Engineering Peptides Using Peptide Epitope Linker Evolution
20250346630 ยท 2025-11-13
Inventors
- Christopher John Brown (Singapore, SG)
- Yuri Frosi (Singapore, SG)
- Simon Ng (Singapore, SG)
- Yen-Chu Lin (Singapore, SG)
Cpc classification
C40B40/10
CHEMISTRY; METALLURGY
C07K2318/10
CHEMISTRY; METALLURGY
G16B15/30
PHYSICS
C12N15/1037
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
G16B20/20
PHYSICS
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to methods of engineering and identifying a peptide aptamer that binds to a target protein of interest, and peptide aptamers engineered and identified using these methods and methods to identify a candidate peptide or nucleic acid that binds to a target protein in a live cell. The peptide aptamers defined herein may be useful for treating a condition associated with dysregulated cap-dependent translation, dysregulated DNA replication, dysregulated DNA repair and/or dysregulated mRNA translation such as cancer, diseases associated with a viral infection and obesity.
Claims
1.-23. (canceled)
24. A peptide aptamer comprising an amino acid sequence selected from the group consisting of TABLE-US-00010 i) (SEQIDNO:24) X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5WX.sub.6X.sub.7SRTPWX.sub.8X.sub.9X.sub.10X.sub.11X.sub.12, ii) (SEQIDNO:25) X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19WX.sub.20X.sub.21SRTPWX.sub.22X.sub.23X.sub.24, and iii) (SEQIDNO:26) X.sub.25X.sub.26X.sub.27WX.sub.28X.sub.29SRTPWX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36; wherein X.sub.1-X.sub.36 is any amino acid; wherein X.sub.1-5, X.sub.13-19 and X.sub.25-29 comprise amino acid sequences for a first linker; wherein X.sub.8-12, X.sub.22-24 and X.sub.30-36 comprise amino acid sequences for a second linker; optionally wherein X.sub.1 and X.sub.15 is an amino acid selected from the group consisting of leucine (L), glutamine (Q), arginine (R), valine (V), tyrosine (Y), glycine (G), alanine (A) and threonine (T); optionally wherein X.sub.2 and X.sub.16 is an amino acid selected from the group consisting of threonine (T), arginine (R), serine (S), proline (P), alanine (A) and isoleucine (I); optionally wherein X.sub.3, X.sub.17 and X.sub.25 is an amino acid selected from the group consisting of tryptophan (W), leucine (L), alanine (A), isoleucine (I), serine (S), threonine (T), glutamine (Q), asparagine (N), glutamate (E), cysteine (C), proline (P), glycine (G), valine (V), arginine (K), arginine (R), phenylalanine (F), and methionine (M); optionally wherein X.sub.4, X.sub.18 and X.sub.26 is an amino acid selected from the group consisting of threonine (T), histidine (H), glutamine (Q), leucine (L), glutamate (E), valine (V), glycine (G), arginine (R), phenylalanine (F), tyrosine (Y), serine (S), isoleucine (I), alanine (A), tryptophan (W) and cysteine (C); optionally wherein X.sub.5, X.sub.19 and X.sub.27 is an amino acid selected from the group consisting of arginine (R), tyrosine (Y), lysine (K), leucine (L), histidine (H), proline (P), serine (S), tryptophan (W) and valine (V); optionally wherein X.sub.6, X.sub.20 and X.sub.28 is an amino acid selected from the group consisting of valine (V), arginine (R), isoleucine (I), histidine (H), leucine (L), serine (S), phenylalanine (F), alanine (A), glycine (G), threonine (T) and lysine (K); optionally wherein X.sub.7, X.sub.21 and X.sub.29 is an amino acid selected from the group consisting of asparagine (N), phenylalanine (F), tryptophan (W), leucine (L), alanine (A), glycine (G), lysine (K), glutamate (E), serine (S), arginine (R), threonine (T), tyrosine (Y) and aspartate (D); optionally wherein X.sub.8, X.sub.22 and X.sub.30 is an amino acid selected from the group consisting of asparagine (N), histidine (H), phenylalanine (F), serine (S), tryptophan (W), alanine (A), threonine (T), tyrosine (Y), lysine (K), arginine (R), valine (V) and cysteine (C); optionally wherein X.sub.9, X.sub.23 and X.sub.31 is an amino acid selected from the group consisting of valine (V), phenylalanine (F), arginine (R), isoleucine (I), asparagine (N), leucine (L), lysine (K), methionine (M), tryptophan (W), threonine (T), glycine (G) and alanine (A); optionally wherein X.sub.10, X.sub.24 and X.sub.32 is an amino acid selected from the group consisting of isoleucine (I), arginine (R), tyrosine (Y), leucine (L), valine (V), lysine (K), histidine (H), alanine (A), methionine (M), tryptophan (W), serine (S), threonine (T) and proline (P); optionally wherein X.sub.11 and X.sub.33 is an amino acid selected from the group consisting of glycine (G), leucine (L), phenylalanine (F), arginine (R), tyrosine (Y), methionine (M), alanine (A), histidine (H), tryptophan (W), asparagine (N), lysine (K), serine (S), threonine (T) and glutamine (Q); optionally wherein X.sub.12 and X.sub.34 is an amino acid selected from the group consisting of phenylalanine (F), threonine (T), histidine (H), arginine (R), tyrosine (Y), valine (V), aspartate (D), alanine (A), serine (S), leucine (L), proline (P), lysine (K) and tryptophan (W); optionally wherein X.sub.13 is an amino acid selected from the group consisting of asparagine (N) and leucine (L); optionally wherein X.sub.14 is an amino acid selected from the group consisting of valine (V), and leucine (L); optionally wherein X.sub.35 is an amino acid selected from the group consisting of (H), arginine (R), methionine (M), threonine (T), lysine (K), valine (V), leucine (L), serine (S), asparagine (N), isoleucine (I) aspartate (D) and glycine (G); and optionally wherein X.sub.36 is an amino acid selected from the group consisting of glutamine (Q), isoleucine (I), leucine (L), phenylalanine (F), proline (P), arginine (R), threonine (T), tyrosine (Y), aspartate (D), lysine (K), glycine (G), serine (S) and alanine (A); wherein the peptide aptamer binds to eIF4A.
25. The peptide aptamer according to claim 24, wherein the peptide aptamer comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
26. The peptide aptamer according to claim 24, wherein the peptide aptamer is not constrained by a disulphide bond.
27. The peptide aptamer according to claim 24, wherein the peptide aptamer is constrained by a disulphide bond.
28. The peptide aptamer according to claim 24, wherein the peptide aptamer comprises a peptide motif and/or one or more linker sequences located in a hypervariable region of a scaffold protein.
29. The peptide aptamer according to claim 28, wherein the scaffold protein is a VH domain.
30. (canceled)
31. A method of treating a condition associated with dysregulated cap-dependent translation, dysregulated DNA replication, dysregulated DNA repair and/or dysregulated mRNA translation, comprising administering the peptide aptamer according to claim 24 to a subject in need thereof, optionally comprising administering the peptide aptamer as a combinatorial treatment with immunotherapy.
32. The method according to claim 31, wherein the condition is selected from the group consisting of cancer, a disease associated with a viral infection and obesity.
33. The method according to claim 32, wherein the cancer is melanoma.
34.-40. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
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DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0103] In a first aspect, the present invention refers to a method of identifying and isolating a peptide aptamer (PA) that is capable of binding to a target protein comprising: [0104] a) identifying a peptide motif that interacts with the target protein from a library of peptides; [0105] b) inserting a hypervariable region in a scaffold protein; [0106] c) inserting the peptide motif into a plurality of positions in the hypervariable region of the scaffold protein to generate one or more library of peptide aptamers, wherein the hypervariable region forms one or more linkers between the peptide motif and the scaffold protein; [0107] d) contacting the library from step c with the target protein to identify the peptide aptamer that is capable of binding to the target protein; and [0108] e) isolating the peptide aptamer from step d.
[0109] The identified peptide aptamer may be further engineered via maturation or chemical modifications to increase the binding affinity to the target protein. Maturation of the peptide aptamer may be post-translational modifications of the peptide aptamers. Post-translational modifications may include phosphorylation, acetylation, hydroxylation and methylation. Chemical modifications may be the addition of chemical groups within the peptide aptamer or at each end of the peptide aptamer. The chemical groups may include biotin, thiol, amide, carboxyl, linear or branched alkyl, lipids, fatty acids. For example, an amide group may be added at N-terminal and/or C-terminal of the peptide aptamer or within the peptide aptamer.
[0110] In one example, the library of peptides includes but is not limited to a phage library, a mRNA display library, a bacterial display library, a synthetic peptide library or combinations thereof. In a preferred example, the library of peptides is a phage library. The phage library may be a linear peptide phage library, a constrained peptide phage library or dodecapeptide library. It will generally be understood that a linear peptide phage library comprises a library of linear peptides or peptide motifs and the constrained peptide phage library comprises a library of peptides or peptide motifs that are structurally constrained. The peptide phage library may comprise a library of peptides constrained with a disulphide bond ranging from 4-mer to 12-mer. The peptides may be 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer or 12-mer. In some examples, the linear peptide phage library may comprise linear peptides or linear peptide motifs ranging from 7-mer to 12-mer. The linear peptides or peptide may be 7-mer, 8-mer, 9-mer, 10-mer, 11-mer or 12-mer. The linear peptides or peptide motifs adopt the conformation when binding to the target protein and the constrained peptides of peptide motifs adopt the conformation prior to binding to the target protein. The constrained peptide phage library includes but is not limited to a disulphide constrained peptide phage library, a cysteine constrained peptide phage library and an a-helical constrained peptide phage library. It will also be generally understood that a dodecapeptide phage library comprises a library of dodecapeptides (12-mer) or dodecapeptide motifs. One or more libraries in various combinations may be used in the methods of the invention. In one example, the phage library is a constrained peptide phage library. In a preferred example, the constrained peptide phage library is a disulphide constrained peptide library.
[0111] The method of isolating a peptide aptamer of the invention also comprises the step of inserting a hypervariable region into a scaffold protein. The hypervariable region is inserted into the loop of the scaffold protein. The hypervariable region may be inserted into any loop of any protein scaffold. The hypervariable region may be inserted into the protein scaffold using conventional molecular biology techniques. The conventional molecular biology techniques comprise restriction enzyme digestion, double stranded DNA cassette ligation and overlapping polymerase chain reaction techniques. The insertion of the hypervariable region into the protein scaffold is randomized which results in the isolation of a peptide aptamer and the peptide aptamer may be synthesized.
[0112] The identified peptide motif is inserted in a plurality of positions in the hypervariable region of the scaffold protein to generate one or more libraries of peptide aptamers comprising the peptide motif and one or more linkers derived from the hypervariable region. Each library may comprise peptide aptamers that comprise the peptide motif and linkers with identical number of amino acid residues at each of the C- and N-terminal. Each of the library may also comprises peptide aptamers that comprise peptide motif and linkers with different number of amino acid residues at each of the C- and N-terminal. For example, one library may comprise peptide aptamers comprising the peptide motif, and linkers comprising 5 amino acid residues long at the C-terminal and 5 amino acid residues long at the N-terminal. In another example, one library may comprise peptide aptamers comprising the peptide motif, and linkers comprising 5 amino acid residues at the C-terminal and 5 amino acid residues at the N-terminal, and peptide aptamers comprising peptide motif, and linkers comprising 7 amino acid residues at the C-terminal and 3 amino acid residues at the N-terminal. In another example, one library may comprise peptide aptamers comprising the peptide motif, and linkers comprising 10 amino acid residues at the C-terminal and 0 amino acid residues at the N-terminal and peptide aptamers comprising peptide motif, and linkers comprising 0 amino acid residues at the C-terminal and 10 amino acid residues at the N-terminal.
[0113] The peptide motif may be inserted randomly in one or more positions in the hypervariable region of the scaffold protein to generate one or more library of peptide aptamers. As such, the peptide motif may be inserted in one position, two positions, three positions, four positions, five positions, six positions, seven positions, eight positions in the hypervariable region of the scaffold protein. For example, the peptide motif may be inserted at three different positions to generate three different libraries of peptide aptamers. The peptide motif may be inserted at five different positions to generate five different libraries of peptide aptamers. In another example, the peptide motif may be inserted at three different positions to generate one library of peptide aptamers. The peptide motif may be inserted in six different positions to generate two libraries of peptide aptamers. The peptide motif may be inserted in five different positions to generate one library of peptide aptamers.
[0114] In some examples, the hypervariable region forms the linker sequences at the C-terminal and/or N-terminal of the peptide motif. The linker sequence may be located at the C-terminal, or the N-terminal, or both C- and N-terminals of the peptide motif. In one example, the linker sequence is from 0 to 10 amino acid residues long. The one or more linker sequences may be 1 amino acid residue long, 2 amino acid residues long, 3 amino acid residues long, 4 amino acid residues long, 5 amino acid residues long, 6 amino acid residues long, 7 amino acid residues long, 8 amino acid residues long, 9 amino acid residues long and 10 amino acid residues long. In some examples, the entire sequence of the hypervariable region may be located at the C-terminal of the peptide motif and the linker sequence at the N-terminal of the peptide motif may not be present. The entire sequence of the hypervariable region may be located at the N-terminal of the peptide motif and the linker sequence at the C-terminal of the peptide motif may not be present. The length of the linker sequences may affect the stability of the scaffold protein. The longer the linker sequences, the weaker the stability of the scaffold protein, thus affecting the sampling of the library.
[0115] In some examples, the linker sequence may be 3 amino acid residues long at the C-terminal and 7 amino acid residues long at the N-terminal. The linker sequence may be 5 amino acid residues long at the C-terminal and 5 amino acid residues long at the N-terminal. The linker sequence may be 7 amino acid residues long at the C-terminal and 3 amino acid residues long at the N-terminal. The linker sequence may be 2 amino acid residues long at the C-terminal and 8 amino acid residues long at the N-terminal. The linker sequence may be 6 amino acid residues long at the C-terminal and 4 amino acid residues long at the N-terminal. The linker sequence may be 10 amino acid residues long at the C-terminal with no linker sequence at the N-terminal. The linker sequence may be absent at the C-terminal and the linker sequence may be 10 amino acid residues long at the N-terminal.
[0116] It will generally be understood that the amino acid residues of the linker sequences are randomized based on where the peptide motif is inserted within the hypervariable region.
[0117] In one example, the hypervariable region is linked to the scaffold protein in a stable confirmation. The stable interaction between the hypervariable region and the scaffold protein allows the peptide motif inserted within the hypervariable region linked to be stably presented to a target protein for binding.
[0118] In one example, the scaffold protein includes but is not limited to VH domain, stefin-A and fibronectin. In a preferred example, the scaffold protein is the VH domain.
[0119] In one example, the library of peptide aptamers is a yeast surface display library. The yeast surface display library may be but not limited to the S. cerevisiae yeast surface display strain EBY100.
[0120] In one example, the peptide aptamer binds to the target protein at a binding affinity measured using a competition binding assay that measures a dissociation constant. The measurement of a dissociation constant defines the binding affinity of the peptide aptamer to the target protein. In one example, the peptide aptamer binds to the target protein with a dissociation constant of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM. In a preferred example, the peptide binds to the target protein with a dissociation constant of less than about 100 nM.
[0121] In another aspect, the present invention refers to a peptide aptamer comprising an amino acid sequence selected from the group consisting of:
TABLE-US-00003 i) (SEQIDNO:1) X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5EMGFFX.sub.6X.sub.7X.sub.8X.sub.9X.sub.10, ii) (SEQIDNO:2) X.sub.11X.sub.12X.sub.13EMGFFX.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20, and iii) (SEQIDNO:3) X.sub.21X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27EMGFFX.sub.28X.sub.29X.sub.30; [0122] wherein X.sub.1-30 is any amino acid; [0123] wherein X.sub.1-5, X.sub.11-13 and X.sub.21-27 comprise amino acid sequences for a first linker; [0124] wherein X.sub.6-10, X.sub.14-20 and X.sub.28-30 comprise amino acid sequences for a second linker; [0125] optionally wherein X.sub.5, X.sub.13 and X.sub.27 is an amino acid selected from the group consisting of proline (P), serine (S) and cysteine (C); [0126] optionally wherein X.sub.6, X.sub.14 and X.sub.28 is an amino acid selected from the group consisting of histidine (H), threonine (T), leucine (L), glutamine (Q), serine (S), lysine (K), alanine (A), glutamate (E) and valine (V); and [0127] optionally where in X.sub.7, X.sub.15 and X.sub.29 is an amino acid selected from the group consisting of asparagine (N), valine (V), threonine (T), aspartate (D), cysteine (C), proline (P) and leucine (L) [0128] wherein the peptide aptamer binds to eIF4E.
[0129] In one example, the peptide aptamer binds to eIF4E in an open conformation. The nucleotide or the cap of the peptide motif of the peptide aptamer is capable of binding to eIF4E. The term open conformation refers to the swinging of W56 and W102 out of the cap-binding site. In contrast, the term closed conformation refers to the stacking of W56 and W102 in parallel on either side of the guanine moiety when W56 and W102 have swung back into the cap binding site.
[0130] In one example, the peptide aptamer is linked to the scaffold protein in a stable conformation. The peptide aptamer linked to the scaffold protein presents the peptide motif binding to eIF4E in a stable conformation. The stable conformation in the context of binding to eIF4E refers to the interaction of R51 and D36 two structured water molecules and the backbone of the CDR3 loop of the hypervariable region. Presentation of the peptide motif in stable conformation allows the high affinity binding of the peptide motif to eIF4E.
[0131] In one example, the peptide aptamer binds to eIF4E at the mRNA 5 binding site. The peptide aptamer binds to eIF4E at the mRNA 5 binding site with high affinity. The peptide aptamer binds to eIF4E at the mRNA 5 binding site with a dissociation constant (Kc) of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM. In one example, the peptide aptamer binds to eIF4E at the mRNA 5 binding site with a Kc of less than 50 nM.
[0132] In one example, the peptide aptamer binding to eIF4E inhibits cap-dependent translation.
[0133] In one example, the peptide aptamer comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. In a preferred example, the peptide aptamer comprises the amino acid sequence set forth in SEQ ID NO: 4.
[0134] In another aspect, the present invention refers to a peptide aptamer comprising an amino acid sequence X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7YPMFX.sub.8X.sub.9X.sub.10 (SEQ ID NO:14); [0135] wherein X.sub.1-X.sub.10 is any amino acid; [0136] wherein X.sub.1-7 comprises amino acid sequences for a first linker; [0137] wherein X.sub.8-10 comprises amino acid sequences for a second linker; [0138] optionally wherein X.sub.1 is serine (S); [0139] optionally wherein X.sub.2 is an amino acid selected from the group consisting of proline (P), glutamine (Q), tyrosine (Y), histidine (H) and arginine (R); [0140] optionally wherein X.sub.3 is an amino acid selected from the group consisting of glycine (G), valine (V), serine (S), arginine (R) and threonine (T); [0141] optionally wherein X.sub.4 is an amino acid selected from the group consisting of threonine (T), asparagine (N), arginine (R), serine (S), proline (P) and glutamate (E); [0142] optionally wherein X.sub.5 is an amino acid selected from the group consisting of histidine (H), proline (P), valine (V), aspartate (D), threonine (T) and arginine (R); [0143] optionally wherein X.sub.6 is an amino acid selected from the group consisting of proline (P), leucine (L) and isoleucine (I); [0144] optionally wherein X.sub.7 is an amino acid selected from the group consisting of lysine (K), valine (V), leucine (L) and phenylalanine (F); [0145] optionally wherein X.sub.8 is an amino acid selected from the group consisting of histidine (H), valine (V), isoleucine (I) and serine (S); [0146] optionally wherein X.sub.9 is an amino acid selected from the group consisting of leucine (L), asparagine (N), arginine (R) and proline (P); and [0147] optionally wherein X.sub.10 is an amino acid selected from the group consisting of arginine (R), histidine (H) and tryptophan (W), [0148] wherein the peptide aptamer binds to proliferating cell nuclear antigen (PCNA).
[0149] In one example, the peptide aptamer comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22. In a preferred example, the peptide aptamer comprises the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In another preferred example, the peptide aptamer comprises the amino acid sequence set forth in SEQ ID NO: 23.
[0150] In one example, the peptide aptamer binding to PCNA inhibits cell proliferation. The peptide aptamer binding to PCNA inhibits cell proliferation in stable transfected inducible cell lines. Examples of stable transfected inducible cell lines include but not limited to HEK293 cells and A375 stable cell lines.
[0151] In another aspect, the present invention refers to a peptide aptamer comprising an amino acid sequence selected from the group consisting of
TABLE-US-00004 i) (SEQIDNO:24) X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5WX.sub.6X.sub.7SRTPWX.sub.8X.sub.9X.sub.10X.sub.11X.sub.12, ii) (SEQIDNO:25) X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19WX.sub.20X.sub.21SRTPWX.sub.22X.sub.23X.sub.24, and iii) (SEQIDNO:26) X.sub.25X.sub.26X.sub.27WX.sub.28X.sub.29SRTPWX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36; [0152] wherein X.sub.1-X.sub.36 is any amino acid; [0153] wherein X.sub.1-5, X.sub.13-19 and X.sub.25-29 comprise amino acid sequences for a first linker; [0154] wherein X.sub.8-12, X.sub.22-24 and X.sub.30-36 comprise amino acid sequences for a second linker; [0155] optionally wherein X.sub.1 and X.sub.15 is an amino acid selected from the group consisting of leucine (L), glutamine (Q), arginine (R), valine (V), tyrosine (Y), glycine (G), alanine (A) and threonine (T); [0156] optionally wherein X.sub.2 and X.sub.16 is an amino acid selected from the group consisting of threonine (T), arginine (R), serine (S), proline (P), alanine (A) and isoleucine (I); [0157] optionally wherein X.sub.3, X.sub.17 and X.sub.25 is an amino acid selected from the group consisting of tryptophan (W), leucine (L), alanine (A), isoleucine (I), serine (S), threonine (T), glutamine (Q), asparagine (N), glutamate (E), cysteine (C), proline (P), glycine (G), valine (V), arginine (K), arginine (R), phenylalanine (F), and methionine (M); optionally wherein X.sub.4, X.sub.18 and X.sub.26 is an amino acid selected from the group consisting of threonine (T), histidine (H), glutamine (Q), leucine (L), glutamate (E), valine (V), glycine (G), arginine (R), phenylalanine (F), tyrosine (Y), serine (S), isoleucine (I), alanine (A), tryptophan (W) and cysteine (C); [0158] optionally wherein X.sub.5, X.sub.19 and X.sub.27 is an amino acid selected from the group consisting of arginine (R), tyrosine (Y), lysine (K), leucine (L), histidine (H), proline (P), serine (S), tryptophan (W) and valine (V); [0159] optionally wherein X.sub.6, X.sub.20 and X.sub.28 is an amino acid selected from the group consisting of valine (V), arginine (R), isoleucine (I), histidine (H), leucine (L), serine (S), phenylalanine (F), alanine (A), glycine (G), threonine (T) and lysine (K); [0160] optionally wherein X.sub.7, X.sub.21 and X.sub.29 is an amino acid selected from the group consisting of asparagine (N), phenylalanine (F), tryptophan (W), leucine (L), alanine (A), glycine (G), lysine (K), glutamate (E), serine (S), arginine (R), threonine (T), tyrosine (Y) and aspartate (D); [0161] optionally wherein X.sub.8, X.sub.22 and X.sub.30 is an amino acid selected from the group consisting of asparagine (N), histidine (H), phenylalanine (F), serine (S), tryptophan (W), alanine (A), threonine (T), tyrosine (Y), lysine (K), arginine (R), valine (V) and cysteine (C); optionally wherein X.sub.9, X.sub.23 and X.sub.31 is an amino acid selected from the group consisting of valine (V), phenylalanine (F), arginine (R), isoleucine (I), asparagine (N), leucine (L), lysine (K), methionine (M), tryptophan (W), threonine (T), glycine (G) and alanine (A); [0162] optionally wherein X.sub.10, X.sub.24 and X.sub.32 is an amino acid selected from the group consisting of isoleucine (I), arginine (R), tyrosine (Y), leucine (L), valine (V), lysine (K), histidine (H), alanine (A), methionine (M), tryptophan (W), serine (S), threonine (T) and proline (P); [0163] optionally wherein X.sub.11 and X.sub.33 is an amino acid selected from the group consisting of glycine (G), leucine (L), phenylalanine (F), arginine (R), tyrosine (Y), methionine (M), alanine (A), histidine (H), tryptophan (W), asparagine (N), lysine (K), serine (S), threonine (T) and glutamine (Q); [0164] optionally wherein X.sub.12 and X.sub.34 is an amino acid selected from the group consisting of phenylalanine (F), threonine (T), histidine (H), arginine (R), tyrosine (Y), valine (V), aspartate (D), alanine (A), serine (S), leucine (L), proline (P), lysine (K) and tryptophan (W); [0165] optionally wherein X.sub.13 is an amino acid selected from the group consisting of asparagine (N) and leucine (L); [0166] optionally wherein X.sub.14 is an amino acid selected from the group consisting of valine (V), and leucine (L); [0167] optionally wherein X.sub.35 is an amino acid selected from the group consisting of (H), arginine (R), methionine (M), threonine (T), lysine (K), valine (V), leucine (L), serine (S), asparagine (N), isoleucine (I), aspartate (D) and glycine (G); and [0168] optionally wherein X.sub.36 is an amino acid selected from the group consisting of glutamine (Q), isoleucine (I), leucine (L), phenylalanine (F), proline (P), arginine (R), threonine (T), tyrosine (Y), aspartate (D), lysine (K), glycine (G), serine (S) and alanine (A); [0169] wherein the peptide aptamer binds to eIF4A.
[0170] In one example, the peptide aptamer comprises the amino acid sequences selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
[0171] In one example, the peptide aptamers of the present invention may not be constrained by a disulphide bond. The peptide aptamers may be engineered to remove the disulphide bonds from the constrained peptide motif and the disulphide bonds are replaced with linkers for increased stability of the peptide aptamers. Increased stability of peptide aptamers results in high affinity binding to the target protein. The peptide aptamers may be expressed in a cell to mimic the peptide motifs constrained by disulphide bonds. In another example, the peptide aptamers of the present invention may be constrained by a disulphide bond. The peptide aptamer comprises a cysteine amino acid in the linker sequence at each of the N-terminal and the C-terminal. The peptide aptamers constrained by a disulphide bond may be employed in diagnostic or purification applications to elute bound substrate using DTT.
[0172] In a preferred example, the peptide aptamer comprises the amino acid sequence set forth in SEQ ID NO: 29, SEQ ID NO: 39 and SEQ ID NO: 61.
[0173] In one example, the peptide aptamers of the present invention comprise a peptide motif and/or one or more linker sequences located in a hypervariable region of a scaffold protein.
[0174] In one example, the scaffold protein includes but is not limited to VH domain, stefin-A and fibronectin. In a preferred example, the scaffold protein is the VH domain.
[0175] In one example, the peptide aptamers of the present invention may be engineered and identified using the method as described herein. It should be clear to the person skilled in the art that the method as described herein can be used to engineer and identify peptide aptamers that bind to the target protein of interest and should not be restricted to the peptide aptamers of the present invention.
[0176] In another aspect, the present invention refers to the peptide aptamer as described herein for use as a medicament.
[0177] In another aspect, the present invention refers to a use of the peptide aptamer as described herein in the manufacture of a medicament for treating a condition associated with dysregulated cap-dependent translation, dysregulated DNA replication, dysregulated DNA repair and/or dysregulated mRNA translation.
[0178] In another aspect, the present invention refers to a method of treating a condition associated with dysregulated cap-dependent translation, dysregulated DNA replication, dysregulated DNA repair and/or dysregulated mRNA translation, comprising administering the peptide aptamer as described herein to a subject in need thereof. The peptide aptamer may in some examples be administered sequentially or simultaneously with one or more therapeutic agents. Examples of therapeutic agents include but are not limited to immunotherapy, chemotherapy or anti-viral therapy. In one example, the peptide aptamer is administered as a combinatorial treatment with immunotherapy. Immunotherapy may comprise therapies that downregulate the STAT1 pathway.
[0179] In one example, the present disclosure refers to the peptide aptamer as described herein for use in treating a condition associated with dysregulated cap-dependent translation, dysregulated DNA replication, dysregulated DNA repair and/or dysregulated mRNA translation.
[0180] In one example, dysregulated cap-dependent translation is a result of aberrant protein expression or aberrant protein activity. In one example, dysregulated cap-dependent translation is a result of aberrant eIF4E expression or aberrant eIF4E activity. In some examples, aberrant eIF4E expression is overexpression of eIF4E and aberrant eIF4E activity is an increase of eIF4E above the normal physiological levels. In one example, the condition associated with eIF4E overexpression is cancer.
[0181] In another example, the condition associated with dysregulated cap-dependent translation is a disease associated with a viral infection. In some examples, the virus invades the cell and drives cap-dependent translation. As a result, the cap-dependent translation may be increased or decreased as compared to the baseline activity.
[0182] In one example, dysregulated DNA replication, dysregulated DNA repair and dysregulated mRNA translation is also a result of aberrant protein expression or aberrant protein activity. For example, dysregulated DNA replication and dysregulated DNA repair is a result of overexpression of PCNA. In another example, dysregulated mRNA translation is a result of the aberrant eIF4A expression. Aberrant eIF4A expression refers to the overexpression of eIF4A and is an increase in eIF4A expression above the normal physiological levels.
[0183] In one example, the condition associated with dysregulated cap-dependent translation, dysregulated DNA replication, dysregulated DNA repair and/or dysregulated mRNA translation may include but is not limited to cancer, a disease associated with a viral infection and obesity. In one example, the cancer includes but is not limited to melanoma, triple negative breast cancer, lung cancer, colorectal cancer and prostate cancer. In a preferred example, the cancer is melanoma.
[0184] In another example, the disease associated with the viral infection is a disease caused by a virus. In one example, the virus is an RNA virus. Examples of the RNA virus include but are not limited to coronavirus, orthomyxovirus, rhabdovirus, reovirus, hantavirus and alphavirus.
[0185] In one aspect, the present invention refers to a method to identify a candidate peptide or nucleic acid that binds to a target protein in a live cell comprising: [0186] a) fusing LargeBiT (LgBiT) to the target protein and fusing Small BiT (SmBiT) to a peptide aptamer that binds to the target protein, or fusing SmBiT to the target protein and fusing LgBiT to a peptide aptamer that binds to the target protein; [0187] b) (i) expressing the fusion proteins from step a in the live cell; measuring the level of signal emitted in step b(i), wherein a signal is emitted when the target protein binds to the peptide aptamer; contacting the cell expressing the bound fusion protein with the candidate peptide or nucleic acid, optionally wherein the cell is permeabilized prior to contacting with the candidate peptide or nucleic acid; or [0188] (ii) expressing the fusion proteins from step a and the candidate peptide or nucleic acid in the live cell; [0189] measuring the level of signal emitted in step b(ii), wherein the signal is emitted when the target protein binds to the peptide aptamer; and [0190] c) detecting a change in level of signal relative to a reference, wherein a decrease in the level of signal emitted is indicative of binding of the candidate peptide or nucleic acid to the target protein.
[0191] The candidate peptide may be an antagonist that binds to the cap-binding site or an antagonist that binds outside the cap-binding site. These antagonists may induce dissociation between the peptide aptamer and target protein.
[0192] In one example, the reference is a live cell which expresses the fusion proteins from step (a) and optionally a peptide or nucleic acid that does not bind to the fusion proteins from step (a) or a live cell which expresses the fusions proteins from step (a) and the candidate peptide or nucleic acid prior to binding to the target protein.
[0193] In one example, the live cell is a mammalian cell. In one example, the mammalian cell is a human cell. In a preferred example, the human cell is a human embryonic kidney (HEK) 293 cell.
[0194] The target protein may by any protein that interacts with the candidate peptide or nucleic acid in a live cell. In one example, the target protein includes but is not limited to eIF4E, PCNA and eIF4A.
[0195] The peptide aptamer fused to the SmallBiT (SmBiT) or LargeBiT (LgBiT) may be any peptide aptamer that binds to a target protein. In one example, the peptide aptamer fused to the SmallBiT (SmBiT) or LargeBiT (LgBiT) is the peptide aptamer as described here.
[0196] The candidate peptide or nucleic acid may be any peptide or nucleic acid that bind to a target protein. In one example, the candidate peptide is the peptide aptamer as described herein.
[0197] The methods as described herein may be utilized to identify any candidate peptide or nucleic acid that display binding affinity to a target protein of interest directly in live cells.
[0198] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms comprising, including, containing, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
[0199] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0200] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
EXPERIMENTAL SECTION
[0201] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
Materials and Methods
Peptide Synthesis
[0202] Peptides were ordered from and synthesized by Mimotopes, Clayton, Australia. Peptides were purified using HPLC to >90% purity. All peptides were amidated at their C-terminus and acetylated at their N-terminus. Peptides were purified using HPLC to >90% purity.
eIF4E Expression and Purification for Crystallisation and Biophysical Assays
[0203] Full-length human eIF4E was expressed and purified as described below. Rossetta pLysS competent bacteria were transformed with the pET11d expression plasmid containing the full-length eIF4E clone. Cells expressing the full-length eIF4E construct were grown in LB medium at 37 C. to an OD600 of 0.6 and eIF4E induction was started with 1 mM IPTG. The culture was immediately placed in a shaker-incubator for 3 h at 37 C. Cells were harvested by centrifugation and the cell pellets were resuspended in 50 mM Tris pH 8.0, 10% sucrose, and were then sonicated. The sonicated sample was centrifuged for 10 min at 17,000 g at 4 C. The resulting pellet was resuspended in Tris/Triton buffer (50 mM Tris pH 8.0, 2 mM EDTA, 100 mM NaCl, 0.5% Triton X-100). The sample was then centrifuged at 25,000 g for 15 min at 4 C. and the pellet was resuspended in Tris/Triton buffer. After re-centrifugation, the remaining pellet was solubilised in 6 M guanidinium hydrochloride, 50 mM Hepes-KOH pH 7.6, 5 mM DTT. The protein concentration of the sample was then adjusted to 1 mg/mL. The denatured protein was refolded via a 1/10 dilution into refolding buffer consisting of 20 mM Hepes-KOH 7.6, 100 mM KCl and 1 mM DTT. The refolded protein was concentrated and desalted using a Amersham PD10 column into refolding buffer. The eIF4E protein sample was run over a monoQ column and eluted with a 1 M KCl gradient. eIF4E eluted as a sharp peak at a 0.3M KCl.
eIF4A Expression and Purification
[0204] eIF4A was cloned into the GST fusion expression vector pGEX-6P1 (GE Lifesciences). BL21 DE3 competent bacteria were then transformed with the GST-tagged fusion constructs. A single colony was picked and grown in LB medium at 37 C. to an OD600 of 0.6 and induction was carried out overnight with 0.3 mM IPTG at 16 C. Cells were harvested by centrifugation, and the cell pellets were resuspended in PBS (Phosphate Buffered Saline, 2.7 mM KCl and 137 mM NaCl, pH 7.4) and then sonicated. The sonicated sample was centrifuged for 60 min at 17,000 g at 4 C. The supernatant was applied to a 5 ml FF GST column (Amersham) pre-equilibrated in PBS buffer with 1 mM DTT. The column was then further washed by 6 volumes of PBS. Proteins were then purified from the column by either 1) cleavage with thrombin (Sigma-Aldrich) protease or 2) elution with glutathione. 1) Ten units of thrombin (Sigma-Aldrich) protease, in one column volume of PBS with 1 mM DTT buffer, were injected into the column. The cleavage reaction was allowed to proceed overnight at 4 C. The cleaved protein was then eluted off the column with wash buffer. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). Protein samples were then dialyzed into a buffer solution containing 20 mM Tris pH 8.0 with 1 mM DTT and loaded onto a mono Q column pre-equilibrated in buffer A (20 mM Tris, pH 8.0, 1 mM DTT). The column was then washed in 6 column volumes of buffer A and bound protein was eluted with a linear gradient of 1 M NaCl over 25 column volumes. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). The cleaved constructs were then purified to 90% purity. Protein concentration was determined using A280. 2) The column was then washed in 6 column volumes of PBS with 1 mM DTT and bound protein was eluted with a linear gradient of 50 mM Tris, pH 8.0, 0.1 M NaCl with 250 mM glutathione over 10 column volumes. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). Concentrated protein was dialysed into PBS with 1 mM DTT and protein concentration determined using A280.
PCNA Expression and Purification.
[0205] A plasmid containing the gene for human tagged to sumo and a hexa his tag was transformed into BL21-DE3 E. coli cells for protein expression. A single colony was picked and inoculated in 1L of LB medium. Upon reaching log phase, cells were induced with 1 mM isopropyl--D-thiogalactopyranoside and harvested after 3 h at 37 C. Pelleted cells were lysed in a buffer containing 25 mM HEPES, pH 7.5, 300 mM NaCl and 5 mM imidazole (buffer A). Cell lysates were clarified at 13 000 rpm for 45 min at 4 C. The supernatant was applied over a 0.22 m filter and loaded onto a chelating column charged with 100 mM nickel sulfate (GE Healthcare). The column was washed with 20 CVs of buffer A and then 50 Unit of sumo was injected onto the detached column in cleavage buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 1 mM DTT). Cleavage was performed overnight at 4 C. and then the column reattached and the cleaved PCNA eluted off. Fractions containing PCNA were pooled and buffer exchanged into 25 mM HEPES, pH 7.5, 100 mM NaCl and 1 mM DTT. These were then further purified using a superdex-75 size exclusion column and then concentrated.
eIF4E and 4EBP1.sup.4ALA Mutants Expression and Purification for Sortase Labelling
[0206] eIF4E and 4EBP1.sup.4ALA mutants were cloned into the GST fusion expression vector pGEX-6P1 (GE Lifesciences). BL21 DE3 competent bacteria were then transformed with the GST-tagged fusion constructs. A single colony was picked and grown in LB medium at 37 C. to an OD600 of 0.6 and induction was carried out overnight with 0.3 mM IPTG at 16 C. Cells were harvested by centrifugation, and the cell pellets were resuspended in PBS (Phosphate Buffered Saline, 2.7 mM KCl and 137 mM NaCl, pH 7.4) and then sonicated. The sonicated sample was centrifuged for 60 min at 17,000 g at 4 C. The supernatant was applied to a 5 ml FF GST column (Amersham) pre-equilibrated in PBS buffer with 1 mM DTT. The column was then further washed by 6 volumes of PBS. Proteins were then purified from the column by 1) cleavage with thrombin (Sigma-Aldrich) protease, 2) elution with glutathione or 3) cleavage with PreScission (GE Lifesciences) protease.
[0207] 1) Ten units of thrombin (Sigma-Aldrich) protease, in one column volume of PBS with 1 mM DTT buffer, were injected into the column. The cleavage reaction was allowed to proceed overnight at 4 C. The cleaved protein was then eluted off the column with wash buffer. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). Protein samples were then dialyzed into a buffer solution containing 20 mM Tris pH 8.0 with 1 mM DTT and loaded onto a mono Q column pre-equilibrated in buffer A (20 mM Tris, pH 8.0, 1 mM DTT). The column was then washed in 6 column volumes of buffer A and bound protein was eluted with a linear gradient of 1 M NaCl over 25 column volumes. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). The cleaved constructs were then purified to 90% purity. Protein concentration was determined using A280.
[0208] 2) The column was then washed in 6 column volumes of PBS with 1 mM DTT and bound protein was eluted with a linear gradient of 50 mM Tris, pH 8.0, 0.1 M NaCl with 250 mM glutathione over 10 column volumes. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). Concentrated protein was dialysed into PBS with 1 mM DTT and protein concentration determined using A280.
[0209] 3) Ten units of PreScission protease, in one column volume of PBS with 1 mM DTT buffer, were injected onto the column. The cleavage reaction was allowed to proceed overnight at 4 C. The cleaved protein was then eluted off the column with wash buffer. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). Protein samples were then dialyzed into a buffer solution containing 20 mM Tris pH 8.0 with 1 mM DTT and loaded onto a mono Q column pre-equilibrated in buffer A (20 mM Tris, pH 8.0, 1 mM DTT). The column was then washed in 6 column volumes of buffer A and bound protein was eluted with a linear gradient of 1 M NaCl over 25 column volumes. Protein fractions were analysed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator (Millipore). The cleaved constructs were then purified to 90% purity. Protein concentration was determined using A280.
Sortase (SrtA.SUP.8M.) Expression and Purification
[0210] The protein sequence corresponding to 61-206 of SrtA (Staphylococcus aureus) containing the following mutations (P94R, D160N, D165A, K190E, K196T, E105K, E108A and G167E) was ordered as a gene fragment from IDT (Integrated DNA technologies). The sequence was PCR amplified and inserted into a pNIC-CH bacterial expression plasmid via ligation independent cloning in frame with a C-terminal 6His tag. The pNIC-CH-(61-206) SrtA.sup.8M (termed SrtA.sup.8M) expression vector was transformed into BL21(DE3) Rosetta competent cells and a single colony was used to inoculate a 20 ml starter culture in TB (terrific broth containing 25 ug/ml of chloramphenicol and 20 ug/ml of kanamycin), which was incubated overnight at 37 C. and shaken at 200 rpm. The starter culture was used to inoculate 750 ml of TB and was incubated at 37 C. until a O.D.sub.600 reading of 2.0 was attained. Next, the temperature of the culture was lowered to 18 C. and protein expression induced with 0.5 mM of IPTG overnight. Cells were harvested by centrifugation, and the cell pellets were resuspended in 20 ml of lysis buffer (100 mM HEPES pH 8.0, 500 mM NaCl, 10 mM Imidazole, 10% glycerol, 0.5 mM TCEP, 1000 u Benzonase (Merck)) and then sonicated. The sonicated sample was centrifuged for 30 min at 17,000 g at 4 C. Supernatants were then filtered through 1.2 m syringe filters and were loaded onto a Ni-nitrilotriacetic acid (NTA) column, pre-equilibrated with 20 mM HEPES pH 7.5, 100 mM NaCl, and 0.5 mM TCEP. The column was then washed with 5 column volumes of the same buffer containing 10 mM Immidazole. Hexahistidine tagged SrtA.sup.8M was then eluted with a 1 M imidazole linear gradient. The protein was further purified by size exclusion chromatography (HiLoad 16/60 Superdex 75 prep grade, Cytiva Lifescience) using a 20 mM HEPES pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, 0.5 mM TCEP buffer. Protein concentration was determined using A280 with an extinction coefficient determined from the primary sequence of the construct determined by ProtPARAM.
N-Terminal Biotin Labelling of eIF4E Mediated by SrtA.sup.8M
[0211] Sortase-mediated ligation was used to specifically label eIF4E at the N-terminal with biotin. Cleavage of the GST-fused eIF4E with thrombin leaves a single glycine at the N-terminus. The ligation was carried out with thrombin cleaved eIF4E at 50 M, SrtA.sup.8M at 1 M, and biotin-KGGGLPET-GG-OHse (Ac)-amide peptide at 200 M in 200 L of ligation buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM TCEP). SortaseA61-206/8M contains mutations that increase ligation efficiency and make it calcium-independent2. The ligation was incubated at room temperature for 4 hours. SrtA.sup.8M, which contains a C-terminal 6His-tag, was removed with Dynabeads His-Tag (cat #10104D, Thermo Fisher). The biotinylated protein was then dialyzed at 4 C. using slide-A-Lyzer cassette (10 k MWCO) against 2 L of an appropriate buffer. The buffer was changed after 4-5 hours and the dialysis was repeated overnight. The biotinylated protein was aliquoted, snap-frozen with liquid nitrogen, and stored at 80 C.
Phage Display
[0212] An M13 phage library (Ph.D.-12, New England Biolabs) encoding random 12-mer peptides at the NH.sub.2 terminus of pIII coat protein (2.710.sup.9 sequences) was used. Biotinylated full length eIF4E was loaded onto 10 l of steptavdin M280 magnetic Dynabeads (Invitrogen). The loaded beads were incubated with blocking buffer (20 mM HEPES pH 7.6, 0.1 M KCL, 0.5% Tween20, 2% BSA) for 1 h at room temperature, washed with buffer W (20 nM HEPES pH 7.6, 0.1 M KCL, 0.5% Tween 20), and incubated in buffer W at room temperature with 410.sup.10 phages. Magnetic M280 beads were washed 8 times in buffer W. Bound phages were eluted with 0.2 M glycine (pH 2.2) and neutralized with 1 M Tris (pH 9.1). The eluted phages were amplified as instructed by the manufacturer. The selection process was repeated for three cycles. Phage plaques from the final round were picked and amplified as described by the manufacturer and sequenced.
[0213] M13 phage library (Ph.D.-C7C, New England Biolabs) encoding random 7-mer peptides flanked by two Cys was used. A 96-well microplate (Corning, #3370) was first coated with streptavidin (100 g/mL) in 100 mM NaHCO.sub.3(pH 8.4) at 4 C. overnight. After washing with 4200 L of binding buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl.sub.2, pH 7.4), the wells were filled with the corresponding biotinylated protein (eIF4E, eIF4A or MDM2, 20 g/mL) in the binding buffer. After incubating at room temperature for 15 min, the microplate was washed with 4200 L of binding buffer and blocked with blocking buffer (binding buffer plus 1% BSA and 0.1% Tween-20) for 1 hour at room temperature. In parallel, the phage library was diluted to 1.010.sup.12 pfu/mL in the blocking buffer. After removal of the blocking buffer from the microplate, phage library (100 L per well) was added, incubated for 1 hour at room temperature, and washed with 4200 L of washing solution (binding buffer+0.1% Tween-20), followed by 2200 L washing solution containing 1 mM streptavidin, and finally with 4200 L of washing solution. The bound phages were eluted for 9 min with 0.2 M glycine (pH 2.2) plus 1% BSA and neutralized with 1 M Tris (pH 9.1). The eluted phages were amplified according to manufacturer's instruction and phage DNA was extracted by QIAprep spin Miniprep kit. The single-stranded DNA was then converted to Illumina-compatible double-stranded DNA amplicon by PCR. Briefly, the single-stranded DNA (50-100 ng) was combined with 1 Phusion buffer, 200 M dNTPs (each), 0.5 M each of forward (F) and reverse (R) primers, and one unit Phusion High-Fidelity DNA Polymerase in a total volume of 50 L. The primer sequences are as follows: forward (F) 5-CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC TXX XXC CTT TCT ATT CTC ACT CT-3 and reverse (R) 5-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC TXX XXA CAG TTT CGG CCG A-3. Of note, XXXX in the primer sequence denotes four-nucleotide-long barcodes used to trace multiple samples in one Illumina sequencing experiment. The temperature cycling protocol for PCR was: 95 C. for 30 s, followed by 25 cycles of 95 C. for 10 s, 60.5 C. for 15 s and 72 C. for 30 s, and a final extension at 72 C. for 5 min before holding at 4 C. The resulting amplicons were pooled (20 ng per sample) together and purified on E-Gel SizeSelect 2% agarose gel (Invitrogen, #G6610-02). Sequencing was performed using the Illumina NextSeq platform (Axil Scientific). Identification of significantly enriched sequences from deep-sequencing data was performed. Heat maps were used to identify sequences isolated from a target selection (eIF4E) that increased significantly in abundance against sequences isolated from the control selection. The copy number of each sequence is normalized through dividing the copy number by the total number of reads in each replicate. Sequences not observed in a specific replicate were assigned a copy number of zero. The ratio of each sequence was calculated through dividing the mean fraction of the particular sequence in the target selection by those in the control screen (e.g., steptavidin). Since the denominator must not be a zero when taking the ratio, sequences with zero copy number found in all three replicates are assigned with 0.3 copy number before taking the normalization. Significance of the ratio was assessed using one-tailed, unequal variance Student t-test. The ratio is considered to be statistically significant if the calculated p-value0.05. Only sequences with ratio5 and p-value0.05 were analysed. Heats maps were plotted showing only the significant sequences and were ranked using their ration values, from highest to lowest. Python scripts were written to perform the described operations.
Fluorescence Anisotropy Competition Assays and K.SUB.d .Determination
[0214] Purified eIF4E and PCNA was titrated against 50 nM carboxyfluorescein (FAM) labelled eIF4E tracer peptide (Ac-KKRYSRDFLLALQK-(FAM)-NH.sub.2) or m.sup.7GTP.sup.5-FAM (Jena Biosciences Cat. No: NU-824-5FM) or PCNA tracer peptide (Ac-SAVLQKKITDYFHPKK-K (5-carboxylysine)-NH2). The K.sub.d (dissociation constant) for the titration of eIF4E and PCNA against the tracer peptide was determined by fitting the experimental data to a 1:1 binding model equation:
[0215] where [P] is the protein concentration, [L] is the labelled peptide concentration, r is the anisotropy measured, r.sub.0 is the anisotropy of the free peptide, r.sub.b is the anisotropy of the eIF4E- or PCNA-tracer peptide complex, [L], is the total FAM labelled peptide or m.sup.7GTP.sup.FAM concentration, and [P] is the total eIF4E or PCNA concentration. The K.sub.ds determined for the interaction of either the tracer peptide or m.sup.7GTP.sup.FAM with eIF4E were 50.3 nM and 149.0 nM respectively. These were used in subsequent K.sub.d determinations in competition experiments to measure binding against the eIF4E cap-binding site and eIF4E:4G interface. The K.sub.d determined for the interaction of the tracer peptide or with PCNA was 150 nM. This K.sub.d was used in subsequent K.sub.d determinations in competition experiments to measure binding against the PCNA protein interaction binding site which is the site where PCNA interacts with more than 200 proteins e.g., p21, p300, CDK2 and others.
[0216] To determine K.sub.ds for compounds that disrupted the eIF4E:4G interfaces and the PCNA: tracer complex, molecules were titrated against eIF4E or PCNA and the labelled peptide. The molecules titrated against eIF4E and the labelled peptide were at set concentrations of 200 nM and 50 nM, respectively and molecules titrated against PCNA and the labelled peptide were at set concentrations of 625 nM and 50 nM, respectively. With respect to compounds that interacted at the eIF4E cap binding site, titrations were performed with eIF4E and m.sup.7GTP.sup.5-FAM at concentrations of 250 nM and 50 nM. Apparent K.sub.d values for both competition assays were determined by fitting the experimental data to the equations shown below:
[0217] [L].sub.st and [L].sub.t denote labelled ligand and total unlabelled ligand input concentrations, respectively. K.sub.d2 is the dissociation constant of the interaction between the unlabelled ligand and the protein. In all competitive types of experiments, it is assumed that [P].sub.t>[L].sub.st, otherwise considerable amounts of free labelled ligand would always be present and would interfere with measurements. K.sub.d1 is the apparent K.sub.d for the labelled peptide used in the respective experiment. The tracer peptide was dissolved in DMSO at 1 mM and diluted into experimental buffer. Readings were carried out with a Envision Multi-label Reader (PerkinElmer). Experiments were carried out in PBS (2.7 mM KCl, 137 mM NaCl, 10 mM Na.sub.2HPO.sub.4 and 2 mM KH.sub.2PO.sub.4 (pH 7.4)) and 0.1% Tween 20 buffer. All titrations were carried out in triplicate. Curve-fitting was carried out using Prism 4.0 (GraphPad).
Tryptophan Quenching
[0218] Tryptophan fluorescence quenching studies were performed using a Envision Multiplate in a black 96 well plate. Protein samples were excited at a wavelength of 290 nm and tryptophan emission was measured at a wavelength of 355 nm. Sample wells contained eIF4E at a concentration of 10 M at a set volume of 100 l with increasing concentrations of the relevant compounds under study. Quenching experiments were performed in PBS buffer (2.7 mM KCl, 137 mM NaCl, 10 mM Na.sub.2HPO.sub.4 and 2 mM KH.sub.2PO.sub.4 (pH 7.4)) with a final DMSO concentration of 1% (v/v).
Construction and Assessment of VH Domains with Rationally Designed Linkers
[0219] VH-DiF clones containing different linker regions flanking the eIF4E cap-site interaction motif (EMGFF) were ordered as Ultramer double stranded oligonucleotides (IDT) containing EagI/HIndIII restriction sites. The double stranded VH-Dif encoding cassettes were then cloned into the pET22b bacteria expression vector via ligation at the EagI/HIndIII cloning sites in frame with the c-terminal polyHis affinity purification tag. VH domain constructs (R1-R3) were purified as out lined in the Bacterial Expression and Purification of VH-Domain constructs section. Purified VH clones were incubated in a 20:1 excess ratio to purified GST-eIF4E (20 M) and incubated for 4 hours at room temperature in PBS with 1 mM DTT (see eIF4E expression and purification for Sortase Labelling). GST-eIF4E:VH domain complexes were pulled down with 20 l of GST-beads (Thermo Fisher). Protein samples were analyzed using SDS-PAGE gel and visualized with Coomassie stain.
Yeast Display PELE Library Construction
[0220] The pCT-CON vector was digested using SalI, NheI, and BamHI restriction enzymes (NEB) to ensure complete linearization and absence of full-length insert, thereby preventing transformation of yeast cells with parental plasmid. The PELE library of Dif-VH domains was constructed by three-step overlap extension PCR (OE-PCR). A set of 9 primers; P1_for, P2_rev to P9_rev were dissolved at 100 M concentration and mixed in an equimolar ratio to prepare three mixed pools containing each primer at a concentration of 10 M. The three mixed pools were denoted Lib1, Lib2, and Lib3 with each containing a primer specifically encoding a designed PELE library, P9a_for, P9b_for, or P9c for, respectively. 1 L was taken from each mixed library and 5-fold dilution series prepared to identify the optimal primer concentration for OE-PCR. 0.4 M of each primer was found to produce optimal yields for OE-PCR for each of the three mixed pools. The full length diF-VH domain product from each library OE-PCR reaction (Lib1, Lib2 and Lib3) was mixed in a 1:1:1 molar ratio (denoted pooled PELE library). 300 ng of the pooled PELE library and 1 g of digested pCT-CON vector were combined with 50-100 L of electrocompetent EBY100 yeast cells and electroporated at 0.54 kV and 25 F using a GenePulser Xcell (Bio-Rad). Homologous recombination of the linearized vector and pooled PELE insert yielded intact plasmid. Cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) for 1 h at 30 C., 250 rpm. The number of total transformants was 5.710.sup.7 cells as determined by serial dilutions plated on SD-CAA plates (0.1 M sodium phosphate, pH 6.0, 182 g/L sorbitol, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 20 g/L glucose). The library was propagated by selective growth in SD-CAA, pH 5.3 (0.07 M sodium citrate, pH 5.3, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 20 g/L glucose, 0.1 g/L kanamycin, 100 kU/L penicillin, and 0.1 g/L streptomycin) at 30 C., 250 rpm.
Selection of Yeast Displayed PELE Library
[0221] 110.sup.10 cells were taken from the propagated library culture and pelleted at 2,500 g for 5 min in 50-ml conical tubes and resuspended in SGCAA media to an absorbance of about 0.5-1 at 600 nm to induce expression of the pooled PELE library. Library induction was maintained for 24 hours at 20 C.
[0222] 110 8 induced yeast cells were pelleted at 2500 g for 5 mins and the supernatant aspirated. Cells were then washed in 25 ml PBSM buffer, re-pelleted, supernatant discarded and re-suspended in 10 ml of PBSM. Biotinylated eIF4E was then added to a final concentration of 1 M. The yeast cell suspension was then incubated at room temperature with gentle rotation on a tube rotator for 60 min, followed by 10 mins on ice.
[0223] Dif-VH domain: eIF4E complexes were then isolated using MidiMACS (Mitenyi Biotec) magnetic separation. An LS column (Mitenyi Biotec) was equilibrated with PBSM buffer at 4 C. The yeast cell suspension was then pelleted at 2500 g for 5 min at 4 C., the supernatant aspirated, the yeast cells washed with 50 ml PBSM buffer and then resuspended in 5 ml PBSM buffer. 200 l of streptavidin microbeads were added to the suspension and incubated on ice with gentle mixing for 10 minutes. Yeast cells were spun down and again washed, before being re-suspended in 50 ml PBSM buffer. Cells were then applied to the LS column in the presence of magnet. Unlabelled cells were washed from the column with 10 ml ice cold PBSM. Cells labelled with streptavidin microbeads were eluted from the column by removal from the magnetic field into a collection tube. Serial dilutions of the sorted cell suspension were plated onto SDCAA plates and incubated at 30 C. to estimate the number of cells captured by MACS. Eluted yeast cells were then propagated with addition of SDCAA (containing 100 units/ml and 100 mg/ml of Penicillin-streptomycin) media to a final volume of 500 ml and incubated overnight at 30 C.
[0224] Magnetic sorting was then followed by 2 rounds of FACs enrichment. 110.sup.8 cells were taken from the propagated library culture and pelleted at 2,500 g for 5 min in 50-ml conical tubes and resuspended in SGCAA media to an absorbance of about 0.5-1 at 600 nm to induce expression of the sorted PELE library. Cells were incubated overnight at 20 C. Induced cells were spun down at 14,000 g for 30 s, supernatant aspirated and cells washed with PBSF buffer. Yeast cells were then labelled with 500 nM of sortase labelled biotinylated eIF4E in 1 ml of PBSF buffer and incubated at room temperature for 30 mins. Cells were then pelleted by centrifugation (14,000 g for 30 s at 4 C.), the supernatant aspirated and then washed with 1 ml ice-cold PBSA. Yeast were resuspended in 500 l PBSF containing Anti-HA Ab Alexa Fluor 488 (Invitrogen, 1:100 fold dilution) and Streptavidin-phycoerythrin (ThermoFisher Scientific, 1:100 fold dilution) and incubated for 30 mins. Cells were then pelleted at 14,000 g for 30 s at 4 C., washed with 1 ml PBSF buffer and resuspended in 2.0 mL PBSF. Cells positive for anti-HA and eIF4E were selected and sorted using an Aria (Becton Dickinson) cytometer. Collected cells were propagated in SDCAA at 30 C. and a second round of FACs selection performed after yeast induction with 110.sup.8 cells as described.
[0225] After the final round of FACs selection, serial dilutions of the sorted cell suspension were plated onto SDCAA plates and incubated at 30 C. until the appearance of yeast colonies. X colonies of yeast were individually picked and then propagated in X mls of SCDAA. Plasmid DNA was then isolated using the Zymoprep kit II (following the manufacturer's instructions), cleaned using the Qiagen PCR Purification kit, and transformed into DH5 (Invitrogen) cells. Purified plasmids were then sequenced using BigDye chemistry.
Assessment of Enriched Yeast PELE Library for Specific Binders to the eIF4E Cap-Binding Site
[0226] Before the second round of FACs sorting an additional subset of 510.sup.7 cells was induced in 5 ml of SGCAA media. Yeast were then pelleted, washed in 1 mL PBSF resuspended in PBSF to a density of 110.sup.7 cell per ml. 1 ml of yeast suspension was added to three individual tubes. Purified sortase biotinylated eIF4E was then added to each sample at a concentration of 2 M and samples were incubated at 20 C. for 1 hour. Purified sortase biotinylated eIF4E was then added to each sample at a concentration of 0.2 M either in combination with 50 M m.sup.7GTP (Sigma-Aldrich), 50 M of purified 4E-BP1.sup.4ALA or 50 M of VH-1C5.sup.M4, followed by sample incubation at 20 C. for 1 hour. 4E-BP1.sup.4ALA and VH-1C5.sup.M4 were then purified. The purification of 4E-BP1.sup.4ALA mutants is described in the Section eIF4E and 4EBP1.sup.4ALA mutants expression and purification for Sortase Labelling
[0227] For purification of VH-1C5.sup.M4, VH-1C5.sup.M4 sequence were ordered as gene fragments from Integrated DNA Technologies (IDT). Both coding sequences were PCR amplified and cloned directly into the bacterial expression vector pET-22b(+) with an in-frame C-terminal six-histidine tag. VH-M4 was directly PCR amplified from the pETCON2 plasmid used in the yeast alanine scanning experiments, whilst VH-S4 was amplified from the plasmid isolated through the affinity maturation selection. Both sequences were then cloned into pET-22b(+) as described earlier. Using VH-M4 (SEQ ID NO: 100) as a template sequence, the in-fusion mutagenesis kit (Takara) was used to generate the following mutants in the pET-22b(+) backbone (VH-1C5.sup.D104A/S108R and VH-1C5.sup.D104A/F120I). Each VH domain plasmid was separately transformed into E. coli BL21(DE3) cells and used to inoculate 10 mls of LB broth (containing 100 g/ml) started culture, which were incubated overnight before being used to seed 1000 ml of fresh LB broth. Bacterial cultures were grown at 37 C. and when they reached a OD600 of 0.6-0.8, the cells were induced with a final concentration 0.5 mM of IPTG and incubated overnight at 25 C. Cells were harvested by centrifugation at 17,000g for 10 min and pellets were resuspended in lysis buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 1 mM DTT) and then sonicated for 5 min. Bacterial supernatants were then filtered through 1.2 m syringe filters. Proteins were purified through a standard two-steps protocol: first, supernatant were loaded onto a 1 ml His Trap column (Cytiva Lifesciences), which was pre-equilibrated then extensively washed with buffer A (25 mM HEPES pH 7.5, 300 mM NaCl, 1 mM DTT) and then eluted with buffer A that also contained 500 mM imidazole; second, the eluted proteins were subjected to gel filtration chromatography on a Superdex 75 column (Cytiva Lifesciences) using PBS buffer containing 1 mM DTT. Protein fractions were analysed by SDS page gel and concentrated. Protein concentration was determined using absorbance at A280 nm.
[0228] Cells were then pelleted by centrifugation (14,000 g for 30 s at 4 C.), the supernatant aspirated and then washed with 1 ml ice-cold PBSA. Yeast were resuspended in 500 l PBSF containing Anti-HA Ab Alexa Fluor 488 (Invitrogen) and Streptavidin-phycoerythrin (ThermoFisher Scientific) and incubated for 30 mins. Cells were then pelleted at 14,000 g for 30 s at 4 C., aspirate supernatant and wash with 1 ml PBSF buffer. Each sample was then analysed by flow cytometry using Aria (Becton Dickinson) cytometer.
VH Domain Expression and Purification Assessment
[0229] VH domains were amplified and cloned into the pET-22b(+) vector (Novagen) using the in-Fusion cloning method (Takara Bio) as described earlier. These VH domains were expressed into E. coli BL21(DE3) cells. Cells were grown at 37 C. and induced protein expression overnight at 25 C. by 0.5 mM Isopropyl-B-D-thiogalactoside (IPTG). For assessment of clones, cells from 20 ml cultures was harvested, and lysed by sonication in lysis buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 1 mM DTT) supplemented with protease inhibitor cocktail. After centrifugation, the supernatant containing soluble proteins was loaded into Ni-NTA spin column (Qiagen). The column was washed twice using lysis buffer and then eluted with 25 mM HEPES at pH 7.5, 300 mM NaCl, 1 mM DTT and 500 mM imidazole. To assess protein solubility of different VH domains, the eluted proteins were analyzed by SDS-PAGE gel and stained with coomassie blue.
Bacterial Expression and Purification of VH-Domain Constructs
[0230] VH-Dif sequences were ordered as gene fragments from Integrated DNA Technologies (IDT). Coding sequences were PCR amplified and cloned directly into the bacterial expression vector pET-22b(+) with an in-frame C-terminal six-hisitidine tag using the BamHI/XhoI cloning site. Each VH-domain plasmid was separately transformed into E. coli BL21(DE3) cells and used to inoculate 10 ml of LB broth (containing 100 ug/ml) started culture, which were incubated overnight before being used to seed 1000 ml of fresh LB broth. Bacterial cultures were grown at 37 C. and when they reached a OD600 of 0.6-0.8, the cells were induced with a final concentration 0.5 mM IPTG and incubated overnight at 25 C. Cells were harvested by centrifugation at 5,000 rpm for 10 minutes and pellets were resuspended in lysis buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 1 mM DTT) and then sonicated for 5 minutes. Bacterial supernatants were then filtered through 1.2 m syringe filters. Proteins were purified through a standard two-step protocol: first, supernatant was loaded onto a pre-equilibrated 1 ml HisTrap column (Cytiva Lifesciences), which was then extensively washed with buffer A (25 mM HEPES pH 7.5, 300 mM NaCl, 1 mM DTT) and then eluted with an imidazole gradient; second, the eluted proteins were subjected to gel filtration chromatography on a Superdex 75 column (Cytiva Lifesciences) using PBS buffer containing 1 mM DTT. Protein fractions were analysed by SDS page gel and concentrated. Protein concentration was determined using absorbance at A280 nm.
Protein Crystallization
[0231] The eIF4E:EE-02 and eIF4E:VH-DiF.sup.CAP-01 complexes were crystallized by vapour diffusion using the hanging drop method. For crystallization, the eIF4E:EE-02 complex was prepared by direct addition of a 100 mM DMSO stock solution of EE-02 to purified eIF4E recombinant protein (dialysed in 10 mM HEPES 7.6, 100 mM KCL buffer) to generate a final solution of 200 M eIF4E and 300 M EE-02 with a residual DMSO concentration of 0.3% (v/v). The sample was then spun down using a tabletop centrifuge at 13,000 g after overnight incubation at 4 C. and the supernatant used for crystallization. The eIF4E:VH-DiF.sup.CAP-01 complex solution for crystallization was prepared by dialysing both proteins into 10 mM HEPES 7.6, 100 mM KCL and 1 mM DTT buffer and mixing them to give final respective concentrations of 100 M and 200 M. Hanging drops were set-up in a pre-greased VDX48 plate (Hampton, USA) with 1 l of the respective crystallization sample mixed with 1 l of the mother-well solution. eIF4E:EE-02 crystals grew over a period of one week in 0.2 M Potassium chloride, 20% (w/v) PEG 3350. eIF4E:VH-Dif.sup.CAP-01 crystals grew over a similar period of time but in 0.1 M TRIS.HCl PH 8.5, 25% (v/v) PEG 550 MME. For X-ray data collection at 100 K, crystals for both sets of crystallization conditions were transferred to an equivalent mother liquor solution containing 25% (v/v) glycerol and then flash frozen in liquid nitrogen.
Data Collection and Refinement
[0232] X-ray diffraction data was collected at the Australian synchrotron (MX1 beamline) using a CCD detector, and integrated and scaled using XDS. The initial phases of the EE02 complexed crystal of eIF4E were solved by molecular replacement with the program PHASER8 using the human eIF4E structure (PDB accession code: 4BEA) as a search model. With respect to the eIF4E:VH-DiF.sup.CAP-01 co-crystal the VH domain structure (PDB accession code: 5TDP, chain B) was also included in the PHASER molecular replacement search as an independent search model. The starting models were subjected to rigid body refinement and followed by iterative cycles of manual model building in Coot and restrained refinement in Refmac 6.0.9 Models were validated using PROCHECK10 and the MOLPROBITY webserver.11 Final models were analysed using PYMOL (Schrdinger). See table 1 for data collection and refinement statistics. The eIF4E complex structures with EE-02 and eIF4E VH-DIF.sup.CAP-01 have been deposited in the PDB under the submission codes 7EZW and 7F07, respectively.
TABLE-US-00005 TABLE 1 Crystallographic data collection and refinement statistics. Highest resolution bin data stated in parentheses. PDB ID: 7EZW 7F07 Resolution () 48.148-2.35, (2.47-2.35) 40.996-2.25, (2.37-2.25) Space Group P6.sub.3 P2.sub.12.sub.12.sub.1 Unit Cell a = 81.14, b = 81.4, c = 66.11, a = 38.52, b = 81.82, c = 122.99, Dimensions () = = 90, = 120 = = = 90 Temp (K) 100 100 Redundancy 11.3, (11.2) 13.4, (14.0) Unique Collected 10511, (1027) 19,226, (2734) Reflections Completeness (%) 99.9, (99.2) 100, (100) R Merge (%) 0.114, (0.711) 0.120, (0.755) I/sigma 13.8, (2.6) 4.7, (1.0) R factor (%) 20.53 22.19 R free (%) 24.76 27.18 RMS Bonds () 0.0023 0.0028 RMS Angles () 1.143 1.216 Wilson B-factor (.sup.2) 30.5 37.6 Average Refined B Factors Chain A 35.29, (eIF4E) 48.34, (eIF4E) Chain B 41.85, (EE-02) 56.6, (VH-DiF.sup.CAP-01) Waters 28.67 46.46, Number of Water 59 48 Molecules Ramachandran Data (Rampage). Number of Residues in (%): Favoured Region 96.7 97.8 Allowed Region 3.3 2.2 Outlier Region 0
Isothermal Titration Calorimetry (ITC)
[0233] ITC measurements were performed with the Affinity ITC (TA Instruments, USA) at 25 C.
[0234] For purified eIF4E, the purified proteins were buffer exchanged into 1PBS, pH 7.2 with 0.001% Tween-20 using 7K MWCO Zeba spin desalting column (ThermoFisher scientific). 10-30 M of eIF4E protein was loaded into the sample cell, and 100-300 M of VH domains were titrated into eIF4E protein, over 15-20 injections of 2.5 L. All experiments were conducted in duplicate. calorimetric data were analysed with NanoAnalyze software using a one-site binding model.
[0235] For purified eIF4A, 10 or 20 M of eIF4A was loaded in the cell with 100 or 200 M of the relevant peptide aptamer in the titrating syringe, depending on the binding affinities of compounds. eIF4A was dialysed into Phosphate Buffered Saline (2.7 mM KCL and 137 mM NaCL, pH 7.4) with 0.05% TWEEN20 using SLIDE-A-LYZER (Pierce) cassettes with a MWCO of 3000. Stock VH domain solutions were dialysed side by side using a different cassette in the same buffer as eIF4A. These were then diluted to their working concentration using the dialysis buffer. The titration experiments were performed at 25 C. with a series of 2.5 l injections (usually 20-30 injections). The spacing between each injection was 300s. The stirring speed during the titration was 75 rpm. Data was analyzed using NanoANALYZE software by fitting to a single-site binding model.
[0236] Correction for the enthalpy of ligand dilution was carried out by subtracting a linear fit from the last three data points of the titration, after the interaction had reached saturation.
eIF4A Surface Plasmon Resonance (SPR) Assays
[0237] For eIF4A immobilisation in the SPR binding assays, the N-terminally sortase biotinylated eIF4A was immobilized on a streptavidin coated CM5 sensor chip. eIF4A at a concentration of 0.5 M was injected across the chip until approximately 100 RU was immobilised. Streptavidin in the reference channel was blocked with free biotin. Six buffer blanks were first injected to equilibrate the instrument fully. Surface Plasmon resonance experiments were performed on a Biacore T100 machine. Stock protein solutions were serially diluted into running buffer immediately prior to analysis. Running buffer consisted of 10 mM Hepes pH 7.6, 0.15 M NaCl, 1 mM DTT and 0.1% Tween20.
[0238] For SPR multi-cycle injection experiments, these injections were performed using a flow rate of 50 l/min. Peptide aptamers were injected for 60 s and dissociation was monitored for 180 s. Individual proteins were injected across streptavidin coated CM5 chips in threefold dilution series using as appropriate concentration range to determine their respective binding constants. Each independent protein injection sampled one concentration only and was immediately followed by a similar injection of SPR buffer to enable the chip surface to be fully regenerated by dissociation. Responses from the target protein surface were transformed by: (i) subtracting the responses obtained from the reference surface that contained no immobilised protein, and (ii) subtracting the responses of the buffer injections from those of the peptide aptamer injections. The last step is known as double referencing, which corrects the systematic artefacts. K.sub.ds were determined using the BiaEvaluation software (Biacore) and calculated from both the response of the eIF4A coated streptavidin CM5 chips at equilibrium and kinetically from the dissociation and association phase data for each of the peptides. Both the equilibrium and kinetic data were fitted to 1:1 binding model. Within each titration, at least two concentration points were duplicated to ensure stability and robustness of the chip surface. Data analysis was performed with Biacore T100 evaluation software (v2.0.4).
Molecular Dynamics Simulations
[0239] The X-ray resolved VH-DIF.sup.CAP-01: eIF4E and EE-02: eIF4E complex state structures, along with the free VH-DIF.sup.CAP-01 domain and EE-02 cyclic peptide derived from the respective complexes were subjected to molecular dynamics simulations in AMBER 1812 using all-atom ff14SB.sup.13 force field parameters. The N-termini of eIF4E and VH-DIF.sup.CAP-01 were capped with the ACE functional group, while the C-termini of VH-DIF.sup.CAP-01 and EE-02 were capped with NME and NHE functional groups respectively. The disulphide bond between residues C2 and C10 in the EE-02 peptide was maintained using the bond command in the tleap module of AMBER 18. All the water molecules resolved in the crystal structures were retained for the simulations. The four systems (VH-DIF.sup.CAP-01: eIF4E, EE-02:eIF4E, free VH-DIF.sup.CAP-01 and free EE-02) were placed inside a truncated octahedral box and solvated with TIP3P.sup.14 water by setting a minimum distance of at-least 8 between any solute atom and the edge of the box. The electroneutrality of the respective systems was achieved by adding appropriate number of counterions. These systems were then energy minimized using steepest descent and conjugate gradient algorithms, heated to a temperature of 300 K in the NVT ensemble and equilibrated for 500 ps in the NPT ensemble with 1 atm pressure. Production dynamics for VH-DIF.sup.CAP-01: eIF4E and EE-02: eIF4E complexes were carried out in triplicates for 200 ns each (cumulative simulation time of 1.2 s) starting with different initial velocities, while that for free VH-DIF.sup.CAP-01 domain and EE-02 cyclic peptide was run for 1 us each. All the simulations in the production stage were carried out under NPT conditions. Electrostatic calculations, regulation of temperature and pressure along with the constraining of bonds to hydrogen atoms during the simulations were employed as previously described. The simulation temperature of 300 K was set using langevin dynamics, with a collision frequency of 1.0 ps.sup.1 and the pressure was maintained at 1 atm using weak-coupling with a pressure relaxation time of 1 ps. Periodic boundary conditions in x, y and z directions were appropriately applied. Particle Mesh Ewald method (PME) was used for treating the long range electrostatic interaction. All bonds involving hydrogen atoms were constrained using the SHAKE algorithm. A time step of 2 fs was used and the coordinates were saved every 1 ps. The first 20 ns of the production run were discarded in the analysis of the trajectories to reduce the biasness caused by the similarity in the starting structures of the different systems.
Analysis of Binding Energy and Water Occupancy During the Molecular Dynamics Simulations
[0240] Residue-wise decomposition analysis was carried out using the MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) 16 scheme through the MMPBSA.py script available in AMBER 18. The 3 simulated trajectories of each complex, ie VH-DIF.sup.CAP-01: eIF4E and EE-2: eIF4E, were concatenated (200 ns per trajectory) into a composite trajectory of 600 ns and from this, 1000 snapshots at equal intervals, were extracted. Water molecules and counterions were removed from these structures and solvation effects were estimated using the implicit Generalized Born Solvation Model (IGB=2) with salt concentration set to 150 mM. The water occupancy map for VH-DIF.sup.CAP-01: eIF4E and free VH-DIF.sup.CAP-01 simulations was generated using the grid command in the CPPTRAJ module of AMBER 18 with cubic grid cells of size 0.5 . The water density within each grid cell was computed and plotted using the volume viewer menu as an isosurface representation in the UCSF Chimera visualization software.
Cell Biology
Plasmid and Reagents
[0241] All plasmids were purchased from Addgene unless indicated otherwise. Mutant VH domains were generated with In-fusion mutagenesis kit (Clontech) and then cloned into either a pCDNA3.1 vector (Thermo Fisher Scientific) harbouring a C-terminal 3 FLAG tag via NheI/BamHI sites or into NanoBit plasmids using the NanoBit PPI starter system (Promega, see manufacturer instruction) to allow mammalian cell expression studies. For lentivirus production, the VH-3FLAG cassette was sub-cloned into the lentiviral expression vector pCW57 via BamHI/AvrII cloning sites. pcDNA3-rLuc-polIRES-fLuc (bicistronic reporter) ret, eIF4E and eIF4G604-646 NanoBIT and 4EBP1.sup.4ALA mutant plasmid generation has been described previously. eIF4E, eIF4G and 4EBP1 mutant cDNAs were synthetized and obtained from IDT (Integrated DNA Technologies). eIF4E and eIF4G604-646 were cloned into NanoBit plasmids using the NanoBit PPI starter system (Promega) using XhoI/EcoRI and NheI/EcoRI cloning sites respectively. 4EBP1 mutants were cloned into pCDNA3.1 vector DNA (Thermo Fisher Scientific) harbouring a C-terminal 3 FLAG tag via NheI/BamHI sites to allow mammalian cell overexpression. For bacterial expression, 4EBP1 mutants were cloned into pGEX6P1 using BamHI/EagI cloning sites. GFP and v-Myc coding sequence residing in a pCMV6 mammalian expression vector were obtained from Origene. The bicistronic luciferase reporter construct pcDNA3-rLuc-polIRES-fLuc was purchased from Addgene.
Cell Culturing Conditions
[0242] All cell lines were cultured in DMEM cell media supplemented with 10% foetal calf serum (FBS) and penicillin/streptomycin. Mammalian cells were maintained in a 37 C. humidified incubator with 5% CO.sub.2 atmosphere.
Immunoprecipitation and m.sup.7GTP Pull Down Experiments
[0243] Twenty-four hours prior to transfection or drugging, cells were seeded at a cell density of 1000.000 (HEK293) cells per well of a six-well plate (ThermoFisher Scientific). Transfections were performed using Lipofectamine 3000 (ThermoFisher Scientific) with either 2 g or the indicated amount of plasmid vectors per well according to the manufacturer's instructions. After a 48-hour incubation period, the cell media was then removed and the cells washed with PBS saline. Cells were directly lysed in the wells with 300 l of lysis buffer containing 20 mM Hepes pH 7.4, 100 mM NaCl, 5 mM MgCl.sub.2, 0.5% NP-40, 1 mM DTT, with protease (Roche) and phosphatase (Sigma-Aldrich) inhibitor cocktail sets added as outlined by the manufacturer's protocols. Cellular debris was removed by centrifugation, and the protein concentration was then determined using the BCA system (Pierce). m.sup.7GTP pulldown and FLAG immunoprecipitation experiments were performed with 200 g of cell lysate, which was either incubated with 20 l of m.sup.7GTP (Jena Bioscience) or anti-FLAG M2 antibody (Roche) immobilised agarose beads for 2-4 hours at 4 C. on a rotator. Beads were then washed four times with lysis buffer containing no protease or phosphatase inhibitors. This was then followed by the addition of Laemlee buffer (2) and the beads boiled for 5 min at 95 C. Samples were centrifuged and the supernatant removed for western blot analysis.
NanoBit Complementation Assay
[0244] For NanoBIT (PROMEGA) system development and validation, opaque 96-well plates were seeded with 30,000 HEK293 cells per well in DMEM and 10% FCS and transfected with 30 ng total DNA of the two NanoBit plasmid vectors and 100 g of the indicated plasmid per well using FUGENE6 (Roche). 48 hours after transfection, the medium was replaced with 100 l of Opti-MEM cell media containing 0% FCS with no added red phenol (Thermo Fisher Scientific). To screen indicated compounds using the NanoBIT system in live and permeabilized cells, 6-well plate was seeded with 1300,000 cells per well in DMEM and 10% FCS and transfected with 2 ug total DNA of the two NanoBit plasmid per well. After 24 hrs, transfected cells were trypsinised and re-suspended in Opti-MEM media with 10% FCS. Cells were then spun down at 1000 rpm for 5 minutes at room temperature. Supernatant was then discarded and cells re-suspended to a density of to 220,000 cells per ml in Opti-MEM I reduced serum containing 10% FCS with no added red phenol. 100 l of cells were added to the wells of a white opaque 96-well plate and incubated for 24 hours at 37 C., 5% CO2. For assessment of permeabilised or live cells, cell medium was replaced with 90 ul of serum free Opti-MEM media that either contained or did not contain 50 g/ml digitonin, respectively. Live or permeabilized cells were then treated with either 10 l of a 10% v/v DMSO vehicle control in FPLC grade water or a suitable 2-fold dilution series of the compound under study in a 10-fold higher stock concentration (containing 10% DMSO and FPLC grade water solution). 96 wells were then incubated for 3 hrs at 37 C., 5% CO2. Luminescence activity was assayed as described elsewhere.sup.18 using an Envision Multi-Plate reader.
Cap-Dependent Translation Assay
[0245] Opaque 96-well plates were seeded with 30,000 HEK293 cells per well in DMEM and 10% FCS. Transfections were performed using FUGENE6 (Roche) with 30 ng of the bicistronic reporter (pcDNA3-rLuc-polIRES-fLuc) plasmid and 75 or 150 ng of the indicated plasmid. 48 hours after transfection, Renilla and firefly luminescence activity was determined using the Dual Glo Luciferase Assay System (PROMEGA). Luminescence readings were performed using an Envision Multi-plate reader (PerkinElmer).
Protein Expression Analysis
[0246] Transfected HEK293 cells (prepared as described in the NanoBit and Cap-dependent translation Experiments sections) were seeded with 30,000 cells per well in 96-well plates. After an incubation period indicated in the relevant figure, cells were washed with PBS and directly lysed in the wells of the plate with 50 l of cell lysis buffer (20 mM Hepes pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM dithiothreitol) containing the protease (Roche) and phosphatase (Sigma-Aldrich) inhibitor cocktail sets (added as outlined by the manufacturer's protocols). Cellular debris was separated by centrifugation. Samples were analysed by western blot without further quantification.
Generation of Stably Transfected A375 Cell Lines Expressing PCNA Interacting Peptide Aptamers
[0247] Confluent HEK293FT cells were used to generate lentivirus for infection of target cells. Packaging cells were transfected using calcium phosphate transfection as described below. 6 g of pCW57 plasmid (Addgene, USA) harbouring either 4EBP1.sup.4ALA or VH-S4 or no insert were co-transfected into HEK293T cells with plasmids encoding pLVSVG (viral envelope), pLP1 (gag-pol) and pLP2 (rev), in a ratio of 2:1:2:2 to generate viral particles. The conditioned medium harbouring viral particles from the transfected HEK293T cells was filtered 48 hours following co-transfection and viral particles were concentrated by ultracentrifugation. A375 cells were seeded in 12-well plates and infected with viral particles over a 12 hour period prior to cell media replacement with fresh medium 72 hours post infection. A375 cells were supplemented with 800 g/ml of geneticin and selections for stably transfected cells were carried out for 2 weeks, replacing the antibiotic-containing media every 3 days. Polyclonal geneticin-resistant pools of cells were then obtained. These were then incubated with 1 g/ml of doxycycline for 24 hours, where upon GFP positive single clones were isolated by FACs into 96-well plates. Monoclonal stable cell lines were verified using western blot and then expanded for subsequent analysis.
Western Blot Analysis
[0248] Samples were resolved on midi or mini Tris-Glycine 4-20% gradient gels (Bio-Rad) according to the manufacturer's protocol. Western transfer was performed with an Immuno-blot PVDF or nitrocellulose membrane (Bio-Rad) using a Trans-Blot Turbo system (Bio-Rad). Western blots were then performed. Antibodies against peIF4E.sup.S209,4EBP1, cyclin D1 and FLAG were purchased from Abcam or Sigma, respectively. All other antibodies used were purchased from Cell Signalling Technology. B-actin levels were measured to ensure equal loading.
Cell Proliferation Assay
[0249] A375 cell lines were plated in 96-well clear bottom plates at a density of 4000 cells per well in 200 l DMEM and 10% FCS medium. After 24 hours, cell media was replaced with 200 l of medium containing doxycycline at 1 g/ml. Cell confluence and cell growth was then measured continuously over 7 days using an IncuCyte FLR instrument (EssenBioscience).
Results
Example 1
Discovery of a Novel eIF4E Cap-Binding Cyclic Peptide Binding Motif
[0250] Two M13 phage peptide libraries (New England Biolabs) either consisting of 7 randomised amino acids constrained by a disulphide bond formed between 2 cysteine residues (ACXXXXXXXC, X=any amino) or a hypervariable linear 12mer sequence were panned against biotinylated eIF4E. Parallel selections were performed concurrently against several negative control biotinylated proteins also. The panning culminated in single round to avoid selection of fast-growing phage clones. Recovered phage populations were then subjected to NGS (Next Generation Sequencing, Illumina NextSEQ technology) sequencing, whereupon differential enrichment analysis was performed to identify peptide sequences that specifically bound eIF4E over the control proteins (
[0251] In the absence of any sequence similarity with described eIF4E interacting proteins (eIF4G1 and the 4E-BP family) and the eIF4E interaction motif (enriched in the 12mer selection), competitive fluorescence anisotropy experiments were performed to delineate the binding sites of the EE-01 to EE-09 peptides on eIF4E utilising either a FAM labelled m.sup.7GTP (m.sup.7GTP.sup.FAM) or a FAM labelled eIF4G/4E-BP1 site interacting peptide (eIF4G.sup.FAM) (
[0252] Tryptophan quenching experiments were performed to compare the binding mechanisms of EE-02 with m.sup.7GTP and PHAGESOL (a phage modified eIF4E interacting peptide that binds at the eIF4G binding site) against eIF4E. It is well established that the binding of m.sup.7GTP to eIF4E results in significant tryptophan fluorescent quenching of W56 and W102 (
TABLE-US-00006 TABLE2 BindingassessmentofdisulphideconstrainedpeptidesisolatedusingM13phage displayagainsteIF4AE(EE-02toEE-09)andalaninescanningmutantsexploring theinteractionprofileofEE-02undernon-reducingconditions.Thebindingsitesof thepeptidesEE-01toEE-09weremappedontoeIF4AEusingtwocompetitivebased fluorescenceanisotropyassays,oneofwhichusedaFAMlabelledm.sup.7GTP(m.sup.7GTP.sup.FAM)to monitorforbindingatthecap-bindingsite,whilsttheotherassayusedaFAMlabelled canonicalsiteinteractingpeptide(eIF4AG.sup.FAM)tomeasurebindingattheeIF4AGinteraction site.Dissociationconstantsweredeterminedusinga1:1bindingmodelandaredescribed inthematerialsandmethods.m.sup.7GTP,m.sup.7GDPandm.sup.7GTP.sup.BIOTINwereusedaspositive controlsforthem.sup.7GTP.sup.FAMcompetitionassay,whilstPHAGESOLwasusedasapositive controlfortheeIF4AG.sup.FAMcompetitionassay.EE-02alaninemutantderivativeswereonly assessedforbindinginthem.sup.7GTP.sup.FAMcompetitionassayND=Notdetermined.K.sub.ds> 20,000weredenotedasnon-binders.Experimentswereperformedintriplicate. CompetitiveAnisotropy(k.sub.d,nM) Sequence m.sup.7GTP.sup.FAM eIF4AG.sup.FAM m.sup.7GDP 200.725.2 NoBinding m.sup.7GTP 66.67.2 NoBinding m.sup.7GTP.sup.BIOTIN 34.74.0 NoBinding EE-01(SEQID NH.sub.2-ACETGFFTGCG- 15,030.03,078.0 NoBinding NO:64) NH.sub.2 EE-02(SEQID NH.sub.2-ACEMGFFQDCG- 406.23.6 NoBinding NO:65) NH.sub.2 EE-03(SEQID NH.sub.2-ACELGYYNDCG- NoBinding NoBinding NO:66) NH.sub.2 EE-04(SEQID NH.sub.2-ACETGFFLKCG- NoBinding NoBinding NO:67) NH.sub.2 EE-05(SEQID NH.sub.2-ACELGFYRLCG- NoBinding NoBinding NO:68) NH.sub.2 EE-06(SEQID NH.sub.2-ACETGFFLRCG- NoBinding NoBinding NO:69) NH.sub.2 EE-07(SEQID NH.sub.2-ACETGYFSQCG- NoBinding NoBinding NO:70) NH.sub.2 EE-08(SEQID NH.sub.2-ACETGFYKTCG- 16,7103,027.0 NoBinding NO:72) NH.sub.2 EE-09(SEQID NH.sub.2-ACEMGYFGNCG- 4,860633.4 NoBinding NO:73) NH.sub.2 PHAGESOL Ac-KKRYSR*QLL*-NH.sub.2 NoBinding 34.056.42 EE-02.sup.E3A(SEQ NH.sub.2-ACAMGFFQDCG- NoBinding ND IDNO:74) NH.sub.2 EE-02.sup.M4A(SEQ NH.sub.2-ACEAGFFQDCG- NoBinding ND IDNO:75) NH.sub.2 EE-02.sup.G5A(SEQ NH.sub.2-ACEMAFFQDCG- NoBinding ND IDNO:76) NH.sub.2 EE-02.sup.F6A(SEQ NH.sub.2-ACEMGAFQDCG- NoBinding ND IDNO:77) NH.sub.2 EE-02.sup.F7A(SEQ NH.sub.2-ACEMGFAQDCG- NoBinding ND IDNO:78) NH.sub.2 EE-02.sup.98A(SEQ NH.sub.2-ACEMGFFADCG- 384.962.1 ND IDNO:79) NH.sub.2 EE-02.sup.D9A(SEQ NH.sub.2-ACEMGFFQACG- 1,682537.4 ND IDNO:80) NH.sub.2 EE-02.sup.N-Del(SEQ NH.sub.2-CEMGFFQDCG- 66.519.5 ND IDNO:81) NH.sub.2 EE-02.sup.C-Del(SEQ NH.sub.2-ACEMGFFQDC- 684.854.8 ND IDNO:82) NH.sub.2 EE-02.sup.NC-Del(SEQ NH.sub.2-CEMGFFQDC- 525.9128.8 ND IDNO:83) NH.sub.2
the Constrained Macrocyclic Peptide EE-02 Interacts with eIF4E Cap-Binding Site Via a Unique Binding Pose
[0253] The structure of the EE-02 complex was solved using X-ray crystallography confirming EE-02 bound to eIF4E at the cap-binding site (
[0254] The EE-02 peptide forms a -hairpin turn-like structure in the binding pocket that allows the side chains of the constrained peptide motif to efficiently interact with eIF4E (
Design and Development of a Novel Miniprotein that Interacts with the Cap-Binding Site of eIF4E
[0255] The EE-02 binding epitope was grafted into the CDR3 loop region of an engineered monomeric VH-domain, termed DiF-VH. The DiF-VH scaffold has several attractive features: 1) relatively large peptide insertions can be made into the CDR3 loop region and 2) the protein scaffold is amenable to expression in E. Coli and mammalian cells. Additionally, the points where the CDR3 loop initiates and terminates itself in the VH domain are spatially close together, suggesting that the protein scaffold can act as structural constraint that mimics the function of the disulphide bond in the cyclic peptide (
[0256] Initial designs where the cyclic eIF4E interaction motif (EMGFF) was introduced at different positions within a 15mer loop in the CDR3 region of the VH-DiF scaffold were tested and none exhibited any binding to eIF4E (
TABLE-US-00007 TABLE3 Dissociationconstantsweredeterminedusingboththem.sup.7GTP.sup.FAMcompetitive anisotropyassayandisothermalcalorimetry(ITC)forselectedconstrained peptidesandpeptideaptamers.ITCwasalsousedtodeterminethefollowing: enthalpyofbinding(H),entropyof(S)bindingandthestoichiometryof theinteraction(N,numberofbindingsites).Experimentswerecarriedout at293K.Experimentswereperformedintriplicate.ND=Notdetermined. Competitive Anisotropy ITC (m.sup.7GTP.sup.FAM, H S Sequence k.sub.d,nM) K.sub.d(nM) (KJ/Mol) (J/mol-K) N EE-02 NH.sub.2-ACEMGFFQDCG-NH.sub.2 406.2 63.9 25.5 16.4 1.3 (SEQ 3.6 18.8 2.1 3.1 0.1 IDNO: 75) EE-02.sup.M4A NH.sub.2-ACEAGFFQDCG-NH.sub.2 NoBinding ND ND ND ND (SEQ IDNO: 65) VH- MHPSAICEMGFFQDC---- NoBinding 2152.5 7.5 83.4 0.99 DiF.sup.CAP_05 184.5 1.4 5.4 0.1 (SEQ IDNO: 8) VH- ----MVPEMGFFEPGLPSP 3,630 421.9 47.1 35.6 1.0 DiF.sup.CAP_09 300 82.6 2.6 10.0 0.1 (SEQ IDNO: 12) VH- ---PLPEMGFFTNIPAMV 501.0 36.3 31.0 39.5 1.0 DiF.sup.CAP_01 58.0 17.0 1.7 10.0 0.1 (SEQ IDNO: 4) VH- --PLYAPEMGFFHVHHL-- NoBinding ND ND ND ND DiF.sup.CAP_02 (SEQ IDNO: 5) VH- ----PLPEAGFFTNIPAMV NoBinding ND ND ND ND DiF.sup.CAP_ 01.sup.Cntrl
VH-DIF.sup.CAP-01 Recapitulates the Interactions of EE-02 with the Cap-Binding Site and Forms Additional Interactions
[0257] Crystallization of the VH-DIF.sup.CAP-01:eIF4E complex (
[0258] The evolved linker regions of VH-DIF.sup.CAP-01, in addition to presenting the EE-02 interaction epitope optimally to interact with eIF4E, also forms multiple additional interactions with eIF4E (
the Conformation of the CDR3 Interaction Loop is Stabilized by an Intricate Set of Interactions
[0259] The strategy of evolving the peptide sequence regions at either side of the cap binding interaction motif to enable optimal presentation of the epitope must also inherently accommodate the presence of residues that occur on the VH domain itself. This is difficult to achieve using the rational design approaches. Several significant features in the linker region of the CDR3 loop that stabilized presentation of the CDR3 loop through packing interactions with the scaffold were noted: 1) the Ile110 sidechain in the linker region forms a hydrophobic core in the CDR3 loop structure that makes multiple hydrophobic contacts with other residues in the linker and the interaction motif, 2) the CDR3 loop when bound to eIF4E forms a folded structure against the R51 and D36 residue of VH-DIF.sup.CAP-01 which form a salt bridge between each other, 3) and that the linker residue T108 also packs directly against W48, which is found in the scaffold (
[0260] However, the most remarkable feature is the presence of two buried structure water molecules that allow the R51: D36 salt bridge to stabilize the fold of the CDR3 loop, when bound to eIF4E (
VH-DIF.sup.CAP-01 Inhibits eiF4F Mediated Cap Dependent Translation by Disrupting the Interplay Between eIF4E and Capped-mRNA
[0261] As predicted from in vitro studies, FLAG-tagged VH-DiF.sup.CAP-01 immuno-precipitated cellular eIF4E more efficiently than VH-DIF.sup.CAP-02 (
Development of a Novel Live Cell Protein-Protein Interaction (PPI) Assay to Measure Antagonism of the eIF4E Cap-Binding Site.
[0262] Currently, there is no live cell-based assay that can evaluate engagement of the cap-binding site by small molecules or other modalities. A site that has been the target of multiple studies to develop cell permeable small molecules for therapeutic development. Fortunately, there is a plethora of suitable technologies that can be used to develop an appropriate assay e.g., split luciferase, BRET and FRET (bio and fluorescence resonance excitation transfer), and cellular localization technologies. The VH-DIF.sup.CAP-01 peptide aptamer in combination with the NanoLuc-based protein complementation system (NanoBit, PCA, Promega) was exploited to develop a PPI assay that can assess antagonism of the m.sup.7GTP cap-binding site in eIF4E in cells. The NanoLuc complementation protein system consists of two components termed LgBiT (18-kDa protein fragment) and SmBiT (11-amino-acid peptide fragment), which have been optimised for minimal self-association and stability. When LgBiT and SmBiT are optimally fused to two interacting proteins, both the fused proteins will be brought into proximity to each other by the resulting interaction, resulting in the formation of the active luciferase.
[0263] The LgBiT-eIF4E and SmBiT-VH-DIF.sup.CAP-01 were identified as the transfection pair that reconstituted the highest luciferase signal without exhibiting background activity in the negative controls, thus confirming that the NanoBit reporter fragments were not spontaneously assembling under the experimental conditions (
[0264] To validate that the NanoBIT eIF4ECAP system can measure small molecule mediated inhibition of the eIF4E cap-binding site, the system was also used to screen two known cap-analogue antagonists (m.sup.7GTP and m.sup.7GDP) and an established cell permeable inhibitor of the eIF4E:4G interface (4EGI1) as a negative control. Unfortunately, both cap-analogue molecules are cell impermeable. Therefore, to circumvent this issue, a sub-CMC (critical micelle concentration) treatment of digitonin was used to permeabilize and enable cellular entry of the cap-analogues into HEK293 cells transfected with the NanoBIT.sup.CAP system. In the permeabilized cells, both m.sup.7GTP and M7GDP disrupted the interaction of LgBiT-eIF4E with SmBiT-VH-DIF.sup.CAP-01 with IC50s of 12.8 M and 34.5 M respectively, whilst 4EGI1 had a negligible effect on the NanoBIT signal, demonstrating that the assay system can measure cap-binding site antagonists (
the Peptide Aptamers 4AM3, 4AM14 and 4AM20 Interact with eIF4a.
[0265] The interaction between the peptide aptamers (4AM3 (SEQ ID NO: 29), 4AM14 (SEQ ID NO: 39) and 4AM20 (SEQ ID NO: 61)) was measured by Surface Plasmon Resonance in PBST running buffer and PBST with 1 mM DTT. Amongst the three peptide aptamers, 4AM3 exhibited the highest K.sub.d of 430 nM in PBS running buffer only (
TABLE-US-00008 TABLE 4 Dissociation constants were determined by Surface Plasmon Resonance in PBST running buffer with and without 1 mM DTT. Dissociation Constant (K.sub.d) PBST Running PBST with Solutions Buffer 1 mM DTT 4AM3 (SEQ ID NO: 29) 430 nM 300 nM 4AM14 (SEQ ID NO: 39) 59 nM 89 nM 4AM20 (SEQ ID NO: 61) 17.5 nM 838 nM
the Peptide Aptamers 4AM14 and 4AM20 Bind to eIF4a.
[0266] The binding of the peptide aptamers 4AM14 and 4AM20 to eIF4A is measured by Isothermal Titration calorimetry (ITC). 4AM14 displayed the K.sub.d of approximately 70 nM without TCEP and approximately 92 nM in the presence of TCEP (
the Peptide Aptamers VH-D1 and VH-31 Inhibit Cell Proliferation by Binding to PCNA.
[0267] To investigate whether the peptide aptamers VH-D1 and VH-31 interact with PCNA, an anti-FLAG immunoprecipitation pull down assay was performed.
[0268] Furthermore, the effect of these peptide aptamers on the confluency of the cells was investigated. Induction of PCNA interacting aptamers (VH-D1 and VH-31) but not their controls reduces the proliferation of human melanoma cancer cells (A375 cells) (
Conclusion
[0269] The present invention shows the development of new methods to engineer and identify peptide aptamers without disulphide bonds that display improved cell permeability and stability whilst retaining high binding affinity to the target protein and peptide aptamers with disulphide bonds which are useful in diagnostic and purification applications. These peptide aptamers are compatible with RNA and DNA delivery technology. These results demonstrate that the PELE process can enable the development of mini-proteins that interact at desired target interaction sites, that can model and assess the potential effects of drug inhibition and allow the construction of critical target engagement assays that can accelerate the identification of lead compounds for drug development.
TABLE-US-00009 TABLE4 Summaryofsequencelisting. Description Polypeptidesequence Aminoacidsequencetemplate1ofpeptide XXXXXEMGFFXXXXX aptamer(eIF4AE)(SEQIDNO:1) Aminoacidsequencetemplate2ofpeptide XXXEMGFFXXXXXXX aptamer(eIF4AE)(SEQIDNO:2) Aminoacidsequencetemplate3ofpeptide XXXXXXXEMGFFXXX aptamer(eIF4AE)(SEQIDNO:3) Aminoacidsequenceofpeptideaptamer1 PLPEMGFFTNIPAMV (eIF4AE)(VH-DiF.sup.CAP-01)(SEQIDNO:4) Aminoacidsequenceofpeptideaptamer2 PLYAPEMGFFHVHHL (eIF4AE)(VH-DiF.sup.CAP-02)(SEQIDNO:5) Aminoacidsequenceofpeptideaptamer3 VNYLPEMGFFHTHSL (eIF4AE)(VH-DiF.sup.CAP-03)(SEQIDNO:6) Aminoacidsequenceofpeptideaptamer4 FASEMGFFLVMTMGF (eIF4AE)(VH-DiF.sup.CAP-04)(SEQIDNO:7) Aminoacidsequenceofpeptideaptamer5 MHPSAICEMGFFQDC (eIF4AE)(VH-DiF.sup.CAP-05)(SEQIDNO:8) Aminoacidsequenceofpeptideaptamer6 PGSEMGFFSCRSLPL (eIF4AE)(VH-DiF.sup.CAP-06)(SEQIDNO:9) Aminoacidsequenceofpeptideaptamer7 RLLPPEMGFFKPYWW (eIF4AE)(VH-DiF.sup.CAP-07)(SEQIDNO:10) Aminoacidsequenceofpeptideaptamer8 PGSEMGFFACLWFYS (eIF4AE)(VH-DiF.sup.CAP-08)(SEQIDNO:11) Aminoacidsequenceofpeptideaptamer9 MVPEMGFFEPGLPSP (eIF4AE)(VH-DiF.sup.CAP-09)(SEQIDNO:12) Aminoacidsequenceofpeptideaptamer10 SEPEMGFFVLCAILQ (eIF4AE)(VH-DiF.sup.CAP-10)(SEQIDNO:13) Aminoacidsequencetemplateofpeptide XXXXXXXYPMFXXX aptamer(PCNA)(SEQIDNO:14) Aminoacidsequenceofpeptideaptamer1 SPGTHPKYPMFHLR (PCNA)(VH-1)(SEQIDNO:15) Aminoacidsequenceofpeptideaptamer2 SQVNPPVYPMFHLH (PCNA)(VH-11)(SEQIDNO:16) Aminoacidsequenceofpeptideaptamer3 SYSRVPLYPMFVNR (PCNA)(VH-18)(SEQIDNO:17) Aminoacidsequenceofpeptideaptamer4 SYRSDLVYPMFIRW (PCNA)(VH-24)(SEQIDNO:18) Aminoacidsequenceofpeptideaptamer5 SHTRHPVYPMFHPR (PCNA)(VH-31)(SEQIDNO:19) Aminoacidsequenceofpeptideaptamer6 SQSPTIFYPMFSLR (PCNA)(VH-34)(SEQIDNO:20) Aminoacidsequenceofpeptideaptamer7 SQSERPVYPMFHLR (PCNA)(VH-D1)(SEQIDNO:21) Aminoacidsequenceofpeptideaptamer8 SRSERPVYPMRFHLR (PCNA)(VH-D2)(SEQIDNO:22) Aminoacidsequenceofpeptideaptamer9 MSEVQLVESGGGLVQP (PCNA)(SEQIDNO:23) GGSLRLSSAISGFSIS STSIDWVRQAPGKGLE WVARISPSSGSTSYAD SVKGRFTISADTSKNT VYLQMNSLRAEDTAVY YTGRSHTRHPVYPMFH PRDYRGQGTLVTVSSG AAEQKLIFEEDL Aminoacidtemplatesequenceofpeptide XXXXXWXXSRTPWXXXXX aptamer1(eIF4A)(SEQIDNO:24) Aminoacidtemplatesequenceofpeptide XXXXXXXWXXSRTPWXXX aptamer2(eIF4A)(SEQIDNO:25) Aminoacidtemplatesequenceofpeptide XXXWXXSRTPWXXXXXXX aptamer3(eIF4A)(SEQIDNO:26) Aminoacidsequenceofpeptideaptamer1 LTWTRWVRSRTPWNVMGF (eIF4A)(SEQIDNO:27) Aminoacidsequenceofpeptideaptamer2 LVQRLTRWRFSRTPWHFW (eIF4A)(SEQIDNO:28) Aminoacidsequenceofpeptideaptamer3 AHYWRFSRTPWFVRLTHQ (eIF4A)(4AM3)(SEQIDNO:29) Aminoacidsequenceofpeptideaptamer4 IQRWRLSRTPWSRSFHRI (eIF4A)(SEQIDNO:30) Aminoacidsequenceofpeptideaptamer5 SLRWINSRTPWWIVRRML (eIF4A)(SEQIDNO:31) Aminoacidsequenceofpeptideaptamer6 TERWHDSRTPWANLYRHF (eIF4A)(SEQIDNO:32) Aminoacidsequenceofpeptideaptamer7 SVKWLNSRTPWTRMMYTL (eIF4A)(SEQIDNO:33) Aminoacidsequenceofpeptideaptamer8 RSAGLWRYSRTPWYLLRT (eIF4A)(SEQIDNO:34) Aminoacidsequenceofpeptideaptamer9 QLRWSTSRTPWAKLAVKP (eIF4A)(SEQIDNO:35) Aminoacidsequenceofpeptideaptamer10 VSSVRWFRSRTPWKMLHH (eIF4A)(SEQIDNO:36) Aminoacidsequenceofpeptideaptamer11 NFRWVWSRTPWHMRADVR (eIF4A)(SEQIDNO:37) Aminoacidsequenceofpeptideaptamer12 CYKWASSRTPWARLWATT (eIF4A)(SEQIDNO:38) Aminoacidsequenceofpeptideaptamer13 ERKWRFSRTPWRWSHSRY (eIF4A)(4AM14)(SEQIDNO:39) Aminoacidsequenceofpeptideaptamer14 SHHWFGSRTPWSWSNTLR (eIF4A)(SEQIDNO:40) Aminoacidsequenceofpeptideaptamer15 STKWSESRTPWALLRLSP (eIF4A)(SEQIDNO:41) Aminoacidsequenceofpeptideaptamer16 YPWLRWRLSRTPWSLRWS (eIF4A)(SEQIDNO:42) Aminoacidsequenceofpeptideaptamer17 GRAYPWRWSRTPWYRTRL (eIF4A)(SEQIDNO:43) Aminoacidsequenceofpeptideaptamer18 NLRAPYRWLKSRTPWANM (eIF4A)(SEQIDNO:44) Aminoacidsequenceofpeptideaptamer19 AIGRWWGWSRTPWRFPLP (eIF4A)(SEQIDNO:45) Aminoacidsequenceofpeptideaptamer20 SSRWTNSRTPWTLISLSD (eIF4A)(SEQIDNO:46) Aminoacidsequenceofpeptideaptamer21 VISWRWSRTPWSLRAKNR (eIF4A)(SEQIDNO:47) Aminoacidsequenceofpeptideaptamer22 VISWRWSRTPWSLRAKNR (eIF4A)(SEQIDNO:48) Aminoacidsequenceofpeptideaptamer23 AARWTNSRTPWSTYRWLK (eIF4A)(SEQIDNO:49) Aminoacidsequenceofpeptideaptamer24 KYVWKNSRTPWRRLTHNT (eIF4A)(SEQIDNO:50) Aminoacidsequenceofpeptideaptamer25 TATERWRNSRTPWTLIKV (eIF4A)(SEQIDNO:51) Aminoacidsequenceofpeptideaptamer26 QLRWLKSRTPWARVMLVL (eIF4A)(SEQIDNO:52) Aminoacidsequenceofpeptideaptamer27 VARRRWLGSRTPWVGKYS (eIF4A)(SEQIDNO:53) Aminoacidsequenceofpeptideaptamer28 GLRWRLSRTPWAAHLARG (eIF4A)(SEQIDNO:54) Aminoacidsequenceofpeptideaptamer29 SLRWRASRTPWNKLFVIL (eIF4A)(SEQIDNO:55) Aminoacidsequenceofpeptideaptamer30 FLRWTLSRTPWTVRAKNP (eIF4A)(SEQIDNO:56) Aminoacidsequenceofpeptideaptamer31 QWRWTWSRTPWRRYRRLS (eIF4A)(SEQIDNO:57) Aminoacidsequenceofpeptideaptamer32 MWRWVFSRTPWRRIQRDT (eIF4A)(SEQIDNO:58) Aminoacidsequenceofpeptideaptamer33 ITRWVNSRTPWHALLLLA (eIF4A)(SEQIDNO:59) Aminoacidsequenceofpeptideaptamer34 NFRWLNSRTPWTRANP (eIF4A)(SEQIDNO:60) Aminoacidsequenceofpeptideaptamer35 GCLWVLSRTPWCMLKLGP (eIF4A)(4AM20)(SEQIDNO:61) Aminoacidscramblesequenceofpeptide YVRPRLPHMSQFES aptamer(PCNA)(SEQIDNO:62) Aminoacidsequenceofpeptideaptamer36 ETCPYVESRTPWCRQ (eIF4A)(EA-6)(SEQIDNO:63) Aminoacidsequenceofpeptidemotif1for CETGFFTGC eIF4AE(EE-01)(SEQIDNO:64) Aminoacidsequenceofpeptidemotif2for CELGYYNDC eIF4AE(EE-02)(SEQIDNO:65) Aminoacidsequenceofpeptidemotif3for CELGYYNDC eIF4AE(EE-03)(SEQIDNO:66) Aminoacidsequenceofpeptidemotif4for CETGFFLKC eIF4AE(EE-04)(SEQIDNO:67) Aminoacidsequenceofpeptidemotif5for CELGFYRLC eIF4AE(EE-05)(SEQIDNO:68) Aminoacidsequenceofpeptidemotif6for CETGFFLRC eIF4AE(EE-06)(SEQIDNO:69) Aminoacidsequenceofpeptidemotif7for CETGYFSQC eIF4AE(EE-07)(SEQIDNO:70) Aminoacidsequenceofpeptidemotif8for CIHSPTSLC eIF4AE(SEQIDNO:71) Aminoacidsequenceofpeptidemotif9for CETGFYKTC eIF4AE(EE-08)(SEQIDNO:72) Aminoacidsequenceofpeptidemotif10for CEMGYFGNC eIF4AE(EE-09)(SEQIDNO:73) Aminoacidsequenceofpeptidemotif11for CAMGFFQDC eIF4AE(EE-02E3A)(SEQIDNO:74) Aminoacidsequenceofpeptidemotif12for CEAGFFQDC eIF4AE(EE-02M4A)(SEQIDNO:75) Aminoacidsequenceofpeptidemotif13for CEMAFFQDC eIF4AE(EE-02G5A)(SEQIDNO:76) Aminoacidsequenceofpeptidemotif14for CEMGAFQDC eIF4AE(EE-02F6A)(SEQIDNO:77) Aminoacidsequenceofpeptidemotif15for CEMGFAQDC eIF4AE(EE-02F7A)(SEQIDNO:78) Aminoacidsequenceofpeptidemotif16for CEMGFFADC eIF4AE(EE-02Q8A)(SEQIDNO:79) Aminoacidsequenceofpeptidemotif17for CEMGFFQAC eIF4AE(EE-02D9A)(SEQIDNO:80) Aminoacidsequenceofpeptidemotif18for CEMGFFQDCG eIF4AE(EE-02N-Del)(SEQIDNO:81) Aminoacidsequenceofpeptidemotif19for ACEMGFFQDC eIF4AE(EE-02C-Del)(SEQIDNO:82) Aminoacidsequenceofpeptidemotif20for CEMGFFQDC eIF4AE(EE-02NC-Del)(SEQIDNO:83) Aminoacidsequenceofpeptideaptamerfor PLPEAGFFTNIPAMV eIF4AE(Control)(VH-DiF.sup.CAP-01Cntrl)(SEQID NO:84) Aminoacidsequenceofpeptide1withpeptide SLTYTREFLRSL motifforeIF4AE(SEQIDNO:85) Aminoacidsequenceofpeptide2withpeptide AQSYTRMELLRL motifforeIF4AE(SEQIDNO:86) Aminoacidsequenceofpeptide3withpeptide HYSRTDLLAYRW motifforeIF4AE(SEQIDNO:87) Aminoacidsequenceofpeptide4withpeptide SGIQQRMIVTWP motifforeIF4AE(SEQIDNO:88) Aminoacidsequenceofpeptide5withpeptide KLWSIPTNFLLP motifforeIF4AE(SEQIDNO:89) 6Aminoacidsequenceofpeptide6with VEGQNLEALVTD peptidemotifforeIF4AE(SEQIDNO:90) Aminoacidsequenceofpeptide7withpeptide TISYDRSTLFLF motifforeIF4AE(SEQIDNO:91) Aminoacidsequenceofpeptide8withpeptide DSAKERLAVSGT motifforeIF4AE(SEQIDNO:92) Aminoacidsequenceofpeptide9withpeptide LPRMMSDGEAGD motifforeIF4AE(SEQIDNO:93) Aminoacidsequenceofpeptide10with YLMQTNQWQIAS peptidemotifforeIF4AE(SEQIDNO:94) Aminoacidsequenceofrationaldesignof GGSEMGFFGGSGG peptideaptamer1foreIF4AE(SEQIDNO:95) Aminoacidsequenceofrationaldesignof GGSGGEMGFFGGS peptideaptamer2foreIF4AE(SEQIDNO:96) Aminoacidsequenceofrationaldesignof GGSGEMGFFGGSG peptideaptamer3foreIF4AE(SEQIDNO:97) AminoacidsequenceofVHdomainofpeptide MSEVQLVESGGGLVQPGGS LRLSSAISGFSISSTSIDWVRQ aptamer1(eIF4AE)(VH-DiF.sup.CAP-01)(SEQIDNO: APGKGLEWVARISPSSGSTS 98) YADSVKGRFTISADTSKNTV YLQMNSLRAEDTAVYYTGR PLPEMGFFTNIPAMVDYRGQ GTLVTVSSGAA AminoacidsequenceofVHdomain(positive MSEVQLVESGGGLVQPGGSLRL control)(eIF4AE)(VH-1C5)(SEQIDNO:99) SSAISGFSISSTSIDWVRQAPGK GLEWVARISPSSGSTSYADSVK GRFTISADTSKNTVYLQMNSLR AEDTAVYYTGRVAKADLNSSSPS FVVNTYSSFGFDYRGQGTLVTVS SGAA AminoacidsequenceofVHdomainthatbinds MSEVQLVESGGGLVQPGGSLRLS attheeIF4AGinteractionsiteofeIF4AE(VH-M4) SAISGFSISSTSIDWVRQAPGKGLE (SEQIDNO:100) WVARISPSSGSTSYADSVKGRFTIS ADTSKNTVYLQMNSLRAEDTAVY YTGRVAKALNSSSPSFVVNTYSSF GFDYRGQGTLVTVSSGAA AminoacidsequenceofVHdomainthat MSEVQLVESGGGLVQPGGSLRLS interactswitheIF4AEattheeIF4AGbinding SAISGFSISSTSIDWVRQAPGKGLE interface(eIF4AE)(VH-S4)(SEQIDNO:101) WVARISPSSGSTSYADSVKGRFTIS ADTSKNTVYLQMNSLRAEDTAVY YTGRVAKALNSRSPSFVVNTYSSIG FDYRGQGTLVTVSSGAA AminoacidsequenceofVHdomainofpeptide MSEVQLVESGGGLVQPGGSLRLSS aptamer1(eIF4AE)withpointmutationof AISGFSISSTSIDWVRQAPGKGLEW M104A(VH-DIF.sup.CAP-01MA)(SEQIDNO:102) VARISPSSGSTSYADSVKGRFTISAD TSKNTVYLQMNSLRAEDTAVYYTG RPLPEAGFFTNIPAMVDYRGQGTLV TVSSGAA AminoacidsequenceofVHdomainofpeptide MSEVQLVESGGGLVQPGGSLRLSSA aptamer1(eIF4AE)withpointmutationof ISGFSISSTSIDWVRQAPGKGLEWV E103A(VH-DIF.sup.CAP-01EA)(SEQIDNO:103) ARISPSSGSTSYADSVKGRFTISADT SKNTVYLQMNSLRAEDTAVYYTGR PLPAMGFFTNIPAMVDYRGQGTLV TVSSGAA AminoacidsequenceofscrambledVH MSEVQLVESGGGLVQPGGSLRLSSA domain(negativecontrol)(VH-1C5.sup.Scrambled) ISGFSISSTSIDWVRQAPGKGLEWVA (SEQIDNO:104) RISPSSGSTSYADSVKGRFTISADTS KNTVYLQMNSLRAEDTAVYYTGRGS VLSVAFKPSVDSFSSFYNTNDYRGQ GTLVTVSSGAA
EQUIVALENTS
[0270] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.