.; (CH.sub.2).sub.0-4C(O)NR.sup..sub.2; C(S)NR.sup..sub.2; C(S)SR.sup.; SC(S)SR.sup., (CH.sub.2).sub.0-4OC(O)NR.sup..sub.2; C(O)N(OR.sup.)R.sup.; C(O)C(O)R.sup.; C(O)CH.sub.2C(O)R.sup.; C(NOR.sup.)R.sup.; (CH.sub.2).sub.0-4SSR.sup.; (CH.sub.2).sub.0-4S(O).sub.2R.sup.; (CH.sub.2).sub.0-4S(O).sub.2OR.sup.; (CH.sub.2).sub.0-4OS(O).sub.2R.sup.; S(O).sub.2NR.sup..sub.2; (CH.sub.2).sub.0-4S(O)R.sup.; N(R.sup.)S(O).sub.2NR.sup..sub.2; N(R.sup.)S(O).sub.2R.sup.; N(OR.sup.)R.sup.; C(NH)NR.sup..sub.2; P(O).sub.2R.sup.; P(O)R.sup..sub.2; OP(O)R.sup..sub.2; OP(O)(OR.sup.).sub.2; SiR.sup..sub.3; OSiR.sup..sub.3; (C.sub.1-4 straight or branched alkylene)ON(R.sup.).sub.2; or (C.sub.1-4 straight or branched alkylene)C(O)ON(R.sup.).sub.2, wherein each R.sup. may be substituted as defined below and is independently hydrogen, C.sub.1-10 (e.g., C.sub.1-9, C.sub.1-6, C.sub.1-5, C.sub.1-4, etc.) aliphatic, C.sub.1-10 (e.g., C.sub.1-9, C.sub.1-6, C.sub.1-5, C.sub.1-4, etc.) heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, CH.sub.2(C.sub.6-14 (e.g., C.sub.6-10, C.sub.6, etc.) aryl), O(CH.sub.2).sub.0-1(C.sub.6-14 (e.g., C.sub.6-10, C.sub.6, etc.) aryl), CH.sub.2-(5-14 (e.g., 5-10, 5-6, 5, 6, 9, 10, 14, etc.) membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur), a 3-10 (e.g., 3-9, 3-7, 3-6, 5-10, 5-6, etc.) membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R.sup., taken together with their intervening atom(s), form a 3-10 (e.g., 3-9, 3-7, 3-6, 5-10, 5-6, etc.) membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.
[0118] Suitable monovalent substituents on R.sup. (or the ring formed by taking two independent occurrences of R.sup. together with their intervening atoms), are independently halogen, (CH.sub.2).sub.0-2R.sup..circle-solid., -(haloR.sup..circle-solid.), (CH.sub.2).sub.0-2OH, (CH.sub.2).sub.0-2OR.sup..circle-solid., (CH.sub.2).sub.0-2CH(OR.sup..circle-solid.).sub.2; O(haloR.sup..circle-solid.), CN, N.sub.3, (CH.sub.2).sub.0-2C(O)R.sup..circle-solid., (CH.sub.2).sub.0-2C(O)OH, (CH.sub.2).sub.0-2C(O)OR.sup..circle-solid., (CH.sub.2).sub.0-2SR.sup..circle-solid., (CH.sub.2).sub.0-2SH, (CH.sub.2).sub.0-2NH.sub.2, (CH.sub.2).sub.0-2NHR.sup..circle-solid., (CH.sub.2).sub.0-2NR.sup..circle-solid..sub.2, NO.sub.2, SiR.sup..circle-solid..sub.3, OSiR.sup..circle-solid..sub.3, C(O)SR.sup..circle-solid., (C.sub.1-4 straight or branched alkylene)C(O)OR.sup..circle-solid., or SSR.sup..circle-solid. wherein each R.sup..circle-solid. is unsubstituted or where preceded by halo is substituted only with one or more halogens, and is independently selected from C.sub.1-4 aliphatic, CH.sub.2Ph, O(CH.sub.2).sub.0-1Ph, or a 3-6 (e.g., 4-6, 5-6, etc.) membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R.sup. include O and S.
[0119] Suitable divalent substituents include the following: O, S, NNR*.sub.2, NNHC(O)R*, NNHC(O)OR*, NNHS(O).sub.2R*, NR*, NOR*, O(C(R*.sub.2)).sub.2-3O, or S(C(R*.sub.2)).sub.2-3S, wherein each independent occurrence of R* is selected from hydrogen, C.sub.1-6 aliphatic which may be substituted as defined below, or an unsubstituted 3-6 (e.g., 4-6, 5-6, etc.) membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an optionally substituted group include: O(CR*.sub.2).sub.2-3O, wherein each independent occurrence of R* is selected from hydrogen, C.sub.1-6 aliphatic which may be substituted as defined below, or an unsubstituted 3-6 (e.g., 4-6, 5-6, etc.) membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0120] Suitable substituents on the aliphatic group of R* include halogen, R.sup..circle-solid., -(haloR.sup..circle-solid.), OH, OR.sup..circle-solid., O(haloR.sup..circle-solid.), CN, C(O)OH, C(O)OR.sup..circle-solid., NH.sub.2, NHR.sup..circle-solid., NR.sup..circle-solid..sub.2, or NO.sub.2, wherein each R.sup..circle-solid. is unsubstituted or where preceded by halo is substituted only with one or more halogens, and is independently C.sub.1-4 aliphatic, CH.sub.2Ph, O(CH.sub.2).sub.0-1Ph, or a 3-6 (e.g., 4-6, 5-6, etc.) membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0121] In some embodiments, suitable substituents on a substitutable nitrogen include R.sup., NR.sup..sub.2, C(O)R.sup., C(O)OR.sup., C(O)C(O)R.sup., C(O)CH.sub.2C(O)R.sup., S(O).sub.2R.sup., S(O).sub.2NR.sup..sub.2, C(S)NR.sup..sub.2, C(NH)NR.sup..sub.2, or N(R.sup.)S(O).sub.2R.sup.; wherein each R.sup. is independently hydrogen, C.sub.1-6 aliphatic which may be substituted as defined below, unsubstituted OPh, or an unsubstituted 3-6 (e.g., 4-6, 5-6, etc.) membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R.sup., taken together with their intervening atom(s) form an unsubstituted 3-12 (e.g., 3-10, 3-7, 3-6, 5-10, 5-7, 5-6, etc.) membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0122] Suitable substituents on the aliphatic group of R.sup. are independently halogen, R.sup..circle-solid., -(haloR.sup..circle-solid.), OH, OR.sup..circle-solid., O(haloR.sup..circle-solid.), CN, C(O)OH, C(O)OR.sup..circle-solid., NH.sub.2, NHR.sup..circle-solid., NR.sup..circle-solid..sub.2, or NO.sub.2, wherein each R.sup..circle-solid. is unsubstituted or where preceded by halo is substituted only with one or more halogens, and is independently C.sub.1-4 aliphatic, CH.sub.2Ph, O(CH.sub.2).sub.0-1Ph, or a 3-6 (e.g., 4-6, 5-6, etc.) membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[0123] Subject: As used herein, the term subject or test subject refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition. In some embodiments, a subject is a human.
[0124] Susceptible to: An individual who is susceptible to a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
[0125] Therapeutic agent: As used herein, the phrase therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
[0126] Therapeutic regimen: A therapeutic regimen, as that term is used herein, refers to a dosing regimen whose administration across a relevant population may be correlated with a desired or beneficial therapeutic outcome.
[0127] Therapeutically effective amount: As used herein, the term therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
[0128] Treat: As used herein, the term treat, treatment, or treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
[0129] Unit dose: The expression unit dose as used herein refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined quantity of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect. A unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described infra. It will be appreciated by those skilled in the art, in many embodiments, a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.
[0130] Unsaturated: The term unsaturated as used herein, means that a moiety has one or more units of unsaturation.
[0131] Wild-type: As used herein, the term wild-type has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a normal (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).
[0132] Unless otherwise specified, salts, such as pharmaceutically acceptable acid or base addition salts, stereoisomeric forms, and tautomeric forms, of provided compound are included. As appreciated by those skilled in the art, agents, compounds, etc. may be provided and/or utilized as various forms including various pharmaceutically acceptable salt forms, solvate forms, etc.
[0133] Unless otherwise clear from context, in the present disclosure, (i) the term a may be understood to mean at least one; (ii) the term or may be understood to mean and/or; (iii) the terms comprising and including may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms about and approximately may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.
Peptide Agents
[0134] Technologies provided by and/or described in the present disclosure particularly relate to peptide agents, e.g., to agents that are or comprise stapled peptides.
[0135] Among other things, the present disclosure provides technologies for developing, identifying, characterizing, and/or making stapled peptides that can modulate one or more functions of a target of interest. In some embodiments, stapled peptides are useful for treating various conditions, disorders, and/or diseases that are associated with the target of interest. Exemplary structural elements of provided stapled peptides are described herein.
[0136] Among other things, as discussed herein, the present disclosure encompasses the insight that biological systems can be utilized to generate peptide agents that include cysteine residues appropriate for and/or amenable to cysteine stapling. The present disclosure further appreciates that use of such biological systems can permit high-throughput production and/or assessment of cysteine stapled peptides (e.g., with respect to one or more (e.g., specificity, affinity, on-rate, off-rate, stability to competition, binding curve over a range of conditions such as concentration, temperature, pH, osmolality, presence or amount of competitor, etc.) characteristics of their binding interaction(s) with one or more targets of interest.
[0137] Thus, in some embodiments, the present disclosure provides peptide agents that include two or more cysteine residues, spaced apart from one another appropriately so as to support cysteine stapling. In some embodiments, provided are collections of such peptide agents. In some embodiments, provided are cysteine stapled peptides and/or collections thereof.
[0138] The present disclosure further appreciates that insights gleaned from producing, screening and/or otherwise analyzing or characterizing one or more cysteine stapled peptides can inform design, production, and/or use of analogous or comparable (e.g., containing the same or substantially the same [e.g., but for one or more conservative substitutions and/or one or a small number of other changes] amino acid sequence except for substitution of those cysteine residue(s) that participate in a staple with non-cysteine residue(s) that can or do participate in an analogous or comparable staple) peptide agents that share with their parent cysteine stapled peptide one or more binding characteristics with a particular target of interest. The present disclosure therefore provides peptide agents (e.g., that are or comprise stapled peptides) that correspond to cysteine stapled peptides (but include non-cysteine residue(s) rather than cysteines that participate in the staple).
[0139] In some embodiments, peptides are stapled through non-cysteine residues.
[0140] In some embodiments, peptide agents are prepared utilizing chemical synthesis technologies. Various technologies for preparing peptides, e.g., solid phase synthesis, may be utilized in accordance with the present disclosure.
[0141] Certain useful technologies for preparing, identifying, characterizing, and using peptide agents and/or collections thereof, including peptide synthesis, staples, etc., are described in U.S. Ser. No. 11/198,713, US 20210179665, WO 2021119537, WO 2021188659, WO 2022020651, or WO 2022020652, the entirety of each of which is incorporated herein by reference.
Amino Acid Sequence
[0142] One particular advantage of technologies provided by the present disclosure is that they permit discovery of and/or define amino acid sequences that are particularly useful for stapled peptides (e.g., that bind to a particular target of interest).
[0143] Thus, in some embodiments, the present disclosure provides amino acid sequences for stapled peptides. In some embodiments, stapled peptides comprising provided amino acid sequences interact with (e.g., directly bind to) a target of interest, and, in some embodiments, such binding displays one or more characteristics as discussed herein.
[0144] As will be appreciated by those skilled in the art reading the present disclosure, in some embodiments, the present disclosure defines useful amino acid sequences from a collection that may be highly diversee.g., that may include two or more amino acid alternatives at any one or collection (including all) of positions along the amino acid chain, except for those that participate in a staple which, in many embodiments, are cysteines.
[0145] In some embodiments, amino acid sequences utilized in peptide agents as described herein may be or comprises, or be derived from, a sequence that is found in nature or in an otherwise appropriate reference polypeptide (e.g., one that may be known to bind to a relevant target of interest, for example via an interaction characterized by one or more features as described herein.
[0146] In some embodiments, an amino acid sequence that is utilized in a peptide agent, or in a collection of peptide agents, as described herein, is a variant of a reference sequence in that (1) it includes a pair of cysteine residues, at least one or which is not found at a corresponding position in the reference sequence, that are amenable to or participate in a cysteine staple; and/or (2) it includes an amino acid substitution at one or more positions of the reference sequence. In some embodiments, a substitution may be a conservative substitution, as understood in the art. In some embodiments, a substitution may involve substitution of a homolog. In some embodiments, a homolog of an amino acid is a naturally occurring or non-naturally occurring amino acid that has one or more similar properties to the amino acid and or amino acid side-chains being replaced, for example, that is typically classified as similar to one another as non-polar, polar, hydrophobic, hydrophilic, basic, acidic, aliphatic, aromatic, and/or similar size.
[0147] For example, in some embodiments, depending on context, a homolog of leucine can be an optionally substituted amino acid selected from isoleucine, alanine, homoleucine, 3-cyclobutylalanine, alpha-neopentylglycine, 3-cyclopropylalanine, alpha-methylleucine, and 3-cyclohexylalanine; a homolog of isoleucine can be an optionally substituted amino acid selected from alanine, leucine, homoleucine, 3-cyclobutylalanine, alpha-neopentylglycine, 3-cyclopropylalanine, L-alloisoleucine, and alpha-methylleucine; a homolog of phenylalanine can be an optionally substituted amino acid residue selected from tryptophan, tyrosine, 3-(1-naphthylalanine), 3-(2-naphthylalanine), 2-chlorophenyalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 4-tert-butylphenylalanine, O-methyl tyrosine, homophenylalanine, 4-fluorophenylalanine, 4-methylphenylalanine, 4-bromophenylalanine, 4-phenyl-L-phenylalanine, 5-chlorotryptophan, 5-hydroxytryptophan, 4-trifluoromethylphenylalanine, 4-guanidino-L-phenylalanine, 2-quinolyl-L-alanine, 3-cyclobutylalanine, alpha-neopentylglycine, and L-2-aminoadipic acid; etc.
[0148] In some embodiments, a homolog of a non-polar amino acid is another non-polar amino acid. In some embodiments, a homolog of an amino acid comprising a non-polar side chain is another non-polar amino acid comprising a non-polar side chain.
[0149] In some embodiments, a homolog of a polar amino acid is another polar amino acid. In some embodiments, a homolog of an amino acid comprising a polar side chain is another polar amino acid comprising a polar side chain. In some embodiments, side chain of a polar amino acid is not charged at about pH 7.4. In some embodiments, side chain of a polar amino acid does not contain a basic or acidic group. In some embodiments, side chain of a polar amino acid comprises OH. In some embodiments, side chain of a polar amino acid comprises an amide group.
[0150] In some embodiments, a homolog of a hydrophobic amino acid is another hydrophobic amino acid. In some embodiments, a homolog of an amino acid comprising a hydrophobic side chain is another amino acid comprising a hydrophobic side chain. In some embodiments, a hydrophobic side chain is optionally substituted C.sub.1-6 aliphatic, wherein each substituent, if any, is a non-polar group. In some embodiments, a hydrophobic side chain is C.sub.1-6 aliphatic. In some embodiments, a hydrophobic side chain is C.sub.1-6 haloaliphatic. In some embodiments, a hydrophobic side chain is C.sub.1-6 alkyl. In some embodiments, a hydrophobic side chain is C.sub.1-6 haloalkyl.
[0151] In some embodiments, a homolog of a hydrophilic amino acid is another hydrophilic amino acid. In some embodiments, a homolog of an amino acid comprising a hydrophilic side chain is another hydrophilic amino acid comprising a hydrophilic side chain.
[0152] In some embodiments, a homolog of a basic amino acid is another basic amino acid. In some embodiments, a homolog of an amino acid comprising a basic side chain is another basic amino acid comprising a basic side chain. In some embodiments, a homolog of an amino acid comprising a side chain comprising a basic group, e.g., an amino group, a guanidine group, etc., is another amino acid comprising a side chain comprising a basic group.
[0153] In some embodiments, a homolog of an acidic amino acid is another acidic amino acid. In some embodiments, a homolog of an amino acid comprising an acidic side chain is another acidic amino acid comprising an acidic side chain. In some embodiments, a homolog of an amino acid comprising a side chain comprising an acidic group, e.g., COOH, is another amino acid comprising a side chain comprising an acidic group.
[0154] In some embodiments, a homolog of an aliphatic amino acid is another aliphatic amino acid. In some embodiments, a homolog of an amino acid comprising an aliphatic side chain is another aliphatic amino acid comprising an aliphatic side chain.
[0155] In some embodiments, a homolog of an aromatic amino acid is another aromatic amino acid. In some embodiments, a homolog of an amino acid comprising an aromatic side chain is another aromatic amino acid comprising an aromatic side chain. In some embodiments, a homolog of an amino acid comprising a side chain comprising an aromatic group, e.g., phenyl, heteroaryl, etc., is another amino acid comprising a side chain comprising an aromatic group.
[0156] In some embodiments, a homolog of an amino acid is sterically similar to the amino acid. In some embodiments, a homolog of an amino acid comprises a side chain that has a similar size to the side chain of the amino acid.
[0157] Provided amino acid sequences and stapled peptides can be various lengths, e.g. 2-100, 5-50, 5-40, 5-35, a range from and including 2, 3, 4, 5, 6, or 7 to and including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues.
[0158] In some embodiments, a length is at least 5 amino acid residues. In some embodiments, a length is at least 6 amino acid residues. In some embodiments, a length is at least 7 amino acid residues. In some embodiments, a length is at least 8 amino acid residues. In some embodiments, a length is at least 9 amino acid residues. In some embodiments, a length is at least 10 amino acid residues. In some embodiments, a length is at least 11 amino acid residues. In some embodiments, a length is at least 12 amino acid residues. In some embodiments, a length is at least 13 amino acid residues. In some embodiments, a length is at least 14 amino acid residues. In some embodiments, a length is at least 15 amino acid residues. In some embodiments, a length is at least 16 amino acid residues. In some embodiments, a length is at least 17 amino acid residues. In some embodiments, a length is at least 18 amino acid residues. In some embodiments, a length is at least 19 amino acid residues. In some embodiments, a length is at least 20 amino acid residues. In some embodiments, a length is at least 21 amino acid residues. In some embodiments, a length is at least 22 amino acid residues. In some embodiments, a length is at least 23 amino acid residues. In some embodiments, a length is at least 24 amino acid residues. In some embodiments, a length is at least 25 amino acid residues. In some embodiments, a length is at least 26 amino acid residues. In some embodiments, a length is at least 27 amino acid residues. In some embodiments, a length is at least 28 amino acid residues. In some embodiments, a length is at least 29 amino acid residues. In some embodiments, a length is at least 30 amino acid residues. In some embodiments, a length is at least 31 amino acid residues. In some embodiments, a length is at least 32 amino acid residues. In some embodiments, a length is at least 33 amino acid residues. In some embodiments, a length is at least 34 amino acid residues. In some embodiments, a length is at least 35 amino acid residues.
[0159] In some embodiments, a length is 5 amino acid residues. In some embodiments, a length is 6 amino acid residues. In some embodiments, a length is 7 amino acid residues. In some embodiments, a length is 8 amino acid residues. In some embodiments, a length is 9 amino acid residues. In some embodiments, a length is 10 amino acid residues. In some embodiments, a length is 11 amino acid residues. In some embodiments, a length is 12 amino acid residues. In some embodiments, a length is 13 amino acid residues. In some embodiments, a length is 14 amino acid residues. In some embodiments, a length is 15 amino acid residues. In some embodiments, a length is 16 amino acid residues. In some embodiments, a length is 17 amino acid residues. In some embodiments, a length is 18 amino acid residues. In some embodiments, a length is 19 amino acid residues. In some embodiments, a length is 20 amino acid residues. In some embodiments, a length is 21 amino acid residues. In some embodiments, a length is 22 amino acid residues. In some embodiments, a length is 23 amino acid residues. In some embodiments, a length is 24 amino acid residues. In some embodiments, a length is 25 amino acid residues. In some embodiments, a length is 26 amino acid residues. In some embodiments, a length is 27 amino acid residues. In some embodiments, a length is 28 amino acid residues. In some embodiments, a length is 29 amino acid residues. In some embodiments, a length is 30 amino acid residues. In some embodiments, a length is 31 amino acid residues. In some embodiments, a length is 32 amino acid residues. In some embodiments, a length is 33 amino acid residues. In some embodiments, a length is 34 amino acid residues. In some embodiments, a length is 35 amino acid residues.
[0160] In some embodiments, a length is no more than 17 amino acid residues. In some embodiments, a length is no more than 18 amino acid residues. In some embodiments, a length is no more than 19 amino acid residues. In some embodiments, a length is no more than 20 amino acid residues. In some embodiments, a length is no more than 21 amino acid residues. In some embodiments, a length is no more than 22 amino acid residues. In some embodiments, a length is no more than 23 amino acid residues. In some embodiments, a length is no more than 24 amino acid residues. In some embodiments, a length is no more than 25 amino acid residues. In some embodiments, a length is no more than 26 amino acid residues. In some embodiments, a length is no more than 27 amino acid residues. In some embodiments, a length is no more than 28 amino acid residues. In some embodiments, a length is no more than 29 amino acid residues. In some embodiments, a length is no more than 30 amino acid residues. In some embodiments, a length is no more than 35 amino acid residues. In some embodiments, a length is no more than 40 amino acid residues. In some embodiments, a length is no more than 50 amino acid residues.
[0161] Both naturally occurring and non-naturally occurring amino acids can be utilized in accordance with the present disclosure. In some embodiments, an amino acid is a compound comprising an amino group that can form an amide group with a carboxyl group and a carboxyl group.
[0162] In some embodiments, an amino acid is a compound having the structure of formula A-I:
NH(R.sup.a1)L.sup.a1-C(R.sup.a2)(R.sup.a3)-L.sup.a2-COOH, A-I
or a salt thereof, wherein: [0163] each of R.sup.a1, R.sup.a2, R.sup.a3 is independently -L.sup.a-R; [0164] each of L.sup.a, L.sup.a1 and L.sup.a2 is independently L; [0165] each L is independently a covalent bond, or an optionally substituted, bivalent C.sub.1-C.sub.25 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O; [0166] each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; [0167] each R is independently R, C(O)R, CO.sub.2R, or SO.sub.2R; [0168] each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or [0169] two R groups are optionally and independently taken together to form a covalent bond, or: [0170] two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or [0171] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0172] In some embodiments, L.sup.a1 is a covalent bond. In some embodiments, a compound of formula A-1 is of the structure NH(R.sup.a1)C(R.sup.a2)(R.sup.a3)-L.sup.a2-COOH.
[0173] In some embodiments, L.sup.a2 is a covalent bond. In some embodiments, a compound of formula A-1 is of the structure NH(R.sup.a1)C(R.sup.a2)(R.sup.a3)-L.sup.a2-COOH.
[0174] In some embodiments, L.sup.a1 is a covalent bond and L.sup.a2 is a covalent bond. In some embodiments, a compound of formula A-1 is of the structure NH(R.sup.a1)C(R.sup.a2)(R.sup.a3)COOH.
[0175] In some embodiments, L.sup.a is a covalent bond. In some embodiments, R is R. In some embodiments, R.sup.a1 is R, wherein R is as described in the present disclosure. In some embodiments, R.sup.a2 is R, wherein R is as described in the present disclosure. In some embodiments, R.sup.a3 is R, wherein R is as described in the present disclosure. In some embodiments, each of R.sup.a1, R.sup.a2, and R.sup.a3 is independently R, wherein R is as described in the present disclosure.
[0176] In some embodiments, R.sup.a1 is hydrogen. In some embodiments, R.sup.a2 is hydrogen. In some embodiments, R.sup.a3 is hydrogen. In some embodiments, R.sup.a1 is hydrogen, and at least one of R.sup.a2 and R.sup.a3 is hydrogen. In some embodiments, R.sup.a1 is hydrogen, one of R.sup.a2 and R.sup.a3 is hydrogen, and the other is not hydrogen.
[0177] In some embodiments, R.sup.a2 is -L.sup.a-R, wherein R is as described in the present disclosure. In some embodiments, R.sup.a2 is -L.sup.a-R, wherein R is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.5-30 aryl, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R.sup.a2 is -L.sup.a-R, wherein R is an optionally substituted group selected from C.sub.6-30 aryl and 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R.sup.a2 is a side chain of an amino acid. In some embodiments, R.sup.a2 is a side chain of a standard amino acid.
[0178] In some embodiments, R.sup.a3 is -L.sup.a-R, wherein R is as described in the present disclosure. In some embodiments, R.sup.a3 is -L.sup.a-R, wherein R is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.5-30 aryl, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R.sup.a3 is -L.sup.a-R, wherein R is an optionally substituted group selected from C.sub.6-30 aryl and 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R.sup.a3 is a side chain of an amino acid. In some embodiments, R.sup.a3 is a side chain of a standard amino acid.
[0179] An amino acid sequence can have various degrees of homology to naturally occurring amino acid sequences. As appreciated by those skilled in the art, the present disclosure can be utilized to design and identify sequences with either high or low homology, or no homology (e.g., sequences completely unrelated to any sequences encoded in a genome). In some embodiments, the present disclosure provides technologies for identifying useful amino acid sequences that share low degree of homology to naturally occurring amino acid sequences, e.g., from libraries designed with many random positions compared to natural amino acid sequences, or from totally random libraries. In some embodiments, an amino acid sequence is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% homologous to naturally occurring amino acid sequence. In some embodiments, an amino acid sequence is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% homologous to naturally occurring amino acid sequence. In some embodiments, the homology is at least 5%. In some embodiments, the homology is at least 10%. In some embodiments, the homology is at least 20%. In some embodiments, the homology is at least 30%. In some embodiments, the homology is at least 40%. In some embodiments, the homology is at least 50%. In some embodiments, the homology is at least 60%. In some embodiments, the homology is at least 70%. In some embodiments, the homology is at least 80%. In some embodiments, the homology is at least 90%. In some embodiments, the homology is at least 95%. In some embodiments, the homology is no more than 5%. In some embodiments, the homology is no more than 10%. In some embodiments, the homology is no more than 20%. In some embodiments, the homology is no more than 30%. In some embodiments, the homology is no more than 40%. In some embodiments, the homology is no more than 50%. In some embodiments, the homology is no more than 60%. In some embodiments, the homology is no more than 70%. In some embodiments, the homology is no more than 80%. In some embodiments, the homology is no more than 90%. In some embodiments, the homology is no more than 95%.
[0180] An amino acid sequence can be various lengths. In some embodiments, a length is or comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 amino acid residues. In some embodiments, a length is or comprises at least 5 amino acid residues. In some embodiments, a length is or comprises at least 6 amino acid residues. In some embodiments, a length is or comprises at least 7 amino acid residues. In some embodiments, a length is or comprises at least 8 amino acid residues. In some embodiments, a length is or comprises at least 9 amino acid residues. In some embodiments, a length is or comprises at least 10 amino acid residues. In some embodiments, a length is or comprises at least 11 amino acid residues. In some embodiments, a length is or comprises at least 12 amino acid residues. In some embodiments, a length is or comprises at least 13 amino acid residues. In some embodiments, a length is or comprises at least 14 amino acid residues. In some embodiments, a length is or comprises at least 15 amino acid residues. In some embodiments, a length is or comprises at least 16 amino acid residues. In some embodiments, a length is or comprises at least 17 amino acid residues. In some embodiments, a length is or comprises at least 18 amino acid residues. In some embodiments, a length is or comprises at least 18 amino acid residues. In some embodiments, a length is or comprises at least 20 amino acid residues. In some embodiments, a length is or comprises at least 25 amino acid residues. In some embodiments, a length is or comprises at least 30 amino acid residues.
[0181] As demonstrated herein, provided technologies can be utilized with or without a starting amino acid sequence (e.g., either totally random libraries or libraries comprising certain pre-determined amino acid residues at certain positions) to identify useful amino acid sequences with desired properties and/or activities. In some embodiments, the present disclosure provide useful amino acid sequences for modulating targets, e.g., proteins associated with various conditions, disorders or diseases. In some embodiments, a provided amino acid sequence comprises X.sub.s1X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.s2, wherein X.sub.s1 and X.sub.s2 are amino acid residues connected via a staple (stapled) or suitable for stapling, and each of X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, and X.sub.6 is independently an amino acid residue. In some embodiments, X.sub.3 and X.sub.4 are A, and X.sub.5 is H. In some embodiments, X.sub.2 is L, X.sub.3 and X.sub.4 are A, and X.sub.5 is H. In some embodiments, X.sub.2 is L. In some embodiments, X.sub.1 is I. In some embodiments, a provided amino acid sequence comprises X.sub.s1X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.s2X.sub.7X.sub.8, wherein X.sub.s1 and X.sub.s2 are amino acid residues connected via a staple (stapled) or suitable for stapling, and each of X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, and X.sub.8 is independently an amino acid residue. In some embodiments, X.sub.3 and X.sub.4 are A, and X.sub.6 is H. In some embodiments, X.sub.2 is an aromatic amino acid residue, X.sub.3 and X.sub.4 are A, and X.sub.6 is H. In some embodiments, X.sub.2 is W. In some embodiments, X.sub.1 is E. In some embodiments, X.sub.7 is an acidic amino acid residue. In some embodiments, X.sub.7 is E. In some embodiments, X.sub.8 is L. In some embodiments, a provided amino acid sequence comprises X.sub.2X.sub.1X.sub.s1X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.s2X.sub.7, wherein X.sub.s1 and X.sub.s2 are amino acid residues connected via a staple, and each of X.sub.2, X.sub.1, X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, and X.sub.7 is independently an amino acid residue. In some embodiments, X.sub.2 is H, X.sub.3 is A, and X.sub.4 is A. In some embodiments, X.sub.2 is W. In some embodiments, X.sub.1 is an acidic amino acid residue. In some embodiments, X.sub.1 is E. In some embodiments, X.sub.1 is D. In some embodiments, X.sub.1 is an acidic amino acid residue. In some embodiments, X.sub.1 is E. In some embodiments, X.sub.1 is D. In some embodiments, X.sub.5 is L or I. In some embodiments, X.sub.5 is L. In some embodiments, X.sub.5 is I. In some embodiments, X.sub.6 is L or I. In some embodiments, X.sub.6 is L. In some embodiments, X.sub.6 is I. In some embodiments, X.sub.7 is an acidic amino acid residue. In some embodiments, X.sub.7 is E. In some embodiments, X.sub.7 is D.
[0182] Certain useful amino acids, including those useful for stapling or are stapled, are described in U.S. Ser. No. 11/198,713, US 20210179665, WO 2021119537, WO 2021188659, WO 2022020651, or WO 2022020652, the entirety of each of which is incorporated herein by reference.
Peptide Libraries/Collections
[0183] In some embodiments, the present disclosure provides collections of peptides. In some embodiments, a collection of peptides is a collection of stapled peptides. In some embodiments, peptides within a collection as described herein may share one or more structural features (e.g. length within a particular range; particular lengths, presence of particular sequence elements such as, for example, a sequence element found in a known interaction partner for a target of interest, a set of amino acids interacting with a target of interest, or a set of amino acids that together support formation of a staple [e.g., two or more cysteine residues, positioned relative to one another so that a cysteine staple as described herein is or may be produced between a pair of them], presence of one or more staples which may, in some embodiments, be of the same type, etc., or any combination thereof, and in some embodiments, a particular collection may be characterized and/or defined by such shared structural feature(s)). In some embodiments, a common structural feature of peptides in a collection of peptides as described herein is at least two stapled residues, or at least two residues suitable for stapling, positioned relative to one another so that a staple as described herein is or may be formed between a pair of them. In some embodiments, two residues are cysteine residues. In some embodiments, such peptides can be reacted with a compound of formula R-I, to produce a collection of stapled peptides.
[0184] In some embodiments, a collection of peptides is a collection of stapled peptides, each of which independently has an amino acid sequence that: [0185] has a length within a range of a* and b*, where a* and b* are each integers independently selected from 2 through 100 and b* is greater than a*; and [0186] includes at least one pair of residues covalently linked with one another via a linker.
[0187] In some embodiments, a* and b* are each integers independently selected from 2 through 50 and b* is greater than a*. In some embodiments, a* and b* are each integers independently selected from 6 through 36 and b* is greater than a*.
[0188] In some embodiments, peptides in a collection are of the same or about the same length. In some embodiments, about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% peptides of a collection are of the same length. In some embodiments, a length is about a* as described herein. In some embodiments, a length is about b* as described herein.
[0189] In some embodiments, a* is 1. In some embodiments, a* is 2. In some embodiments, a* is 3. In some embodiments, a* is 4. In some embodiments, a* is 5. In some embodiments, a* is 6. In some embodiments, a* is 7. In some embodiments, a* is 8. In some embodiments, a* is 9. In some embodiments, a* is 10. In some embodiments, a* is 11. In some embodiments, a* is 12. In some embodiments, a* is 13. In some embodiments, a* is 14. In some embodiments, a* is 15. In some embodiments, a* is 16. In some embodiments, a* is 17. In some embodiments, a* is 18. In some embodiments, a* is 19. In some embodiments, a* is 20. In some embodiments, a* is 21. In some embodiments, a* is 22. In some embodiments, a* is 23. In some embodiments, a* is 24. In some embodiments, a* is 25. In some embodiments, a* is 26. In some embodiments, a* is 27. In some embodiments, a* is 28. In some embodiments, a* is 29. In some embodiments, a* is 30. In some embodiments, a* is 31. In some embodiments, a* is 32. In some embodiments, a* is 33. In some embodiments, a* is 34. In some embodiments, a* is 35. In some embodiments, a* is 36. In some embodiments, a* is 37. In some embodiments, a* is 38. In some embodiments, a* is 39. In some embodiments, a* is 40. In some embodiments, a* is 41. In some embodiments, a* is 42. In some embodiments, a* is 43. In some embodiments, a* is 44. In some embodiments, a* is 45. In some embodiments, a* is 46. In some embodiments, a* is 47. In some embodiments, a* is 48. In some embodiments, a* is 49.
[0190] In some embodiments, b* is 2. In some embodiments, b* is 3. In some embodiments, b* is 4. In some embodiments, b* is 5. In some embodiments, b* is 6. In some embodiments, b* is 7. In some embodiments, b* is 8. In some embodiments, b* is 9. In some embodiments, b* is 10. In some embodiments, b* is 11. In some embodiments, b* is 12. In some embodiments, b* is 13. In some embodiments, b* is 14. In some embodiments, b* is 15. In some embodiments, b* is 16. In some embodiments, b* is 17. In some embodiments, b* is 18. In some embodiments, b* is 19. In some embodiments, b* is 20. In some embodiments, b* is 21. In some embodiments, b* is 22. In some embodiments, b* is 23. In some embodiments, b* is 24. In some embodiments, b* is 25. In some embodiments, b* is 26. In some embodiments, b* is 27. In some embodiments, b* is 28. In some embodiments, b* is 29. In some embodiments, b* is 30. In some embodiments, b* is 31. In some embodiments, b* is 32. In some embodiments, b* is 33. In some embodiments, b* is 34. In some embodiments, b* is 35. In some embodiments, b* is 36. In some embodiments, b* is 37. In some embodiments, b* is 38. In some embodiments, b* is 39. In some embodiments, b* is 40. In some embodiments, b* is 41. In some embodiments, b* is 42. In some embodiments, b* is 43. In some embodiments, b* is 44. In some embodiments, b* is 45. In some embodiments, b* is 46. In some embodiments, b* is 47. In some embodiments, b* is 48. In some embodiments, b* is 49.
[0191] In some embodiments, a pair of residues covalently linked with one another via a linker is covalently linked via a staple. In some embodiments, a pair of residues covalently linked with one another via a linker covalently linked via a non-hydrocarbon staple. In some embodiments, a pair of residues covalently linked with one another via a linker is covalently linked with via a cysteine staple. In some embodiments, a cysteine staple has the structure as described in the present disclosure. In some embodiments, a staple has the structure of -L.sup.s1-S-L.sup.s2-S-L.sup.s3- wherein each variable is independently as described herein. In some embodiments, a staple has the structure of L as described herein.
[0192] In some embodiments, the present disclosure provides a collection of stapled peptides, each of which independently has an amino acid sequence that: has a length within a range of a* and b*, where a* and b* are integers selected from 2 through 100 inclusive and b* is greater than a*; comprises a pair of residues covalently linked with one another via a linker; and residues of the pair are separated by c* residues, where c* is an integer selected from 0 through 12.
[0193] In some embodiments, a linker is a staple as described herein. In some embodiments, a linker is -L.sup.s1-S-L.sup.s2-S-L.sup.s3- wherein each variable is independently as described herein. In some embodiments, a linker is L as described herein.
[0194] In some embodiments, the present disclosure provides a collection of stapled peptides, each of which independently has an amino acid sequence that: [0195] has a length within a range of a* and b*, where a* and b* are integers selected from 2 through 100 inclusive and b* is greater than a*; [0196] includes at least one pair of cysteine residues covalently linked with one another via a linker comprising a moiety S-L.sup.s2-S, where each S is independently a sulfur atom of a cysteine residue; [0197] L.sup.s2 is described in the present disclosure; and [0198] cysteine residues of a pair are independently separated by c* residues, where c* is an integer selected from 0 through 12.
[0199] In some embodiments, a pair of cysteine residues covalently linked with one another via linker are separated by c* residues, wherein c* is an integer 1 to 12 inclusive. In some embodiments, c* is 1. In some embodiments, c* is 2. In some embodiments, c* is 3. In some embodiments, c* is 4. In some embodiments, c* is 5. In some embodiments, c* is 6. In some embodiments, c* is 7. In some embodiments, c* is 8. In some embodiments, c* is 9. In some embodiments, c* is 10. In some embodiments, c* is 11. In some embodiments, c* is 12.
[0200] In some embodiments, the present disclosure provides a collection of stapled peptides of the structure: [0201] [X.sup.1].sub.p1[X.sup.2].sub.p2[X.sup.3].sub.p3[X.sup.4].sub.p4[X.sup.5].sub.p5[X.sup.6].sub.p6[X.sup.7].sub.p7[X.sup.8].sub.p8[X.sup.9].sub.p9[X.sup.10].sub.p10[X.sup.11].sub.p11[X.sup.12].sub.p12[X.sup.13].sub.p13-X.sup.14X.sup.15X.sup.16X.sup.17X.sup.18X.sup.19-[X.sup.20].sub.p20[X.sup.21].sub.p21[X.sup.22].sub.p22[X.sup.23].sub.p23[X.sup.24].sub.p24[X.sup.25].sub.p25[X.sup.26].sub.p26[X.sup.27].sub.p27[X.sup.28].sub.p28[X.sup.29].sub.p29[X.sup.30].sub.p30[X.sup.31].sub.p31[X.sup.32].sub.p32, [0202] wherein: [0203] each of p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, p11, p12, p13, p20, p21, p22, p23, p24, p25, p26, p27, p28, p29, p30, p31, p32 is independently 0 or 1; [0204] each of X.sup.1 to X.sup.32 is independently an amino acid residue; [0205] at least two of X.sup.1 to X.sup.32 are covalently linked with one another via a linker.
[0206] In some embodiments, the present disclosure provides a collection of stapled peptides of the structure: [0207] [X.sup.1].sub.p1[X.sup.2].sub.p2[X.sup.3].sub.p3[X.sup.4].sub.p4[X.sup.5].sub.p5[X.sup.6].sub.p6[X.sup.7].sub.p7[X.sup.8].sub.p8[X.sup.9].sub.p9[X.sup.10].sub.p10[X.sup.11].sub.p11[X.sup.12].sub.p12[X.sup.13].sub.p13-X.sup.14X.sup.15X.sup.16X.sup.17X.sup.18X.sup.19-[X.sup.20].sub.p20[X.sup.21].sub.p21[X.sup.22].sub.p22[X.sup.23].sub.p23[X.sup.24].sub.p24[X.sup.25].sub.p25[X.sup.26].sub.p26[X.sup.27].sub.p27[X.sup.28].sub.p28[X.sup.29].sub.p29[X.sup.30].sub.p30[X.sup.31].sub.p31[X.sup.32].sub.p32, [0208] wherein: [0209] each of p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, p11, p12, p13, p20, p21, p22, p23, p24, p25, p26, p27, p28, p29, p30, p31, p32 is independently 0 or 1; [0210] each of X.sup.1 to X.sup.32 is independently an amino acid residue; [0211] at least two of X.sup.1 to X.sup.32 comprise cysteine chains that are optionally linked together to form a cysteine staple.
[0212] In some embodiments, each X.sup.j is independently selected from the group of standard amino acids, wherein j is an integer from 1 to 32 inclusive. In some embodiments, X.sup.j is selected from a subset of standard amino acids. In some embodiments, X.sup.j is selected from a group of all natural amino acids except cysteine. In some embodiments, X.sup.j is selected from fewer than 20 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 19 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 18 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 17 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 16 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 15 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 14 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 13 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 12 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 11 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 10 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 9 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 8 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 7 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 6 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 5 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 4 of the standard amino acids. In some embodiments, X.sup.j is selected from fewer than 3 of the standard amino acids. In some embodiments.
[0213] In some embodiments, each X.sup.j is independently selected from 20 of the standard amino acids. In some embodiments, X.sup.j is selected from 19 of the standard amino acids. In some embodiments, X.sup.j is selected from 18 of the standard amino acids. In some embodiments, X.sup.j is selected from 17 of the standard amino acids. In some embodiments, X.sup.j is selected from 16 of the standard amino acids. In some embodiments, X.sup.j is selected from 15 of the standard amino acids. In some embodiments, X.sup.j is selected from 14 of the standard amino acids. In some embodiments, X.sup.j is selected from 13 of the standard amino acids. In some embodiments, X.sup.j is selected from 12 of the standard amino acids. In some embodiments, X.sup.j is selected from 11 of the standard amino acids. In some embodiments, X.sup.j is selected from 10 of the standard amino acids. In some embodiments, X.sup.j is selected from 9 of the standard amino acids. In some embodiments, X.sup.j is selected from 8 of the standard amino acids. In some embodiments, X.sup.j is selected from 7 of the standard amino acids. In some embodiments, X.sup.j is selected from 6 of the standard amino acids. In some embodiments, X.sup.j is selected from 5 of the standard amino acids. In some embodiments, X.sup.j is selected from 4 of the standard amino acids. In some embodiments, X.sup.j is selected from 3 of the standard amino acids. In some embodiments, X.sup.j is selected from 2 of the standard amino acids. In some embodiments, X.sup.j is selected from 1 of the standard amino acids.
[0214] In some embodiments, j is 1. In some embodiments, j is 2. In some embodiments, j is 3. In some embodiments, j is 4. In some embodiments, j is 5. In some embodiments, j is 6. In some embodiments, j is 7. In some embodiments, j is 8. In some embodiments, j is 9. In some embodiments, j is 10. In some embodiments, j is 11. In some embodiments, j is 12. In some embodiments, j is 13. In some embodiments, j is 14. In some embodiments, j is 15. In some embodiments, j is 16. In some embodiments, j is 17. In some embodiments, j is 18. In some embodiments, j is 19. In some embodiments, j is 20. In some embodiments, j is 21. In some embodiments, j is 22. In some embodiments, j is 23. In some embodiments, j is 24. In some embodiments, j is 25. In some embodiments, j is 26. In some embodiments, j is 27. In some embodiments, j is 28. In some embodiments, j is 29. In some embodiments, j is 30. In some embodiments, j is 31. In some embodiments, j is 32.
[0215] Those skilled in the art, reading the present disclosure, will appreciate that peptide collections as described herein can be prepared, provided and/or utilized in a variety of formats. In some embodiments, a peptide collection is prepared, provided, and or utilized in a format such as, for example, phage display, yeast display, bacteria display, ribosome display, mRNA display, on a solid support, on a solid phase, on a resin, in liquid solution, as a dried sample or set thereof, etc.
[0216] In some embodiments, a collection of peptides can be provided and or utilized in phage display. In some embodiments, a collection of peptides are fused to a phage protein. In some embodiments, a collection of peptides are fused to a phage coat protein. In some embodiments, a collection of peptides are fused to a phage coat protein pIII.
[0217] Peptide collections may be displayed using a number of technologies. In some embodiments, peptides of libraries are displayed on p4 of M13 phage. In some embodiments, peptides of libraries are displayed on p7 of M13 phage. In some embodiments, peptides of libraries are displayed on p8 of M13 phage. In some embodiments, peptides of libraries are displayed on p9 of M13 phage.
[0218] In some embodiments, libraries, e.g., phage libraries, may be prepared with one or more barcodes within the DNA sequence of the library members, for example, comprising silent mutations (e.g., distinct codons that all encode the same amino acid), amino acid mutations, etc. In some embodiments, barcodes are used to identify certain features of library members and/or certain features of uses, e.g., experiments they are screened in, so that those features may be associated with the library member screening results, e.g., during analyses of DNA sequencing outputs of the screens. For example, in some embodiments, barcodes are incorporated into multiple libraries so that those libraries can be screened together, and their results are identified (and separated) by use of the barcodes. In some embodiments, barcodes are used to identify features of processes, e.g., experiments that a library member were screened in. Exemplary features include targets (e.g., proteins, nucleic acids, cells, etc.), buffer conditions, binding partners, competitors included in screens, temperatures of experiments, duration of experiments, washing procedures, and/or other features of experimental procedures. In some embodiments, barcodes are utilized to identify crosslinkers, e.g., those used to prepare chemically modified phage libraries comprising stapled peptides.
[0219] Various technologies can be utilized to incorporate unnatural amino acids into peptides, collections (e.g., various libraries described herein), etc., in accordance with the present disclosure. In some embodiments, incorporation comprises the use of codon suppression and/or aminoacyl-tRNA synthetase /tRNA pairs that result in the incorporation of unnatural amino acids. In some embodiments, useful methods comprise the addition of glyphosate (or other agents that selectively suppress the biosynthesis of one or more amino acids) and unnatural amino acid(s) in growth media. In some embodiments, useful methods comprise the use of cell lines lacking the ability to synthesize certain amino acids and the addition of unnatural amino acid(s) in growth media. In some embodiments, useful methods comprise the addition of unnatural amino acid(s) in growth media. In some embodiments, barcodes are used to identify the incorporation of unnatural amino acids into library designs.
[0220] In some embodiments, known phage or DNA sequence(s) are added during a step of a phage screen, for the purpose of scaling or normalizing DNA sequencing data using the known sequence(s) (spike-in samples).
[0221] In some embodiments, in the context of a collection of peptides (or proteins, etc.; e.g., of a provided library), diversity may refer to either a) the actual number of unique amino acid sequences present in said collection of peptides or b) the theoretical number of unique amino acid sequences that could exist, e.g., based on design and/or preparation of the collection. In some embodiments, diversity is actual diversity, e.g., as measured and described in the Examples.
[0222] In some embodiments, a peptide collection as described herein is characterized by an actual diversity of at least 110.sup.4 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.5 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.6 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.7 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.8 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.9 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.10 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.11 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.12 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.13 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.14 unique peptide sequences. In some embodiments, a peptide collection is characterized by an actual diversity of at least 110.sup.15 unique peptide sequences. In some embodiments, a preferred actual diversity for a peptide collection e.g., of a library, is 10.sup.8-10.sup.9 unique peptide sequences. In some embodiments, such a collection may have a theoretical diversity that is often 1,000-10,000-fold greater than the actual diversity, for example, 10 positions with 16 amino acid possibilities at each position can have a theoretical diversity of 10.sup.12; depending on procedures, purposes, etc., a collection of peptides, e.g., of a library may be prepared with a subset of the theoretical diversity, e.g., of 10.sup.8 unique sequences.
[0223] In some embodiments, one or more (e.g., about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% of all amino acid residues), or each of the amino acid residue positions are randomized (e.g., at degenerate positions). In some embodiments, at a randomized position a variety of amino acid residues are independently presented in a collection of peptides (e.g., a library described in an example). In some embodiments, each position is randomized. In some embodiments, a collection is provided without amino acid residue preferences at any positions (e.g., in some embodiments, a naive or unbiased collection/library). As appreciated by those skilled in the art, incorporation of randomized positions in some embodiments can be done by encoding a degenerate codon in an oligonucleotide primer used to generate a library, for example, NNN or NNK where N=A, T, C, G and K=T, G. Alternatively, randomized positions can be incorporated by the use of trimer phosphoroamidite mixtures, e.g., those available from Glen research, wherein a defined mixture of trimer codons that encode a corresponding defined mixture of amino acids are incorporated in the oligonucleotide primer used to generate the library. For example, a library could be constructed using a mixture of all 20 naturally occurring amino acids, or alternatively by using a subset of all 20 naturally occurring amino acids (e.g., A, D, E, F, H, I, L, M, N, Q, R, S, T, V, W, Y, etc.). In some embodiments, non-natural amino acids may also be incorporated using various technologies.
[0224] In some embodiments, randomized positions can be incorporated through chemical synthesis, e.g., by utilizing mixtures of amino acids at such positions. In some embodiments, amino acids are present at the same levels in mixtures. In some embodiments, certain amino acids may have higher levels than others, e.g., in view of synthesis efficiency.
[0225] In some embodiments, an amino acid residue at a randomized, degenerate, or not biased or enriched position (e.g., in a cluster as described herein) is selected from alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.
[0226] In some embodiments, peptides of a collection, e.g., a library, comprise fixed or a subset of preferred amino acids at one or more positions for one or more desired property and/or activity, e.g., in some embodiments, to favor an alpha-helical conformation. For example, incorporation of alanine residues at defined positions in the middle of the peptide (e.g. library designs b), e), and h) above), a proline at the N-terminus (e.g. library designs g), h), i) above), or an aspartic acid followed by a proline at the N-terminus (e.g. library designs d), e), f) in the list above). In some embodiments, a desired property or activity is binding to a particular target (e.g., protein, nucleic acid, etc.). In some embodiments, a desired property or activity is cell penetration. In some embodiments, a desired property or activity is stability (e.g., to proteases or other types of degradation). In some embodiments, a desired property or activity is low immunogenicity. In some embodiments, a desired property or activity is improved physicochemical property. In some embodiments, a desired property or activity is improved pharmacokinetic properties. In some embodiments, a desired property or activity is selectivity for a target or tissue. As those skilled in the art will appreciate, fixed and/or a subset of preferred amino acids may be independently presented at each of one or more positions to provide one or more desired properties and/or activities.
[0227] In some embodiments, peptides of a collection comprise stapled residues or residues suitable for stapling at certain positions.
[0228] In some embodiments, peptides of a collection comprise enriched amino acid residues useful for binding to a target of interest. In some embodiments, such enriched amino acid residues interact with a target of interest. In some embodiments, peptides of such a collection comprise one or more, e.g., about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, randomized positions. In some embodiments, such a collection are useful for identifying agents that can bridge two targets of interest, wherein the enriched amino acid residues facility interactions with a first target of interest (e.g., a presenter protein), wherein the randomized positions can provide diversity for binding to a second target of interest.
[0229] In some embodiments, collections, e.g., peptide libraries, can be designed on the basis of one or more preferred or parent sequences. In some embodiments, a parent sequence is systematically diversified to give rise to many collection members. In some embodiments, a parent sequence is subjected to an alanine scan where each residue within the sequence is changed to alanine, either individually or in combination with other changes, mutations and/or modifications, to provide a number of peptides in a collection. In some embodiments, alanine scan is useful for assessing importance of one or more residues for certain properties and/or activities, e.g., binding, interactions, stability, physicochemical properties, cell penetration, immunogenicity, selectivity (e.g., for a target (e.g., protein, nucleic acid, etc.), tissue, etc.), pharmacokinetic properties, etc. A parent sequence could similarly be systematically changed/mutated to proline or glycine e.g., for assessing the effect of structural and/or conformational changes. A parent sequence could also be systematically changed/mutated to charged residues, e.g., for interrogating roles of charge in binding, stability, physicochemical properties, cell penetration, immunogenicity, selectivity, pharmacokinetic properties, etc. A parent sequence could also be systematically changed/mutated at each position to a variety of amino acids, e.g., for identifying point-mutations with improved properties and/or activities, e.g., binding, stability, physicochemical properties, cell penetration, immunogenicity, selectivity, pharmacokinetic properties, etc. As those skilled in the art will appreciate, collections can be designed using various established methods of library mutagenesis and directed evolution in accordance with the present disclosure.
[0230] In some embodiments, a parent sequence is pancreatic polypeptide, neuropeptide Y, or peptide YY, or a fragment thereof. In some embodiments, a fragment has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, amino acid residues. For example, in some embodiments, a sequence is selected from:
TABLE-US-00003 TABLEQ-1 Species Sequence Homosapiens PSKPDNPGEDAPAEDMARYYSALGHYINLIT(SEQIDNO:6) Ictaluruspunctatus PSKPENPGEDAAPEELAKYYTALRHYINLIT(SEQIDNO:7) Marmotamarmota PLEPVYPGDNATPEQMAQYAAELRRYINMLT(SEQIDNO:8) Myotislucifugus PSKPEKPGENASAEELARYYSALRHYINLIT(SEQIDNO:9) Bettasplendens PPKPENPGEDAPPEELAKYYTALRHYINLIT(SEQIDNO:10) Notechisscutatus PPKPESPGENASPEEMAKYLADLRHYINLVT(SEQIDNO:11) Orcinusorca PAKPEAPGSHASPEELKRYYLSLRHFLNLVT(SEQIDNO:12) Calypteanna PPKPETPGDEASPEEVAKYFSALRHYINLVT(SEQIDNO:13) Podarcismuralis PQQPEHPGEDASAEEMARYLSALRHYLNLVT(SEQIDNO:14) Vombatusursinus PSKPKPPSENASREELSRYYAALRQYLNLVT(SEQIDNO:15) Ornithorhynchusanatinus PVKPQPPPDNATPEELAQYFASLRHYLNLVT(SEQIDNO:16) Otolemurgarnettii PLEPVYPGENATPEQMAQYAAELRRYINMLT(SEQIDNO:17) Gulogulo PSKPDNPGEDAPAEDMARYYSALRHYINLIT(SEQIDNO:18) Crocodylusporosus PSKPDNPGEDAPAEDMARYYSALRHYINLIT(SEQIDNO:915)
[0231] In some embodiments, a parent sequence is a sequence that shares certain level of homology with another sequence, e.g., pancreatic polypeptide sequences above. In some embodiments, a level is 85%, 90%, 95% or more. In some embodiments, a level is 90%. In some embodiments, a level is 95%. In some embodiments, amino acid sequences of stapled peptides are the same as parent sequences except one or more residues of the parent sequences are replaced with residues for stapling (e.g., cysteine residues for cysteine stapling as described herein). In some embodiments, amino acid sequences of stapled peptides are the same as parent sequences except two or more residues of the parent sequences are replaced with residues for stapling. In some embodiments, amino acid sequences of stapled peptides are the same as parent sequences except two residues of the parent sequences are replaced with residues for stapling.
[0232] In some embodiments, collections of peptides, e.g., various libraries, are based on pancreatic polypeptides. In some embodiments, libraries of pancreatic polypeptides comprise randomization of one or more residues in an alpha-helical region. In some embodiments, libraries of pancreatic polypeptide, neuropeptide Y, or peptide YY comprise randomization of one or more residues in the PPII region. In some embodiments, libraries of pancreatic polypeptide, neuropeptide Y, or peptide YY comprise randomization of one or more residues in a loop region. In some embodiments, libraries of pancreatic polypeptide, neuropeptide Y, or peptide YY comprise randomization of one or more residues in two or more regions of the peptide. In some embodiments, such libraries are prepared by randomizing residues one or more regions.
[0233] In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Homo sapiens. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Ictalurus punctatus. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Marmota marmota. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Myotis lucifugus. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Betta splendens. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Notechis scutatus. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Orcinus orca. In some embodiments, pancreatic polypeptide, neuropeptide Y, or peptide YY is from Calypte anna. In some embodiments, protein folds with higher thermal stability are preferred scaffolds for randomization/generation of libraries. In some embodiments, libraries, e.g., of pancreatic polypeptides, are prepared by randomizing residues of a naturally occurring pancreatic polypeptide fold with high thermal stability. In some embodiments, libraries of pancreatic polypeptides are prepared by randomizing residues of a pancreatic polypeptide fold that has been modified to improve thermal stability.
[0234] In some embodiments, the present disclosure provides methods for optimizing a peptide of a parent sequence. In some embodiments, the present disclosure provides methods comprising: [0235] modifying a peptide by replacing each of one or more amino acid residues independently with a different amino acid residue to provide a modified peptide, wherein the modified peptide comprises at least two residues suitable for stapling; and [0236] stapling the two residues suitable for stapling.
[0237] In some embodiments, a peptide is of or comprises a parent sequence as described herein (e.g., a peptide YY sequence or a fragment thereof). In some embodiments, the two residues suitable for stapling are cysteine residues. In some embodiments, two cysteine residues are stapled as described herein, e.g., using reagents described herein. In some embodiments, a modified peptide provides an improved properties and/or activity, e.g., improved affinity and/or selectivity for target binding, improved stability, improved helix formation, improved cell penetration, etc. In some embodiments, a collection of modified peptides are prepared, e.g., by randomizing one or more residues as described herein. In some embodiments, collections of modified peptides are provided as phase display libraries. In some embodiments, members of such collections/libraries have staples, e.g., cysteine staples, as described herein. In some embodiments, the present disclosure provides, comprising contacting provided collections, libraries or modified peptides with a target of interest so that one or more stapled peptides of the collection binds to the target, and determining amino acid sequences of stapled peptides that bind to the target as described herein.
[0238] As will be appreciated by those skilled in the art, provided technologies are applicable to various parent sequences and/or helical scaffolds. In some embodiments, a parent sequence is, or is a fragment (comprising, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues), or is a helical scaffold of, exendin-4, trp-cage peptides, retro trp-cage peptides, conotoxins, scyllatoxins, scorpion toxins, charybdotoxin, villin headpiece, beta-alpha-beta motif peptides, beta-beta-alpha motif peptides, mastoparan, zinc fingers, helix-linker-helix scaffolds, leucine zipper scaffolds, pancreatic polypeptide scaffolds, neuropeptide Y scaffolds, peptide YY scaffolds, mutants of the aforementioned scaffolds that have been modified for the purpose of increased helix stabilization, and other helical display scaffolds in the art. In some embodiments, peptides of provided collections comprise helix-initiating or helix-termination sequences. In some embodiments, peptides of provided collections comprise an N-terminal leader sequence such as AAA, DPA, NPA, APA, PA, P, or AP, and/or a C-terminal leader sequence such as R, RR, RP, RG, GR, PR, G, GG, P, PP, GP, or PG.
[0239] In some embodiments, cysteines are incorporated into sequences of peptides, e.g. displayed peptides using various display technologies. In some embodiments, peptides which bind a target peptide or protein at a site within close distance of one or more cysteines on the target protein may be identified via the formation a covalent bond between the cysteine on a peptide and a cysteine of the target protein (e.g., on its surface), either directly or through a linker (e.g., through using various reagents suitable for cross-linking cysteine residues), which in some embodiments can lead to an observed high-affinity and/or slow off-rate interactions in an assay (e.g., a screen assay assessing binding). Among other things, identification of such cysteines are useful for various purposes, e.g., development of agents that can interact the targets (e.g., covalent target inhibitors), use of disulfide tethering for library synthesis, compound optimization, etc. Those skilled in the art will appreciates that various uses of target cysteines, e.g., surface cysteines on targets, are available in the art.
[0240] Peptides of the present disclosure can have various sequences. Useful sequences can have various percentage and/or numbers of one or more amino acid residues. For example, in some embodiments, a sequence comprises 2 and no more than 2 cysteine residues (either stapled or not). In some embodiments, a sequence comprises more than 2 cysteine residues (either stapled or not). In some embodiments, a sequence comprises 2 and no more than 2 stapled cysteine residues. In some embodiments, a sequence comprises more than 2 stapled cysteine residues. In some embodiments, a percentage of stapled cysteine residues in a sequence is 5%-50%. In some embodiments, a percentage is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%. In some embodiments, a percentage is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%. In some embodiments, a percentage is about or no more than about 5%. In some embodiments, a percentage is about or no more than about 6%. In some embodiments, a percentage is about or no more than about 7%. In some embodiments, a percentage is about or no more than about 8%. In some embodiments, a percentage is about or no more than about 9%. In some embodiments, a percentage is about or no more than about 10%. In some embodiments, a percentage is about or no more than about 11%. In some embodiments, a percentage is about or no more than about 12%. In some embodiments, a percentage is about or no more than about 13%. In some embodiments, a percentage is about or no more than about 14%. In some embodiments, a percentage is about or no more than about 15%. In some embodiments, a percentage is about or no more than about 20%.
[0241] In some embodiments, collections of peptides, e.g., provided peptide libraries, are utilized to screen for peptides of certain properties and/or activities. Various screening technologies are described in the art and can be utilized in accordance with the present disclosure. In some embodiments, a library is screened against a target, e.g., a protein, a nucleic acid, etc. In some embodiments, all amino acid residues in a target are D configured amino acids (e.g., a mirror-image display). In some embodiments, libraries are screened against cells, e.g., cell samples, cells grown in culture, etc., or in vivo, using various technologies described in the art. In some embodiments, libraries are screened against human cells. In some embodiments, libraries are screened in a living mammal. In some embodiments, libraries are screened in a living primate.
[0242] In some embodiments, libraries are screened for binding affinity to targets, e.g., proteins, nucleic acids, etc. In some embodiments, libraries are screened for selectivity for one target (e.g., a protein) over another target (e.g., a protein). In some embodiments, libraries are screened for one or more desired characteristics such as stability, physicochemical properties, cell penetration, immunogenicity (e.g., low immunogenicity), pharmacokinetic properties, etc. In some embodiments, libraries are screened for affinity to lipids, for optimizing physicochemical properties, and/or cell penetration. In some embodiments, libraries are screened against immobilized lipids, for optimizing physicochemical properties and/or cell penetration. In some embodiments, libraries are screened for crossing blood-brain-barrier or neural barriers, either in vivo or with in vitro models of a blood-brain-barrier or neural barriers. In some embodiments, libraries are screened for crossing intestine or other relevant epithelial cells, either in vivo or with in vitro models of intestine or other relevant epithelial cells. In some embodiments, libraries are screened using organoid models.
[0243] In some embodiments, screens are performed in buffers that have been optimized, e.g., for targets (e.g., proteins, cells. etc.) that are subjects of the screens. In some embodiments, screens are performed in buffers that reduce nonspecific binding, for example, buffers comprising bovine serum albumin, bovine gamma-globulin, collagen and sheared collagen, milk proteins, randomized mixtures of synthetic peptides, lysates of bacteria or mammalian cells, lysates or protein preparations subjected to limited proteolysis, poly(deoxyinosinic-deoxycytidylic) acid, salmon sperm DNA, sheared DNA, polylysine, glycerol, trehalose, detergents such as Triton X-100, NP-40, Tween-20, Tween-80, Pleuronic F-127, octyl beta-D-glucopyranoside, etc. Additional additives are known to those skilled in the art and can be utilized in accordance with the present disclosure.
[0244] Using technologies described herein, e.g., peptide collections (libraries), screens, etc., peptide binders for diverse protein targets (e.g., beta-catenin (CTNNB1), CBL, CBLB, TEAD4, ERG) were identified.
[0245] In some embodiments, stapled peptides or collections thereof may be assessed relative to reference peptides and/or collections thereof. In some embodiments, a reference peptide is an unstapled peptide. In some embodiments, a reference peptide is an unstapled peptide of the same or a comparable sequence. In some embodiments, a peptide is a peptide comprising cysteine stapling, and a reference peptide does not have such cysteine stapling. In some embodiments, a reference peptide do not contain cysteines utilized in cysteine stapling. In some embodiments, a reference peptide does not contain a PPII region of pancreatic polypeptide, neuropeptide Y, or peptide YY which can be utilized to stabilize helical structures.
[0246] In some embodiments, a peptide collection as described herein is characterized by a certain redundancy. In some embodiments, redundancy in the context of a collection of peptides (or proteins, etc.; e.g., of a provided library) refers to the number of copies of a unique amino acid sequence within said collection of peptides. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 10000. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 5000. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 1000. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 500. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 100. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 50. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 10. In some embodiments, a peptide collection as described herein is characterized by a redundancy of at least 1. In some embodiments, a preferred redundancy with respect to an actual diversity is 100-1000 phages for each unique sequence in a given screening sample, providing a good oversampling of each sequence. For example, a library of 10.sup.8 sequences and 1000 phages per sequence will contain 10.sup.11 phage particles; in a typical screen of 48 samples, there would therefore be 4.8*10.sup.12 total phages.
[0247] In some embodiments, a collection of peptides comprises a spiked-in standard. In some embodiments, a spiked-in standard is added to a collection of peptides after screening and prior to sequencing of identifying nucleic acid sequences (e.g., those encoding the peptides) of said collection of peptides. In some embodiments, a spiked-in standard is a spiked-in phage. In some embodiments, a spiked-in standard is a spiked-in nucleotide sequence. In some embodiments, a spiked-in standard serves an internal standard. In some embodiments, a spiked-in standard allows for measurement of enrichment of certain amino acid sequences.
[0248] In some embodiments, a collection of peptides can be provided and/or utilized in a phage display format. In some embodiments, the present disclosure provides a method comprising the steps of: [0249] i) expressing in a cell a nucleic acid encoding a peptide having an amino acid sequence that: has a length within a range of a* to b* amino acid residues, includes at least one pair of cysteine residues separated by 2 to 15 amino acid residues, and wherein the nucleic acid encodes a fusion protein that is incorporated into a phage particle; [0250] ii) isolating phage particles from the cell; and [0251] iii) contacting phage particles with a cross-linking agent, e.g., having the structure of R.sup.x-L.sup.s2-R.sup.x or a salt thereof or formula R-I:
R.sup.x-L.sup.x1-C(O)Q-L-QC(O)-L.sup.x2-R.sup.x R-I, [0252] or a salt thereof, wherein: [0253] a*, b*, and R.sup.x, Q, and L are described in the present disclosure.
[0254] In some embodiments, a cross-linking agent is a reagent as described herein. In some embodiments, a cross-linking agent is a compound having the structure of R.sup.E-L.sup.s2-R.sup.E or a salt thereof. In some embodiments, a cross-linking agent is a compound having the structure of R.sup.E-L.sup.x1-C(O)Q-L-QC(O)-L.sup.x2-R.sup.E or a salt thereof. In some embodiments, a cross-linking agent is a compound having the structure of R.sup.E-C(O)Q-L-QC(O)-R.sup.E or a salt thereof. In some embodiments, a cross-linking agent is a compound having the structure of R.sup.x-L.sup.s2-R.sup.x or a salt thereof. In some embodiments, a cross-linking agent is a compound having the structure of R.sup.x-L.sup.x1-C(O)Q-L-QC(O)-L.sup.x2-R.sup.x or a salt thereof.
[0255] In some embodiments, the present disclosure provides a method further comprising the steps of: [0256] iv) contacting the collection of peptides with a target of interest so that one or more stapled peptides of the collection binds to a target of interest; and [0257] v) determining amino acid sequences of stapled peptides that bind to a target of interest.
[0258] In some embodiments, each amino acid sequence is discretely associated with an identifier so that each amino acid sequence can be independently identified. In some embodiments, each amino acid sequence is independently associated with an identifier that comprises a nucleic acid sequence that encodes an amino acid sequence or a portion (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more optionally consecutive amino acid residues, or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, of the amino acid sequence) thereof. In some embodiments, association is covalent. In some embodiments, association is through a discrete system comprising an amino acid sequence and its identifier. In some embodiments, a system is a cell, phage, etc. that comprises and/or expresses an amino acid sequence and its identifier. In some embodiments, determination of an amino acid sequence comprises determining nucleotide sequences that encode peptides of a fusion protein incorporated into a phage particle. In some embodiments, determining amino acid sequences comprises the use of high-throughput sequencing of nucleic acids encoding the amino acid sequences. In some embodiments, high-throughput sequencing comprises single-molecule real-time sequencing, ion semiconductor sequencing (e.g. Ion Torrent Sequencing), pyrosequencing, sequencing by synthesis, sequencing by ligation (e.g. SOLiD sequencing), nanopore sequencing, etc. As appreciated by those skilled in the art, various high-throughput sequencing technologies can be utilized in accordance with the present disclosure. In some embodiments, the present disclosure recognizes that technologies prior to the present disclosure can be severely and inherently limited and in many instances cannot be successfully performed when libraries containing large numbers of diverse amino acid sequences are utilized as e.g., amino acid sequences with desired properties and/or activities cannot be readily identified from background noise, e.g., due to the limited numbers of sequences that can be individually assessed. Among other things, the present disclosure demonstrates that the combination of peptide libraries and high-throughput sequencing of the corresponding encoding nucleic acid sequences are particularly powerful for screening peptide libraries comprising large numbers of diverse sequences and identifying from such libraries useful amino acid sequences, in some instances, amino acid sequences very different or totally different from natural amino acid sequences. In some embodiments, provided technologies greatly improve throughput and/or enable analysis/assessment of many candidate sequences at unprecedented level.
[0259] In some embodiments, a peptide can be identified through mass spectrometry. In some embodiments, mass spectrometry identifies peptides by their mass. In some embodiments, mass spectrometry identifies peptides by sequencing. In some embodiments, collections are designed and many or all peptides have unique mass.
[0260] In some embodiments, the present disclosure provides a method further comprising the steps of: [0261] vi) synthesizing a new collection of peptides guided by patterns observed during the determination of amino acid sequences of stapled peptides that bind to a target of interest.
[0262] In some embodiments, the present disclosure provides a method comprising identifying or characterizing a binding agent specific for a target of interest, the method comprising steps of contacting the target of interest with members of a collection of stapled peptides; and detecting specific binding by one or more of the members to the target.
[0263] In some embodiments, a collection of peptides is a collection of polypeptides. In some embodiments, a collection of peptides is a collection of polypeptides with secondary structure. In some embodiments, a collection of peptides is a collection of polypeptides with a tertiary structure. In some embodiments, a collection of polypeptides is a collection of proteins. In some embodiments, a collection of proteins is a collection of stapled proteins.
[0264] As described in the present disclosure, in some embodiments, peptides in a library comprise an amino acid sequences, e.g., for screening. In some embodiments, such amino acid sequences comprise stapled amino acid residues. In some embodiments, such amino acid sequences have the same length, and/or have the staples at the same positions. In some embodiments, the amino acid sequences in a collection of peptides of a library comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 random positions, the amino acid residue at each of such positions can independently be any amino acid residue (e.g., natural amino acid residue). In some embodiments, there are at least 5 random positions. In some embodiments, there are at least 6 random positions. In some embodiments, there are at least 7 random positions. In some embodiments, there are at least 8 random positions. In some embodiments, there are at least 9 random positions. In some embodiments, there are at least 10 random positions. In some embodiments, there are at least 11 random positions. In some embodiments, there are at least 12 random positions. In some embodiments, there are at least 13 random positions. In some embodiments, there are at least 14 random positions. In some embodiments, there are at least 15 random positions. In some embodiments, there are at least 16 random positions. In some embodiments, there are at least 17 random positions. In some embodiments, each amino acid residue except the stapled residues is independently random. In some embodiments, at a random position there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different amino acid residues with in a collection of peptides of a library. In some embodiments, there are at least 2 different amino acid residues. In some embodiments, there are at least 3 different amino acid residues. In some embodiments, there are at least 4 different amino acid residues. In some embodiments, there are at least 5 different amino acid residues. In some embodiments, there are at least 6 different amino acid residues. In some embodiments, there are at least 7 different amino acid residues. In some embodiments, there are at least 8 different amino acid residues. In some embodiments, there are at least 9 different amino acid residues. In some embodiments, there are at least 10 different amino acid residues. In some embodiments, there are at least 15 different amino acid residues. In some embodiments, there are at least 20 different amino acid residues.
Library Scaffold
[0265] In some embodiments, the present disclosure provides a collection of peptides of the structure: [0266] [X.sup.0].sub.p0[X.sup.1].sub.p1[X.sup.2].sub.p2[X.sup.3].sub.p3[X.sup.4].sub.p4[X.sup.5].sub.p5[X.sup.6].sub.p6[X.sup.7].sub.p7[X.sup.8].sub.p8[X.sup.9].sub.p9[X.sup.10].sub.p10[X.sup.11].sub.p11[X.sup.12].sub.p12[X.sup.13].sub.p13-X.sup.14X.sup.15X.sup.16X.sup.17X.sup.18X.sup.19-[X.sup.20].sub.p20[X.sup.21].sub.p21[X.sup.22].sub.p22[X.sup.23].sub.p23[X.sup.24].sub.p24[X.sup.25].sub.p25[X.sup.26].sub.p26[X.sup.27].sub.p27[X.sup.28].sub.p28[X.sup.29].sub.p29[X.sup.30].sub.p30[X.sup.31].sub.p31[X.sup.32].sub.p32,
wherein: [0267] X.sup.0 is a scaffold constant region; [0268] each of p0, p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, p11, p12, p13, p20, p21, p22, p23, p24, p25, p26, p27, p28, p29, p30, p31, p32 is independently 0 or 1; [0269] each of X.sup.1 to X.sup.32 is independently an amino acid residue; [0270] at least two of X.sup.1 to X.sup.32 comprise side chains that are optionally linked together to form a staple.
[0271] In some embodiments, p0 is 0. In some embodiments, p0 is 1.
[0272] In some embodiments, a scaffold constant region provides a peptide sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% homologous to naturally occurring amino acid sequence. In some embodiments, the homology is at least 50%. In some embodiments, the homology is at least 60%. In some embodiments, the homology is at least 70%. In some embodiments, the homology is at least 80%. In some embodiments, the homology is at least 90%. In some embodiments, the homology is at least 95%. In some embodiments, a scaffold constant region comprises the amino acid sequence AGPAKPEAGEDASP (SEQ ID NO: 19).
Targets of Interest
[0273] In some embodiments, the present disclosure provides reagents, peptide agents, and combinations of peptides that interact with a target of interest. In some embodiments, a target of interest is a biomolecule (e.g. protein, RNA, DNA, etc.), a tissue, or a cell. In some embodiments, a target of interest has an activity or characteristic that is associated with a disease, disorder or condition. In some embodiments, a target of interest is a protein. In some embodiments, a target of interest is a protein associated with a condition, disorder or disease, e.g., a protein associated with cancer. Many targets associated with various conditions, disorders or diseases are known in the art and can be targeted using technologies of the present disclosure.
[0274] In some embodiments, a target of interest may be an entity that occurs in a biological system or organism (e.g., a human). In some embodiments, a target of interest may have a known interaction partner. In some embodiments, a target of interest may not have any known interaction partner, or may not have any known interaction partner whose binding interaction with the target is characterized by one or more features as described herein.
[0275] In some embodiments, a target of interest may be or comprise one or more of beta-catenin and Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b). In some embodiments, a target of interest is beta-catenin. In some embodiments, a target of interest is Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b).
Reagents
[0276] In some embodiments, the present disclosure provides reagents useful in the production, identification, characterization and/or use of stapled peptides (e.g., cysteine stapled peptides). In some embodiments, provided reagents are particularly useful, for example, they provide high yields and/or purities when used in combination of a biological system (e.g., a system that expresses amino acid sequences).
[0277] In some embodiments, the present disclosure provides compounds (e.g., that may be useful as reagents) having the structure of R.sup.E-L.sup.s2-R.sup.E or a salt thereof, wherein each variable is as described in the present disclosure. In some embodiments, R.sup.E is or comprises an electrophilic group. As appreciated by those skilled in the art, various electrophilic groups are known and utilized in the art and can be utilized in accordance with the present disclosure. In some embodiments, an electrophilic group reacts with SH, e.g., SH of an amino acid side chain, under suitable conditions (e.g., certain pH conditions utilized in the art) so that a reaction occurs between the SH group and the electrophilic group forming a covalent bond between the S and the electrophilic group. In some embodiments, an electrophilic group comprises a leaving group bonded to a carbon atom, e.g., CH.sub.2R.sup.x, wherein R.sup.x is a leaving group. In some embodiments, SH reacts with CH.sub.2R.sup.x to form CH.sub.2S. In some embodiments, an electrophilic group is or comprises a double or triple bond. In some embodiments, a double or triple bond is bonded to one or more electron-withdrawing groups (e.g., one or more C(O) groups). In some embodiments, an electrophilic group is a Michael accepter. In some embodiments, an electrophilic group is or comprises
##STR00002##
In some embodiments, R.sup.E is
##STR00003##
In some embodiments, R.sup.E is R.sup.x-L.sup.x1-. In some embodiments, two R.sup.E in the same molecule is the same. In some embodiments, they are different. In some embodiments, useful compounds as reagents have the formula of R.sup.E-L.sup.x1-EWG-Q-L-Q-EWG-L.sup.x2-R.sup.E or a salt thereof, wherein each EWG is independently an electron-withdrawing group moiety (e.g., being or comprising C(O), S(O), S(O).sub.2, etc.) and each other variable is independently as described herein. In some embodiments, useful compounds as reagents have the formula of R.sup.E-L.sup.x1-C(O)Q-L-QC(O)-L.sup.x2-R.sup.E or a salt thereof, wherein each variable is independently as described herein. In some embodiments, useful compounds as reagents have the formula of R.sup.E-L.sup.x1-SO.sub.2-Q-L-Q-SO.sub.2-L.sup.x2-R.sup.E or a salt thereof, wherein each variable is independently as described herein. In some embodiments, useful compounds as reagents have the formula of R.sup.E-EWG-Q-L-Q-EWG-R.sup.E or a salt thereof, wherein each EWG is independently an electron-withdrawing group moiety (e.g., being or comprising C(O), S(O), S(O).sub.2, etc.) and each other variable is independently as described herein. In some embodiments, useful compounds as reagents have the formula of R.sup.EC(O)Q-L-QC(O)-R.sup.E or a salt thereof, wherein each variable is independently as described herein. In some embodiments, useful compounds as reagents have the formula of R.sup.E-SO.sub.2-Q-L-Q-SO.sub.2-R.sup.E or a salt thereof, wherein each variable is independently as described herein. In some embodiments, useful compounds as reagents have the formula of R.sup.E-Q-L-Q-R.sup.E or a salt thereof, wherein each variable is independently as described herein. In some embodiments, the present disclosure provides compounds (e.g., that may be useful as reagents) having the structure of R.sup.x-L.sup.s2-R.sup.x or a salt thereof, wherein each variable is as described in the present disclosure. In some embodiments, the present disclosure provides compounds (e.g., that may be useful as reagents) having the formula R-I:
R.sup.x-L.sup.x1-C(O)Q-L-QC(O)-L.sup.x2-R.sup.x R-I
or a salt thereof, wherein: [0278] each R.sup.x is independently a leaving group; [0279] each Q is independently selected from a covalent bond, N(R), O, and S; [0280] each of L.sup.x1, L.sup.x2, and L is independently a covalent bond, or an optionally substituted, bivalent C.sub.1-C.sub.20 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O; [0281] each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; [0282] each R is R, C(O)R, CO.sub.2R, or SO.sub.2R; [0283] each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or [0284] two R groups are optionally and independently taken together to form a covalent bond; or [0285] two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or [0286] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
[0287] Various leaving groups are known in the art and may be utilized in accordance with the present disclosure, e.g., as embodiments for R.sup.x. In some embodiments, a leaving group, e.g., R.sup.x, is a halogen. In some embodiments R.sup.x is Cl. In some embodiments R.sup.x is Br. In some embodiments R.sup.x is I. In some embodiments, a leaving group is OSO.sub.2R (e.g., wherein R is optionally substituted alkyl (e.g., perfluoroalkyl such as CF.sub.3)), optionally substituted phenyl, etc.). In some embodiments, a leaving group is -OTs or -OMs. In some embodiments, R.sup.x is bonded to CH.sub.2. In some embodiments, R.sup.x is boned to a propargyl or allylic carbon atom, or carbon bonded to an aromatic ring, e.g., a benzylic carbon atom.
[0288] In some embodiments, R.sup.x groups within the same compound are the same. In some embodiments, R.sup.x groups within the same compound are different.
[0289] In some embodiments, L.sup.x1 is an optionally substituted methylene group. In some embodiments, L.sup.x1 is CH.sub.2. In some embodiments, L.sup.x2 is an optionally substituted methylene group. In some embodiments, L.sup.x2 is CH.sub.2. In some embodiments, L.sup.x1 and L.sup.x2 are the same; in other embodiments, they are different. In some embodiments, both L.sup.x1 and L.sup.x2 are CH.sub.2.
[0290] In some embodiments, Q is a covalent bond. In some embodiments, Q is selected from a covalent bond, N(R), O, and S. In some embodiments, Q is N(R), wherein R is described in the present disclosure. In some embodiments, Q is O. In some embodiments, Q is S. In some embodiments, each Q is a covalent bond. In some embodiments, each Q is independently selected from N(R), O, and S. In some embodiments, each Q is independently N(R), wherein R is described in the present disclosure. In some embodiments, each Q is independently NH. In some embodiments, each Q is independently O. In some embodiments, each Q is independently S.
[0291] In some embodiments, Cy is an optionally substituted bivalent C.sub.3-20 cycloaliphatic ring. In some embodiments, Cy is an optionally substituted bivalent C.sub.6-20 aryl ring. In some embodiments, Cy is an optionally substituted bivalent phenyl ring. In some embodiments, Cy is an optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Cy is an optionally substituted 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Cy is monocyclic. In some embodiments, Cy is bicyclic. In some embodiments, Cy is polycyclic. In some embodiments, Cy comprises one or more optionally substituted heterocyclic rings, wherein each of the heterocyclic rings independently comprises one or more nitrogen atoms each of which is independently bonded to Q, or C(O) when Q is a covalent bond. In some embodiments, Cy is monocyclic. In some embodiments, Cy is bicyclic. In some embodiments, Cy is polycyclic.
[0292] In some embodiments, for a bicyclic or polycyclic ring, each of the monocyclic rings is independently a 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered saturated or partially unsaturated ring having 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur) or a 5-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered aromatic ring having 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur).
[0293] In some embodiments, L is or comprises Cy. In some embodiments, L is Cy.
[0294] In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.15 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.10 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.9 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.8 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.7 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.6 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.5 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.3 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.2 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1 aliphatic. In some embodiments, L is CH.sub.2. In some embodiments, L is (CH.sub.2).sub.2. In some embodiments, L is (CH.sub.2).sub.3. In some embodiments, L is (CH.sub.2).sub.4. In some embodiments, L is (CH.sub.2).sub.5. In some embodiments, L is (CH.sub.2).sub.6. In some embodiments, L is (CH.sub.2).sub.7. In some embodiments, L is (CH.sub.2).sub.8.
[0295] In some embodiments, L is optionally substituted bivalent C.sub.6-20 aryl ring. In some embodiments, L is optionally substituted bivalent C.sub.6-14 aryl ring. In some embodiments, L is optionally substituted bivalent C.sub.6-10 aryl ring. In some embodiments, L is optionally substituted bivalent C.sub.6 aryl ring. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one electron-withdrawing group as appreciated by those skilled in the art. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with four halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with four fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with four chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted at with least one O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is bivalent C.sub.6 aryl substituted with four O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is optionally substituted
##STR00004##
In some embodiments, L is
##STR00005##
In some embodiments, L is substituted
##STR00006##
In some embodiments, L is di-substituted
##STR00007##
In some embodiments, L is 2,5-di-substituted
##STR00008##
[0296] In some embodiments, L comprises or is bivalent 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is bivalent 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is bivalent 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, L is bivalent 6 membered heteroaryl ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, L is bivalent 6 membered heteroaryl ring having 2 nitrogen.
[0297] In some embodiments, L is optionally substituted bivalent C.sub.3-20 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-15 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-10 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-9 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-8 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-7 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-6 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-5 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-4 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.4 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.5 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.5 cycloalkyl ring. In some embodiments, L is optionally substituted bivalent C.sub.5 cycloalkenyl ring. In some embodiments, L is optionally substituted bivalent C.sub.6 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.6 cycloalkyl ring.
[0298] In some embodiments, L.sup.s2 comprises N(R)-L-N(R)- and L is a covalent bond. In some embodiments L.sup.s2 comprises N(R)N(R), wherein: [0299] each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or [0300] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0301] In some embodiments L.sup.s2 comprises N(R)N(R), wherein: [0302] each R is independently optionally substituted C.sub.1-30 aliphatic; or [0303] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered monocyclic ring.
[0304] In some embodiments, L.sup.s2 is -L.sup.x1-C(O)Q-L-QC(O)-L.sup.x1-, wherein each variable is independently as described in the present disclosure. In some embodiments, L.sup.s2 is CH.sub.2C(O)Q-L-QC(O)CH.sub.2, wherein each CH.sub.2 is independently and optionally substituted. In some embodiments, L.sup.s2 is CH.sub.2C(O)Q-L-QC(O)CH.sub.2. In some embodiments, a provided compound, e.g., a compound of R.sup.x-L.sup.s2-R.sup.x, is selected from the group consisting of: Table 1.
TABLE-US-00004 TABLE 1
In some embodiments, a provided compound, e.g., a compound of R.sup.x-L.sup.s2-R.sup.x, has the structure of R.sup.x-L.sup.x1-C(O)Q-L-QC(O)-L.sup.x2-R.sup.x or R.sup.xCH.sub.2C(O)Q-L-QC(O)CH.sub.2-R.sup.x and is selected from
TABLE-US-00005 TABLE 2
In some embodiments, a compound is not
##STR00048##
In some embodiments, a compound is not
##STR00049##
In some embodiments, a compound is not
##STR00050##
In some embodiments, a compound is not
##STR00051##
In some embodiments, a compound is not
##STR00052##
In some embodiments, L.sup.x2 or CH.sub.2C(O)Q-L-QC(O)CH.sub.2 is as described in such compounds, wherein R.sup.x is Br. In some embodiments, L.sup.s or L.sup.s2 is
##STR00053##
or
##STR00054##
In some embodiments, L.sup.s or L.sup.s2 is not
##STR00055##
In some embodiments, L.sup.s or L.sup.s2 is not
##STR00056##
In some embodiments, L.sup.s or L.sup.s2 is not
##STR00057##
In some embodiments, L.sup.s or L.sup.s2 is
##STR00058##
In some embodiments, L.sup.s or L.sup.s2 is
##STR00059##
In some embodiments, L.sup.s or L.sup.s2 is substituted
##STR00060##
In some embodiments, L.sup.s or L.sup.s2 is substituted
##STR00061##
In some embodiments, L.sup.s or L.sup.s2 or -L.sup.x1-C(O)Q-L-QC(O)-L.sup.x1- or CH.sub.2C(O)Q-L-QC(O)CH.sub.2 is
##STR00062##
in some embodiments, it is substituted
##STR00063##
in some embodiments, it is
##STR00064##
in some embodiments, it is substituted
##STR00065##
in some embodiments, it is
##STR00066##
in some embodiments, it is substituted
##STR00067##
in some embodiments, it is
##STR00068##
in some embodiments, it is substituted
##STR00069##
in some embodiments, it is optionally substituted
##STR00070##
in some embodiments, it is substituted
##STR00071##
in some embodiments, it is
##STR00072##
wherein the bivalent phenyl ring is optionally substituted; in some embodiments, it is
##STR00073##
in some embodiments, it is optionally substituted
##STR00074##
in some embodiments, it is
##STR00075##
wherein the bivalent phenyl ring is optionally substituted; in some embodiments, it is not
##STR00076##
in some embodiments, it is
##STR00077##
in some embodiments, it is substituted
##STR00078##
in some embodiments, it is optionally substituted
##STR00079##
in some embodiments, it is
##STR00080##
wherein the bivalent phenyl ring is optionally substituted; in some embodiments, it is
##STR00081##
in some embodiments, it is substituted
##STR00082##
in some embodiments, it is
##STR00083##
in some embodiments, it is
##STR00084##
in some embodiments, it is
##STR00085##
in some embodiments, it is
##STR00086##
in some embodiments, it is
##STR00087##
in some embodiments, it is substituted
##STR00088##
in some embodiments, it is
##STR00089##
in some embodiments, it is substituted
##STR00090##
in some embodiments, it is
##STR00091##
in some embodiments, it is substituted
##STR00092##
in some embodiments, it is
##STR00093##
in some embodiments, it is substituted
##STR00094##
in some embodiments, it is
##STR00095##
in some embodiments, it is substituted
##STR00096##
in some embodiments, it is
##STR00097##
in some embodiments, it is substituted
##STR00098##
in some embodiments, it is
##STR00099##
in some embodiments, it is substituted
##STR00100##
in some embodiments, it is
##STR00101##
in some embodiments, it is substituted
##STR00102##
[0305] In some embodiments, a useful compound is selected from:
TABLE-US-00006 TABLE 3
[0306] In some embodiments, L.sup.s or L.sup.s2 or -L.sup.s1-C(O)Q-L-QC(O)-L.sup.x1- or CH.sub.2C(O)Q-L-QC(O)CH.sub.2 comprise an optionally substituted saturated or partially unsaturated 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic or polycyclic ring having 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur); in some embodiments, it comprises an optionally substituted 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered cycloaliphatic ring; in some embodiments, it is
##STR00129##
in some embodiments, it is substituted
##STR00130##
in some embodiments, it is
##STR00131##
in some embodiments, it is substituted
##STR00132##
in some embodiments, it is
##STR00133##
in some embodiments, it is substituted
##STR00134##
in some embodiments, it is
##STR00135##
in some embodiments, it is substituted
##STR00136##
in some embodiments, it is
##STR00137##
in some embodiments, it is substituted
##STR00138##
in some embodiments, it comprises an optionally substituted 6-20 (e.g., 6, 10, or 14) membered monocyclic, bicyclic or polycyclic aryl ring; in some embodiments, it is optionally substituted
##STR00139##
in some embodiments, it is
##STR00140##
wherein the phenyl ring is optionally substituted; in some embodiments, it is
##STR00141##
in some embodiments, it is substituted
##STR00142##
in some embodiments, it is
##STR00143##
in some embodiments, it is substituted
##STR00144##
in some embodiments, it is
##STR00145##
in some embodiments, it is substituted
##STR00146##
in some embodiments, it is
##STR00147##
in some embodiments, it is substituted
##STR00148##
in some embodiments, it is
##STR00149##
in some embodiments, it is substituted
##STR00150##
in some embodiments, it comprises an optionally substituted 5-20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic or polycyclic heteroaryl ring having 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur); in some embodiments, it is
##STR00151##
in some embodiments, it is substituted
##STR00152##
in some embodiments, it is
##STR00153##
in some embodiments, it is substituted
##STR00154##
in some embodiments, it is
##STR00155##
in some embodiments, it is substituted
##STR00156##
in some embodiments, it comprises two or more optionally substituted rings, each of which is independently a 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered saturated or partially unsaturated ring or 5-10 membered aromatic ring, and each of which independently has 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur); in some embodiments, it is
##STR00157##
in some embodiments, it is substituted
##STR00158##
in some embodiments, it comprises an optionally substituted 5-20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered bicyclic or polycyclic ring having 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur), wherein each of the monocyclic rings is independently a 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered saturated or partially unsaturated ring having 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur) or a 5-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered aromatic ring having 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms (e.g., in some embodiments, selected from nitrogen, oxygen and sulfur); in some embodiments, it is
##STR00159##
in some embodiments, it is substituted
##STR00160##
in some embodiments, it is
##STR00161##
in some embodiments, it is substituted
##STR00162##
in some embodiments, it is
##STR00163##
in some embodiments, it is substituted
##STR00164##
in some embodiments, it is
##STR00165##
in some embodiments, it is substituted
##STR00166##
in some embodiments, it is
##STR00167##
in some embodiments, it is substituted
##STR00168##
in some embodiments, it is
##STR00169##
in some embodiments, it is substituted
##STR00170##
in some embodiments, it is
##STR00171##
in some embodiments, it is substituted
##STR00172##
in some embodiments, it is
##STR00173##
in some embodiments, it is substituted
##STR00174##
in some embodiments, it is
##STR00175##
in some embodiments, it is substituted
##STR00176##
In some embodiments, L.sup.s or L.sup.s2 is
##STR00177##
In some embodiments, L.sup.s or L.sup.s2 is substituted
##STR00178##
In some embodiments, L.sup.s or L.sup.s2 is
##STR00179##
In some embodiments, L.sup.s or L.sup.s2 is substituted
##STR00180##
[0307] As described herein, rings can be of various sizes. In some embodiments, non-aromatic rings are 3-20 membered. In some embodiments, aromatic rings are 5-20 membered. In some embodiments, a ring is 3-10 membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring is 8-membered. In some embodiments, a ring is 9-membered. In some embodiments, a ring is 10-membered. In some embodiments, a ring is 11-membered. In some embodiments, a ring is 12-membered. In some embodiments, a ring is 13-membered. In some embodiments, a ring is 14-membered. In some embodiments, a ring is 15-membered. Rings may also have various numbers and types of heteroatoms. In some embodiments, a ring has 0-10 heteroatoms. In some embodiments, a ring has 0 heteroatom. In some embodiments, a ring has 1 heteroatom. In some embodiments, a ring has 2 heteroatoms. In some embodiments, a ring has 3 heteroatoms. In some embodiments, a ring has 4 heteroatoms. In some embodiments, a ring has 5 heteroatoms. In some embodiments, a ring has 6 heteroatoms. In some embodiments, a ring has 7 heteroatoms. In some embodiments, a ring has 8 heteroatoms. In some embodiments, a ring has 9 heteroatoms. In some embodiments, a ring has 10 heteroatoms. In some embodiments, a heteroatom is selected from nitrogen, oxygen and sulfur. In some embodiments, at least one heteroatom is nitrogen. In some embodiments, at least one heteroatom is oxygen. In some embodiments, at least one heteroatom is sulfur. In some embodiments, a ring is saturated. In some embodiments, a ring is partially unsaturated. In some embodiments, a ring is aromatic. In some embodiments, a ring is a cycloaliphatic ring. In some embodiments, a ring is a cycloalkyl ring. In some embodiments, a ring is a heteroaliphatic ring. In some embodiments, a ring is a heterocyclyl ring. In some embodiments, a ring is a heterocycloalkyl ring. In some embodiments, a ring is an aryl ring. In some embodiments, a ring is a heteroaryl ring. As appreciated by those skilled in the art, rings are independently and optionally substitutedeither substituted or unsubstituted.
[0308] Many additional technologies are useful for preparing stapled peptides in accordance with the present disclosure. For example, in some embodiments, amino acid residues having side chains comprising double or triple bonds and optionally various heteroatoms may be utilized to construct various types of staples, e.g., hydrocarbon staples, amino- or carbamate-containing staples, etc.
[0309] As those skilled in the art will appreciate, after stapling using reagents described herein, portions of reagents' structures are incorporated into staples in the products. Thus, various embodiments of L.sup.s, L.sup.s2, -L.sup.x1-C(O)Q-L-QC(O)-L.sup.x1-, CH.sub.2C(O)Q-L-QC(O)CH.sub.2, L.sup.x1, Q, L, etc. described for reagents can be utilized in staples and vice versa.
Stapled Peptides
[0310] Stapled peptides as described herein are peptides in which two or more amino acids of a peptide chain are linked through bonding of two peptide backbone atoms of the amino acid residues and, as is understood by those skilled in the art, the resulting linker is not through the peptide backbone between the linked amino acid residues. In some embodiments, a stapled peptide comprises a staple as described herein. A staple as described herein is a linker that can link one amino acid residue to another amino acid residue through bonding two peptide backbone atoms of the amino acid residues and, as is understood by those skilled in the art, the resulting bond is not through the peptide backbone between the linked amino acid residues. In some embodiments, a staple bonds to the peptide backbone by replacing one or more hydrogen and/or substituents (e.g., side chains, O, S, etc.) on peptide backbone atoms (e.g., C, N, etc.).
[0311] As will be appreciated by those of ordinary skill in the art, a variety of peptide stapling technologies are available, including both hydrocarbon-stapling and non-hydrocarbon-stapling technologies. Certain technologies are described in U.S. Ser. No. 11/198,713, US 20210179665, WO 2021119537, WO 2021188659, WO 2022020651, or WO 2022020652, the entirety of each of which is incorporated herein by reference, and can be utilized in accordance with the present disclosure.
[0312] In some embodiments, a staple as described herein is a hydrocarbon staple. In some embodiments, a staple as described herein is a non-hydrocarbon staple. In some embodiments, a non-hydrocarbon staple comprises one or more chain heteroatoms wherein a chain of a staple is the shortest covalent connection within the staple from one end of the staple to the other end of the staple. In some embodiments, a non-hydrocarbon staple is a comprises at least one sulfur atom derived from an amino acid residue of a polypeptide. In some embodiments, a non-hydrocarbon staple comprises two sulfur atom derived from two different amino acid residues of a polypeptide. In some embodiments, a non-hydrocarbon staple comprises two sulfur atoms derived from two different cysteine residues of a polypeptide. In some embodiments, a staple is a cysteine staple. In some embodiments, a staple is a non-cysteine staple.
[0313] In some embodiments, the present disclosure provides a stapled peptide agent having a structure: [0314] [X.sup.1].sub.p1[X.sup.2].sub.p2[X.sup.3].sub.p3[X.sup.4].sub.p4[X.sup.5].sub.p5[X.sup.6].sub.p6[X.sup.7].sub.p7[X.sup.8].sub.p8[X.sup.9].sub.p9[X.sup.10].sub.p10[X.sup.11].sub.p11[X.sup.12].sub.p12[X.sup.13].sub.p13-X.sup.14X.sup.15X.sup.16X.sup.17X.sup.18X.sup.19-[X.sup.20].sub.p20[X.sup.21].sub.p21[X.sup.22].sub.p22[X.sup.23].sub.p23[X.sup.24].sub.p24[X.sup.25].sub.p25[X.sup.26].sub.p26[X.sup.27].sub.p27[X.sup.28].sub.p28[X.sup.29].sub.p29[X.sup.30].sub.p30[X.sup.31].sub.p31[X.sup.32].sub.p32, [0315] wherein: [0316] each of p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, p11, p12, p13, p20, p21, p22, p23, p24, p25, p26, p27, p28, p29, p30, p31, p32 is independently 0 or 1; [0317] each of X.sup.1 to X.sup.32 is independently an amino acid residue; [0318] at least two of X.sup.1 to X.sup.32 comprise side chains that are optionally linked together to form a staple.
[0319] In some embodiments, a provided peptide is a stapled peptide, and at least two of X.sup.1 to X.sup.32 comprise side chains that are linked together to form a staple. In some embodiments, a provided peptide is an unstapled peptide, wherein at least two of X.sup.1 to X.sup.32 comprise side chains that can be linked together to form a staple. In some embodiments, a stapled peptide, or an unstapled peptide once stapled, interacts with a target of interest at one or more sites on the target of interest.
[0320] In some embodiments, each of X.sup.1 to X.sup.32 is independently an amino acid residue of an amino acid having the structure of formula A-I.
[0321] In some embodiments, X.sup.i and X.sup.i+m, each independently comprises a side chain comprising a thiol, and the two side chains can be linked together to form a staple. In some embodiments, X.sup.i and X.sup.i+m, each independently comprises a cysteine side chain, and the two side chains can be linked together to form a cysteine staple.
[0322] In some embodiments, X.sup.i and X.sup.i+m, each independently comprises a side chain that comprises an olefin. In some embodiments, both of the olefins are terminal olefins. In some embodiments, at least one of X.sup.i and X.sup.i+m comprises a side chain comprising an olefin and a nitrogen atom. In some embodiments, at least one of X.sup.i and X.sup.i+m comprises C(R.sup.2a)(R.sup.3a) being -C(-L.sup.a-R)(R.sup.3a), wherein at least one methylene unit of L.sup.a is replaced with N(R) and R comprises an olefin. In some embodiments, at least one of X.sup.i and X.sup.i+m comprises C(R.sup.2a)(R.sup.3a) being C(-L.sup.a-CHCH.sub.2)(R.sup.3a), wherein at least one methylene unit of L.sup.a is replaced with N(R).
[0323] In some embodiments, i is an integer of 1 to 31 inclusive, and is m an integer of 1 to 12 inclusive.
[0324] In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. In some embodiments, i is 1. In some embodiments, i is 2. In some embodiments, i is 3. In some embodiments, i is 4. In some embodiments, i is 5. In some embodiments, i is 6. In some embodiments, i is 7. In some embodiments, i is 8. In some embodiments, i is 9. In some embodiments, i is 10. In some embodiments, i is 11. In some embodiments, i is 12. In some embodiments, i is 13. In some embodiments, i is 14. In some embodiments, i is 15. In some embodiments, i is 16. In some embodiments, i is 17. In some embodiments, i is 18. In some embodiments, i is 17. In some embodiments, i is 18. In some embodiments, i is 19. In some embodiments, i is 20. In some embodiments, i is 21. In some embodiments, i is 22. In some embodiments, i is 23. In some embodiments, i is 24. In some embodiments, i is 25. In some embodiments, i is 26. In some embodiments, i is 27. In some embodiments, i is 28. In some embodiments, i is 29. In some embodiments, i is 30. In some embodiments, i is 31. In some embodiments, i is 32.
[0325] In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12.
[0326] In some embodiments, a stapled peptide comprise one or more staples. In some embodiments, a stapled peptide comprises one and no more than one staple. In some embodiments, a stapled peptide comprises one and no more than one staples from cysteine stapling. In some embodiments, a stapled peptide comprises one and no more than one staples having the structure of
##STR00181##
In some embodiments, a stapled peptide comprises one and no more than one staples having the structure of
##STR00182##
In some embodiments, a stapled peptide comprises one and no more than one staples having the structure of
##STR00183##
In some embodiments, a stapled peptide comprises one and no more than one staples having the structure of
##STR00184##
In some embodiments, a stapled peptide comprises no staples having the structure of
##STR00185##
In some embodiments, a stapled peptide comprises no staples having the structure of
##STR00186##
In some embodiments, a stapled peptide comprises no staples having the structure of
##STR00187##
In some embodiments, a stapled peptide comprises no staples having the structure of
##STR00188##
[0327] In some embodiments, peptides, e.g., staple peptides, of the present disclosure is or comprises a helix structure. As those skilled in the art will appreciate, helixes can have various lengths. In some embodiments, lengths of helixes range from 5 to 30 amino acid residues. In some embodiments, a length of a helix is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more, amino acid residues. In some embodiments, a length of a helix is 6 amino acid residues. In some embodiments, a length of a helix is 8 amino acid residues. In some embodiments, a length of a helix is 10 amino acid residues. In some embodiments, a length of a helix is 12 amino acid residues. In some embodiments, a length of a helix is 14 amino acid residues. In some embodiments, a length of a helix is 16 amino acid residues. In some embodiments, a length of a helix is 17 amino acid residues. In some embodiments, a length of a helix is 18 amino acid residues. In some embodiments, a length of a helix is 19 amino acid residues. In some embodiments, a length of a helix is 20 amino acid residues.
[0328] In some embodiments, stapled peptides form or comprise alpha-helix structures. In some embodiments, stapling promotes and/or enhances formation of alpha-helix structures. In some embodiments, hydrophilic collapse cloaks amide protons. In some embodiments, helix formation expels high-energy water molecules. In some embodiments, helix formation retains weakly-bound water molecules. In some embodiments, stapled peptides cross membranes at higher efficiencies relative to comparable non-stapled peptides. In some embodiments, staples constrain peptide backbones of stapled peptides or portions thereof to adopt alpha-helical structures. In some embodiments, alpha-helical structures present side chains that confer target-specific recognition and/or drug-like properties. In some embodiments, technologies of present disclosure provide high levels of cytoplasmic and/or nuclear exposure, in some embodiments, by passive membrane permeability. In some embodiments, for increased permeability a hydrophobic amino acid residue may be utilized over a polar residue or a charged residue. In some embodiments, for increased permeability a polar amino acid residue may be utilized over a charged residue. In some embodiments, it is observed that replacing carbon-carbon double bond with a carbon-carbon single bond does not significantly impact cell permeability. In some embodiments, it is observed that staple type and/or positioning impacts cytosolic exposure. In some embodiments, longer staples provide higher cytosolic exposure. In some embodiments, less polar staples may provide higher cytosolic exposure. In some embodiments, stapled peptides provide suppressed renal clearance and long-circulating half-lives. In some embodiments, stapled peptides have high proteolytic stability, e.g., in cells and in vivo relative to comparable non-stapled peptides. In some embodiments, stapled peptides are non- or low-immunogenic. In some embodiments, stapled peptides do not bind MHC class I and/or class II receptor. In some embodiments, stapled peptides provide oral bioavailability, in some cases including and especially in left-handed (D-configurated) form.
Cysteine Stapling
[0329] In some embodiments, the present disclosure provides useful technologies relating to cysteine stapling. Among other things, the present disclosure appreciates that peptides amenable to cysteine stapling and/or comprising one or more cysteine staples, can be produced and/or assessed in a biological system. The present disclosure further appreciates that certain such systems permit development, production, and/or assessment of cysteine stapled peptides having a range of different structures (e.g., different amino acid sequences), and in fact can provide a user with complete control over selection and implementation of amino acid sequences to be incorporated into stapled peptides.
[0330] Cysteine stapling, as described herein, involves linking one cysteine residue to another cysteine residue, where the resulting bond is not through the peptide backbone between the linked cysteine residues.
[0331] In some embodiments, a stapled peptide as described herein comprises a staple which staple is L.sup.s, wherein: [0332] L.sup.s is -L.sup.s1-S-L.sup.s2-S-L.sup.s3-; [0333] L.sup.s1, L.sup.s2 and L.sup.s3 are each independently L; [0334] each L is independently a covalent bond, or an optionally substituted, bivalent C.sub.1-C.sub.25 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O; [0335] each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; [0336] each R is independently R, C(O)R, CO.sub.2R, or SO.sub.2R; [0337] each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or [0338] two R groups are optionally and independently taken together to form a covalent bond; or [0339] two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or [0340] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0341] In some embodiments, L.sup.s2 is L and comprises at least one C(O)-.
[0342] As described herein, various linker moieties or staples can be L. In some embodiments, L is a covalent bond. In some embodiments, L is an optionally substituted, bivalent C.sub.1-C.sub.25 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O-. In some embodiments, L is an optionally substituted, bivalent C.sub.1-C.sub.20 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2-, S(O).sub.2N(R), C(O)S, or C(O)O-. In some embodiments, L is an optionally substituted, bivalent C.sub.1-C.sub.15 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O-. In some embodiments, L is an optionally substituted, bivalent C.sub.1-C.sub.10 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O. In some embodiments, L is an optionally substituted, bivalent C.sub.1-C.sub.5 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O.
[0343] In some embodiments, L is independently a bivalent C.sub.1-C.sub.25 aliphatic group. In some embodiments, L is independently a bivalent C.sub.1-C.sub.20 aliphatic group. In some embodiments, L is independently a bivalent C.sub.1-C.sub.10 aliphatic group. In some embodiments, L is independently a bivalent C.sub.1-C.sub.5 aliphatic group. In some embodiments, L is independently a bivalent C.sub.1 aliphatic group. In some embodiments, L is optionally substituted CH.sub.2. In some embodiments, L is CH.sub.2.
[0344] In some embodiments, L.sup.s1 is L as described herein. In some embodiments, L.sup.s1 is optionally substituted CH.sub.2. In some embodiments, L.sup.s1 is CH.sub.2. In some embodiments, L.sup.s3 is L as described herein. In some embodiments, L.sup.s3 is optionally substituted CH.sub.2. In some embodiments, L.sup.s3 is CH.sub.2. In some embodiments, L.sup.s1 and L.sup.s3 are both optionally substituted CH.sub.2. In some embodiments, L.sup.s1 and L.sup.s3 are both CH.sub.2. In some embodiments, L.sup.s is CH.sub.2S-L.sup.s2-SCH.sub.2 wherein L.sup.s2 is as described herein.
[0345] In some embodiments, L.sup.s2 is L as described herein. In some embodiments, L.sup.s2 comprises C(R).sub.2-L-C(R).sub.2, wherein L is described in the present disclosure. In some embodiments, L.sup.s2 is -L.sup.x1-C(O)Q-L-QC(O)-L.sup.x1-, wherein each variable is independently as described in the present disclosure. In some embodiments, L.sup.s2 is CH.sub.2C(O)Q-L-QC(O)CH.sub.2, wherein each CH.sub.2 is independently and optionally substituted. In some embodiments, L.sup.s2 is CH.sub.2C(O)Q-L-QC(O)CH.sub.2.
[0346] In some embodiments, L.sup.s2 In some embodiments, L.sup.s2 is L and comprises at least one C(O)-. In some embodiments, L.sup.s2 is L and comprises at least two -C(O)-. In some embodiments, L.sup.s2 is L and comprises at least one C(O)Q-, wherein Q is selected from the group consisting of: a covalent bond, N(R), O, and S. In some embodiments, L.sup.s2 is L and comprises at least one C(O)Q-, wherein Q is selected between N(R) and O. In some embodiments, L.sup.s2 is L and comprises at least two C(O)Q-, wherein Q is selected from the group consisting of: N(R), O, and S. In some embodiments, L.sup.s2 is L and comprises at least two C(O)Q-, wherein Q is selected between N(R) and O. In some embodiments, L.sup.s2 is L and comprises at least one C(O)N(R). In some embodiments, L.sup.s2 is L and comprises at least two C(O)N(R). In some embodiments, L.sup.s2 is L and comprises at least one C(O)O. In some embodiments, L.sup.s2 is L and comprises at least two C(O)O.
[0347] In some embodiments, L.sup.s2 comprises -Q-L-Q-, wherein Q is independently selected from the group consisting of: N(R), O, and S, wherein L is described in the present disclosure.
[0348] In some embodiments, L.sup.s2 comprises -Q-L-Q-, wherein Q is independently selected between N(R) and O, wherein L is described in the present disclosure. In some embodiments, L.sup.s2 comprises C(O)Q-L-QC(O), wherein Q is independently selected from the group consisting of: N(R), O, and S, wherein L is described in the present disclosure. In some embodiments, L.sup.s2 comprises C(O)Q-L-QC(O), wherein Q is independently selected between N(R) and -0, wherein L is described in the present disclosure. In some embodiments, L.sup.s2 comprises C(R).sub.2C(O)Q-L-QC(O)C(R).sub.2, wherein Q is independently selected from the group consisting of: N(R), O, and S, wherein L is described in the present disclosure. In some embodiments, L.sup.s2comprises C(R).sub.2C(O)Q-L-QC(O)C(R).sub.2, wherein Q is independently selected between N(R) and -0, wherein L is described in the present disclosure.
[0349] In some embodiments, L.sup.s2 comprises N(R)-L-N(R), wherein L is described in the present disclosure. In some embodiments, L.sup.s2 comprises C(O)N(R)-L-N(R)C(O)-, wherein L is described in the present disclosure. In some embodiments, L.sup.s2 is C(R).sub.2C(O)N(R)-L-N(R)C(O)C(R).sub.2, wherein L is described in the present disclosure.
[0350] In some embodiments, L.sup.s2 comprises O(R)-L-O(R), wherein L is described in the present disclosure. In some embodiments, L.sup.s2 comprises C(O)O-L-OC(O)-, wherein L is described in the present disclosure. In some embodiments, L.sup.s2 is C(R).sub.2C(O)O-L-OC(O)C(R).sub.2, wherein L is described in the present disclosure.
[0351] In some embodiments, R is an optionally substituted C.sub.1-30 aliphatic. In some embodiments, R is an optionally substituted C.sub.1-15 aliphatic. In some embodiments, R is an optionally substituted C.sub.1-10 aliphatic. In some embodiments, R is an optionally substituted C.sub.1-5 aliphatic. In some embodiments, R is hydrogen.
[0352] In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.15 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.10 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.9 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.8 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.7 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.6 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.5 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.3 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.2 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1 aliphatic. In some embodiments, L is CH.sub.2. In some embodiments, L is (CH.sub.2).sub.2. In some embodiments, L is (CH.sub.2).sub.3. In some embodiments, L is (CH.sub.2).sub.4. In some embodiments, L is (CH.sub.2).sub.5. In some embodiments, L is (CH.sub.2).sub.6. In some embodiments, L is (CH.sub.2).sub.7. In some embodiments, L is (CH.sub.2).sub.8.
[0353] In some embodiments, L is optionally substituted bivalent C.sub.6-20 aryl ring. In some embodiments, L is optionally substituted bivalent C.sub.6-14 aryl ring. In some embodiments, L is optionally substituted bivalent C.sub.6-10 aryl ring. In some embodiments, L is optionally substituted bivalent C.sub.6 aryl ring. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with four halogen. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with four fluorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least one chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted with four chlorine. In some embodiments, L is bivalent C.sub.6 aryl substituted at with least one O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least two O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is bivalent C.sub.6 aryl substituted with at least three O(CH.sub.2).sub.0-4CH.sub.3. In some embodiments, L is bivalent C.sub.6 aryl substituted with four O(CH.sub.2).sub.0-4CH.sub.3.
[0354] In some embodiments, L is bivalent 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is bivalent 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is bivalent 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, L is bivalent 6 membered heteroaryl ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, L is bivalent 6 membered heteroaryl ring having 2 nitrogen.
[0355] In some embodiments, L is optionally substituted bivalent C.sub.320 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-15 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-10 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-9 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-8 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-7 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-6 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-5 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3-4 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.3 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.4 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.5 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.5 cycloalkyl ring. In some embodiments, L is optionally substituted bivalent C.sub.5 cycloalkenyl ring. In some embodiments, L is optionally substituted bivalent C.sub.6 cycloaliphatic ring. In some embodiments, L is optionally substituted bivalent C.sub.6 cycloalkyl ring.
[0356] In some embodiments, L.sup.s2 comprises N(R)-LN(R) and L is a covalent bond. In some embodiments L.sup.s2 comprises N(R)N(R), wherein: [0357] each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or [0358] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0359] In some embodiments L.sup.s2 comprises N(R)N(R), wherein: [0360] each R is independently optionally substituted C.sub.1-30 aliphatic; or [0361] two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered monocyclic ring.
[0362] In some embodiments, L.sup.s2 is a staple selected from the group consisting of:
##STR00189## ##STR00190##
[0363] As those skilled in the art will appreciate, provided technologies can be utilized to prepare collection of peptides using non-cysteine residues and suitable chemistry therefor. For example, in some embodiments, cysteine stapling is replaced with lysine stapling, wherein the cysteine residues for cysteine stapling are replaced with lysine residues for lysine stapling (e.g., using agents that can crosslink two lysine residues, for example, through reactions with side chain amino groups). In some embodiments, for lysine stapling, R.sup.E in various formulae is or comprises an activated carboxylic acid group (e.g., NHS ester group), an imidoester group, etc. Suitable reagents are widely known in the art including many commercially available ones. In some embodiments, cysteine stapling is replaced with methionine stapling. In some embodiments, cysteine residues for cysteine stapling are replaced with methionine residues for methionine stapling. In some embodiments, cysteine stapling is replaced with tryptophan stapling. In some embodiments, cysteine residues for cysteine stapling are replaced with tryptophan residues for tryptophan stapling. As those skilled in the art will appreciate, various technologies (e.g., reagents, reactions, etc.) are described in the art and can be utilized in accordance with the present disclosure for, e.g., methionine stapling, tryptophan stapling, etc. In some embodiments, such stapling can be performed using reagents having various formulae described herein, wherein R.sup.E is or comprises a group that are suitable for methionine and/or tryptophan stapling. In some embodiments, stapling may be performed using one residue at a first position, and a different residue at a second position. Useful reagents for such stapling may comprise a first reactive group for stapling at a first position (e.g., through a first R.sup.E), and a second reactive group for stapling at a second position (e.g., through a second R.sup.E).
[0364] In some embodiments, for various types of stapling (e.g., cysteine stapling, or non-cysteine stapling), stapling is between residues (e.g., cysteine residues for cysteine stapling) separated by two residues (i+3 stapling). In some embodiments, stapling is between residues separated by three residues (i+4 stapling). In some embodiments, stapling is between residues separated by six residues (i+7 stapling).
[0365] As appreciated by those skilled in the art, in some embodiments, more than two residues can be stapled at the same time. For example, in some embodiments, three or more cysteines are stapled using crosslinking reagents containing three or more reactive groups (e.g., R.sup.E groups).
Non-Cysteine Stapling
[0366] In some embodiments, the present disclosure provides useful technologies relating to non-cysteine stapling. Among other things, the present disclosure appreciates that peptides amenable to cysteine stapling and/or comprising one or more non-cysteine staples, can have its cysteine residues and cysteine staple replaced with other amino acids and staples (e.g. hydrocarbon and other non-hydrocarbon amino acid and staples). In some embodiments, the resulting non-cysteine stapled peptide maintains the same or similar interaction with a target of interest when compared to a reference cysteine stapled peptide. Described herein are non-cysteine amino acids and non-cysteine staples involving such non-cysteine amino acid residues.
[0367] In some embodiments, an amino acid of formula A-I is a compound having the structure of formula A-II:
NH(R.sup.a1)-L.sup.a1-C(-L.sup.a-CHCH.sub.2)(R.sup.a3)-L.sup.a2-COOH, A-II
or a salt thereof, wherein each variable is independently as described in the present disclosure.
[0368] In some embodiments, an amino acid of formula A-I is a compound having the structure of formula A-III:
NH(R.sup.a1)-C(-L.sup.a-CHCH.sub.2)(R.sup.a3)COOH, A-III
or a salt thereof, wherein each variable is independently as described in the present disclosure.
[0369] In some embodiments, L.sup.a comprises at least one N(R) wherein R is independently as described in the present disclosure.
[0370] In some embodiments, an amino acid of formula A-I is a standard amino acid. In some embodiments, an amino acid of formula A-I is selected from Tables A-I, A-II, and A-III:
TABLE-US-00007 TABLE A-I Exemplary amino acids (Fmoc-Protected). Monomer A (M.sub.A)
TABLE-US-00008 TABLE A-II Exemplary amino acids (Fmoc-Protected). Monomer D (M.sub.D)
TABLE-US-00009 TABLE A-III Exemplary amino acids (Fmoc-Protected). S.sub.3
[0371] In some embodiments, an amino acid is an alpha-amino acid. In some embodiments, an amino acid is an L-amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, the alpha-carbon of an amino acid is achiral. In some embodiments, an amino acid is a beta-amino acid. In some embodiments, an amino acid is a gamma-amino acid.
[0372] In some embodiments, a provided amino acid sequence contains two or more amino acid residues whose side chains are linked together to form one or more staples. In some embodiments, a provided amino acid sequence contains two or more amino acid residues, each of which independently has a side chain comprising an olefin. In some embodiments, a provided amino acid sequence contains two or more amino acid residues, each of which independently has a side chain comprising a terminal olefin. In some embodiments, a provided amino acid sequence contains two and no more than two amino acid residues, each of which independently has a side chain comprising an olefin. In some embodiments, a provided amino acid sequence contains two and no more than two amino acid residues, each of which independently has a side chain comprising a terminal olefin. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid that comprises an olefin and a nitrogen atom other than the nitrogen atom of its amino group. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid that comprises a terminal olefin and a nitrogen atom other than the nitrogen atom of its amino group. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid that has a side chain than comprises a terminal olefin and a nitrogen atom. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid of formula A-I, wherein R.sup.a2 comprising an olefin and a N(R) moiety, wherein R is as described in the present disclosure (including, in some embodiments, optionally taken together with R.sup.a3 and their intervening atoms to form an optionally substituted ring as described in the present disclosure). In some embodiments, R.sup.a2 comprising a terminal olefin and a N(R) moiety wherein R is as described in the present disclosure. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid selected from Table A-I. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid selected from Table A-II. In some embodiments, a provided amino acid sequence comprises at least one residue of an amino acid selected from Table A-III. In some embodiments, two olefins from two side chains are linked together through olefin metathesis to form a staple. In some embodiments, a staple is preferably formed by side chains of amino acid residues that are not at the corresponding positions of a target of interest. In some embodiments, a formed staple does not disrupt interaction between the peptide and a target of interest.
[0373] In some embodiments, a provided staple is a hydrocarbon staple. In some embodiments, a hydrocarbon staple comprises no chain heteroatoms wherein a chain of a staple is the shortest covalent connection within the staple from one end of the staple to the other end of the staple.
[0374] In some embodiments, a provided staple is a non-hydrocarbon staple. In some embodiments, a non-hydrocarbon staple comprises one or more chain heteroatoms wherein a chain of a staple is the shortest covalent connection within the staple from one end of the staple to the other end of the staple. In some embodiments, a non-hydrocarbon staple is a carbamate staple in that it comprises a N(R)-C(O)-O moiety in its chain. In some embodiments, a non-hydrocarbon staple is an amino staple in that it comprises a N(R) moiety in its chain, wherein the N(R) moiety is not part of N(R)-C(O)-O-. In some embodiments, a non-hydrocarbon staple is an amino staple in that it comprises a N(R) moiety in its chain, wherein the N(R) moiety is not bonded to a carbon atom that additionally forms a double bond with a heteroatom (e.g., C(O), C(S), C(N-R), etc.).
[0375] In some embodiments, a provided stapled peptide comprises a staple which staple is L.sup.s, wherein L.sup.s is -L.sup.s1-L.sup.s2-L.sup.s3-, each of L.sup.s1, L.sup.s2, and L.sup.s3 is independently L, wherein each L is independently as described in the present disclosure. In some embodiments, a provided staple is L.sup.s.
[0376] In some embodiments, L.sup.s1 comprises at least one N(R), wherein R is as described in the present disclosure. In some embodiments, the N(R) is bonded to two carbon atoms, wherein neither of the two carbon atoms forms a double bond with a heteroatom. In some embodiments, the N(R) is not bonded to C(O). In some embodiments, the N(R) is not bonded to C(S). In some embodiments, the N(R) is not bonded to C(NR). In some embodiments, L.sup.s1 is -L-N(R), wherein L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic. In some embodiments, L.sup.s1 is -L-N(CH.sub.3), wherein L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic.
[0377] In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is C.sub.1-6 alkyl. In some embodiments, R is methyl. In some embodiments, the peptide backbone atom to which L.sup.s1 is bonded is also bonded to R.sup.1, and R and R.sup.1 are both R and are taken together with their intervene atoms to form an optionally substituted ring as described in the present disclosure. In some embodiments, a formed ring has no additional ring heteroatoms in addition to the nitrogen atom to which R is bonded. In some embodiments, a formed ring is 3-membered. In some embodiments, a formed ring is 4-membered. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6-membered.
[0378] In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.20 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.15 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.10 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.9 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.8 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.7 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.6 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.5 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.4 aliphatic. In some embodiments, L is optionally substituted alkylene. In some embodiments, L is optionally substituted alkenylene. In some embodiments, L is unsubstituted alkylene. In some embodiments, L is CH.sub.2. In some embodiments, L is (CH.sub.2).sub.2. In some embodiments, L is (CH.sub.2).sub.3. In some embodiments, L is (CH.sub.2).sub.4. In some embodiments, L is (CH.sub.2).sub.5. In some embodiments, L is (CH.sub.2).sub.6. In some embodiments, L is (CH.sub.2).sub.7. In some embodiments, L is (CH.sub.2).sub.8. In some embodiments, L is bonded to a peptide backbone atom. In some embodiments, L is optionally substituted alkenylene. In some embodiments, L is unsubstituted alkenylene. In some embodiments, L is CH.sub.2CHCH-CH.sub.2.
[0379] In some embodiments, L.sup.s1 comprises at least one N(R)C(O), wherein R is as described in the present disclosure. In some embodiments, L.sup.s1 is -L-N(R)C(O), wherein each of L and R is independently as described in the present disclosure. In some embodiments, L.sup.s1 is -L-N(CH.sub.3)C(O), wherein L is independently as described in the present disclosure.
[0380] In some embodiments, L.sup.s1 is a covalent bond.
[0381] In some embodiments, L.sup.s1 is L, wherein L is as described in the present disclosure.
[0382] In some embodiments, L.sup.s2 is L, wherein L is as described in the present disclosure. In some embodiments, L.sup.s2 is L, wherein L is as described in the present disclosure. In some embodiments, L.sup.s2comprises CH.sub.2CHCH-CH.sub.2. In some embodiments, L.sup.s2 is CH.sub.2CHCH-CH.sub.2. In some embodiments, L.sup.s2 comprises (CH.sub.2).sub.4. In some embodiments, L.sup.s2 is (CH.sub.2).sub.4.
[0383] In some embodiments, L.sup.s3 comprises at least one N(R), wherein R is as described in the present disclosure. In some embodiments, the N(R) is bonded to two carbon atoms, wherein neither of the two carbon atoms forms a double bond with a heteroatom. In some embodiments, the N(R) is not bonded to C(O). In some embodiments, the N(R) is not bonded to C(S). In some embodiments, the N(R) is not bonded to C(NR). In some embodiments, L.sup.s3 is -L-N(R), wherein L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic. In some embodiments, L.sup.s3 is -L-N(CH.sub.3), wherein L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic.
[0384] In some embodiments, L.sup.s3 comprises at least one N(R)C(O), wherein R is as described in the present disclosure. In some embodiments, L.sup.s3 is -L-N(R)C(O), wherein each of L and R is independently as described in the present disclosure. In some embodiments, L.sup.s3 is -L-N(CH.sub.3)C(O), wherein L is independently as described in the present disclosure.
[0385] In some embodiments, L.sup.s3 is L, wherein L is as described in the present disclosure. In some embodiments, L.sup.s3 is optionally substituted alkylene. In some embodiments, L.sup.s3 is unsubstituted alkylene.
[0386] In some embodiments, L.sup.s comprises at least one N(R), wherein R is as described in the present disclosure. In some embodiments, the N(R) is bonded to two carbon atoms, wherein neither of the two carbon atoms forms a double bond with a heteroatom. In some embodiments, the N(R) is not bonded to C(O). In some embodiments, the N(R) is not bonded to C(S). In some embodiments, the N(R) is not bonded to C(NR). In some embodiments, L.sup.s comprises at least one N(R)C(O), wherein R is as described in the present disclosure.
[0387] In some embodiments, L comprises at least one N(R), wherein R is as described in the present disclosure. In some embodiments, the N(R) is bonded to two carbon atoms, wherein neither of the two carbon atoms forms a double bond with a heteroatom. In some embodiments, the N(R) is not bonded to C(O). In some embodiments, the N(R) is not bonded to C(S). In some embodiments, the N(R) is not bonded to C(NR). In some embodiments, L is -L-N(R), wherein L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic. In some embodiments, L is -L-N(CH.sub.3), wherein L is optionally substituted bivalent C.sub.1-C.sub.19 aliphatic.
[0388] In some embodiments, L comprises at least one N(R)C(O), wherein R is as described in the present disclosure. In some embodiments, L is -L-N(R)C(O), wherein each of L and R is independently as described in the present disclosure. In some embodiments, L is -L-N(CH.sub.3)C(O), wherein L is independently as described in the present disclosure.
[0389] In some embodiments, L is L, wherein L is as described in the present disclosure. In some embodiments, L is optionally substituted alkylene. In some embodiments, L is unsubstituted alkylene.
[0390] In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.25 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.20 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.15 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.10 aliphatic. In some embodiments, Lis optionally substituted bivalent C.sub.1-C.sub.9 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.8 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.7 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.6 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.5 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-C.sub.4 aliphatic. In some embodiments, L is optionally substituted alkylene. In some embodiments, L is optionally substituted alkenylene. In some embodiments, L is unsubstituted alkylene. In some embodiments, L is CH.sub.2. In some embodiments, L is (CH.sub.2).sub.2. In some embodiments, L is (CH.sub.2).sub.3. In some embodiments, L is (CH.sub.2).sub.4. In some embodiments, L is (CH.sub.2).sub.5. In some embodiments, L is (CH.sub.2).sub.6. In some embodiments, L is (CH.sub.2).sub.7. In some embodiments, L is (CH.sub.2).sub.8. In some embodiments, L is bonded to a peptide backbone atom. In some embodiments, L is optionally substituted alkenylene. In some embodiments, L is unsubstituted alkenylene. In some embodiments, L is CH.sub.2CHCH-CH.sub.2.
[0391] In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-30 (e.g., C.sub.1-20, C.sub.1-15, C.sub.1-10, etc.) aliphatic, C.sub.1-30 (e.g., C.sub.1-20, C.sub.1-15, C.sub.1-10, etc.) heteroaliphatic having 1-10 (e.g., 1-8, 1-6, 1-5, 1-4, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 (e.g., C.sub.6-20, C.sub.6-14, C.sub.6-10, etc.) aryl, C.sub.6-30 (e.g., C.sub.6-20, C.sub.6-14, C.sub.6-10, etc.) arylaliphatic, C.sub.6-30 (e.g., C.sub.6-20, C.sub.6-14, C.sub.6-10, etc.) arylheteroaliphatic having 1-10 (e.g., 1-8, 1-6, 1-5, 1-4, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 (e.g., 5-20, 5-14, 5-10, 5-6, 5, 6, 9, 10, etc.) membered heteroaryl having 1-10 (e.g., 1-8, 1-6, 1-5, 1-4, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 (e.g., 3-20, 3-15, 3-10, 3-9, 3-6, 5-10, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) membered heterocyclyl having 1-10 (e.g., 1-8, 1-6, 1-5, 1-4, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two R groups are optionally and independently taken together to form a covalent bond. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 (e.g., 3-20, 3-15, 3-10, 3-9, 3-6, 5-10, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 (e.g., 1-10, 1-8, 1-6, 1-5, 1-4, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 (e.g., 3-20, 3-15, 3-10, 3-9, 3-6, 5-10, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 (e.g., 1-10, 1-8, 1-6, 1-5, 1-4, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-10 aliphatic, C.sub.1-10 heteroaliphatic having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-14 aryl, C.sub.6-14 arylaliphatic, C.sub.6-14 arylheteroaliphatic having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-14 membered heteroaryl having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-10 membered heterocyclyl having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond; or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-10 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-10 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0392] In some embodiments, one end of a staple is connected to an atom A.sup.n1 of the peptide backbone, wherein A.sup.n1 is optionally substituted with R.sup.1 and is an atom of an amino acid residue at amino acid position n.sup.1 of the peptide from the N-terminus, and the other end is connected to an atom A.sup.n2 of the peptide backbone, wherein A.sup.n2 is optionally substituted with R.sup.2 (in some embodiments, R.sup.1 and/or R.sup.2 is R which can be hydrogen) and is an atom of an amino acid residue at amino acid position n.sup.2 of the peptide from the N-terminus, wherein each of n.sup.1 and n.sup.2 is independently an integer, and n.sup.2=n.sup.1+m, wherein m is 3-12.
[0393] In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, a staple is referred to a (i, i+m) staple.
[0394] In some embodiments, A.sup.n1 is a carbon atom. In some embodiments, A.sup.n1 is achiral. In some embodiments, A.sup.n1 is chiral. In some embodiments, A.sup.n1 is R. In some embodiments, A.sup.n1 is S.
[0395] In some embodiments, A.sup.n2 is a carbon atom. In some embodiments, A.sup.n2 is achiral. In some embodiments, A.sup.n2 is chiral. In some embodiments, A.sup.n2 is R. In some embodiments, A.sup.n2 is S.
[0396] In some embodiments, A.sup.n1 is achiral and A.sup.n2 is achiral. In some embodiments, A.sup.n1 is achiral and A.sup.n2 is R. In some embodiments, A.sup.n1 is achiral and A.sup.n2 is S. In some embodiments, A.sup.n1 is R and A.sup.n2 is achiral. In some embodiments, A.sup.n1 is R and A.sup.n2 is R. In some embodiments, A.sup.n1 is R and A.sup.n2 is S. In some embodiments, A.sup.n1 is S and A.sup.n2 is achiral. In some embodiments, A.sup.n1 is S and A.sup.n2 is R. In some embodiments, A.sup.n1 is S and A.sup.n2 is S.
[0397] In some embodiments, provided stereochemistry at staple-backbone connection points and/or combinations thereof, optionally together with one or more structural elements of provided peptide, e.g., staple chemistry (hydrocarbon, non-hydrocarbon), staple length, etc. can provide various benefits, such as improved preparation yield, purity, and/or selectivity, improved properties (e.g., improved solubility, improved stability, lowered toxicity, improved selectivities, etc.), improved activities, etc. In some embodiments, provided stereochemistry and/or stereochemistry combinations are different from those typically used, e.g., those of U.S. Pat. No. 9,617,309, US 2015-0225471, US 2016-0024153, US 2016-0215036, US 2016-0244494, WO 2017/062518, and provided one or more of benefits described in the present disclosure.
[0398] In some embodiments, a staple can be of various lengths, in some embodiments, as represent by the number of chain atoms of a staple. In some embodiments, a chain of a staple is the shortest covalent connection in the staple from a first end (connection point with a peptide backbone) of a staple to a second end of the staple, wherein the first end and the second end are connected to two different peptide backbone atoms. In some embodiments, a staple comprises 5-30 chain atoms, e.g., 5, 6, 7, 8, 9, or 10 to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chain atoms. In some embodiments, a staple comprises 5 chain atoms. In some embodiments, a staple comprises 6 chain atoms. In some embodiments, a staple comprises 7 chain atoms. In some embodiments, a staple comprises 8 chain atoms. In some embodiments, a staple comprises 9 chain atoms. In some embodiments, a staple comprises 10 chain atoms. In some embodiments, a staple comprises 11 chain atoms. In some embodiments, a staple comprises 12 chain atoms. In some embodiments, a staple comprises 13 chain atoms. In some embodiments, a staple comprises 14 chain atoms. In some embodiments, a staple comprises 15 chain atoms. In some embodiments, a staple comprises 16 chain atoms. In some embodiments, a staple comprises 17 chain atoms. In some embodiments, a staple comprises 18 chain atoms. In some embodiments, a staple comprises 19 chain atoms. In some embodiments, a staple comprises 20 chain atoms. In some embodiments, a staple has a length of 5 chain atoms. In some embodiments, a staple has a length of 6 chain atoms. In some embodiments, a staple has a length of 7 chain atoms. In some embodiments, a staple has a length of 8 chain atoms. In some embodiments, a staple has a length of 9 chain atoms. In some embodiments, a staple has a length of 10 chain atoms. In some embodiments, a staple has a length of 11 chain atoms. In some embodiments, a staple has a length of 12 chain atoms. In some embodiments, a staple has a length of 13 chain atoms. In some embodiments, a staple has a length of 14 chain atoms. In some embodiments, a staple has a length of 15 chain atoms. In some embodiments, a staple has a length of 16 chain atoms. In some embodiments, a staple has a length of 17 chain atoms. In some embodiments, a staple has a length of 18 chain atoms. In some embodiments, a staple has a length of 19 chain atoms. In some embodiments, a staple has a length of 20 chain atoms. In some embodiments, a staple has a length of 8-15 chain atoms. In some embodiments, a staple has 8-12 chain atoms. In some embodiments, a staple has 9-12 chain atoms. In some embodiments, a staple has 9-10 chain atoms. In some embodiments, a staple has 8-10 chain atoms. In some embodiments, length of a staple can be adjusted according to the distance of the amino acid residues it connects, for example, a longer staple may be needed for a (i, i+7) staple than a (i, i+4) staple. Staple lengths may be otherwise described. For example, in some embodiments, staple lengths may be described as the total number of chain atoms and non-chain ring atoms, where a non-chain ring atom is an atom of the staple which forms a ring with one or more chain atoms but is not a chain atom in that it is not within the shortest covalent connection from a first end of the staple to a second end of the staple. In some embodiments, staples formed using Monomer A (which comprises a azetidine moiety), Monomer B (which comprises a pyrrolidine moiety), and/or Monomer C (which comprises a pyrrolidine moiety) may comprise one or two non-chain ring atoms as illustrated in the exemplary stapled peptides.
[0399] In some embodiments, a staple has no heteroatoms in its chain. In some embodiments, a staple comprises at least one heteroatom in its chain. In some embodiments, a staple comprises at least one nitrogen atom in its chain.
[0400] In some embodiments, a staple is L.sup.s, wherein L.sup.s is an optionally substituted, bivalent C.sub.8-14 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O. In some embodiments, a staple is L.sup.s, wherein L.sup.s is an optionally substituted, bivalent C.sub.9-13 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O. In some embodiments, a staple is L.sup.s, wherein L.sup.s is an optionally substituted, bivalent C.sub.10-15 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O. In some embodiments, a staple is L.sup.s, wherein L.sup.s is an optionally substituted, bivalent C.sub.11-14 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O. In some embodiments, a staple is a (i, i+4) staple in that not including the two amino acid residues that are directly connected to the staple, there are three amino acid residues between the two amino acid residues that are directly connected to the staple. In some embodiments, a staple is a (i, i+7) staple in that not including the two amino acid residues that are directly connected to the staple, there are six amino acid residues between the two amino acid residues that are directly connected to the staple.
[0401] In some embodiments, for each of L.sup.s, L.sup.s1, L.sup.s2, and L.sup.s3, any replacement of methylene units, if any, is replaced with N(R) or N(R)C(O).
[0402] In some embodiments, an olefin in a staple is a Z-olefin. In some embodiments, an olefin in a staple in an E-olefin. In some embodiments, a provided composition comprises stapled peptides comprising a staple that contains a Z-olefin and stapled peptides comprising a staple that contains an E-olefin. In some embodiments, a provided composition comprises stapled peptides comprising a staple that contains a Z-olefin. In some embodiments, a provided composition comprises stapled peptides comprising a staple that contains an E-olefin. In some embodiments, otherwise identical stapled peptides that differ only in the E/Z configuration of staple olefin demonstrate different properties and/or activities as demonstrated herein. In some embodiments, stapled peptides with E-olefin in a staple may provide certain desirable properties and/or activities given the context. In some embodiments, stapled peptides with Z-olefin in a staple may provide certain desirable properties and/or activities given the context.
[0403] In some embodiments, two staples may be bonded to the same atom of the peptide backbone, forming a stitch structure.
[0404] In some embodiments, a staple is Pro-lock in that one end of the staple is bonded to the alpha-carbon of a proline residue.
[0405] In some embodiments, an exemplary staple is a staple as illustrated below in Tables S-1, S-2, S-3, and S-4 (with exemplary peptide backbone illustrated for clarity (can be applied to other peptide backbone), X being amino acid residues). In some embodiments, the olefin is Z. In some embodiments, the olefin is E. In some embodiments, an (i, i+4) staple is selected from Table S-1. In some embodiments, an (i, i+4) staple is selected from Table S-2. In some embodiments, an (i, i+7) staple is selected from Table S-3. In some embodiments, an (i, i+7) staple is selected from Table S-4.
TABLE-US-00010 TABLE S-1 Exemplary staples.
TABLE-US-00011 TABLE S-2 Exemplary staples.
TABLE-US-00012 TABLE S-3 Exemplary staples.
TABLE-US-00013 TABLE S-4 Exemplary staples.
[0406] As described herein, in various instances, a group is or comprises an optionally substituted ring. For example, in some embodiments, R or a group that can be R (e.g., R) is or comprises an optionally substituted ring as described herein. In some embodiments, two or more R groups, or two or more groups that are or can be R (e.g., R, R.sup.2, R.sup.3, etc.,), can be taken together with their intervening atom(s) to form an optionally substituted ring as described herein. In some embodiments, a ring is substituted (in addition to groups attached to the intervening atom(s)). In some embodiments, a ring is unsubstituted. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring is 8-membered. In some embodiments, a ring is 9-membered. In some embodiments, a ring is 10-membered. In some embodiments, a ring is saturated. In some embodiments, a ring is partially unsaturated. In some embodiments, a ring is aromatic. In some embodiments, a ring is monocyclic. In some embodiments, it is bicyclic. In some embodiments, it is polycyclic. In some embodiments, each monocyclic unit is independently a 3-10 (e.g., 3-8, 3-6, 5-6, or 3, 4, 5, 6, 7, 8, 9, or 10, etc.) membered ring which is independently saturated, partially unsaturated or aromatic and has 0-4 (e.g., 0, 1, 2, 3, or 4) heteroatoms. In some embodiments, each monocyclic unit is independently a 3-10 (e.g., 3-10, 3-8, 3-6, 5-6, or 3, 4, 5, 6, 7, 8, 9, or 10, etc.) membered ring which is independently saturated, partially unsaturated or aromatic and has 0-4 (e.g., 0, 1, 2, 3, or 4, etc.) heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments, each monocyclic ring unit is independently 3-7 membered. In some embodiments, each monocyclic ring unit is independently 3-6 membered. In some embodiments, each monocyclic ring unit is independently 5-7 membered. In some embodiments, each monocyclic unit is independently saturated or partially unsaturated. In some embodiments, at least one monocyclic unit is saturated. In some embodiments, at least one monocyclic unit is partially unsaturated. In some embodiments, at least one monocyclic unit is aromatic. In some embodiments, a ring has, in addition to the intervening atom(s), 0-4 (e.g., 0, 1, 2, 3, or 4, etc.) heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, there are no additional heteroatoms. In some embodiments, there is one additional heteroatom. In some embodiments, there are 2 additional heteroatoms. In some embodiments, there are 3 additional heteroatoms. In some embodiments, there are 4 additional heteroatoms. In some embodiments, there are 5 additional heteroatoms. In some embodiments, there are 6 or more additional heteroatoms. In some embodiments, an additional heteroatom is nitrogen. In some embodiments, an additional heteroatom is oxygen. In some embodiments, an additional heteroatom is sulfur.
[0407] In some embodiments, R or a group that can be R has at least one but no more than about 10 (e.g., 1-9, 1-8, 1-6, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) carbon atoms and no more than 6 (e.g., 0-5, 1-5, 1, 2, 3, 4, 5, 6, etc.) heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments, a bivalent moiety, e.g., L, L, etc., has no more than about 10 (e.g., 1-9, 1-8, 1-6, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) carbon atoms and no more than 6 (e.g., 0-5, 1-5, 1, 2, 3, 4, 5, 6, etc.) heteroatoms independently selected from nitrogen, oxygen and sulfur.
[0408] As described herein, various groups may be optionally substituted. Substituents are routinely utilized in chemistry including in development of various therapeutics. Many substituents can be utilized in accordance with the present disclosure. In some embodiments, an optionally substituted group is unsubstituted. In some embodiments, an optionally substituted group is substituted. Substituents are preferably those that result in the formation of compounds for a desired property, activity, use, etc., as described herein. In some embodiments, compounds are stable for therapeutic use as described herein. The term stable, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a substituent is a hydrocarbon group. In some embodiments, a substituent comprises a heteroatom. In some embodiments, a substituent comprises multiple heteroatoms. In some embodiments, each atom in a substituent is independently selected from hydrogen, carbon, halogen, nitrogen, oxygen, sulfur, phosphorus and silicon. In some embodiments, each atom in a substituent is independently selected from hydrogen, carbon, halogen, nitrogen, oxygen, and sulfur. In some embodiments, each atom in a substituent is independently selected from hydrogen, carbon, fluorine, chlorine, bromine, iodine, nitrogen, oxygen, and sulfur. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in a substituent is about or no more than about 1; in some embodiments, it is no more than about 2; in some embodiments, it is no more than about 3; in some embodiments, it is no more than about 4; in some embodiments, it is no more than about 5; in some embodiments, it is no more than about 6; in some embodiments, it is no more than about 7; in some embodiments, it is no more than about 8; in some embodiments, it is no more than about 9; in some embodiments, it is no more than about 10; in some embodiments, it is no more than about 11; in some embodiments, it is no more than about 12; in some embodiments, it is no more than about 13; in some embodiments, it is no more than about 14; in some embodiments, it is no more than about 15; in some embodiments, it is no more than about 20. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 20. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 15. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 10. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 6. In some embodiments, each optional substituent on a substitutable group is independently halogen, C.sub.1-4 alkyl, OH, CN, NO.sub.2, C.sub.1-4 haloalkyl (e.g., CF.sub.3), OR.sup.SB, N(R.sup.SB).sub.2, C(O)OR.sup.SB, C(O)N(R.sup.SB).sub.2, or S(O).sub.2N(R.sup.SB).sub.2 wherein each R.sup.SB is independently H, C.sub.1-4 alkyl or C.sub.1-4 haloalkyl. In some embodiments, each optional substituent on a substitutable group is independently halogen, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, or OH. In some embodiments, each optional substituent on a substitutable group is independently halogen or C.sub.1-4 alkyl.
Peptide Characterization
[0409] In some embodiments, peptides as described herein (e.g., stapled peptides, e.g., cysteine stapled peptides, non-cysteine stapled peptides that are variants of cysteine stapled peptides, as described herein, and/or collections thereof) are characterized with respect to, for example, one or more characteristics selected from the group consisting of: binding characteristicse.g., with respect to a particular target of interest; stability characteristics, for example in solution or in dried form; cell permeability characteristics, etc., and combinations thereof.
[0410] In some embodiments, a binding characteristic may be or comprise specificity, affinity, on-rate, off-rate, etc, optionally under (or over a range of) specified conditions such as, for example, concentration, temperature, pH, cell type, presence or level of a particular competitor, etc.
[0411] As will be appreciated by those skilled in the art, assessments of characteristics as described herein may involve comparison with an appropriate reference (e.g., a positive or negative control) which may, in some embodiments, be a contemporaneous reference or, in some embodiments, a historical reference.
[0412] In some embodiments, desirable peptide characteristics may be, for example: binding to a target of interest (e.g., binding affinity of about or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 M, and preferably no more than 10, 1 0.5, 0.1, 0.05 or 0.01 M); cell penetration (e.g., as measured by fluorescence-based assays or mass spectrometry of cellular fractions, etc.); activity (e.g., modulating one or more functions of a target, which may be assessed in a cellular reporter assay (e.g., with an IC50 of no more than a concentration, e.g., about 10, 1 M, 500 nM, etc.), an animal model and/or a subject; stability, which may be assessed using a number of assays (e.g., in a rat pharmacokinetic study (e.g., administered via oral, iv, ip, etc.) with a terminal half-life of greater than a suitable time, e.g., 1 hour); low toxicity, which might be assessed by a number of assays (e.g., a standard ADME/toxicity assays); and/or low levels of cytotoxicity (e.g., low levels of lactate dehydrogenase (LDH) released from cells when treated at a suitable concentration, e.g., about 10 M of a peptide).
Peptide Production
[0413] Various technologies are known in the art for producing stapled peptides of may be utilized in accordance with the present disclosure. Those skilled in the art, reading the present disclosure, will well appreciate which such technologies are applicable in which aspects of the present disclosure. Certain technologies are described U.S. Ser. No. 11/198,713, US 20210179665, WO 2021119537, WO 2021188659, WO 2022020651, or WO 2022020652, the entirety of each of which is incorporated herein by reference and can be utilized in accordance with the present disclosure.
[0414] In some embodiments, as described herein, certain stapled peptides, and in particular cysteine stapled peptides, may be provided in and/or produced by a biological system and reacting with a provided reagent, e.g., one having the structure of R.sup.x-L.sup.s2-R.sup.x formula R-I, or a salt thereof.
[0415] In some embodiments, peptides are prepared on solid phase on a synthesizer using, typically, Fmoc chemistry.
[0416] In some embodiments, staples are formed by olefin metathesis. In some embodiments, a product double bond of metathesis is reduced/hydrogenated. In some embodiments, CO.sub.2 are extruded from a carbamate moiety of a staple. In some embodiments, provided stapled peptides are further modified, and/or conjugated to other entities. Conditions and/or reagents of these reactions are widely known in the art and can be performed in accordance with the present disclosure to provide stapled peptides.
[0417] Properties and/or activities of provided stapled peptides can be readily assessed in accordance with the present disclosure, for example, through use of one or more methods described in the examples.
[0418] In some embodiments, technologies for preparing and/or assessing provided stapled peptides include those described in U.S. Pat. No. 9,617,309, US 2015-0225471, US 2016-0024153, US 2016-0215036, US2016-0244494, WO 2017/062518, etc.
[0419] In some embodiments, a provided agent, e.g., a provided peptide, has a purity of 60%-100%. In some embodiments, a provided agent has a purity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, a purity is at least 60%. In some embodiments, a purity is at least 70%. In some embodiments, a purity is at least 80%. In some embodiments, a purity is at least 85%. In some embodiments, a purity is at least 90%. In some embodiments, a purity is at least 91%. In some embodiments, a purity is at least 92%. In some embodiments, a purity is at least 93%. In some embodiments, a purity is at least 94%. In some embodiments, a purity is at least 95%. In some embodiments, a purity is at least 96%. In some embodiments, a purity is at least 97%. In some embodiments, a purity is at least 98%. In some embodiments, a purity is at least 99%. In some embodiments, a purity is at least 99.5%.
[0420] In some embodiments, provided methods provide high yields. In some embodiments, a yield is 50%-100%. In some embodiments, a yield is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, a yield is at least 60%. In some embodiments, a yield is at least 65%. In some embodiments, a yield is at least 70%. In some embodiments, a yield is at least 75%. In some embodiments, a yield is at least 80%. In some embodiments, a yield is at least 85%. In some embodiments, a yield is at least 90%. In some embodiments, a yield is at least 91%. In some embodiments, a yield is at least 92%. In some embodiments, a yield is at least 93%. In some embodiments, a yield is at least 94%. In some embodiments, a yield is at least 95%. In some embodiments, a yield is at least 96%. In some embodiments, a yield is at least 97%. In some embodiments, a yield is at least 98%. In some embodiments, a yield is at least 99%.
[0421] In some embodiments, a provided method delivers high E/Z selectivity for olefin. In some embodiments, provided selectivity favors the E isomer. In some embodiments, provided selectivity favors the Z isomer. In some embodiments, a E:Z ratio is at least 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, or 100:1. In some embodiments, a Z:E ratio is at least 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 80:1, 90:1, 95:1, 99:1, or 100:1. In some embodiments, a ratio is at least 1:1. In some embodiments, a ratio is at least 1.5:1. In some embodiments, a ratio is at least 2:1. In some embodiments, a ratio is at least 3:1. In some embodiments, a ratio is at least 4:1. In some embodiments, a ratio is at least 5:1. In some embodiments, a ratio is at least 6:1. In some embodiments, a ratio is at least 7:1. In some embodiments, a ratio is at least 8:1. In some embodiments, a ratio is at least 9:1. In some embodiments, a ratio is at least 10:1. In some embodiments, a ratio is at least 20:1. In some embodiments, a ratio is at least 30:1. In some embodiments, a ratio is at least 40:1. In some embodiments, a ratio is at least 50:1. In some embodiments, a ratio is at least 80:1. In some embodiments, a ratio is at least 90:1. In some embodiments, a ratio is at least 95:1. In some embodiments, a ratio is at least 99:1. In some embodiments, a ratio is at least 100:1.
[0422] In some embodiments, a provide method comprises a period of time at a temperature higher than room temperature. In some embodiments, a temperature is about 25-200 C. In some embodiments, a temperature is about 25 C. In some embodiments, a temperature is about 30 C. In some embodiments, a temperature is about 35 C. In some embodiments, a temperature is about 40 C. In some embodiments, a temperature is about 45 C. In some embodiments, a temperature is about 50 C. In some embodiments, a temperature is about 55 C. In some embodiments, a temperature is about 60 C. In some embodiments, a temperature is about 65 C. In some embodiments, a temperature is about 70 C. In some embodiments, a temperature is about 75 C. In some embodiments, a temperature is about 80 C. In some embodiments, a temperature is about 85 C. In some embodiments, a temperature is about 90 C. In some embodiments, a temperature is about 95 C. In some embodiments, a temperature is about 100 C. In some embodiments, a temperature is about 150 C. In some embodiments, a temperature is higher than about 150 C.
Peptide Compositions
[0423] Among other things, the present disclosure provides compositions that comprise or otherwise relate to peptides, e.g., stapled peptides, as described herein.
[0424] For example, in some embodiments, provided compositions are or comprise elements of a phage display system that encodes and/or expresses stapled peptides (e.g., cysteine stapled peptides), or a collection thereof, as described herein.
[0425] In some embodiments, provided compositions are or comprise an assay system for characterizing (and optionally including) a stapled peptide as described herein.
[0426] In some embodiments, provided compositions are pharmaceutical compositions e.g., that comprise or deliver one or more stapled peptides (e.g., in particular one or more non-cysteine stapled peptides that may, in some embodiments, correspond to and/or be a variant of a parent cysteine stapled peptide as described herein).
[0427] In some embodiments, a pharmaceutical composition comprises a peptide agent and a pharmaceutically acceptable carrier.
[0428] In some embodiments, a peptide composition may include or deliver a particular form (e.g., a particular optical isomer, diastereomer, salt form, covalent conjugate form [e.g., covalently attached to a carrier moiety], etc., or combination thereof) of a peptide agent as described herein). In some embodiments, a peptide agent included or delivered by a pharmaceutical composition is described herein is not covalently linked to a carrier moiety.
[0429] In some embodiments, a provided therapeutic composition may comprise one or more additional therapeutic agents and/or one or more stabilizing agents and/or one or more agents that alters (e.g., extends or limits to a particular tissue, location or site) rate or extent of delivery over time.
Uses and Applications
[0430] In some embodiments, the present disclosure provides certain stapled peptides and/or other technologies (e.g., collections of stapled peptides, and/or a biological system adapted to express or display and/or expressing or displaying such stapled peptide(s), etc.) for the development and/or use of stapled peptides (e.g., that bind a target of interest).
[0431] In some embodiments, the present disclosure provides collections of peptides, wherein the collection of peptides comprise one or more (e.g., about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) degenerate positions and two or more residues stapled or suitable for stapling. In some embodiments, two or more residues are stapled. In some embodiments, two or more residues are suitable for stapling and can be stapled in accordance with the present disclosure. In some embodiments, a collection of peptides comprises one or more positions at each of which a single amino acid or a selected set of amino acids are utilized. In some embodiments, a collection of peptides comprises a set of enriched amino acid residues at a position (an enriched position). In some embodiments, a collection of peptides comprises a set of enriched amino acid residues independently at two or more enriched positions. In some embodiments, a set of enriched amino acid residues comprise about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unique amino acid residues. In some embodiments, there is a single amino acid residue in a set. In some embodiments, there are two or more amino acid residues in a set. In some embodiments, compared to at a degenerate position, there are fewer unique amino acid residues independently at each enriched position. In some embodiments, a set of enriched amino acid residues is selected for interacting with a target of interest, e.g., in some embodiments, a first target of interest. In some embodiments, a set of enriched amino acid residues occurs at higher frequency in stapled peptides binding to a target of interest compared to stapled peptides that do not bind to a target of interest, or an initial collection of stapled peptides for screening.
[0432] In some embodiments, the present disclosure provides certain stapled peptides, or collections thereof (e.g., including collections in which individual peptides may be fused with a phage coat protein), and/or a biological system adapted to express or display and/or expressing or displaying such cysteine stapled peptide(s). In some embodiments, certain provided stapled peptides and/or other technologies can be utilized to identify and/or characterize one or more desirable structural features (e.g., amino acid sequence, staple location and/or structure [e.g., length, composition, degree of constraint, etc.]) of a stapled peptide that interacts with (e.g., binds to) a target of interest. In some embodiments, certain provided stapled peptides and/or other technologies are useful for the development of stapled peptides (e.g., non-cysteine stapled peptides) that correspond to (e.g., share significant structural identity with, and optionally structural identity with, a reference cysteine stapled peptide, except for substitution of the cysteine(s) for other non-cysteine staple-forming residue(s)).
[0433] In some embodiments, a provided peptide or collection thereof, whose amino acid sequence, includes at least two appropriately spaced cysteine residues, is used to prepare a cysteine stapled peptide, or collection thereof (e.g., by reaction with a compound of formula R-I. certain provided stapled peptides and/or other technologies
[0434] In some embodiments, certain provided stapled peptides can be identified and synthesized by technologies and examples described in the present disclosure. In some embodiments, a certain provided stapled peptide is a cysteine stapled peptide. In some embodiments, a cysteine stapled peptide that binds a target of interest may have a cysteine staple and corresponding cysteine residues replaced by a non-cysteine staple and amino acids necessary to facilitate such a non-cysteine staple.
[0435] In some embodiments, a provided collection of peptides (or nucleic acids that encode them) is characterized in that peptides of the collection all include cysteine residues (e.g. a pair of cysteine residues), spaced relative to one another to permit cysteine stapling as described herein, but otherwise have independent amino acid sequences and, optionally, in that peptides of the collection all have the same length. In some embodiments, degeneracy and/or bias is introduced in one or more positions through genetic engineering and/or expression of selected nucleic acid sequences in a biological system. In some embodiments, degree of degeneracy or bias at one or more positions in peptides of a peptide collection or library as described here is informed and/or selected by prior assessment one or more binding characteristics of a related library or collection (e.g., with comparable cysteine residues). In some embodiments, such prior assessment is by high-throughput analysis (e.g., screening) of a collection or collections of stapled peptides against a target of interest and the use of high-throughput sequencing to decode the genotypes of a subset of the collection of stapled peptides can inform the production of a biased library.
[0436] In some embodiments, a collection of stapled peptides comprises cysteine stapled peptides fused to another molecule for use in a biological system (e.g. phage display) or non-biological system. In some embodiments, after screening a collection of stapled peptides in the context of a biological system or non-biological system, high throughput sequencing will identify particular cysteine stapled peptides that interact with a target of interest. In some embodiments, a particular cysteine stapled peptide, when not fused to another molecule for use in a biological system, will exhibit the same or similar interaction with a target of interest as the particular cysteine stapled peptide when fused to said molecule. In some embodiments, a particular cysteine stapled peptide, when not fused to another molecule for use in a biological system, can have its cysteine residues and cysteine staple replaced with other amino acids and staples. In some embodiments, the resulting peptide maintains the same or similar interaction with a target of interest.
[0437] In some embodiments, a collection of stapled peptides comprises non-cysteine staples.
[0438] In some embodiments, certain provided stapled peptides and/or other technologies as described herein may be useful to modulate one or more biological events or statuses, e.g., by binding with a relevant target of interest.
[0439] In some embodiments, the present disclosure provides a method for modulating an activity of a target of interest, comprising contacting the target of interest with an agent described herein. In some embodiments, the present disclosure provides a method for modulating interaction of a target of interest with a partner, comprising contacting the target of interest with an agent described herein. In some embodiments, the present disclosure provides a method for modulating an activity of a target of interest in a system comprising the target of interest, comprising administering or delivering to the system an agent described herein. In some embodiments, the present disclosure provides a method for modulating interaction a target of interest with a partner in a system comprising the target of interest, comprising administering or delivering to the system an agent described herein. In some embodiments, a system expresses a target of interest. In some embodiments, a system is in vivo. In some embodiments, a system is in vitro. In some embodiments, a system is or comprises a cell. In some embodiments, a system is or comprises a population of cells. In some embodiments, a system is or comprises a tissue. In some embodiments, a system is or comprises an organ. In some embodiments, a system is a subject. In some embodiments, a partner is a polypeptide. In some embodiments, a partner is a protein. In some embodiments, an activity is inhibited. In some embodiments, an interaction is reduced. In some embodiments, an interaction is enhanced. In some embodiments, an agent comprises a stapled peptide. In some embodiments, an agent is a stapled peptide.
[0440] In some embodiments, provided technologies enables de novo design of stapled peptides for targets of interest including protein targets without prior information on their alpha-helix binding properties, which has significantly limited the proteins and diseases for which stapled peptides or other alpha-helical structure could be discovered. In some embodiments, provided technologies can be utilized to identify, characterize, and produce stapled peptides that can bind to and modulate activities of various targets of interest, including many that have not been reported to bind to isolated a-helical peptides. In some embodiments, provided technologies provide distinct a-helix recognition sites. Among other things, provided technologies can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization.
[0441] Certain agents, collections, methods, etc. are described below as examples. In some embodiments, at a position where there is no strong preference or enrichment, an amino acid residue, e.g., X (such as X.sup.31, X.sup.2, etc. if no enrichment), the amino acid is selected from natural amino acids. In some embodiments, it is selected from alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.
[0442] In some embodiments, a sequence may be truncated. In some embodiments, a truncated sequence may be part of optimization, e.g., to increase affinity. In some embodiments, one or more (e.g., 1, 2, or 3) or all residues to the N-terminus side of a stapled residue (e.g., X.sup.4), e.g., X.sup.31, X.sup.32, X.sup.33, X.sup.34, X.sup.35, X.sup.41, X.sup.42, X.sup.43, X.sup.44, X.sup.45, X.sup.46, X.sup.47, X.sup.48, X.sup.51, X.sup.52, X.sup.53, X.sup.54, X.sup.61, X.sup.62, X.sup.2, X.sup.3, etc. are absent. In some embodiments, one or more (e.g., 1, 2, or 3) or all residues to the C-terminus side of a stapled residue (e.g., X.sup.11), e.g., X.sup.12, X.sup.13, X.sup.14, etc. are absent. In some embodiments, X.sup.31, X.sup.32, X.sup.33, X.sup.34, X.sup.35, X.sup.41, X.sup.42, X.sup.43, X.sup.44, X.sup.45, X.sup.46, X.sup.47, X.sup.48, X.sup.51, X.sup.52, X.sup.53, X.sup.54, X.sup.61, or X.sup.62 is absent. In some embodiments, X.sup.2 is absent. In some embodiments, X.sup.3 is absent. In some embodiments, X.sup.12 is absent. In some embodiments, X.sup.13 is absent. In some embodiments, X.sup.14 is absent.
Beta-Catenin
[0443] In some embodiments, the present disclosure provides agents that can bind to and/or modulate activities of beta-catenin. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 31, cluster 32, cluster 33 and cluster 43, compete with axin for beta-catenin interaction. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 31, cluster 32, cluster 33 and cluster 43, compete with TCF for beta-catenin interaction. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 31, cluster 32, cluster 33 and cluster 43, compete with both axin and TCF for beta-catenin interaction. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 35, compete with TCF for beta-catenin interaction. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 35, compete with TCF but not axin for beta-catenin interaction.
Cluster C31
[0444] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C31 as described herein. In some embodiments, an agent comprises X.sup.31X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.31, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0445] In some embodiments, the present disclosure provides an agent comprising
X.sup.31X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0446] each of X.sup.31, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0447] X.sup.3 comprises a side chain comprising an acidic group; [0448] X.sup.5 comprises a hydrophobic side chain; [0449] X.sup.6 comprises a hydrophobic side chain; [0450] X.sup.9 comprises a side chain comprising an aromatic group or a basic group; and [0451] X.sup.10 comprises a hydrophobic side chain.
[0452] Various amino acid residues may be utilized for X.sup.31. For example, in some embodiments, X.sup.31 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.31. In some embodiments, X.sup.31 comprises a side chain comprising COOH. In some embodiments, X.sup.31 is D. In some embodiments, X.sup.31 is E. In some embodiments, X.sup.31 comprises a hydrophobic side chain. In some embodiments, X.sup.31 is V. In some embodiments, X.sup.31 comprises a side chain comprising a polar group. In some embodiments, X.sup.31 is Q. In some embodiments, X.sup.31 is N.
[0453] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an acidic group. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.2 is D. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, X.sup.2 is A. In some embodiments, X.sup.2 is V. In some embodiments, X.sup.2 comprises a side chain comprising a polar group. In some embodiments, X.sup.2 is Q. In some embodiments, X.sup.2 is N.
[0454] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a hydrophobic side chain. In some embodiments, X.sup.3 is I. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.3 is Q. In some embodiments, X.sup.3 is N. In some embodiments, X.sup.3 comprises a side chain comprising OH. In some embodiments, X.sup.3 is S. In some embodiments, X.sup.3 is T.
[0455] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00374##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0456] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is I. In some embodiments, X.sup.5 is V. In some embodiments, X.sup.5 is M. In some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is T.
[0457] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1 6alkyl. In some embodiments, X.sup.6 is L. In some embodiments, X.sup.6 is M. In some embodiments, X.sup.6 is I.
[0458] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0459] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0460] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.9. In some embodiments, X.sup.9 is H. In some embodiments, X.sup.9 comprises a side chain comprising a polar group. In some embodiments, X.sup.9 comprises a side chain comprising OH. In some embodiments, X.sup.9 is Y. In some embodiments, X.sup.9 is Q.
[0461] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 is C.sub.1 6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.10 comprises an aromatic group. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is F. In some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues comprising an acidic group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is E.
[0462] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.12. In some embodiments, X.sup.12 is R. In some embodiments, X.sup.12 is H. In some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.12. In some embodiments, X.sup.12 is Q. In some embodiments, X.sup.12 is N. In some embodiments, X.sup.12 is A.
[0463] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.13 comprises an aromatic group. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is L. In some embodiments, X.sup.13 is A. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is W. In some embodiments, X.sup.13 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.13. In some embodiments, X.sup.13 is H. In some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, X.sup.13 is N.
[0464] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.14 comprises an aromatic group. In some embodiments, X.sup.14 is I. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is W. In some embodiments, X.sup.14 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising COOH. In some embodiments, X.sup.14 is D. In some embodiments, X.sup.14 is E.
[0465] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of SEQ ID NO: 1 or amino acid residues corresponding thereto: H219, H223, S250, F253, Y254, T257, N261, H260, L264, K292, A295, I296, N299, Y333, K335, W338, and T339. In some embodiments, it interacts with H219 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H223 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with S250 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with F253 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y254 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T257 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N261 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H260 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with L264 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K292 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with A295 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with I296 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N299 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y333 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K335 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with W338 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T339 of SEQ ID NO: 1 or an amino acid residue corresponding thereto.
TABLE-US-00014 (SEQIDNO:1) MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGK GNPEEEDVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAM FPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAE LATRAIPELTKLLNDEDQVVVNKAAVMVHQLSKKEASRHAIMRSPQMVS AIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAIFKSGGIPALVKMLG SPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFL AITTDCLQILAYGNQESKLIILASGGPQALVNIMRTYTYEKLLWTTSRV LKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAA TKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQV GGIEALVRTVLRAGDREDITEPAICALRHLTSRHQEAEMAQNAVRLHYG LPVVVKLLHPPSHWPLIKATVGLIRNLALCPANHAPLREQGAIPRLVQL LVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRI VIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGAT APLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTE PMAWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMM EHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDL
[0466] In some embodiments, an agent comprising a cluster 31 sequence competes with axin for interaction with beta-catenin.
Cluster C32
[0467] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C32 as described herein. In some embodiments, an agent comprises X.sup.32X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.32, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0468] In some embodiments, the present disclosure provides an agent comprising
X.sup.32X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0469] each of X.sup.32, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0470] X.sup.2 comprises a hydrophobic side chain; [0471] X.sup.3 comprises a side chain comprising an acidic group; [0472] X.sup.5 comprises a side chain comprising an aromatic group; [0473] X.sup.6 comprises a hydrophobic side chain; and [0474] X.sup.10 comprises a side chain comprising an acidic group.
[0475] Various amino acid residues may be utilized for X.sup.32. For example, in some embodiments, X.sup.32 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.32. In some embodiments, the side chain of X.sup.32 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.32 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.32 comprises an aromatic group. In some embodiments, X.sup.32 is I. In some embodiments, X.sup.32 is V. In some embodiments, X.sup.32 is F. In some embodiments, X.sup.32 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.32. In some embodiments, X.sup.32 is T. In some embodiments, X.sup.32 comprises a side chain comprising a basic group. In some embodiments, X.sup.32 is H.
[0476] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.2. In some embodiments, X.sup.2 is L. In some embodiments, X.sup.2 is F.
[0477] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.3. In some embodiments, X.sup.3 is A. In some embodiments, X.sup.3 is N.
[0478] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00375##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0479] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.5. In some embodiments, X.sup.5 is H. In some embodiments, X.sup.9 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.5. In some embodiments, X.sup.5 is Q. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T.
[0480] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is I. In some embodiments, X.sup.6 is L. In some embodiments, X.sup.6 is M. In some embodiments, X.sup.6 is V.
[0481] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0482] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.8 is A.
[0483] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1 6 alkyl. In some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, X.sup.9 is W. In some embodiments, X.sup.9 is I In some embodiments, X.sup.9 is L. In some embodiments, X.sup.9 is M.
[0484] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is D. In some embodiments, X.sup.10 is E. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprises polar groups at X.sup.10. In some embodiments, X.sup.10 is N. In some embodiments, X.sup.10 is T.
[0485] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 is Y. In some embodiments, X.sup.12 is W. In some embodiments, X.sup.12 is S.
[0486] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 is E. In some embodiments, X.sup.13 is D.
[0487] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 is I. In some embodiments, X.sup.14 is E. In some embodiments, X.sup.14 is W.
[0488] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of SEQ ID NO: 1 or amino acid residues corresponding thereto: H219, H223, S250, F253, Y254, T257, N261, H260, L264, K292, A295, I296, N299, Y333, K335, W338, and T339. In some embodiments, it interacts with H219 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H223 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with S250 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with F253 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y254 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T257 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N261 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H260 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with L264 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K292 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with A295 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with I296 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N299 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y333 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K335 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with W338 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T339 of SEQ ID NO: 1 or an amino acid residue corresponding thereto.
[0489] In some embodiments, an agent comprising a cluster 32 sequence competes with axin for interaction with beta-catenin.
Cluster C33
[0490] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C33 as described herein. In some embodiments, an agent comprises X.sup.33X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.33, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0491] In some embodiments, the present disclosure provides an agent comprising
X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0492] each of X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0493] X.sup.5 comprises a side chain comprising an acidic group; [0494] X.sup.6 comprises a side chain comprising an aromatic group; [0495] X.sup.10 comprises a side chain comprising an aromatic group; [0496] X.sup.13comprises a hydrophobic side chain; and [0497] X.sup.14 comprises a hydrophobic side chain.
[0498] Various amino acid residues may be utilized for X.sup.33. For example, in some embodiments, X.sup.33 is T. In some embodiments, X.sup.33 is S. In some embodiments, X.sup.33 is absent.
[0499] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 is H. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 is absent.
[0500] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 is R. In some embodiments, X.sup.3 is L. In some embodiments, X.sup.3 is absent.
[0501] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00376##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0502] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is E. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.5. In some embodiments, X.sup.5 is Q. In some embodiments, X.sup.5 is N.
[0503] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is W. In some embodiments, X.sup.6 is M.
[0504] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0505] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0506] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising COOH. In some embodiments, X.sup.9 is E. In some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is L. In some embodiments, X.sup.9 is V. In some embodiments, X.sup.9 is F. In some embodiments, X.sup.9 is M.
[0507] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise basic groups at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising an aromatic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise aromatic groups at X.sup.10. In some embodiments, X.sup.10 is H. In some embodiments, X.sup.10 is Y.
[0508] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is E. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.12. In some embodiments, X.sup.12 is Q. In some embodiments, X.sup.12 is S.
[0509] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is L.
[0510] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, X.sup.14 is I. In some embodiments, X.sup.14 is L. In some embodiments, X.sup.14 is V.
[0511] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of SEQ ID NO: 1 or amino acid residues corresponding thereto: H219, H223, S250, F253, Y254, T257, N261, H260, L264, K292, A295, I296, N299, Y333, K335, W338, and T339. In some embodiments, it interacts with H219 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H223 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with S250 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with F253 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y254 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T257 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N261 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H260 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with L264 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K292 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with A295 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with I296 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N299 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y333 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K335 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with W338 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T339 of SEQ ID NO: 1 or an amino acid residue corresponding thereto.
[0512] In some embodiments, an agent comprising a cluster 33 sequence competes with axin for interaction with beta-catenin.
Cluster C34
[0513] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C34 as described herein. In some embodiments, an agent comprises X.sup.34X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.34, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0514] In some embodiments, the present disclosure provides an agent comprising
X.sup.34X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0515] each of X.sup.34, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0516] X.sup.2 comprises a side chain comprising an aromatic group; [0517] X.sup.5 comprises a side chain comprising an acidic side chain; [0518] X.sup.6 comprises a side chain comprising a basic group or an aromatic group; [0519] X.sup.9 comprises a hydrophobic side chain; and [0520] X.sup.10 comprises a hydrophobic side chain.
[0521] Various amino acid residues may be utilized for X.sup.34. For example, in some embodiments, X.sup.34 is D. In some embodiments, X.sup.34 is L. In some embodiments, X.sup.34 is absent.
[0522] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.2. In some embodiments, X.sup.2 is W.
[0523] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 is Q.
[0524] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00377##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0525] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is E. In some embodiments, X.sup.5 is D.
[0526] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise basic groups at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise aromatic groups at X.sup.6. In some embodiments, X.sup.6 is H.
[0527] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0528] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0529] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is L.
[0530] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is L. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.10. In some embodiments, X.sup.10 is N. In some embodiments, X.sup.10 is T.
[0531] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 is H. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is absent.
[0532] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 is Y. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is absent.
[0533] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 is W. In some embodiments, X.sup.14 is H. In some embodiments, X.sup.14 is absent.
[0534] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of SEQ ID NO: 1 or amino acid residues corresponding thereto: H219, H223, S250, F253, Y254, T257, N261, H260, L264, K292, A295, I296, N299, Y333, K335, W338, and T339. In some embodiments, it interacts with H219 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H223 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with S250 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with F253 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y254 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T257 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N261 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H260 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with L264 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K292 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with A295 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with I296 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with N299 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y333 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with K335 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with W338 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T339 of SEQ ID NO: 1 or an amino acid residue corresponding thereto.
[0535] In some embodiments, an agent comprising a cluster 34 sequence competes with axin for interaction with beta-catenin.
Cluster C35
[0536] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C35 as described herein. In some embodiments, an agent comprises X.sup.35X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.35, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0537] In some embodiments, the present disclosure provides an agent comprising
X.sup.35X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0538] each of X.sup.35, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0539] X.sup.5 comprises a side chain comprising an aromatic group; [0540] X.sup.6 comprises a side chain comprising an acidic group; [0541] X.sup.9 comprises a side chain comprising an aromatic group; and [0542] X.sup.13 comprises a side chain comprising an aromatic group.
[0543] Various amino acid residues may be utilized for X.sup.35. For example, in some embodiments, X.sup.35 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.35. In some embodiments, the side chain of X.sup.35 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.35 is C.sub.1-6 alkyl. In some embodiments, X.sup.35 is I. In some embodiments, X.sup.35 is V. In some embodiments, X.sup.35 is M
[0544] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, X.sup.2 is I. In some embodiments, X.sup.2 is L. In some embodiments, X.sup.2 is M. In some embodiments, X.sup.2 is V.
[0545] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 is H. In some embodiments, X.sup.3 is E.
[0546] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00378##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0547] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, the side chain of X.sup.5 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.5. In some embodiments, the side chain of X.sup.5 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is Y.
[0548] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is E. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.6. In some embodiments, X.sup.6 is Q.
[0549] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0550] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0551] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.9. In some embodiments, X.sup.9 is F. In some embodiments, X.sup.9 is Y.
[0552] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 is F. In some embodiments, X.sup.10 is I.
[0553] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise polar groups at X.sup.12. In some embodiments, X.sup.12 is Q. In some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues comprising an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is E. In some embodiments, X.sup.12 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.12. In some embodiments, X.sup.12 is H.
[0554] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.13. In some embodiments, the side chain of X.sup.13 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is Y. In some embodiments, X.sup.13 is N.
[0555] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.14 comprises an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is M. In some embodiments, X.sup.14 is I. In some embodiments, X.sup.14 is V. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is Q.
[0556] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) or all of the following amino acid residues of SEQ ID NO: 1 or amino acid residues corresponding thereto: L519, I579, H578, R582, E620, C619, R661, F660, A657, T653, Y654, R612, G575, and C613. In some embodiments, it interacts with L519 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with I579 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with H578 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with R582 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with E620 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with C619 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with R661 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with F660 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with A657 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with T653 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Y654 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with R612 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with G575 of SEQ ID NO: 1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with C613 of SEQ ID NO: 1 or an amino acid residue corresponding thereto.
[0557] In some embodiments, an agent comprising a cluster 35 sequence does not compete with axin for interaction with beta-catenin.
RNF31
[0558] In some embodiments, the present disclosure provides agents that can bind to and/or modulate activities of RNF31. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster C41, cluster C42, cluster C43, cluster C44, and cluster C45, compete with Otulin for RNF31 interaction. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 46, cluster C47, and cluster C48, compete with Sharpin/SIPL1 for RNF31 interaction. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster C41, cluster C42, cluster C43, cluster C44, and cluster C45, bind to RNF31 PUB domain. In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster 46, cluster C47, and cluster C48 bind to RNF31 UBA domain. In some embodiments, the present disclosure provides methods for reducing interactions with relevant partners, or reducing interactions at PUB or UBA domain, comprising contacting RNF31 with stapled peptides that compete with the partner or interact with RNF at PUB or UBA domain, or comprising administering or delivering to a system comprising RNF31 stapled peptides that compete with the partner or interact with RNF at PUB or UBA domain.
Cluster C41
[0559] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C41 as described herein. In some embodiments, an agent comprises X.sup.41X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.41, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0560] In some embodiments, the present disclosure provides an agent comprising
X.sup.41X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0561] each of X.sup.41, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0562] X.sup.6 comprises a side chain comprising an aromatic group; [0563] X.sup.9 comprises a hydrophobic side chain; [0564] X.sup.10 comprises a side chain comprising an aromatic group; and [0565] X.sup.12 comprises a side chain comprising an acidic group.
[0566] Various amino acid residues may be utilized for X.sup.41. Various amino acid residues may be utilized for X.sup.41. For example, in some embodiments, X.sup.41 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.41. In some embodiments, the side chain of X.sup.41 comprises an aromatic group. In some embodiments, X.sup.41 is I. In some embodiments, X.sup.41 is Y. In some embodiments, X.sup.41 is W. In some embodiments, X.sup.41 is L. In some embodiments, X.sup.41 is A.
[0567] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 is D. In some embodiments, X.sup.2 comprises a side chain comprising a polar group. In some embodiments, X.sup.2 is T. In some embodiments, X.sup.2 is V.
[0568] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.3 is Q.
[0569] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00379##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0570] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 is E. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is A.
[0571] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.6. In some embodiments, X.sup.6 is W. In some embodiments, X.sup.6 is D.
[0572] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0573] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0574] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is L. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is M.
[0575] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is Y.
[0576] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is E. In some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, X.sup.12 is N. In some embodiments, X.sup.12 is S.
[0577] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising COOH. In some embodiments, X.sup.13 is D. In some embodiments, X.sup.13 is E. In some embodiments, X.sup.13 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is M.
[0578] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 is Q.
[0579] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Thr74, Asn77, Ile78, Lys81, Tyr82, Asn85, Leu86, Pro92, Tyr94, Trp95, Val98, Asn102, Val104, Thr108, and Tyr124. In some embodiments, it interacts with Thr74 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn77 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile78 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys81 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr82 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn85 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu86 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Pro92 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr94 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp95 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val98 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn102 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val104 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr108 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr124 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
TABLE-US-00015 (SEQIDNO:2) MPGEEEERAFLVAREELASALRRDSGQAFSLEQLRPLLASSLPLAARYL QLDAARLVRCNAHGEPRNYLNTLSTALNILEKYGRNLLSPQRPRYWRGV KFNNPVFRSTVDAVQGGRDVLRLYGYTEEQPDGLSFPEGQEEPDEHQVA TVTLEVLLLRTELSLLLQNTHPRQQALEQLLEDKVEDDMLQLSEFDPLL REIAPGPLTTPSVPGSTPGPCFLCGSAPGTLHCPSCKQALCPACDHLFH GHPSRAHHLRQTLPGVLQGTHLSPSLPASAQPRPQSTSLLALGDSSLSS PNPASAHLPWHCAACAMLNEPWAVLCVACDRPRGCKGLGLGTEGPQGTG GLEPDLARGRWACQSCTFENEAAAVLCSICERPRLAQPPSLVVDSRDAG ICLQPLQQGDALLASAQSQVWYCIHCTFCNSSPGWVCVMCNRTSSPIPA QHAPRPYASSLEKGPPKPGPPRRLSAPLPSSCGDPEKQRQDKMREEGLQ LVSMIREGEAAGACPEEIFSALQYSGTEVPLQWLRSELPYVLEMVAELA GQQDPGLGAFSCQEARRAWLDRHGNLDEAVEECVRTRRRKVQELQSLGF GPEEGSLQALFQHGGDVSRALTELQRQRLEPFRQRLWDSGPEPTPSWDG PDKQSLVRRLLAVYALPSWGRAELALSLLQETPRNYELGDVVEAVRHSQ DRAFLRRLLAQECAVCGWALPHNRMQALTSCECTICPDCFRQHFTIALK EKHITDMVCPACGRPDLTDDTQLLSYFSTLDIQLRESLEPDAYALFHKK LTEGVLMRDPKFLWCAQCSFGFIYEREQLEATCPQCHQTFCVRCKRQWE EQHRGRSCEDFQNWKRMNDPEYQAQGLAMYLQENGIDCPKCKFSYALAR GGCMHFHCTQCRHQFCSGCYNAFYAKNKCPEPNCRVKKSLHGHHPRDCL FYLRDWTALRLQKLLQDNNVMENTEPPAGARAVPGGGCRVIEQKEVPNG LRDEACGKETPAGYAGLCQAHYKEYLVSLINAHSLDPATLYEVEELETA TERYLHVRPQPLAGEDPPAYQARLLQKLTEEVPLGQSIPRRRK
[0580] In some embodiments, an agent comprising a cluster 41 sequence interacts with RNF31 PUB domain.
Cluster C42
[0581] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C42 as described herein. In some embodiments, an agent comprises X.sup.42X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.42, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0582] In some embodiments, the present disclosure provides an agent comprising
X.sup.42X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0583] each of X.sup.42, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0584] X.sup.2 comprises a side chain comprising an aromatic group; [0585] X.sup.5 comprises a hydrophobic side chain; and [0586] X.sup.6 comprises a side chain comprising an aromatic group.
[0587] Various amino acid residues may be utilized for X.sup.42. For example, in some embodiments, X.sup.42 comprises a side chain comprising an aromatic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise aromatic groups at X.sup.42. In some embodiments, X.sup.42 is W. In some embodiments, X.sup.42 is Y. In some embodiments, X.sup.42 is F.
[0588] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, X.sup.2 is W. In some embodiments, X.sup.2 is M. In some embodiments, X.sup.2 is H.
[0589] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.3 is N.
[0590] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00380##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0591] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is L. In some embodiments, X.sup.5 is I. In some embodiments, X.sup.5 is M.
[0592] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is Y.
[0593] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0594] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0595] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 is W.
[0596] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is D. In some embodiments, X.sup.10 is E. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, X.sup.10 is T. In some embodiments, X.sup.10 is S.
[0597] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is Y.
[0598] Various amino acid residues may be utilized for X.sup.13.
[0599] Various amino acid residues may be utilized for X.sup.14.
[0600] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Thr74, Asn77, Ile78, Lys81, Tyr82, Asn85, Leu86, Pro92, Tyr94, Trp95, Val98, Asn102, Val104, Thr108, and Tyr124. In some embodiments, it interacts with Thr74 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn77 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile78 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys81 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr82 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn85 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu86 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Pro92 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr94 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp95 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val98 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn102 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val104 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr108 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr124 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
[0601] In some embodiments, an agent comprising a cluster 42 sequence interacts with RNF31 PUB domain.
Cluster C43
[0602] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C43 as described herein. In some embodiments, an agent comprises X.sup.43X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.43, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0603] In some embodiments, the present disclosure provides an agent comprising
X.sup.43X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0604] each of X.sup.43, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0605] X.sup.2 comprises a side chain comprising an acidic group; [0606] X.sup.3 comprises a side chain comprising an aromatic group; [0607] X.sup.5 comprises a hydrophobic side chain; [0608] X.sup.6 comprises a side chain comprising a polar group or an aromatic group; and [0609] X.sup.10 comprises a side chain comprising an acidic group.
[0610] Various amino acid residues may be utilized for X.sup.43. For example, in some embodiments, the side chain of X.sup.43 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.43. In some embodiments, X.sup.43 is Y. In some embodiments, X.sup.43 is W. In some embodiments, X.sup.43 is H.
[0611] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.2 is D. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 is W.
[0612] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, the side chain of X.sup.3 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.3. In some embodiments, X.sup.43 is W. In some embodiments, X.sup.3 is Y.
[0613] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00381##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0614] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is L. In some embodiments, X.sup.5 is M.
[0615] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is Y.
[0616] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0617] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0618] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 is D. In some embodiments, X.sup.9 is Y. In some embodiments, X.sup.9 is E.
[0619] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is D. In some embodiments, X.sup.10 is E. In some embodiments, X.sup.10 is N.
[0620] Various amino acid residues may be utilized for X.sup.12.
[0621] Various amino acid residues may be utilized for X.sup.13.
[0622] Various amino acid residues may be utilized for X.sup.14.
[0623] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Thr74, Asn77, Ile78, Lys81, Tyr82, Asn85, Leu86, Pro92, Tyr94, Trp95, Val98, Asn102, Val104, Thr108, and Tyr124. In some embodiments, it interacts with Thr74 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn77 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile78 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys81 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr82 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn85 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu86 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Pro92 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr94 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp95 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val98 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn102 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val104 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr108 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr124 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
[0624] In some embodiments, an agent comprising a cluster 43 sequence interacts with RNF31 PUB domain.
Cluster C44
[0625] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C44 as described herein. In some embodiments, an agent comprises X.sup.44X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.4, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0626] In some embodiments, the present disclosure provides an agent comprising
X.sup.44X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.1,
wherein: [0627] each of X.sup.44, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0628] X.sup.10 comprises a hydrophobic side chain; [0629] X.sup.13 comprises a side chain comprising a polar group; and [0630] X.sup.14 comprises a side chain comprising s polar group or an aromatic group.
[0631] Various amino acid residues may be utilized for X.sup.44. Various amino acid residues may be utilized for X.sup.44. For example, in some embodiments, X.sup.44 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.44. In some embodiments, the side chain of X.sup.44 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.44 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.44 comprises an aromatic group. In some embodiments, X.sup.44 is I. In some embodiments, X.sup.44 is F. In some embodiments, X.sup.44 is A.
[0632] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.3 is T. In some embodiments, X.sup.3 is Q.
[0633] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00382##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0634] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 is Q.
[0635] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, X.sup.6 is L. In some embodiments, X.sup.6 is I. In some embodiments, X.sup.6 is V. In some embodiments, X.sup.6 is F.
[0636] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0637] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0638] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, X.sup.9 is A. In some embodiments, X.sup.9 is M. In some embodiments, X.sup.9 is L. In some embodiments, X.sup.9 is Q. In some embodiments, X.sup.9 is D.
[0639] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is T.
[0640] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising OH. In some embodiments, X.sup.12 is S. In some embodiments, X.sup.12 is T. In some embodiments, X.sup.12 is M. In some embodiments, X.sup.12 is A.
[0641] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is T.
[0642] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, the side chain of X.sup.14 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising OH. In some embodiments, X.sup.14 is Y.
[0643] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Thr74, Asn77, Ile78, Lys81, Tyr82, Asn85, Leu86, Pro92, Tyr94, Trp95, Val98, Asn102, Val104, Thr108, and Tyr124. In some embodiments, it interacts with Thr74 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn77 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile78 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys81 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr82 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn85 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu86 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Pro92 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr94 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp95 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val98 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn102 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val104 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr108 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr124 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
[0644] In some embodiments, an agent comprising a cluster 44 sequence interacts with RNF31 PUB domain.
Cluster C45
[0645] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C45 as described herein. In some embodiments, an agent comprises X.sup.45X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.45, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0646] In some embodiments, the present disclosure provides an agent comprising
X.sup.45X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0647] each of X.sup.45, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0648] X.sup.6 comprises a side chain comprising an acidic group; [0649] X.sup.9 comprises a hydrophobic side chain; [0650] X.sup.12 comprises a side chain comprising a polar group; and [0651] X.sup.13 comprises a side chain comprising a polar group or an aromatic group.
[0652] Various amino acid residues may be utilized for X4.sup.5.
[0653] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 is D.
[0654] Various amino acid residues may be utilized for X.sup.3.
[0655] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00383##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0656] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.5 comprises an aromatic group. In some embodiments, X.sup.5 is W. In some embodiments, X.sup.5 is L. In some embodiments, X.sup.5 is I. In some embodiments, X.sup.5 is F.
[0657] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.6. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 is E. In some embodiments, the side chain of X.sup.6 comprises a polar group. In some embodiments, X.sup.6 is N. In some embodiments, X.sup.6 is S.
[0658] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0659] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0660] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is V. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is M.
[0661] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is M. In some embodiments, X.sup.10 is L. In some embodiments, X.sup.10 is A.
[0662] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising OH. In some embodiments, X.sup.12 is T. In some embodiments, X.sup.12 is S.
[0663] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is Y.
[0664] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising OH. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is T. In some embodiments, X.sup.14 is S.
[0665] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Thr74, Asn77, Ile78, Lys81, Tyr82, Asn85, Leu86, Pro92, Tyr94, Trp95, Val98, Asn102, Val104, Thr108, and Tyr124. In some embodiments, it interacts with Thr74 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn77 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile78 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys81 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr82 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn85 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu86 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Pro92 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr94 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp95 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val98 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn102 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val104 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr108 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr124 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
[0666] In some embodiments, an agent comprising a cluster 45 sequence interacts with RNF31 PUB domain.
Cluster C46
[0667] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C46 as described herein. In some embodiments, an agent comprises X.sup.46X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.46, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0668] In some embodiments, the present disclosure provides an agent comprising
X.sup.46X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0669] each of X.sup.46, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0670] X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [0671] X.sup.6 comprises a side chain comprising a polar group; [0672] X.sup.9 comprises a hydrophobic side chain [0673] X.sup.10 comprises a side chain comprising an aromatic group;
[0674] Various amino acid residues may be utilized for X.sup.46. For example, in some embodiments, X.sup.46 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.46. In some embodiments, the side chain of X.sup.46 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.46 is C.sub.1-6 alkyl. In some embodiments, X.sup.46 is M. In some embodiments, X.sup.46 is L. In some embodiments, X.sup.46 is A. In some embodiments, X.sup.46 is Q.
[0675] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 is Q. In some embodiments, X.sup.2 is R. In some embodiments, X.sup.2 is M.
[0676] Various amino acid residues may be utilized for X.sup.3. In some embodiments, X.sup.2 is R.
[0677] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00384##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0678] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.5 comprises an aromatic group. In some embodiments, X.sup.5 is L. In some embodiments, X.sup.5 is F. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is Y. In some embodiments, X.sup.5 is T.
[0679] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is S.
[0680] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0681] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0682] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, X.sup.9 is V. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is F. In some embodiments, X.sup.9 is M.
[0683] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.10. In some embodiments, X.sup.10 is Y. In some embodiments, X.sup.10 is W. In some embodiments, X.sup.10 is F.
[0684] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is Q. In some embodiments, X.sup.12 is A. In some embodiments, X.sup.12 is S. In some embodiments, X.sup.12 is N.
[0685] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.13 comprises an aromatic group. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is M. In some embodiments, X.sup.13 is F.
[0686] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is S. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is H.
[0687] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Glu506, Phe509, Ser510, Leu524, Arg525, Leu528, Tyr530, Val531, Met534, Leu538, Trp558, His562, Gly563, Gly563, Gly563, and Leu565. In some embodiments, it interacts with Glu506 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe509 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser510 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu524 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg525 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu528 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr530 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val531 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met534 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu538 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp558 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His562 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly563 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly563 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly563 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu565 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
[0688] In some embodiments, an agent comprising a cluster 41 sequence interacts with RNF31 UBA domain.
Cluster C47
[0689] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C47 as described herein. In some embodiments, an agent comprises X.sup.47X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.47, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0690] In some embodiments, the present disclosure provides an agent comprising
X.sup.47X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0691] each of X.sup.47, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0692] X.sup.2 comprises a side chain comprising a polar group; [0693] X.sup.5 comprises a hydrophobic side chain; [0694] X.sup.6 comprises a side chain comprising a polar group or an aromatic group; and [0695] X.sup.9 comprises a hydrophobic side chain.
[0696] Various amino acid residues may be utilized for X.sup.47. For example, in some embodiments, X.sup.47 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.47. In some embodiments, the side chain of X.sup.47 comprises an aromatic group. In some embodiments, the side chain of X.sup.47 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.47 is C.sub.1-6 alkyl. In some embodiments, X.sup.47 is F. In some embodiments, X.sup.47 is L.
[0697] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, the side chain of X.sup.2 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising OH. In some embodiments, X.sup.2 is S. In some embodiments, X.sup.2 is Y. In some embodiments, X.sup.2 is F.
[0698] Various amino acid residues may be utilized for X.sup.3. In some embodiments, X.sup.3 is T. In some embodiments, X.sup.3 is Q. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 is R.
[0699] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00385##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0700] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is V. In some embodiments, X.sup.5 is I.
[0701] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is Y.
[0702] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0703] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0704] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. in some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, X.sup.9 is V. In some embodiments, X.sup.9 is F. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is L.
[0705] Various amino acid residues may be utilized for X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, X.sup.10 is F.
[0706] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is E. In some embodiments, X.sup.12 is D.
[0707] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 is D.
[0708] Various amino acid residues may be utilized for X.sup.14.
[0709] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) or all of the following amino acid residues of SEQ ID NO: 2 or amino acid residues corresponding thereto: Glu506, Phe509, Ser510, Leu524, Arg525, Leu528, Tyr530, Val531, Met534, Leu538, Trp558, His562, Gly563, Gly563, Gly563, and Leu565. In some embodiments, it interacts with Glu506 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe509 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser510 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu524 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg525 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu528 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr530 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val531 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met534 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu538 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp558 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His562 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly563 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly563 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly563 of SEQ ID NO: 2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu565 of SEQ ID NO: 2 or an amino acid residue corresponding thereto.
[0710] In some embodiments, an agent comprising a cluster 47 sequence interacts with RNF31 UBA domain.
Cluster C48
[0711] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C33 as described herein. In some embodiments, an agent comprises X.sup.48X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.48, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0712] In some embodiments, the present disclosure provides an agent comprising
X.sup.48X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0713] each of X.sup.48, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0714] X.sup.3 comprises a side chain comprising a basic group or an aromatic group; [0715] X.sup.10 comprises a side chain comprising an aromatic group; [0716] X.sup.12 comprises a hydrophobic side chain; and [0717] X.sup.13 comprises a hydrophobic side chain.
[0718] Various amino acid residues may be utilized for X.sup.48. In some embodiments, X.sup.48 is R.
[0719] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 is E.
[0720] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.3. In some embodiments, the side chain of X.sup.3 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.3. In some embodiments, X.sup.3 is H.
[0721] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00386##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0722] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 is T.
[0723] Various amino acid residues may be utilized for X.sup.6. In some embodiments, X.sup.6 is A. In some embodiments, X.sup.6 is S.
[0724] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0725] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0726] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 is A.
[0727] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 comprises an aromatic group. In some embodiments, X.sup.10 is W. In some embodiments, X.sup.10 is F.
[0728] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is Y. In some embodiments, X.sup.12 is W. In some embodiments, X.sup.12 is H.
[0729] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is L. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is V.
[0730] Various amino acid residues may be utilized for X.sup.114. For example, in some embodiments, X.sup.14 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, X.sup.14 is L. In some embodiments, X.sup.14 is M. In some embodiments, X.sup.14 is I.
[0731] In some embodiments, an agent comprising a cluster 48 sequence interacts with RNF31 UBA domain.
CDK2
[0732] In some embodiments, the present disclosure provides agents that can bind to and/or modulate activities of CDK2. In some embodiments, provided stapled peptides selectively bind CDK2 or CDK2 in complex with its partner, e.g., CyclinE1, over CDK1 or CDK1 in complex with its partner, e.g., CyclinA2 (C51 and C52). In some embodiments, a stapled peptide does not compete with ATP. In some embodiments, a stapled peptide bind to CDK2 in the presence of other ATP-competitive CDK2-binding agents, e.g., proteins, Dinaciclib, Zotiraciclib, etc. In some embodiments, a stapled peptide does not inhibit the kinase activity of CDK2. In some embodiments, a stapled peptide does not inhibit the kinase activity of CDK2 in a luminescence-based assay that detects ADP production.
Cluster C51
[0733] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C51 as described herein. In some embodiments, an agent comprises X.sup.51X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.51, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0734] In some embodiments, the present disclosure provides an agent comprising
X.sup.51X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0735] each of X.sup.51, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0736] X.sup.1 comprises a side chain comprising an aromatic group; [0737] X.sup.2 comprises a side chain comprising an aromatic group; [0738] X.sup.6 comprises a hydrophobic side chain; [0739] X.sup.9 comprises a hydrophobic side chain; and [0740] X.sup.10 comprises a side chain comprising an acidic group.
[0741] Various amino acid residues may be utilized for X.sup.51. For example, in some embodiments, X.sup.51 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.51. In some embodiments, the side chain of X.sup.51 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.51. In some embodiments, X.sup.51 is W.
[0742] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.2. In some embodiments, X.sup.2 is W. In some embodiments, X.sup.2 is Y
[0743] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 is R. In some embodiments, X.sup.3 is V. In some embodiments, X.sup.3 is T. In some embodiments, X.sup.3 is A. In some embodiments, X.sup.3 is N.
[0744] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00387##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0745] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is I. In some embodiments, X.sup.5 is V. In some embodiments, X.sup.5 is A. In some embodiments, the side chain of X.sup.5 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T.
[0746] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is I. In some embodiments, X.sup.6 is V.
[0747] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0748] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0749] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is L. In some embodiments, X.sup.9 is M. In some embodiments, X.sup.9 is V.
[0750] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is E. In some embodiments, X.sup.10 is D. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, X.sup.10 is Q.
[0751] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is E. In some embodiments, X.sup.12 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising a basic groups at X.sup.12. In some embodiments, X.sup.12 is H. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, X.sup.12 is Q. In some embodiments, X.sup.12 is S.
[0752] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising COOH. In some embodiments, X.sup.13 is D. In some embodiments, X.sup.13 is E. In some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, X.sup.13 is T. In some embodiments, X.sup.13 is S.
[0753] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising OH. In some embodiments, X.sup.14 is T. In some embodiments, X.sup.14 is S. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is Q. In some embodiments, X.sup.14 is V.
[0754] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of SEQ ID NO: 3 or amino acid residues corresponding thereto: Gln5, Val7, Tyr19, Ala21, Asn23, Glu28, Val30, Leu32, Lys34, Arg36, Leu67, Asp68, Ile70, Thr72, Lys75, Tyr77, and Val79. In some embodiments, it interacts with Gln5 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val7 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr19 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala21 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn23 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu28 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val30 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu32 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys34 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg36 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu67 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asp68 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile70 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr72 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys75 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr77 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val79 of SEQ ID NO: 3 or an amino acid residue corresponding thereto.
TABLE-US-00016 (SEQIDNO:3) MENFQKVEKIGEGTYGVVYKARNKLTGEVVALKKIRLDTETEGVPSTAI REISLLKELNHPNIVKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTG IPLPLIKSYLFQLLQGLAFCHSHRVLHRDLKPQNLLINTEGAIKLADFG LARAFGVPVRTYTHEVVTLWYRAPEILLGCKYYSTAVDIWSLGCIFAEM VTRRALFPGDSEIDQLFRIFRTLGTPDEVVWPGVTSMPDYKPSFPKWAR QDFSKVVPPLDEDGRSLLSQMLHYDPNKRISAKAALAHPFFQDVTKPVP HLRL
[0755] In some embodiments, an agent comprising a cluster 51 sequence demonstrates selectivity for CDK2 and a CDK2-CCNE1 complex over CDK1 or a CDK1-CCNA2 complex.
Cluster C52
[0756] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C52 as described herein. In some embodiments, an agent comprises X.sup.52X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.52, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0757] In some embodiments, the present disclosure provides an agent comprising
X.sup.52X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0758] each of X.sup.52, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0759] X.sup.5 comprises a hydrophobic side chain; [0760] X.sup.8 comprises a side chain comprising an aromatic group; and [0761] X.sup.9 comprises a side chain comprising an aromatic group.
[0762] Various amino acid residues may be utilized for X.sup.52. For example, in some embodiments, X.sup.52 is P. In some embodiments, X.sup.52 is absent.
[0763] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, X.sup.2 is P. In some embodiments, X.sup.2 is F. In some embodiments, X.sup.2 is M. In some embodiments, X.sup.2 is Y.
[0764] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.3. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.3 comprises an aromatic group. In some embodiments, X.sup.3 is P. In some embodiments, X.sup.3 is A. In some embodiments, X.sup.3 is Y.
[0765] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00388##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0766] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is L. In some embodiments, X.sup.5 is V. In some embodiments, X.sup.5 is M.
[0767] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 is E. In some embodiments, the side chain of X.sup.6 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is S.
[0768] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0769] Various amino acid residues may be utilized for X.sup.8. For example, in some embodiments, X.sup.8 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.8. In some embodiments, the side chain of X.sup.8 comprises an aromatic group. In some embodiments, X.sup.8 is F. In some embodiments, X.sup.8 is Y.
[0770] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, X.sup.9 is F.
[0771] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.19. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is S. In some embodiments, X.sup.10 is T. In some embodiments, X.sup.10 is A.
[0772] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 is V. In some embodiments, X.sup.12 is absent.
[0773] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 is absent.
[0774] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is absent.
[0775] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) or all of the following amino acid residues of SEQ ID NO: 3 or amino acid residues corresponding thereto: His84, Gln85, Lys89, Phe90, Ala93, Ser94, Thr97, Ile99, Pro100, Leu103, Tyr107, Ile135, Thr137, Thr137, Thr137, and Gly139. In some embodiments, it interacts with His84 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln85 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys89 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe90 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala93 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser94 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr97 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile99 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Pro100 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu103 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr107 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile135 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr137 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr137 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr137 of SEQ ID NO: 3 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly139 of SEQ ID NO: 3 or an amino acid residue corresponding thereto.
PPIA
[0776] In some embodiments, the present disclosure provides agents that can bind to and/or modulate activities of PPIA. In some embodiments, a stapled peptide competes with CsA for PPIA interaction.
Cluster C53
[0777] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C53 as described herein. In some embodiments, an agent comprises X.sup.53X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.53, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0778] In some embodiments, the present disclosure provides an agent comprising
X.sup.53X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0779] each of X.sup.53, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0780] X.sup.3 comprises a side chain comprising an acidic group; [0781] X.sup.5 comprises a side chain comprising a basic or aromatic group; [0782] X.sup.6 comprises a hydrophobic side chain; and [0783] X.sup.9 comprises a side chain comprising an aromatic group or a polar group.
[0784] Various amino acid residues may be utilized for X.sup.53. For example, in some embodiments, X.sup.53 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.53. In some embodiments, X.sup.53 is R. In some embodiments, the side chain of X.sup.53 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.53. In some embodiments, X.sup.53 is T. In some embodiments, X.sup.53 is Q. In some embodiments, X.sup.53 is S. In some embodiments, X.sup.53 is A. In some embodiments, X.sup.53 is N.
[0785] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 is Q. In some embodiments, X.sup.2 is H. In some embodiments, X.sup.2 is Y. In some embodiments, X.sup.2 is T. In some embodiments, X.sup.2 is A.
[0786] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, the side chain of X.sup.3 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising OH. In some embodiments, X.sup.3 is S. In some embodiments, X.sup.3 is N.
[0787] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00389##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0788] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise basic groups at X.sup.5. In some embodiments, X.sup.5 is H. In some embodiments, X.sup.5 is R. In some embodiments, X.sup.5 is Q.
[0789] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is V. In some embodiments, X.sup.6 is I.
[0790] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0791] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0792] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.9. In some embodiments, X.sup.9 is Y. In some embodiments, X.sup.9 is W. In some embodiments, X.sup.9 is F.
[0793] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.10 comprises an aromatic group. In some embodiments, X.sup.10 is H. In some embodiments, X.sup.10 is W. In some embodiments, X.sup.10 is F. In some embodiments, X.sup.10 is L. In some embodiments, X.sup.10 is Q.
[0794] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.12. In some embodiments, the side chain of X.sup.12 is C.sub.1 6 aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.12 comprises an aromatic group. In some embodiments, X.sup.12 is W. In some embodiments, X.sup.12 is Y. In some embodiments, X.sup.12 is I. In some embodiments, X.sup.12 is H.
[0795] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 is Q. In some embodiments, X.sup.13 is H. In some embodiments, X.sup.13 is F. In some embodiments, X.sup.13 is W.
[0796] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 is R. In some embodiments, X.sup.14 is Q.
[0797] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) or all of the following amino acid residues of SEQ ID NO: 4 or amino acid residues corresponding thereto: His54, Ile57, Phe60, Met61, Gln63, Thr73, Lys82, Ala101, Ala103, Gln111, Phe113, Trp121, Leu122, and His126. In some embodiments, it interacts with His54 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile57 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe60 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met61 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln63 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr73 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys82 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala101 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala103 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln111 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe113 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp121 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu122 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His126 of SE ID NO: 4 or an amino acid residue corresponding thereto.
TABLE-US-00017 (SEQIDNO:4) MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYK GSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGIL SMANAGPNINGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGS RNGKTSKKITIADCGQLE
[0798] In some embodiments, an agent comprising a cluster 53 sequence competes with cyclosporine for PPIA binding. In some embodiments, an agent comprising a cluster 53 sequence inhibit PPIA peptidyl-prolyl cis-trans isomerase activity. In some embodiments, an agent comprising a cluster 53 sequence inhibit PPIA peptidyl-prolyl cis-trans isomerase activity but with higher IC.sub.50 than CsA.
Cluster C54
[0799] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C54 as described herein. In some embodiments, an agent comprises X.sup.54X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.54, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0800] In some embodiments, the present disclosure provides an agent comprising
X.sup.54X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0801] each of X.sup.54, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0802] X.sup.5 comprises a side chain comprising an aromatic group or a basic group; [0803] X.sup.6 comprises a hydrophobic side chain; [0804] X.sup.8 comprises a hydrophobic side chain; and [0805] X.sup.9 comprises a side chain comprising an aromatic group.
[0806] Various amino acid residues may be utilized for X.sup.54. For example, in some embodiments, X.sup.54 is P. In some embodiments, X.sup.54 is absent.
[0807] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, X.sup.2 is P. In some embodiments, X.sup.2 is Y. In some embodiments, X.sup.2 is H.
[0808] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, the side chain of X.sup.3 comprises a polar group. In some embodiments, X.sup.3 is N. In some embodiments, X.sup.3 is T. In some embodiments, X.sup.3 is Y.
[0809] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00390##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0810] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.5. In some embodiments, the side chain of X.sup.5 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.5. In some embodiments, X.sup.5 is H.
[0811] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is I. In some embodiments, X.sup.6 is V. In some embodiments, X.sup.6 is L.
[0812] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A. In some embodiments, X.sup.7 is R.
[0813] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0814] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.9. In some embodiments, X.sup.9 is Y. In some embodiments, X.sup.9 is W. In some embodiments, X.sup.9 is F.
[0815] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.1 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.10 comprises an aromatic group. In some embodiments, X.sup.10 is Y. In some embodiments, X.sup.10 is M. In some embodiments, X.sup.10 is F. In some embodiments, X.sup.10 is V.
[0816] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 is H.
[0817] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 is absent.
[0818] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is absent.
[0819] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) or all of the following amino acid residues of SEQ ID NO: 4 or amino acid residues corresponding thereto: His54, Ile57, Phe60, Met61, Gln63, Thr73, Lys82, Ala101, Ala103, Gln111, Phe113, Trp121, Leu122, and His126. In some embodiments, it interacts with His54 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile57 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe60 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met61 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln63 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr73 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys82 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala101 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala103 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln11I of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe113 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp121 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu122 of SEQ ID NO: 4 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His126 of SEQ ID NO: 4 or an amino acid residue corresponding thereto.
[0820] In some embodiments, an agent comprising a cluster 54 sequence competes with cyclosporine for PPIA binding. In some embodiments, an agent comprising a cluster 54 sequence inhibit PPIA peptidyl-prolyl cis-trans isomerase activity. In some embodiments, an agent comprising a cluster 54 sequence inhibit PPIA peptidyl-prolyl cis-trans isomerase activity but with higher IC.sub.50 than CsA.
PD-Li
[0821] In some embodiments, the present disclosure provides agents that can bind to and/or modulate activities of PD-L1. In some embodiments, a stapled peptide reduces interaction between PD-L1 and PD1. In some embodiments, a stapled peptide induces PD-L1 dimerization. In some embodiments, a stapled peptide binds to extracellular domain of PD-L1.
Cluster C61
[0822] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C61 as described herein. In some embodiments, an agent comprises X.sup.61X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.0X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.61, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0823] In some embodiments, the present disclosure provides an agent comprising
X.sup.61X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0824] each of X.sup.61, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X, X.sup.1, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0825] X.sup.61 comprises a side chain comprising an aromatic group; [0826] X.sup.6 comprises a hydrophobic side chain and/or a side chain comprising an aromatic group; and [0827] X.sup.9 comprises a side chain comprising a basic group.
[0828] Various amino acid residues may be utilized for X.sup.61. For example, in some embodiments, X.sup.61 is L.
[0829] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.2. In some embodiments, X.sup.2 is W.
[0830] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 is Q.
[0831] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00391##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0832] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, X.sup.5 is V.
[0833] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.6. In some embodiments, X.sup.6 is F.
[0834] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0835] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0836] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.9. In some embodiments, X.sup.9 is R.
[0837] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 is S.
[0838] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 is Y.
[0839] Various amino acid residues may be utilized for X.sup.13. In some embodiments, side chain of X.sup.13 comprises an acidic group, e.g., COOH. In some embodiments, X.sup.13 is E.
[0840] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, side chain of X.sup.14 comprises an acidic group, e.g., COOH. In some embodiments, X.sup.14 is E. In some embodiments, X.sup.14 is absent.
[0841] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) or all of the following amino acid residues of SEQ ID NO: 5 or amino acid residues corresponding thereto: Ile54, Tyr56, Glu58, Asp61, Asn63, Gln66, Val68, Val76, His78, Arg113, Met115, Ser117, Ala121, and Tyr123. In some embodiments, it interacts with Ile54 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr56 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu58 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asp61 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn63 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln66 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val68 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val76 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His78 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg113 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met115 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser117 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala121 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr123 of SEQ ID NO: 5 or an amino acid residue corresponding thereto.
TABLE-US-00018 (SEQIDNO:5) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLD LAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAA LQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTST LRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILG AILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET
Cluster C62
[0842] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C62 as described herein. In some embodiments, an agent comprises X.sup.62X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.62, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0843] In some embodiments, the present disclosure provides an agent comprising
X.sup.62X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0844] each of X.sup.62, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0845] X.sup.3 comprises a side chain comprising a polar group or an acidic group; [0846] X.sup.6 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [0847] X.sup.9 comprises a side chain comprising an aromatic group.
[0848] Various amino acid residues may be utilized for X.sup.62. For example, in some embodiments, X.sup.62 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.62. In some embodiments, the side chain of 62 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.62 is C.sub.1-6 alkyl. In some embodiments, X.sup.62 is I. In some embodiments, X.sup.62 is V. In some embodiments, the side chain of X.sup.62 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.62. In some embodiments, X.sup.62 comprises a side chain comprising OH. In some embodiments, X.sup.62 is S. In some embodiments, X.sup.62 is Y.
[0849] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.2 is D. In some embodiments, X.sup.2 is E.
[0850] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprising acidic groups at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, the side chain of X.sup.3 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising OH. In some embodiments, X.sup.3 is S. In some embodiments, X.sup.3 is T. In some embodiments, X.sup.3 is Y.
[0851] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00392##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0852] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.5. In some embodiments, X.sup.5 is R. In some embodiments, X.sup.5 comprises a side chain comprising an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.5. In some embodiments, X.sup.5 is W. In some embodiments, X.sup.5 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.62. In some embodiments, the side chain of X.sup.62 is C.sub.1 6 aliphatic. In some embodiments, the side chain of X.sup.62 is C.sub.1-6 alkyl. In some embodiments, X.sup.62 is A.
[0853] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 comprises an aromatic group. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is W. In some embodiments, X.sup.6 is F. In some embodiments, X.sup.6 is V.
[0854] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0855] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0856] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.9. In some embodiments, X.sup.9 is F. In some embodiments, the side chain of X.sup.9 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising OH. In some embodiments, X.sup.9 is T. In some embodiments, X.sup.9 is Y.
[0857] Various amino acid residue may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 is L.
[0858] Various amino acid residue may be utilized for X.sup.11.
[0859] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.12. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, X.sup.12 is M. In some embodiments, X.sup.12 is A. In some embodiments, the side chain of X.sup.12 comprises a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a side chain comprising a basic group at X.sup.12. In some embodiments, X.sup.12 is R.
[0860] Various amino acid residue may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 is V.
[0861] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, the side chain of X.sup.14 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising OH. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is S.
[0862] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) or all of the following amino acid residues of SEQ ID NO: 5 or amino acid residues corresponding thereto: Ile54, Tyr56, Glu58, Asp61, Gln66, Val68, Arg113, Met115, Ser117, Ala121, Tyr123, and Arg125. In some embodiments, it interacts with Ile54 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr56 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu58 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asp61 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln66 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val68 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg113 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met115 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser117 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala121 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr123 of SEQ ID NO: 5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg125 of SEQ ID NO: 5 or an amino acid residue corresponding thereto.
HECT E3 Family
[0863] In some embodiments, the present disclosure provides agents that can bind to HECT E3 ligases. In some embodiments, provided agents can bind to WWP1. In some embodiments, provided agents can bind to WWP2. In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C71, are capable of binding both an inactive form of WWP1 (e.g., WWP1.sup.WW-HECT), a form of WWP1 that may assume active and inactive states (e.g., WWP1.sup.HECT), and a form of WWP2 that may assume active and inactive states (e.g., WWP2.sup.HECT). In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C72, are capable of selectively binding to WWP2 (e.g., WWP2.sup.HCT) over WWP1 (e.g., WWP1.sup.HECT and/or WWP1.sup.WW-HECT). In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C73, are capable of binding an active form of WWP2 (e.g., WWP2.sup.HECT) and/or selectively binding to an active form of WWP1 (e.g., WWP1.sup.HECT) over an inactive form WWP1 (e.g., WWP1.sup.WW-HECT). In some embodiments, stapled peptides comprising sequences of certain clusters, e.g., cluster C74, are capable of selectively binding to an inactive form of WWP1 (e.g., WWP1.sup.WW-HECT) over an active form of WWP1 (e.g., WWP1.sup.HECT) or an active form of WWP2 (e.g., WWP2.sup.HECT) In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C71 and cluster C73, are capable of binding to the HECT domain of WWP1. In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C71, interact with the N-lobe of the HECT domain of WWP1. In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C71 and cluster C73, interact with the N-lobe and the C-lobe of the HECT domain of WWP2. In some embodiments, agents comprising sequences of certain clusters, e.g., cluster C72, interact with the C-lobe of the HECT domain of WWP2. In some embodiments, the present disclosure provides methods for modulating a function or interaction of an HECT E3 ligases, comprising contacting the ligase with a provided agent.
Cluster C71
[0864] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C71 as described herein. In some embodiments, an agent comprises X.sup.71X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.71, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0865] In some embodiments, the present disclosure provides an agent comprising
X.sup.71X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0866] each of X.sup.71, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0867] X.sup.10 comprises a hydrophobic side chain.
[0868] Various amino acid residues may be utilized for X.sup.71. For example, in some embodiments, X.sup.71 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a hydrophobic side chain at X.sup.71. In some embodiments, X.sup.71 is M. In some embodiments, the side chain of X.sup.71 is aliphatic. In some embodiments, the side chain of X.sup.71 is C.sub.1-6 alkyl. In some embodiments, X.sup.71 is A. In some embodiments, X.sup.71 is V. In some embodiments, X.sup.71 is I. In some embodiments, X.sup.71 is L. In some embodiments, X.sup.71 is Q.
[0869] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 is M.
[0870] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.3. In some embodiments, X.sup.3 is R. In some embodiments, X.sup.3 is H. In some embodiments, X.sup.3 comprises an acidic side chain. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.3 comprises a side chain comprising OH. In some embodiments, X.sup.3 is S. In some embodiments, X.sup.3 is T. In some embodiments, X.sup.3 is Y.
[0871] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00393##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0872] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T. In some embodiments, X.sup.5 is M. In some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is T. In some embodiments, X.sup.5 is R.
[0873] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic polar at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is Q. In some embodiments, X.sup.6 is E.
[0874] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0875] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0876] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.9. In some embodiments, X.sup.9 is R. In some embodiments, X.sup.9 is H. In some embodiments, X.sup.9 comprises a side chain comprising a polar group. In some embodiments, X.sup.9 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.9 is N. In some embodiments, X.sup.9 is Q. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an aromatic group at X.sup.9. In some embodiments, X.sup.9 is Y. In some embodiments, X.sup.9 is W. In some embodiments, X.sup.9 is F
[0877] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 may be Y
[0878] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues comprising a basic group at X.sup.12. In some embodiments, X.sup.12 is R. In some embodiments, X.sup.12 is H. In some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues comprising an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is E. In some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising OH. In some embodiments, X.sup.12 is S. In some embodiments, X.sup.12 may be F.
[0879] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is L. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is M.
[0880] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, the side chain of X.sup.14 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, X.sup.14 is V. In some embodiments, X.sup.14 is L. In some embodiments, X.sup.14 is H.
[0881] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) or all of the following amino acid residues of WWP1 or amino acid residues corresponding thereto: Glu702, Phe703, Ser706, Leu707, Trp709, Ile710, Glu717, Cys718, Gly719, Leu720, Glu721, Met722, Val726, and Met760. In some embodiments, it interacts with Glu702 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe703 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser706 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu707 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp709 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile710 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu717 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Cys718 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly719 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu720 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu721 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met722 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val726 of WWP1 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met760 of WWP1 or an amino acid residue corresponding thereto.
[0882] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) or all of the following amino acid residues of WWP2 or amino acid residues corresponding thereto: Glu650, Phe651, Asn653, Ser654, Ile655, Trp657, Ile658, Asn661, Asn662, Glu665, Cys666, Gly667, Leu668, Glu669, Leu670, Gln674, Tyr704, and Leu708. In some embodiments, it interacts with Glu650 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe651 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn653 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser654 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile655 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp657 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile658 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn661 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn662 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu665 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Cys666 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly667 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu668 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu669 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu670 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln674 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr704 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu708 of WWP2 or an amino acid residue corresponding thereto.
[0883] In some embodiments, an agent comprising a cluster C71 sequence is capable of binding to WWP1. In some embodiments, an agent comprising a cluster C71 sequence is capable of binding to an autoinhibited form of WWP1. In some embodiments, an agent comprising a cluster C71 sequence is capable of binding to WWP2.
[0884] In some embodiments, a cluster comprising a cluster C71 sequence is capable of selectively binding to an active form of WWP1 over an autoinhibited form thereof. In some embodiments, a cluster comprising a cluster C71 sequence is capable of selectively binding to an active form of WWP2 over an active form of WWP1.
Cluster C72
[0885] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C72 as described herein. In some embodiments, an agent comprises X.sup.72X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.72, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0886] In some embodiments, the present disclosure provides an agent comprising
X.sup.72X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0887] each of X.sup.72, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0888] X.sup.9 comprises a side chain comprising a polar group; [0889] X.sup.13 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [0890] X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[0891] Various amino acid residues may be utilized for X.sup.72. For example, in some embodiments, the side chain of X.sup.72 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.72. In some embodiments, the side chain of X.sup.72 is aliphatic. In some embodiments, the side chain of X.sup.72 is C.sub.1-6 alkyl. In some embodiments, X.sup.72 is A. In some embodiments, X.sup.72 is I. In some embodiments, X.sup.72 is L.
[0892] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.2 is N. In some embodiments, X.sup.2 is Q. In some embodiments, the side chain of X.sup.2 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, the side chain of X.sup.2 is aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, X.sup.2 is V. In some embodiments, X.sup.2 is I. In some embodiments, X.sup.2 is L. In some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is H. In some embodiments, X.sup.2 is Y. In some embodiments, X.sup.2 is W.
[0893] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.3 is H. In some embodiments, X.sup.3 is F. In some embodiments, X.sup.3 is Y. In some embodiments, X.sup.3 is W.
[0894] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00394##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0895] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.5. In some embodiments, X.sup.5 is R. In some embodiments, X.sup.5 is H. In some embodiments, the side chain of X.sup.5 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is L.
[0896] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is A. In some embodiments, X.sup.6 is V.
[0897] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0898] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0899] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising COOH. In some embodiments, X.sup.9 is D. In some embodiments, X.sup.9 is E.
[0900] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise polar groups at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is T. In some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is L.
[0901] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.13 is F.
[0902] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is W.
[0903] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of WWP2 or amino acid residues corresponding thereto: Tyr499, Phe495, Arg496, His500, Arg503, Phe504, His507, Ser508, Gly619, Lys620, Phe621, Leu747, Met748, Met752, and Glu789. In some embodiments, it interacts with Tyr499 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe495 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg496 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His500 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg503 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe504 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His507 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser508 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly619 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys620 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe621 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu747 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met748 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met752 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu789 of WWP2 or an amino acid residue corresponding thereto.
[0904] In some embodiments, an agent comprising a cluster C72 sequence is capable of binding to WWP2. In some embodiments, an agent comprising a cluster C72 sequence is capable of selectively binding to WWP2 over WWP1.
Cluster C73
[0905] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C73 as described herein. In some embodiments, an agent comprises X.sup.73X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.73, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0906] In some embodiments, the present disclosure provides an agent comprising
X.sup.73X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0907] each of X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; and [0908] X.sup.2 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[0909] Various amino acid residues may be utilized for X.sup.73. For example, in some embodiments, X.sup.73 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.73. In some embodiments, X.sup.73 comprises a side chain comprising OH. In some embodiments, X.sup.73 is T. In some embodiments, the side chain of X.sup.73 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.73. In some embodiments, the side chain of X.sup.73 is aliphatic. In some embodiments, the side chain of X.sup.73 is C.sub.1-6 alkyl. In some embodiments, X.sup.73 is A. In some embodiments, X.sup.73 is V. In some embodiments, X.sup.73 is I. In some embodiments, X.sup.73 is L.
[0910] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is W.
[0911] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E.
[0912] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00395##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0913] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.5. In some embodiments, X.sup.5 is R.
[0914] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH2. In some embodiments, X.sup.6 is N. In some embodiments, X.sup.6 is Q. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is Y. In some embodiments, X.sup.6 is W.
[0915] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0916] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0917] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is H. In some embodiments, X.sup.9 is F. In some embodiments, X.sup.9 is W.
[0918] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is D. In some embodiments, X.sup.10 is E. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.10 is N. In some embodiments, X.sup.10 is Q.
[0919] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.12 is F. In some embodiments, X.sup.12 is Y. In some embodiments, X.sup.12 is W.
[0920] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, X.sup.13 is M. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is L.
[0921] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.14 is N. In some embodiments, X.sup.14 is Q.
[0922] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) or all of the following amino acid residues of WWP2 or amino acid residues corresponding thereto: Phe495, Arg496, Tyr499, His500, Arg561, Glu562, Phe565, Leu566, Gly619, Lys620, Phe621, Ile622, Asp623, Leu747, Cys750, Gly751, Met752, Gln753, Glu754, Asp787, Glu789, and Arg803. In some embodiments, it interacts with Phe495 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg496 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr499 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His500 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg561 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu562 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe565 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu566 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly619 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys620 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe621 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile622 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asp623 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu747 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Cys750 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly751 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met752 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln753 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu754 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asp787 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu789 of WWP2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg803 of WWP2 or an amino acid residue corresponding thereto.
[0923] In some embodiments, an agent comprising a cluster C73 sequence is capable of binding to WWP2. In some embodiments, an agent comprising a cluster C73 sequence is capable of binding to WWP1. In some embodiments, an agent comprising a cluster C73 sequence is capable of selectively binding to an active WWP1 over an autoinhibited form of WWP1.
Cluster C74
[0924] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C74 as described herein. In some embodiments, an agent comprises X.sup.74X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.74, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0925] In some embodiments, the present disclosure provides an agent comprising
X.sup.74X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0926] each of X.sup.34, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0927] X.sup.10 comprises a hydrophobic side chain; and [0928] X.sup.12 comprises a side chain comprising a basic group or an aromatic group.
[0929] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, X.sup.2 is M. In some embodiments, the side chain of X.sup.2 is aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, X.sup.2 is V. In some embodiments, X.sup.2 is I. In some embodiments, X.sup.2 is L.
[0930] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00396##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0931] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T. In some embodiments, the side chain of X.sup.5 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is A. In some embodiments, X.sup.5 is V.
[0932] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, X.sup.6 is M. In some embodiments, the side chain of X.sup.6 is aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is V. In some embodiments, X.sup.6 is L. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is H. In some embodiments, X.sup.6 is F. In some embodiments, X.sup.6 is W.
[0933] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0934] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0935] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.9 is N. In some embodiments, X.sup.9 is Q. In some embodiments, the side chain of X.sup.9 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is V.
[0936] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is L. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is V.
[0937] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.12 is H.
[0938] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is S. In some embodiments, X.sup.13 is T. In some embodiments, X.sup.13 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.13 is N. In some embodiments, X.sup.13 is Q.
[0939] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising COOH. In some embodiments, X.sup.14 is D. In some embodiments, X.sup.14 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising OH. In some embodiments, X.sup.14 is S. In some embodiments, X.sup.14 is T.
[0940] In some embodiments, an agent comprising a cluster C74 sequence is capable of selectively binding to an autoinhibited form of WWP1 over an active form thereof. In some embodiments, an agent comprising a cluster C74 sequence is capable of selectively binding to an autoinhibited form of WWP1 over WWP2.
Cullin-RING Family
[0941] In some embodiments, the present disclosure provides agents that can bind to Cullin-Ring E3 ligases. In some embodiments, the present disclosure provides methods for modulating a function or interaction of such an E3 ligase, comprising contacting the ligase with a provided agent.
Cluster C75
[0942] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C75 as described herein. In some embodiments, an agent comprises X.sup.75X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.75, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0943] In some embodiments, the present disclosure provides an agent comprising
X.sup.75X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0944] each of X.sup.75, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0945] X.sup.75 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [0946] X.sup.12 comprises a side chain comprising an acidic group.
[0947] Various amino acid residues may be utilized for X.sup.35. In some embodiments, X.sup.3s is W.
[0948] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is H. In some embodiments, X.sup.2 is Y. In some embodiments, X.sup.2 is W.
[0949] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00397##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0950] Various amino acid residues may be utilized for X.sup.5. In some embodiments, the side chain of X.sup.5 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, X.sup.5 is M. In some embodiments, the side chain of X.sup.5 is aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is V. In some embodiments, X.sup.5 is I. In some embodiments, X.sup.5 is L. For example, in some embodiments, the side chain of X.sup.5 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.5. In some embodiments, the side chain of X.sup.5 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is Y. In some embodiments, X.sup.5 is F.
[0951] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D.
[0952] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0953] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0954] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D.
[0955] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 comprises an aromatic group. In some embodiments, a cluster is enriched for amino acid residues comprising an aromatic group at X.sup.13. In some embodiments, the side chain of X.sup.13 comprises a polar group. In some embodiments, a cluster is enriched for amino acid residues comprising a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is Y. In some embodiments, X.sup.13 is N.
[0956] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a hydrophobic side chain. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, the side chain of X.sup.14 comprises an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is M. In some embodiments, X.sup.14 is I. In some embodiments, X.sup.14 is V. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is Q.
[0957] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of VHL or amino acid residues corresponding thereto: Pro59, Arg60, Val62, Leu63, Arg64, Val66, Gly114, His115, Leu116, Thr133, Glu134, Leu135, Val137, Leu201, Thr202, Arg205, and Ile206. In some embodiments, it interacts with Pro59 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg60 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val62 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu63 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg64 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val66 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly114 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with His115 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu116 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr133 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu134 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu135 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val137 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu201 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr202 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg205 of VHL or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile206 of VHL or an amino acid residue corresponding thereto.
[0958] In some embodiments, an agent comprising a cluster C73 sequence is capable of binding to VHL.
Cluster C76
[0959] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C76 as described herein. In some embodiments, an agent comprises X.sup.76X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.76, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0960] In some embodiments, the present disclosure provides an agent comprising
X.sup.76X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0961] each of X.sup.76, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0962] X.sup.76 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [0963] X.sup.12 comprises a side chain comprising an acidic group.
[0964] Various amino acid residues may be utilized for X.sup.76. In some embodiments, X.sup.76 is W.
[0965] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00398##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0966] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, the side chain of X.sup.5 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is A.
[0967] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is E.
[0968] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0969] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0970] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a hydrophobic side chain. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W.
[0971] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or all of the following amino acid residues of CUL5 or amino acid residues corresponding thereto: Val35, Thr36, Lys37, Trp40, Phe41, Phe44, His48, Ile106, Lys109, Cys112, and Gln113. In some embodiments, it interacts with Val35 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr36 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys37 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Trp40 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe41 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe44 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His48 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile106 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys109 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Cys112 of CUL5 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln113 of CUL5 or an amino acid residue corresponding thereto.
[0972] In some embodiments, an agent comprising a cluster C76 sequence is capable of binding to CUL5.
Cluster C77
[0973] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C77 as described herein. In some embodiments, an agent comprises X.sup.77X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.77, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0974] In some embodiments, the present disclosure provides an agent comprising
X.sup.77X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0975] each of X.sup.77, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0976] X.sup.6 comprises a hydrophobic side chain; and [0977] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[0978] Various amino acid residues may be utilized for X.sup.77. For example, in some embodiments, X.sup.77 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.77. In some embodiments, X.sup.77 comprises a side chain comprising COOH. In some embodiments, X.sup.77 is D. In some embodiments, X.sup.77 is E. In some embodiments, X.sup.77 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.77. In some embodiments, X.sup.77 comprises a side chain comprising OH. In some embodiments, X.sup.77 is S. In some embodiments, X.sup.77 is T.
[0979] Various amino acid residues may be utilized for X.sup.2. In some embodiments, wherein X.sup.2 comprises a side chain comprising a basic group. In some embodiments, X.sup.2 is R.
[0980] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00399##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0981] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 is E.
[0982] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 is M. In some embodiments, the side chain of X.sup.6 is aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is L. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is F.
[0983] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[0984] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[0985] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W.
[0986] Various amino acid residues may be utilized for X.sup.10. In some embodiments, X.sup.10 comprises a hydrophobic side chain. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is A. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is I.
[0987] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, the side chain of X.sup.12 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.12. In some embodiments, the side chain of X.sup.12 is aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, X.sup.12 is A. In some embodiments, X.sup.12 is V. In some embodiments, X.sup.12 is I. In some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is E.
[0988] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, the side chain of X.sup.14 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 comprises a side chain comprising OH. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is W. In some embodiments, X.sup.14 is T.
[0989] In some embodiments, an agent comprising a cluster C77 sequence is capable of binding to CUL4B.
Cluster C78
[0990] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C78 as described herein. In some embodiments, an agent comprises X.sup.78X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.78, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[0991] In some embodiments, the present disclosure provides an agent comprising X.sup.78X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [0992] each of X.sup.78, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [0993] X.sup.5 comprises a side chain comprising an acidic group; and [0994] X.sup.13 comprises an aliphatic side chain.
[0995] Various amino acid residues may be utilized for X.sup.78. For example, in some embodiments, X.sup.78 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.78. In some embodiments, X.sup.78 comprises a side chain comprising COOH. In some embodiments, X.sup.78 is E.
[0996] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00400##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[0997] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 is E.
[0998] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, X.sup.6 is M. In some embodiments, the side chain of X.sup.6 is aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is V. In some embodiments, X.sup.6 is I. In some embodiments, X.sup.6 is L. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is H. In some embodiments, X.sup.6 is Y. In some embodiments, X.sup.6 is W.
[0999] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1000] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1001] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is A.
[1002] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is A. In some embodiments, X.sup.10 is L. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.10 is F. In some embodiments, X.sup.10 is Y. In some embodiments, X.sup.10 is W.
[1003] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is A. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is L.
[1004] In some embodiments, an agent comprising a cluster C78 sequence is capable of binding to CUL1.
Cluster C79
[1005] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C79 as described herein. In some embodiments, an agent comprises X.sup.79X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14 wherein each of X.sup.79, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1006] In some embodiments, the present disclosure provides an agent comprising
X.sup.79X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [1007] each of X.sup.79, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1008] X.sup.13 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [1009] X.sup.14 comprises a side chain comprising an aromatic group.
[1010] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, the side chain of X.sup.2 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, X.sup.2 is M. In some embodiments, the side chain of X.sup.2 is aliphatic. In some embodiments, the side chain of X.sup.2 is C.sub.1-6 alkyl. In some embodiments, X.sup.2 is I. In some embodiments, X.sup.2 is L. In some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is F. In some embodiments, X.sup.2 is Y. In some embodiments, X.sup.2 is W.
[1011] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, the side chain of X.sup.3 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.3. In some embodiments, X.sup.3 is M. In some embodiments, the side chain of X.sup.3 is aliphatic. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 alkyl. In some embodiments, X.sup.3 is A. In some embodiments, X.sup.3 is V. In some embodiments, X.sup.3 is I. In some embodiments, X.sup.3 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.3 is F. In some embodiments, X.sup.3 is Y. In some embodiments, X.sup.3 is W.
[1012] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00401##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1013] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 is E.
[1014] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 is E. In some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is N.
[1015] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1016] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1017] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising COOH. In some embodiments, X.sup.9 is D. In some embodiments, X.sup.9 is E. In some embodiments, the side chain of X.sup.9 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, the side chain of X.sup.9 is aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is L.
[1018] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is L.
[1019] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 is E.
[1020] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is A. In some embodiments, X.sup.13 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.13 is F. In some embodiments, X.sup.13 is W.
[1021] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is W.
[1022] In some embodiments, an agent comprising a cluster C79 sequence is capable of binding to FBXW7.
RING/U-BOX Family
Cluster C80
[1023] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C80 as described herein. In some embodiments, an agent comprises X.sup.80X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.80, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1024] In some embodiments, the present disclosure provides an agent comprising
[X.sup.80]p1[X.sup.2]p2[X.sup.3]p3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.3X.sup.4
wherein: [1025] each of p1, p2 and p3 is independently 0 or 1; [1026] each of X.sup.80, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1027] X.sup.7 comprises a hydrophobic side chain; and [1028] X.sup.9 comprises a hydrophobic side chain.
[1029] Various amino acid residues may be utilized for X.sup.80. In some embodiments, X.sup.80 is absent. In some embodiments, X.sup.80 is P.
[1030] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 is absent. In some embodiments, X.sup.2 is P.
[1031] Various amino acid residues may be utilized for X.sup.3. In some embodiments, X.sup.3 is absent. In some embodiments, X.sup.2 is P. In some embodiments, X.sup.2 comprises a hydrophobic side chain. In some embodiments, X.sup.2 is I.
[1032] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00402##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1033] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 is comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is W. In some embodiments, X.sup.5 is Y. In some embodiments, X.sup.5 is F.
[1034] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise acidic groups at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 is E. In some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is S. In some embodiments, X.sup.6 is T. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is N. In some embodiments, X.sup.6 is Q.
[1035] Various amino acid residues may be utilized for X.sup.7. For example, in some embodiments, the side chain of X.sup.7 is aliphatic. In some embodiments, the side chain of X.sup.7 is C.sub.1-6 alkyl. In some embodiments, X.sup.7 is A.
[1036] Various amino acid residues may be utilized for X.sup.8. For example, in some embodiments, the side chain of X.sup.8 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.8. In some embodiments, X.sup.8 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.8 is W.
[1037] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 is M. In some embodiments, the side chain of X.sup.9 is aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is V. In some embodiments, X.sup.9 is I. In some embodiments, X.sup.9 is L.
[1038] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is E.
[1039] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising OH. In some embodiments, X.sup.12 is S. In some embodiments, X.sup.12 is T.
[1040] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) or all of the following amino acid residues of CHIP or amino acid residues corresponding thereto: Phe37, Val38, Tyr49, Val61, Asn65, Leu68, Leu71, Lys72, Ser93, Val94, Lys95, Phe98, Phe99, Gln102, Glu106, Gln127, Leu129, Asn130, Phe131, Gly132, Asp134, and Ile135. In some embodiments, it interacts with Asn34 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe37 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val38 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr49 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn65 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu68 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu71 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys72 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ser93 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val94 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys95 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe98 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe99 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln102 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu106 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu129 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asn130 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe131 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly132 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Asp134 of CHIP or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile135 of CHIP or an amino acid residue corresponding thereto.
[1041] In some embodiments, an agent comprising a cluster C80 sequence is capable of binding to CHIP.
Cluster C81
[1042] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C81 as described herein. In some embodiments, an agent comprises X.sup.81X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.3X.sup.14, wherein each of X.sup.81, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1043] In some embodiments, the present disclosure provides an agent comprising
X.sup.81X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [1044] each of X.sup.81, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1045] X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1046] X.sup.6 comprises a side chain comprising a polar group; [1047] X.sup.10 comprises a side chain comprising an acidic group; and [1048] X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[1049] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00403##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1050] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is F.
[1051] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is S. In some embodiments, X.sup.6 is T. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is Q.
[1052] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1053] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1054] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W.
[1055] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is D.
[1056] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.12 is Q.
[1057] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.13 is F. In some embodiments, X.sup.13 is Y.
[1058] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is M. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is W.
[1059] In some embodiments, an agent comprising a cluster C81 sequence is capable of binding to MDM2.
Cluster C82
[1060] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C82 as described herein. In some embodiments, an agent comprises X.sup.82X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.3X.sup.14, wherein each of X.sup.82, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1061] In some embodiments, the present disclosure provides an agent comprising
X.sup.82X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [1062] each of X.sup.82, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1063] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [1064] X.sup.13 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[1065] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00404##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1066] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T. In some embodiments, X.sup.5 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.5 is N. In some embodiments, X.sup.5 is Q.
[1067] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 is E.
[1068] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1069] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1070] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W. In some embodiments, X.sup.9 is F.
[1071] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.10 is W.
[1072] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, the side chain of X.sup.12 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.12 is H. In some embodiments, X.sup.12 is F. In some embodiments, X.sup.12 is Y. In some embodiments, X.sup.12 is W.
[1073] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising an aromatic group. In some embodiments X.sup.13 is W.
[1074] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, the side chain of X.sup.14 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, X.sup.14 is M. In some embodiments, the side chain of X.sup.14 is aliphatic. In some embodiments, the side chain of X.sup.14 is C.sub.1-6 alkyl. In some embodiments, X.sup.14 is V. In some embodiments, X.sup.14 is I. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is W.
[1075] In some embodiments, an agent comprising a cluster C82 sequence is capable of binding to MDM2.
Cluster C83
[1076] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C83 as described herein. In some embodiments, an agent comprises X.sup.83X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.83, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1077] In some embodiments, the present disclosure provides an agent comprising
X.sup.83X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [1078] each of X.sup.83, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1079] X.sup.83 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1080] X.sup.5 comprises a side chain comprising a polar group; and [1081] X.sup.9 comprises a hydrophobic side chain.
[1082] Various amino acid residues may be utilized for X.sup.83. For example, in some embodiments, X.sup.83 comprises a side chain comprising an aromatic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an aromatic group at X.sup.83. In some embodiments, X.sup.83 is W.
[1083] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising COOH. In some embodiments, X.sup.2 is D. In some embodiments, X.sup.2 is E. In some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is W.
[1084] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.3. In some embodiments, X.sup.3 is R.
[1085] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00405##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1086] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 is Q.
[1087] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is F. In some embodiments, X.sup.6 is Y. In some embodiments, X.sup.6 is W.
[1088] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1089] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1090] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 is aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is A. In some embodiments, X.sup.9 is V. In some embodiments, X.sup.9 is I.
[1091] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is S. In some embodiments, X.sup.10 is T. In some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M.
[1092] In some embodiments, an agent comprising a cluster C83 sequence is capable of binding to CHIP.
Cluster C84
[1093] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C84 as described herein. In some embodiments, an agent comprises X.sup.84X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.84, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1094] In some embodiments, the present disclosure provides an agent comprising
X.sup.84X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [1095] each of X.sup.84, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1096] X.sup.10 comprises a hydrophobic side chain; [1097] X.sup.13 comprises a hydrophobic side chain; and [1098] X.sup.14 comprises a side chain comprising an acidic group.
[1099] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, the side chain of X.sup.3 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.3. In some embodiments, X.sup.3 is M. In some embodiments, the side chain of X.sup.3 is aliphatic. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 alkyl. In some embodiments, X.sup.3 is A. In some embodiments, X.sup.3 is V. In some embodiments, X.sup.3 is I. In some embodiments, X.sup.3 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.3 is F. In some embodiments, X.sup.3 is Y. In some embodiments, X.sup.3 is W.
[1100] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00406##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1101] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T. In some embodiments, X.sup.5 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is H. In some embodiments, X.sup.5 is F. In some embodiments, X.sup.5 is Y. In some embodiments, X.sup.5 is W.
[1102] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is F. In some embodiments, X.sup.6 is Y. In some embodiments, X.sup.6 is W.
[1103] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1104] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1105] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.9. In some embodiments, X.sup.9 is H. In some embodiments, X.sup.9 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising COOH. In some embodiments, X.sup.9 is D. In some embodiments, X.sup.9 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising OH. In some embodiments, X.sup.9 is S. In some embodiments, X.sup.9 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.9 is N. In some embodiments, X.sup.9 is Q.
[1106] Various amino acid residues may be utilized for X.sup.10. In some embodiments, X.sup.10 is I.
[1107] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, the side chain of X.sup.12 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.12. In some embodiments, X.sup.12 is M. In some embodiments, X.sup.12 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising COOH. In some embodiments, X.sup.12 is D. In some embodiments, X.sup.12 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.12 is W.
[1108] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is I.
[1109] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is D.
[1110] In some embodiments, an agent comprising a cluster C84 sequence is capable of binding to CHIP.
Cluster C85
[1111] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C85 as described herein. In some embodiments, an agent comprises X.sup.85X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14, wherein each of X.sup.85, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue.
[1112] In some embodiments, the present disclosure provides an agent comprising
X.sup.85X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14,
wherein: [1113] each of X.sup.85, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14 is independently an amino acid residue; [1114] X.sup.3 comprises a side chain comprising an acidic or polar group; [1115] X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1116] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [1117] X.sup.12 comprises a hydrophobic side chain.
[1118] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH or a salt form thereof. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising -C(O)NH.sub.2. In some embodiments, X.sup.3 is N. In some embodiments, X.sup.3 is Q.
[1119] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00407##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1120] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 is comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is F.
[1121] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising OH. In some embodiments, X.sup.6 is S. In some embodiments, X.sup.6 is T. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is N. In some embodiments, X.sup.6 is Q.
[1122] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1123] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1124] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 is comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W.
[1125] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 is M. In some embodiments, the side chain of X.sup.12 is aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, X.sup.12 is A. In some embodiments, X.sup.12 is V. In some embodiments, X.sup.12 is I.
[1126] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, the side chain of X.sup.13 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.13. In some embodiments, X.sup.13 is M. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is A. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.13 is W.
[1127] In some embodiments, an agent comprising a cluster C85 sequence is capable of binding to MDM2.
Trimerizers
[1128] In some embodiments, the present disclosure provides technologies for identification, characterization, production, and/or use of stapled peptide agents that can generate or enhance interactions of multiple targets of interest. In some embodiments, stapled peptide agents can bridge two targets of interests. In some embodiments, stapled peptide agents can enhance interactions of two targets of interest. In some embodiments, two targets of interest do not interact with each other absence of stapled peptide agents. In some embodiments, absence of provided stapled peptide agents, interactions of the two targets of interest are of low level or cannot be detected. In some embodiments, two targets of interest and a stapled peptide form a trimer (trimerize). In some embodiments, a stapled peptide forms a trimer with two targets of interest (such a stapled peptide may be referred to as a trimerizer or molecular glue).
[1129] In some embodiments, a trimerizer interacts with two targets of interest to cooperatively bind the targets of interest (e.g., polypeptides) together. In some embodiments, a trimerizer interacts with two protein surfaces to cooperatively bind the proteins together. In some embodiments, a trimerizer can bind targets of interest with greater affinity. In some embodiments, a trimerizer can bind sites or targets that a typical agent cannot bind. In some embodiments, a trimerizer is useful for inducing protein degradation.
[1130] In some embodiments, the present disclosure provides technologies for identifying trimerizers.
[1131] In some embodiments, the present disclosure provides a method comprising: [1132] contacting a first collection of stapled peptides with a first target of interest so that a number of stapled peptides of the first collection binds to the target; [1133] collecting stapled peptides of the first collection that bind to the first target; [1134] contacting the collected stapled peptides with a second target of interest so that one or more stapled peptides binds to the second target; and [1135] determining amino acid sequences of stapled peptides that bind to the second target.
[1136] In some embodiments, contact with a second target of interest is performed in the presence of a first target of interest. In some embodiments, contact in the presence of both targets of interest reduce or prevent enrichment of stapled peptides that can bind to a second target of interest but cannot bridge both target of interest.
[1137] In some embodiments, after contacting a first collection of stapled peptides with a first target of interest, sequences of stapled peptides of the first collection that bind to the first target are determined, and a second collection of stapled peptides is generated based on sequences of stapled peptides of the first collection that bind to the first target. For example, in some embodiments, enriched amino acid residues shared by a cluster of stapled peptides of the first collection that bind to the first target at one or more or all positions are determined, and when generating a second collection of stapled peptides, such enriched amino acid residues c utilized at one or more or all corresponding positions. At each position where enrichment is observed, one or more of the enriched amino acid residues at such a position based on the first collection can be independently and optionally utilized. In some embodiments, at each position that is important for interacting with a first target of interest, one or more of the enriched amino acid residues at such a position based on the first collection are independently and optionally utilized. In some embodiments, at each position that is important for interacting with a first target of interest, one or more of the enriched amino acid residues at such a position based on the first collection are independently utilized.
[1138] In some embodiments, the present disclosure provides comprising: [1139] contacting a first collection of stapled peptides with a first target of interest so that a number of stapled peptides of the first collection binds to the target; [1140] determining amino acid sequences of stapled peptides of the first collection that bind to the first target; [1141] identifying one or more enriched amino acid residues shared by a cluster of stapled peptides of the first collection that bind to the first target independently at one or more enriched positions (first collection enriched amino acid residues); [1142] contacting a second collection of stapled peptides with a second target of interest so that one or more stapled peptides of the second collection binds to the second target, wherein the second collection is enriched for one or more first collection enriched amino acid residues independently at one or more enriched positions or positions corresponding thereto; and [1143] determining amino acid sequences of stapled peptides that bind to the second target.
[1144] In some embodiments, the present disclosure provides a method, comprising: [1145] contacting a first collection of stapled peptides with a first target of interest so that a number of stapled peptides of the first collection binds to the target; [1146] determining amino acid sequences of stapled peptides of the first collection that bind to the first target; [1147] identifying one or more enriched amino acid residues shared by a cluster of stapled peptides of the first collection that bind to the first target independently at one or more enriched positions (first collection enriched amino acid residues); providing a second collection of stapled peptides, which stapled peptides are enriched for one or more of the enriched amino acid residue independently at one or more enriched positions or positions corresponding thereto, and are randomized at one or more positions; [1148] contacting a second collection of stapled peptides with a second target of interest so that one or more stapled peptides of the second collection binds to the second target; and [1149] determining amino acid sequences of stapled peptides that bind to the second target.
[1150] In some embodiments, the present disclosure provides a method, comprising: [1151] contacting a collection of stapled peptides with a target of interest so that a number of stapled peptides of the collection binds to the target, wherein the collection is enriched for one or more enriched amino acid residues independently at one or more enriched positions; and [1152] determining amino acid sequences of stapled peptides that bind to the target; wherein the one or more enriched amino acid residues are characterized in that when a collection of stapled peptides, which are randomized independently at the one or more enriched positions or positions corresponding thereto, is contacted with another target of interest, the stapled peptides that bind to the another target is enriched for one or more of the enriched amino acid residues independently at one or more of the enriched positions.
[1153] In some embodiments, a target of interest is a second target of interest as described herein. In some embodiments, an another target of interest is a first target of interest as described herein.
[1154] In some embodiments, first collection enriched amino acid residues are identified from a first collection of stapled peptides. In some embodiments, one or more or all of such enriched amino acid residues are utilized to generate a second collection of stapled peptides, biased/enriched for one or more or all of the enriched amino acid residues at relevant positions. In some embodiments, each of the first collection enriched amino acid residues is preferred or required for binding to a first target of interest. In some embodiments, each of the first collection enriched amino acid residues biased/enriched for in a second collection is preferred or required for binding to a first target of interest. In some embodiments, contacting with a second target of interest is performed in the presence of the first target. In some embodiments, stapled peptides that can bridge the first and second targets of interest is selected over stapled peptides that only binds to a second target of interest, only binds to a second target of interest, or can bind to both the first and the second targets of interest but cannot bridge them together. In some embodiments, a stapled peptide is a trimerizer. In some embodiments, a first target of interest is cytosolic. In some embodiments, a first target of interest is more abundant than a second target of interest. In some embodiments, a first target of interest is a polypeptide. In some embodiments, a first target of protein is considered an abundant protein by those skilled in the art. In some embodiments, a first target of interest may be referred to as a presenter protein. In some embodiments, a second target of interest is a polypeptide. In some embodiments, one of the target of interest is an E3 ubiquitin ligase, e.g., MDM2. In some embodiments, a presenter protein is an E3 ubiquitin ligase. In some embodiments, the other target of interest is degraded.
[1155] For example, in some embodiments, a first collection of stapled peptides is or comprises a naive library targeting a first target of interest. In some embodiments, a first target of interest is or comprises a polypeptide. In some embodiments, a first target of interest is or comprises an E3 ligase. In some embodiments, a first target of interest is CHIP. In some embodiments, a first target of interest is MDM2. In some embodiments, a first collection enriched amino acid residues comprises enriched amino acid residues from a cluster, e.g., enriched amino acid residues in various clusters as described herein (see, e.g., cluster C84 and C85 in
[1156] In some embodiments, the present disclosure provides a method for reducing level of a polypeptide, e.g., beta-catenin, comprising administering to a system comprising the peptide a stapled peptide, wherein the stapled peptide bridges the polypeptide to an E3 ubiquitin ligase. In some embodiments, a present disclosure provides trimerizers for beta-catenin and MDM2.
[1157] Among other things, provided technologies can recognize a polypeptide surface. In some embodiments, provided technologies can reprogram a polypeptide surface. In some embodiments, the present disclosure provides a method for recognizing or reprogramming a polypeptide surface, comprising contacting the polypeptide with an agent of the present disclosure. In some embodiments, contacting is performed in the presence of another polypeptide, wherein the agent binds to the polypeptide and the another polypeptide cooperatively. In some embodiments, the present disclosure provides a method for reprograming a polypeptide to cooperatively bind a target polypeptide, comprising contacting the polypeptides with an agent present disclosure.
[1158] In some embodiments, provided technologies can modulate polypeptide interactions. In some embodiments, the provided technologies can enhance certain interactions. In some embodiments, the provided technologies can induce new interactions. In some embodiments, provided technologies inhibit certain interactions. In some embodiments, provided technologies remove certain interactions. In some embodiments, the present disclosure provides a method for modulating polypeptide interaction, comprising contacting two polypeptides with an agent of present disclosure, wherein the agent binds to the two polypeptides cooperatively.
[1159] In some embodiments, the present disclosure provides technologies for modulating polypeptide functions. In some embodiments, the present disclosure provides technologies for modulating polypeptide levels. In some embodiments, the present disclosure provides technologies for reducing polypeptide levels. In some embodiments, the present disclosure provides technologies for targeting a polypeptide for degradation. In some embodiments, provided technologies comprise modulating interactions of polypeptides. In some embodiments, provided technologies comprise contacting a polypeptide with an agent, wherein a complex forms comprising the polypeptide, the agent, and another polypeptide. In some embodiments, the present disclosure provides technologies for forming a complex, comprising contacting an agent of present disclosure with polypeptides. In some embodiments, the present disclosure provides complexes comprising an agent of present disclosure and two polypeptides.
[1160] In some embodiments, an agent modulate polypeptide interactions as described herein. In some embodiments, two polypeptides interact with each other absence of the agent. In some embodiments, an agent provides new interactions between the polypeptides (which interactions does not exist absence of an agent). In some embodiments, an agent enhances an interaction between the polypeptides. In some embodiments, an agent reduces an interaction between the polypeptides. In some embodiments, the polypeptides do not interact with each absence of the agent. As those skilled in the art appreciates, polypeptides may interaction each other through residues, domains, surfaces, etc. In some embodiments, an agent binds to polypeptides simultaneously. In some embodiments, an agent binds to polypeptides cooperatively. In some embodiments, an agent interacts with one polypeptide through a set of residues and interacts with another polypeptide through another set of residues. In some embodiments, an agent binds to a polypeptide at a reduced level or weaker, or does not bind, compared to in the presence of another polypeptide.
[1161] In some embodiments, provided technologies modulate interactions, including inducing new interactions, between various target polypeptides with polypeptides associated with polypeptide degradation, e.g., E3 ligase. In some embodiments, a polypeptide is or comprises an E3 ligase. In some embodiments, an E3 ligase is or comprises an RBR E3 ligase. In some embodiments, an E3 ligase is or comprises a HECT E3 ligase. In some embodiments, an E3 ligase is or comprises an E3 ligase of a Cullin-RING (CRL) multi-subunit E3 family. In some embodiments, an E3 ligase is or comprises an E3 ligase of a RING/U-Box family. In some embodiments, an E3 ligase is or comprises an RBR E3 ligase. In some embodiments, an E3 ligase is or comprises RNF31. In some embodiments, an E3 ligase is or comprises WWP1. In some embodiments, an E3 ligase is or comprises WWP2. In some embodiments, an E3 ligase is or comprises WWP1.sup.WW-HECT. In some embodiments, an E3 ligase is or comprises WWP1.sup.HECT. In some embodiments, an E3 ligase is or comprises WWP2.sup.HECT. In some embodiments, an E3 ligase is or comprises STUB1. In some embodiments, an E3 ligase is or comprises CUL1. In some embodiments, an E3 ligase is or comprises CUL2. In some embodiments, an E3 ligase is or comprises CUL5. In some embodiments, an E3 ligase is or comprises CRBN. In some embodiments, an E3 ligase is or comprises BIRC2. In some embodiments, an E3 ligase is or comprises FBXW7. In some embodiments, an E3 ligase is or comprises VHL. In some embodiments, an E3 ligase is or comprises XIAP. In some embodiments, an E3 ligase is or comprises MDM2. In some embodiments, an E3 ligase is or comprises a NEDD4-like E3 ligase.
[1162] Various target of interests may be targeted by provided technologies. In some embodiments, a target of interest is or comprises a polypeptide. In some embodiments, a target polypeptide is or comprises an enzyme. In some embodiments, a target peptide is or comprises transcription factor. In some embodiments, a target peptide is or comprises transcription coactivator. In some embodiments, a target polypeptide is PPIA. In some embodiments, a target polypeptide is TEAD4. In some embodiments, a target polypeptide is beta-catenin.
[1163] In some embodiments, the present disclosure provides technologies, e.g., agents and uses thereof, that can modulate polypeptide interactions. In some embodiments, provided technologies can enhance or induce new polypeptide interactions between a target of interest and another polypeptide. In some embodiments, a target of interest is or comprises a polypeptide. In some embodiments, an another polypeptide is or comprises an E3 ligase. In some embodiments, provided technologies can enhance or induce new interactions of a polypeptide with a target of interest, wherein the polypeptide is or comprises an E3 ligase. In some embodiments, the present disclosure provides complexes comprising an agent as described herein, a polypeptide that is or comprises an E3 ligase, and a target polypeptide. Among other things, provided technologies are useful for degradation of polypeptides. Certain technologies are presented below as examples.
CHIP Trimerizers
[1164] In some embodiments, an E3 ligase is or comprises CHIP. In some embodiments, an E3 ligase is or comprises CHIP.sup.23-303. In some embodiments, the present disclosure provides agents that can form complexes with CHIP and a target polypeptide. In some embodiments, the present disclosure provides agents that can form complexes with CHIP.sup.23-3.sup.03 and a target polypeptide.
Cluster C86
[1165] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C86 as described herein. In some embodiments, an agent comprises X.sup.86 X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.86, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1166] In some embodiments, the present disclosure provides an agent comprising:
X.sup.86X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1167] each of X.sup.86, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1168] X.sup.5 comprises a side chain comprising a polar group; [1169] X.sup.6 comprises a hydrophobic side chain; [1170] X.sup.9 comprises a side chain comprising a polar group; [1171] X.sup.10 comprises a hydrophobic side chain; [1172] X.sup.12 comprises a hydrophobic side chain; [1173] X.sup.13 comprises a hydrophobic side chain; and [1174] X.sup.14 comprises a side chain comprising an acidic group.
[1175] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00408##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1176] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising OH. In some embodiments, X.sup.5 is S. In some embodiments, X.sup.5 is T.
[1177] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 is aliphatic. In some embodiments, the side chain of X.sup.6 is C.sub.1-6 alkyl. In some embodiments, X.sup.6 is A. In some embodiments, X.sup.6 is V. In some embodiments, X.sup.6 is I.
[1178] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1179] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1180] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, X.sup.9 comprises a side chain comprising OH. In some embodiments, X.sup.9 is S. In some embodiments, X.sup.9 is T.
[1181] Various amino acid residues may be utilized for X.sup.10. In some embodiments, X.sup.10 is I.
[1182] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is M. In some embodiments, X.sup.12 is W.
[1183] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is I.
[1184] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is D.
[1185] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.15 is G.
[1186] Various amino acid residues may be utilized for X.sup.17. For example, in some embodiments, X.sup.17 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.17. In some embodiments, X.sup.17 comprises a side chain comprising OH. In some embodiments, X.sup.17 is S. In some embodiments, X.sup.17 is T. In some embodiments, the side chain of X.sup.17 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.17. In some embodiments, the side chain of X.sup.17 is aliphatic. In some embodiments, the side chain of X.sup.17 is C.sub.1-6 alkyl. In some embodiments, X.sup.17 is A. In some embodiments, X.sup.17 is V. In some embodiments, X.sup.17 is I. In some embodiments, X.sup.17 is L.
[1187] In some embodiments, an agent comprising a cluster C86 sequence is capable of binding to CHIP. In some embodiments, an agent comprising a cluster C86 sequence is capable of promoting an interaction between CHIP and PPIA. In some embodiments, an agent comprising a cluster C86 sequence is capable of binding to PPIA in the presence of CHIP.
[1188] In some embodiments, the present disclosure provides a complex comprising PPIA, CHIP and an agent of cluster C86. In some embodiments, the present disclosure provides methods for reducing levels of PPIA in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C86. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with PPIA, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C86.
Cluster C87
[1189] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C87 as described herein. In some embodiments, an agent comprises X.sup.87X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.87, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1190] In some embodiments, the present disclosure provides an agent comprising:
X.sup.87X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.3X.sup.4X.sup.15X.sup.16X.sup.17,
wherein: [1191] each of X.sup.87, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1192] X.sup.0 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1193] X.sup.1 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1194] X.sup.9 comprises a hydrophobic side chain.
[1195] Various amino acid residues may be utilized for X.sup.87. For example, in some embodiments, X.sup.87 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.87. In some embodiments, X.sup.87 comprises a side chain comprising OH. In some embodiments, X.sup.87 is T. In some embodiments, the side chain of X.sup.87 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.87. In some embodiments, the side chain of X.sup.87 is aliphatic. In some embodiments, the side chain of X.sup.87 is C.sub.1-5 alkyl. In some embodiments, X.sup.87 is V. In some embodiments, X.sup.87 is I.
[1196] Various amino acid residues may be utilized for X.sup.1. For example, in some embodiments, X-1 is P.
[1197] Various amino acid residues may be utilized for X.sup.0. In some embodiments, X.sup.0 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.0 is F. In some embodiments, X.sup.0 is Y.
[1198] Various amino acid residues may be utilized for X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising an aromatic group. For example, in some embodiments, X.sup.1 is F. In some embodiments, X.sup.1 is Y.
[1199] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, the side chain of X.sup.2 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is W.
[1200] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, X.sup.3 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D. In some embodiments, X.sup.3 is E. In some embodiments, X.sup.3 is P.
[1201] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00409##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1202] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.5 is Q.
[1203] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, the side chain of X.sup.6 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.6 is H. In some embodiments, X.sup.6 is F. In some embodiments, X.sup.6 is Y.
[1204] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1205] Various amino acid residues may be utilized for X.sup.8. In some embodiments, the side chain of X.sup.8 is aliphatic. In some embodiments, the side chain of X.sup.8 is C.sub.1-6 alkyl. In some embodiments, X.sup.8 is A.
[1206] Various amino acid residues may be utilized for X.sup.9. For example, in some embodiments, the side chain of X.sup.9 is aliphatic. In some embodiments, the side chain of X.sup.9 is C.sub.1-6 alkyl. In some embodiments, X.sup.9 is A. In some embodiments, X.sup.9 is I.
[1207] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is T. In some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M.
[1208] In some embodiments, an agent comprising a cluster C87 sequence is capable of binding to CHIP. In some embodiments, an agent comprising a cluster C87 sequence is capable of promoting an interaction between CHIP and TEAD4. In some embodiments, an agent comprising a cluster C87 sequence is capable of binding to TEAD4 in the presence of CHIP.
[1209] In some embodiments, the present disclosure provides a complex comprising TEAD4, CHIP and an agent of C87. In some embodiments, the present disclosure provides methods for reducing levels of TEAD4 in a system, comprising administering or delivering to the system an effective amount of an agent of C87. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with TEAD4, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of C87.
Cluster C88
[1210] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C88 as described herein. In some embodiments, an agent comprises X.sup.88X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.88, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1211] In some embodiments, the present disclosure provides an agent comprising: X.sup.88X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1212] each of X.sup.88, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1213] X.sup.2 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1214] X.sup.3 comprises a side chain comprising a polar group; [1215] X.sup.5 comprises a hydrophobic side chain; [1216] X.sup.6 comprises a side chain comprising an acidic group; [1217] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1218] X.sup.10 comprises a hydrophobic side chain; [1219] X.sup.12 comprises a side chain comprising an acidic group; [1220] X.sup.13 comprises a side chain comprising a polar group; [1221] X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [1222] X.sup.15 comprises a hydrophobic side chain.
[1223] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.2 is W.
[1224] Various amino acid residues may be utilized for X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising a polar group. In some embodiments, X.sup.3 comprises a side chain comprising OH. In some embodiments, X.sup.3 is S.
[1225] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00410##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1226] Various amino acid residues may be utilized for X.sup.5. In some embodiments, the side chain of X.sup.5 is aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is A. In some embodiments, X.sup.5 is V.
[1227] Various amino acid residues may be utilized for X.sup.6. In some embodiments, X.sup.6 is E.
[1228] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1229] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1230] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is Y. In some embodiments, X.sup.9 is F.
[1231] Various amino acid residues may be utilized for X.sup.10. In some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is I. In some embodiments, X.sup.10 is L.
[1232] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is E.
[1233] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.13 is N. In some embodiments, X.sup.13 is Q.
[1234] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is H. In some embodiments, X.sup.14 is Y.
[1235] Various amino acid residues may be utilized for X.sup.15. In some embodiments, the side chain of X.sup.15 is aliphatic. In some embodiments, the side chain of X.sup.15 is C.sub.1-6 alkyl. In some embodiments, X.sup.15 is V.
[1236] Various amino acid residues may be utilized for X.sup.17. For example, in some embodiments, X.sup.17 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.17. In some embodiments, X.sup.17 comprises a side chain comprising OH. In some embodiments, X.sup.17 is S. In some embodiments, X.sup.17 is T. In some embodiments, the side chain of X.sup.17 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.17. In some embodiments, the side chain of X.sup.17 is aliphatic. In some embodiments, the side chain of X.sup.17 is C.sub.1-6 alkyl. In some embodiments, X.sup.17 is A. In some embodiments, X.sup.17 is V. In some embodiments, X.sup.17 is I.
[1237] In some embodiments, an agent comprising a cluster C88 sequence is capable of binding to CHIP. In some embodiments, an agent comprising a cluster C88 sequence is capable of promoting an interaction between CHIP and PPIA. In some embodiments, an agent comprising a cluster C88 sequence is capable of binding to PPIA in the presence of CHIP.
[1238] In some embodiments, the present disclosure provides a complex comprising PPIA, CHIP and an agent of cluster C88. In some embodiments, the present disclosure provides methods for reducing levels of PPIA in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C88. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with PPIA, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C88.
Cluster C89
[1239] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C89 as described herein. In some embodiments, an agent comprises X.sup.89X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.89, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1240] In some embodiments, the present disclosure provides an agent comprising: X.sup.89X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1241] each of X.sup.89, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1242] X.sup.9 comprises a side chain comprising a polar group; [1243] X.sup.10 comprises a hydrophobic side chain; [1244] X.sup.12 comprises a hydrophobic side chain; [1245] X.sup.13 comprises a hydrophobic side chain; and [1246] X.sup.14 comprises a side chain comprising an acidic group.
[1247] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00411##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1248] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 is G.
[1249] Various amino acid residues may be utilized for X.sup.6. In some embodiments, X.sup.6 is P. In some embodiments, a cluster is enriched for P at X.sup.6.
[1250] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1251] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1252] Various amino acid residues may be utilized for X.sup.9. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise OH at X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising OH. In some embodiments, X.sup.9 is S. In some embodiments, X.sup.9 is T.
[1253] Various amino acid residues may be utilized for X.sup.10. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is I.
[1254] Various amino acid residues may be utilized for X.sup.12. For example, in some embodiments, X.sup.12 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.12 is W.
[1255] Various amino acid residues may be utilized for X.sup.13. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is V. In some embodiments, X.sup.13 is I.
[1256] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising COOH. In some embodiments, X.sup.14 is D.
[1257] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.15 is G.
[1258] Various amino acid residues may be utilized for X.sup.16. For example, in some embodiments, the side chain of X.sup.16 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.16. In some embodiments, X.sup.16 is M. In some embodiments, the side chain of X.sup.16 is aliphatic. In some embodiments, the side chain of X.sup.16 is C.sub.1-6 alkyl. In some embodiments, X.sup.16 is V. In some embodiments, X.sup.16 is I. In some embodiments, X.sup.16 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.16 is F. In some embodiments, X.sup.16 is Y.
[1259] Various amino acid residues may be utilized for X.sup.17. For example, in some embodiments, the side chain of X.sup.17 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.17. In some embodiments, the side chain of X.sup.17 is aliphatic. In some embodiments, the side chain of X.sup.17 is C.sub.1-6 alkyl. In some embodiments, X.sup.17 is V. In some embodiments, X.sup.17 is I.
[1260] In some embodiments, an agent comprising a cluster C89 sequence is capable of binding to CHIP. In some embodiments, an agent comprising a cluster C89 sequence is capable of promoting an interaction between CHIP and PPIA. In some embodiments, an agent comprising a cluster C89 sequence is capable of binding to PPIA in the presence of CHIP.
[1261] In some embodiments, the present disclosure provides a complex comprising PPIA, CHIP and an agent of cluster C89. In some embodiments, the present disclosure provides methods for reducing levels of PPIA in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C89. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with PPIA, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C89.
Cluster C90
[1262] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C90 as described herein. In some embodiments, an agent comprises X.sup.90X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.0X.sup.1X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.90, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1263] In some embodiments, the present disclosure provides an agent comprising:
X.sup.90X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1264] each of X.sup.90, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1265] X.sup.1 comprises a hydrophobic side chain; [1266] X.sup.2 comprises a side chain comprising an acidic group; [1267] X.sup.3 comprises a hydrophobic side chain; [1268] X.sup.6 comprises a side chain comprising an aromatic group; [1269] X.sup.9 comprises a side chain comprising an acidic group; [1270] X.sup.10 comprises a hydrophobic side chain; [1271] X.sup.12 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1272] X.sup.13 comprises a hydrophobic side chain; and [1273] X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[1274] Various amino acid residues may be utilized for X.sup.1. For example, in some embodiments, X.sup.1 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising COOH. In some embodiments, X.sup.1 is D. In some embodiments, X.sup.1 is E.
[1275] Various amino acid residues may be utilized for X.sup.0. For example, in some embodiments, X.sup.0 is P. In some embodiments, X.sup.0 is G. In some embodiments, X.sup.0 is E.
[1276] Various amino acid residues may be utilized for X.sup.1. In some embodiments, X.sup.1 is M. In some embodiments, X.sup.1 is F. In some embodiments, the side chain of X.sup.1 is aliphatic. In some embodiments, the side chain of X.sup.1 is C.sub.1-6 alkyl. In some embodiments, X.sup.1 is V. In some embodiments, X.sup.1 is I.
[1277] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 is D.
[1278] Various amino acid residues may be utilized for X.sup.3. In some embodiments, X.sup.3 is M. In some embodiments, the side chain of X.sup.3 is aliphatic. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 alkyl. In some embodiments, X.sup.3 is V. In some embodiments, X.sup.3 is I. In some embodiments, X.sup.3 is L.
[1279] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00412##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1280] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, X.sup.5 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising COOH. In some embodiments, X.sup.5 is D. In some embodiments, X.sup.5 is E.
[1281] Various amino acid residues may be utilized for X.sup.6. In some embodiments, X.sup.6 is H. In some embodiments, X.sup.6 is F. In some embodiments, X.sup.6 is Y.
[1282] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1283] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1284] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising COOH. In some embodiments, X.sup.9 is D. In some embodiments, X.sup.9 is E.
[1285] Various amino acid residues may be utilized for X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.1 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is I.
[1286] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is H. In some embodiments, X.sup.12 is W.
[1287] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.13 is W. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is L. In some embodiments, X.sup.13 is I.
[1288] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is L. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is W.
[1289] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.15 comprises a hydrophobic side chain. In some embodiments, X.sup.15 is M. In some embodiments, the side chain of X.sup.15 is aliphatic. In some embodiments, the side chain of X.sup.15 is C.sub.1-6 alkyl. In some embodiments, X.sup.15 is A. In some embodiments, X.sup.15 is V. In some embodiments, X.sup.15 is I. In some embodiments, X.sup.15 is L.
[1290] Various amino acid residues may be utilized for X.sup.16. For example, in some embodiments, X.sup.16 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.16. In some embodiments, X.sup.16 comprises a side chain comprising COOH. In some embodiments, X.sup.16 is D. In some embodiments, X.sup.16 is E.
[1291] In some embodiments, an agent comprising a cluster C90 sequence is capable of binding to CHIP. In some embodiments, an agent comprising a cluster C90 sequence is capable of promoting an interaction between CHIP and TEAD4. In some embodiments, an agent comprising a cluster C90 sequence is capable of binding to TEAD4 in the presence of CHIP.
[1292] In some embodiments, the present disclosure provides a complex comprising TEAD4, CHIP and an agent of C90. In some embodiments, the present disclosure provides methods for reducing levels of TEAD4 in a system, comprising administering or delivering to the system an effective amount of an agent of C90. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with TEAD4, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of C90.
Cluster C94
[1293] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C94 as described herein. In some embodiments, an agent comprises X.sup.94X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.94, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1294] In some embodiments, the present disclosure provides an agent comprising:
X.sup.94X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1295] each of X.sup.94, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1296] X.sup.9 comprises a side chain comprising an acid group; [1297] X.sup.10 comprises a hydrophobic side chain; [1298] X.sup.12 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1299] X.sup.13 comprises a hydrophobic side chain; [1300] X.sup.14 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1301] X.sup.15 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1302] X.sup.16 comprises a hydrophobic side chain; and [1303] X.sup.17 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[1304] Various amino acid residues may be utilized for X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising OH. In some embodiments, X.sup.2 is S. In some embodiments, X.sup.2 is T. In some embodiments, X.sup.2 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.2 is N. In some embodiments, X.sup.2 is Q.
[1305] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00413##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1306] Various amino acid residues may be utilized for X.sup.5. For example, in some embodiments, the side chain of X.sup.5 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.5. In some embodiments, the side chain of X.sup.5 is aliphatic. In some embodiments, the side chain of X.sup.5 is C.sub.1-6 alkyl. In some embodiments, X.sup.5 is A. In some embodiments, X.sup.5 is V. In some embodiments, X.sup.5 is I. In some embodiments, X.sup.5 is L. In some embodiments, X.sup.5 is P.
[1307] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.6. In some embodiments, X.sup.6 is H. In some embodiments, X.sup.6 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising COOH. In some embodiments, X.sup.6 is D. In some embodiments, X.sup.6 is E. In some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is N. In some embodiments, X.sup.6 is Q.
[1308] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1309] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1310] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising COOH. In some embodiments, X.sup.9 is D. In some embodiments, X.sup.9 is E.
[1311] Various amino acid residues may be utilized for X.sup.10. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is V. In some embodiments, X.sup.10 is I.
[1312] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 is W.
[1313] Various amino acid residues may be utilized for X.sup.13. In some embodiments, the side chain of X.sup.13 is aliphatic. In some embodiments, the side chain of X.sup.13 is C.sub.1-6 alkyl. In some embodiments, X.sup.13 is I. In some embodiments, X.sup.13 is L.
[1314] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is Y.
[1315] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.15 is W.
[1316] Various amino acid residues may be utilized for X.sup.16. In some embodiments, the side chain of X.sup.16 is aliphatic. In some embodiments, the side chain of X.sup.16 is C.sub.1-6 alkyl. In some embodiments, X.sup.16 is V. In some embodiments, X.sup.16 is L.
[1317] Various amino acid residues may be utilized for X.sup.17. In some embodiments, X.sup.17 is H. In some embodiments, X.sup.17 is Y.
[1318] In some embodiments, an agent comprising a cluster C94 sequence is capable of binding to CHIP. In some embodiments, an agent comprising a cluster C94 sequence is capable of promoting an interaction between CHIP and PPIA. In some embodiments, an agent comprising a cluster C94 sequence is capable of binding to PPIA in the presence of CHIP.
[1319] In some embodiments, the present disclosure provides a complex comprising PPIA, CHIP and an agent of cluster C94. In some embodiments, the present disclosure provides methods for reducing levels of PPIA in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C94. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with PPIA, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C94.
MDM2 Trimerizers
[1320] In some embodiments, an E3 ligase is or comprises MDM2. In some embodiments, an E3 ligase is or comprises MDM2.sup.25-109. In some embodiments, the present disclosure provides agents that can form complexes with MDM2 and a target polypeptide. In some embodiments, the present disclosure provides agents that can form complexes with MDM2.sup.25-109 and a target polypeptide.
Cluster C91
[1321] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C91 as described herein. In some embodiments, an agent comprises X.sup.91X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.91, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1322] In some embodiments, the present disclosure provides an agent comprising:
X.sup.91X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1323] each of X.sup.91, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1324] X.sup.3 comprises a hydrophobic side chain; [1325] X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1326] X.sup.6 comprises a side chain comprising a polar group; [1327] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1328] X.sup.12 comprises a hydrophobic side chain; [1329] X.sup.14 comprises a side chain comprising an acidic group; [1330] X.sup.15 comprises a side chain comprising an acidic group; and [1331] X.sup.16 comprises a hydrophobic side chain or a side chain comprising an aromatic group.
[1332] Various amino acid residues may be utilized for X.sup.91. In some embodiments, the side chain of X.sup.91 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.91. In some embodiments, X.sup.91 is M. In some embodiments, the side chain of X.sup.91 is aliphatic. In some embodiments, the side chain of X.sup.91 is C.sub.1-6 alkyl. In some embodiments, X.sup.91 is V. In some embodiments, X.sup.91 is I. In some embodiments, X.sup.91 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.91 is F. In some embodiments, X.sup.91 is Y. In some embodiments, X.sup.91 is W.
[1333] Various amino acid residues may be utilized for X.sup.1. For example, in some embodiments, X.sup.1 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising COOH. In some embodiments, X.sup.1 is E. In some embodiments, X.sup.1 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.1 is Q.
[1334] Various amino acid residues may be utilized for X.sup.3. In some embodiments, the side chain of X.sup.3 is aliphatic. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 alkyl. In some embodiments, X.sup.3 is V. In some embodiments, X.sup.3 is I.
[1335] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00414##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1336] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is F. In some embodiments, X.sup.5 is Y.
[1337] Various amino acid residues may be utilized for X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is Q.
[1338] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1339] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1340] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W.
[1341] Various amino acid residues may be utilized for X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is T. In some embodiments, X.sup.10 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.10 is Q. In some embodiments, the side chain of X.sup.10 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.10. In some embodiments, X.sup.10 is M. In some embodiments, the side chain of X.sup.10 is aliphatic. In some embodiments, the side chain of X.sup.10 is C.sub.1-6 alkyl. In some embodiments, X.sup.10 is L. In some embodiments, X.sup.10 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.10 is Y.
[1342] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.12 is F. In some embodiments, the side chain of X.sup.12 is aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, X.sup.12 is V. In some embodiments, X.sup.12 is L.
[1343] Various amino acid residues may be utilized for X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is S. In some embodiments, X.sup.13 is T.
[1344] Various amino acid residues may be utilized for X.sup.14. In some embodiments, X.sup.14 is L. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is W.
[1345] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.14 comprises a side chain comprising COOH. In some embodiments, X.sup.14 is D. In some embodiments, X.sup.14 is E.
[1346] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.15 comprises a side chain comprising COOH. In some embodiments, X.sup.15 is D. In some embodiments, X.sup.15 is E.
[1347] Various amino acid residues may be utilized for X.sup.16. In some embodiments, X.sup.16 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.16 is Y. In some embodiments, X.sup.16 is W.
[1348] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of MDM2 or amino acid residues corresponding thereto: Thr26, Met50, Leu54, Leu57, Gly58, Ile61, Met62, Tyr67, Gln72, His73, Val75, Val93, Lys94, His96, Ile99, Tyr100, and Tyr104. In some embodiments, it interacts with Thr26 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met50 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu54 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu57 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly58 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile61 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met62 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr67 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln72 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His73 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val75 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val93 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys94 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His96 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile99 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr100 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr104 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) or all of the following amino acid residues of beta-catenin or amino acid residues corresponding thereto: Tyr432, Arg474, His475, Arg515, Leu519, His578, Arg582, Arg612, Cys619, Glu620, Gln623, Gly650, Thr653, Tyr654, Ala656, Ala657, Phe660, and Arg661. In some embodiments, it interacts with Tyr432 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg474 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with His475 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg515 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu519 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with His578 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg582 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg612 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu620 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Cys619 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln623 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly650 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr653 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr654 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala656 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala657 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe660 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe661 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, in a complex comprising an agent, beta-catenin and MDM2, one or more of Asn430, Tyr432, Lys433, Arg474, His475, Gln476, Glu479 and Arg582 of beta-catenin or amino acid residues corresponding thereto interact with one or more of Glu25, Thr26, His96, Arg97, Tyr104, Val109 and Val110 of MDM2 or amino acid residues corresponding thereto.
[1349] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of MDM2 or amino acid residues corresponding thereto: Thr26, Met50, Leu54, Leu57, Gly58, Ile61, Met62, Tyr67, Gln72, His73, Val75, Val93, Lys94, His96, Ile99, Tyr100, and Tyr104. In some embodiments, it interacts with Thr26 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met50 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu54 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu57 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly58 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile61 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met62 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr67 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln72 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His73 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val75 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val93 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys94 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His96 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile99 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr100 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr104 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or all of the following amino acid residues of beta-catenin or amino acid residues corresponding thereto: Arg474, His475, Arg515, Leu519, His578, Arg582, Arg612, Glu620, Gln623, Gly650, Thr653, Tyr654, Ala656, Ala657, and Phe660. In some embodiments, it interacts with Arg474 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with His475 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg515 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu519 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with His578 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg582 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Arg612 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu620 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln623 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly650 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr653 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr654 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala656 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala657 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe660 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, in a complex comprising an agent, beta-catenin and MDM2, one or more of Tyr432, Arg474, His475, Gln476, Als478, Glu479, Arg582 and Glu649 of beta-catenin or amino acid residues corresponding thereto interact with one or more of Glu25, Lys51, His96, Arg97, Tyr104, and Val109 of MDM2 or amino acid residues corresponding thereto.
[1350] In some embodiments, an agent comprising a cluster C91 sequence is capable of binding to MDM2. In some embodiments, an agent comprising a cluster C91 sequence is capable of promoting an interaction between MDM2 and beta-catenin. In some embodiments, an agent comprising a cluster C91 sequence is capable of binding to beta-catenin in the presence of MDM2.
[1351] In some embodiments, the present disclosure provides a complex comprising beta-catenin, MDM2 and an agent of cluster C91. In some embodiments, the present disclosure provides methods for reducing levels of beta-catenin in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C91. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with beta-catenin, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C91.
Cluster C92
[1352] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C92 as described herein. In some embodiments, an agent comprises X.sup.92X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.92, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.5, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1353] In some embodiments, the present disclosure provides an agent comprising:
X.sup.92X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1354] each of X.sup.92, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1355] X.sup.3 comprises a side chain comprising an acidic group; [1356] X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1357] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1358] X.sup.12 comprises a hydrophobic side chain; and [1359] X.sup.15 comprises a hydrophobic side chain.
[1360] Various amino acid residues may be utilized for X.sup.92. For example, in some embodiments, X.sup.92 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.92. In some embodiments, X.sup.92 comprises a side chain comprising COOH. In some embodiments, X.sup.92 is D. In some embodiments, X.sup.92 is E.
[1361] Various amino acid residues may be utilized for X-1. For example, in some embodiments, X-1 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising OH. In some embodiments, X.sup.1 is S. In some embodiments, X.sup.1 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.1 is Q.
[1362] Various amino acid residues may be utilized for X.sup.0. For example, in some embodiments, the side chain of X.sup.0 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.0. In some embodiments, the side chain of X.sup.0 is aliphatic. In some embodiments, the side chain of X.sup.0 is C.sub.1-6 alkyl. In some embodiments, X.sup.0 is A. In some embodiments, X.sup.0 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.0 is H. In some embodiments, X.sup.0 is F. In some embodiments, X.sup.0 is Y. In some embodiments, X.sup.0 is W.
[1363] Various amino acid residues may be utilized for X.sup.1. For example, in some embodiments, X.sup.1 comprises a side chain comprising a basic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a basic group at X.sup.1. In some embodiments, X.sup.1 is R. In some embodiments, X.sup.1 is H. In some embodiments, X.sup.1 is K.
[1364] Various amino acid residues may be utilized for X.sup.3. In some embodiments, X.sup.3 comprises a side chain comprising COOH. In some embodiments, X.sup.3 is D.
[1365] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00415##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1366] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is F. In some embodiments, X.sup.5 is Y.
[1367] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1368] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1369] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W. In some embodiments, X.sup.9 is F.
[1370] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising COOH. In some embodiments, X.sup.10 is D. In some embodiments, X.sup.10 is E. In some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising OH. In some embodiments, X.sup.10 is S. In some embodiments, X.sup.10 is T. In some embodiments, X.sup.10 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.10 is N. In some embodiments, X.sup.10 is Q.
[1371] Various amino acid residues may be utilized for X.sup.12. In some embodiments, the side chain of X.sup.12 is aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, X.sup.12 is I. In some embodiments, X.sup.12 is L.
[1372] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, the side chain of X.sup.14 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.14 is F. In some embodiments, X.sup.14 is Y. In some embodiments, X.sup.14 is W.
[1373] Various amino acid residues may be utilized for X.sup.15. In some embodiments, X.sup.15 is M. In some embodiments, the side chain of X.sup.15 is aliphatic. In some embodiments, the side chain of X.sup.15 is C.sub.1-6 alkyl. In some embodiments, X.sup.15 is I. In some embodiments, X.sup.15 is L.
[1374] Various amino acid residues may be utilized for X.sup.16. In some embodiments, X.sup.16 comprises a hydrophobic side chain or a side chain comprising an aromatic group. In some embodiments, X.sup.16 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.16 is H. In some embodiments, X.sup.16 is Y. In some embodiments, X.sup.16 is W.
[1375] Various amino acid residues may be utilized for X.sup.17. For example, in some embodiments, X.sup.17 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.17. In some embodiments, X.sup.17 comprises a side chain comprising COOH. In some embodiments, X.sup.17 is D. In some embodiments, X.sup.17 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.17. In some embodiments, X.sup.17 comprises a side chain comprising OH. In some embodiments, X.sup.17 is T. In some embodiments, X.sup.17 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.17 is N. In some embodiments, X.sup.17 is Q.
[1376] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) or all of the following amino acid residues of MDM2 or amino acid residues corresponding thereto: Met50, Lys51, Leu54, Phe55, Leu57, Gly58, Ile61, Met62, Tyr67, Gln72, Val75, Val93, Lys94, His96, Ile99, Tyr100, and Tyr104. In some embodiments, it interacts with Met50 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys51 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu54 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe55 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu57 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly58 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile61 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Met62 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr67 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln72 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val75 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val93 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys94 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His96 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile99 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr100 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr104 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, or 6) or all of the following amino acid residues of beta-catenin or amino acid residues corresponding thereto: Glu620, Thr653, Tyr654, Ala656, Ala657, and Phe660. In some embodiments, it interacts with Glu620 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr653 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr654 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala656 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala657 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe660 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, in a complex comprising an agent, beta-catenin and MDM2, one or more of Arg582, Val584, Arg587, Cys619, Gln623, Ala656, Ala657, Phe660, Glu664 and Asp665 of beta-catenin or amino acid residues corresponding thereto interact with one or more of Gln71, His73, Val93, Lys94, Glu95, His96 and Arg97 of MDM2 or amino acid residues corresponding thereto.
[1377] In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) or all of the following amino acid residues of MDM2 or amino acid residues corresponding thereto: Met50, Lys51, Leu54, Leu57, Gly58, Ile61, Gln72, Val75, Val93, Lys94, His96, and Tyr100. In some embodiments, it interacts with Met50 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys51 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu54 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Leu57 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gly58 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ile61 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Gln72 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val75 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Val93 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Lys94 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with His96 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr100 of MDM2 or an amino acid residue corresponding thereto. In some embodiments, an agent described herein, e.g., a stapled peptide, interacts with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) or all of the following amino acid residues of beta-catenin or amino acid residues corresponding thereto: Arg582, Glu620, Thr653, Tyr654, Ala656, Ala657, and Phe660. In some embodiments, it interacts with Arg582 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Glu620 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Thr653 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Tyr654 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala656 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Ala657 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, it interacts with Phe660 of beta-catenin or an amino acid residue corresponding thereto. In some embodiments, in a complex comprising an agent, beta-catenin and MDM2, one or more of Arg582, Val584, Arg587, Cys619, Gln623, Ala656, Ala657, Val658, Phe660 and Arg661 of beta-catenin or amino acid residues corresponding thereto interact with one or more of His73, Val93, Lys94, Glu95, His96 and Arg97 of MDM2 or amino acid residues corresponding thereto.
[1378] In some embodiments, an agent comprising a cluster C92 sequence is capable of binding to MDM2. In some embodiments, an agent comprising a cluster C92 sequence is capable of promoting an interaction between MDM2 and beta-catenin. In some embodiments, an agent comprising a cluster C92 sequence is capable of binding to beta-catenin in the presence of MDM2.
[1379] In some embodiments, the present disclosure provides a complex comprising beta-catenin, MDM2 and an agent of cluster C92. In some embodiments, the present disclosure provides methods for reducing levels of beta-catenin in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C92. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with beta-catenin, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C92.
Cluster C93
[1380] In some embodiments, the present disclosure provides agents comprising one or more (e.g., 1-14, 1-10, 1-5, 5-10, 5-9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) residues of cluster C93 as described herein. In some embodiments, an agent comprises X.sup.93X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17, wherein each of X.sup.93, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue.
[1381] In some embodiments, the present disclosure provides an agent comprising:
X.sup.93X.sup.1X.sup.0X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16X.sup.17,
wherein: [1382] each of X.sup.93, X.sup.1, X.sup.0, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14, X.sup.15, X.sup.16, and X.sup.17 is independently an amino acid residue; [1383] X.sup.5 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1384] X.sup.9 comprises a hydrophobic side chain or a side chain comprising an aromatic group; [1385] X.sup.11 comprises a hydrophobic side chain; [1386] X.sup.16 comprises a hydrophobic side chain or a side chain comprising an aromatic group; and [1387] X.sup.17 comprises an acidic side chain.
[1388] Various amino acid residues may be utilized for X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising OH. In some embodiments, X.sup.1 is T. In some embodiments, the side chain of X.sup.1 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.1. In some embodiments, the side chain of X.sup.1 is aliphatic. In some embodiments, the side chain of X.sup.1 is C.sub.1-6 alkyl. In some embodiments, X.sup.1 is I. In some embodiments, X.sup.1 is L. In some embodiments, X.sup.1 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.1 is W.
[1389] Various amino acid residues may be utilized for X.sup.0. For example, in some embodiments, X.sup.0 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.0. In some embodiments, X.sup.0 comprises a side chain comprising OH. In some embodiments, X.sup.0 is S. In some embodiments, X.sup.0 is T. In some embodiments, X.sup.0 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.0 is N. In some embodiments, X.sup.0 is Q.
[1390] Various amino acid residues may be utilized for X.sup.1. For example, in some embodiments, X.sup.1 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising COOH. In some embodiments, X.sup.1 is D. In some embodiments, X.sup.1 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.1. In some embodiments, X.sup.1 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.1 is Q.
[1391] Various amino acid residues may be utilized for X.sup.2. For example, in some embodiments, X.sup.2 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.2. In some embodiments, X.sup.2 comprises a side chain comprising OH. In some embodiments, X.sup.2 is S. In some embodiments, X.sup.2 is T. In some embodiments, X.sup.2 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.2 is N. In some embodiments, X.sup.2 is Q.
[1392] Various amino acid residues may be utilized for X.sup.3. For example, in some embodiments, the side chain of X.sup.3 is hydrophobic. In some embodiments, a cluster is enriched for amino acid residues comprising a hydrophobic side chain at X.sup.3. In some embodiments, the side chain of X.sup.3 is aliphatic. In some embodiments, the side chain of X.sup.3 is C.sub.1-6 alkyl. In some embodiments, X.sup.3 is A. In some embodiments, X.sup.3 is V. In some embodiments, X.sup.3 is I. In some embodiments, X.sup.3 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.3 is Y. In some embodiments, X.sup.3 is W.
[1393] In some embodiments, X.sup.4 is a residue for stapling. In some embodiments, X.sup.4 is stapled with an amino acid residue. In some embodiments, X.sup.11 is a residue for stapling. In some embodiments, X.sup.11 is stapled with an amino acid residue. In some embodiments, X.sup.4 is stapled with X.sup.11. Various staples can be utilized in accordance with the present disclosure. In some embodiments, a staple is L.sup.s as described herein. In some embodiments, X.sup.4 and X.sup.11 are cysteine residues stapled through SH. In some embodiments, the two SH groups are linked through L.sup.s2 as described herein. In some embodiments, L.sup.s2 is
##STR00416##
In some embodiments, X.sup.4 and X.sup.11 are stapled through a non-cysteine staple.
[1394] Various amino acid residues may be utilized for X.sup.5. In some embodiments, X.sup.5 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.5 is F. In some embodiments, X.sup.5 is W.
[1395] Various amino acid residues may be utilized for X.sup.6. For example, in some embodiments, X.sup.6 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.6. In some embodiments, X.sup.6 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.6 is Q. In some embodiments, X.sup.6 is P.
[1396] Various amino acid residues may be utilized for X.sup.7. In some embodiments, X.sup.7 is A.
[1397] Various amino acid residues may be utilized for X.sup.8. In some embodiments, X.sup.8 is A.
[1398] Various amino acid residues may be utilized for X.sup.9. In some embodiments, X.sup.9 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.9 is W.
[1399] Various amino acid residues may be utilized for X.sup.10. For example, in some embodiments, X.sup.10 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.10. In some embodiments, X.sup.10 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.10 is Q. In some embodiments, X.sup.10 is M.
[1400] Various amino acid residues may be utilized for X.sup.12. In some embodiments, X.sup.12 comprises a hydrophobic side chain. In some embodiments, the side chain of X.sup.12 is aliphatic. In some embodiments, the side chain of X.sup.12 is C.sub.1-6 alkyl. In some embodiments, X.sup.12 is V. In some embodiments, X.sup.12 is I. In some embodiments, X.sup.12 is L.
[1401] Various amino acid residues may be utilized for X.sup.13. For example, in some embodiments, X.sup.13 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.13. In some embodiments, X.sup.13 comprises a side chain comprising OH. In some embodiments, X.sup.13 is S. In some embodiments, X.sup.13 is T. In some embodiments, X.sup.13 comprises a side chain comprising C(O)NH.sub.2. In some embodiments, X.sup.13 is Q.
[1402] Various amino acid residues may be utilized for X.sup.14. For example, in some embodiments, X.sup.14 comprises a side chain comprising an acidic group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise an acidic group at X.sup.14. In some embodiments, X.sup.14 comprises a side chain comprising COOH. In some embodiments, X.sup.14 is D.
[1403] Various amino acid residues may be utilized for X.sup.15. For example, in some embodiments, X.sup.15 comprises a side chain comprising a polar group. In some embodiments, a cluster is enriched for amino acid residues whose side chains comprise a polar group at X.sup.15. In some embodiments, X.sup.15 comprises a side chain comprising OH. In some embodiments, X.sup.15 is T.
[1404] Various amino acid residues may be utilized for X.sup.16. In some embodiments, X.sup.16 comprises a side chain comprising an aromatic group. In some embodiments, X.sup.16 is F. In some embodiments, X.sup.16 is Y. In some embodiments, X.sup.16 is W.
[1405] Various amino acid residues may be utilized for X.sup.17. In some embodiments, X.sup.17 comprises a side chain comprising COOH. In some embodiments, X.sup.17 is D. In some embodiments, X.sup.17 is E.
[1406] In some embodiments, an agent comprising a cluster C93 sequence is capable of binding to MDM2. In some embodiments, an agent comprising a cluster C93 sequence is capable of promoting an interaction between MDM2 and beta-catenin. In some embodiments, an agent comprising a cluster C93 sequence is capable of binding to beta-catenin in the presence of MDM2.
[1407] In some embodiments, the present disclosure provides a complex comprising beta-catenin, MDM2 and an agent of cluster C93. In some embodiments, the present disclosure provides methods for reducing levels of beta-catenin in a system, comprising administering or delivering to the system an effective amount of an agent of cluster C93. In some embodiments, the present disclosure provides a method for preventing or treating a condition, disorder or disease associated with beta-catenin, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of cluster C93.
[1408] Among other things, provided technologies can provide various benefits and advantages, e.g., selective binding. In some embodiments, a trimerizer agent selectively forms a complex with two polypeptides compared to each of the polypeptide alone. In some embodiments, a trimerizer agent binds to an individual polypeptide weakly or do not bind. In some embodiments, a trimerizer agent selectively forms complex with a pair of polypeptide compared to another pair. In some embodiments, the present disclosure provides technologies that bind VHL-ELOBC. In some embodiments, the present disclosure provides technologies that bind VHL-ELOBC selectively over SOCS2-ELOBC. In some embodiments, provided technologies do not compete with HXC78.
Griptides
[1409] In some embodiments, the present disclosure provides technologies for identification, characterization, production, and/or use of griptide agents. In some embodiments, a griptide comprises two paired alpha-helices. In some embodiments, a griptide comprises two stapled peptides portions, each forming an alpha-helix structure. In some embodiments, a griptide provides a larger surface compared to agent comprising a single alpha-helix structure. In some embodiments, a griptide can engage a target with high affinity and/or specificity. In some embodiments, a griptide can engage a challenging target with high affinity and specificity, e.g., compared to an agent comprising a single alpha-helix structure.
[1410] In some embodiments, the present disclosure provides a method comprising: [1411] contacting a collection of stapled peptides with a target of interest so that a number of stapled peptides of the collection binds to the target; and [1412] determining amino acid sequences of stapled peptides that bind to the target.
[1413] In some embodiments, one or more stapled peptides, e.g., about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% of all stapled peptides, independently comprise two portions, wherein the peptide backbones of the two portions do not overlap, and each portion independently is or comprises an alpha-helical structure. In some embodiments, one or more stapled peptides, e.g., about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% of all stapled peptides, independently comprise two portions, wherein the peptide backbones of the two portions do not overlap, and each portion independently comprises two residues stapled or suitable for stapling. In some embodiments, one or more stapled peptides, e.g., about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% of all stapled peptides, independently comprise two portions, wherein the peptide backbones of the two portions do not overlap, and each portion independently comprises two stapled residues. In some embodiments, each portion independently forms or comprises or is part of an alpha-helical structure. In some embodiments, each portion is independently a moiety of a stapled peptide as described herein. In some embodiments, each portion independently comprises an amino acid sequence and a staple as described herein. For example, in some embodiments, each portion is independently about 5-30, about 5-20, about 8-20, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues in length. In some embodiments, each portion is independently about 8-20 amino acid residues in length.
[1414] In some embodiments, each of the two portions independently comprises an amino acid residue suitable for crosslinking. In some embodiments, the two portions are crosslinked. In some embodiments, an amino acid residue suitable for crosslinking is cysteine. In some embodiments, two portions are crosslinked through formation of a disulfide bond between two cysteine residues.
[1415] In some embodiments, the two portions are of two different peptide chains. In some embodiments, the two portions are ports of the same peptide chains. In some embodiments, peptide backbones of the two portions are two portions of the same peptide backbone. In some embodiments, the two portions are tandem. In some embodiments, each of them is independently part of two peptide backbones.
[1416] In some embodiments, the present disclosure provides a method comprising: [1417] contacting a collection of stapled peptides with a target of interest so that a number of stapled peptides of the collection binds to the target; and [1418] determining amino acid sequences of stapled peptides that bind to the target; [1419] wherein one or more stapled peptides independently comprise an amino acid residue suitable for crosslinking with other stapled peptides, or one or more stapled peptides are independently crosslinked with one or more other stapled peptides.
[1420] In some embodiments, an amino acid residue suitable for crosslinking is cysteine. In some embodiments, one or more stapled peptides are independently crosslinked with one or more other stapled peptides. In some embodiments, crosslinking is through cysteine residues. In some embodiments, about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 93%, 95%, 96%, 97%, 98%, or 99% of all stapled peptides in the collection are crosslinked.
[1421] In some embodiments, a peptide is a dimer. In some embodiments, a peptide is a homodimer. In some embodiments, a peptide is a heterodimer.
Treatment of Conditions, Disorders or Diseases
[1422] In some embodiments, certain stapled peptides and/or other agents as described herein are useful for preventing or treating various conditions, disorders or diseases. In some embodiments, a therapeutic composition as described herein, that comprises and/or delivers a stapled peptide (e.g., a non-cysteine stapled peptide, for example corresponding to a parent cysteine stapled peptide identified and/or characterized as described herein) is administered to a subject in need thereof. In some embodiments, a subject may have received and/or be receiving other therapy so that, in some embodiments, therapy with a provided peptide agent may be administered in combination with other therapy; in some such embodiments, a peptide agent as described herein may be administered via a composition that contains and/or delivers another therapeutic agent; in some embodiments, separate compositions are employed.
[1423] Various strategies for administering stapled peptides are available in the art and can be utilized in accordance with the present disclosure. In some embodiments, provided peptides have enhanced cell penetration properties compared otherwise identical but non-stapled peptides.
[1424] In some particular embodiments, certain provided stapled peptides and/or other technologies are useful to bind to and/or modulate one or more activities or effects of, beta-catenin.
[1425] In some embodiments, certain provided stapled peptides and/or other technologies are useful in treatment of a condition, disorder, or disease associated with one or more components involved in Wnt/beta-catenin signaling and/or specifically with one or more beta-catenin functions.
[1426] In some embodiments, certain provided stapled peptides and/or other technologies are useful in modulating, e.g., reducing the interaction between beta-catenin and another protein, for example, Axin, APC, BCL9, TCF4/TCF7L2, TCF3/TCF7L1, and TCF7.
[1427] In some embodiments, a condition, disorder or disease is associated with beta-catenin. In some embodiments, a condition, disorder or disease is associated with RNF31. In some embodiments, a condition, disorder or disease is associated with CDK2. In some embodiments, a condition, disorder or disease is associated with PPIA. In some embodiments, a condition, disorder or disease is associated with PD-L1. In some embodiments, a condition, disorder or disease is associated with an E3 ligase. Various conditions, disorders or diseases associated with such polypeptides have been reported and can be prevented or treated in accordance with the present disclosure.
[1428] In some embodiments, a condition, disorder or disease is selected from cancer, cardiac disease, dilated cardiomyopathy, fetal alcohol syndrome, depression, and diabetes. In some embodiments, a condition, disorder, or disease is a heart condition, disorder, or disease. In some embodiments, a condition, disorder, or disease is cancer. In some embodiments a cancer is selected from: colon cancer, colorectal cancer, rectal cancer, prostate cancer familial adenomatous polyposis (FAP), Wilms Tumor, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, primary hetpatocellular carcinoma, ovarial carcinoma, breast cancer, lung cancer, glioblastoma, pliomatrixoma, medulloblastoma, thyroid tumors, ovarian neoplasms. In some embodiments, a cancer is colorectal cancer. In some embodiments, a cancer is hepatocellular cancer. In some embodiments, a cancer is prostate cancer. In some embodiments, a cancer is melanoma. In some embodiments, a cancer is associated with Wnt/beta-catenin signaling. In some embodiments, a cancer is associated with beta-catenin. As appreciated by those skilled in the art, provided technologies are applicable to various targets, including many protein targets associated with cancer.
EXEMPLIFICATION
[1429] Those skilled in the art appreciate that various technologies are available for designing, identifying, characterizing, manufacturing, assessing, using, etc., provided agents, collections, methods, etc. in accordance with the present disclosure. Described below are certain such useful technologies.
Example 1. Technologies for Preparing, Assessing and Identifying Stapled Peptides that can Bind Various Targets and Collections Thereof
[1430] Recent advances in identifying human disease targets have not been matched by advances in the ability to drug these targets. This actionability gap is largely due to the fact that neither of the two main classes of approved therapeuticsbiologics and small moleculescan simultaneously address target accessibility and selective target engagement. Biologics, despite an impressive ability to engage diverse target proteins, are largely restricted to an extracellular operating theatre, as their size and polarity renders them unable to cross biological membranes. Small molecules, in contrast, can access the intracellular space, but cannot bind with high affinity and specificity to the vast majority of proteins that are found there.
[1431] Among other things, the present disclosure encompasses the disconnect between the ability to identify disease targets and the ability to drug them with high strength and specificity. In some embodiments, the present disclosure provides technologies that can provide new classes of drugsones that can engage intracellular proteins that lack the deep hydrophobic pocket ordinarily required for small-molecule binding.
[1432] In nature, such undruggable proteins are often targeted with macrocyclic molecules, frequently peptidic in structure, whose large size compared to small molecules enables them to bind with high affinity and specificity to protein surfaces. Significant efforts have been made to elucidate the mechanisms of cell entry for these natural products, which typically possess molecular weights of 700-1,200 Da or higher, well beyond the typical range for cell penetration in small-molecule drug discovery. While the mechanisms of cell entry are complex and vary from molecule to molecule, certain research on peptidic macrocycles has been reported to highlight the importance of desolvating amide protons and reducing their exposure to the membrane interior as a key driver in passive, thermal diffusion across the lipid bilayera phenomenon that may be referred to as amide-proton cloaking. In some embodiments, the amide proton, present between residues in a polypeptide chain, is highly electropositive and forms a strong hydrogen-bonding interaction with water. In some embodiments, this poses a substantial hurdle for membrane permeability, since tightly bound solvent water molecules are shed prior to entering the lipid bilayer. In some embodiments, exposed amide groups incur a further energetic penalty upon membrane entry due to unfavorable electrostatic interactions with the low-dielectric environment of the membrane interior. Consequently, many peptides and proteins are unable to cross membranes.
[1433] In some embodiments, the present disclosure provides agents, e.g., stapled peptides, that can cross membranes. In some embodiments, provided agents comprises staples. In some embodiments, staples facilitate formations and/or maintenance, or stabilize alpha-helical structures.
[1434] Among other things, the present disclosure encompasses the recognition that design of stapled peptides can be challenging in view of the inability to systematically define the alpha-helix binding sites on a protein's surface, and to identify stapled peptides that bind to those sites. In some embodiments, this limitation has restricted development of stapled peptides and their uses by many third parties to only those protein targets for which naturally occurring or previously characterized alpha-helical binders were known, with stapled peptides generated from fragments of the known binders. Among other things, the present disclosure provide a rapid, high-throughput platform, in some embodiments, utilizing phage display, that enables an unbiased mapping of interactome of a target of interest, e.g., a protein, with agents such as stapled peptides, without requiring prior knowledge of the structure or known binding partners of a target of interest. Among other things, provided technologies are capable of identifying alpha-helix-binding sites on the surfaces of a range of protein folds, including many for which no alpha-helical binders are reported. In some embodiments, stapled peptides that bind these sites are able to impact diverse functions, including inhibiting interactions (e.g., protein-protein interactions), inhibiting enzymatic activity, inducing dimerization, and inducing conformational changes. In some embodiments, a range of binding modes can be adopted. In some embodiments, a mode is side-on, i.e., mediated exclusively by stapled peptide side-chains rather than involving main chain amide interactions. Among other things, provided technologies significantly expands the universe of targets of interest including proteins that can be bound by stapled peptides, and furthers the pursuit of targeting undruggable targets of interest including proteins.
[1435] Among other things, the present disclosure provides technologies for preparing, assessing, characterizing and identifying stapled peptides that can bind various targets. In some embodiments, provided stapled peptides comprise alpha-helical structures. In some embodiments, stapled peptides may be referred to as Helicons. In some embodiments, the present disclosure provides unbiased screening platform for identifying stapled peptide binders. In some embodiments, the present disclosure provides libraries comprising a number of (e.g., about or at least about 100, 500, 1000, 5000, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, etc.) stapled peptides. Various technologies can be utilized to screen provided libraries. In one example, a phage display technology was utilized for preparing and/or screening libraries comprising stapled peptides. In some embodiments, a phage display technology can provide i) high library diversities, ii) tolerance to chemical manipulation, and/or iii) rapid and inexpensive library generation. In some embodiments, the present disclosure utilizes technologies that are compatible with primary amines and other reactive groups present on phage particles. In some embodiments, a stapling system based on crosslinking cysteine residues with electrophiles, e.g., alkyl bromides, was utilized. In some embodiments, such chemistry can be performed under aqueous conditions and in the presence of other biomacromolecules. Various bifunctional electrophiles are available and may be utilized in accordance with the present disclosure, e.g., those described in WO 2020/041270, the stapling technologies, e.g., electrophiles, of which are incorporated herein by reference. A panel of bifunctional alkyl bromides was utilized for stapling. In one example, N,N-(1,4-phenylene)bis(2-bromoacetamide) was utilized for crosslinking pairs of cysteines at i,i+7 positioning. In some embodiments, incorporating this staple into a diverse panel of peptides, e.g., those are 14 amino acid residues in length, consistently led to an increase in alpha-helical secondary structure, as assessed by circular dichroism.
[1436] In some embodiments, this chemistry was performed on phage-displayed Helicons. M13 bacteriophage was prepared with a model sequence containing a pair i,i+7 cysteine residues fused to a pIII coat protein. In some embodiments, one or more phage-displayed polypeptides with pairs of cysteines were independently present in the oxidized, disulfide-bonded state. In some embodiments, timing and concentration of phage was optimized. In some embodiments, choice of reducing agent was optimized. In some embodiments, choice of crosslinker was optimized. In one example, on-phage crosslinking was achieved by briefly treating the phage with dithiothreitol (DTT), followed by rapid dialysis, incubation with a bifunctional electrophile, e.g., a bifunctional alkyl bromide such as N,N-(1,4-phenylene)bis(2-bromoacetamide), and a final capping and quenching step. Stapling of desired cysteine residues was achieved despite the presence of multiple structural disulfide bonds in the pIII protein. In some embodiments, presence of properly crosslinked Helicons was confirmed by trypsinization of these phage followed by mass spectrometry (MS) analysis. In some embodiments, phage prepared in this way remained viable and compatible with downstream screening and sequencing. In some embodiments, stapled peptides interact with their targets with alpha-helical modes. In some embodiments, stapled peptides interact with their targets with non-alpha-helical modes. In some embodiments, it was observed that in many co-crystal structures solved of stapled peptides discovered using phage libraries prepared with this crosslinker, a majority of stapled peptides bind their targets with mostly or entirely alpha-helical modes.
[1437] Agents, e.g., stapled peptides, can be characterized utilizing various technologies in accordance with the present disclosure. In some embodiments, stapled peptides and unstapled peptides were characterized by HPLC, mass spectrometry, etc. In some embodiments, crosslinked peptides displayed on phage were characterized by mass spectrometry, confirming on-phage cysteine stapling. In some embodiments, sequencing of 12 phage library members indicates that 11 of 12 sequences match the expected library design (with one sequence containing an additional amino acid insertion).
[1438] As demonstrated herein, provided technologies allow for swift, unbiased screening of large Helicon libraries, e.g., using phage display. Among other things, provided technologies are able to identify previously unknown alpha-helix binding sites on a range of targets of interest including protein folds and types, including a transcriptional regulator (beta-catenin), two structurally dissimilar domains from an E3 ubiquitin ligase (RNF31), a kinase (CDK2), a peptidyl-prolyl cis-trans isomerase (PPIA), and an extracellular receptor (PD-L1). X-ray co-crystal structures indicate that many of these sites are located on surfaces not previously known to bind alpha-helices. Among other things, biochemical and structural experiments demonstrate diverse functional impacts by the Helicons which bind these sites, including inhibition of protein-protein interactions, inhibition of enzymatic activity, induced conformational rearrangement, and induced dimerization. Additional screens have been successfully performed with a wide range of small molecule, peptidic, and protein binding partners, and phosphorylated vs. non-phosphorylated, glycosylated vs. non-glycosylated, apo vs. ligand-bound, mouse vs. human, monomeric vs. multimeric proteins, etc. were also assessed. In some embodiments, one or more panning rounds prior to screen, e.g., the multiwell screen, can be introduced to enrich low-abundance library members and provide clearer signals to select binders with, and the use of monovalent phagemid systems can avoid the avidity effects of the multivalent phage system that can complicate the interpretation of apparent on-phage affinities.
[1439] Among other things, it was observed many Helicons were alpha-helical in the region of the molecule involved in engaging the target, with few examples of fraying at the ends of Helicons outside of the stapled region. In some embodiments, significant exposure of amide protons in a Helicon structure, except those at the N-terminus of each Helicon, is not observed. In some embodiments, Helicons engage target surface, e.g., protein surface, by side chains and not by any Helicon main-chain amides, e.g., as shown in crystal structures.
[1440] In some embodiments, the present disclosure provides technologies for establishment of structure-activity relationships to identify which features are critical for binding and activity. In the absence of a co-crystal structure or other direct structural information, establishing structure-activity relationships can requires extensive empirical testing of compound analogs. Due to the relatively large number of Helicons in many of the clusters discovered in the provided technologies, in some embodiments, sufficient information in clusters can be utilized to predict which amino acids are involved, e.g., in target binding. As demonstrated herein, comparison of clusters with co-crystal structures confirmed that in almost all cases, highly conserved amino acids were directly participating in protein binding.
[1441] Certain stapled peptides that were identified to bind their targets are described herein as examples. Certain agents including stapled peptides are presented below as examples (including some controls).
TABLE-US-00019 TABLE1 Certainagentsincludingstapledpeptides. SEQ Expected Observed Target FOGID Sequence IDNO: Mass Mass Stapletype CTNNB1 FP01567 Ac-DPATHRCEWAALHCELV-NH2 46 2180.4 2180.2 Cys- stapled CTNNB1 FP49332 Ac-DPATHRCEWAALHCELV-NH2 47 1992.2 1989.2 unstapled CTNNB1 FP49250 Ac-DPAAHRCEWAALHCELV-NH2 48 2150.4 2149.0 Cys- stapled CTNNB1 FP49251 Ac-DPATARCEWAALHCELV-NH2 49 2114.4 2112.8 Cys- stapled CTNNB1 FP49252 Ac-DPATHACEWAALHCELV-NH2 50 2095.3 2094.0 Cys- stapled CTNNB1 FP49253 Ac-DPATHRCAWAALHCELV-NH2 51 2122.4 2120.8 Cys- stapled CTNNB1 FP49254 Ac-DPATHRCEAAALHCELV-NH2 52 2065.3 2063.8 Cys- stapled CTNNB1 FP49255 Ac-DPATHRCEWAAAHCELV-NH2 53 2138.3 2136.8 Cys- stapled CTNNB1 FP49257 Ac-DPATHRCEWAALHCALV-NH2 54 2122.4 2120.8 Cys- stapled CTNNB1 FP49258 Ac-DPATHRCEWAALHCEAV-NH2 55 2138.3 2136.8 Cys- stapled CTNNB1 FP49259 Ac-DPATHRCEWAALHCELA-NH2 56 2152.4 2150.8 Cys- stapled CTNNB1 FP01822 Ac-DPASELCEWAAIHCDLV-NH2 57 2101.3 2100.2 Cys- stapled CTNNB1 FP49333 Ac-DPASELCEWAAIHCDLV-NH2 58 1913.1 1912.1 unstapled CTNNB1 FP01838 Ac-DPAILECHIAAWNCYEI-NH2 59 2190.5 2189.0 Cys- stapled CTNNB1 FP49194 |Ac-DPATLDCHIAAWDCWDE-NH2 60 2190.3 2188.8 Cys- stapled CTNNB1 FP49193 Ac-DPAILACHLAAMDCSDW-NH2 61 2061.3 2060.4 Cys- stapled CTNNB1 FP49432 Ac-DPAILACHLAAMDCSDW-NH2 62 1873.1 1871.2 unstapled CTNNB1 FP49196 Ac-DPANANCILAAHECRIW-NH2 63 2126.4 2124.8 Cys- stapled CTNNB1 FP49197 Ac-DPAEVECMLAAHVCRAF-NH2 64 2091.4 2089.8 Cys- stapled CTNNB1 FP01848 Ac-DPAQDDCILAAHVCALW-NH2 65 2070.3 2069.1 Cys- stapled CTNNB1 FP49433 Ac-DPAQDDCILAAHVCALW-NH2 66 1882.1 1881.1 unstapled CTNNB1 FP49199 Ac-DPADWECEHAALLCHYW- 67 2288.5 2286.9 Cys- NH2 stapled CTNNB1 FP49200 Ac-DPALWQCEHAALLCDVH-NH2 68 2150.4 2149.2 Cys- stapled CTNNB1 FP49431 Ac-DPALWQCEHAALLCDVH-NH2 69 1962.2 1959.5 CTNNB1 FP05863 Ac-DPAIIHCYEAAFFCQYI-NH2 70 2233.5 2233.4 Cys- stapled CTNNB1 FP05874 Ac-DPAVMECYEAAFICHYV-NH2 71 2190.5 2189.3 Cys- stapled CTNNB1 FP04872 5FAM-bAla- 72 5245.3 5242.2 Cys- DDLGANDELISFKDEGEQEEKSSE stapled NSSAERDLADVKSSLVNESE-NH2 CTNNB1 FP00013 FITC-PEG1-PQ-S5-ILD-S5- 73 2358.8 2357.8 S5-S5 (fStAx- HVRRVWR stapled 33.sup.(10)) RNF31 FP06635 Ac-DPAEWICRMAAMNCLYQ-NH2 74 2244.6 2244.4 Cys- (PUB) stapled RNF31 FP49434 Ac-DPAEWICRMAAMNCLYQ-NH2 75 2056.4 2054.2 unstapled (PUB) RNF31 FP06641 Ac-DPARDWCLYAAYDCYTA- 76 2226.4 2226.3 Cys- (PUB) NH2 stapled RNF31 FP49435 Ac-DPARDWCLYAAYDCYTA- 77 2038.2 2035.9 unstapled (PUB) NH2 RNF31 FP06649 Ac-DPAFTDCQLAAAVCMTY-NH2 78 2049.3 2049.5 Cys- (PUB) stapled RNF31 FP06652 Ac-DPAIVQCAWAALYCDMQ-NH2 79 2127.4 2127.3 Cys- (PUB) stapled RNF31 FP16923 FITC-NHHex- 80 2824.0 2822.0 Cys- (PUB) AEHEEDMYRAADEIEKEKE-NH2 stapled RNF31 FP06655 Ac-DPAMQRCFSAAVYCAIS-NH2 81 2062.3 2062.3 Cys- (UBA) stapled RNF31 FP12122 5FAM-bAla- 82 2449.7 2449.8 Cys- (UBA) DPAMQRCFSAAVYCAIS-NH2 stapled CDK2 FP24322 Ac-FECLDAFFSC-NH2 83 1410.6 1409.8 Cys- stapled CDK2 FP19711 Ac-DPAWWVCAIAAIECSDV-NH2 84 2078.3 2077.4 Cys- stapled CDK2 FP33215 Ac-WWVCAIAAIECSD-NH2 85 1695.9 1695.0 Cys- stapled PPIA FP29103 Ac-PDCHIRAYVCH-NH2 86 1542.7 1542.1 Cys- stapled PPIA FP29092 Ac-DPANQDCHVAAWHCWQR- 87 2266.4 2265.8 Cys- NH2 stapled PPIA FP29102 Ac-PECHIEAYWCI-NH2 88 1592.8 1591.8 Cys- stapled PD-L1 FP30790 Ac-DPAAADCQWAAFLCRVY-NH2 89 2129.4 2128.0 Cys- stapled PD-L1 FP28132 Ac-DPALWQCVFAARSCYEE-NH2 90 2217.4 2217.0 Cys- stapled PD-L1 FP28141 Ac-DPALAQCVFAARSCYEE-NH2 91 2102.3 2102.5 Cys- stapled PD-L1 FP28135 Ac-DPALWMCVFAARQCYES-NH2 92 2219.5 2218.9 Cys- stapled PD-L1 FP28136 Ac-DPALWQCVFAARYCYEE-NH2 93 2293.5 2293.2 Cys- stapled PRKCI FP47831 5FAM-QRFARKGALRQKNV-NH2 94 2028.0 2029.2 unstapled (5FAM- PKC iota/zeta- blocking peptide) NA FP47842 5FAM-dGlu-dGln-dLys-dLeu-dIle- 95 1559.7 1561.1 unstapled (F5AM- dSer-dGlu-dGlu-dAsp-dLeu-NH2 D-Myc peptide) NA FP47843 5FAM-dTyr-dPro-dTyr-dAsp-dVal- 96 1458.5 1459.8 unstapled (5FAM- dPro-dAsp-dTyr-dAla-NH2 D-HA peptide) MDM2 FP47839 Ac-[5FAM]-Lys-Gln-Ser-Gln-Gln- 97 2594.3 2596.4 R8-S5.sup.(11) (5FAM- Thr-Phe-R8-Asn-Leu-Trp-Arg-Leu- stapled SAH-p53-8) Leu-S5-Gln-Asn-NH2 MDM2 FP01413 Ac-Leu-Thr-Phe-R8-Glu-Tyr-Trp- 98 1743.9 1745.0 R8-S5 (ATSP- Ala-Gln-Cba-S5-Ser-Ala-Ala-NH2 stapled 7041) CTNNB1 FP07855 Ac-Val-nLeu-Glu-R8-Tyr-Glu-Ala- 99 1786.0 1785.7 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07856 Ac-Val-nLeu-Glu-R8-Tyr-Glu-Ala- 99 1786.0 1785.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07859 Ac-Ile-Leu-Gln-R8-Tyr-Glu-Ala- 100 1801.9 1801.6 R8-S5 Ala-Phe-Ser-S5-His-Tyr-Gln-NH2 stapled CTNNB1 FP07860 Ac-Ile-Leu-Gln-R8-Tyr-Glu-Ala- 100 1801.9 1801.7 R8-S5 Ala-Phe-Ser-S5-His-Tyr-Gln-NH2 stapled CTNNB1 FP07863 Ac-Val-nLeu-Gln-R8-Tyr-Glu-Ala- 101 1785.0 1784.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07864 Ac-Val-nLeu-Gln-R8-Tyr-Glu-Ala- 101 1785.0 1784.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07867 Ac-Val-nLeu-Glu-R8-Tyr-Gln-Ala- 102 1785.0 1784.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07868 Ac-Val-nLeu-Glu-R8-Tyr-Gln-Ala- 102 1785.0 1784.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07871 Ac-Val-nLeu-Gln-R8-Tyr-Gln-Ala- 103 1784.0 1783.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP07872 Ac-Val-nLeu-Gln-R8-Tyr-Gln-Ala- 103 1784.0 1783.8 R8-S5 Ala-Phe-Ile-S5-His-Tyr-Val-NH2 stapled CTNNB1 FP08011 Ac-Ile-Ile-His-R8-Tyr-Glu-Ala- 104 1847.0 1847.2 R8-S5 Ala-Phe-Phe-S5-Gln-Tyr-Ile-NH2 stapled CTNNB1 FP08012 Ac-Ile-Ile-His-R8-Tyr-Glu-Ala- 104 1847.0 1847.4 R8-S5 Ala-Phe-Phe-S5-Gln-Tyr-Ile-NH2 stapled CTNNB1 FP08017 Ac-Ile-Leu-Glu-R8-Tyr-Glu-Ala- 105 1821.0 1821.1 R8-S5 Ala-Phe-Glu-S5-Gln-Tyr-nLeu-NH2 stapled CTNNB1 FP08018 Ac-Ile-Leu-Glu-R8-Tyr-Glu-Ala- 105 1821.0 1821.1 R8-S5 Ala-Phe-Glu-S5-Gln-Tyr-nLeu-NH2 stapled CTNNB1 FP08023 Ac-Ile-nLeu-Ala-R8-Tyr-Gln-Ala- 106 1820.0 1820.1 R8-S5 Ala-Phe-Trp-S5-Gln-Tyr-Asn-NH2 stapled CTNNB1 FP08024 Ac-Ile-nLeu-Ala-R8-Tyr-Gln-Ala- 106 1820.0 1820.1 R8-S5 Ala-Phe-Trp-S5-Gln-Tyr-Asn-NH2 stapled CTNNB1 FP08029 Ac-Ile-Leu-Thr-R8-Tyr-Glu-Ala- 107 1779.9 1780.1 R8-S5 Ala-Phe-Thr-S5-Gln-Tyr-Gln-NH2 stapled CTNNB1 FP08030 Ac-Ile-Leu-Thr-R8-Tyr-Glu-Ala- 107 1779.9 1780.1 R8-S5 Ala-Phe-Thr-S5-Gln-Tyr-Gln-NH2 stapled
Example 2. Technologies for Screening Stapled Peptide Collections
[1442] In some embodiments, the present disclosure provides technologies for identifying stapled peptides that can bind to targets of interest using stapled peptide collections. Among other things, the present disclosure provides a naive screening library. In some embodiments, a library comprises a collection of cysteine-stapled peptides, wherein the collection comprises random amino acid residues at one or more positions. In some embodiments, various stapled peptides in the library comprise alpha-helical structures. In one design, cysteine-stapled peptides are displayed as N-terminal fusions to a pIII protein on M13 bacteriophage. In some embodiments, stapled peptides are designed to contain one or more or all of the following features: a) a cap containing a proline residue at the N-terminus, or a N-terminal cap containing N-DPAA-C(SEQ ID NO: 20) sequence; b) two amino acid residues suitable for stapling, e.g., cysteine residues, placed at i,i+7 positions; c) one or more, e.g., three, randomized residues flanking each amino acid residue suitable for stapling, e.g., cysteine; d) a pair of randomized residues towards the inside of the stapled section between the two stapling residues, two alanine residues at the center of the stapled section (i,i+3 and i,i+4, numbered from the first amino acid residue suitable for stapling, e.g., cysteine) (without the intention to be limited by theory, in some embodiments, to minimize steric hindrance with the staple); and e) a short (8-residue) glycine-rich linker between the last randomized residue and the N-terminus of the pIII coat protein. Those skilled in the art will appreciate that if other technologies are utilized to generate a collection, a linker to a pIII coated protein may not be needed.
[1443] In some embodiments, a randomized peptide library was achieved by synthesizing and using degenerate phage library primers using a mix, e.g., an equimolar mix, of trimer phosphoroamidites that corresponded to various amino acid residues, for example, in some embodiments 16 of the 20 naturally occurring amino acids: alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenyalanine, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, a X residue, e.g., X.sup.31, X.sup.2, X.sup.3, etc., is selected from alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenyalanine, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, cysteine and lysine were omitted to avoid intramolecular competition with the crosslinking between the two fixed stapling cysteine residues, and proline and glycine were omitted to avoid destabilization of the alpha-helical structure. In one embodiment, 10 randomized residues corresponds to a theoretical diversity of 16.sup.1010.sup.12 possible sequences. In some embodiments, the number of unique sequences after transformation and amplification of such library was estimated to be approximately 10.sup.8 library members, in which case the constructed library explores roughly 0.01% of a theoretical sequence space of about 10.sup.12 possible sequences.
[1444] Phage display has been reported to be performed by panning a library against a protein target and then eluting and amplifying the bound phage by infection of E. coli, followed by additional rounds of selection and amplification. Among other things, the present disclosure recognizes that while this approach can enable the detection of weak or initially low-abundance binders, it is not practical to perform multiple selections and enrichments on dozens or hundreds of selections in parallel, and/or it inherently prevents quantitative comparisons between different selections after the first round due to a number of factors. For example, in some embodiments, input phage libraries are no longer identical and/or there can be biases in elution efficiency, in growth, and/or infection rates.
[1445] In some embodiments, the present disclosure describes technologies for screening stapled peptide libraries. In some embodiments the present disclosure utilized a single-round approach wherein a library of stapled peptides, e.g., displayed on phages, are incubated with one or more protein targets. In some embodiments, NGS is utilized to identify and/or quantify stapled peptides that can bind to targets of interest. In some embodiments, individual protein targets have been immobilized, e.g., on magnetic beads, followed by protein target isolation, e.g., bead isolation and washing, and then identification of stapled peptides bound to target proteins. In some embodiments, after bead isolation and washing, direct boiling, PCR amplification, and next-generation sequencing (NGS) of phage DNA were performed, followed by hierarchical statistical clustering. In some embodiments, one or more phage screens (e.g., 48 or 96) are run in parallel, e.g., in multi-well plates, against a range of targets of interest, e.g., protein targets. In some embodiments, each target, e.g., each target in a well, receives approximately multiple, e.g., 100, copies of each library member, e.g., of each of the 10 library members in a library (10.sup.10 phage total). In some embodiments, provided technologies allow results of a target to be compared to others. In some embodiments, following target binding and washes, phage DNA is directly extracted, amplified and sequenced. In some embodiments, following the binding and washing steps, but prior to boiling and PCR, a defined quantity of a known sequence, e.g., phage sequence was spiked into each well, which can enable the normalization of NGS counts in each well, e.g., by dividing the number of reads for each library member by the number of reads for the spike-in, then multiplying the result by the number of spike-in copies that were originally added. In some embodiments, NGS counts in each well are normalized by dividing the number of reads for each library member by the number of reads for the spike-in, then multiplying the result by the number of spike-in copies that were originally added. In some embodiments, binding of each of the 10i library members is expressed in terms of eluted phage particles rather than as sequence reads.
[1446] In some embodiments, approximately the same number of phage particles were added to each well. In some embodiments, a quantitative comparison among one or more samples, for all library members can be performed, allowing stapled peptides to be selected for specific criteria in silico rather than by having to perform actual enrichment or depletion experiments. In some embodiments, non-specific or bead binders can be flagged by screening multiple proteins as well as blank beads, since such binders will appear in most or all samples, whereas highly specific binders will appear only in samples corresponding to one or more desired targets. In some embodiments, screens can be performed to search for stapled peptides with specific properties, such as their ability to compete with a known interaction or function of a target. In some embodiments, competition for binding sites can be assessed by screening a target in both the presence and absence of a binding partner or ligand, and searching the screening data for stapled peptides whose phage binding is affected when the partner or ligand is present. In some embodiments, these screens occur in vitro. In some embodiments, one or more or dozens or even hundreds of screens can be run in parallel. Among other things, the provided technologies can provide considerable flexibility in screen design and conditions. In some embodiments, screens were performed with a wide range of small molecule, peptidic, and protein binding partners. In some embodiments, proteins with different sequences (e.g., from different species), modifications, forms (with or without ligands, monomeric or polymeric forms, with or without binding partners, etc.), etc., can be compared. In some embodiments, proteins with or without a modification are compared. In some embodiments, phosphorylated vs. non-phosphorylated, proteins were compared. In some embodiments, glycosylated vs. non-glycosylated proteins were compared. In some embodiments, apo vs. ligand-bound proteins were compared. In some embodiments, mouse vs. human proteins were compared. In some embodiments, monomeric vs. multimeric proteins were compared.
[1447] In some embodiments, certain stapled peptides are selected based on their target-binding and selectivity properties. In some embodiments, certain stapled peptides are grouped into families of related sequences using hierarchical clustering. In some embodiments, it was observed certain positions appear to be highly conserved (enriched positions; the conserved amino acid residues, enriched amino acid residues). In some embodiments, only one or a small number of amino acids (enriched amino acid residues) are found at a conserved position across all members of a cluster. In some embodiments, these conserved positions are strongly predictive of which residues are forming direct binding interactions with their protein targets. In some embodiments, one or more conserved residues directly interact with protein targets. In some embodiments, direct interactions can and have been confirmed using suitable technologies, e.g., X-ray co-crystal structures of stapled peptides and their binding proteins. In some embodiments, conserved residues can be used to define a conserved pharmacophore and/or predict structure-activity relationships for a cluster.
[1448] Certain useful technologies are described in
Example 3. Stapled Peptides Binding Beta-Catenin at Various Binding Sites
[1449] In some embodiments, provided technologies were utilized to discover stapled peptides that bind to therapeutic targets. In some embodiments, beta-catenin was screened against stapled peptide libraries. In some embodiments, the present disclosure provides stapled peptides that can bind beta-catenin at multiple binding sites. In some embodiments, the present disclosure provides stapled peptides that can modulate beta-catenin activities.
[1450] beta-Catenin is reported to be a key hub in the Wnt signaling pathway that is an attractive intracellular target for therapeutic intervention in many Wnt-driven cancers, such as those with APC or CTNNB1 mutations. beta-Catenin is reported to be engaged in numerous functional protein-protein interactions that have been difficult to target with traditional small molecules and biologics, but several examples of stapled peptide PPI inhibitors based on native binders have been described for beta-catenin and other undruggable targets. In one example, beta-catenin was screened at multiple concentrations, and in the presence and absence of an Axin-derived stapled peptide (which binds beta-catenin as a short alpha-helix) and the beta-catenin-binding domain of the TCF4 transcription factor. In some embodiments, certain stapled peptides that bind to beta-catenin competed with both Axin and TCF4. In some embodiments, certain stapled peptides that bind to beta-catenin competed with TCF4 but not with Axin.
[1451] In some embodiments, clusters of stapled peptides that competed with both Axin and TCF4 possessed different sequence patterns. In some embodiments, there were pairs of clusters that bore similar patterns of conserved residues, but that were shifted to different positions within the sequence, for example, Clusters C31 and C32 or C33 and C34. In some embodiments, members of a cluster may be binding beta-catenin in a similar way, but pharmacophores may be presented in different positions, or register, relative to the position of a staple. In some embodiments, such clusters are referred to as shifts of each other. In some embodiments, there were clusters whose patterns of conserved residues resembled the conserved residues in other clusters, , but were in the reverse orientation (N->C vs. C->N), for example, C32 and C33. In some embodiments, a similar binding mode is achieved from a peptide whose N- and C-termini have been inverted relative to another peptide, and side chains are able to form similar interactions, despite a different presentation angle from the main chain of the peptide. In some embodiments, analysis of co-crystal structures confirmed similar interactions despite inverted N- and C-termini. In some embodiments, conserved residues for several of these clusters, such as C31 or C32, bore close resemblance to the alpha-helical sequence in Axin that is known to bind beta-catenin.
[1452] In some embodiments, binding to beta-catenin, e.g., at the Axin site for certain stapled peptides were further assessed. In some embodiments, stapled peptides were synthesized and assessed in surface plasmon resonance (SPR) assays. In some embodiments, stapled peptides were synthesized and assessed in competition fluorescence polarization (FP) binding assays. In some embodiments, stapled peptides members of the C31-C35 clusters were synthesized and assessed in SPR and/or competition fluorescence polarization (FP) binding assays.
[1453] In one embodiment, SPR and competition FP data demonstrated binding to beta-catenin, as well as competition with both an Axin-derived Helicon and the full beta-catenin-binding domain of the TCF4 transcription factor. In some embodiment, SPR and competition FP data is consistent with the predicted binding behavior based on screens against beta-catenin. In some embodiments, direct interactions can and have been confirmed using suitable technologies, e.g., X-ray co-crystal structures of stapled peptides and their binding proteins. In some embodiments, beta-catenin binding site of stapled peptides from C33 cluster, e.g. FP01567, that was predicted to be a flip of the natural Axin sequence, was confirmed using suitable technologies, e.g. X-ray co-crystal structures and an alanine-screening analysis. In some embodiments, binding mode of FP01567 with beta-catenin was shown to be similar to binding mode of Axin but with an inverted/flipped N-to-C alpha-helical orientation and sidechains which were projected from a different direction. The crystal structure with FP01567, combined with an alanine-scanning analysis, further confirmed the importance of the conserved logo residues. Certain data are presented in
TABLE-US-00020 IC.sub.50 IC.sub.50 K.sub.D PeptideID Cluster Sequence (M,Axin) (M,TCF) (M,SPR) FP01838 C31 DPAILECHIAAWNCYEI 1.8 >10 0.4 FP49194 C31 DPATLDCHIAAWDCWDE 2.9 >10 3 FP49193 C31 DPAILACHLAAMDCSDW >10 >10 n.d. FP49196 C32 DPANANCILAAHECRIW 1 >10 0.1 FP01848 C32 DPAQDDCILAAHVCALW 1.3 >10 0.14 FP01848-unstapled C32 DPAQDDCILAAHVCALW N.D. >10 >10 FP01567 C33 DPATHRCEWAALHCELV 7.3 >10 3 FP01822 C33 DPASELCEWAAIHCDLV 3.7 >10 3 FP01822-unstapled C33 DPASELCEWAAHCDLV >10 >10 >10 FP49199 C34 DPADWECEHAALLCHYW 1.1 8.1 0.3 FP49200 C34 DPALWQCEHAALLCDVH 0.78 5.3 0.4 FP49200-unstapled C34 DPALWQCEHAALLCDVH >10 >10 >10 FP05863 C35 DPAIIHCYEAAFFCQYI >10 1.0 0.7 FP05874 C35 DPAVMECYEAAFICHYV >10 1.5 0.15
[1454] In some embodiments, stapled peptides compete with TCF, but not Axin. In some embodiments, biochemical assays performed on members of Cluster C35 confirmed competition in vitro with TCF but not Axin. In some embodiments, co-crystal structures of member of C35 cluster, e.g., FP05877, indicated direct interactions with beta-catenin at a similar site as the naturally occurring beta-catenin binder ICAT. Certain results are presented in Table 2 as examples.
[1455] In some embodiments, cysteine staples are replaced with other types of staples. In some embodiments, Helicons with non-cysteine staples maintained binding to beta-catenin while also demonstrating cell entry, e.g., as assessed by mass spectrometry quantification of intact peptide in cells after treatment and washing. Certain results are presented in Table 2 as examples.
TABLE-US-00021 TABLE2 Certaindataasexamples. -catenin CellTiter- Helicon Binding Glofold Parent SEQ incells IC.sub.50.sup.b(TCF change.sup.c FOGID Cluster peptidesequence IDNO: SD(%) probe,uM) SD FP47839 (Assay 5FAM-SAH-p53-8.sup.d 2.10.1 1.30.01 Positive Control) FP01413 (Assay ATSP-7041.sup.d 3.70.4 1.20.07 Positive Control) FP47831 (Assay 5FAM-PKCI.sup.d 0.00.0 1.10.02 Negative Control) FP47842 (Assay 5FAM-D-Mycpeptide.sup.d 0.00.0 1.20.01 Negative Control) FP47843 (Assay 5FAM-D-HApeptide.sup.d 0.00.0 1.20.02 Negative Control) FP07855 C35 DPAVMECYEAAFICHYV 108 16.40.6 4.00.1 1.20.10 (CTNNB1) FP07856 C35 DPAVMECYEAAFICHYV 108 15.50.9 6.60.9 1.30.12 (CTNNB1) FP07859 C35 DPAILQCYEAAFSCHYQ 109 0.10.0 >10 1.20.02 (CTNNB1) FP07860 C35 DPAILQCYEAAFSCHYQ 109 0.30.0 >10 1.20.03 (CTNNB1) FP07863 C35 DPAVMECYEAAFICHYV 108 23.56.5 8.71.6 1.20.02 (CTNNB1) FP07864 C35 DPAVMECYEAAFICHYV 108 18.511.4 7.61.1 1.10.05 (CTNNB1) FP07867 C35 DPAVMECYEAAFICHYV 108 8.60.4 >10 1.20.03 (CTNNB1) FP07868 C35 DPAVMECYEAAFICHYV 108 10.31.4 >10 1.20.10 (CTNNB1) FP07871 C35 DPAVMECYEAAFICHYV 108 7.00.3 >10 1.20.03 (CTNNB1) FP07872 C35 DPAVMECYEAAFICHYV 108 11.71.6 >10 1.10.02 (CTNNB1) FP08011 C35 DPAIIHCYEAAFFCQYI 110 6.50.5 >10 1.20.01 (CTNNB1) FP08012 C35 DPAIIHCYEAAFFCQYI 110 5.40.9 >10 1.10.04 (CTNNB1) FP08017 C35 DPAILECYEAAFECQYM 111 0.60.1 >10 1.20.01 (CTNNB1) FP08018 C35 DPAILECYEAAFECQYM 111 1.00.1 >10 1.10.05 (CTNNB1) FP08023 C35 |DPAIMACYQAAFWCQYN 112 3.00.3 >10 1.20.03 (CTNNB1) FP08024 C35 |DPAIMACYQAAFWCQYN 112 6.50.5 >10 1.20.00 (CTNNB1) FP08029 C35 DPAILTCYEAAFTCQYQ 113 0.20.0 8.01.1 1.10.03 (CTNNB1) FP08030 C35 DPAILTCYEAAFTCQYQ 113 0.30.0 >10 1.2.02 (CTNNB1) .sup.aThe percentage of Helicons in cells after treatment and wash. n =2; data are presented as mean SD. .sup.bIn vitro assay for competition with beta-catenin-binding TCF peptide. .sup.cMeasure of ATP levels, reflective of cell health. .sup.dFull sequences of control peptides can be found in Table 1.
[1456] Stapled peptides of certain beta-catenin binding clusters are presented below as examples.
TABLE-US-00022 ClusterC31 TargetConcentration Competitors Sequence SEQIDNO 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAEHDCMLAAHFCRHF 114 8 55 242 194 337 260 0 2 0 0 0 36 DPAQDDCILAAHVCALW 115 44 161 560 800 809 743 0 3 0 0 0 206 DPAVEDCVLAAHVCAWF 116 0 2 47 39 49 73 0 0 0 0 0 0 DPAEAECILAAHICNIS 117 10 0 37 39 51 52 0 0 0 0 0 0 DPAEVDCIMAAHICVWD 118 0 0 31 42 40 51 0 0 0 0 4 0 DPADNECILAAHICTLA 119 0 5 24 36 45 30 0 0 0 0 4 0 DPAQQDCTLAAHVCRVF 120 0 0 19 21 70 11 0 0 0 0 0 0 DPAEADCMMAAHICSNI 121 0 0 21 15 46 22 0 0 0 0 0 0 DPADYECVIAAQVCQHF 122 0 1 11 18 19 14 0 4 0 0 0 15 DPANANCILAAHECRIW 123 87 199 501 899 945 680 0 2 0 0 25 173 DPAFESCFLAAHICYWE 124 2 0 10 7 18 16 0 0 0 0 0 0 DPAEAICILAAHECWWD 125 19 38 139 235 322 228 0 0 0 0 0 28 DPAEVECMLAAHVCRAF 126 8 26 67 109 142 130 0 0 0 0 0 1 DPADYDCILAAHICTNI 127 2 18 70 147 118 122 0 0 0 0 19 0 DPADTECILAAHFCHYF 128 0 1 39 81 78 49 0 2 0 0 0 0 DPAVDECIIAAHICHIW 129 0 4 21 44 31 66 0 0 0 0 0 0 DPAESDCILAAYICYVE 130 0 1 22 51 69 40 0 2 0 0 0 0 DPADEDCILAAHVCRHF 131 0 6 11 29 25 21 0 0 0 0 0 1 DPAADDCVLAAHVCAYF 132 8 46 144 397 407 343 7 1 0 0 0 0 DPARHECILAAHICEWE 133 41 151 612 1704 1683 1509 1 2 2 0 2 144 ClusterC32 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAILDCQIAAIECEFA 134 0 22 76 68 139 56 0 0 0 1 0 3 DPAILDCHIAAIQCFHD 135 2 1 6 8 1 44 0 0 0 3 0 0 ClusterC32 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAIFNCTMAALECTQI 136 0 0 2 3 19 0 0 0 0 0 0 0 DPAILECHIAAWNCYEI 137 0 2 50 70 57 97 0 0 0 0 0 1 DPAHLMCHYAAWDCRDE 138 0 0 11 13 12 23 0 0 0 0 0 0 DPAILDCQLAAMDCSTW 139 0 6 22 21 25 55 0 0 0 0 15 0 DPAFLTCHIAAWACSEQ 140 0 1 21 15 52 25 0 2 1 0 0 0 DPAFLDCHIAAWTCNEF 141 0 1 10 15 28 15 0 0 0 0 0 0 DPAILDCQLAAIDCVYT 142 3 21 67 168 201 114 0 0 0 0 0 9 DPAILDCSIAAIDCAYS 143 0 0 16 42 39 29 0 0 0 0 0 0 DPAILECHMAAFECYIE 144 8 2 20 59 86 41 0 0 0 0 0 0 DPAVFECSLAAIDCLQI 145 0 0 10 28 10 81 0 0 0 0 4 0 DPAVLECHIAAWECRMD 146 0 0 24 83 91 45 0 0 0 0 0 0 DPAILECHVAALDCQMW 147 0 2 40 55 98 81 0 2 0 0 0 0 DPAILNCHMAAWDCNFY 148 0 7 14 57 37 59 0 0 1 0 0 0 DPATLDCHIAAWDCWDE 149 14 10 42 104 176 104 0 0 0 0 0 0 DPAILACHLAAMDCSDW 150 0 1 11 98 103 83 0 0 0 0 0 0 DPAVLECHIAAWECNHT 151 0 1 22 67 140 55 0 0 0 0 0 0 DPAVLDCHIAALDCHAH 152 0 1 0 10 39 3 0 0 0 0 0 0 DPAILNCHVAAWNCHSF 153 0 0 1 8 4 23 0 0 0 0 0 0 ClusterC33 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAELACEWAAIHCELV 154 0 2 41 94 69 75 0 0 0 0 0 17 DPASELCEWAAIHCDLV 155 19 41 163 495 605 424 1 2 3 1 0 3 DPADDFCMWAAEHCELL 156 10 12 81 226 273 227 0 0 1 0 0 1 DPAWQNCTWAAFHCELL 157 0 10 52 194 158 163 0 1 0 0 0 39 DPAQNICEWAALHCDLI 158 0 13 37 109 177 82 0 1 0 0 0 10 ClusterC33 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAEFYCEMAAVHCELL 159 2 1 17 99 61 107 0 0 0 0 0 0 DPAVHRCFWAAEHCDLI 160 0 2 11 86 95 62 0 2 0 0 0 0 DPAWILCDWAAMHCELV 161 0 0 2 3 1 18 0 0 0 0 0 0 DPAMIQCQWAAEHCELI 162 0 0 4 33 19 42 0 1 0 0 0 0 DPAIADCDWAAEHCDLL 163 0 6 118 266 325 417 0 1 4 1 0 1 DPAVQDCEWAAIYCELI 164 0 9 10 158 104 157 0 1 0 1 0 12 DPAWSDCIWAAEHCQLI 165 0 3 5 127 109 85 0 1 0 0 0 0 DPAMSQCNWAAVHCQLL 166 6 6 6 13 15 23 0 0 0 0 0 0 DPANVECEWAAIHCSLV 167 13 2 19 158 164 187 0 0 0 0 0 3 DPAQSDCDWAAEHCNLI 168 0 1 0 23 9 57 0 0 0 0 2 1 DPAAQRCTWAAIHCELI 169 0 0 7 39 25 93 0 0 2 0 0 1 DPAYTYCQWAAEHCSLL 170 5 5 3 88 115 137 2 2 1 2 0 0 DPAHWSCEWAAEHCRLI 171 0 1 0 31 19 64 0 0 0 0 0 0 DPAENTCSWAAEHCALV 172 0 0 0 26 28 31 0 0 0 0 0 0 DPATWHCDWAAEHCELL 173 2 10 81 147 203 280 0 0 0 0 0 3 ClusterC34 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPALWNCDHAALLCDHQ 174 0 0 10 8 13 10 0 0 0 0 0 0 DPAEWDCMHAALICEVH 175 0 10 55 54 67 89 0 0 0 0 0 0 DPASWRCEHAALNCQID 176 0 6 20 18 21 57 0 0 0 0 0 0 DPAWWQCDHAALICIAE 177 28 127 422 637 830 731 0 1 2 0 32 113 DPAEWQCEHAALLCEQQ 178 78 201 907 1681 1740 1609 0 2 0 6 36 154 DPAQWACEHAALLCSMD 179 6 35 226 419 326 432 0 1 0 0 0 25 DPALWQCEHAALLCDVH 180 13 27 122 158 246 246 0 2 1 0 0 0 DPADWICEHAALTCQEF 181 0 2 6 15 12 14 0 1 0 0 0 0 ClusterC34 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAHWVCEHAALNCDQN 182 0 2 47 96 104 118 0 1 0 0 0 0 DPADWECEHAALLCHYW 183 11 18 90 179 198 226 0 2 0 0 0 1 DPAEWICEHAALLCSVL 184 0 0 11 33 33 27 0 0 0 0 0 0 DPAEWACEHAALTCWDQ 185 6 24 148 448 431 449 1 4 0 0 0 33 DPASWDCDHAALLCTLD 186 3 6 86 257 283 238 0 0 0 1 0 0 DPAQFYCEHAALLCRWE 187 0 0 5 11 15 11 0 0 0 0 0 0 DPAHWRCEHAALLCWYN 188 13 6 51 132 140 149 0 0 2 0 0 0 DPAEWYCEHAALVCTNH 189 0 12 47 156 148 155 2 0 0 1 0 0 DPAMWICEHAALNCEIR 190 2 6 46 158 145 152 0 0 0 0 0 7 DPAYWDCTHAALLCNLD 191 3 14 152 42 543 409 0 1 2 0 0 19 DPAHWHCEHAALLCASV 192 2 15 60 231 177 234 1 0 0 0 0 1 DPAYWVCEHAALLCTDY 193 5 18 85 337 283 227 3 3 1 0 0 4 ClusterC35 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAIIHCYEAAFFCQYI 110 0 0 15 20 22 28 1 0 0 1 27 1 DPAILECYEAAFECQYM 111 7 8 20 49 41 39 0 0 0 0 87 0 DPAIMACYQAAFWCQYN 112 0 0 4 27 29 18 1 0 1 0 38 0 DPATIRCYEAAFMCQYW 194 0 0 3 34 21 38 0 0 0 0 23 0 DPAILTCYEAAFTCQYQ 113 0 7 18 71 69 105 0 1 0 0 107 0 DPAIMACYEAAYLCDYI 195 2 4 29 85 111 150 1 0 0 1 130 0 DPAIIRCYEAAFYCQNY 196 0 3 10 67 93 94 0 0 1 0 124 0 DPAVIQCYQAAFHCDYQ 197 0 0 0 13 12 30 0 0 3 0 14 0 DPAEIICYEAAFLCQYQ 198 0 0 0 15 38 26 0 0 0 0 40 0 DPAALSCYQAAFHCQYY 199 0 0 1 5 14 17 0 0 0 0 16 0 DPARMTCYEAAFRCQYY 200 0 2 2 3 11 9 0 0 0 0 4 0 ClusterC35 TargetConcentration Competitors Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Axin_FP0597c_300 TCF4_300 DPAVVECYEAAYTCQYI 201 0 0 0 0 0 13 0 0 0 0 0 0 DPAVMECYEAAFICHYV 108 0 27 16 79 51 74 0 0 0 0 120 0 DPAILQCYEAAFSCHYQ 109 0 9 40 54 39 42 0 0 0 0 47 0 DPAVLQCYEAAFICEYM 202 0 0 1 6 4 5 0 0 0 0 2 0 DPAVMACYEAAFQCAYW 203 0 0 0 4 12 0 0 0 1 0 36 0 DPAVIACYDAAFVCEYF 204 0 0 0 0 3 3 0 1 0 0 0 0 DPAVFMCYEAAFECQYF 205 0 0 0 1 2 7 0 2 1 0 13 0 DPAIIHCYQAAFQCQYM 206 0 0 0 0 7 0 0 0 0 0 0 0 DPAMVTCYEAAYVCQYF 207 0 0 0 0 7 0 0 0 0 0 0 0
Example 4. Stapled Peptides Binding the E3 Ubiquitin Ligase RNF31 at Various Binding Sites
[1457] In some embodiments, the present disclosure provides technologies useful for identifying, characterizing, manufacturing, assessing, using, etc., stapled peptide binders to targets of interest, e.g., proteins, with few or no known alpha-helical binding sites. In some embodiments, the present disclosure provides stapled peptide agents that bind E3 ubiquitin ligase RNF31. In some embodiments, the present disclosure provides stapled peptide agents that modulate activities of RNF31. RNF31 is reported to be an integral component of the linear ubiquitin chain assembly complex (LUBAC). It is reported to be a 1,072-amino acid protein comprised of multiple folded and disordered regions. In some embodiments, two of the non-enzymatic folded domains of RNF31, the PUB and UBA domains, were screened, each at multiple concentrations and in the presence or absence of a known binding partner.
[1458] Screens for both UBA and PUB domains afforded multiple distinct clusters of binders, and several instances of shift clusters were observed. In some embodiments, most hits for the RNF31 PUB domain could be grouped into two families of related clusters, (C41-C43 and C44-C45), both of which competed with the natural binding partner Otulin, and both of which appeared to contain a critical tyrosine residuealso present in Otulinbut that had other dissimilar structure features. In some embodiments, X-ray co-crystal structures of member from a representative cluster of each family revealed that the two Helicons, FP06649 from Cluster C44 and FP06652 from C41, bound the same site in the protein surface, but that they engaged this site using completely different binding modes, with the critical tyrosine residues oriented differently. Among other other things, the present disclosure provides distinct binding solutions to a common site, e.g., for RNF31 and other targets of interest.
[1459] In some embodiments, binders to the RNF31 UBA domain were observed to compete with its partner Sharpin/SIPL1. In one embodiment, these binders could be grouped into at least two families. One of these families, comprised of Cluster C46 and C47, was characterized by a shared compact pharmacophore containing conserved leucine/phenylalanine, serine, valine, and tyrosine residues that was observed in two shifted registers. In some embodiments, biochemical characterization of members from C46 revealed that several stapled peptides bound with mid-nanomolar affinity. In some embodiments, analysis of a co-crystal structure of FP06655, from C46 cluster, in complex with the RNF31 UBA domain, revealed an large conformational rearrangement when compared to the co-crystal structure of the RNF31 UBA domain in complex with its natural binding partner Sharpin. In some embodiments, binding of FP06655 with this target involves a significant rotation of the bundle of three N-terminal a-helices of the RNF31 UBA domain to form a groove in which the conserved leucine/phenylalanine and valine residues of FP06655 engage one wall of the groove, and the conserved serine and tyrosine residues engage the opposite wall, thereby stabilizing the RNF31 UBA domain in this induced fit. In some embodiments, it was showed that Sharpin UBL indeed competes with 5FAM-labeled FP06655 in solution. In some embodiments, binding of stapled peptides to their targets lead to significant induced fits.
[1460] Stapled peptides of certain RNF31 binding clusters are presented below as examples.
TABLE-US-00023 ClusterC41 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3455_300 DPAIVQCAWAALYCDMQ 208 0 0 0 7 22 20 0 0 0 0 0 0 DPALEDCQWAALYCDEY 209 0 0 1 2 7 9 1 1 1 0 0 1 DPAYEDCAWAALYCDMT 210 0 0 0 10 70 64 0 0 0 0 0 0 DPAYYTCDWAAIYCDYH 211 0 0 0 1 6 6 0 1 0 0 0 0 DPAYRDCDWAALYCDDF 212 0 0 1 2 12 12 0 3 0 0 1 0 DPAWTQCDWAALYCEDQ 213 0 1 0 1 7 10 0 0 0 0 0 0 DPAEDECDWAALYCDMQ 214 0 0 0 1 7 6 0 0 0 0 0 0 DPAYEDCADAAMYCDMD 215 0 0 0 0 11 10 1 0 0 0 0 0 DPAWTDCDWAALYCEFQ 216 1 0 0 0 6 6 0 0 0 1 0 0 DPAWTDCDWAALYCDES 217 0 0 0 0 8 4 0 0 0 0 0 0 DPALVTCTWAAIYCDDF 218 1 1 2 1 4 10 0 1 0 2 0 0 DPAYTLCSWAAMYCDSM 219 0 1 0 0 5 6 0 1 1 4 2 2 DPAYSSCSWAAIYCNWT 220 1 1 5 2 7 4 1 1 4 2 1 1 DPAADDCEWAALYCSFH 221 2 0 0 3 3 8 2 9 0 2 3 0 ClusterC42 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3455_300 DPAWNDCLYAAETCISW 222 1 1 1 3 11 1 2 1 1 0 0 0 DPAWWNCLYAANTCVED 223 0 1 0 5 7 5 1 2 1 1 1 1 DPAQHECIYAAWDCDML 224 1 1 1 5 6 8 3 2 0 1 0 5 DPAWWDCMYAASHCDTV 225 0 0 0 3 12 2 2 0 0 0 0 0 DPAWMQCMYAAWDCNIE 226 0 0 0 4 6 5 0 0 0 0 0 1 DPAYWDCMYAAFYCDDI 227 0 0 0 3 3 8 0 0 0 0 2 0 DPAYWDCLYAAADCAFY 228 0 0 0 4 8 5 2 0 0 0 0 0 DPAYWDCLYAAWSCDIL 229 0 1 0 4 9 7 1 1 1 0 0 1 DPAWWSCMYAAWTCDDT 230 1 10 0 7 14 15 0 2 2 1 1 2 DPAWLDCIYAAYDCVTD 231 1 1 1 3 6 4 1 1 1 0 2 1 DPAYWQCIYAAWDCDQM 232 0 0 1 4 13 6 1 2 2 1 0 4 DPAWWNCLYAAYECNHA 233 1 0 0 3 9 5 0 0 0 2 0 0 DPALWDCLYAADACITS 234 0 1 0 4 8 12 0 2 1 0 0 0 DPAFWECMYAASDCDAS 235 0 0 1 2 6 4 1 0 0 0 2 0 DPAFWACLYAAEECVVD 236 0 0 0 3 6 9 1 0 0 0 1 0 DPAWMDCLYAAYDCQHM 237 2 2 0 9 29 24 1 1 1 0 0 1 DPAYWHCLYAAASCDDI 238 0 0 0 2 8 4 0 3 0 0 2 0 DPAEWDCLYAAFDCFAN 239 2 2 0 3 15 7 0 0 0 0 0 0 DPADWDCMYAAQDCFED 240 0 0 1 1 11 1 0 0 0 0 0 0 DPAWWNCLYAADECWSW 241 0 0 0 3 13 6 0 0 1 0 0 0 ClusterC43 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3455_300 DPAYDWCMYAAHECVDQ 242 0 0 0 3 3 8 0 0 0 1 0 1 DPALDYCLYAAYDCMYA 243 0 0 0 3 5 12 0 3 0 0 0 3 DPATWWCLYAAEDCVNI 244 0 1 0 2 6 6 0 0 0 0 4 0 DPAYDWCLYAAYQCDQN 245 0 0 0 6 13 40 0 0 0 0 0 0 DPAWEWCLYAAAECIED 246 0 0 0 2 12 5 0 0 0 0 0 0 DPAEDWCMYAAWDCQME 247 1 0 0 4 22 10 0 0 0 0 0 0 DPAQDWCMYAAYECDAH 248 0 0 0 2 7 9 0 0 2 1 1 0 DPAWDYCMYAAYDCNHS 249 2 0 1 8 46 29 0 1 0 0 1 2 DPARDWCLYAAYDCYTA 250 0 0 4 61 350 353 1 0 3 1 0 2 DPAHDWCLYAAYDCRVY 251 0 0 1 2 15 8 0 0 0 0 0 0 DPAWEHCLYAAYDCTHE 252 0 1 0 2 10 15 0 4 0 0 0 4 DPAYDWCMYAAYNCVED 253 2 1 1 6 41 38 0 0 0 0 0 0 DPAWESCMYAAYDCDNE 254 0 1 0 2 14 5 0 0 0 0 0 0 DPAEQFCLYAAYDCYSE 255 2 0 0 2 18 9 1 3 1 0 0 0 DPAYDWCLYAAEYCHTD 256 0 0 0 1 7 4 0 0 1 0 0 0 DPAYDYCLYAAQDCVWW 257 0 0 0 1 6 5 0 0 0 0 0 0 DPAFDWCLYAADDCAET 258 1 0 0 3 31 12 0 0 0 0 1 0 DPAHTWCMYAAFDCFES 259 1 0 0 1 9 4 1 2 2 0 0 0 DPAYDYCLYAAYDCTQT 260 0 0 1 5 48 38 1 0 0 1 0 0 ClusterC44 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3455_300 DPAQYQCDIAAQVCSTY 261 0 0 0 4 12 10 0 0 0 0 0 0 DPAFAQCEVAAMVCTTY 262 0 0 0 2 5 6 0 0 0 0 0 0 DPAIQECLLAAATCWTY 263 0 0 0 2 8 4 0 0 0 0 0 0 DPAHANCQLAADVCSTY 264 0 0 0 13 49 33 1 0 0 0 0 0 DPAWQECVIAALVCDTY 265 0 0 0 3 13 8 0 0 1 4 0 0 DPAIYQCSLAADICSTY 266 1 3 3 3 7 8 2 2 0 0 0 1 DPAIWECVSAAAVCMTY 267 0 0 0 2 7 7 0 0 0 1 0 0 DPAIQECYLAAQVCTTY 268 0 0 0 4 14 13 0 0 0 0 0 0 DPAAWECHIAAFVCSTY 269 1 1 0 2 8 7 0 0 0 0 0 0 DPAAEDCHIAAMVCMTY 270 0 4 0 12 46 67 0 0 0 0 0 0 DPAIRECMLAALVCSTY 271 0 1 3 10 42 49 0 0 0 1 0 1 DPAFTDCQLAAAVCMTY 272 0 0 3 31 161 145 1 2 0 0 0 1 DPAADQCDIAAFVCSTY 273 0 0 0 4 26 17 0 0 0 0 0 0 DPAFMDCVFAAAVCATY 274 0 0 0 2 8 8 0 1 1 0 0 0 DPALSHCTVAAMVCSTY 275 1 1 1 11 96 69 0 0 2 0 0 1 DPANFTCRVAADVCSTY 276 0 0 3 3 26 12 0 0 1 0 0 0 DPAIAQCMLAAQVCSTY 277 2 0 0 5 54 34 1 0 0 0 0 0 DPAMQDCQLAALVCTTY 278 0 1 0 1 6 6 0 0 0 0 0 0 DPAFHDCQFAAAVCSTY 279 0 0 0 3 26 26 0 0 0 0 0 0 DPAMIDCFLAAQVCATY 280 0 0 0 1 6 6 0 0 0 0 0 0 ClusterC45 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3455_300 DPAFQFCVEAAVACSYY 281 0 0 0 6 9 5 0 0 0 0 4 0 DPAEDECQDAAILCSYN 282 1 3 0 7 19 6 1 0 0 0 1 1 DPALRDCLDAAIMCTYT 283 0 1 0 4 5 7 0 0 0 0 0 0 DPAQYQCWNAAMSCSYS 284 1 1 3 3 7 4 1 0 0 3 2 0 DPAQMECIDAAIMCSYT 285 0 0 0 3 5 5 0 0 0 0 0 0 DPAQDQCISAAVLCTYT 286 0 0 0 22 44 37 0 0 1 0 0 0 DPAQEACWDAAIMCTYR 287 0 0 0 29 56 80 0 0 0 0 0 0 DPAEEYCLEAAILCTYD 288 0 0 1 14 44 32 0 0 0 0 0 0 DPAMQECMDAAIMCTYA 289 0 0 0 4 14 8 0 2 0 0 0 0 DPAMEWCWDAAVFCTYS 290 1 1 0 6 18 16 2 1 0 1 1 0 DPAVVDCWDAAVQCTYY 291 0 0 0 4 15 11 0 0 0 2 0 1 DPAIDACYDAAVMCTYV 292 0 0 0 6 27 15 0 0 0 0 4 0 DPASHMCLEAAVMCTYD 293 0 0 0 4 10 13 0 0 0 0 0 0 DPATEACFDAAIECTYY 294 0 0 0 2 7 5 0 0 0 0 0 0 DPAYDLCLNAAVACTYV 295 0 2 2 19 62 71 1 2 1 0 0 0 DPAYSLCISAAVECTYY 296 0 1 0 2 6 7 0 0 0 0 0 0 DPARRECWDAAILCTYY 297 0 0 1 12 40 44 0 0 1 0 0 0 DPASEWCLDAAVLCTYS 298 0 0 3 18 63 65 2 0 0 0 0 0 DPALEICWDAAIQCSYV 299 2 3 4 47 186 171 2 0 0 3 1 1 DPAQEECFEAAVFCSYY 300 2 0 2 7 36 20.49 0 0 0 0 1 0 ClusterC46 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3456s_300 DPAMQRCFSAAVYCAIS 301 11 44 101 150 156 159 0 0 1 0 1 158 DPAQMSCFSAAVYCRLF 302 0 0 11 10 10 17 0 0 0 0 0 4 DPAQRQCLSAAVWCATF 303 10 57 121 199 201 203 1 0 2 1 0 215 DPAALACLSAAVYCEMY 304 6 49 132 200 199 246 0 1 2 0 1 203 DPALQSCLSAAVYCTVS 305 0 0 6 10 9 10 0 0 0 0 0 12 DPAVQSCLSAAVYCAFR 306 2 9 68 106 117 118 0 1 2 0 1 92 DPAQMFCYSAAVYCAAF 307 0 0 4 5 8 4 0 0 0 0 0 5 DPAASQCFSAAVYCAWI 308 0 0 8 13 16 13 0 0 0 1 0 9 DPAEQRCLSAAVYCAWH 309 1 3 16 18 36 26 0 1 1 0 0 30 DPAHVRCTSAALWCNIF 310 0 0 3 4 5 7 0 0 0 0 0 2 DPAAQACLSAAVFCAWY 311 5 19 45 86 88 97 0 0 0 0 0 98 DPALRTCLSAAVFCAVA 312 0 6 10 18 15 25 0 0 1 0 1 24 DPASASCLSAAIYCSMF 313 5 17 122 255 247 297 0 0 1 0 0 235 DPAQLNCLSAAMYCALY 314 2 19 49 95 104 111 0 0 1 0 0 87 DPAQRQCLSAAVFCATL 315 3 24 137 266 292 319 0 1 1 0 1 266 DPALTNCFSAAVYCAMF 316 8 39 131 239 297 291 0 1 1 0 0 279 DPASMVCLSAAVYCAMH 317 1 15 38 77 82 91 0 2 0 0 1 87 DPAQQICLSAAVFCSIF 318 0 0 4 10 11 7 0 0 0 0 0 6 DPAMIQCLSAAVYCVHY 319 1 3 8 13 21 13 0 0 0 0 0 13 DPAQRDCFSAAVYCSFL 320 210 40 88 98 99 1 0 1 0 0 98 ClusterC47 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3456s_300 DPAFSQCVYAAFFCREI 321 1 0 2 20 28 18 0 0 0 0 0 11 DPAFSTCVYAAVFCQNT 322 0 0 1 8 7 12 0 0 0 0 0 4 DPAFSTCVYAAIICNDT 323 0 0 7 29 46 36 0 0 0 0 0 12 DPAFSTCIYAAVMCMDQ 324 0 0 3 19 21 29 0 0 0 0 0 14 DPAFSECVYAAIYCVHS 325 0 0 0 7 9 16 0 0 0 0 2 5 DPAFSQCVYAAVVCEVQ 326 2 0 11 55 91 102 1 0 1 1 1 54 DPAFSRCVYAAVACEDH 327 0 1 3 31 57 56 0 0 2 0 0 27 DPAFSLCVYAALFCEQQ 328 0 0 8 20 36 43 0 0 0 0 0 14 DPAFSACVYAAYMCNQD 329 0 0 1 4 6 11 0 5 0 0 0 0 DPAFSQCVYAAWHCESL 330 1 0 2 7 14 15 0 1 0 0 0 5 DPALSTCVYAAVMCQDV 331 0 0 2 4 8 8 0 0 0 0 0 3 DPAFSMCVYAAVTCVST 332 0 0 0 4 6 13 0 0 0 0 0 2 DPAFSRCVYAAFTCQTF 333 0 0 1 10 21 30 1 0 0 0 0 8 DPAFSECVYAAFSCSWI 334 1 2 3 9 22 23 0 0 2 1 0 4 DPAFFTCVYAAVTCEMT 335 0 0 0 3 8 6 0 0 1 0 2 3 DPAFYECVYAAVTCTQF 336 0 0 0 2 7 6 0 1 0 0 0 1 DPALSQCVYAAVFCQNL 337 0 2 0 4 14 11 3 0 1 0 1 1 DPAFSLCVYAAVECDHA 338 0 0 0 1 9 3 0 0 0 0 0 2 DPAFSQCIYAAFQCDMQ 339 0 0 0 2 11 6 0 0 1 2 0 2 DPAFSECVYAATMCLNH 340 2 0 1 1 9 10 0 0 0 0 0 2 ClusterC48 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK FP3456s_300 DPAYEHCVAAAYWCYLL 341 1 0 11 30 32 36 0 0 0 0 0 27 DPAREHCDSAAIFCYIL 342 0 0 1 14 13 14 0 0 1 0 0 8 DPAREHCAAAATWCOLL 343 1 0 1 28 31 33 0 1 0 0 0 20 DPAHEHCTAAASWCYIL 344 0 0 3 8 11 12 0 0 0 0 0 8 DPAQNHCDSAAVFCYIL 345 0 0 1 8 10 11 0 0 0 0 0 3 DPAHVHCQAAAQWCVLL 346 0 1 1 6 10 9 1 0 0 0 0 4 DPAREHCDSAAAWCWVL 347 0 0 1 4 11 6 0 0 4 0 2 6 DPAITHCTAAAMWCHLL 348 1 0 9 80 153 169 0 0 0 1 0 88 DPASSHCQSAAIWCYLM 349 1 0 6 35 67 81 0 0 2 0 0 52 DPASIHCAAAALWCDIL 350 0 0 1 8 25 15 0 0 0 0 0 10 DPANEHCIAAAWWCQIL 351 0 0 0 3 11 5 0 0 0 0 0 6 DPALTHCTAAAYWCYVM 352 0 0 0 2 6 6 0 0 0 0 0 3 DPALQHCAAAASWCWLM 353 1 2 4 20 56 52 2 0 2 0 1 16 DPAHHHCQAAAAWCWVM 354 2 0 4 19 44 65 0 0 0 1 1 22 DPATQHCLAAAMWCHLI 355 0 0 1 3 9 9 0 0 0 1 0 1 DPAHEHCTSAAAWCMLL 356 0 0 0 2 6 5 0 0 0 0 0 4 DPAQQHCSAAAAWCVVL 357 0 0 1 10 29 31 0 0 1 1 1 7 DPAEYHCDAAAVWCYLM 358 0 0 2 19 65 55 0 0 0 0 0 30 DPAQVHCRSAAAWCILM 359 0 0 0 2 7 7 0 0 0 0 0 1 DPARYHCEAAAAWCELL 360 0 0 0 8 33 24 1 0 0 0 0 12 ClusterC49 TargetConcentration Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK DPADLICQLAAANCIYL 361 0 0 2 30 100 79 0 0 0 0 0 DPAQWVCHMAAMNCLYQ 362 0 1 0 4 10 14 0 0 0 4 0 DPAEWLCQMAAHNCIYL 363 0 1 2 13 62 55 0 0 2 3 1 DPAMYICEMAAANCLYQ 364 0 1 1 6 29 28 0 0 0 0 0 DPADYICYLAANNCLYH 365 3 1 4 19 117 106 1 1 0 0 1 DPAEWICRMAAMNCLYQ 366 0 0 4 10 54 80 0 0 0 2 0 DPAAFVCEMAANDCLYT 367 0 1 0 2 10 8 0 0 0 0 0 DPATLVCTMAANNCIYR 368 0 0 0 1 13 10 0 0 0 0 0 DPARWVCELAAQNCLYR 369 0 1 1 1 18 14 0 0 1 0 0 DPAEWICEMAAHNCLYM 370 0 1 1 1 14 22 0 0 0 0 0 DPASIICMLAADNCLYE 371 0 0 0 0 7 7 0 0 0 0 0 DPADLLCHMAAQNCLYR 372 1 0 0 0 15 4 0 0 2 0 0 DPAAYLCDMAAHNCLYL 373 1 0 0 0 6 4 0 0 0 0 0 DPAHILCEMAARNCLYN 374 3 1 1 1 10 2 2 3 1 3 2 DPADWTCVLAAHNCLYT 375 0 0 0 1 3 9 0 4 4 2 4
[1461] Certain data are presented in
Example 5. Stapled Peptides Binding CDK2
[1462] In some embodiments, the present disclosure provides stapled peptides that bind and modulate the function of enzymes. In some embodiments, the present disclosure provides stapled peptides that bind cyclin-dependent kinase 2, CDK2. In some embodiments, the present disclosure provides stapled peptides that modulate CDK2 activities. CDK2 is reported to be involved in regulation of the cell cycle. CDK2 is reportedly a protein target of high therapeutic interest, e.g., due to its role in cell cycle progression and its implication in CyclinE1-mediated resistance to CDK4/6-inhibitor treated cancers, but it has historically been challenging to develop CDK2-selective inhibitors, e.g., due to the close similarity of CDK2 with other CDK family members, particularly CDK1. Whereas the ATP-binding pockets of CDK2 and CDK1 are highly similar, the present disclosure encompasses the recognition that their surfaces share significantly less sequence identity so that Helicons can engage the CDK2 surface and differentiate between the two family members.
[1463] Among other things, two distinct clusters of Helicons that bound both CDK2 and CDK2 in complex with its partner CyclinE1, but not to CDK1 or CDK1 in complex with its partner CyclinA2 (C51 and C52). In some embodiments, co-crystal structures of Helicons from each of these clusters, FP19711 from Cluster C51 and FP24322 from C52, showed two allosteric binding sites with respect to the ATP pocket. A FP19711-CDK2 co-structure indicated that the N-terminal and C-terminal-most peptide residues may be not directly involved in the CDK2 interaction. In some embodiments, truncation of both the N- and C-termini to generate FP33215 improved the affinity of FP19711 to 300 nM. In some embodiments, one or more (e.g., 1, 2 or 3) or all residues to the N-terminus side of X.sup.4 are absent. In some embodiments, one or more (e.g., 1, 2 or 3) or all residues to the C-terminus side of X.sup.11 are absent. It was also confirmed that FP19711 does not compete with ATP, and can bind to CDK2 in the presence of other ATP-competitive CDK2-binding proteins. In some embodiments, neither FP19711 nor FP33215 appeared to inhibit the kinase activity of CDK2 in a luminescence-based assay that detects ADP production. It was confirmed that FP19711 binding site can be a region on the CDK2 surface where there is high divergence from CDK1. These results highlight the utility of exploiting surface binding to achieve selectivity between closely related proteins when selectivity is difficult to achieve otherwise, e.g., with small-molecule ligands.
[1464] Certain results are presented in
[1465] Stapled peptides of certain CDK2 binding clusters are presented below as examples:
TABLE-US-00024 ClusterC51 TargetConcentration Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK BLANK BLANK DPAWWVCSIAAIECESS 376 0 0 10 85 65 77 0 0 0 0 0 0 0 DPAWWMCTIAAIECETT 377 0 0 73 973 909 998 2 0 0 0 0 0 0 DPAWWRCVVAALDCVDY 378 0 12 8 262 162 409 2 0 0 0 0 0 0 DPAWWHCSIAALECHTT 379 0 18 25 216 195 358 2 0 0 0 0 0 0 DPAWWRCSVAAIDCHWN 380 0 10 3 93 65 230 0 0 0 0 0 0 0 DPAWWECIVAALECSDR 381 0 0 0 62 76 90 0 0 0 0 0 0 0 DPAWWHCVIAALDCETQ 382 0 15 5 93 119 141 0 0 0 0 0 0 0 DPAWYECVVAAIECHAY 383 0 12 0 286 411 473 0 0 0 0 0 0 0 DPAWWVCAIAAMDCHEQ 384 0 0 0 139 216 422 0 0 0 0 0 0 0 DPAWWSCVIAAMECQDI 385 0 0 0 39 87 90 0 0 0 0 0 0 0 DPAWWTCSVAAIQCWDS 386 0 0 10 69 119 243 0 12 0 0 0 0 0 DPAWWRCSIAAVDCEMQ 387 0 0 0 93 238 217 0 0 0 0 0 0 0 DPAWWRCSIAAMECHHD 388 0 0 13 39 119 102 0 0 0 0 0 0 0 DPAWYACIVAAVDCEHS 389 0 0 8 46 206 115 0 0 0 0 0 0 0 DPAWWVCAIAAIECSDV 390 38 634 2330 12368 7673 8315 8 0 0 0 0 0 0 DPAWWVCAVAAIECQEY 391 3 47 258 1258 822 819 0 0 0 0 0 0 0 DPAWWTCIIAALECEEQ 392 0 0 51 193 195 102 0 0 0 0 0 0 0 DPAWYECIVAAIECVEY 393 0 0 79 131 54 115 0 0 0 0 0 0 0 ClusterC52 TargetConcentration Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK BLANK BLANK BLANK PECLEAFFDCR 396 0 9 0 84 344 328 0 0 0 0 0 0 0 MACVDAFFSC 397 0 0 1 17 320 16 0 0 0 0 0 0 0 PCLDAFFYCVT 398 0 0 0 7 18 51 0 0 0 0 0 0 0 PCLEAFFSCVY 399 0 0 0 61 314 257 0 0 0 0 0 0 0 ECLSAFFTCV 400 0 0 0 57 452 284 0 0 0 0 0 0 0 PMACLSAFFTC 401 0 0 0 16 114 176 0 0 0 0 0 0 0 FECLDAFFSC 402 0 0 0 49 982 405 0 0 0 0 0 0 0 CLDAFFACER 403 0 0 0 4 7 408 0 0 0 0 0 0 0 PECLVAFFSCV 404 0 1 9 9 422 142 0 0 0 0 0 0 0 PSCLSAFFSCY 405 0 1 0 14 722 485 0 0 0 0 0 0 0 PCMDAFFSCVW 406 0 0 1 0 39 8 0 0 0 0 0 0 0 PFECLSAFFSC 407 0 0 0 1 212 44 0 0 0 0 0 0 0 PTCLNAFFACE 408 0 0 0 1 109 35 0 0 0 0 0 0 0 YECLEAFFNC 409 0 0 0 2 208 193 0 0 0 0 0 0 0 PACVSAFFACL 410 0 0 0 0 151 19 0 0 0 0 0 0 0 CLSAFFMCSS 411 0 0 0 0 90 50 0 0 0 0 0 0 0 WSCLSAYFSC 412 0 0 0 0 19 189 0 0 0 0 0 0 0 CLSAFFSCTY 413 29 0 0 0 111 330 0 0 0 0 0 0 0
Example 6. Stapled Peptides Binding PPIA
[1466] In some embodiments, the present disclosure provides stapled peptides that bind peptidyl-prolyl cis-trans isomerase cyclophilin A, PPIA. In some embodiments, the present disclosure provides stapled peptides that modulate PPIA activities. PPIA is reported to bind non-alpha-helical peptide substrates and catalyzes the cis-trans isomerization of proline residues.
[1467] In some embodiments, peptidyl-prolyl cis-trans isomerases (PPIases) are reported to be a family of enzymes that recognize a diverse range of proline-containing polypeptide substrates, and are targets of the trimerizer natural products CsA and Sanglifehrin A that bind PPIA to form ternary complexes with the proteins Calcineurin and IMPDH2, respectively.
[1468] In some embodiments, the present disclosure provides a pair of shifted clusters that competed with CsA both in the phage screen and in in vitro biochemical assays. Co-crystal structures of Helicons from both Clusters C53 and C54 confirmed that they can bind the site ordinarily occupied by peptide substrates or natural products, providing another instance where provided technologies were able to identify an alpha-helical binding solution to a site ordinarily recognized by peptides in a non-alpha-helical conformation. A PPIase assay showed that these Helicons inhibit PPIA activity, as expected given their orthosteric binding mode, demonstrating the ability of Helicons to directly block enzymatic function.
[1469] Certain results are presented in
[1470] Stapled peptides of certain PPIA binding clusters are presented below as examples:
TABLE-US-00025 ClusterC53 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK Cyclosporin DPAYYDCHIAAYFCRTQ 414 16 62 77 106 93 131 0 0 0 0 0 DPARADCHVAAYWCYHR 415 0 1 6 6 7 6 0 0 0 0 0 DPASYDCHVAAYFCNFA 416 6 10 176 197 190 227 0 0 0 0 0 DPAAFSCHVAAWQCHTL 417 0 1 156 199 142 190 0 0 0 0 0 DPALFDCHIAAYQCIFY 418 0 0 25 29 20 55 0 0 0 0 0 DPARSDCHVAAYWCYHQ 419 0 0 4 5 6 6 0 0 0 0 0 DPATMNCHVAAYFCIVE 420 0 0 10 13 10 19 0 0 0 1 0 DPARADCQVAAYWCYHQ 421 0 3 13 16 20 21 0 0 0 0 0 DPARTDCHVAAYWCYHQ 422 0 5 13 14 18 25 0 0 0 0 0 DPAQLNCHVAAFFCFQS 423 0 0 21 27 125 8 0 0 0 0 0 DPARADCHVAAYWCYQQ 424 0 1 6 8 10 10 0 0 0 0 0 DPARADCRVAAYWCYHQ 425 0 1 6 6 11 7 0 0 0 0 0 DPAAHDCHIAAYWCLNY 426 0 7 116 208 201 121 0 0 0 0 0 DPAQHDCHVAAYQCIWE 427 0 52 273 416 616 456 0 0 0 0 0 DPARADCHVAAYWCHHQ 428 0 0 4 5 8 7 0 0 0 0 0 DPARYSCHVAAYECILN 429 0 0 17 14 41 27 0 0 0 0 0 DPARADCHVAAYWCYHE 430 0 1 4 7 9 7 0 0 0 0 0 DPAQADCHIAAYLCLFD 431 0 0 10 19 25 18 0 0 0 0 0 DPARAACHVAAYWCYHQ 432 0 1 3 7 6 7 0 0 0 0 0 ClusterC54 TargetConcentration Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK PNCHVMAYWCW 434 0 0 1 8 10 7 0 0 0 PNCHVEAYFCM 435 0 1 8 68 98 63 0 0 0 PHDCHVNAWYC 436 0 0 0 26 21 45 0 0 0 PDCHVAAYICY 437 0 0 3 38 54 44 0 0 0 PDCHLNAYWCM 438 0 0 1 47 47 115 0 0 0 PTCHIDAWVCY 439 0 0 0 12 18 26 0 0 0 PFCHIEAYYCW 440 0 0 0 14 46 22 0 0 0 PNCHLYAWECY 441 0 0 0 4 5 19 0 0 0 PDCHIYAFYCL 442 0 0 1 5 8 18 0 0 0 PDCHVAAWMCF 443 0 0 0 8 16 26 0 0 0 PTCHIHAYHCY 444 0 0 3 5 31 8 0 0 0 PDCHIASWFCS 445 0 0 0 3 25 5 0 0 0 PDCHIQAYICH 446 0 0 0 4 6 35 0 0 0 PDCHIRAYVCH 447 0 0 1 8 11 120 0 0 0 PNCHVEAWYCI 448 0 0 0 1 12 12 0 0 0 PDCHIRAYMCN 449 0 0 0 0 20 26 0 0 0 PDCHIAAWYCR 450 0 0 0 0 11 10 0 0 0 PECHVEAYFCE 451 0 0 0 0 24 22 0 0 0 PTCHVQAWYCW 452 0 0 0 0 10 29 0 0 0 PYDCHIAAWMC 453 0 0 0 0 20 35 0 0 0
Example 7. Stapled Peptides Binding PD-L1
[1471] In some embodiments, the present disclosure provides stapled peptides that bind PD-L1. In some embodiments, the present disclosure provides stapled peptides that modulate PD-L1 activities. In some embodiments, the present disclosure provides stapled peptides that bind extracellular domains or (ECDs). In some embodiments, the present disclosure provides stapled peptides that bind extracellular domains or (ECDs) of PD-L1.
[1472] It has been reported that ECD of the transmembrane protein Programmed cell death 1 ligand 1 (PD-L1) can bind to the ECD of Programmed cell death protein 1 (PD-1) to suppress T-cell function. To the Applicant's knowledge, the PD-L1 ECD has not been shown to bind a-helices. In some embodiments, the present disclosure provides two clusters of Helicons (C61 and C62) that bind PD-L1. In some embodiments, they compete with PD-1. In some embodiments, on-phage competition was confirmed. Among other things, in vitro competition enzyme-linked immunoassay (ELISA) confirmed that FP28132 from Cluster C61 compete with PD-L1. FP30790 from Cluster C62 did not show competition in the utilized ELISA format, but competition SPR confirmed that PD1 blocks its binding to PD-L1. Co-crystal structures of PD-L1 with these two Helicons showed that the two clusters both engage the PD-1 binding surface of PD-L1, but occupy distinct alpha-helical sites on it.
[1473] It was observed that an asymmetric unit of the FP28132/PD-L1 co-crystal structure contained a symmetric dimer of two FP28132/PD-L1 complexes. Two other Helicons, FP28135 and FP28136 derived from the same PD-L1-binding phage cluster, were also structurally characterized and a similar assembly was observed. An extensive series of contacts between both the two Helicon protomers and the two PD-L1 protomers were shown. To assess whether this is an indication that the FP28132/PD-L1 complex is a dimer in solution, analytical size-exclusion chromatography (SEC) and time-resolved fluorescence energy transfer (TR-FRET) experiments were performed with PD-L1 in the presence and absence of FP28132, FP30790, a non-PD-L1 binding mutant of FP28132 that differs by one residue (FP28141), and BMS-1001, a small-molecule PD-L1 binder that has also been shown to induce protein dimerization. Evidence of PD-L1 dimerization in the presence of both BMS-1001 and FP28132, but not in the presence of FP30790 or FP28141, was observed. Among other things, these findings demonstrate that the provided technologies can provide multiple a-helix binding sites to a relatively small ECD that was not previously known to have any, as well as Helicons that can induce protein dimerization.
[1474] Certain results are presented in
[1475] Stapled peptides of certain PD-L1 binding clusters are presented below as examples:
TABLE-US-00026 ClusterC61 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK PD1_300 DPALWQCVFAARSCYEE 454 667 1034 1256 1411 599 730 6 0 0 0 496 DPAVWACTFAARYCHEA 455 0 26 269 259 105 91 0 0 0 0 0 DPATWRCVFAARACSMQ 456 0 0 0 121 7 75 0 0 0 0 0 DPALWMCVFAARQCYES 457 97 3 3 77 7 83 1 0 0 0 4 ClusterC62 TargetConcentration Competitor Sequence SEQIDNO: 10 30 100 300 1000 1000 BLANK BLANK BLANK BLANK PD1_300 DPAAADCQWAAFLCRVY 458 0 0 12 47 60 62 0 0 0 0 0 DPAYQTCWWAAYICAQY 459 0 0 0 5 6 5 0 0 0 0 0 DPAVDDCWVAATICRIM 460 0 0 0 13 12 31 0 0 0 0 0 DPASEDCRWAAFLCAHY 461 0 0 9 110 191 289 0 0 0 0 0 DPAIEECRFAAFQCMYA 462 0 0 0 2 7 5 0 0 0 0 0 DPAIADCRFAAFRCMYA 463 0 0 0 2 12 35 0 0 0 0 0 DPAVQSCWWAAYTCAIQ 464 0 0 0 2 23 38 0 0 0 0 0 DPASSDCSWAAFRCMFS 465 0 0 0 1 15 14 0 0 0 0 0 DPAVDYCRFAAFSCQWA 466 0 12 0 0 13 19 0 0 0 0 0 DPAIRDCAWAAFLCMHE 467 0 0 0 5 18 17 0 3 0 2 0
Example 8. Trimerizers
[1476] In some embodiments, the present disclosure provides technologies for designing, identifying, characterizing, producing and using stapled peptides that can bridge more than one target of interest. In some embodiments, the present disclosure provides trimerizers that can bridge targets of interest. Certain useful technologies are presented below as examples.
[1477] Naive Phage Library: In some embodiments, collections of stapled peptides, etc. phage-displayed peptide libraries are constructed using the filamentous bacteriophage vector M13KE (New England Biolabs, Ipswich, MA). In some embodiments, protocol guidelines in the New England Biolabs Ph.D. Peptide Display Cloning System kit were utilized. Briefly, library oligonucleotides are chemically synthesized using a mix of trimer phosphoramides (Glen Research, Sterling, VA) without codons for cysteine, lysine, proline, and glycine, annealed, extended, and ligated into a digested M13KE vector. KpnI and EagI sites are used to digest M13KE for 5 h at 37 C. The insert coding strand contains the library sequence, 5-CATGCCCGGGTACCTTTCTATTCTCACTCTGCGGATCCGGCGXXXTGCXXGCAGCAXXTGTXXXGGTGGTTCTGGCTGGGGTCGTGGTTC-3 (SEQ ID NO: 21), where X represents a single trimer phosphoramide incorporation, flanked by KpnI site. The antisense strand complements the 3-end of the insert coding strand to allow Klenow extension, 5-CATGTTTCGGCCGAACCACGACCTGCGCCAGAACCAC-3 (SEQ ID NO: 22). The antisense strand possesses an EagI site. Annealed library inserts are digested with EagI and KpnI for 5 h at 37 C. The digested products are purified using Monarch PCR and DNA cleanup kit (New England Biolabs, Ipswich, MA), followed by T4 ligation (New England Biolabs, Ipswich, MA). The resulted library-containing vector is transformed into E. coli strain ER2738 (Lucigen, Middleton, WI) by electroporation. Post-rescue culture is used to determine library diversity. Phage particles containing library members are amplified by adding the post-rescue electroporated cells to a 500 mL robustly growing at 37 C. E. coli culture at early-lag phase (OD600=0.01). Phage propagation continues for 5 h in LB media supplemented with 100 uM of MgCl.sub.2 and CaCl.sub.2, with shaking at 37 C. E. coli cells are pelleted at 5000g and the phage particles are precipitated from the supernatant by addition of 1/5 volume of 20% (w/v) polyethylene glycol 8000, 2.5 M NaCl, followed by overnight incubation at 4 C., pelleted the second time at 5000g, and resuspended in Tris-buffered saline (TBS). Phage-displayed peptide libraries are further purified by repeating the precipitation, pelleting, and resuspension steps. Phage-displayed peptide libraries are covalently crosslinked (stapled) by diluting the phage particle solution in 1TBS to an OD.sub.600 of 1.0 and adding dithiothreitol to a concentration of 1 mM, followed by dialysis against 100 volumes of 20 mM NH.sub.4CO.sub.3, 2 mM EDTA, pH -8 for 30-60 min, followed by addition of the dialyzed phage to a solution of crosslinker prepared in 20 mM NH.sub.4CO.sub.3, 2 mM EDTA, pH -8 (final crosslinker concentration is 200 uM, note that crosslinker may not dissolve completely, in some embodiments, brief sonication immediately prior to mixing with phage is performed to disperse the solid into a fine suspension) and incubation with rotation for 2 h at 32 C. Excess crosslinker is removed first by pelleting at 5000g and decanting, followed by addition of dithiothreitol to a concentration of 0.25 mM with incubation for 10 min, and then addition of iodoacetamide to a concentration of 0.75 mM with incubation for a further 10 min. Ellman's reagent (5,5-dithiobis-(2-nitrobenzoic acid) can be used to track the quenching of DTT until all thiols are capped. Phage particles are further purified by repeating the precipitation, pelleting, and resuspension steps described above, then are stored as solutions in 50% v/v glycerol in TBS at 80 C. at >10.sup.12 pfu/mL. Finally, individual phage library members are characterized by DNA sequencing. Well-separated blue plaques are picked from the LB/IPTG/Xgal Agar plates in 50 L of water. 2 L of a resuspended template is mixed with 23 L of the amplification master mix containing OneTaq DNA polymers (NEB, Ipswitch, MA) and two 10 uM M13KE sequence-specific amplification primers (NEB, Ipswitch, MA). Routine PCR is performed, and samples are submitted for standard Sanger sequencing (GENEWIZ, Cambridge, MA). Prior to library screening, deep sequencing (as described below) of the library is preformed to ensure that it is high in sequence diversity.
[1478] Trimerizer Library: Following identification of peptide clusters specific for a first target of interest, e.g., a presenter protein of interest, based on a naive presenter screen, trimerizer library oligonucleotides are designed. First target of interest, e.g., presenter, specific clusters of various sizes can be used, ranging in size from 10-mer to 20-mer. As an example of a Trimerizer library design, a design based on a presenter specific 20-mer cluster, X.sub.1X.sub.2X.sub.3X.sub.4W.sub.5E.sub.6C.sub.7X.sub.8E.sub.9A.sub.10A.sub.11(F/I/L/M).sub.12X.sub.13C.sub.14X.sub.15(F/Y).sub.16(F/Y).sub.17X.sub.18X.sub.19X.sub.20 (SEQ ID NO: 23), is used. Briefly, codons of conserved or semi-conserved residues (enriched amino acid residues for a first target of interest) responsible for binding with a first target of interest, e.g., a presenter protein, are fixed or partially randomized to bias the library for retained affinity towards a first target of interest, e.g., a chosen presenter protein. In some embodiments, for partial randomization, semi-degenerate codons are used to randomize to sample a fixed subset of residues conserved within that position reducing the total sequence space required for full randomization at every position. For example, in position 12, which has four potential residues (F/L/I/M), semi-degenerate codon, WTK, is used where W represents A or T and K represents G or T, coding for phenylalanine (TTT), leucine (TTG), methionine (ATG), and isoleucine (ATT), thus representing all residues observed within a identified cluster specific for a first target of interest, e.g. a presenter. For position 16 and 17 within the example cluster, semi-degenerate codons, TWT, are used coding for tyrosine (TAT) and phenylalanine (TTT) in a similar manner presenting all residues observed within an identified cluster specific for a first target of interest, e.g., a presenter. All other residues within the displayed peptide with no apparent preference for binding to a first target of interest, e.g., a presenter, (represented as Xs within the cluster) are randomized to any other amino acid, e.g., except cysteine, lysine, proline and glycine. Trimerizer libraries are built using multiple oligonucleotides from various designs based on specific binding sequences for a first target of interest, e.g., a presenter. Library oligonucleotides can be chemically synthesized using a mix of trimer phosphoramides (Glen Research, Sterling, VA) lacking codons for amino acids that are not to be included, e.g., cysteine, lysine, proline, and glycine, annealed, extended, and ligated into a digested with KpnI and EagI restriction enzymes M13KE vector. Example oligonucleotide insert coding strand containing the Trimerizer library sequence is shown below. 5-CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXTGGGAATGTXGAAGCAGCAWTKXTGTXTWTTWTXXXGGTGGTTCTGGCGCAGGTCGTGGTTC-3 (SEQ ID NO: 24), where X represents a single trimer phosphoramide incorporation, flanked by KpnI site. The antisense strand complements the 3 end of the sense strand to allow Klenow extension, 5-CATGTTTCGGCCGAACCACGACCTGCGCCAGAACCAC-3 (SEQ ID NO: 22). The antisense strand possesses an EagI site. Trimerizer Library construction protocol can follow the previously described Naive Phage Library construction protocol.
[1479] Naive Library Presenter Screening: In some embodiments, phage screening is performed using biotinylated proteins bound to streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher Scientific, Waltham, MA). About 10.sup.10 phage particles are added to each phage screening sample, to ensure approximately 100 copies of each of the 10 library members. Peptide-displayed phage libraries are incubated with streptavidin magnetic beads for 1 h at room temperature in a buffer of 1TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 5% (w/v) nonfat milk to remove bead-binding library members. For each screening condition, 100 L of 2 uM biotinylated desired presenter protein is captured with 0.5 mg of streptavidin-coated magnetic beads that have been previously blocked with 1% BSA, 0.1% Tween-20, 2% glycerol in 1TBS pH 7.4 at room temperature for 15 min in 96-well plates, then the supernatant is removed using a plate magnet and the beads are promptly but gently resuspended in 50 L of the same buffer. 150 L of the depleted phage library is added to each well for 200 uL final volume, plates are sealed, and the screening reactions are incubated at room temperature for 45 min, with rotation to maintain beads in solution. Inspection of these solutions is performed to confirm that beads have not aggregated or crashed out of solution, which can be indicative of protein aggregation. Biochemical tests should be performed to ensure that target proteins are stable under the conditions of the screen, and conditions should be adjusted accordingly if required. Following binding, beads are washed 5 times with ice-cold 1TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 2% (w/v) glycerol. This can be performed with an automated bead handler such as a KingFisher (Thermo Fisher) or manually. Target-bound phage library members are then directly processed for NGS.
[1480] Trimerizer Phage Screening: In some embodiments, trimerizer phage screening is performed using the similar procedure described above. In some embodiments, a key difference is that Trimerizer library is incubated with a first target of interest, e.g., a presenter protein, after removal of the bead-binding phage library members prior to the incubation with biotinylated proteins bound to streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher Scientific, Waltham, MA). To identify first target of interest (e.g., presenter)-dependent peptide members, in some embodiments, a target protein (e.g., a second target of interest) is screened with depleted peptide library in the presence and absence of a first target of interest, e.g. a presenter protein. In some embodiments, prior to addition to captured protein, depleted peptide-displayed phage library is split into two portions, and a first target of interest, e.g., a presenter protein is added to one portion to a final concentration of 10 uM. 150 L of the depleted peptide-displayed phage library without presenter protein is then added to replicate highest concentration of a target protein and blank wells for 200 uL final volume. To remaining wells, 150 L of the depleted peptide-displayed phage library mixed with 10 uM of a first target of interest, e.g., a presenter protein, is added to 50 L of a target of interest, plates are sealed, and the screening reactions are incubated at room temperature for 45 min, with rotation to maintain beads in solution. The rest of the experiment is performed similar to the above described phage screening with the exception of the addition of a first target of interest at a suitable concentration, e.g., a presenter protein at a final concentration of 10 uM to the wash buffer (ice-cold 1TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 2% (w/v) glycerol).
[1481] Next-Generation Sequencing: To sequence the phage-displayed peptide library members, phage particles are denatured from beads at 95 C. for 15 min in 25 mM Tris pH 8, 50 mM NaCl, 0.5% Tween-20. Prior to boiling, 10,000 copies of a phage clone of known sequence (not a library member) are spiked in to each well to enable cross-well normalization of sequence reads. A two-step low-cycled PCR is performed to introduce Illumina adaptors and 10 bp TruSeq DNA UD Indexes (Illumina, San Diego, CA) to the 3 and 5 ends of amplicons with M13KE Forward and M13KE Reverse primers (M13KE Forward: 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCGCAATTCCTTTAGTGG-3 (SEQ ID NO: 25) and M13KE Reverse: 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATTTTCTGTATGGGATTTTGCTAA-3 (SEQ ID NO: 26)) similar to Illumina's 16S Metagenomic Sequencing Library Preparation protocol. The NGS library is sequenced by an Illumina NovaSeq platform using a 2150 bp high-output kit (Illumina, San Diego, CA).
[1482] Hit ID and Clustering: NGS reads are trimmed for quality (Phred score >18) and filtered for sequences that matched the design of the phage library. Counts for each unique sequence are tallied, and then normalized by the counts of the spike-in sequence added to each sample. A metric called Hit Strength is computed for each sequence as the fold change between the normalized counts in the highest target concentration sample with presenter and the normalized counts in the target (no presenter) samples (averaged across experimental replicates). By using target wells with no first target of interest, e.g., presenter as blanks, presenter-dependent binding is observed. This approach eliminates sequences that show binding to target (e.g., a second target of interest) alone or binding to a free first target of interest, e.g., presenter, alone. 0 counts are observed for a sequence in target only blank samples, a count of 0.5 is used to prevent dividing by zero.
[1483] Sequences with a hit strength greater than 5 are subjected to hierarchical clustering to identify sequence families. Pairwise distances between sequences i and j are computed using
where score.sub.ij is the alignment scores based on a modified BLOSUM62 substitution matrix. In some embodiments, tryptophan-tryptophan match score was decreased from 11 to 7 in the BLOSUM62 substitution matrix to prevent overly-biasing clustering towards tryptophans. Hierarchical clustering using average linkage is used to group the sequences into families. To avoid clustering using a large number of sequences, which is computationally intensive and can make it difficult to identify small clusters, in some embodiments, multiple rounds of clustering were performed. First, sequences were sort by descending hit strength. The top 1000 (first round of clustering), top 2000 (2.sup.nd round of clustering), or top 3000 (3.sup.rd round of clustering) sequences, were then taken, and was subjected to clustering as described above. Clusters of sequences with high sequence similarity (sequence families) are identified at each round and removed from the pool of sequence for subsequent rounds. Sequences subjected to 3 rounds of clustering without falling into a sequence family are similarly dropped from subsequent rounds of clustering under the assumption that they do not belong to a sequence family. The process is halted after 10 rounds of clustering, or when no sequences remained in the list.
[1484] Certain useful technologies and data are described in
Example 9. Griptides
[1485] In some embodiments, the present disclosure provides technologies for designing, identifying, characterizing, producing and using stapled peptides, e.g., those comprising two stapled peptide moieties or alpha-helical structures linked together. In some embodiments, such griptides can provide high affinity. In some embodiments, hydrophobic interface residues support dimer formation. In some embodiments, surface-exposed residues are diversified into a library as described herein, e.g., with about 10 members or more. Certain useful technologies are presented below as examples.
[1486] Griptide Phage Library construction: In some embodiments, collections of griptides, e.g., phage-displayed Griptide libraries are constructed using a modified phagemid vector according to manufacturer's protocol (Antibody Design Labs, San Diego, CA). pADL-100 phagemid vector (Antibody Design Labs, San Diego, CA), is modified to include a FLAG-tag (DYKDDDDK (SEQ ID NO: 29)) sequence, two additional restriction sites (KpnI and Sac7), a placeholder sequence, and an extended linker sequence between the 3-end of the leader sequence and 5-end of the minor coat protein, pIII, by utilizing available restriction cutting sites NocI and SpeI. Griptide DNA-library oligonucleotides are chemically synthesized using trimer phosphoramides technology (Glen Research, Sterling, VA) without codons for cysteine, annealed, extended, and ligated into a digested modified pADL-100 phagemid vector. The antisense strand contains the library sequence, 5-GGGCCGGTACCGGCGGCCCGAGCCAGCCGGCGTGTCCGGGCGATGATGCGAGCATTXGATCTGXXTATXXXCTGXXTATCTGXGCGGTGGCGGGTGGTGAGCTCATTTT-3 (SEQ ID NO: 30), where X represents a single trimer phosphoramides, flanked by introduced SacI site. The sense strand complements the 3 end of the antisense strand to allow Klenow extension, 5-AAAATGGTACCGGCGGCCCGAGCCAGCCGGCGTGTCCGGGCGATGATGCGAGCATT-3 (SEQ ID NO: 31). The sense strand possesses introduced KpnI site. Annealed and extended Griptide DNA-library inserts together with the modified phagemid vector are digested with KpnI and SacI for 5 h at 37 C., and the digested products are purified using Monarch PCR and DNA cleanup kit (New England Biolabs, Ipswich, MA). The digested modified phagemid vector dephosphorylated using calf intestine phosphatase, CIP, (New England Biolabs, Ipswich, MA) and purified using Monarch PCR and DNA cleanup kit (New England Biolabs, Ipswich, MA). Purified DNA-library inserts and modified phagemid vector ligated by T4 ligase (New England Biolabs, Ipswich, MA). The resulted Griptide DNA-library-containing phagemid vector is transformed into E. coli electrocompetent TG1 cells (Lucigen, Middleton, WI) using established electroporation procedures. Following a recovery step, a portion of transformed TG1 containing Griptide DNA-libraries is serial diluted and plated to determine library diversity. The remaining recovered TG1 containing Griptide DNA-libraries are plated on LB-agar containing ampicillin (100 g/mL) and glucose (2% w/v) overnight at 37 C. The following day, TG1 containing Griptide DNA-libraries scrapped from the plate into 2YT media containing 2% (w/v) glucose and subcultured in 62.5 mL 2YT containing ampicillin (100 g/mL) and 2% glucose with shaking at 37 C. until OD.sub.600 of 0.4 to 0.5. TG1 containing Griptide DNA-libraries superinfected with pIII-deficient helper phage, CM13d3 (Antibody Design Labs, San Diego, CA), at a MOI of 10 for 30 min infection without shaking at 37 C., followed by 30 min infection with shaking at 37 C. Infected TG1 containing Griptide DNA-libraries are pelleted to remove glucose and resuspended in 500 mL 2YT containing kanamycin (50 ug/mL), ampicillin (100 g/mL) and 0.1 mM IPTG. Phage-displayed Griptide libraries are amplified by expanding the E. coli culture for 5 h with shaking at 30 C., pelleting E. coli cells at 5000g, precipitating phage particles from the supernatant by addition of 1/5 volume of 20% (w/v) polyethylene glycol 8000, 2.5 M NaCl, followed by overnight incubation at 4 C., pelleting at 5000g, and resuspending in Tris-buffered saline (TBS). Phage-displayed Griptide libraries are further purified by repeating the precipitation, pelleting, and resuspension steps. Purified phage-displayed Griptide libraries are stored as solutions in 50% (v/v) glycerol in TBS at 80 C. at >10.sup.12 pfu/mL. Individual phage library members are characterized by Sanger sequencing followed by NGS. Well-separated colonies are picked from the library tittering plate in 20 L of water. 2 L of a resuspended template is mixed with 23 L of the amplification master mix containing OneTaq DNA polymers (NEB, Ipswitch, MA) and two 10 uM phagemid sequence-specific amplification primers (Forward: 5-GTGGAATTGTGAGCGGATAACAATTTG-3 (SEQ ID NO: 32) and Reverse: 5-GCGTAACGATCTAAAGTTTTGTCG-3 (SEQ ID NO: 33)). Routine PCR is performed. Samples are submitted for standard Sanger sequencing (GENEWIZ, Cambridge, MA). Prior to library screening, deep sequencing (as described below) of the library is performed to ensure that it is high in sequence diversity and is not dominated by a small number of individual sequences, which can occur if construction is not performed properly.
[1487] Phage Screen: In some embodiments, phage screening is performed using biotinylated proteins bound to streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher Scientific, Waltham, MA). 10.sup.10 phage particles are added to each phage screening sample, to ensure approximately 100 copies of each of the 10 library members. Phage-display libraries are incubated with streptavidin magnetic beads for 1 h at room temperature in a buffer of 1TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 5% (w/v) nonfat milk to remove bead-binding library members. For each screening condition, 100 L of 2 uM biotinylated protein is captured with 0.5 mg of streptavidin-coated magnetic beads that have been previously blocked with 1% BSA, 0.1% Tween-20, 2% glycerol in 1TBS pH 7.4 at room temperature for 15 min in 96-well plates, then the supernatant is removed using a plate magnet and the beads are promptly but gently resuspended in 50 L of the same buffer. 150 L of the depleted phage library is added to each well for 200 uL final volume, plates are sealed, and the screening reactions are incubated at room temperature for 45 min, with rotation to maintain beads in solution. Inspection of these solutions is performed to confirm that beads have not aggregated or crashed out of solution, which can be indicative of protein aggregation. Biochemical tests should be performed to ensure that target proteins are stable under the conditions of the screen, and conditions should be adjusted accordingly if required. Following binding, beads are washed 5 times with ice-cold 1TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 2% (w/v) glycerol. This can be performed with an automated bead handler such as a KingFisher (Thermo Fisher) or manually. Target-bound phage library members are then directly processed for NGS.
[1488] Next Generation Sequencing: To sequence the phage-displayed Griptide library members, phage particles are denatured from beads at 95 C. for 15 min in 25 mM Tris pH 8, 50 mM NaCl, 0.5% Tween-20. Prior to boiling, 12,500 copies of a phage clone of known sequence (not a library member) are spiked in to each well to enable cross-well normalization of sequence reads. A two-step low-cycled PCR is performed to introduce Illumina adaptors and 10 bp TruSeq DNA UD Indexes (Illumina San Diego, CA) to the 3 and 5 ends of amplicons with modified phagemid vector forward and pADL-100 reverse primers (Forward: 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCGGATTATAAAGATGACGATGACAAAGG-3 (SEQ ID NO: 34) and Reverse: 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGTTAGTAAATGAATTTTCTGTATGAGG-3 (SEQ ID NO: 35)) similar to Illumina's 16S Metagenomic Sequencing Library Preparation protocol. The NGS library is sequenced by an Illumina NovaSeq platform using a 2150 bp high-output kit (Illumina San Diego, CA).
[1489] Hit ID and Clustering: NGS reads are trimmed for quality (Phred score >18) and filtered for sequences that matched the design of the phage library. Counts for each unique sequence are tallied, and then normalized by the counts of the spike-in sequence added to each sample. A metric called Hit Strength is computed for each sequence as the fold change between the normalized counts in the highest target concentration sample and the normalized counts in the blank bead samples (averaged across experimental replicates). When 0 counts are observed for a sequence in blank bead samples, a count of 0.5 is used to prevent dividing by zero. Sequences with a hit strength greater than 5 are subjected to hierarchical clustering to identify sequence families. Pairwise distances between sequences i and j are computed using
where score.sub.ij is the alignment scores based on a modified BLOSUM62 substitution matrix. Tryptophan-tryptophan match score is decreased from 11 to 7 in the BLOSUM62 substitution matrix to prevent overly-biasing clustering towards tryptophans. Hierarchical clustering using average linkage is used to group the sequences into families. To avoid clustering using a large number of sequences, which is computationally intensive and can make it difficult to identify small clusters, multiple rounds of clustering is performed. First, sequences are sorted by descending hit strength. The top 1000 (first round of clustering), top 2000 (2.sup.nd round of clustering), or top 3000 (3.sup.rd round of clustering) sequences, are then taken and are subjected to clustering as described above. Clusters of sequences with high sequence similarity (sequence families) are identified at each round and removed from the pool of sequence for subsequent rounds. Sequences subjected to 3 rounds of clustering without falling into a sequence family are similarly dropped from subsequent rounds of clustering under the assumption that they do not belong to a sequence family. The process is halted after 10 rounds of clustering, or when no sequences remained in the list.
[1490] Certain useful technologies and data are described in
Example 10. Certain Useful Technologies
[1491] Various technologies can be utilized to design, identify, characterize, manufacture, assess, and use provided technologies in accordance with the present disclosure. Certain technologies are described in U.S. Ser. No. 11/198,713, US 20210179665, WO 2021119537, WO 2021188659, WO 2022020651, or WO 2022020652, the entirety of each of which is incorporated herein by reference. Certain useful technologies are described below as examples.
[1492] Crosslinker Synthesis: N,N-(1,4-phenylene)bis(2-bromoacetamide) was synthesized by adding bromoacetyl bromide (1.1 mmol) to a solution of p-phenylenediamine (0.5 mmol) in dichloromethane (DCM) (5 mL), dry pyridine (1.2 mmol), and 4-(dimethylamino)pyridine (13 umol) at 0 C. The mixture was stirred at 25 C. for 3 hours. The reaction mixture was filtered and washed with DCM to obtain N,N-(1,4-phenylene)bis(2-bromoacetamide) as an off-white solid. .sup.1H NMR (DMF-d7, 500 MHz): 4.12 (s, 4H), 6 7.67 (s, 4H), 6 10.47 (s, 2H).
[1493] Helicon Synthesis and Cysteine Bisalkylation: Helicons were synthesized at 100 umol scale on Rink Amide resin (0.5 mmol/g) using standard Fmoc-based solid phase peptide synthesis workflows. Specifically, the Fmoc-protected resin was swollen using N,N-dimethylformamide (DMF) before coupling the first amino acid. Fmoc-deprotection was performed by treating the resin with 20% (v/v) piperidine in DMF. The amino acid coupling was performed in DMF with 4 equivalents of Fmoc-protected amino acid, 8 equivalents of ethyl cyanohydroxyiminoacetate (oxyma), and 4 equivalents of N,N-diisopropylcarbodiimide (DIC). The peptides were globally deprotected and cleaved off the resin by treating the resin with a cleavage cocktail composed of 92.5% (v/v) trifluoroacetic acid (TFA), 2.5% (v/v) water, 2.5% (v/v) triisopropylsilane, and 2.5% (v/v) mercaptopropionic acid for 2 hours. The crude peptide was precipitated by adding ice-cold isopropyl ether to the concentrated cleavage cocktail. The precipitated peptide was pelleted by centrifugation and dried under nitrogen.
[1494] The crude peptide was dissolved in DMSO before proceeding with the cysteine bisalkylation/stapling reaction. The DMSO stock was diluted in a 2:1 solvent mixture of acetonitrile and 50 mM ammonium hydroxide. The pH of the solution was adjusted to 8.5 using N, N-Diisopropylethylamine (DIPEA). 1.3 equivalents of the alkylating agent, N, N-(1,4-phenylene)bis(2-bromoacetamide) in DMF were added to the crude peptide solution. The reaction mixture was stirred at room temperature for at least two hours or until the reaction had been completed. The progress of the reaction was monitored by analytical HPLC and mass spectrometry. The final reaction was quenched by 0-mercaptoethanol before lyophilizing the reaction mixture. The lyophilized reaction mixture was dissolved in DMSO for purification.
[1495] The crude peptide mixtures were purified by preparatory HPLC (solvent A: water with 0.1% (v/v) FA; solvent B: acetonitrile with 0.1% (v/v) FA) using a C18 column. Analytical HPLC and mass spectrometry were used to characterize the peptides. The observed masses of certain final peptide products are presented in Table 1 as examples.
[1496] Circular Dichroism (CD) Spectroscopy: CD spectra of stapled and unstapled peptide pairs were obtained using Aviv Biomedical, Inc. 420 CD spectrometer with peptide concentrations at 25 uM or 50 uM in 20 mM phosphate buffer at pH=7.4. The CD measurements were obtained using a cuvette with a 1 mm pathlength at a fixed temperature of 25 C. Three scans were obtained at every 1-nm interval in the wavelengths ranging from 190 to 260 nm. The buffer background was subtracted from each CD spectrum, followed by smoothing the curves by the moving-means method with a convolution width of 2 data points. Next, the smoothed baseline between 250-260 nm was subtracted from the smoothed spectrum of the sample. Finally, the CD measurements were converted to mean residue molar ellipticity (deg.Math.cm.sup.2.Math.dmol.sup.1) for data visualization. As DPA residues were used as an N-terminal nonhelical cap, they were ignored for the mean residue molar ellipticity calculations. The percent helicity of peptides was calculated using the ratio of [].sub.222/[].sub.max. [].sub.max for the peptides was estimated to be 26964.28 using the formula below.
[1497] Phage Library construction (primers, protocol, crosslinking, and DNA sequencing): Phage-displayed peptide libraries were constructed using the filamentous bacteriophage vector M13KE (New England Biolabs, Ipswich, MA). Protocol guidelines in the New England Biolabs Ph.D. Peptide Display Cloning System kit were followed. Briefly, library of oligonucleotides were chemically synthesized using a mix of trimer phosphoramides (Glen Research, Sterling, VA) lacking cysteine, lysine, proline, and glycine, then annealed, extended, and ligated into a digested M13KE vector. The sense strand contains the library sequence, 5-CATGCCCGGGTACCTTTCTATTCTCACTCTGCGGATCCGGCGXXXTGCXXGCAGCAXXTGTXXXGGTGGTTCTGGCTGGGGTCGTGGTTC-3 (SEQ ID NO: 21), where X represents a single trimer phosphoramide incorporation, flanked by KpnI site. The antisense strand complements the 3 end of the sense strand to allow Klenow extension, 5-CATGTTTCGGCCGAACCACGACCTGCGCCAGAACCAC-3 (SEQ ID NO: 22). The antisense strand possesses an EagI site. Annealed library inserts, along with the M13KE vector, were digested with EagI and KpnI for 5 hours, and the digested products were purified using Monarch PCR and DNA cleanup kit (New England Biolabs, Ipswich, MA), followed by T4 ligation (New England Biolabs, Ipswich, MA). The resulting library-containing vector was transformed into E. coli strain ER2738 (Lucigen, Middleton, WI) by electroporation, reserving and plating post-rescue to determine library diversity. Between 510.sup.7 and 110.sup.8 individual phage clones (established by a traditional plaque assay) were used for library amplification. Phages were amplified by adding the post-rescue electroporated cells to a robustly growing E. coli culture at early-log phase for 5 h in 500 mL LB media supplemented with 100 uM each of MgCl.sub.2 and CaCl.sub.2, with shaking at 37 C. E. coli cells were pelleting at 5000g and supernatant was removed. Phage particles were collected from the supernatant by addition of 1/5 volume of 20% (w/v) polyethylene glycol 8000, 2.5 M NaCl, followed by overnight incubation at 4 C., pelleted at 5000g, and resuspended in Tris-buffered saline (TBS). Phage-displayed Helicon libraries were further purified by repeating the precipitation, pelleting, and resuspension steps. After the final resuspension step, a plaque assay was performed to assess the overall titer of the phage display library. In some embodiments, between 510.sup.12 and 110.sup.13 phage particles in each amplified library were observed.
[1498] Phage-displayed Helicon libraries were covalently crosslinked (stapled) by diluting the phage particle solution in TBS to an OD.sub.600 of 1.0 and adding dithiothreitol to a concentration of 1 mM, followed by dialysis against 100 volumes of 20 mM NH.sub.4CO.sub.3, 2 mM EDTA, pH 8 for 30-60 min, followed by addition of the dialyzed phage to a solution of crosslinker prepared in 20 mM NH.sub.4CO.sub.3, 2 mM EDTA, pH 8 (final crosslinker concentration is 200 uM. As the crosslinker does not completely dissolve in buffer, the solution was sonicated immediately prior to mixing with phage to disperse the solid into a fine suspension) and incubation with rotation for 2 hours at 32 C. Excess crosslinker was removed first by pelleting at 5000g and decanting, followed by addition of dithiothreitol to a concentration of 0.25 mM with incubation for 10 minutes, and then addition of iodoacetamide to a concentration of 0.75 mM with incubation for a further 10 minutes. Ellman's reagent (5,5-dithiobis-(2-nitrobenzoic acid) was used to track the quenching of DTT until all thiols were capped. Phage particles were further purified by repeating the precipitation, pelleting, and resuspension steps described above for purification from E. coli culture, then are stored as solutions in 50% v/v glycerol in TBS at 80 C. at >10.sup.12 pfu/mL. Next-generation sequencing was performed to assess the library quality (details can be found in the Phage NGS section). In some embodiments, between 10.sup.6-10.sup.7 phage particles were sequenced and found that on average, between 95%-98% of all reads have the correct library structure.
[1499] Mass spectrometry analysis of crosslinked phage was performed by adding 15 L of phage samples to 2.5 uL of a solution of Trypsin at 1.0 mg/ml freshly prepared in 5 mM acetic acid and then pH adjusted by mixing 1:1 with 100 mM Tris pH 8. After 1 hour of digestion at room temperature, each cleavage reaction was quenched with 15 L of 20% ACN+1% formic acid, and analyzed a Q-Exactive Plus mass spectrometer equipped with an Ultimate 3000 LC system (Thermo Electron) and a Aeris C18 column (Phenomenex). Finally, individual phage library members were characterized by DNA sequencing. Well-separated blue plaques were picked from the LB/IPTG/Xgal Agar plates in 50 L of water. 2 L of resuspended template was mixed with 23 L of the amplification master mix containing OneTaq DNA polymers (NEB, Ipswitch, MA) and two 10 uM M13KE sequence-specific amplification primers (NEB, Ipswitch, MA). Routine PCR was performed, and samples were submitted for standard Sanger sequencing (GENEWIZ, Cambridge, MA). Prior to library screening, deep sequencing (as described below) of the library were performed to ensure that it is high in sequence diversity and is not dominated by a small number of individual sequences.
[1500] Phage Library Screening: Phage screening was performed using biotinylated proteins bound to streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, Thermo Fisher Scientific, Waltham, MA). About 10.sup.10 phage particles were added to each phage screening sample, to ensure approximately 100 copies of each of the 10.sup.8 library members. Phage display libraries were incubated with streptavidin magnetic beads for 1 hour at room temperature in a buffer made of 1TBS, 1 mM MgCl.sub.2, 1% w/v BSA, 0.1% Tween-20, 0.02% w/v sodium azide, 5% w/v nonfat milk to remove bead-binding library members. Briefly, Dynabeads were prepared in a 15 ml Falcon tube according to a manufacturing protocol from Thermo Fisher Scientific, diluted phage display libraries were added to the magnetic beads. After an incubation period, the tube was placed on a magnet for 1 min to separate bead-bound and non-bead-bound phage library members. Supernatant containing bead depleting phage library was collected and beads were discarded. For each screening condition, 100 L of 2 uM biotinylated protein was captured with 0.5 mg of streptavidin-coated magnetic beads that have been previously blocked with 1% BSA, 0.1% Tween, 2% glycerol in 1TBS pH 7.4 at room temperature for 15 minutes in 96-well plates, followed by removal of the supernatant using a plate magnet and prompt but gentle resuspension of the beads in 50 L of the same buffer. Next, 150 L of the depleted phage library was added to each well for 200 uL final volume, the plates were sealed, and the screening reactions incubated at room temperature for 45 minutes, with rotation to maintain beads in solution. These solutions was inspected to confirm that beads had not aggregated or crashed out of solution, which can be indicative of protein aggregation. Following binding, beads were washed 5 with ice-cold 1TBS, 1 mM MgCl.sub.2, 1% w/v BSA, 0.1% Tween-20, 0.02% w/v sodium azide, 2% w/v glycerol. Washing steps can be performed, as in our case, with an automated bead handler such as a KingFisher (Thermo Fisher) or manually. Target-bound phage library members are directly processed for NGS.
[1501] Phage Next-Generation Sequencing (NGS): In some embodiments, the protocol below is used to perform NGS for newly built phage display libraries and to identify target-bound phage library members after a phage screen. To sequence the phage-displayed peptide library members, phage particles are removed from the beads by a denaturation step at 95 C. for 15 min in 25 mM Tris pH 8, 50 mM NaCl, 0.5% Tween-20. Prior to boiling, 10,000 copies of a phage clone of known sequence (not a library member) are spiked in to each well to enable cross-well normalization of sequence reads. The sequence of the spike-in clone is TCTCACTCTGCGCCGGAATGCATTCTGGATTGCCATGTGGCGCGCGTGTGGGGTGGTTCT (SEQ ID NO: 38). A two-step low-cycled PCR is performed to introduce Illumina adaptors and 10 bp TruSeq DNA UD Indexes (Illumina, San Diego, CA) to the 3 and 5 ends of amplicons with M13KE Forward and M13KE Reverse primers (M13KE Forward: 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCGCAATTCCTTTAGTGG-3 (SEQ ID NO: 25) and M13KE Reverse: 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATTTTCTGTATGGGATTTTGCTAA-3 (SEQ ID NO: 26)) similar to Illumina's 16S Metagenomic Sequencing Library Preparation protocol. The NGS libraries are sequenced by an Illumina NovaSeq platform using a 2x150-bp high-output kit (Illumina, San Diego, CA).
[1502] Hit ID and Clustering: NGS reads were trimmed for quality (Phred score >18) and filtered for sequences that matched the design of the phage library. Counts for each unique sequence were tallied, and then normalized by the counts of the spike-in sequence added to each sample. A metric called Hit Strength was computed for each sequence as the fold change between the normalized counts in the highest target concentration sample and the normalized counts in the blank bead samples (averaged across experimental replicates). When 0 counts were observed for a sequence in blank bead samples, a count of 0.5 was used to prevent dividing by zero. Sequences with a hit strength greater than 5 were then subjected to hierarchical clustering to identify sequence families. Pairwise distances between sequences i and j were computed using
where score.sub.ij is the alignment scores based on a modified BLOSUM62 substitution matrix. Tryptophan-tryptophan match score is reduced from 11 to 7 in the BLOSUM62 substitution matrix to prevent overly biasing clustering towards tryptophans. Hierarchical clustering using average linkage was used to group the sequences into families. To avoid clustering using a large number of sequences, which is computationally intensive and can make it difficult to identify small clusters, multiple rounds of clustering were performed. First, sequences were sorted by descending hit strength. The top 1000 (first round of clustering), top 2000 (2.sup.nd round of clustering), or top 3000 (>3.sup.rd round of clustering) sequences, were taken and subjected to clustering as described above. Clusters of sequences with high sequence similarity (sequence families) were identified at each round and removed from the pool of sequence for subsequent rounds. Sequences subjected to three rounds of clustering without falling into a sequence family were similarly dropped from subsequent rounds of clustering under the assumption that they did not belong to a sequence family. The process was halted after 10 rounds of clustering, or when no sequences remained in the list.
[1503] beta-Catenin Surface Plasmon Resonance (SPR): SPR experiments were performed on a Biacore 8K (Cytiva) instrument at 25 C. Test peptides were diluted into running buffer (50 mM Tris pH 8.0, 300 mM NaCl, 2% glycerol, 0.5 mM TCEP, 0.5 mM EDTA, 0.005% Tween-20, 1% DMSO). Compounds were diluted to 10 uM or 1 uM and serially diluted 1:3 for seven concentrations and two blanks (7-point three-fold peptide dilution series with top concentration=10 mM). Biotinylated -catenin residues 134-665 (Uniprot ID P35222) was immobilized to the active surface of the sensor chip for 25 seconds at 10 uL/min using the Biotin CAPture Kit, Series S (Cytiva) and compounds were injected over the reference and active surfaces for 180 seconds at 65 uL/min then allowed to dissociate for 400 seconds. Results were analyzed using the Biacore Insight Evaluation software, with double-referencing and fitted to a 1:1 binding affinity model.
[1504] beta-Catenin-TCF Competition by Fluorescence Polarization: Compounds at 10 mM in DMSO were serially diluted 1:3 in DMSO for a total of 11 concentrations using a Mosquito LV (SPT Labtech), then diluted 1000-fold in buffer (50 mM HEPES, pH 7.5, 125 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.05% v/v pluronic acid) in duplicate by the Mosquito LV (SPT Labtech) into a black polystyrene 384-well plate (Corning) (11-point three-fold peptide dilution series with top concentration=10 mM) Probe solution (10 nM full-length -catenin (Uniprot ID P35222), mixed with 10 nM 5FAM-labeled TCF4 residues 10-53 (Uniprot ID Q9NQB0) peptide (FP04872) in buffer) was prepared and plated using the MultiDrop Combi (Thermo Fisher) for a total reaction pool of 40 mL. The plate was incubated and protected from light for 1 hour at room temperature prior to read. Reads were performed on a CLARIOstar plate reader (BMG Labtech) with excitation at 485 nm, emission at 525 nm, and cutoff at 504 nm. Data were fitted to a 1:1 binding model with Hill slope using an in-house script.
[1505] beta-Catenin-Axin Competition by Fluorescence Polarization: Compounds at 10 mM in DMSO were serially diluted 1:3 in DMSO for a total of 11 concentrations using the Mosquito LV (SPT Labtech), then diluted 1000-fold in buffer (50 mM HEPES, pH 7.5, 125 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.05% v/v pluronic acid) in duplicate by the Mosquito LV (SPT Labtech) into a black polystyrene 384-well plate (Corning). Probe solution (15 nM full-length -catenin (Uniprot ID P35222), mixed with 20 nM FITC labeled fStAx-33 peptide (FP00013) in buffer) was prepared and plated using the MultiDrop Combi (Thermo Fisher) for a total reaction pool of 40 mL. The plate was incubated protected from light for 1 hour at room temperature prior to read. Reads were performed on a CLARIOstar plate reader (BMG Labtech) with excitation at 485 nm, emission at 525 nm, and cutoff at 504 nm. Data were fitted to a 1:1 binding model with Hill slope using an in-house script.
[1506] Measurement of the Cell Association of Helicons: A Source plate of 47 test compounds was prepared at a concentration of 1 mM in 90% DMSO in a 500 uL 96-well plate (2 replicates for each compound with 2 DMSO blanks). The 96-well format with one compound per well was maintained for all transfers throughout the protocol. A 2000 uL 96-well v-bottom plate (Cells plate) was used to dilute 1.25 uL of compounds from the Source plate into 500 L of Expi293 Expression Medium. Each well also received 500 L of Expi293 cells (Thermo Fisher) for a total concentration of 110.sup.6 cells/mL in 1 mL of Expi293 Expression Medium. Baseline cell health at the time of compound addition was measured using CellTiter-Glo 2.0 Reagent (CTG) and a GloMax Discover reader (Promega). The Cells plate was incubated in an Infors HT Multitron Pro shaking incubator at 1000 rpm, 37 C., 8.0% CO.sub.2, 55% humidity, for 22 hours, along with four plates of DI water to maintain humidity. After 22 hours, the cells were sampled again for post-incubation CTG analysis of cell health. CellTiter-Glo fold-change is calculated as the luminescence readout at T.sub.final divided by the luminescence readout at T.sub.inital (time at which peptide was added). The cells were washed twice with 200 L of Dulbecco's Phosphate Buffered Saline (DPBS) and transferred to a 500 uL 96-well Final Assay plate. After the second wash, the cells were resuspended in 80 L of buffer (90% DPBS, 10% dimethyl sulfoxide (DMSO), 10 uM of a mixture of nonstapled 14-mer peptides with randomized sequences) in the Final Assay plate. Cell lysis was induced by the addition of 240 L of ammonium hydroxide and 2 hours of shaking at 37 C. All solvents were removed via 23 hours in a SpeedVac vacuum concentrator (Thermo Fisher). The dried compounds and cell debris were resuspended using 180 L of resuspension buffer (47.5% Acetonitrile (ACN) with 0.1% formic acid (FA), 47.5% H.sub.2O with 0.1% FA, 5% DMSO) and shaking for 3 hours at 600 rpm. Once resuspended, the cell debris was separated from the resuspended compounds via centrifugation at 3220 rcf for 20 min. A portion of compound-containing supernatant from each well was transferred to a corresponding well in a 384-well plate for mass spectrometry analysis. Matching cell-free wells for all cells wells were plated in the same 384-well plate. The cell-free wells were prepared by adding 1 L of 0.1 mM compounds from the Source plate to 19 L of input buffer (47.5% ACN with 0.1% FA, 47.5% H.sub.2O with 0.1% FA, 5% DMSO, 10 uM of a mixture of nonstapled 14-mer peptides with randomized sequences), then adding 2 L of this 5 uM-dilution to 58 L of input buffer in the 384-well mass spectrometry plate. All samples were analyzed using mass spectrometry. The percentage of compound in cells after treatment and wash was computed as the percentage of the total compound added to the Cells samples that was present in the cell fraction after removal of the extracellular media. Compounds were quantitated by mass spectrometry, and the signal in the cell-free samples were used as a single-point calibration curve to convert from signal intensity in the Cells sample to the percentage of the total amount added.
[1507] beta-Catenin protein production: beta-catenin protein (residues 134-665) with a N-terminal His6-yBBr-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) pLysS cells (Thermo Fisher) from pET28a vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM isopropyl P-D-1-thiogalactopyranoside (IPTG) for 16 hours at 16 C., then harvested and resuspended in 25 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 20 mM imidazole, 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 30 minutes at 4 C., then the supernatant was purified with HisTrap HP columns (Cytiva), eluting with 250 mM imidazole. For crystallography efforts, protein was TEV-cleaved by adding TEV protease at a ratio of 1:10 protease to protein and incubated overnight at 4 C. For biochemical assays, protein was biotinylated via the yBBr reaction according to standard procedures. All proteins were concentrated using Amicon spin filters (Millipore Sigma) then diluted into 25 mM Tris, pH 8.8, 1 mM DTT, 10% glycerol and loaded onto a Q HP (Cytiva) column. Proteins were eluted with a gradient from 50 mM to 600 mM NaCl. Protein-containing fractions were pooled, concentrated and loaded onto a Superdex 10/300 200 pg (Cytiva) SEC column. Purified proteins were eluted isocratically in 25 mM Tris-HCl, pH 8.8, 10% glycerol, 300 mM NaCl and fractions containing pure protein were collected and pooled.
[1508] RNF31 Protein production (UBA, PUB, Sharpin). UBA Domain: RNF31 UBA domain protein (residues 480-639) with N-terminal Thioredoxin-TEV-6his-yBBr-3C tags (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) pLysS cells (Thermo Fisher) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.25 mM IPTG for 16 hours at 16 C., then harvested and resuspended in 50 mM HEPES pH 7.5, 500 mM NaCl, 15 mM imidazole, 1 mM TCEP, 2 mM ATP, 10 mM MgCl.sub.2, 0.1 BugBuster, 5% glycerol 25 U/mL Ready-Lyse, 25 U/mL Omnicleave, and 1 tablet Roche cOmplete EDTA-free per 50 mL. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 40 minutes at 4 C., then the supernatant was purified with HisTrap HP columns (Cytiva), eluting with 500 mM imidazole. For crystallography efforts, protein was cleaved by adding 3C protease at a ratio of 1:40 protease to protein and incubating overnight at 4 C. For biochemical experiments, protein was cleaved by adding TEV protease at a ratio of 1:10 protease to protein and incubating overnight at 4 C. overnight. TEV cleaved proteins were then biotinylated via the yBBr reaction. Proteins were concentrated and injected over a Superdex HiLoad 16/600 75 pg SEC column pre-equilibrated with 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP and 5% Glycerol. Proteins were eluted isocratically and fractions containing pure protein were collected and pooled. PUB domain: RNF31 PUB domain protein (residues 1-179) with N-terminal MBP-TEV-6His-YBBR-3C tags (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) pLysS cells (Thermo Fisher) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 16 hours at 16 C., then harvested and resuspended in 50 mM HEPES pH 7.5, 500 mM NaCl, 1 mM TCEP, 2 mM ATP, 5 mM MgCl.sub.2, 5% Glycerol, and 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 30 minutes at 4 C., then the supernatant was purified with Ni-NTA resin (Qiagen), eluting with 250 mM imidazole. For crystallography efforts, protein was cleaved by adding 3C protease at a ratio of 1:40 protease to protein and incubating overnight at 4 C. For biochemical experiments, protein was cleaved by adding TEV protease at a ratio of 1:10 protease to protein and incubating overnight at 4 C. overnight. TEV cleaved proteins were then biotinylated via the yBBr reaction. Proteins were concentrated and injected over a Superdex HiLoad 16/600 75 pg SEC column pre-equilibrated with 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP. Proteins were eluted isocratically and fractions containing pure protein were collected and pooled.
[1509] Sharpin: Sharpin ubiquitin-like domain protein (residues 206-309) with N-terminal Thioredoxin-6His-Thrombin-yBBr-TEV tags (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) pLysS cells (Thermo Fisher) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.2 mM IPTG for 16 hours at 16 C., then harvested and resuspended in 50 mM HEPES pH 7.5, 500 mM NaCl, 15 mM imidazole, 1 mM TCEP, 2 mM ATP, 10 mM MgCl2, 0.1 BugBuster, 5% glycerol 25 U/mL Ready-Lyse, 25 U/mL Omnicleave, and 1 tablet Roche Complete EDTA-free per 50 mL. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 30 minutes at 4 C., then the supernatant was purified with HisTrap HP columns (Cytiva), eluting with 500 mM imidazole. For biochemical experiments, protein was cleaved by adding TEV protease at a ratio of 1:10 protease to protein and incubating overnight at 4 C. overnight. TEV cleaved proteins were then biotinylated via the yBBr reaction. Proteins were concentrated and injected over a Superdex HiLoad 16/600 75 pg SEC column pre-equilibrated with 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP and 5% Glycerol. Proteins were eluted isocratically and fractions containing pure protein were collected and pooled.
[1510] RNF31 UBA and PUB SPR: All SPR experiments were performed on a Biacore 8K (Cytiva) instrument at 25 C. For kinetics experiments, the instrument was primed with 10 mM HEPES, pH7.5, 150 mM NaCl, 0.05% Tween 20, 1% DMSO. A CAP Series S sensor chip was docked and pre-conditioned with 3 injections of 1CAP regeneration solution to remove unbound capture reagent from the surface. Biotinylated RNF31 UBA and PUB domain proteins were diluted to 1 uM in running buffer. FP06655 was diluted to 1 uM in running buffer and serially diluted 1:3 for a total of 8 concentrations and a blank (8-point four-fold peptide dilution series with top concentration=1 mM.). Otulin and test peptides were diluted to 10 uM in running buffer and serially diluted 1:3 for a total of 8 concentrations and a blank. Proteins were captured to the active surface of the sensor chip for 60 seconds at 5 L/min and the peptides were injected over the reference and active surfaces for 180 seconds at 50 L/min then allowed to dissociate for 360 seconds. Surface was regenerated with a 120-second injection of CAP regeneration solution each cycle. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and fit to 1:1 binding affinity model.
[1511] RNF31-Otulin competition. Fluorescence Polarization: RNF31 PUB domain was diluted to 1.6 uM in assay buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20) and pipetted into a 384-well black microplate (Corning) in a final volume of 20 uL. Test peptides were added to the plate (40 nL each) serially diluted 3-fold from 10 mM and the plate was incubated at room temperature for 20 minutes (11-point three-fold peptide dilution series with top concentration=10 mM). FITC-labeled Otulin peptide (residues 49-67) (FP16923) was diluted to 40 nM in assay buffer, then 20 L of the stock was added to the plate for a final volume of 40 L. The plate was incubated for 60 minutes at room temperature, then fluorescence anisotropy was recorded on a CLARIOstar (BMG LabTech) with excitation at 485 nm, emission at 525 nm, and cutoff at 515 nm. Data were plotted using Prism (Graphpad) and fit to a one-site specific binding model with Hill coefficient. SPR ABA Competition: All SPR experiments were performed on a Biacore 8K (Cytiva) instrument at 25 C. For kinetics experiments, the instrument was primed with 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1% DMSO. A SA Series S sensor chip was docked and pre-conditioned with three injections of 50 mM NaOH/1M NaCl to remove unbound streptavidin from the surface. Biotinylated RNF31 PUB domains were diluted to 2 uM in running buffer. FP06649 and FP06652 were diluted to 10 uM in running buffer. Otulin peptide (residues 49-67) was diluted to 10 uM in running buffer. Proteins were captured on the active surface of the sensor chip for 300 seconds at 1 uL/min. For each injection, compounds were injected over the surface for 120 seconds at 30 uL/min to achieve equilibrium binding. Otulin was then injected for 60 seconds at 30 uL/min in the absence or presence of competing compound over the surface. Surface was regenerated with an injection of 1M sodium chloride after each cycle. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1512] RNF31-Sharpin competition by fluorescence polarization: RNF31 UBA domain, and FAM-labeled RNF31-binding peptide, (FP12122), were diluted to 400 nM and 40 nM, respectively in assay buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20) and pipetted into a 384-well black microplate (Corning) in a final volume of 20 uL. Recombinant Sharpin/SIPL1 UBL protein (residues 153-256), as well as selected control peptides, were added to the plate (20 mL each), serially diluted 3-fold from 10 mM (10-point three-fold peptide or Sharpin dilution series with top concentration=3.3 mM). The plate was incubated for 60 minutes at room temperature, then fluorescence anisotropy was recorded on a CLARIOstar (BMG LabTech) with excitation at 485 nm, emission at 525 nm, and cutoff at 515 nm. Data were plotted using Prism (Graphpad) and fit to a one-site specific binding model with Hill coefficient.
[1513] CDK2 and PPIA protein production. CDK2: Full length CDK2 (residues 1-298) with an N-terminal GST-3C-6his-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in Sf9 cells according to the Bac-to-Bac protocol (Thermo Fisher). Briefly, Sf9 cells were plated at 110.sup.6 cells in 2 mL Sf-900 II media (Thermo Fisher) into a 6-well cell culture plate. Cells were transfected with purified bacmid diluted in OptiMem media using Cellfectin reagent. Cells were incubated at 27 C. for 5 days. Cells and supernatant were removed from plate and centrifuged. P1 virus was collected and cell pellet was evaluated for protein expression by Western blot. P2 virus was generated by infecting 210.sup.6 cells/mL Sf9 in 50 mL Sf-900 II media with 500 mL P1 virus. Cells were incubated with shaking at 27 C. for 5 days. Cells were centrifuged at 1500 rpm at room temperature for 5 minutes. Supernatant was stored at 4 C. as P2 virus stock and pellet was evaluated by Western blot for protein expression. Protein was expressed by seeding Sf9 cells at 210.sup.6 cells/mL in Sf-900 II media and infecting at an MOI of 1:200. Cultures were incubated with shaking for 72 hours at 27 C. Cells were harvested and supernatant was discarded. Pellets were resuspended in 25 mM HEPES, pH 7.5, 300 NaCl, 10% glycerol, 0.5 mM PMSF and then sonicated with a tip sonicator. Lysates were centrifuged at 22,000g for 30 minutes at 4 C. Clarified lysate was purified with a GSTrap (Cytiva) pre-equilibrated in 25 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol. Protein was eluted with 25 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol, 10 mM GSH. Eluted protein was cleaved by combining protein with TEV protease at a ratio of 1:10 protease to protein and incubating at room temperature for 40 hours. Cleaved protein was then dialyzed into 25 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol and re-injected over a GSTrap to remove cleaved tags. Purified protein was concentrated and centrifuged at 22,000g for 10 minutes at 4 C. to remove soluble aggregates. Protein was then loaded onto a Superdex HiLoad 16/600 75 pg SEC column pre-equilibrated with 20 mM HEPES, pH 7.5, 150 mM NaCl, 2% glycerol, 2 mM DTT. Protein was eluted isocratically at 0.5 mL/min. Finally, protein was centrifuged at 22,000g for 10 minutes at 4 C. to remove soluble aggregates, then aliquoted and frozen.
[1514] CDK2 (pT160): CDK2 with T160 phosphorylation was obtained through the co-expression of GST-3C-6his-TEV-CDK2 (HHHHHH disclosed as SEQ ID NO: 39) above with Saccharomyces cerevisiae GST-Cak1. The phosphorylation of pT160 was confirmed with phospho-CDK2 (Thr160) antibody (Cell Signaling #2561) and mass spectrometry.
[1515] CDK2 (pT160)/CCNE1: GST-CCNE1 (residues 81-363) was expressed in E. coli BL21 (DE3) pLys S cells. Briefly, cells were grown at 37 C. to OD.sub.60o reached 0.8 and induced with 0.1 mM IPTG overnight at 20 C. Cells were lysed in 10 mM HEPES, 150 mM NaCl, pH 7.5 and pelleted with centrifugation. The cell lysate was incubated with glutathione-Sepharose 4B beads with purified CDK2 harboring pT160. After elution with 20 mM glutathione, pCDK2/GST-cyclin E1 was digested with GST-3C protease overnight. The pCDK2/cyclin E1 complex was collected and loaded onto a Superdex 16/600 75 pg (Cytiva) SEC column pre-equilibrated with 20 mM Tris pH 7.4, 200 mM NaCl, 10% glycerol, 1.0 mM DTT. Purified proteins were eluted isocratically. Protein fractions were collected, concentrated, aliquoted and frozen.
[1516] PPIA: Full length PPIA (residues 1-165) with an N-terminal 6his-yBBR-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) CodonPlus RIPL cells (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM isopropyl P-D-1-thiogalactopyranoside (IPTG) for 16 hours at 16 C., then harvested and resuspended in PBS pH 7.4 with 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 30 minutes at 4 C. Supernatant was collected then centrifuged again at 22,000g for 30 minutes at 4 C. The supernatant was purified with Ni-NTA resin (Qiagen), eluting with 250 mM imidazole. Protein was TEV cleaved by adding TEV protease at a ratio of 1:10 protease to protein and incubated for 4 hours at 4 C. Protein was then concentrated and diluted into 20 mM HEPES pH 7.0, 5% glycerol and centrifuged at 22,000g for 10 min at 4 C. The supernatant was loaded onto a SP HP (Cytiva) column pre-equilibrated with 20 mM HEPES pH 7.0, 5% glycerol. Purified protein was eluted with a gradient from 0 mM to 1 mM NaCl. Protein fractions were pooled, concentrated then centrifuged at 22,000g for 10 min at 4 C. Supernatant was collected and loaded onto a Superdex 16/600 75 pg (Cytiva) SEC column pre-equilibrated with PBS pH 7.4. Purified proteins were eluted isocratically in PBS pH 7.4. Protein fractions were collected, concentrated, aliquoted and frozen.
[1517] SPR for CDK2 and the CDK2/CCNE1 complex: SPR experiments were performed on a Biacore S200 or 8K (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. A SA Series S sensor chip was docked and pre-conditioned with three injections of 50 mM NaOH/1 M NaCl to remove unbound streptavidin from the surface. CDK2 protein, or CDK2: CCNE1 complex, was diluted to g/mL in running buffer and immobilized to channels 1 through 8 at 5 uL/min for 50-80 seconds for a final immobilization level of 1800 RU. Peptides were diluted to 5 uM in running buffer then serially diluted 2-fold for a total of seven concentrations with one blank (7-point two-fold peptide dilution series with top concentration=5 uM.). Compounds were injected over the immobilized and reference surfaces at 30 uL/min for 60 seconds and then allowed to dissociate for 180 seconds without surface regeneration. Sensorgrams were double-referenced and fit to a 1:1 steady state affinity model.
[1518] ATP competition of CDK2: For ATP competition experiments, 50 mM of selected CDK2-binding compounds were added into 20 nM Bodipy-ATP- yS (Thermo Fisher) and 2 mM CDK2 in an assay buffer contained 20 mM Tris pH 8, 300 mM NaCl, 10% (v/v) glycerol, 2 mM TCEP, and 10 mM MgCl.sub.2, and pipetted into a 384-well black microplate (Corning) in a final volume of 40 L. The plate was incubated for 60 minutes at room temperature, then fluorescence anisotropy was recorded on a CLARIOstar (BMG LabTech) with excitation at 485 nm, emission at 525 nm, and cutoff at 515 nm. Final data were normalized against DMSO control and Bodipy-ATP-yS-free control.
[1519] SPR for PPIA, including cyclosporine competition: All SPR experiments were performed on a Biacore 8K (Cytiva) instrument at 25 C. For kinetics experiments, the instrument was primed with 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM DTT, 1% DMSO. A SA Series S sensor chip was docked and pre-conditioned with 3 injections of 50 mM NaOH/1 M NaCl to remove unbound streptavidin from the surface. PPIA protein was diluted to 5 g/mL in running buffer and immobilized to channels 1 through 8 at 5 uL/min for 50 seconds for a final immobilization level of -1900 R U. Peptides were diluted to 5 uM in running buffer then serially diluted 2-fold for a total of seven concentrations with one blank (7-point two-fold peptide dilution series with top concentration=5 uM.). Compounds were injected over the immobilized and reference surfaces at 30 uL/min for 60 seconds then allowed to dissociate for 180 seconds. The surface was regenerated after each cycle with an injection of 1 M sodium chloride. Sensorgrams were double-referenced and fit to a 1:1 steady state affinity model. For ABA competition experiments, PPIA was immobilized similarly to a level of -850 RU. Compounds were diluted to 10 uM in running buffer and cyclosporine A (CsA) was diluted to 100 nM in running buffer. For each injection, peptides were injected over the surface for 120 seconds at 30 uL/min to achieve equilibrium binding. CsA was then injected for 60 seconds at 30 uL/min in the absence or presence of competing compound over the surface. Surface was regenerated after each cycle with an injection of 1 M sodium chloride. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1520] PPIA inhibition assay: PPIA inhibition assays were performed at Eurofins Discovery (Ongar, Essex, United Kingdom). Briefly, a 1.5 mL of assay buffer (35 mM HEPES pH 7.8, 50 mM DTT) is pipetted into a 3-mL glass cuvette and cooled to 10 C. with stirring. Test compounds are diluted in 100% DMSO then added to the buffer to establish a blank. PPIA is then added at a final concentration of 2 nM and substrate is added to a final concentration of 60 uM. The absorbance at 330 nm is measured for 300 seconds. The resulting data were fit to a first order rate equation and the catalytic rate was calculated. An exponential curve was generated using the catalytic rate versus the inhibitor concentration to obtain a Ki value. CsA is included at a single concentration as a positive control.
[1521] Production of PDL1. E. coli Protein: For crystallography, human PD-L1 protein (residues 18-134) with a C-terminal 6His tag (SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) CodonPlus RIPL cells (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 1.0 mM IPTG for 4 hours at 37 C., then harvested and resuspended in 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF, DNase I. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 50 minutes at 4 C. Protein was located in the inclusion bodies, so the cell pellet was collected and resuspended in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM EDTA, 10 mM P-mercaptoethanol (f-ME), 0.5% Triton X-100 and stirred by magnetic stir bar at room temperature for 30 minutes. The suspension was centrifuged at 22,000g for 30 minutes at 4 C. The process was repeated three times. The pellet was resuspended in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM EDTA, 10 mM f-ME and stirred by magnetic stir bar at room temperature overnight. The suspension was centrifuged at 22,000g for 30 minutes at 4 C. and the supernatant was collected. Supernatant was loaded onto a HisTrap (Cytiva) pre-equilibrated with 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 8 M urea, 10 mM f-ME and eluted with 250 mM imidazole. Protein was refolded by diluting into 100 mM Tris-HCl pH 8.0, 1 M L-Arginine, 0.235 mM GSH, 0.25 mM GSSG with incubation overnight at 4 C. Protein was then dialyzed against PBS pH 7.4 for 4 hours at 4 C. Dialysis was repeated three times. Protein was concentrated and injected onto a Superdex HiLoad 16/600 75 pg SEC column pre-equilibrated with 10 mM Tris-HCl pH 8.0, 20 mM NaCl. Fractions containing pure protein were collected and pooled. Mammalian Protein :For biochemical assays, human PD-Li (residues 18-239) with a C-terminal human IgG Fc-Avi-tag or a C-terminal TEV-10his-Avi tag (HHHHHHHHHH disclosed as SEQ ID NO: 40) were recombinantly co-expressed with BirA in Expi293 cells from pcDNA-derived plasmid (Thermo Fisher) using the Expifectamine 293 expression system. For Fc-tagged protein, cells were harvested after 5 days of expression and supernatant was collected and passed over a MabSelect sure affinity column (Cytiva) pre-equilibrated with PBS pH 7.4. Protein was eluted with 100 mM sodium citrate, pH 3.0 and neutralized in 1M Tris-base, pH 9.0. Elution was concentrated and injected over a Superdex HiLoad 16/600 200 pg pre-equilibrated with PBS pH 7.4. Fractions containing pure protein were collected and pooled. For 10his-tagged (SEQ ID NO: 40) protein, cells were harvested after 5 days of expression and supernatant was collected and passed over a Ni-NTA affinity column (Qiagen) pre-equilibrated with PBS pH 7.4. Protein was eluted with 300 mM imidazole. Elution was concentrated and injected over a Superdex HiLoad 16/600 75 pg pre-equilibrated with PBS pH 7.4. Fractions containing pure protein were collected and pooled.
[1522] SPR for PDL1, including PD1 competition: All SPR analysis was performed on a Biacore S200 (Cytiva) in at 25 C. BMS PD-Li interacting small molecules (BMSpep-57, BMS-1, BMS-1001) were obtained from MedChem Express. A Protein G Series S Sensor Chip (Cytiva) was docked into the instrument primed with PBS pH 7.4 with 0.05% Tween 20 and 1% DMSO. PD-L1-Fc was diluted in running buffer to 50 nM and captured on the surface for 60 seconds at 5 uL/min. Compounds were diluted to 2 uM then serially diluted 3-fold in running buffer (7-point two-fold dilution series with top concentration=5 mM (FP30790), or 6-point two-fold dilution series with top concentration=2 mM (others)). Diluted compounds were injected over the surface at 30 uL/min for 180 seconds and allowed to dissociate for 360 seconds. The surface was regenerated every cycle with a 60 second injection of 10 mM glycine-HCl, pH 2.5. The resulting sensorgrams were double-referenced and fit to a 1:1 binding model using Biacore Insight Evaluation software (Cytiva). For ABA competition experiments, PD-L1 was immobilized on a Streptavidin Series S Sensor Chip (Cytiva) to -250 RU. Compounds were diluted to 10 uM in running buffer and PD-1 was diluted to 400 nM in running buffer. For each injection, compounds were injected over the surface for 120 seconds at 30 uL/min to achieve equilibrium binding. PD-1 was then injected for 60 seconds at 30 uL/min in the absence or presence of competing compound over the surface. The surface was regenerated every cycle with a 60-second injection of 10 mM glycine-HCl, pH 2.5. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1523] ELISA for PDL1: PD-1/PD-L1 ELISA competition assays were performed according to manufacturer's instruction (Acro Biosystems). Human PD-L1 was diluted to 2 g/mL in PBS+0.05% Tween 20. High-binding 96-well plates (Corning) were coated with PD-L1 at 2 g/100 uL per well. Human PD-1-Avi was diluted to 0.6 g/mL in ELISA wash buffer (PBS+0.05% Tween 20+0.5% BSA+0.09% DMSO) and added to the coated wells to form complexes. Test peptides were diluted in ELISA wash buffer to 20 uM then serially diluted 4-fold and added to the PD-L1/PD-1-avi complexes. After incubation with the ligand, the plate was washed, and the bound ligand was detected with the addition of streptavidin-HRP, followed by development with 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher). The HRP reaction was stopped by adding ELISA stop solution (Thermo Fisher). Absorbance at 450 nm was determined on a CLARIOstar plate reader. Samples were blank-subtracted and normalized using in-plate controls. Data were plotted using Prism (Graphpad) and fit to a one-site specific binding model with Hill coefficient.
[1524] PD-L1 Dimerization Assays, analytical SEC and TR-FRET. Analytical SEC: All analytical SEC methods were performed at Viva Biotech (Shanghai, China) on an Agilent Bio-1260 Infinity II HPLC system. Complexes were prepared by mixing PD-L1 and peptide at a 1:2 protein to peptide ratio. Complexes were injected in separate analyses onto a Superdex Increase 5/150 200 pg column pre-equilibrated with 10 mM Tris-HCl pH 8.0, 20 mM NaCl containing 1 uM of corresponding peptide (for complexes) or no peptide (for apo-protein). Data was processed using Agilent ChemStation software to determine retention time shifts. TR-FRET: For TR-FRET dimerization experiments, biotinylated, mammalian PD-L1 was diluted to 50 nM, Alexa Fluor 488 labeled PD-L1 was diluted to 120 nM and Terbium-labeled streptavidin (Cis-Bio) was diluted to 20 nM in assay buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween20) in a final volume of 40 L per well of a black 384-well plate (Costar). Compounds were serially diluted in 90% DMSO and 80 nL of compound (11-point three-fold peptide dilution series with top concentration=20 mM) was added to the plate and the samples were incubated for 60 minutes at room temperature. FRET signal was determined using a LanthaScreen filter on a PheraStar (BMG Biotech) plate reader (Ex: 337 nm; Em1: 490 nM; Em2: 520 nM). The ratio of Em.sub.520 to Em.sub.490 was calculated and plotted against compound concentration. Resulting data was fit to a four-parameter dose-response curve with variable slope.
[1525] Crystallography. X-Ray Crystallography and Structure Determination. beta-Catenin/FP01567 complex crystals were obtained in 0.1 M Sodium phosphate monobasic monohydrate, 0.1 M Potassium phosphate monobasic, 0.1 M MES monohydrate pH 6.5, 2.0 M Sodium chloride. beta-Catenin/FP05874 complex crystals were obtained in 0.05 M MgCl.sub.2, 0.1 M MES 6.5 5% (w/v) PEG 4000. RNF31/FP06649 complex crystals were obtained in 0.03 M Magnesium chloride hexahydrate; 0.03 M Calcium chloride dihydrate, Buffer System 1 pH 6.5 (0.0555M MES, 0.0445 Imidazole), 12.5% MPD, 12.5% PEG 1000, 12.5% PEG 3350. RNF31/FP06652 complex crystals were obtained in 0.02M DL-Glutamic acid monohydrate, 0.02 M DL-Alanine, 0.02 M Glycine, 0.02 M DL-Lysine monohydrochloride, 0.02 M DL-Serine, Buffer System 1 pH 6.5 (0.0555 M MES, 0.0445 M Imidazole), 20% v/v PEG 500 MME, 10% w/v PEG 20000. RNF31/FP06655 complex crystals were obtained in 10% w/v glycerol, 20% w/v ethanol. CDK2/FP19711 complex crystals were obtained in 0.1 M Tris pH 8.5, 25% (w/v) PEG 2,000 MME. CDK2/FP24322 complex crystals were obtained in 0.1 M Bicine pH 9.2, 2% (v/v) 1,4-Dioxane, 6% (w/v) PEG 20000. PPIA/FP29092 complex crystals were obtained in 0.15 M Potassium bromide, 30% (w/v) Polyethylene glycol monomethyl ether 2,000. PPIA/FP29102 complex crystals were obtained in 0.01 M Nickel (II) chloride hexahydrate, 0.1 M Tris pH 8.5, 20% (w/v) Polyethylene glycol monomethyl ether 2,000. PDL1/FP28135 complex crystals were obtained in 1.1 M Sodium malonate pH 7.0, 0.1 M HEPES pH 7.0, 0.5% (v/v) Jeffamine ED-2001 pH 7.0. PDL1/FP30790 complex crystals were obtained in 0.1 M Sodium acetate pH 4.6, 2.0 M Sodium formate. PPIA/FP29103 complex crystals were obtained in 0.2 M Sodium acetate, 0.1 M Sodium cacodylate pH 6.5, 30% (w/v) PEG 8000. PDL1/FP28132 complex crystals were obtained in 0.1 M MES pH 6.5, 1.6 M Magnesium sulfate. PDL1/FP28136 complex crystals were obtained in 0.1 M TRIS pH 8, 25% (v/v) PEG 350 MME.
[1526] Crystals were obtained by either the sitting hanging drop or hanging drop vapor diffusion methods at room temperature. Crystals were cryo-protected followed by flash-freezing in liquid nitrogen. Diffraction datasets were collected at 100K. Data was processed in XDS & XSCALE, AIMLESS, and/or STARANISO, all part of the autoPROC suite. Molecular replacement solutions were obtained using PHASER with previously deposited high resolution PDB structures as search models. Complete models were built through iterative cycles of manual model building in COOT and structure refinement was carried out using either REFMAC or PHENIX. Structure model figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 2.4, Schrdinger, LLC.). Certain atomic coordinates and structure factors are in the Protein Data Bank, www.pdb.org. (e.g., PDB IDs: 7UWI, 7UWO, 7UX5, 7UXI, 7UXJ, 7UXK, 7UXM, 7UXN, 7UXO, 7UXP, 7UXQ, 7UY2, 7UYJ, and 7UYJ).
Example 11. Provided Technologies for Recognition and Reprogramming of Polypeptide Surfaces
[1527] In some embodiments, the present disclosure provides technologies (e.g., agents, methods, etc.) for modulating interactions between polypeptides. In some embodiments, the present disclosure provides technologies for inducing interactions between two polypeptides. In some embodiments, two polypeptides do not interact with each other absence of technologies described herein, e.g., agents such as stapled peptide agents. In some embodiments, two polypeptide interact each other absence of technologies described herein but provided technologies provide new interactions, e.g., between and among residues, domains, surfaces, etc., that do not interact, and/or interact in different ways, absence of provided technologies. In some embodiments, two polypeptides interact each other absence of technologies described herein but provided technologies enhance one or more interactions, in some cases create one or more interactions, and/or reduce one or more interactions, in some cases remove one or more interactions, between and among residues, domains, surfaces, etc., of the polypeptides. In some embodiments, the provided technology provides technologies for recognition of polypeptide surfaces. In some embodiments, the present disclosure provides technologies for reprogramming polypeptide surfaces. In some embodiments, a polypeptide is or comprises an E3 ligase or a characteristic portion thereof.
[1528] Agents that modulate interactions or induce novel interactions between proteins are useful for various purposes including the study of biological systems, therapeutics uses, etc. In many cases, their discovery can be limited by the complexities of rationally designing interactions between three components. Technologies reported by others typically require known binders to each protein to inform initial designs. In some embodiments, the present disclosure provides general technologies for developing agents, in some embodiments, a-helically constrained (Helicon) polypeptides, that cooperatively induce the interaction between two target proteins without a requirement for previously known binders or an intrinsic affinity between the proteins in the absence of such agents. In some embodiments, such agents are or comprise stapled peptides. For example, in some embodiments, the present disclosure provides agents, e.g., Helicons, that are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. In some embodiments, the present disclosure provides technologies for developing agents and such agents that can induce interactions between polypeptides and E3 ubiquitin ligases. In some embodiments, the present disclosure provides phage-based screening technologies for developing trimerizers, e.g., trimerizer Helicons, that can induce interactions between polypeptides and E3s. In some embodiments, the present disclosure provides technologies that reprogram E3s to cooperatively bind a target polypeptide. In some embodiments, a target polypeptide is or comprises an enzyme (e.g., PPIA). In some embodiments, the present disclosure provides technologies for reducing levels of a target polypeptide, comprising contacting the target polypeptide with an agent that can induce interactions between the target polypeptide and an E3 ubiquitin ligase. In some embodiments, a target polypeptide is or comprises a transcription factor (e.g., TEAD4). In some embodiments, a target polypeptide is or comprises a transcriptional coactivator (e.g., -catenin).
[1529] In some embodiments, polypeptide-polypeptide interactions (PPIs), e.g., protein-protein interactions, play a central role in many biological processes, e.g., binding of protein or peptide ligands to their receptors, modulation of activity and specificity of polypeptides such as enzymes, scaffolding of signaling cascades and other functional complexes, etc., which can become dysregulated in various conditions, disorders or diseases. Among other things, the present disclosure provides technologies for modulating PPIs.
[1530] Historically, efforts have largely focused on agents that disrupt PPIs. Reports has been made in recent years to induce the formation of novel PPIs, including in the context of reprogramming E3 ubiquitin ligases to recognize novel substrate proteins and mark them for proteasomal degradation. However, a key constraint facing many reported technologies, e.g., rational design of molecules that induce novel interactions between proteins, is the typical requirement to possess known binding ligands to each, or a known interaction between the two proteins or closely related relatives, to serve as a starting point for designs. Because many proteins cannot be effectively bound by small molecules (the druggability problem), this constraint significantly limits the proteins for which small molecule-based PPI-inducing agents can be developed. This limitation is particularly acute for E3 ubiquitin ligases, of which only a handful can be bound by small molecules. There has been reports of peptide-based solutions to modulating therapeutically relevant PPIs, given their ability to engage significantly larger surfaces than small molecules. A critical challenge in these efforts has been the delivery of peptides, which generally possess large numbers of exposed polar and charged groups, into cells.
[1531] Among other things, in some embodiments, the present disclosure provides technologies that overcome existing limits and challenges. In some embodiments, the present disclosure provides technologies, e.g., stapled peptide agents and uses thereof, that can target various polypeptides including those that cannot be effectively targeted by reported technologies. In some embodiments, agents of the present disclosure comprise a-helical structure. In some embodiments, agents of the present disclosure comprise a-helical structure reinforced, e.g., by stapling. In some embodiments, reinforced a-helical structures increase agents' ability to cross cellular membranes. In some embodiments, technologies of the present disclosure, e.g., stapled peptide agents, provide increased stability and membrane permeability, and are capable of binding large, flat protein surfaces that are inaccessible to targeting by small molecules. In some embodiments, technologies of the present disclosure, e.g., stapled peptide agents, with their expanded surface area and ability to engage surfaces that small molecules cannot, can induce PPIs, in some cases, novel PPIs.
[1532] A general method for developing useful agents, e.g., Helicons that cooperatively lead to the interaction between two proteins, which may be referred to as trimerizer Helicons, in some cases without relying on previously known binders to either, is described below as an example. In some embodiments, provided technologies, which are accessible to laboratories with standard molecular biology and DNA sequencing technologies, can be utilized to develop trimerizer Helicons that induce the binding of E3 ligases to target proteins, including for which they have no intrinsic affinity, thereby reprogramming their surfaces. Among other things, the present example confirms various technical effects, including benefits and advantages, of provided technologies.
[1533] It is reported that there are an estimated 600 human E3s, in four sub-families. Only a small number of these have been reported to be successfully co-opted into novel PPIs for applications of targeted protein degradation (TPD), using small molecules called molecular glues, or degraders if they induce TPD. Agents that can engage surfaces, in some embodiments, novel surfaces, on various targets, including an RBR E3 ligase, RNF31, are provided in the present disclosure including in various Examples (e.g., Example 4). The present Example provides agents, e.g., stapled peptide agents such as Helicons, that bind to eight additional E3 proteins from the three remaining E3 ligase families. The present Example further describes a useful screening approach that enables the direct discovery of cooperative, molecular glue-like binders, trimerizer Helicons, that can lead to cooperative ternary complex formation between E3 ligases and target proteins that have been considered undruggable by small molecules. Among other things, the present Example confirm that provided technologies can be utilized to formation of various complexes including between various E3 ligases and target polypeptides.
[1534] In an example, from the first set of high-throughput screens, Applicant identified Helicons that bind to these E3s from a naive 10.sup.8-member phage display library of polypeptide comprising stapled peptides, e.g., Helicons (in some embodiments, 14-mer). In some embodiments, the present disclosure provides agents that bind members of the HECT family (e.g., WWP1 and WWP2). In some embodiments, the present disclosure provides agents that bind members of the Cullin-RING (CRL) multi-subunit E3 family (e.g., Cullin proteins paired with their canonical adaptor proteins (CUL1-FBXW7, CUL2-VHL, and CUL5-SOCS2)). In some embodiments, the present disclosure provides agents that bind RING/U-Box family members (e.g., MDM2 and CHIP/STUB1). In some embodiments, characterizing Helicon-E3 co-structures and possible mechanisms of action, the present disclosure identified new a-helical binding sites on the E3 surfaces, as well as potential probes of the disease-linked E3, WWP1, highlighting the generality of provided technologies against a therapeutically important target class.
[1535] In some embodiments, these findings were then used for the generation of subsequent focused libraries, e.g., of 20-mer Helicons, wherein the E3-binding residues were fixed and the remainder were randomized. By screening these focused libraries against target proteins in the presence and absence of the E3 presenter protein, provided technologies were able to directly identify trimerizer Helicons that cooperatively bind the targets only in the presence of the E3 presenter protein. In some embodiments, the present disclosure provides trimerizers that induce interactions between the E3 ubiquitin ligase CHIP and the peptidyl-prolyl cis-trans isomerase Cyclophilin A (PPIA). In some embodiments, the present disclosure provides trimerizers that induce interactions between CHIP and the transcription factor TEAD. In some embodiments, the present disclosure provides trimerizers that induce interactions between the E3 ubiquitin ligase MDM2 and the transcriptional coactivator -catenin. Biochemical and biophysical assessment of various trimerizers confirmed their cooperative binding and their ability to inhibit protein-protein interactions. In some embodiments, x-ray co-crystal structures of two trimerizers between MDM2 and -catenin revealed the structural basis for the interactions both between the Helicon and each protein and between the two proteins themselves. Among other things, provided technologies are useful for developing agents that induce the interaction of polypeptides, e.g., proteins, that are challenging to engage with small molecules.
Stapled Peptide Agents Targeting Various E3 Ligases
[1536] Of the estimated 600 human E3s, only a select few have been reported to be successfully targeted by small-molecule molecular glue-like molecules, e.g., molecules that co-opt the activity of Von Hippel-Lindau (VHL) and CRBN, which are members of the multi-subunit Cullin-RING (CRL) family, and of MDM2 and IAP, which are members of the single-subunit RING-finger/U-Box family. In some embodiments, the present disclosure provides agents targeting E3 ligases with a range of substrate recruitment capabilities, substrate specificities, and tissue distribution profiles. In some embodiments, the present disclosure provides agents that target members of each of the four major E3 families. Certain technologies useful for developing stapled peptide agents, e.g., for screening for Helicons that target members of each of the four major E3 families, are described herein as examples. See, e.g.
Stapled Peptide Agents that Bind the HECT E3 Family
[1537] In some embodiments, the present disclosure provides technologies targeting NEDD4-like E3 ligases. It is reported that such E3 ligases can make up approximately a third of the HECT family. Such ligases include WWP1 and WWP2, which according to some reports each comprise four tandem WW domains and a catalytic HECT domain at the C-terminus that in some embodiments transfers ubiquitin from a bound E2 first to itself and subsequently to the target substrate (see, e.g.,
[1538] In some embodiments, the present disclosure provides HECT-containing protein constructs that either stabilize the autoinhibited, inactive state of WWP1 (WWP1.sup.WW-HECT). In some embodiments, the present disclosure provides WWP1 constructs that are able to adopt both the active and inactive conformations (WWP1.sup.HECT). In some embodiments, the present disclosure provides WWP2 constructs that are able to adopt both the active and inactive conformations (WWP2.sup.HECT). The WWP1.sup.WW-HECT construct that locks WWP1 in an inactive state comprises WW domains 2-4, an inhibitory linker region that connects WW domains 2 and 3, and the HECT domain. In some embodiments, these three recombinant proteins were screened in vitro in parallel against a single naive phage library that displays 10.sup.8 14-mer Helicons and hierarchical statistical clustering was utilized to group certain binders into families of related sequences, represented as pharmacophore logos (see, e.g.,
[1539] Several binding profiles were illustrated in the present Example (see, e.g.,
TABLE-US-00027 TABLEE11-1 Certainagents. SEQ Helicon Cluster ID Expected Observed Target Name Name Sequence NO: Modification Mass Mass WWP1/WWP2 H301 C71 Ac-DPADRRCIQAARVCAVL-NH2 468 Cys-stapled 2086.395 2086.4 WWP1/WWP2 H302 C71 Ac-DPASMRCHRAAHICSVL-NH2 469 Cys-stapled 2096.413 2094.9 WWP1/WWP2 H303 C71 Ac-DPAMYHCEQAANVCRVL-NH2 470 Cys-stapled 2149.426 2148.0 WWP1/WWP2 H304 C71 Ac-DPAQMYCREAAFYCFMH-NH2 471 Cys-stapled 2310.911 2312.2 WWP1/WWP2 H305 C72 Ac-DPATIMCRAAADTCTFF-NH2 472 Cys-stapled 2063.331 2062.6 WWP1/WWP2 H306 C72 Ac-DPAYHHCLAAADLCSFF-NH2 473 Cys-stapled 2110.324 2109.8 WWP1/WWP2 H307 C72 Ac-DPADQWCLAAADVCHFF-NH2 474 Cys-stapled 2138.334 2138.2 WWP1/WWP2 H308 C73 Ac-DPANWECRYAAFNCFIQ-NH2 475 Cys-stapled 2277.489 2277.5 WWP1/WWP2 H309 C73 Ac-DPAAWDCLYAAWDCYIN-NH2 476 Cys-stapled 2219.404 2218.6 WWP1/WWP2 H310 C74 Ac-DPALQACTHAANLCHHF-NH2 477 Cys-stapled 2078.288 2077.2 WWP1/WWP2 H311 C74 Ac-DPALMQCAHAAVLCHQF-NH2 478 Cys-stapled 2084.398 2084.1 WWP1/WWP2 H312 C74 Ac-DPATQRCSMAAYMCHTT-NH2 479 Cys-stapled 2116.375 2116.2 VHL H313 C75 Ac-DPAWWNCFSAAQQCDAM-NH2 480 Cys-stapled 2173.360 2172.6 CUL5 H314 C76 Ac-DPAWYDCADAAWICTFQ-NH2 481 Cys-stapled 2205.377 2206.8 CUL4B H316 C77 Ac-DPADRWCELAAWTCDTF-NH2 482 Cys-stapled 2229.400 2228.2 CHIP H317 C80 Ac-CWEAWLLCET-NH2 483 Cys-stapled 1482.677 1482.7 CHIP H318 C80 Ac-PCYEAWVLCEY-NH2 484 Cys-stapled 1604.798 1604.8 MDM2 H319 C81 Ac-DPANHACFQAAWDCQFF-NH2 485 Cys-stapled 2200.364 2200.4 MDM2 P320 Ac-LTF-R8-EYWAQ-Cba- 486 R8-S5 1745.020 1745.3 S5-SAA-NH2 stapled CHIP-TEAD4 H321 C87 Ac-PVPFFWECQYAAATCQTPRIK-NH2 487 Cys-stapled 2686.066 2685.5 CHIP-TEAD4 H322 C87 Ac-PVPFFWECQYAAATCQ-NH2 488 Cys-stapled 2090.333 2089.4 CHIP-TEAD4 H323 C87 Ac-PTPFFWECQFAAATCTAPRVQ-NH2 489 Cys-stapled 2600.919 2600.8 CHIP-TEAD4 H324 C87 Ac-PVPFFWDCQFAAATCDAPQRR-NH2 490 Cys-stapled 2655.957 2655.9 CHIP-TEAD4 P325 Ac-LWWPDGSGSGGSPGQVPMRKRQLPA 491 unstapled 3638.026 3637.5 SFWEEPR-NH2 CHIP-PPIA H326 C88 Ac-PAQDDWSCVEAAYLCENYVRV-NH2 492 Cys-stapled 2660.883 2661.0 CHIP-PPIA H327 C89 Ac-PILQGMACGPAATICWIDGII-NH2 493 Cys-stapled 2372.819 2372.8 CHIP-PPIA H328 C89 Ac-PDMLAPMCGPAASICWIDGVI-NH2 494 Cys-stapled 2389.827 2389.8 MDM2--catenin H329 C91 Ac-PMEQQAICFQAAWMCLADDWT-NH2 27 Cys-stapled 2688.037 2688.4 MDM2--catenin H330 C91 Ac-PWKYEQVCYQAAWQCLSDDWD- 28 Cys-stapled 2864.078 2863.7 NH2 MDM2--catenin H331 C91 Ac-QAICFQAAWMCLADDWT-NH2 495 Cys-stapled 2202.484 2201.6 MDM2--catenin H332 C92 Ac-PISAANDCFKAAWQCIIWLHQ-NH2 496 Cys-stapled 2645.017 2645.1 MDM2--catenin H333 C92 Ac-PSENARDCFWAAWDCLYFIYQ-NH2 497 Cys-stapled 2828.091 2827.6 MDM2--catenin H334 C93 Ac-PNLTGADCFPAAWQCLQFLWD-NH2 498 Cys-stapled 2625.925 2625.8 MDM2--catenin P335 Ac-PD-cyclopentylalanine- 43 Cys-stapled 3760.075 3758.7 CDDAAFNC-3Thi- benzothienylalanine- QGSGS-bAla- LTFEHYWAQLTS-NH2
[1540] Selected Helicons from representative clusters from each group were synthesized, crosslinked, and biochemically validated. Most of the phage hits bound the proteins with M affinity, as assessed by surface plasmon resonance (SPR). See, e.g.,
TABLE-US-00028 TABLEE11-2 Certainagentsandinteractiondata. WWP1-C71 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAQQTCSQAAWVCSIL 500 0 55 443 363 397 477 0 0 DPASMRCHRAAHICSVL 501 0 21 288 218 244 281 0 0 DPAIQRCSTAAQVCRYL 502 0 6 202 236 306 197 0 0 DPAMYHCEQAANVCRVL 503 32 86 259 381 306 337 0 1 DPAATLCNQAANVCALL 504 0 0 132 145 153 197 0 0 DPARLDCTAAAYVCSVL 505 0 0 109 91 153 140 0 0 DPAIMSCMYAAEVCRVL 506 0 1 147 109 183 253 0 0 DPALQTCMDAAQVCRLL 507 0 0 107 163 153 197 0 0 DPAVRDCSDAAIVCELV 508 5 5 147 91 306 197 0 0 DPALASCQQAAWVCEVL 509 0 43 173 291 306 309 0 0 DPAMRRCHQAAWVCEVL 510 0 1 98 182 183 140 0 0 DPAHTACNQAARVCHTL 511 1 4 104 200 153 253 0 0 DPADRRCIQAARVCAVL 512 30 30 95 73 153 225 0 0 DPAIFDCEFAAHVCSVL 513 0 26 222 436 275 618 0 3 DPARWACTEAAHVCDTL 514 0 2 112 182 183 281 0 0 WWP2-C72 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPARVDCLVAADMCHMF 515 165 532 522 555 466 573 0 0 DPATIMCRAAADTCTFF 516 38 83 92 106 86 79 0 0 DPAYHHCLAAADLCSFF 517 59 97 107 147 101 141 0 0 DPATYYCEAAAETCRFF 518 37 57 70 94 78 123 0 0 DPALWECMTAADWCMFM 519 1 24 17 18 28 32 0 0 DPADQWCLAAADVCHFF 520 112 222 322 349 344 392 0 0 DPARVNCHAAADTCFFF 521 6 83 76 103 112 123 0 0 DPALWHCLAAAETCAMF 522 4 38 24 49 47 76 0 0 DPAIHSCYAAAETCQFF 523 43 87 89 139 127 194 0 0 DPAANYCEAAADTCQFF 524 0 179 254 297 283 378 0 0 DPALLHCMAAAQTCSFF 525 0 47 26 37 59 123 0 0 DPAQDTCRAAADTCAFF 526 8 159 267 241 232 373 0 0 DPAQNMCQAAADVCMFF 527 2 67 51 125 120 127 0 0 DPALIHCRAAADTCAFW 528 87 318 384 608 419 565 0 0 DPAIQNCRSAADVCMFF 529 0 65 96 109 117 127 0 0 WWP2-C73 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPANWECRYAAFNCFIQ 530 0 40 41 40 55 86 0 0 DPAVWECRWAAFQCWLD 531 0 0 88 86 97 107 0 0 DPAAWDCLYAAWDCYIN 532 1 17 382 567 427 516 0 0 DPATWDCNVAAHRCWFL 533 1 0 12 60 40 75 0 0 DPAVWDCRYAAWDCYTN 534 0 0 0 28 33 15 0 0 DPATWDCRNAAWLCLLN 535 0 1 4 8 12 12 0 0 DPALWECRNAAWQCWMH 536 0 0 0 24 35 27 0 0 DPAMWDCRYAALDCYMT 537 0 0 6 23 62 24 0 0 DPANWDCQQAAWLCIIQ 538 0 0 0 8 7 27 0 0 DPAMWSCRYAAWECWVL 539 0 0 0 8 7 28 0 0 DPAIWDCLYAATNCYIN 540 0 0 0 8 12 20 0 0 DPATWDCQQAAHHCWVL 541 0 0 2 23 35 66 1 0 DPATWDCIWAAERCWRY 542 0 0 1 4 9 13 0 0 DPATWACINAAHNCWRN 543 0 0 0 6 23 15 0 0 DPAAWDCRYAALACYIH 544 0 2 11 66 34 0 0 WWP1-C74 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPALQACTHAANLCHHF 545 0 11 40 218 244 169 0 0 DPATHVCTTAAVMCHQT 546 0 0 6 327 275 365 2 0 DPATMNCTLAANICHSH 547 0 0 3 363 458 197 0 0 DPAFLTCTLAASLCHQD 548 0 0 0 145 153 253 0 0 DPATETCAQAAVLCHSD 549 1 12 3 200 519 449 0 0 DPAWMACAWAANLCHSS 550 0 18 14 91 244 253 0 0 DPAIIRCTSAAMMCHNT 551 0 4 0 55 183 169 0 0 DPAHQTCAFAAVLCHQN 552 0 0 32 18 153 309 0 0 DPAMIACSHAAQMCHWS 553 0 8 3 454 275 281 0 1 DPAFIQCTHAANMCHSS 554 0 3 43 381 336 562 0 0 DPAWVSCVRAANLCHVS 555 33 1 0 0 183 140 0 0 DPALMQCAHAAVLCHQF 556 0 2 0 400 550 281 8 0 DPATQRCSMAAYMCHTT 557 1 47 155 509 672 1067 40 0 DPAQLSCTVAAHVCHST 558 0 25 0 254 275 140 9 0 DPAFHYCTMAANMCHQD 559 0 0 3 73 153 169 0 4 VHL-C75 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAWWHCVAAATQCDQR 560 0 20 56 79 24 0 0 DPAWYACVDAAYFCDYQ 561 0 5 34 35 31 0 0 DPAWWHCVDAALQCDYY 562 0 10 59 40 104 0 0 DPAWWDCFNAARRCDDI 563 0 1 19 43 6 0 0 DPAWWQCFDAAAYCDVH 564 0 1 24 7 77 0 0 DPAWWYCMDAATMCDQQ 565 0 1 39 19 51 0 0 DPAWWYCLDAAYDCDHT 566 2 2 45 97 23 0 0 DPAWWNCIDAAILCDDY 567 0 1 14 18 16 0 0 DPAWYACYDAARLCDNF 568 0 3 43 78 46 0 0 DPAWWNCFSAAQQCDAM 569 8 16 24 27 65 0 0 DPAWYACMDAARVCDRT 570 8 2 30 158 23 0 0 DPAWHQCFRAAYMCDNH 571 0 2 9 57 7 0 0 DPAWHACFQAAYICDQA 572 0 3 11 31 18 0 0 DPAWWSCFAAAHHCDAL 573 0 0 20 73 35 0 0 DPAWWDCFNAAMRCDIS 574 0 0 10 59 12 0 0 CUL5-C76 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAWRECAHAAWQCYIQ 75 0 50 47 21 93 0 0 DPAWYDCADAAWICTFQ 576 7 193 188 231 389 0 0 DPAWAICAEAAWDCFMI 577 0 14 13 58 11 0 0 DPAYYVCAEAAWNCLFE 578 4 46 104 100 76 0 0 DPAIQQCAEAAWSCYVE 579 0 13 30 40 14 0 0 DPAWHMCAEAAWQCAFW 580 5 61 106 119 152 0 0 DPAWFTCAQAAWDCVFH 581 0 65 112 96 224 0 0 DPAWFDCAEAAWQCVFI 582 2 35 91 170 31 0 0 DPAWRACAQAAWDCIIA 583 1 167 267 385 368 0 0 DPAWQWCAEAAWQCITH 584 14 59 284 284 265 0 0 DPATFACANAAWECYAR 585 0 6 25 11 35 0 0 DPAWMRCATAAWECVMT 586 16 22 151 104 222 0 0 DPAWWECAEAAWQCQVL 587 0 6 44 20 59 0 0 DPAWMTCATAAWECYLA 588 6 6 62 48 55 0 0 DPAWYECASAAWVCYSM 589 0 13 94 60 171 0 0 CUL4B-C77 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPADRWCELAAWTCDTF 590 1 25 191 321 234 0 1 DPATTECILAAWICDRF 591 0 0 15 31 18 0 0 DPASRLCELAAWVCDSF 592 4 15 139 319 251 0 0 DPASRMCEMAAWFCENY 593 0 3 11 13 78 0 0 DPAMNHCVLAAWVCDTF 594 0 0 6 37 9 0 0 DPAQRACQFAAWICERF 595 1 0 13 56 73 0 0 DPADRQCIFAAWMCEAF 596 0 0 3 9 34 0 0 DPADRYCDMAAWVCERF 597 1 1 15 248 340 0 0 DPAERHCQLAAWVCHQF 598 0 0 0 99 29 0 0 DPADRLCELAAWVCQSF 599 0 0 0 48 9 0 0 DPADRWCELAAWICDEF 600 0 0 0 6 17 0 0 DPATRHCIFAAWACDRF 601 0 0 47 20 12 0 0 DPATRMCELAAWTCEVW 602 0 0 1 31 9 0 0 DPADRQCLFAAWYCEHT 603 0 10 0 6 11 2 0 CUL1-C78 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAYDWCDVAALFCLVE 604 0 0 57 36 57 151 0 0 DPAYFACDIAALMCTVH 605 0 2 131 135 403 120 0 0 DPAMLQCDMAALWCMVS 606 1 3 77 64 143 126 0 0 DPAQILCDLAALYCVVE 607 0 7 61 73 111 119 0 0 DPAQNDCEVAAFFCWLD 608 1 1 233 428 586 468 0 0 DPAEQECDHAALWCIVA 609 14 19 340 937 1137 743 0 0 DPAEFVCDWAALACLVD 610 4 32 183 247 517 300 0 0 DPAEMQCDYAAILCIVR 611 0 0 32 95 114 71 0 0 DPAIIQCDQAALWCVVT 612 0 0 28 83 80 61 0 0 DPAEAACDIAALMCIID 613 15 13 65 211 203 195 0 0 DPAVMECDWAATICDIF 614 0 2 154 402 477 419 0 0 DPAEVHCDMAALYCIIR 615 3 7 65 242 366 119 0 0 DPAEQMCDIAAVWCLAH 616 0 0 35 135 151 88 0 0 FBXW7-C79 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPADLTCEDAAELCEFW 617 2 80 352 535 1005 510 0 0 DPAFWYCDEAAEMCEFW 618 0 0 20 28 91 17 0 0 DPANIACDDAALLCAFW 619 0 0 2 7 5 8 0 0 DPANIACDDAALLCELW 620 0 1 1 7 8 6 0 0 DPANIACDDAALLCEFW 621 4 131 203 946 1140 1464 0 0 DPADMYCRDAAELCEFW 622 1 23 187 302 505 554 0 0 DPAAFFCDDAAERCEFW 623 4 39 35 163 245 449 0 0 DPATYFCYDAAELCDWW 624 0 0 1 5 5 82 0 0 DPAITTCLDAAELCDFW 625 1 26 61 34 215 132 0 0 DPAAWICEEAAEICEFW 626 0 0 5 4 15 36 0 0 DPAYFSCEDAAELCEWY 627 0 0 1 3 13 38 0 0 DPADMMCENAALLCEFW 628 1 3 68 20 282 228 0 0 DPAMTWCLNAAELCEFW 629 0 0 0 3 17 83 0 0 DPAIRMCRDAAELCEFW 630 0 0 1 4 66 94 0 0 DPANWVCMDAAELCDFY 631 0 0 5 2 99 15 0 0 CHIP-C80 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK PCWDAWMLCTD 632 19 142 542 632 701 581 0 0 ICYNARLECE 633 1 2 13 16 21 20 0 0 ICYTAWLECE 634 0 1 5 9 7 12 0 0 ICYSAWLECE 635 1 3 13 25 22 28 0 0 PCYSAWITCES 636 0 134 286 457 563 538 0 0 ICYNAWLECA 637 0 1 5 7 11 9 0 0 ICYNAWFECE 638 0 2 9 11 18 14 0 0 PCWNAWMVCSV 639 2 44 334 474 690 691 0 0 ICYNAWVECE 640 1 2 10 21 19 23 0 0 PCFEAWVMCEI 641 4 83 121 285 375 297 0 0 PCYSAWIVCSD 642 4 81 123 444 549 468 0 0 PCWNAWLYCSN 643 0 0 11 105 155 58 0 0 ICWAAWVSCS 644 0 0 0 25 42 17 0 0 PCWSAWLACEL 645 0 81 72 130 220 206 0 0 PMCSDAWLFCE 646 0 0 0 74 143 57 0 0 PCWEAWLWCSM 647 0 0 4 124 194 145 0 0 MDM2-C81 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAYASCFSAAWDCQFW 648 0 0 19 21 13 38 0 0 DPAFDSCFSAAWDCMYW 649 3 0 27 92 65 84 0 0 DPAENECFTAAWDCMFF 650 0 0 7 51 70 40 0 0 DPANHACFQAAWDCQFF 651 11 154 643 1045 776 1088 1 0 DPASEACFQAAWDCVSW 652 2 57 241 559 336 447 1 0 DPATQQCFQAAWDCQFM 653 1 60 324 683 382 679 8 0 DPADNRCFQAAWDCAFW 654 1 0 51 22 20 23 0 0 DPADSECFQAASDCQYW 655 7 11 0 0 5 6 1 0 MDM2-C82 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAINTCQDAAFWCYWS 656 0 131 203 226 185 315 0 0 DPAETDCSQAAFWCYWQ 657 1 15 170 133 176 188 0 0 DPAHNLCFDAAWWCHWQ 658 0 0 9 7 8 11 0 0 DPARVHCNQAAFMCHWV 659 0 0 9 6 14 8 0 0 DPALMQCTEAAFWCYWE 660 0 0 24 36 24 32 0 0 DPALFSCDSAAFSCYWY 661 0 0 25 17 25 57 0 0 DPATESCEDAAFWCHWI 662 0 0 30 30 30 70 0 0 DPAYTQCSDAAFMCWWW 663 0 19 32 48 65 62 0 0 DPAQHHCQQAAFWCQWW 664 0 0 13 13 36 18 0 0 DPARLRCQDAAFACMWY 665 0 0 17 31 32 37 0 0 DPARRECMDAAFWCQWV 666 0 53 106 232 148 261 0 0 DPAHYECTDAAFECYWF 667 0 2 33 74 57 82 0 0 DPAQEDCHAAAFWCIWD 668 0 0 15 37 23 48 0 0 DPADIVCQDAAFQCFWW 669 0 1 12 33 46 18 0 0 DPAAVQCNTAAFQCYWF 670 0 0 23 56 53 69 0 0 CHIP-C83 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAFEVCQFAAATCSSN 671 0 0 8 10 6 30 0 0 DPAFWSCQFAAATCSHL 672 0 0 4 6 11 8 0 0 DPAMWHCQYAAIMCNAN 673 0 0 0 15 7 29 0 0 DPAFVRCQFAAASCHTI 674 0 0 0 22 27 19 0 0 DPAFEYCQWAAATCQMD 675 0 12 0 43 46 90 0 0 DPAFEQCQFAAAMCLHH 676 0 6 1 17 42 21 0 0 DPAFTACQFAAAMCDSS 677 0 0 0 3 8 5 0 0 DPAFWRCQFAAATCEHT 678 2 3 210 321 597 726 1 0 DPAFWQCQFAAATCAHY 679 0 0 19 16 70 44 0 0 DPAFERCQLAAAFCYDS 680 0 0 2 15 60 47 0 0 DPAFDACQFAAAMCDHQ 681 0 30 23 39 80 248 0 0 DPALWNCQYAAVMCTQL 682 0 0 0 14 34 75 0 0 DPAFWVCQYAAAMCQDV 683 8 3 160 86 331 323 1 0 DPAFDRCQWAAALCMMN 684 0 0 4 13 31 76 0 0 DPAFTRCQFAAAVCNFT 685 0 3 0 6 19 28 0 0 CHIP-C84 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAEYACYHAASICNVD 686 0 0 54 61 31 91 0 0 DPAIIWCSQAASICTVD 687 0 0 19 15 23 24 0 0 DPAYDVCHFAAHICWID 688 0 0 22 19 20 62 0 0 DPANHICHYAAHICDVD 689 0 0 3 15 6 49 0 0 DPALEECTQAAHICMVD 690 0 0 3 14 26 10 0 0 DPAETFCWDAADICMVD 691 0 0 1 17 7 32 0 0 DPAVHNCSQAASICDVD 692 0 0 0 22 9 39 0 0 DPAVRWCAAAAAICMVD 693 0 0 0 21 16 22 0 0 DPASQWCQRAASICWID 694 0 0 0 15 18 8 0 0 DPAELFCTEAADICMVD 695 0 0 0 18 13 22 0 0 DPAQYNCYFAANICTVD 696 0 0 0 48 79 17 0 0 DPAINMCYRAAQICDVD 697 0 0 0 31 28 27 0 0 DPAFFECVRAASICMVD 698 0 0 0 51 41 54 0 0 DPAQDNCHYAAHICMVD 699 0 0 0 47 25 57 0 0 MDM2-C85 SEQID TargetConcentration Sequence NO: 10 30 100 300 1000 1000 BLANK BLANK DPAMSNCFMAAWECLIQ 701 49 104 206 192 185 176 0 0 DPAYSDCFQAAWTCMIH 702 0 78 135 155 110 150 0 0 DPASAECFFAAWQCIAT 703 0 8 28 27 33 14 0 0 DPAHRQCFTAAWRCMMT 704 0 42 149 156 202 149 0 0 DPADRDCFSAAWACVIR 705 0 37 188 209 160 215 0 0 DPASQDCFSAAWQCLQT 706 0 31 138 165 94 231 0 0 DPAVTECFQAAWRCMVM 707 0 77 184 276 269 268 0 0 DPAQQDCFLAAWDCVWQ 708 0 12 132 166 104 237 0 0 DPARQDCFSAAWMCMAI 709 0 1 94 107 120 114 0 0 DPAQSDCFQAAWECVAN 710 0 0 35 47 52 29 0 0 DPAERNCFTAAWQCMVW 711 0 23 158 230 135 225 0 0 DPAAQECFRAAWRCMQD 712 0 0 25 37 34 29 0 0 DPASADCFNAAWDCMWI 713 0 19 102 165 124 209 0 0 DPAHQQCFMAAWRCVVH 714 1 48 230 375 356 327 0 0 DPAYEDCFFAAWNCMMV 715 0 5 18 17 23 39 0 0 Blank =Media only. Unless otherwise noted, concentration in nM.
[1541] Crystal structures of four Helicons representing each of the three HECT-binding clusters in complex with WWP2.sup.HECT, and one Helicon from Group 1, H302, with WWP1.sup.HECT were solved. See, e.g.,
[1542] In some embodiments, although they belonged to different clusters, the Group 2 Helicons H304 and H308 were observed to bind WWP2.sup.HECT using similar residues, interacting with N- and C-lobes of the HECT domain and overlapping the binding site of WWP2's linker domain, which is reported to be necessary for its auto-inhibition (see, e.g.,
[1543] In some embodiments, it was observed that Group 1 Helicons H302 (WWP1.sup.HECT-binding) and H301 (WWP2.sup.HECT-binding) showed similar binding modes, making contact only with the N-lobe of either WWP1.sup.HECT (see, e.g.,
[1544] Having confirmed that provided technologies, e.g., screening platform can successfully discover novel binding sites and Helicon binders against the HECT family of E3 ligases, Applicant further confirmed that provided technologies can be utilized against the larger Cullin-RING (CRL) family. It has been reported that, among other things, as the HECT family has been shown to be coopted by viral oncoproteins, exemplified by human papilloma virus E6 which binds to host E3 E6AP to induce degradation of the tumor suppressor p53, there are numerous reported examples of viral proteins that can recruit and rewire the substrate specificities of host CRLs to induce degradation of host immunity proteins. In some embodiments, the present disclosure provides technologies, e.g., Helicons, that can modulate CRL structures and/or functions. In some embodiments, the present disclosure provides technologies that can inhibit or promote PPIs of CRL.
[1545] According to some reports, CRL is the largest E3 family, and in some cases is unique in that its members are modularCullin (CUL) proteins form the central scaffold that recruits an E2 enzyme via a RING-Box protein (typically RBX1 or RBX2) to its N-terminus and a CUL-specific adaptor that bridges the C-terminus of the CUL protein to the substrate protein (see, e.g.,
[1546] In some embodiments, screens were performed in parallel to find agents, e.g., Helicons, that bind to CRLs comprising Cullin proteins paired with their canonical adaptor proteins. In some embodiments, to identify and to assess agents, e.g., Helicons, that interact specifically with CRL1, CRL2, or CRL5, the N-terminal domains of CUL1, CUL2, and CUL5 were purified and screened in parallel with a counter-target CUL4B. In some embodiments, also included were the corresponding CRL substrate-recognition adaptors, including VHL and the ElonginB/ElonginC complex (ELOBC) for CUL2. In some embodiments, the present disclosure provides agents, e.g., Helicons, that form VHL-ELOBC-specific clusters and CUL5-specific clusters (see, e.g.,
[1547] Various technologies can be utilized to characterize and assess agents of the present disclosure. In some embodiments, biochemical and biophysical approaches were utilized to confirm that the clusters and agents were target-specific and can bind to biologically relevant sites. Among the VHL-ELOBC-specific binding clusters, we identified Helicon H313 (C76) that binds VHL-ELOBC, but does not compete with the previously reported fluorescent VHL-binding probe, HXC78, and it did not bind SOCS2-ELOBC that acted as a counter-target for VHL-ELOBC in the CRL phage screens (Table E11-2). In some embodiments, the present disclosure provides technologies that bind VHL-ELOBC. In some embodiments, the present disclosure provides technologies that bind VHL-ELOBC selectively over SOCS2-ELOBC. In some embodiments, provided technologies do not compete with HXC78. In some embodiments, provided technologies compete with VH298. Using SPR, specificity of H313 for VHL-ELOBC over SOCS2-ELOBC was confirmed: the K.sub.D for the former was calculated to be 4.1 M (see, e.g.,
[1548] In some embodiments, X-ray crystallography was utilized to characterize or assess provided technologies. A structure of H314 with the CUL5 N-terminal domain (CUL5.sup.NTD) was solved at 2.8 resolution (see, e.g.,
[1549] Consistent with its disruption of the CUL5 interaction with ELOBC in SPR assays, H314 binds at the very N-terminus of CUL5, the binding site of the adaptor complex SOCS2-ELOBC (see, e.g.,
[1550] In some embodiments, the distinct binding modes and Helicons described here can be further developed to provide additional probes and tools for this important E3 family. For instance, both the H314-binding site on CUL5 and the H313-binding site on VHL face the Ub-E2 binding sites of the cognate Cullin CTDs (see, e.g.,
Stapled Peptide Agents that Bind the RING/U-Box E3 Family
[1551] In some embodiments, the present disclosure provides technologies targeting RING/U-Box family (see, e.g.,
[1552] In some embodiments, agents of the same cluster may interact with targets similarly. In some embodiments, agents of the same cluster may interact with targets similarly but with differences. For example, in some embodiments, while Helicons H317 and H318 belonged to the same cluster, several differences in the co-structures with CHIP were observed, reflecting unique conformational changes and binding surfaces revealed by the Helicons. For instance, the side chain of Glu9 in H317 directly interacts with Lys30 of CHIP.sup.TPR while Glu10 from H318 does not (see, e.g.,
Trimerizer Technologies
[1553] In some embodiments, the present disclosure provides technologies for identifying, developing, optimizing, characterizing and/or assessing trimerizer agents, e.g., trimerizer Helicons. In some embodiments, based on their ability to present a much larger surface than small molecules that have been traditionally used to facilitate binding of E3s with substrate targets, provided agents comprising stapled peptides, e.g., Helicons, are engineered to cooperatively promote specific interactions between E3s and a range of substrate targets. In some embodiments, the present disclosure provides technologies to exploit large library diversity of a phage screening platform to explore a vast number of design hypotheses and/or to directly discover trimerizer Helicons, in some embodiments, without a need for structure-based design (see, e.g.,
[1554] In some embodiments, a provided approach for discovering trimerizer Helicons involves two successive screens of 10.sup.8-membered Helicon libraries, wherein the first is a naive binding screen (as described above for E3 ligases) and the second is a screen of a focused library that is designed based on hits from the naive screen, in some embodiments, comprises one or more enriched amino acid residues from a first screen. As confirmed herein, in some embodiments, hit clusters for the RING/U-Box family E3s CHIP and MDM2 were selected, and focused libraries were designed based on fixing these conserved binding residues and diversifying the exposed Helicon residues (see, e.g.,
[1555] Once constructed, these libraries were screened against target proteins in both the presence and absence of the E3 presenter they were designed to bind. In some embodiments, inspection of the phage binding data revealed two types of target binding behavior: binding that occurred regardless of the presence of the E3 presenter protein, and binding that occurred only when the E3 was present. In some embodiments, these latter, E3-dependent binders were selected for synthesis and validation. In some embodiments, E3 dependence indicated a cooperative binding mode to their target (see, e.g.,
Construction of Trimerizer Libraries for CHIP and MDM2
[1556] In some embodiments, focused phage libraries were designed and constructed based on selected CHIP and MDM2 hit clusters, including those exemplified by H319 for MDM2 and H317 and H318 for CHIP. In some embodiments, four total stapling and scaffolding residues across the 14-mer Helicons were fixed, and consensus residues responsible for binding defined by the cluster logos were also fixed (see, e.g.,
[1557] For trimerizer phage screening, purified, bead-immobilized target proteins were incubated with the phage librariesfor example, the focused libraries built for discovery of CHIP and MDM2 trimerizersbut also included non-immobilized E3 presenter proteins free in solution during the screen (see, e.g.,
Provided Technologies can Induce the Interaction Between CHIP and PPIA or TEAD4
[1558] In some embodiments, the present disclosure provides agents that can promote interactions between CHIP and a target polypeptide. In some embodiments, the present disclosure provides agents that can induce interactions between CHIP and a target polypeptide. In some embodiments, a polypeptide is PPIA. In some embodiments, a polypeptide is TEAD4.
[1559] According to some reports, TEAD4 is a member of the transcriptional enhancer factor (TEF) family of transcription factors, and through its interactions with YAP/TAZ, acts as an effector of the Hippo signaling pathway, which is implicated in cell proliferation and migration, organ development, and resistance to specific cancer treatments.
[1560] Various technologies can be utilized to assess complex formation induced by agents of present disclosure. For example, in some embodiments, capability of CHIP-TEAD4 trimerizers to promote ternary complex formation were assessed using time-resolved fluorescence energy transfer (TR-FRET), SPR (ABA mode), and fluorescence polarization (FP) assays (see, e.g.,
[1561] For TR-FRET, Helicon H321 from C85 and a C-terminally truncated version of H321, H322 were assessed (see, e.g.,
[1562] In some embodiments, both H321 and H322 could disrupt the interaction between recombinant TEAD4 and a fluorescently labeled YAP1 fragment in the presence of CHIP in a competition FP assay (see, e.g.,
[1563] Among other things, technologies of the present disclosure provides versatility. For example, in some embodiments, versatility of CHIP related technologies to engage other targets beyond TEAD4 was confirmed. In some embodiments, the present disclosure provides trimerizers between CHIP and another polypeptide, e.g., PPIA (Cyclophilin A). PPIA is reported to be a peptidyl-prolyl cis-trans isomerase (PPIase) that plays a widespread role in the folding of nascent proteins. In some embodiments, screens with a CHIP-based trimerizer library identified several CHIP-dependent PPIA clusters, including C86, C88, C89, and C94 (see, e.g.,
Provided Technologies can Induce the Interaction Between MDM2 and -Catenin
[1564] In some embodiments, the present disclosure provides technologies that can induce interactions between MDM2 and target polypeptides. In some embodiments, a target polypeptide is beta-catenin. Certain useful technologies that modulate interactions between MDM2 and target polypeptides are described herein as examples, confirming, among other things, generality of provided technologies.
[1565] In some embodiments, MDM2-based libraries were screened against -catenin, a key component of the canonical Wnt signaling pathway that is reported to be often dysregulated in cancer. In some embodiments, MDM2-focused 20-mer Helicon libraries were screened against -catenin and hits were identified that bound -catenin only in the presence of MDM2, which belonged to multiple clusters, including C91-C93 (see, e.g.,
[1566] A direct FP assay of ternary complex formation confirmed that H329 and H330 from C91, H332 and H333 from C92, H334 from C93, and an N-terminally truncated version of H329 (H331) could all promote a cooperative interaction between MDM2 and the Armadillo domain of -catenin with EC.sub.50 values ranging from 10-100 nM, but not between MDM4 and -catenin (see, e.g.,
[1567] An inverted SPR experiment further confirmed the H330 trimerizer activity, where MDM2 was immobilized with -catenin as the free analyte (see, e.g.,
Structural Characterization Confirms Direct Interactions Between all Three Molecules of the Complex
[1568] Various technologies can be utilized to characterize, assess and confirm formation of complexes formed by agents of the present disclosure and polypeptides, e.g., E3 ligase proteins and target proteins. In some embodiments, X-ray crystallography was utilized to characterize the MDM2-O-catenin complexes induced by trimerizer agents, e.g., H329 and H330 from C91 and H332 and H333 from C92. In some embodiments, calculated electron density maps indicated that residues 6-21 of H330 and residues 5-21 of H332 were well-resolved. (see, e.g.,
[1569] Using the PDBePISA explorer to define the macromolecular interfaces between Helicons H330 and H332 and MDM2 or -catenin, and between MDM2 and -catenin, we observed that both Helicons induced similarly sized MDM2-O-catenin interfaces, though H330 did so with a much more extensive set of interactions with -catenin than H332 did (see, e.g., Table E11-3; and
[1570] In the 2.6 ternary structure between MDM2, Helicon H330, and -catenin, we found that the sidechains of Tyr9, Trp13 and Leu16 of H330 insert deeply into a hydrophobic cleft on the MDM2 surface, reminiscent of the endogenous p53-MDM2 interaction mediated by p53 residues Phe19, Trp23, and Leu26 (see, e.g.,
[1571] Similar to the H330-bridged ternary complex, the 3.9 ternary structure between MDM2, Helicon H332, and -catenin also revealed the importance of Helicon residues Phe9, Trp13, and Ile16 for interacting with MDM2, but revealed a similar solution for MDM2-Helicon-binding to -catenin, with notable differences at the -catenin-binding interface (see, e.g.,
[1572] In total, these structures reveal the structural basis for the trimerizer Helicon-induced molecular recognition events that promote cooperativity in binding between the E3 and target proteins. In some embodiments, provided technologies can allosterically modify surfaces of E3 ligases to shift their substrate selectivity.
[1573] Among other things, as confirmed herein the present disclosure provides technologies for targeting the E3 ligase family, which was considered largely undruggable by traditional small molecules, and even with the traction provided by TPD approaches, still today only a select handful of this large family have been targeted. In some embodiments, the present disclosure provides high-throughput screening platform to identify Helicons that bind, and in some cases modulate the function of diverse E3s across all four families, including the discovery of WWP and CUL binders as well as alternatives to existing VHL binders. These E3 ligases have diverse tissue distributions. In some embodiments, provided technologies can improve cellular selectivity and limit on-target but off-tissue toxicity of candidate therapeutics.
[1574] Among other things, various provided technologies, e.g., platform technologies, are rapid, affordable, scalable, and as confirmed with structurally diverse targets, they are generalizable, including here against a very large protein family. Beyond representing additional binding sites on E3s for their optimization into direct inhibitors and degrader-like tools, the trimerizer Helicons of the present disclosure confirmed that Helicons can bridge two proteins by increasing the surface area and cooperative interactions between them.
[1575] Provided technologies can overcome several shortcomings of other TPD tools such as PROTACs. As a drug modality, TPD tools such as PROTACs have several attractive features including superior selectivity and a catalytic-type mechanism of action that can result in total elimination of the targetincluding of disease-relevant point mutants and traditionally undruggable targets. As well, they are not subject to cellular resistance mechanisms, and they represent unique probes that can be used to gain new biological insights into the function of their targets. Unfortunately, PROTACs have several shortcomings. They are difficult to develop given the often-imbalanced determinants of ternary complex formation, proper distance and orientation to allow ubiquitin transfer between E2, E3 and the target protein, and cellular permeability. Typically, PROTAC design and development involves optimizing the linker between the E3- and target-engaging small molecules used to make them. Given the nature of linking these disparate molecules, it is difficult to program cooperative interactions, so PROTACs are prone to the hook effect, a phenomenon that limits ternary complex formation in in vitro assays used during the optimization process. Further, not all PROTACs that direct ternary complex formation lead to degradation. Target binding and ternary complex formation are not the only considerations for PROTAC development. The identity of the recruited E3 ligase also plays a critically important role in determining their degradation capacity.
[1576] Provided technologies have various features as tools for TPD and other applications requiring proximity-induced interactions. For example, they can promote cooperative interactions between polypeptides, e.g., E3s and target proteins to drive specificity. Provided technologies can also be developed across the entire E3 family, providing attractive alternatives to the E3s VHL, CRBN, MDM2, and IAP that are used in a majority of TPD applications. This includes the ability to precisely target a protein of interest in its native tissue with an E3 with a matched tissue distribution. Further, provided technologies significantly expand the range of E3-recruiting ligands that can be used. Generally, common E3-recruiting ligands, such as the molecular glues thalidomide and its derivatives are used across most TPD applications. Still further, provided technologies can present an orientation of the E3 ligase that allows for efficient ubiquitin transfer to target proteins.
[1577] In some embodiments, provided technologies can identify specific trimerizer hits across a broad range of E3-target interactions with a single round of focused screening. In some embodiments, leads are optimized for the cellular penetration of the leads. It is noted a number of peptides have been used as PPI inhibitors, including ALRN-6924, an analog of ATSP-7041, the chimeric control utilized herein. It is reported that ALRN-6924 inhibits the degradation of the tumor suppressor p53 by targeting the E3s MDM2 and MDM4. There are also reports that viral proteins such as HIV-1 Vif can recruit host E3s and rewire their substrate specificities to counteract immunity. The F-box domains from HIV-1 Vif, VHL and SOCS2 have been reported to share a high structural homology and all bind the RING E3 ligase CUL5-ELOBC complex.
[1578] In some embodiments, directed evolution are utilized to exploit the E3-binding capability of Helicons to increase the surface area for binding target proteins. In some embodiments, the present disclosure provides focused libraries from E3-binding hits. In some embodiments, such libraries are screened against therapeutically important target proteins for those that bind only in the presence of the E3 presenter protein. In some embodiments, the successful identification of trimerizers that bridge E3s with target proteins greatly expands the range of tools available for applications requiring induced PPIs, and may eliminate the potential dose-limiting viabilities of current molecules in this space.
[1579] PPIs are among the most intractable targets for probes and drugs, and being able to use Helicons to not only disrupt these but also to generate novel PPIs, highlights certain strength of provided technologies to advance these fields simultaneously.
[1580] Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes, e.g., 8EIC, 8EHZ, 8EI0, 8EI1, 8EI2, 8EI3, 8EI4, 8EI5, 8EI6, 8EI7, and 8EI8. Certain results are also presented below:
TABLE-US-00029 TABLE E11-3 Certain macromolecular interfaces driven by trimerizer Helicons. Buried Interface (.sup.2) Helicon:-catenin Helicon:MDM2 MDM2:-catenin H330-mediated 640 700 340 MDM2:-catenin interaction H332-mediated 280 780 400 MDM2:-catenin interaction
Certain Useful Technologies
[1581] Those skilled in the art appreciate that many technologies may be utilized for preparing, characterizing, assessing and/or using agents of the present disclosure. Certain technologies are described herein as examples.
Recombinant Protein Expression.
[1582] Unless otherwise stated, all protein constructs correspond to human protein sequences. [1583] WWP.sup.WW-HECT, WWP1.sup.HECT and WWP2.sup.HECT
[1584] The expression and purification of human WWP1 (Uniprot ID: Q9HOMO) and WWP2 (Uniprot ID: 000308) fragments were adapted from previous work (Wang, Z. et al. A multi-lock inhibitory mechanism for fine-tuning enzyme activities of the HECT family E3 ligases. Nat Commun 10, 3162 (2019)). Briefly, WWP1.sup.WW-HECT (residues 379-922), WWP1.sup.HECT (residues 546-917) and WWP2.sup.HECT (residues 492-865) were individually cloned into pET-based expression vectors to generate the final constructs GST-TEV-WWP1.sup.379-922-yBBr, His-TEV-WWP1.sup.546-917-yBBr, and GST-TEV-WWP2.sup.492-865-yBBr, respectively, for phage screening and SPR analysis; and His-Thrombin-WWP1.sup.546-917, His-3C-WWP2.sup.492-865 for ELISA and crystallography. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) (New England Biolabs). After induction at 16 C. for 16 hrs with 1 mM isopropyl R-D-1-thiogalactopyranoside (IPTG), the cells were harvested and resuspended in buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride (PMSF). For purification, the pellet was lysed with a tip sonicator, and centrifuged at 22,000g for 30 mins at 4 C. The supernatant was purified with Pierce Glutathione Agarose or Ni-NTA resin (Qiagen), eluting with 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM tris(2-carboxyethyl) phosphine (TCEP), 10% glycerol, with 10 mM reduced glutathione (GSH) or 250 mM imidazole. Eluted proteins were pooled, concentrated, and cleaved by adding the corresponding protease at a protease to protein ratio of 1:10 and incubated overnight at 4 C. yBBr-tag-containing proteins were biotinylated via the yBBr reaction according to standard procedures.sup.62. Final proteins were loaded onto a Superdex 10/300 75 pg or 200 pg (Cytiva) size exclusion chromatography (SEC) column and eluted in 50 mM Tris, pH 8.0, 200 mM NaCl, 10% glycerol, 1 mM DTT, and 1 mM EDTA. Fractions containing target protein were collected and pooled. GST contaminants were removed with an additional GST-purification step with target protein collected in the flow-through. Final protein fractions were concentrated and stored at 80 C.
[1585] The yBBr reaction was carried out as previously described (Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc National Acad Sci 102, 15815-15820 (2005)) with 100 M target protein tagged with ybbR13 (DSLEFIASKLA (SEQ ID NO: 41)), incubated with 150 M CoA-PEG11-biotin, 5 M Sfp, and 10 mM MgCl.sub.2 in protein storage buffer at room temperature for 1 hr. Excess CoA-conjugates and Sfp enzymes were removed by follow-up SEC.
Chemical Structure of CoA-PEG11-Biotin:
[1586] ##STR00417##
N-Terminal Domains of CUL1, CUL2, CUL4B, and CUL5
[1587] For proteins used in phage screening and SPR: the N-terminal domains of CUL1 (Uniprot ID: Q13616, residue 15-410, V367R/L371D), CUL2 (Uniprot ID Q13617, residues 8-384, V340R/L344D), CUL4B (Uniprot ID: Q13620, residues 206-557, V516R/L520D), and CUL5 (Uniprot ID: Q93034, residues 1-386, V341R/L345D) with N-terminal GST-TEV tags and C-terminal AVI tags were cloned into a pET-derived expression vector (Novagen). For proteins used in crystallography: the N-terminal domains of CUL5 (residues 8-384, V340R/L344D) or CUL4B (residues 206-557, V516R/L520D) with N-terminal GST-TEV tags were cloned into pET21b. Proteins were recombinantly expressed in E. coli BL21 CodonPlus cells (Agilent). The cells were induced at OD=0.6 with 1 mM IPTG for 4 hrs at 37 C., then harvested and resuspended in buffer, 20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, and 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, toggling between 3 sec on and 3 sec off for 20 min, and pelleted at 22,000g for 30 min. at 4 C. The supernatant was purified using Pierce Glutathione Agarose resin eluting with 20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, and 10 mM GSH. Eluted proteins were pooled, concentrated, and cleaved by adding TEV protease at a ratio of 1:10 protease to protein and incubated overnight at 4 C. TEV-cleaved proteins were biotinylated with the published AviTag technology (Tykvart, J. et al. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II. Protein Expres Purif 82, 106-115 (2012)). Briefly, purified target proteins were incubated with BirA biotin ligase with a 20:1 molar ratio, in a reaction buffer containing 50 M biotin, M ATP and 10 mM MgCl.sub.2 at 4 C. for 16 hrs proteins were loaded onto a Superdex 10/300 75 pg (Cytiva) SEC column and eluted in 20 mM Tris pH 7.4, 200 mM NaCl, 2 mM DTT, and 5% glycerol. Fractions containing target protein were collected and pooled. GST contaminants were removed with an additional GST-purification step with target protein collected in the flow-through. Final protein fractions were concentrated and stored at 80 C.
VHL, SOCS2, and FBWX7
[1588] For protein used in phage display screens and SPR: FBXW7 (Uniprot ID: Q969H0, residues 263-706) with an N-terminal GST-TEV tag and C-terminal AVI tag were co-expressed with full-length SKP1 (Uniprot ID: P63208, residue 1-163) in the pETDuet-1 plasmid. SOCS2 (Uniprot ID: 014508, residues 32-198) or VHL (Uniprot ID: P40337, residues 54-213) with an N-terminal 6His-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) cloned into pET21b and co-expressed with C-term AVI-tagged ELOB (Uniprot ID: Q15370, residues 1-104) and ELOC (Uniprot ID: Q15369, residues 17-112) cloned in pCDFDuet-1. For protein used in competition SPR (ABA mode) and x-ray crystallography: SOCS2 (residues 32-198) or VHL (residues 54-213) with an N-terminal 6His-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) cloned in pET21b was co-expressed with full-length ELOB (residues 1-118) and ELOC (residues 17-112) cloned in pCDFDuet-1. Recombinant proteins were expressed in E. coli BL21 (DE3) host cells and purified and biotinylated as for the WWP and Cullin proteins above.
MDM2 and MDM4
[1589] For proteins used in the phage display screens and SPR: the p53-binding domain of MDM2 (Uniprot ID: Q00987, residues 25-109) with an N-terminal 6His-yBBr-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 CodonPlus cells (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 1 mM IPTG for 4 hrs at 37 C., then harvested and resuspended in 25 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, and pelleted at 22,000g for 30 mins at 4 C. The pellets were washed three times with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 M urea, 1.0% Triton X-100, and dissolved in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 8 M urea, and 2 mM B-mercaptoethanol (B-me). The supernatant was purified using a Ni-NTA resin (Qiagen), and eluted with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 8 M urea, 2 mM B-me, and 250 mM imidazole. Protein elutes were diluted to 0.1 mg/mL and dialyzed into buffers containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM B-me, with 4, 2, 1, or 0 M urea, at 4 C. for 8 hr for each urea gradient. Urea-free proteins were concentrated with Amicon spin filters (Millipore Sigma) to 1 mg/mL and biotinylated via the yBBr reaction according to standard procedures (Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc National Acad Sci 102, 15815-15820 (2005)), as above. Biotinylated proteins were pooled, concentrated, and loaded onto a Superdex 10/300 75 pg (Cytiva) SEC column and eluted in 20 mM HEPES pH 7.0, 200 mM NaCl, 5% glycerol, 0.5 mM TCEP. Fractions containing pure protein were collected, pooled, concentrated to 1 mg/mL and stored at 80 C.
[1590] For protein used in crystallography and other biochemical assays: p53-binding domains of MDM2 (residues 17-111, with C17S substitution; MDM2.sup.17-111) with an N-terminal 6His-TEV tag (HHHHHH disclosed as SEQ ID NO: 39), and MDM4 (Uniprot ID: O15151, residues 14-111, with C17S substitution; MDM4.sup.14-111) with an N-terminal 6His-yBBr-3C tag (HHHHHH disclosed as SEQ ID NO: 39) were recombinantly expressed in E. coli BL21 (DE3) cells (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 16 hr at 37 C., then harvested and resuspended in 50 mM Tris, pH 8.0, 200 mM NaCl, 10% glycerol, 1 mM TCEP, and 20 mM imidazole. For purification, the pellet was lysed with a tip sonicator, toggling between 3 sec on and 3 sec off for 20 min, and then centrifuged at 22,000g for 30 min at 4 C. The supernatant was purified using a Ni-NTA resin (Qiagen), and eluted with 50 mM Tris, pH 8.0, 200 mM NaCl, 10% glycerol, 1 mM TCEP, and cleaved by adding protease (TEV or PreScission protease) at a protease to protein ratio of 1:10 and incubated overnight at 4 C. Cleaved proteins were loaded onto a Superdex 10/300 75 pg (Cytiva) SEC column and eluted in 50 mM Tris, pH 8.0, 200 mM NaCl, 10% glycerol, and 1 mM TCEP. Fractions containing pure protein were collected, pooled, concentrated to 8 mg/mL and stored at 80 C.
[1591] For protein labeling: Purified tag-free MDM2.sup.17-111 and MDM4.sup.14-111 were loaded onto a Superdex 10/300 75 pg (Cytiva) SEC column and eluted in 25 mM HEPES pH 7.5 and 250 mM NaCl, with pooled protein factions with the concentration at 300 M. The protein was then mixed with Alexa Fluor 488 NHS Ester (Thermo Scientific) prepared as 100 mM stock, with a final protein to NHS ratio of 1:0.8. The reaction was carried out at room temperature and quenched with 50 mM hydroxylamine before the final SEC purification in 25 mM HEPES pH 7.5, 250 mM NaCl and 1 mM TCEP buffer. Fractions containing Alexa488-labeled protein were collected, pooled, and stored at 80 C.
CHIP
[1592] For protein used in phage screen and SPR: N-term truncated CHIP (also known as STUB1, Uniprot ID: Q9UNE7, residues 23-303; CHIP.sup.23-3.sup.03) or the TPR domain of CHIP (residues 23-154; CHIP.sup.23-15) with an N-terminal 6His-yBBr-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 CodonPlus cells (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 1 mM IPTG for 4 hrs at 37 C., then harvested and resuspended in 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 10% glycerol, and 10 mM 0-me. For purification, the pellet was lysed with a tip sonicator and centrifuged at 22,000g for 30 mins at 4 C. The supernatant was purified with Ni-NTA resin (Qiagen), eluted with 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM R-me and 250 mM imidazole, and biotinylated via the yBBr reaction according to standard procedures as above. Biotinylated proteins were pooled, concentrated, and loaded onto a Superdex 10/300 75 pg (Cytiva) SEC column, and eluted in 20 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 2 mM DTT. Fractions containing pure protein were collected, pooled, concentrated to 1.2 mg/mL and stored at 80 C.
[1593] For proteins used in crystallography and other biochemical assays: CHIP TPR domain, CHIP.sup.21-154 or CHIP.sup.23-1.sup.54, or CHIP.sup.23-3.sup.03 each with an N-terminal 6His-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) were recombinantly expressed in E. coli BL21 CodonPlus cells (Agilent) from a pET21b-derived expression vector (Novagen). The cells were induced at OD.sub.600=0.6 with 1 mM IPTG for 4 hrs at 37 C. or 16 hrs at 16 C., then harvested and resuspended in 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 10% glycerol, and 10 mM B-me. For purification, the pellet was lysed with a tip sonicator, toggling between 3 sec on and 3 sec off for 20 min, centrifuged at 22,000g for 30 mins at 4 C. The supernatant was purified with Ni-NTA resin (Qiagen), eluting with 250 mM imidazole. Eluted proteins were pooled, concentrated, and the TEV tag cleaved off by adding TEV protease at a protease to protein ratio of 1:10 and incubated overnight at 4 C. Cleaved/untagged proteins were loaded onto a Superdex 10/300 75 pg (Cytiva) SEC column, and eluted in 50 mM HEPES, pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM DTT. Fractions containing pure protein were collected, pooled, concentrated to 30 mg/mL and stored at 80 C.
[1594] For Alexa488 labeling: Purified tag-free CHIP.sup.23-3.sup.03 was labeled as MDM2.sup.17111 above, with the final SEC purification in buffer: 20 mM Tris pH 7.5, 250 NaCl, and 1 mM DTT. Fractions containing Alexa488-labeled protein were collected, pooled, concentrated to 0.6 mg/mL, and stored at 80 C.
PPIA
[1595] Full length PPIA (Uniprot ID: P62937, residues 1-165) with an N-terminal 6His-yBBR-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) CodonPlus RIPL cells (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 16 hr at 16 C., then harvested and resuspended in PBS pH 7.4 with 1 mM PMSF. For purification, cell pellets were lysed with a tip sonicator and centrifuged at 22,000g for 30 min at 4 C. The resulting supernatant was collected then centrifuged again at 22,000g for 30 min at 4 C. The supernatant was purified with Ni-NTA resin (Qiagen) and eluted with 250 mM imidazole. TEV was cleaved from the recombinant proteins by adding TEV protease at a protease to protein ratio of 1:10 and incubation for 4 hr at 4 C. Protein was then concentrated and diluted into 20 mM HEPES pH 7.0, 5% glycerol, and centrifuged at 22,000g for 10 min at 4 C. The supernatant was loaded onto an SP HP (Cytiva) column pre-equilibrated with 20 mM HEPES pH 7.0, 5% glycerol. Purified protein was eluted with a gradient from 0 mM to 1 M NaCl. Protein fractions were pooled, concentrated then centrifuged at 22,000g for 10 min at 4 C. The supernatant was collected and loaded onto a Superdex 16/600 75 pg (Cytiva) SEC column pre-equilibrated with PBS pH 7.4. Purified proteins were eluted isocratically in PBS pH 7.4. Protein fractions were collected, concentrated, aliquoted and frozen.
TEAD4
[1596] YAP/TAZ binding domain of TEAD4 (Uniprot ID: Q15561, residues 217-434) with an N-terminal 6His-yBBr-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21-CodonPlus cells (DE3) (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 16 hrs at 16 C., then harvested and resuspended in 50 mM Tris pH 7.4, 200 mM NaCl, 5% glycerol, 1 mM TCEP, 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 30 min at 4 C., then the supernatant was purified with an Ni-NTA column (Cytiva), eluting with 250 mM imidazole. Protein-containing fractions were pooled, concentrated, and loaded onto a Superdex 75 10/300 (Cytiva) SEC column. Purified proteins were eluted isocratically in 50 mM Tris pH 7.4, 200 mM NaCl, 5% glycerol, 1 mM TCEP, and fractions containing pure protein were collected, pooled and frozen.
-catenin (CTNNB1)
[1597] -catenin protein (encoded by CTNNB1, Uniprot ID: P35222) Armadillo domain (residues 134-665) with a N-terminal 6His-yBBr-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) pLysS cells (Thermo Fisher) from pET28a vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 16 hrs at 16 C., then harvested and resuspended in 25 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 20 mM imidazole, 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, centrifuged at 22,000g for 30 min at 4 C., then the supernatant was purified with HisTrap HP columns (Cytiva), eluting with 250 mM imidazole. For crystallography and selected biochemical assays, protein was TEV-cleaved by adding TEV protease at a protease to protein ratio of 1:10 and incubated overnight at 4 C. For phage display screening and SPR analysis, protein was biotinylated via the yBBr reaction according to standard procedures. All proteins were concentrated using Amicon spin filters (Millipore Sigma) then diluted into 25 mM Tris, pH 8.8, 1 mM DTT, 10% glycerol and loaded onto a Q HP (Cytiva) column. Proteins were eluted with a gradient from 50 mM to 600 mM NaCl. Protein-containing fractions were pooled, concentrated and loaded onto a Superdex 10/300 200 pg (Cytiva) SEC column. Purified proteins were eluted isocratically in 25 mM Tris-HCl, pH 8.8, 10% glycerol, 300 mM NaCl, and fractions containing pure protein were collected and pooled.
[1598] Full-length -catenin protein (CTNNB1, residues 1-781) with N-terminal 6His-thrombin-T7-TEV tag (HHHHHH disclosed as SEQ ID NO: 39) was recombinantly expressed in E. coli BL21 (DE3) pLysS cells (Thermo Fisher) from pET28a vectors (Novagen). E. coli cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 20 hrs at 16 C., shaking at 180 rpm, then harvested and resuspended in 20 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol. For purification, the pellet was lysed with a sonicator, pelleted at 130,000 rpm for 30 mins at 4 C., then the supernatant was purified with a Ni-NTA column (Cytiva), eluting with 250 mM imidazole. Protein was then diluted into 20 mM HEPES, pH 7.5, 10% glycerol, 1 mM DTT and loaded onto a Q HP column (Cytiva), and was eluted with a NaCl gradient of concentration 0 to 1.0 M. Protein-containing fractions were pooled, concentrated, and loaded onto a Superdex 10/300 200 pg (Cytiva) SEC column. Purified protein was eluted isocratically in 20 mM HEPES, pH7.5, 300 mM NaCl, 10% glycerol and 1 mM DTT, and fractions containing pure protein were collected and pooled.
ICAT (CTNNBIP1)
[1599] Full-length ICAT (CTNNBIP1, Uniprot ID: Q9NSA3, residues 1-81) with an N-terminal GST-TEV tag was recombinantly expressed in E. coli BL21-CodonPlus cells (DE3) (Agilent) from pET-derived expression vectors (Novagen). The cells were induced at OD.sub.600=0.6 with 0.15 mM IPTG for 16 hrs at 16 C., then harvested and resuspended in 20 mM Tris pH 7.4, 200 mM NaCl, 10% glycerol, 0.5 mM TCEP, 1 mM PMSF. For purification, the pellet was lysed with a tip sonicator, pelleted at 22,000g for 30 min at 4 C., then the supernatant was purified with a GST column (Cytiva), eluting with 10 mM glutathione. Protein-containing fractions were pooled, concentrated, and loaded onto a Superdex 75 10/300 (Cytiva) SEC column. Purified proteins were eluted isocratically in 20 mM Tris pH 7.4, 200 mM NaCl, 10% glycerol, 0.5 mM TCEP and fractions containing pure protein were collected, pooled and frozen.
[1600] Trimerizer Phage Library construction (e.g., primers, protocol, crosslinking, and DNA sequencing, etc.)
[1601] Certain useful technologies are described in Li, K. et al. De novo mapping of -helix recognition sites on protein surfaces using unbiased libraries. Proc National Acad Sci 119, e2210435119 (2022), and can be utilized in accordance with the present disclosure.
[1602] Naive Phage Library: Naive phage-displayed Helicon libraries were constructed using described methods. Briefly, the Peptide Display Cloning System kit from New England Biolabs was used to construct M13KE-based libraries (New England Biolabs, Ipswich, MA). Library oligonucleotides were chemically synthesized using a mix of trimer phosphoramides (Glen Research, Sterling, VA) without codons encoding cysteine, lysine, proline, or glycine, then annealed, extended, and ligated into a digested M13KE vector. All DNA products were purified using Monarch PCR and DNA cleanup kit (New England Biolabs, Ipswich, MA). The resulting library-containing phage vector was transformed into E. coli strain ER2738 (Lucigen, Middleton, WI) by electroporation and amplified by adding the post-rescue electroporated cells to a 500 mL E. coli culture at early-log phase (OD.sub.600=0.01). Phage propagation, purification, and stapling were conducted as described.
Trimerizer Library
[1603] Following identification of Helicon clusters specific for a presenter protein of interest based on screening the naive library, trimerizer library oligonucleotides were designed. Presenter-specific clusters of various sizes were used, ranging in size, e.g., from 10-mer to 20-mer. As an illustrative example of the design of a trimerizer library, a presenter-specific 20-mer cluster, X.sub.1X.sub.2X.sub.3X.sub.4W.sub.5E.sub.6C.sub.7X.sub.8E.sub.9A.sub.10A.sub.11(F/I/L/M).sub.12X.sub.13C.sub.14X.sub.15(F/Y).sub.16(F/Y).sub.17X.sub.18X.sub.19X.sub.20 (SEQ ID NO: 23), is used. Briefly, codons of conserved or semi-conserved residues responsible for binding with a presenter protein are fixed or partially randomized in the primer to bias the library for retained affinity towards the chosen presenter protein (see, e.g.,
[1604] Certain useful technologies are described in the Table below.
TABLE-US-00030 TABLEE11-4 Certaintechnologies. SEQ ID Name Sequence NO: Presenter PR01 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXXg 716 CHIP cagcaASCATTTGTATGGTTGATXXXggtggttctggcgcaggtcgtggttc PR02 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXXg 717 CHIP cagcaASCATTTGTTGGATTGATXXXggtggttctggcgcaggtcgtggttc PR03 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXXg 718 CHIP cagcaGAGRTTTGTTGGMTTTATXXXggtggttctggcgcaggtcgtggttc PR04 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXCA 719 CHIP TgcagcaGATRTKTGTTGGMTTTWTXXXggtggttctggcgcaggtcgtggttc PR05 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXTWTGAKXT 720 CHIP GTXXgcagcaATTAKGTGTMTTGTTXXXXggtggttctggcgcaggtcgtggttc PR06 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXTTTTGGXTG 721 CHIP TCAGTWTgcagcaGCGAYGTGTXXXXXXggtggttctggcgcaggtcgtggttc PR07 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXTATXTGTA 722 CHIP TGGAGgcagcaSTGCTGTGTAYGXXXXXggtggttctggcgcaggtcgtggttc PR08 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXTGGAGCTG 723 CHIP TGYGGAGgcagcaTWTMTRTGTGAKXYWTXXXggtggttctggcgcaggtcgtggttc PR09 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXASTATTTGG 724 CHIP TGTRTTGATgcagcaXXTGTXXXXXXggtggttctggcgcaggtcgtggttc PR10 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXMTGTGT 725 CHIP ATTTGGgcagcaGATGATTGTXXXXXXggtggttctggcgcaggtcgtggttc PR11 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXASCGATXTG 726 CHIP TGTGTATgcagcaTATTTCTGTGAKXXXXXggtggttctggcgcaggtcgtggttc PR12 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXXG 727 CHIP CGAGCMTTTGGTGTRTTGATXXXXggtggttctggcgcaggtcgtggttc PR13 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTTGG 728 CHIP GAKGCGTGGVTTXTGTGAAXXXXXggtggttctggcgcaggtcgtggttc PR14 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTTWT 729 MDM2 XGCAGCATTTXTGTVTTXGATXXXggtggttctggcgcaggtcgtggttc PR15 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXGATTGTT 730 MDM2 TTXGCAGCATGGXTGTWTKXXXXXggtggttctggcgcaggtcgtggttc PR16 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXSAAXXTGTT 731 MDM2 WTCAAGCAGCATGGXTGTVTTXGATXXXggtggttctggcgcaggtcgtggttc PR17 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTTTT 732 MDM2 TGGGCAGCATGGXTGTXXXXXXggtggttctggcgcaggtcgtggttc PR18 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTTTT 733 MDM2 CAGGCAGCATGGGATTGTCAGTWTTGGXXXggtggttctggcgcaggtcgtggttc PR19 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTTTT 734 MDM2 CAGGCAGCATGGGATTGTCAGTWTTTTXXXggtggttctggcgcaggtcgtggttc PR20 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXGA 735 MDM2 TGCAGCATTTXTGTXTWTTATXXXggtggttctggcgcaggtcgtggttc PR21 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXTGGGAATG 736 MDM2 TXGAAGCAGCAWTKXTGTXTWTTWTXXXggtggttctggcgcaggtcgtggttc PR22 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXTGGXTGTT 737 MDM2 GGGAKGCAGCAXXTGTXGAKXXXXggtggttctggcgcaggtcgtggttc PR23 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTXXG 738 MDM2 CAGCATTTXTGTXTGGXXXXggtggttctggcgcaggtcgtggttc PR24 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXXXTGTTWT 729 MDM2 XGCAGCATTTXTGTVTTXGATXXXggtggttctggcgcaggtcgtggttc PR25 CATGCCCGGGTACCTTTCTATTCTCACTCTGCGCCGXXXXTGGXTGTA 739 MDM2 CCASTGCAGCARTTXTGTXTWTXXXXggtggttctggcgcaggtcgtggttc
TABLE-US-00031 Mixed-base code Mixed Bases R A, G Y C, T M A, C K G, T S G, C W A, T H A, C, T B G, C, T V A, C, G D A, G, T N A, C, G, T
Phage Library Screening.
Naive Library Presenter Screening.
[1605] To conduct phage library screening, previously described procedures were utilized in accordance with the present disclosure. Briefly, Helicon-displayed phage libraries were incubated with streptavidin magnetic beads for 1 hr at room temperature in a buffer of 1TBS, 1 mM MgCl.sub.2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 5% (w/v) nonfat milk. For each screening condition, 100 L of 2 M biotinylated protein was captured with streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher Scientific, Waltham, MA) that had been previously blocked with 1% BSA, 0.1% Tween-20, 2% glycerol in 1TBS pH 7.4 at room temperature for 15 min, the supernatant was removed using a plate magnet and the beads are resuspended in 50 L of the blocking buffer. 150 L of the depleted phage library is added to each well for 200 L final volume, plates are sealed, and the screening reactions are incubated at room temperature for 45 min, with rotation to maintain beads in solution. Following binding, beads were washed 5 times with ice-cold washing buffer (1TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 2% (w/v) glycerol), beads containing protein-bound phage were collected and directly processed for NGS.
Trimerizer Phage Screening.
[1606] Trimerizer phage screening is performed as for the naive library screening described above, with the key practical difference being that the Trimerizer library is incubated with a presenter protein after removal of the bead-binding phage library members and prior to the incubation with biotinylated proteins bound to streptavidin magnetic beads. To identify presenter-dependent phage-displayed Helicon members, target proteins were screened with the phage library in both the presence and absence of a presenter protein. Prior to addition of the bead-bound targets, sequences that bind to the target alone or to the presenter alone were removed: the phage library was split into two portions, and the presenter protein was added to one portion to a final concentration of 10 M. 150 L of the phage library without presenter protein was then added to a well containing 50 L of the highest concentration of the target protein, and also to a blank (beads-only) well, both for a final volume of 200 L. To the remaining wells, 150 L of the phage library mixed with 10 M presenter protein was added to wells containing 50 L of the target of interest at a range of concentrations, and also to a blank (beads-only) well. The plate was sealed, and the screening reactions were incubated at room temperature for 45 min, with rotation to maintain beads in solution. The rest of the experiment was performed similar to the procedure described above with one exceptionthe addition of a presenter protein at a final concentration of 10 M to the wash buffer (ice-cold iX TBS, 1 mM MgCl2, 1% (w/v) BSA, 0.1% Tween-20, 0.02% (w/v) sodium azide, 2% (w/v) glycerol).
Next-Generation Sequencing.
[1607] Next-generation sequencing was performed as described in accordance with the present disclosure. Briefly, phage particles were denatured from magnetic beads at 95 C. for 15 min with an added spike-in sequence (a non-library member) that is used to enable cross-well normalization of sequence reads, followed by a two-step low-cycled PCR to introduce Illumina adaptors and 10 bp TruSeq DNA UD Indexes (Illumina, San Diego, CA) according to Illumina's 16S Metagenomic Sequencing Library Preparation protocol. The NGS library was sequenced with an Illumina NovaSeq platform using a 2x150 bp high-output kit (Illumina, San Diego, CA).
Hit ID and Clustering.
[1608] Hit ID and Clustering is performed according to a described procedure in accordance with the present disclosure. Briefly, NGS reads were trimmed for quality (Phred score >18) and filtered for sequences that matched the design of the phage library. Counts for each unique sequence were tallied, and then normalized by the counts of the spike-in sequence added to each sample. A metric called Hit Strength was computed for each sequence as the fold-change between the normalized counts in the highest target concentration sample with presenter and the normalized counts in the target (no presenter) samples (averaged across experimental replicates). By using target wells with no presenter as target blanks, presenter-dependent binding could be identified. This approach eliminates sequences that show binding to target alone, or binding to a free presenter alone. When 0 counts are observed for a sequence in target only target blank samples, a count of 0.5 is used to prevent dividing by zero (Supplementary Dataset 1). In some embodiments, sequences with a hit strength greater than 5 were subjected to hierarchical clustering to identify sequence families.
Helicon synthesis.
[1609] Various technologies are available for preparing stapled peptides and can be utilized in accordance with the present disclosure. In some embodiments, technologies for synthesis of cysteine-stapled Helicons was utilized. Briefly, linear peptides containing two cysteine residues were synthesized at 100 or 250 mol scale on Rink Amide resin (-0.5 mmol/g) using standard Fmoc-based solid phase peptide synthesis workflows. The peptides were globally deprotected and cleaved off-resin, then dissolved in DMSO. The DMSO stock was diluted in a 2:1 solvent mixture of acetonitrile and 50 mM ammonium hydroxide. The pH of the solution was adjusted to 8.5 using N, N-Diisopropylethylamine (DIPEA). For crosslinking of cysteine residues, 1.3 equivalents of the alkylating agent, N, N-(1,4-phenylene)bis(2-bromoacetamide) in DMF were added to the crude peptide solution for two hours at room temperature. The crude helicons were purified by preparatory HPLC, and the purity of the final products were analyzed with analytical UPLC. The R8-S5 stapled peptides, including ATSP-7041 (P320), were synthesized according to reported procedures.
[1610] Certain agents and data are presented in Table E11-5 as examples.
TABLE-US-00032 TABLEE11-5 Certaintrimerizeragentsanddata. CHIP-PPIAC86 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PGPMRAECTVAATICMVDGAL 740 9 108 334 272 300 0 9 0 0 PLIALEDCTVAATICMVDGVV 741 0 26 108 105 78 0 0 0 0 PLQQEHLCSVAATICWIDGRI 742 5 84 386 432 321 0 5 0 0 PQQQIQYCTVAASICMVDGIT 743 0 31 50 136 44 0 0 0 0 PWDNEGFCTVAATICMVDGTV 744 0 8 56 45 54 0 0 0 0 PYSLQTACTVAATICWIDGTI 745 0 17 134 166 78 0 0 0 0 PYEEHTMCSVAATICWIDGPV 746 0 22 204 174 163 0 0 0 3 PKYFTADCSVAATICMVDGSS 747 0 6 38 42 41 0 0 0 0 PIEMMVECTAAATICMVDGWV 748 0 0 42 39 34 0 0 0 0 PMAEPEQCTVAATICMVDGMS 749 0 0 58 49 75 0 0 0 0 PMYMTGQCTVAATICMVDGTH 750 0 1 71 73 78 0 0 0 0 PYRDNVECGVAASICMVDGSS 751 0 0 29 32 31 0 0 0 0 PGPVYQHCTVAASICMVDGIT 752 0 4 38 28 70 0 0 0 0 PMATVQQCTVAATICWIDGII 753 0 0 15 25 13 0 0 0 0 PQYKINHCTVAASICMVDGRA 754 0 3 23 36 28 0 0 0 0 CHIP-TEAD4C87 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PVPFFWECQYAAAMCQQWYNK 755 0 0 56 29 114 0 0 6 0 PTPFFWDCQFAAAMCTKQPLR 756 0 6 38 36 65 0 0 0 0 PVPFFWECQYAAATCQTPRIK 757 4 40 434 499 572 0 0 0 0 PVPFFWECQYAAATCPLVTAK 758 0 3 69 61 112 0 0 0 0 PTNFYDPCVKAAIRCIVTDFE 759 0 0 9 9 14 4 0 3 0 PTPFFWECQFAAATCTAPRVQ 760 13 244 1155 1428 1982 0 0 0 0 PTPFFWDCQFAAATCYWSSHK 761 3 2 15 22 19 0 0 0 0 PVPFFWECQFAAATCIHVYPA 762 0 0 50 60 90 0 0 0 0 PVPFFWDCQFAAATCDAPQRR 763 0 11 200 343 360 0 0 0 0 PIPFFWECQFAAATCNRWTQW 764 0) 0 12 13 32 0 0 0 3 PVPFFWPCQFAAATCAGTATQ 765 0 0 18 15 68 0 0 0 0 PTPFFWECQYAAAMCNWDRTH 766 0 5 37 60 91 0 0 0 0 PVPFFDECAQAAIRCIVTQVD 767 0 0 8 7 32 0 0 0 0 PMPFFWECQFAAATCDRRVNW 768 0 0 11 29 25 0 0 0 0 PVPFFWDCQYAAAMCVAPHRF 769 0 0 17 44 40 0 0 0 0 CHIP-PIPAC88 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PNWMYWSCVEAAYLCENYVMV 770 0 8 6 14 16 0 0 4 0 PIDRHWSCVEAAYICENYVLT 771 0 15 29 15 44 0 0 0 0 PTMFSWSCVEAAYLCENYVLI 772 0 18 42 17 62 0 0 0 0 PKMQMWSCVEAAFLCENYVFI 773 5 29 117 98 140 0 0 0 0 PGYEMWSCAEAAFLCENYVWA 774 5 40 171 95 191 0 0 0 3 PHNGKWSCVEAAYICENYVYT 775 0 26 384 286 328 0 0 0 0 PAQDDWSCVEAAYLCENYVRV 776 1 5 311 265 266 0 0 0 0 PEHEQWSCVEAAYLCENYVMT 777 3 41 294 293 375 0 0 0 0 PHVSPWSCVEAAYLCENYVRT 778 0 0 33 24 34 0 0 0 0 PMQRQWSCVEAAYLCENYVMV 779 0 13 111 115 147 0 0 0 0 PPKSSWSCVEAAFICENYVRI 780 0 8 73 72 106 0 0 0 0 PIMETWSCVEAAYLCENYVKS 781 0 0 175 204 196 0 0 0 0 PHIQQWSCAEAAYMCENHVAT 782 0 3 13 17 18 2 0 0 0 PPYHSWSCVEAAYLCENYVRA 783 0 1 48 69 80 0 0 0 0 PPAPSWSCVEAAYLCENYVWV 784 0 5 88 145 147 0 0 0 0 CHIP-PIPAC89 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PVRAYAMCGPAASICWIDGII 785 3 39 219 197 235 0 0 0 0 PILQGMACGPAATICWIDGII 786 5 34 207 160 212 0 0 0 0 PDMLAPMCGPAASICWIDGVI 787 8 19 123 72 122 0 0 0 0 PPFHMMMCGPAASICMVDGVV 788 0 13 240 212 222 2 0 0 0 PDMEFITCGPAASICMVDGMT 789 0 0 17 15 34 0 5 0 0 PTINMNNCGPAASICWIDGVV 790 0 4 50 62 122 0 0 0 0 PTSEFVVCGAAASICWIDGKV 791 0 0 6 10 16 0 0 0 0 PMYMTDACGPAATICMVDGVV 792 0 8 104 236 199 0 0 0 0 PYPWMHDCGVAATICWIDGVI 793 2 10 60 136 127 0 1 0 3 PDHRFVYCGPAATICMVDGSV 794 0 3 19 84 59 0 0 0 0 PHPVDYMCGPAATICMVDGYI 795 0 2 6 29 57 0 0 0 0 PMSYTKACGPAASICWIDGFI 796 3 0 0 20 21 0 0 0 0 PWGRPVECGPAATICMVDGKI 797 18 402 5468 6954 6280 0 6 0 26 PDPVHVMCGPAASICMVDGVV 798 14 189 1028 1016 667 0 0 13 0 PMWQWPECGPAATICWIDGMI 799 0 27 159 135 80 0 0 0 0 CHIP-TEAD4C90 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PDDPMDLCEYAAEVCWIYIDS 800 0 26 45 60 35 0 0 0 0 PQEGVDLCEYAAEVCWIYIKE 801 0 0 13 25 16 0 0 0 0 PDLPVDICAHAADVCWLYLDH 802 0 6 74 121 198 0 0 0 0 PPWPVDICQHAAEVCWLYLPI 803 0 0 5 12 16 0 0 0 0 PYEPFWLCQFAAATCHRWMEV 804 0 0 2 7 9 6 2 0 0 PIEYIDVCDYAAEICWIYAES 805 0 0 6 28 56 0 0 0 0 PLHPIDVCEYAAEVCWIYLDD 806 0 0 1 9 12 0 0 0 0 PDEPIDLCEHAAEICWLYDQS 807 0 0 3 12 83 0 0 0 0 PQWPVDMCNHAADVCWIYMDY 808 4 0 1 19 21 0 0 3 0 PYEEVDLCEHAADVCWLYLDV 809 0 0 0 15 19 0 0 0 0 PEPGPDICAHAADVCWIYVEE 810 0 0 0 13 12 0 0 0 0 PHSFIDLCEHAADVCWLYGDD 811 0 10 0 31 44 0 2 0 0 PVMPVWSCVEAAYMCEWLLDP 812 0 5 0 7 18 0 0 0 0 MDM2-beta-cateninC91 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PPVKEAICFQAAWQCLSDDWA 813 0 22 26 18 51 0 0 0 0 PIEMQSVCFQAAWTCLSDTWY 814 5 31 28 62 46 0 0 0 0 PIYHTNDCFGAAWYCFSEEWV 815 8 22 28 42 37 0 0 0 1 PFKDERVCFQAAWMCVSDDYN 816 12 31 44 74 46 0 0 0 0 PTNIEQPCFQAAWQCVTDDWT 817 0 11 17 21 20 0 0 0 0 PMEQQAICFQAAWMCLADDWT 818 17 58 121 124 92 0 0 0 0 PWKYEQVCYQAAWQCLSDDWD 819 40 105 179 209 287 0 0 3 0 PNLPQRVCFQAAWLCLSDDWT 820 0 6 24 29 15 0 0 0 0 PRNSENICYQAAWYCLTDDWI 821 0 6 29 19 41 0 0 0 0 PGWKQGICFQAAWMCVADEWI 822 3 10 30 35 36 0 0 0 0 PFMSQEVCFQAAWLCVMDDYV 823 4 11 25 33 35 0 0 0 0 PIKTEHVCYQAAWQCLVDDEN 824 2 4 29 14 46 0 0 2 0 PYVHQDICFQAAWMCVKDEWM 825 10 17 42 60 51 0 0 0 0 MDM2-beta-cateninC92 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PTQFKPDCFGAAFTCIHDMYM 827 6 5 5 6 7 0 0 0 0 PDQHHHDCFGAAWHCLLFLED 828 0 10 11 5 24 0 0 0 0 PAKYQQDCFRAAWGCLEYLYY 829 9 86 99 134 162 0 0 0 0 PEQFQKDCFPAAWQCLVYIYG 830 0 5 7 13 7 0 0 0 0 PISAANDCFKAAWQCIIWLHQ 831 12 20 69 58 62 0 0 0 0 PSENARDCFWAAWDCLYFIYQ 832 8 17 63 58 76 0 4 0 0 PEPYIADCFDAAWRCLMYIYD 833 0 4 10 9 18 0 0 7 0 PKVAPPDCFEAAWNCLYFIYN 834 0 0 9 6 9 0 0 0 0 PHQDKQDCFGAAWTCIEWIYQ 835 0 37 89 94 167 0 0 0 0 PVQDHPDCFFAAWDCLDWLYT 836 0 0 10 7 22 0 10 0 0 PSRAKDDCFKAAWNCLEYIWH 837 0 3 10 27 13 0 0 0 0 PEERKNDCFDAAWECIQYLYD 838 7 24 108 226 176 0 0 0 2 PDQNEEDCYQAAWTCILDLYR 839 0 0 4 8 8 0 0 0 4 PEEWRGDCFSAAWDCLNYIYD 840 0 0 7 16 11 0 0 0 0 MDM2-beta-cateninC93 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 1000 BLANK BLANK BLANK BLANK BLANK PNLTGADCFPAAWQCLQFLWD 841 83 128 146 142 126 0 0 0 0 PKGLQSICYQAAWQCLVDTWD 842 30 56 111 137 101 0 0 0 3 PAWEQMYCFQAAWQCITDIWD 843 3 4 11 9 22 0 0 0 0 PQITKADCFPAAWECIMVIFD 844 0 0 6 5 8 0 0 0 0 PDTQQNTCFQAAWMCVSDDWD 845 0 10 27 27 51 0 0 0 0 PITQESVCFQAAWQCLKDPWE 846 0 0 5 5 7 0 0 0 0 PQWEESVCFQAAWHCLADTWD 847 0 0 10 25 43 0 3 0 0 PLLSQYPCFQAAWQCISDIFD 848 0 3 3 6 24 0 0 0 0 PDPQETWCFQAAWQCVADTWD 849 0 0 3 12 10 4 0 0 0 PMTTQQVCFQAAWQCVGDTWD 850 0 0 1 16 20 0 0 0 0 PEIKEWACFQAAWQCLDDTWD 851 0 0 0 6 16 0 0 0 0 PKIIQSVCFQAAWQCLTDTYF 852 0 0 0 6 14 0 0 0 0 PKTMQPVCFQAAWMCVTDHWD 853 4 0 3 14 17 0 3 0 0 PKLNQQECFQAAWWCVRDPWD 854 0 3 0 13 6 0 0 0 0 CHIP-PPIAC94 SEQID Presenter+Target TARGET TARGET Sequence NO: 30 100 300 BLANK BLANK BLANK BLANK BLANK BLANK PPRHENECIHAADICWLYWLY 855 0 6 86 41 171 0 0 0 0 PYSIPTMCQHAADICWLYWLY 856 0 6 33 51 28 0 0 0 0 PLMLDQECTDAAEVCWIYWLY 857 0 5 33 44 31 0 0 0 0 PLWDDQYCDHAADVCWLYWLY 858 0 8 42 60 41 0 3 0 0 PDWQATHCPHAADVCWLYWLY 859 0 8 27 46 28 0 0 0 0 PDVVEVPCLNAAEVCWLYWLY 860 0 12 38 48 62 0 0 0 0 PWGSTDICVYAAYFCESEGLY 861 0 0 8 8 13 0 0 0 0 PLEQFQQCVDAAEVCWLYWLY 862 0 0 10 11 16 0 0 0 0 PGPQQDKCAQAAEICWLYWLY 863 0 2 15 10 41 0 0 0 0 PETEYPQCQDAAEVCWLYWVY 864 0 0 19 29 21 0 0 0 0 PKLEPSNCNHAADVCWLYWLY 865 0 21 142 212 321 0 0 0 0 PEPEYSACSNAAEICWLYWVY 866 0 0 10 14 26 0 0 0 0 PIADTQLCVEAAEICWLYWLY 867 0 0 15 29 31 0 0 0 0 PTPSSGQCPHAADVCWLYWLY 868 6 31 163 271 434 0 0 0 0 PAQVQHNCPDAAEICWLYWLH 869 0 0 15 32 28 0 0 0 0 Blank =Media only. Target Blank =target only (no presenter). Unless otherwise noted, target and presenter concentrations in nM.
Surface Plasmon Resonance (SPR) Spectroscopy.
[1611] SPR of E3-binding helicons: To confirm Helicon binding to all selected E3 ligases and E3-related proteins, SPR experiments were performed on a Biacore 8K (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. A SA Series S sensor chip was docked and pre-conditioned with three injections of 50 mM NaOH/1 M NaCl to remove unbound streptavidin from the surface. Biotinylated proteins, including CUL4B.sup.NTD, CUL5.sup.NTD, WWP1.sup.WW-HECT, WWP1.sup.HECT, WWP2.sup.HECT, VHL-ELOBC, SOCS2-ELOBC, CHIP.sup.23-154, CHIP.sup.23-303, and MDM2.sup.25-109, were each diluted to 5-10 g/mL in running buffer and immobilized to channels 1 through 8 at 5 L/min for 50-80 sec for a final immobilization level of 500-2000 R U. Helicons were diluted to 5 M in running buffer and then serially diluted 2-fold for a total of seven concentrations with one blank (7-point two-fold Helicon dilution series with top concentration=5 M and bottom concentration=78 nM). Compounds were injected over the immobilized and reference surfaces at 30 L/min for 60 sec and then allowed to dissociate for 180 sec without surface regeneration (N=1-2). Data were analyzed using Biacore Insight Evaluation Software (Cytiva). Sensorgrams were double referenced, with most of them fitted to a 1:1 steady-state affinity model, with a few fitted with both the steady-state affinity model and the kinetics model.
[1612] SPR analysis of the trimerizer Helicon-dependent -catenin:MDM2 interaction: To probe the trimerizer Helicon-dependent CTNNB1: MDM2 interaction, SPR experiments were performed on a Biacore S200 (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. CTNNB1.sup.134-665 was immobilized using the Biotin CAPture Kit, Series S (Cytiva) to 600-1000 RU. Tag-free MDM2.sup.17-111 was diluted to 625 nM then serially diluted 2-fold for a total of seven concentrations with one blank (7-point two-fold Helicon dilution series with top concentration=625 nM and bottom concentration=9.8 nM), in running buffer in the absence or presence of 1 M trimerizer Helicon (n=3). MDM2-binding Helicons were injected over the immobilized and reference surfaces at 30 L/min for 90 sec and then allowed to dissociate for 270 sec. Surface was regenerated with a 120-second injection of CAP regeneration solution each cycle. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1613] SPR analysis of trimerizer Helicons against -catenin: To understand how the trimerizer Helicons interact with CTNNB1 by themselves, SPR experiments were performed on a Biacore S200 (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. CTNNB1.sup.34-665 was immobilized using the Biotin CAPture Kit, Series S (Cytiva) to -600-1000 RU, while Helicons were diluted to 10 M in running buffer then serially diluted 2-fold for a total of four concentrations with one blank (4-point two-fold Helicon dilution series with top concentration=10 M and bottom concentration=1.25 M). Data was analyzed using Biacore Insight Evaluation software (Cytiva).
[1614] SPR competition, ABA mode: CUL5: SOCS2-ELOBC. To probe the H314-binding site on CUL5, SPR ABA experiments were performed on a Biacore S200 (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. Biotinylated CUL5 (residues 1-186) was immobilized using the Biotin CAPture Kit, Series S (Cytiva) to -150 RU. For each injection, H314 in 10 mM DMSO stock was diluted to 10 M in SPR running buffer and was injected over the surface for 120 sec at 30 L/min to achieve equilibrium binding. 100 nM tag-free SOCS2-ELOBC was then injected for 60 sec at 30 L/min in the absence or presence of H314 over the surface. Surface was regenerated with a 120-second injection of CAP regeneration solution each cycle. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1615] SPR, ABA mode: TEAD4: CHIP. To confirm the ternary complex formation of TEAD4: CHIP, SPR ABA experiments were performed on a Biacore S200 (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. Biotinylated CHIP.sup.23-3.sup.03 was immobilized using the Biotin CAPture Kit, Series S (Cytiva) to -200 RU. For each injection, Helicons in 10 mM DMSO stock were diluted to 10 M in SPR running buffer and were injected over the surface for 120 sec at 30 L/min to achieve equilibrium binding (A). 300 nM tag-free TEAD4.sup.217434 protein was then injected for 60 sec at 30 L/min (B) in the absence or presence of Helicons over the surface. The surface was regenerated with a 120-second injection of CAP regeneration solution each cycle. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1616] SPR, ABA mode: MDM2:0-catenin. To confirm the ternary complex formation of MDM2:-catenin, SPR ABA experiments were performed on a Biacore S200 (Cytiva) instrument at 25 C. in 1HBS-P+ buffer (Cytiva) with 1% DMSO. Biotinylated MDM2.sup.25-109 was immobilized using the Biotin CAPture Kit, Series S (Cytiva) to 200 RU. For each injection, Helicons in 10 mM DMSO stock were diluted to 10 M in SPR running buffer and were injected over the surface for 120 sec at 30 L/min to achieve equilibrium binding. 150 nM tag-free CTNNB1.sup.34-665 protein was then injected for 60 sec at 30 L/min in the absence or presence of Helicons over the surface. Surface was regenerated with a 120-second injection of CAP regeneration solution each cycle. Data was analyzed using Biacore Insight Evaluation software (Cytiva). Sensorgrams were double-referenced and evaluated for competition.
[1617] Auto-ubiquitylation of WWP2 (ELISA). E3LITE Customizable Ubiquitin Ligase Kit (LifeSensors, UC101) was used to assess the autoubiquitination activity of HECT domain of WWP2 (WWP2.sup.HECT). The ELISAs were performed with steps following the manufacturer protocol, with all solutions freshly made before the start of the experiment and protein components carefully stored on ice before addition (n=3). The concentration of the catalytic HECT domain was 50 nM. The Helicon inhibitors were used at a concentration of 10 M, with DMSO as the negative control. Relative Luminescence Units (RLUs) were recorded with a GloMax Discover luminometer.
[1618] Assessment of ternary complex formation with Time-Resolved Fluorescence Energy Transfer (TR-FRET) and Fluorescence Polarization (FP).
[1619] TR-FRET analysis of the TEAD4: CHIP complex. For the TR-FRET ternary complex formation of the TEAD4: CHIP pair, biotinylated recombinant TEAD4.sup.217-434 was diluted to 100 nM, Alexa Fluor 488 labeled CHIP.sup.23-303 was diluted to 150 nM and Terbium-labeled streptavidin (Cis-Bio) was diluted to 2 nM in assay buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween-20) in a final volume of 20 L per well of a black 384-well plate (Costar). Compounds were serially diluted in 90% DMSO and 40 nL of compound (11-point three-fold Helicon dilution series with top concentration=20 M) was added to the plate and the samples were incubated for 60 mins at room temperature (n=2). FRET signal was determined using a LanthaScreen filter on a PheraStar (BMG Biotech) plate reader (Ex: 337 nm; Emi: 490 nM; Em.sub.2: 520 nM). The ratio of Em.sub.520 to Em.sub.490 was calculated and plotted against compound concentration. Resulting data was fit to a four-parameter dose-response curve with variable slope. The positive control chimeric compound, P325, Ac-LWWPDGSGSGGSPGOVPMRKROLPASFWEEPR-NH.sub.2 (SEQ ID NO: 42), is a designed bi-functional molecule with its N-terminus adapted from the CHIPopt peptide that interacts with CHIP (Ravalin, M. et al. Specificity for latent C termini links the E3 ubiquitin ligase CHIP to caspases. Nat Chem Biol 15, 786-794 (2019)), and the C-terminus derived from the FAM181A fragment that interacts with TEAD4 (Bokhovchuk, F. et al. Identification of FAM181A and FAM181B as new interactors with the TEAD transcription factors. Protein Sci 29, 509-520 (2020)). The curve for the positive control was fitted with a Biphasic Curve function in Prism 9 (GraphPad).
[1620] TR-FRET analysis of the PPIA: CHIP complex. For the TR-FRET ternary complex formation of the PPIA: CHIP pair, biotinylated recombinant full length PPIA was diluted to 100 nM, Alexa Fluor 488 labeled CHIP.sup.23-303 was diluted to 150 nM and Terbium-labeled streptavidin (Cis-Bio) was diluted to 2.3 nM in assay buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween-20) in a final volume of 20 L per well of a black 384-well plate (Costar). Compounds were serially diluted in 90% DMSO, and 40 nL of compound (11-point three-fold Helicon dilution series with top concentration=20 M) was added to the plate. To probe the trimerizer interface of PPIA, CHIP, 2 M CHIP-binding Helicon H318 or 2 M cyclosporine A (CsA) was added to the assay buffer. Assay plates (n=2) were incubated for 60 mins at room temperature. FRET signal was determined using a LanthaScreen filter on a PheraStar (BMG Biotech) plate reader (Ex: 337 nm; Em.sub.1: 490 nM; Em.sub.2: 520 nM). The ratio of Em.sub.520 to Em.sub.490 was calculated and plotted against compound concentration. The resulting data was fitted to a four-parameter dose-response curve with variable slope.
[1621] FP analysis of the -catenin: MDM2 and -catenin:MDM4 complexes: For the FP analysis of -catenin ternary complex formation, compounds at 10 mM in DMSO were serially diluted 1:3 in DMSO for a total of 11 concentrations using a Mosquito LV (SPT Labtech), then diluted 1000-fold in buffer (50 mM HEPES, pH 7.5, 125 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.05% v/v pluronic acid) in duplicate by the Mosquito LV (SPT Labtech) into a black polystyrene 384-well plate (Corning) (11-point three-fold Helicon dilution series with top concentration=10 M). Protein-probe solution includes 80 nM full-length -catenin or 130 nM -catenin residues 134-665, mixed with 25 nM MDM2.sup.17-111 or MDM4.sup.14-111 labeled with Alexa Fluor 488. Protein-probe solution was plated into the Helicon plate using the MultiDrop Combi (Thermo Fisher) for a total reaction pool of 40 L. The plate was incubated and protected from light for 1 hr at room temperature prior to reading. (n=2) reads were performed on a CLARIOstar plate reader (BMG Labtech) with excitation at 485 nm, emission at 525 nm, and cutoff at 504 nm, and the resulting data was fit to a four-parameter dose-response curve with variable slope. To probe the trimerizer interface of MDM2:0-catenin, 2 M ALRN-6924 peptide.sup.11 or 100-400 nM ICAT (residues 1-81) recombinant proteins were added to the assay buffer, and the plates were prepared similarly. Positive control Helicon, P335, Ac-PD-cyclopentylalanine-CDDAAFNC-3Thi-benzothienylalanine-QGSGS-bAla-LTFEHYWAQLTS-NH.sub.2 (SEQ ID NO: 43) (Cys-stapled), is a designed bi-functional molecule comprising at its N-terminus a -catenin-interacting Helicon.sup.19 and at its C-terminus, a p53-derived peptide that interacts with MDM2 (Czarna, A. et al. High affinity interaction of the p53 peptide-analogue with human Mdm2 and Mdmx. Cell Cycle 8, 1176-1184 (2009)). The curve for the positive control was fitted with the Biphasic Curve function in Prism 9.
Competition Fluorescence Polarization.
[1622] Competition FP of TEAD4: For the competition FP of TEAD4, Helicons at 10 mM in DMSO were serially diluted 1:3 in DMSO for a total of 11 concentrations using a Mosquito LV (SPT Labtech), then diluted 1000-fold in buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 0.05% v/v pluronic acid) in duplicate by the Mosquito LV (SPT Labtech) into a black polystyrene 384-well plate (Corning) (11-point three-fold Helicon dilution series with top concentration=10 M). Probe solution (40 nM TEAD4, mixed with 10 nM 5FAM-labeled YAP1 residues 10-53, Uniprot ID: Q9NQB0, in buffer) with or without 10 M CHIP.sup.23-303 recombinant protein as the presenter was prepared and plated using the MultiDrop Combi (Thermo Fisher) for a total reaction pool of 40 L. The plate was incubated and protected from light for 1 hr at room temperature prior to reading. Reads were performed on a CLARIOstar plate reader (BMG Labtech) with excitation at 485 nm, emission at 525 nm, and cutoff at 504 nm. Data were fitted to a 1:1 binding model with Hill slope using an in-house script.
[1623] Competition FP of CHIP: For the competition FP of CHIP, the assay was performed similarly to the competition FP of TEAD4. The assay was performed in buffer: 1HBS-P+(Cytiva), with 400 nM CHIP.sup.23-3.sup.03 recombinant protein as target, and 20 nM CHIP-binding peptide as probe (5FAM-bAla-SSGPTIEEVD (SEQ ID NO: 44), derived from HSP70 (Smith, M. C. et al. The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex. Biochemistry-us 52, 5354-5364 (2013)). The plates were incubated and protected from light for 1 hr at room temperature, and resulting data were fitted to a 1:1 binding model with Hill slope using an in-house script.
[1624] Competition FP of MDM2: For the competition FP of MDM2, the assay was performed similarly to the competition FP of TEAD4. The assay was performed in buffer: lx PBS with 0.01% Tween, with 30 nM MDM2.sup.17-1 recombinant protein as target, and 3 nM MDM2-binding peptide as probe (5FAM-bAla-LTFEHYWAQLTS-NH.sub.2 (SEQ ID NO: 45), derived from p53). The plates were incubated and protected from light for 1 hr at room temperature, and resulting data were fitted to a 1:1 binding model with Hill slope using an in-house script.
[1625] Competition FP of VHL: For the competition FP of VHL, the assay was performed similarly to the competition FP of TEAD4. The assay was performed in the buffer: 10 mM HEPES, pH 7.5, 50 mM NaCl, 0.05% v/v pluronic acid, 15 nM VHL-ELOBC recombinant protein, and 5 nM VHL tracer, HXC78 (Han, X. et al. Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor (AR) for the Treatment of Prostate Cancer. J Med Chem 62, 941-964 (2019)). A structurally similar small molecule VHL binder, VH298 (Sigma SML1896) (Frost, J. et al. Potent and selective chemical probe of hypoxic signalling downstream of HIF- hydroxylation via VHL inhibition. Nat Commun 7, 13312 (2016)) was included in the competition FP assay as a positive control. The plates were incubated and protected from light for 1 hr at room temperature, and resulting data were fitted to a 1:1 binding model with Hill slope using an in-house script. HXC78 systematic name: (2S,4R)-N-((S)-1-(3,6-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9-xanthen]-5-yl)-17-(4-(4-methylthiazol-5-yl)phenyl)-1,15-dioxo-5,8,11-trioxa-2,14-diazaheptadecan-17-yl)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide; VHL298 systematic name: (2S,4R)-1-((S)-2-(1-Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.
Crystal Structure Data Collection.
[1626] To obtain the structures of the protein-Helicon complexes, briefly, 10 mM of Helicon stock in 90% DMSO were added to the protein stocks to a final 1:1.25 protein: Helicon molar ratio or 1:1:1.25 protein-A: protein-B: Helicon and screened against commercially available crystallization screens. Crystals were obtained by hanging or sitting hanging drop vapor diffusion methods at room temperature, with their crystallization conditions detailed in, e.g., Table E11-6. Crystals were cryo-protected with glycerol or ethylene glycol, followed by flash-freezing in liquid nitrogen. Diffraction datasets were collected at 100K. Data were processed in XDS & XSCALE, AIMLESS, and/or STARANISO, all parts of the autoPROC suite. Molecular replacement solutions were obtained using PHASER with previously deposited high-resolution PDB structures as search models. Complete models were built through iterative cycles of manual model building in COOT and structure refinement was carried out using either REFMAC or PHENIX. All the structure model figures in the paper were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 2.4, Schrdinger, LLC.). The atomic coordinates and structure factors have been deposited in the Protein Data Bank. Certain technologies are presented below as examples.
[1627] Certain observed interactions are described below (Residues involves in Interaction (with 4.5A)).
[1628] -catenin_MDM2_Helicon332 (PDB code: 8EI9). beta-Catenin by H332: Glu620, Thr653, Tyr654, Ala656, Ala657 and Phe660. MDM2 by H332: Met50, Lys51, Leu54, Phe55, Leu57, Gly58, Ile61, Met62, Tyr67, Gln72, Val75, Val93, Lys94, His96, Ile99, Tyr100 and Tyr104. beta-Catenin by MDM2: Arg582, Val584, Arg587, Cys619, Gln623, Ala656, Ala657, Phe660, Glu664 and Asp665. MDM2 by beta-catenin: Gln71, His73, Val93, Lys94, Glu95, His96 and Arg97.
[1629] -catenin_MDM2_Helicon333 (PDB code: 8EIA). beta-Catenin by H333: Arg582, Glu620, Thr653, Tyr654, Ala656, Ala657 and Phe660. MDM2 by H333: Met50, Lys51, Leu54, Leu57, Gly58, Ile61, Gln72, Val75, Val93, Lys94, His96 and Tyr100. beta-Catenin by MDM2: Arg582, Val584, Arg587, Cys619, Gln623, Ala656, Ala657, Val658, Phe660 and Arg661. MDM2 by beta-catenin: His73, Val93, Lys94, Glu95, His96 and Arg97.
[1630] -catenin_MDM2_Helicon329 (PDB code: 8EIB). beta-Catenin by H329: Arg474, His475, Arg515, Leu519, His578, Arg582, Arg612, Glu620, Gln623, Gly650, Thr653, Tyr654, Ala656, Ala657 and Phe660. MDM2 by H329: Thr26, Met50, Leu54, Leu57, Gly58, Ile61, Met62, Tyr67, Gln72, His73, Val75, Val93, Lys94, His96, Ile99, Tyr100 and Tyr104. beta-Catenin by MDM2: Tyr432, Arg474, His475, Gln476, Ala478, Glu479, Arg582 and Glu649. MDM2 by beta-catenin: Glu25, Lys51, Phe55, His96, Arg97, Tyr104, Arg105 and Val109.
[1631] -catenin_MDM2_Helicon330 (PDB code: 8EIC). beta-Catenin by H330: Tyr432, Arg474, His475, Arg515, Leu519, His578, Arg582, Arg612, Cys619, Glu620, Gln623, Gly650, Thr653, Tyr654, Ala656, Ala657, Phe660 and Arg661. MDM2 by H330: Thr26, Met50, Leu54, Leu57, Gly58, Ile61, Met62, Tyr67, Gln72, His73, Val75, Val93, Lys94, His96, Ile99, Tyr100 and Tyr104. beta-Catenin by MDM2: Asn430, Tyr432, Lys433, Arg474, His475, Gln476, Glu479 and Arg582. MDM2 by beta-catenin: Glu25, Thr26, His96, Arg97, Tyr104, Val109 and Val110.
[1632] CHIP_Helicon317 (PDB code: 8EHZ). CHIP by Helicon317: LYS30, ASN34, PHE37, VAL38, TYR49, VAL61, ASN65, LEU68, LEU71, LYS72, LYS95, PHE98, PHE99, GLN102, GLN127, LEU129, ASN130, PHE131, ASP134 and ILE135.
[1633] CHIP_Helicon318 (PDB code: 8EI0). CHIP by Helicon318: Asn34, Phe37, Val38, Tyr49, Asn65, Leu68, Leu71, Lys72, Ser93, Val94, Lys95, Phe98, Phe99, Gln102, Glu106, Leu129, Asn130, Phel31, Gly132, Asp134 and Ile135.
[1634] CUL5_Helicon314 (PDB code: 8EI2). CUL5 by Helicon314: Val35, Thr36, Lys37, Trp40, Phe41, Phe44, His48, Ile106, Lys109, Cys112 and Gln113.
[1635] VHL_Helicon313 (PDB code: 8EI3). VHL by Helicon313: Pro59, Arg60, Val62, Leu63, Arg64, Val66, Gly114, His115, Leu116, Thr133, Glu134, Leu135, Val137, Leu201, Thr202, Arg205 and Ile206.
[1636] WWP1_Helicon302 (PDB code: 8EI4). WWP1 by Helicon302: Glu702, Phe703, Ser706, Leu707, Trp709, Ile710, Glu717, Cys718, Gly719, Leu720, Glu721, Met722, Val726 and Met760.
[1637] WWP2_Helicon301 (PDB code: 8EI5). WWP2 by Helicon301: Glu650, Phe651, Asn653, Ser654, Ile655, Trp657, Ile658, Asn661, Asn662, Glu665, Cys666, Gly667, Leu668, Glu669, Leu670, Gln674, Tyr704 and Leu708.
[1638] WWP2_Helicon305 (PDB code: 8EI6). WWP2 by Helicon305: Tyr499, Phe495, Arg496, His500, Arg503, Phe504, His507, Ser508, Gly619, Lys620, Phe621, Leu747, Met748, Met752 and Glu789.
[1639] WWP2_Helicon304 (PDB code: 8EI7). WWP2 by Helicon304: Phe495, Arg496, Tyr499, His500, Gly557, Arg561, Phe565, His569, Gly619, Lys620, Phe621, Ile622, Asp623, Leu747, Met748, Cys750, Gly751, Met752, Gln753, Glu754, Glu789, Arg803, Leu804, Pro805 and Val806. WWP2_Helicon308 (PDB code: 8EI8). WWP2 by Helicon308: Phe495, Arg496, Tyr499, His500, Arg561, Glu562, Phe565, Leu566, Gly619, Lys620, Phe621, Ile622, Asp623, Leu747, Cys750, Gly751, Met752, Gln753, Glu754, Asp787, Glu789 and Arg803.
TABLE-US-00033 TABLE E11-6 Certain structural data collection and refinement information PDB code 8EI9 8EIA 8EIB Structure Name -catenin_MDM2_Helicon332 -catenin_MDM2_Helicon333 -catenin_MDM2_Helicon329 Full Name Crystal structure of beta-catenin and Crystal structure of beta-catenin and Crystal structure of beta-catenin and the MDM2 p53-binding domain in the MDM2 p53-binding domain in the MDM2 p53-binding domain in complex with H332, a Helicon complex with H333, a Helicon complex with H329, a Helicon Polypeptide Polypeptide Polypeptide Wavelength () 0.920 0.920 0.920 Resolution range () 48.01-3.90 (4.36-3.90) 48.03-3.60 (3.94-3.60) 20.17-3.76 (4.20-3.76) Space group P 21 21 21 P 1 21 1 P 21 21 21 Unit cell a, b, c () 87.04 95.32 166.72 49.81 96.07 76.79 55.62 69.83 165.47 , , () 90 90 90 90 106.62 90 90 90 90 Total reflections 76659 (19433) 41220 (9723) 84610 (22310) Unique reflections 13180 (3642) 7517 (1803) 6955 (1935) Multiplicity 5.8 (5.3) 5.5 (5.4) 12.2 (11.5) Completeness (%) 99.8 (99.4) 92.8 (94.2) 99.3 (99.9) Mean I/sigma (I) 3.2 (1.1) 5.3 (1.7) 5.1 (2.7) Wilson B-factor (.sup.2) 63.57 56.79 56.12 R-merge 0.545 (1.54) 0.241 (0.912) 0.698 (1.27) R-meas 0.661 (1.89) 0.295 (1.11) 0.758 (1.39) R-pim 0.369 (1.09) 0.167 (0.625) 0.294 (0.560) CC1/2 0.923 (0.428) 0.978 (0.491) 0.972 (0.794) Reflections used in refinement 13130 (1261) 7500 (759) 6915 (666) Reflections used for R-free 582 (70) 750 (75) 691 (67) R-work 0.266 (0.324) 0.319 (0.349) 0.245 (0.262) R-free 0.313 (0.404) 0.351 (0.329) 0.260 (0.258) Number of non-hydrogen atoms 4817 4553 4757 macromolecules 4803 4539 4743 ligands 14 14 14 solvent Protein residues 621 586 614 RMS (bonds) () 0.005 0.006 0.009 RMS (angles) () 0.72 1.04 1.37 Ramachandran favored (%) 89.27 85.81 82.31 Ramachandran allowed (%) 9.27 10.73 12.07 Ramachandran outliers (%) 1.46 3.46 5.62 Rotamer outliers (%) 0.95 2.03 2.51 Clashscore 7.36 22.81 19.55 Average B-factor (.sup.2) 84.49 84.34 80.46 macromolecules 84.43 84.24 80.37 ligands 105.47 114.94 111.61 solvent Crystallization conditions 0.1M MES Sodium Salt pH 6.5, 0.1M Potassium chloride, 0.1M 0.2M Calcium acetate pH 7.5, 20% 10% v/v 2-Propanol, 25% v/v HEPES pH 7, 15% w/v PEG 5000 w/v PEG 3350 PEG 400 MME PDB code 8EIC 8EHZ 8EI0 Structure Name -catenin_MDM2_Helicon330 CHIP_Helicon317 CHIP_Helicon318 Full Name Crystal structure of beta-catenin and Crystal structure of the STUB1 TPR Crystal structure of the STUB1 TPR the MDM2 p53-binding domain in domain in complex with H317, a domain in complex with H318, a complex with H330, a Helicon Helicon Polypeptide Helicon Polypeptide Polypeptide Wavelength () 0.979 1.000 1.033 Resolution range () 46.36-2.62 (2.74-2.62) 43.08-2.06 (2.12-2.06) 43.11-1.47 (1.50-1.47) Space group P 21 21 21 P 32 C 2 Unit cell a, b, c () 56.11 71.54 164.56 63.21 63.21 69.82 82.56 46.96 53.62 , , () 90 90 90 90 90 120 90 126.48 90 Total reflections 267056 (31979) 202213 (15637) 139635 (6682) Unique reflections 20670 (2482) 19293 (1471) 27179 (1284) Multiplicity 12.9 (12.9) 10.5 (10.6) 5.1 (5.2) Completeness (%) 99.9 (99.8) 100.0 (100.0) 96.7 (94.5) Mean I/sigma (I) 14.4 (2.4) 15.2 (2.6) 18.3 (2.2) Wilson B-factor (.sup.2) 47.13 41.14 19.02 R-merge 0.139 (1.30) 0.078 (0.796) 0.036 (0.650) R-meas 0.150 (1.41) 0.087 (0.884) 0.045 (0.815) R-pim 0.057 (0.538) 0.038 (0.382) 0.027 (0.487) CC1/2 0.999 (0.685) 0.998 (0.938) 0.999 (0.758) Reflections used in refinement 20615 (2023) 19236 (1897) 26777 (2611) Reflections used for R-free 992 (107) 1935 (185) 1362 (121) R-work 0.224 (0.303) 0.241 (0.344) 0.194 (0.322) R-free 0.262 (0.346) 0.267 (0.342) 0.210 (0.338) Number of non-hydrogen atoms 4800 2279 1350 macromolecules 4775 2227 1189 ligands 14 34 31 solvent 11 18 130 Protein residues 619 278 144 RMS (bonds) () 0.01 0.006 0.008 RMS (angles) () 1.05 0.79 1.07 Ramachandran favored (%) 95.59 94.03 97.84 Ramachandran allowed (%) 3.92 5.97 2.16 Ramachandran outliers (%) 0.49 0 0 Rotamer outliers (%) 1.73 0 1.64 Clashscore 10.7 3.81 5.02 Average B-factor (.sup.2) 71.4 60.79 26.37 macromolecules 71.4 60.71 25.27 ligands 86.83 71.8 37.04 solvent 52.39 49.92 33.9 Crystallization conditions 4M Na Form 0.1M Sodium acetate, 0.1M MES 0.2M Ammonium sulfate, pH 6.0, 15% w/v PEG 4000 30% w/v PEG 8000. PDB code 8EI1 8EI2 8EI3 Structure Name CUL4B_Helicon316 CUL5_Helicon314 VHL_Helicon313 Full Name Crystal structure of the N-terminal Crystal structure of the N-terminal Crystal structure of VHL in domain of CUL4B in complex with domain of CUL5 in complex with complex with H313, a Helicon H316, a Helicon Polypeptide H314, a Helicon Polypeptide Polypeptide Wavelength () 0.976 1.000 1.000 Resolution range () 52.36-2.89 (3.00-2.89) 47.88-2.80 (2.95-2.80) 47.08-3.49 (3.82-3.49) Space group P 43 21 2 P 6 P 41 Unit cell a, b, c () 99.65 99.65 366.49 173.03 173.03 57.48 47.08 47.08 359.58 , , () 90 90 90 90 90 120 90 90 90 Total reflections 1134593 (121904) 318680 (48322) 89660 (21010) Unique reflections 42685 (4410) 24154 (3518) 9977 (2380) Multiplicity 26.6 (27.6) 13.2 (13.7) 9.0 (8.8) Completeness (%) 100.0 (100.0) 98.2 (99.3) 100.0 (100.0) Mean I/sigma (I) 23.9 (2.4) 5.0 (1.8) 6.5 (2.6) Wilson B-factor (.sup.2) 72.97 47.98 53.11 R-merge 0.110 (1.84) 0.322 (1.12) 0.287 (0.726) R-meas 0.114 (1.90) 0.346 (1.20) 0.326 (0.829) R-pim 0.030 (0.498) 0.125 (0.430) 0.152 (0.389) CC1/2 1.000 (0.882) 0.988 (0.888) 0.987 (0.897) Reflections used in refinement 42538 (4147) 16542 (442) 9896 (1033) Reflections used for R-free 2101 (225) 845 (31) 476 (53) R-work 0.212 (0.360) 0.339 (0.407) 0.216 (0.305) R-free 0.255 (0.419) 0.358 (0.574) 0.282 (0.405) Number of non-hydrogen atoms 11809 3067 5748 macromolecules 11745 3053 5734 ligands 64 14 14 solvent Protein residues 1427 373 709 RMS (bonds) () 0.005 0.009 0.003 RMS (angles) () 0.85 1 0.58 Ramachandran favored (%) 96.24 90.19 91.9 Ramachandran allowed (%) 2.91 8.45 6.22 Ramachandran outliers (%) 0.85 1.36 1.88 Rotamer outliers (%) 2.8 0.6 0.62 Clashscore 7.41 13.14 7.15 Average B-factor (.sup.2) 90.35 20.32 68.24 macromolecules 90.15 20.22 68.19 ligands 126.15 41.2 87.57 solvent Crystallization conditions 0.2M Lithium sulfate, 0.1M Tris 30% w/v PEG 8000, 0.1M MES 0.1M Tris pH 7.0, 20% w/v pH 8.5, 30% w/v PEG 4000 Sodium Salt pH 6.5, 0.2M PEG 1000 Ammonium Sulfate, 4% v/v 1,3-Propanediol PDB code 8EI4 8EI5 8EI6 Structure Name WWP1_Helicon302 WWP2_Helicon301 WWP2_Helicon305 Full Name Crystal structure of the WWP1 Crystal structure of the WWP2 Crystal structure of the WWP2 HECT domain in complex with HECT domain in complex with HECT domain in complex with H302, a Helicon Polypeptide H301, a Helicon Polypeptide H305, a Helicon Polypeptide Wavelength () 1.181 1.033 1.000 Resolution range () 45.18-2.43 (2.52-2.43) 45.40-2.60 (2.68-2.60) 49.07-3.60 (3.94-3.60) Space group P1 P 21 P 21 21 21 Unit cell a, b, c () 45.42 51.21 58.64 120.66 63.10 122.26 67.62 71.32 211.57 , , () 112.09 99.61 102.51 90 96.78 90 90 90 90 Total reflections 43024 (3472) 289797 (23732) 163370 (39244) Unique reflections 16258 (1472) 56697 (4596) 12498 (2921) Multiplicity 2.6 (2.4) 5.1 (5.2) 13.1 (13.4) Completeness (%) 94.0 (80.3) 99.9 (99.9) 99.9 (99.9) Mean I/sigma (I) 11.3 (2.1) 7.6 (2.2) 10.2 (2.7) Wilson B-factor (.sup.2) 35.41 29.51 99.13 R-merge 0.054 (0.310) 0.139 (0.616) 0.190 (1.056) R-meas 0.077 (0.438) 0.175 (0.773) 0.205 (1.139) R-pim 0.054 (0.310) 0.103 (0.460) 0.077 (0.425) CC1/2 0.997 (0.872) 0.995 (0.827) 0.998 (0.979) Reflections used in refinement 15431 (1326) 56014 (5551) 9414 (101) Reflections used for R-free 785 (64) 2658 (246) 957 (12) R-work 0.220 (0.328) 0.214 (0.270) 0.259 (0.328) R-free 0.280 (0.477) 0.261 (0.306) 0.279 (0.275) Number of non-hydrogen atoms 3210 13513 6492 macromolecules 3145 13036 6464 ligands 30 158 28 solvent 35 319 Protein residues 381 1542 766 RMS (bonds) () 0.009 0.006 0.004 RMS (angles) () 1.06 0.97 0.85 Ramachandran favored (%) 95.44 97.38 91.78 Ramachandran allowed (%) 2.95 2.36 7.96 Ramachandran outliers (%) 1.61 0.26 0.27 Rotamer outliers (%) 0.59 4.45 0.43 Clashscore 8.29 5.93 9.37 Average B-factor (.sup.2) 45.6 39.63 78.69 macromolecules 45.51 39.61 78.63 ligands 56.1 51.4 92.73 solvent 44.83 34.75 Crystallization conditions 0.2M Sodium acetate, 0.1M 0.1M Sodium acetate, 0.1M sodium 18% v/v 2-Propanol, 0.1M Sodium Sodium citrate pH 5.5, 5% w/v citrate pH 6.0, 10% PEG 4000 citrate pH 5.5, 20% w/v PEG 4,000 PEG 4000 PDB code 8EI7 8EI8 Structure Name WWP2_Helicon304 WWP2_Helicon308 Full Name Crystal structure of the WWP2 Crystal structure of the WWP2 HECT domain in complex with HECT domain in complex with H304, a Helicon Polypeptide H308, a Helicon Polypeptide Wavelength () 1.033 1.000 Resolution range () 44.69-2.22 (2.29-2.22) 43.77-2.90 (3.08-2.90) Space group P 21 C 2 2 21 Unit cell a, b, c () 62.16 64.30 103.69 111.38 117.81 65.39 , , () 90 90 90 90 90 90 Total reflections 206480 (18955) 129994 (21614) Unique reflections 40576 (3697) 9862 (1564) Multiplicity 5.1 (5.1) 13.2 (13.8) Completeness (%) 99.8 (99.8) 100.0 (100.0) Mean I/sigma (I) 13.1 (2.3) 8.0 (2.4) Wilson B-factor (.sup.2) 35.97 46.92 R-merge 0.075 (0.709) 0.248 (1.08) R-meas 0.093 (0.882) 0.268 (1.16) R-pim 0.054 (0.517) 0.101 (0.430) CC1/2 0.998 (0.720) 0.992 (0.814) Reflections used in refinement 40032 (3950) 9858 (968) Reflections used for R-free 2013 (180) 486 (63) R-work 0.200 (0.271) 0.236 (0.312) R-free 0.263 (0.352) 0.303 (0.457) Number of non-hydrogen atoms 6764 3355 macromolecules 6481 3306 ligands 89 49 solvent 194 Protein residues 763 391 RMS (bonds) () 0.011 0.006 RMS (angles) () 1.27 0.9 Ramachandran favored (%) 97.35 92.76 Ramachandran allowed (%) 2.12 6.46 Ramachandran outliers (%) 0.53 0.78 Rotamer outliers (%) 2.84 1.12 Clashscore 8.46 10.57 Average B-factor (.sup.2) 47.92 61.15 macromolecules 48.04 61.05 ligands 49.64 68.02 solvent 43.11 Crystallization conditions 0.2M magnesium chloride, 0.1M 0.2M Magnesium Chloride, 0.1M Bis-tris pH 7.5, 30% PEG 3350 Bis-Tris pH 6.5, 20% PEG 3350
[1640] While various embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described in the present disclosure, and each of such variations and/or modifications is deemed to be included. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be example and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described in the present disclosure. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, provided technologies, including those to be claimed, may be practiced otherwise than as specifically described and claimed. In addition, any combination of two or more features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.