PEPTIDE INHIBITORS OF FOCAL ADHESION KINASE ACTIVITY AND USES THEREOF
20220306692 · 2022-09-29
Inventors
Cpc classification
C07K14/705
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
C07K7/64
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K7/64
CHEMISTRY; METALLURGY
A61K38/12
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
Abstract
This disclosure provides peptides which have an affinity for the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). In particular, the peptides are modified and derived from the sequence of the LD2 alpha helical domain of paxillin (e.g., LD2 peptides), the LD4 domain of paxillin (e.g., LD4 peptides), and CD8 peptides. These peptides are capable of blocking an interaction between paxillin and FAK, thereby inhibiting FAK activity related to FAK-paxillin interaction. The invention further provides uses for such peptides as therapeutics for the treatment of cancer and other diseases characterized with FAK activity and/or expression (e.g., fibrosis).
Claims
1. A compound having Formula I: ##STR00010## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein Y.sub.C, Y.sub.T, Z.sub.1, R.sub.1, L.sub.1, Q.sub.1, R.sub.2, and Z.sub.2 independently include any chemical moiety that permits the resulting compound to bind with the FAT domain of FAK and inhibit FAK's interaction with a paxillin protein.
2. The compound of claim 1, wherein the compound is capable of binding with one or more of the helix1-4 and helix2-3 portion of the FAT domain of the FAK protein.
3. The compound of claim 1, wherein the compound is capable of binding with one or more of the following amino acid residues within a wild type FAK protein: V928, 1936, R962, and K955.
4. The compound of claim 1, wherein the compound is capable of inhibiting interaction between FAK and paxillin.
5. The compound of claim 1, wherein the compound is an isolated polypeptide.
6. The compound of claim 1, wherein the compound is capable of one or more of the following: disrupting FAK non-catalytic activity upon inhibition of interaction between FAK and paxillin, disrupting FAK catalytic activity through direct binding of the FAT domain, inhibiting FAK related scaffolding function, inhibiting FAK protein-protein interactions mediated by the FAT domain, inhibiting binding of paxillin with the helix 1-4 region of the FAT domain of FAK, inhibiting binding of paxillin with the helix 2-3 region of the FAT domain of FAK, inhibiting FAK related apoptosis, proliferation, invasion, and/or metastasis, inhibiting FAK-paxillin interaction resulting inhibiting of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and/or invasion, inhibiting FAK-Leupaxin interaction through binding the FAT domain of FAK, inhibiting FAK-CD4 interaction through binding the FAT domain of FAK, inhibiting FAK-CD8 interaction through binding the FAT domain of FAK, inhibiting FAK-DCC interaction through binding the FAT domain of FAK, inhibiting paxillin LD2 and LD4 binding with respective binding partners, inhibiting CD4 and CD8 binding with respective binding partners, inhibiting CD4 and CD8 binding with Lck, inhibiting Leupaxin binding with respective binding partners, inhibiting DCC binding with respective binding partners, inhibiting interactions of FAK-related molecules including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git½, Pax3, hic-5, and ARF.
7. The compound of claim 1, wherein Q.sub.1 is a chain of 2 (-[Aa1]-[Aa2]-), 3 (-[Aa1]-[Aa2]-[Aa3]-), 6 (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) amino acids resulting in motifs, respectively, of (i, i+3); (i, i+4); (i, i+7) and (i, i+11); wherein “i” refers to the alpha-amino acid backbone residue towards the N-terminus of the macrocycle and the “number” in “i+[number]” refers to the amino acid residue [number] away from i towards the C-terminus; wherein Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, Aa10 are independent of each other and are each a natural or unnatural alpha-amino acid.
8. The compound of claim 1, wherein Z.sub.1 and Z.sub.2 are independent of each other and are each a chain of natural or unnatural amino acids between 0 and 200 units in length.
9. The compound of claim 1, wherein Y.sub.C is an optional moiety such as biotin or a dye or molecular probe, fluorescent or otherwise, or a chemically reactive moiety including but not limited to an azide, alkyne, or photoreactive species (examples can be found in, but are not limited to the selection in, the Molecular Probes Handbook, Eleventh edition, lain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), any moiety that renders the resulting compound a bifunctional compound capable of recruiting endogenous proteins to an E3 Ubiquitin Ligase for degradation, any other therapeutic agent, a FAK kinase inhibiting agent, a covalent derivative of other kinase inhibitors (e.g. Src, EGFR, HER2, etc.), a covalent derivative of a GPCR compound, a covalent derivative of a nuclear receptor compound, a covalent derivative of an E3 ubiquitin ligase targeting ligand, a covalent derivative of a protein-protein interaction inhibitor, a covalent derivative of a radioactive moiety, a covalent derivative of a drug transporter-binding ligand, a covalent derivative of a cell penetrating moiety/sequence (e.g. TAT, etc.), a covalent derivative of a chemotherapeutic agent, a covalent derivative of a lipid moiety, a covalent derivative of a pro-drug moiety facilitating favorable bioavailability and/or pharmacokinetics, and a covalent derivative of an electrophilic moiety for covalent binding to the target protein.
10. The compound of claim 1, wherein Y.sub.T is an optional chemical tether between Y.sub.C and Z.sub.1, which might form an alkyl or amide (e.g., carbamide, sulfonamide or phosphoramide) group if present.
11. The compound of claim 10, wherein the chemical tether is selected from beta-alanine, 6-aminohexanoic acid and an amino acid where the amine and acid functionalities are separated by a poly(ethyleneglycol) (PEG) monomer, oligomer or polymer, and/or wherein Y.sub.T may or may not contain one or more carbon atoms, or one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or one or more carbo- or heterocyclic rings.
12. The compound of claim 1, wherein R.sub.1 is a hydrogen or lower alkyl or substituted methyl group.
13. The compound of claim 1, wherein R.sub.2 is a hydrogen or lower alkyl or substituted methyl group.
14. The compound of claim 1, wherein L.sub.1 is typically a hydrocarbon chain containing a single double bond, of cis- or trans-geometry, or a mixture thereof, typically containing 8 or 11 atoms, but may be a different integer, and/or may be a disulfide staple or triazole staple.
15. The compound of claim 1, wherein the compound is at least 60% identical with one of SEQ ID Nos: 1-38.
16. The compound of claim 1, wherein the compound is at least 75% identical with one of SEQ ID Nos: 1-38.
17. The compound of claim 1, wherein the compound is at least 80% identical with one of SEQ ID Nos: 1-38.
18. The compound of claim 1, wherein the compound is at least 85% identical with one of SEQ ID Nos: 1-38.
19. The compound of claim 1, wherein the compound is at least 90% identical with one of SEQ ID Nos: 1-38.
20. The compound of claim 1, wherein the compound is at least 95% identical with one of SEQ ID Nos: 1-38.
21. The compound of claim 1, wherein the compound is at least 98% identical with one of SEQ ID Nos: 1-38.
22. The compound of claim 1, wherein the compound is at least 99% identical with one of SEQ ID Nos: 1-38.
23. The compound of claim 1, wherein the compound comprises, consists of, or consists essentially of one of SEQ ID Nos: 1-38.
24. A compound having Formula II: ##STR00011## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein Y.sub.C, Y.sub.T, Z.sub.1, R.sub.1, Q.sub.1, L.sub.2, R.sub.2, Z.sub.3, R.sub.3, Q.sub.2, L.sub.3, R.sub.4 and Z.sub.2 include any chemical moiety that permits the resulting compound to bind with the FAT domain of FAK and inhibit FAK's interaction with a paxillin protein.
25. The compound of claim 24, wherein the compound is capable of binding with one or more of the helix1-4 and helix2-3 portion of the LD2 domain of a paxillin protein.
26. The compound of claim 24, wherein the compound is capable of binding with one or more of the following amino acid residues within a wild type FAK protein: V928, 1936, R962, and K955.
27. The compound of claim 24, wherein the compound is capable of inhibiting interaction between FAK and paxillin.
28. The compound of claim 24, wherein the compound is an isolated polypeptide.
29. The compound of claim 24, wherein the compound is capable of one or more of the following: disrupting FAK non-catalytic activity upon inhibition of interaction between FAK and paxillin, disrupting FAK catalytic activity through direct binding of the FAT domain, inhibiting FAK related scaffolding function, inhibiting FAK protein-protein interactions mediated by the FAT domain, inhibiting binding of paxillin with the helix 1-4 region of the FAT domain of FAK, inhibiting binding of paxillin with the helix 2-3 region of the FAT domain of FAK, inhibiting FAK related apoptosis, proliferation, invasion, and/or metastasis, inhibiting FAK-paxillin interaction resulting inhibiting of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and/or invasion, inhibiting FAK-Leupaxin interaction through binding the FAT domain of FAK, inhibiting FAK-CD4 interaction through binding the FAT domain of FAK, inhibiting FAK-CD8 interaction through binding the FAT domain of FAK, inhibiting FAK-DCC interaction through binding the FAT domain of FAK, inhibiting paxillin LD2 and LD4 binding with respective binding partners, inhibiting CD4 and CD8 binding with respective binding partners, inhibiting CD4 and CD8 binding with Lck, inhibiting Leupaxin binding with respective binding partners, inhibiting DCC binding with respective binding partners, inhibiting interactions of FAK-related molecules including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git½, Pax3, hic-5, and ARF.
30. The compound of claim 24, wherein Q.sub.1 is a chain of 2 (-[Aa1]-[Aa2]-), 3 (-[Aa1]-[Aa2]-[Aa3]-), 6 (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) amino acids resulting in motifs, respectively, of (i, i+3); (i, i+4); (i, i+7) and (i, i+11); wherein Q.sub.2 is a chain of 2 (-[Aa11]-[Aa12]-), 3 (-[Aa11]-[Aa12]-[Aa13]-), 6 (-[Aa11]-[Aa12]-[Aa13]-[Aa14]-[Aa15]-[Aa16]-) or 10 (-[Aa11]-[Aa12]-[Aa13]-[Aa14]-[Aa15]-[Aa16]-[Aa17]-[Aa18]-[Aa19]-[Aa20]-) amino acids resulting in motifs, respectively, of (i, i+3); (i, i+4); (i, i+7) and (i, i+11); wherein “i” refers to the alpha-amino acid backbone residue towards the N-terminus of the macrocycle and the “number” in “i+[number]” refers to the amino acid residue [number] away from i towards the C-terminus; wherein Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, Aa10 are independent of each other and are each a natural or unnatural alpha-amino acid; wherein Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, Aa20 are independent of each other and are each a natural or unnatural alpha-amino acid.
31. The compound of claim 24, wherein Z.sub.1 and Z.sub.2 and Z.sub.3 are independent of each other and are each a chain of natural or unnatural amino acids between 0 and 200 units in length.
32. The compound of claim 24, wherein Y.sub.C is an optional moiety such as biotin or a dye or molecular probe, fluorescent or otherwise, or a chemically reactive moiety including but not limited to an azide, alkyne, or photoreactive species (examples can be found in, but are not limited to the selection in, the Molecular Probes Handbook, Eleventh edition, lain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), any moiety that renders the resulting compound a bifunctional compound capable of recruiting endogenous proteins to an E3 Ubiquitin Ligase for degradation, any other therapeutic agent, a FAK kinase inhibiting agent, a covalent derivative of other kinase inhibitors (e.g. Src, EGFR, HER2, etc.), a covalent derivative of a GPCR compound, a covalent derivative of a nuclear receptor compound, a covalent derivative of an E3 ubiquitin ligase targeting ligand, a covalent derivative of a protein-protein interaction inhibitor, a covalent derivative of a radioactive moiety, a covalent derivative of a drug transporter-binding ligand, a covalent derivative of a cell penetrating moiety/sequence (e.g. TAT, etc.), a covalent derivative of a chemotherapeutic agent, a covalent derivative of a lipid moiety, a covalent derivative of a pro-drug moiety facilitating favorable bioavailability and/or pharmacokinetics, and a covalent derivative of an electrophilic moiety for covalent binding to the target protein.
33. The compound of claim 24, wherein Y.sub.T is an optional chemical tether between Y.sub.C and Z.sub.1, which might form an alkyl or amide (carbamide, sulfonamide or phosphoramide) group if present.
34. The compound of claim 24, wherein the chemical tether is selected from beta-alanine, 6-aminohexanoic acid and an amino acid where the amine and acid functionalities are separated by a poly(ethyleneglycol) (PEG) monomer, oligomer or polymer, and/or wherein Y.sub.T may or may not contain one or more carbon atoms, or one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or one or more carbo- or heterocyclic rings.
35. The compound of claim 24, wherein R.sub.1 and R.sub.2 are independently a hydrogen or lower alkyl or substituted methyl group.
36. The compound of claim 24, wherein R.sub.3 and R.sub.4 are independently a hydrogen or lower alkyl or substituted methyl group.
37. The compound of claim 24, wherein L.sub.2 is typically a hydrocarbon chain containing a single double bond, of cis- or trans-geometry, or a mixture thereof, typically containing 8 or 11 atoms, but may be a different integer, and/or a disulfide staple or a triazole staple.
38. The compound of claim 24, wherein L.sub.3 is typically a hydrocarbon chain containing a single double bond, of cis- or trans-geometry, or a mixture thereof, typically containing 8 or 11 atoms, but may be a different integer.
39. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 60% identical with one of SEQ ID Nos: 1-38.
40. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 75% identical with one of SEQ ID Nos: 1-38.
41. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 80% identical with one of SEQ ID Nos: 1-38.
42. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 85% identical with one of SEQ ID Nos: 1-38.
43. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 90% identical with one of SEQ ID Nos: 1-38.
44. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 95% identical with one of SEQ ID Nos: 1-38.
45. The compound of claim 24 wherein at least a portion of the compound encompasses an amino acid sequence that is at least 98% identical with one of SEQ ID Nos: 1-38.
46. The compound of claim 24, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 99% identical with one of SEQ ID Nos: 1-38.
47. The compound of claim 24, wherein at least a portion of the compound comprises one of SEQ ID Nos: 1-38.
48. A compound having Formula III: ##STR00012## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein Y.sub.C, Y.sub.T, Z.sub.1, R.sub.1, Q.sub.1, L.sub.4, Q.sub.4, L.sub.5, R.sub.5, and Z.sub.2 independently include any chemical moiety that permits the resulting compound to bind with the FAT domain of FAK and inhibit FAK's interaction with a paxillin protein.
49. The compound of claim 48, wherein the compound is capable of binding with one or more of the helix1-4 and helix2-3 portion of the LD2 domain of a paxillin protein.
50. The compound of claim 48, wherein the compound is capable of binding with one or more of the following amino acid residues within a wild type FAK protein: V928, 1936, R962, and K955.
51. The compound of claim 48, wherein the compound is capable of inhibiting interaction between FAK and paxillin.
52. The compound of claim 48, wherein the compound is an isolated polypeptide.
53. The compound of claim 48, wherein the compound is capable of one or more of the following: disrupting FAK non-catalytic activity upon inhibition of interaction between FAK and paxillin, disrupting FAK catalytic activity through direct binding of the FAT domain, inhibiting FAK related scaffolding function, inhibiting FAK protein-protein interactions mediated by the FAT domain, inhibiting binding of paxillin with the helix 1-4 region of the FAT domain of FAK, inhibiting binding of paxillin with the helix 2-3 region of the FAT domain of FAK, inhibiting FAK related apoptosis, proliferation, invasion, and/or metastasis, inhibiting FAK-paxillin interaction resulting inhibiting of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and/or invasion, inhibiting FAK-Leupaxin interaction through binding the FAT domain of FAK, inhibiting FAK-CD4 interaction through binding the FAT domain of FAK, inhibiting FAK-CD8 interaction through binding the FAT domain of FAK, inhibiting FAK-DCC interaction through binding the FAT domain of FAK, inhibiting paxillin LD2 and LD4 binding with respective binding partners, inhibiting CD4 and CD8 binding with respective binding partners, inhibiting CD4 and CD8 binding with Lck, inhibiting Leupaxin binding with respective binding partners, inhibiting DCC binding with respective binding partners, inhibiting interactions of FAK-related molecules including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git½, Pax3, hic-5, and ARF.
54. The compound of claim 48, wherein Q.sub.1 is a chain of 2 (-[Aa1]-[Aa2]-), 3 (-[Aa1]-[Aa2]-[Aa3]-), 6 (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) amino acids resulting in motifs, respectively, of (i, i+3); (i, i+4); (i, i+7) and (i, i+11); wherein Q.sub.4 is a chain of 2 (-[Aa11]-[Aa12]-), 3 (-[Aa11]-[Aa12]-[Aa13]-), 6 (-[Aa11]-[Aa12]-[Aa13]-[Aa14]-[Aa15]-[Aa16]-) or 10 (-[Aa11]-[Aa12]-[Aa13]-[Aa14]-[Aa15]-[Aa16]-[Aa17]-[Aa18]-[Aa19]-[Aa20]-) amino acids resulting in motifs, respectively, of (i, i+3); (i, i+4); (i, i+7) and (i, i+11); wherein “i” refers to the alpha-amino acid backbone residue towards the N-terminus of the macrocycle and the “number” in “i+[number]” refers to the amino acid residue [number] away from i towards the C-terminus; wherein Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, Aa10 are independent of each other and are each a natural or unnatural alpha-amino acid; wherein Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, Aa20 are independent of each other and are each a natural or unnatural alpha-amino acid.
55. The compound of claim 48, wherein Z.sub.1 and Z.sub.2 are independent of each other and are each a chain of natural or unnatural amino acids between 0 and 200 units in length.
56. The compound of claim 48, wherein Y.sub.C is an optional moiety such as biotin or a dye or molecular probe, fluorescent or otherwise, or a chemically reactive moiety including but not limited to an azide, alkyne, or photoreactive species (examples can be found in, but are not limited to the selection in, the Molecular Probes Handbook, Eleventh edition, lain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), any moiety that renders the resulting compound a bifunctional compound capable of recruiting endogenous proteins to an E3 Ubiquitin Ligase for degradation, any other therapeutic agent, a FAK kinase inhibiting agent, a covalent derivative of other kinase inhibitors (e.g. Src, EGFR, HER2, etc.), a covalent derivative of a GPCR compound, a covalent derivative of a nuclear receptor compound, a covalent derivative of an E3 ubiquitin ligase targeting ligand, a covalent derivative of a protein-protein interaction inhibitor, a covalent derivative of a radioactive moiety, a covalent derivative of a drug transporter-binding ligand, a covalent derivative of a cell penetrating moiety/sequence (e.g. TAT, etc.), a covalent derivative of a chemotherapeutic agent, a covalent derivative of a lipid moiety, a covalent derivative of a pro-drug moiety facilitating favorable bioavailability and/or pharmacokinetics, and a covalent derivative of an electrophilic moiety for covalent binding to the target protein.
57. The compound of claim 48, wherein Y.sub.T is an optional chemical tether between Y.sub.C and Z.sub.1, which might form an alkyl or amide (carbamide, sulfonamide or phosphoramide) group if present.
58. The compound of claim 57, wherein the chemical tether is selected from beta-alanine, 6-aminohexanoic acid and an amino acid where the amine and acid functionalities are separated by a poly(ethyleneglycol) (PEG) monomer, oligomer or polymer, and/or wherein Y.sub.T may or may not contain one or more carbon atoms, or one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or one or more carbo- or heterocyclic rings.
59. The compound of claim 48, wherein R.sub.1 and R.sub.5 are independently selected from a hydrogen or lower alkyl or substituted methyl group.
60. The compound of claim 48, wherein L.sub.4 and L5 are independently selected from a hydrocarbon chain containing a single double bond, of cis- or trans-geometry, or a mixture thereof, typically containing 8 or 11 atoms, but may be a different integer, and/or a disulfide staple or a triazole staple.
61. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 60% identical with one of SEQ ID Nos: 1-38.
62. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 75% identical with one of SEQ ID Nos: 1-38.
63. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 80% identical with one of SEQ ID Nos: 1-38.
64. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 85% identical with one of SEQ ID Nos: 1-38.
65. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 90% identical with one of SEQ ID Nos: 1-38.
66. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 95% identical with one of SEQ ID Nos: 1-38.
67. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 98% identical with one of SEQ ID Nos: 1-38.
68. The compound of claim 48, wherein at least a portion of the compound encompasses an amino acid sequence that is at least 99% identical with one of SEQ ID Nos: 1-38.
69. The compound of claim 48, wherein at least a portion of the compound comprises one of SEQ ID Nos: 1-38.
70. A compound having Formula IV: ##STR00013## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein SPA—NH2 and SPB—NH2 are independently a compound of Formula I (as recited in claim 1) excluding Y.sub.C—Y.sub.T—, or a compound of Formula II (as recited in claim 24) excluding Y.sub.C—Y.sub.T—, or a compound of Formula III (as recited in claim 48) excluding Y.sub.C—Y.sub.T—; wherein T.sub.1 is a tether or 0-400 atoms in length, which typically, but not necessarily, might include a poly(ethyleneglycol) chain and which is typically, but not necessarily, connected to SPA—NH2 and SPB—NH2 as an amide functionality and which may or may not contain an internal structure such as a carbocycle or heterocycle which may or may not function as a dye or chromophore, may or may not branch to a pendant moiety such as biotin or a dye or chemical probe or reactive group, and may or may not contain one or more carbon atoms, or one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms.
71. The compound of claim 70, wherein the tether (T.sub.1) is selected from an ether (including a polyether such as poly(ethyleneglycol)), ester, amide, thioether, thioester or a hydrocarbon chain, which may or may not contain internal unsaturation (such as a single or multiple double bonds, or one or more alkynes) and may or may not contain an internal structure such as a carbocycle or heterocycle which may or may not function as a dye or chromophore and may or may not branch to a pendant moiety such as biotin or a dye or chemical probe or reactive group or E3 ligase ligand.
72. A compound having Formula V: ##STR00014## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein Y.sub.C—Y.sub.T—SPA—NH2 and Y.sub.C—Y.sub.T—SPB—NH2 are independently a compound of Formula I (as recited in claim 1), or a compound of Formula II (as recited in claim 24), or a compound of Formula III (as recited in claim 48), wherein the side chain from an individual member of [Aa1], [Aa2], [Aa3], [Aa4], [Aa5], [Aa6], [Aa7], [Aa8], [Aa9], [Aa10], [Aa11], [Aa12], [Aa13], [Aa14], [Aa15], [Aa16], [Aa17], [Aa18], [Aa19] or [Aa20]; or from an amino acid that is part of Z.sub.1; or from an amino acid that is part of Z.sub.2; or from an amino acid that is part of Z.sub.3, as found on Y.sub.C—Y.sub.T—SPA—NH2, is connected via a tether (T.sub.2) to the side chain from an individual member of [Aa1], [Aa2], [Aa3], [Aa4], [Aa5], [Aa6], [Aa7], [Aa8], [Aa9], [Aa10], [Aa11], [Aa12], [Aa13], [Aa14], [Aa15], [Aa16], [Aa17], [Aa18], [Aa19] or [Aa20]; or from an amino acid that is part of Z.sub.1; or from an amino acid that is part of Z.sub.2; or from an amino acid that is part of Z.sub.3, as found on Y.sub.C—Y.sub.T—SPB—NH2 via chemical ligation.
73. The compound of claim 72, wherein the tether (T.sub.2) is selected from an ether (including a polyether such as poly(ethyleneglycol)), ester, amide, thioether, thioester or a hydrocarbon chain, which may or may not contain internal unsaturation (such as a single or multiple double bonds, or one or more alkynes) and may or may not contain an internal structure such as a carbocycle or heterocycle which may or may not function as a dye or chromophore and may or may not branch to a pendant moiety such as biotin or a dye or chemical probe or reactive group or E3 ligase ligand.
74. The compound of claim 73, wherein the tether is selected from an ether (including a polyether such as poly(ethyleneglycol)), ester, amide, thioether, thioester or a hydrocarbon chain, which may or may not contain internal unsaturation (such as a single or multiple double bonds, or one or more alkynes) and may or may not contain an internal structure such as a carbocycle or heterocycle which may or may not function as a dye or chromophore and may or may not branch to a pendant moiety such as biotin or a dye or chemical probe or reactive group may or may not contain one or more carbon atoms, or one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms.
75. A compound having Formula VI: ##STR00015## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein SPA—NH2 is a compound of Formula I (as recited in claim 1) excluding Y.sub.C—Y.sub.T—, or a compound of Formula II (as recited in claim 24) excluding Y.sub.C—Y.sub.T—, or a compound of Formula III (as recited in claim 48) excluding Y.sub.C—Y.sub.T—, wherein the N-terminus of SPA—NH2 is connected via linker L.sub.6 to an internal amino acid (individually a member of [Aa1], [Aa2], [Aa3], [Aa4], [Aa5], [Aa6], [Aa7], [Aa8], [Aa9], [Aa10], [Aa11], [Aa12], [Aa13], [Aa14], [Aa15], [Aa16], [Aa17], [Aa18], [Aa19] or [Aa20]; or from an amino acid that is part of Z.sub.1; or from an amino acid that is part of Z.sub.2; or from an amino acid that is part of Z.sub.3) from SPA—NH2, wherein the linker might be a hydrocarbon chain containing a cis- or trans-alkene, or a mixture thereof; or it might be an ether (including a polyether such as poly(ethyleneglycol)), ester, amide, thioether, thioester or a hydrocarbon chain, which may or may not contain internal unsaturation (such as a single or multiple double bonds, or one or more alkynes).
76. The compound of claim 1, 24 or 48, wherein the amino acid is selected from a standard amino acid, L-α-tert-butylglycine, D-α-tert-butylglycine, β-(2-thienyl)-L-alanine, L-allo-isoleucine, 4,5-dehydro-L-leucine, D-homoleucine, L-homoleucine, 1-aminocyclopentane-1-carboxylic acid, D-allo-isoleucine, 3-(4-thiazolyl)-L-alanine, L-Homoarginine, 5,5,5-Trifluoro-DL-leucine, γ-carboxy γ-(di-tert-butyl ester)-L-glutamic acid, γ-carboxy γ-(di-tert-butyl ester)-D-glutamic acid, L-α-aminobutyric acid, D-α-aminobutyric acid, α,β-dehydro-2-aminobutyric acid, 4-nitro-L-phenylalanine, 4-chloro-L-phenylalanine, 4-chloro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-fluoro-D-phenylalanine, L-homophenylalanine, D-homophenylalanine, 3,4-dichloro-D-phenylalanine, 3-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, p-phenyl-L-Phenylalanine, p-phenyl-D-Phenylalanine, 4-bromo-L-phenylalanine, 4-bromo-D-phenylalanine, 2-chloro-L-phenylalanine, 2-chloro-D-phenylalanine, 3-cyano-L-phenylalanine, and 3-cyano-D-phenylalanine.
77. The compound of claim 1, claim 24 or claim 48, wherein the compound is further conjugated with an additional therapeutic agent.
78. An alpha-helical stapled peptide comprising hydrophobic amino acids and hydrophilic amino acids, wherein two or more amino acids of the peptide are connected to each other, wherein the peptide is capable of binding with the FAT domain of FAK and inhibit FAK's interaction with a paxillin protein.
79. The stapled peptide of claim 78, wherein the peptide is capable of binding with one or more of the helix1-4 and helix2-3 portion of the LD2 domain of a paxillin protein.
80. The stapled peptide of claim 78, wherein the compound is capable of binding with one or more of the following amino acid residues within a wild type FAK protein: V928, 1936, R962, and K955.
81. The stapled peptide of claim 78, wherein the peptide is capable of inhibiting interaction between FAK and paxillin.
82. The stapled peptide of claim 78, wherein the peptide is capable of disrupting FAK non-catalytic activity upon inhibition of interaction between FAK and paxillin.
83. The stapled peptide of claim 78, wherein the peptide is capable of one or more of the following: disrupting FAK non-catalytic activity upon inhibition of interaction between FAK and paxillin, disrupting FAK catalytic activity through direct binding of the FAT domain, inhibiting FAK related scaffolding function, inhibiting FAK protein-protein interactions mediated by the FAT domain, inhibiting binding of paxillin with the helix 1-4 region of the FAT domain of FAK, inhibiting binding of paxillin with the helix 2-3 region of the FAT domain of FAK, inhibiting FAK related apoptosis, proliferation, invasion, and/or metastasis, inhibiting FAK-paxillin interaction resulting inhibiting of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and/or invasion, inhibiting FAK-Leupaxin interaction through binding the FAT domain of FAK, inhibiting FAK-CD4 interaction through binding the FAT domain of FAK, inhibiting FAK-CD8 interaction through binding the FAT domain of FAK, inhibiting FAK-DCC interaction through binding the FAT domain of FAK, inhibiting paxillin LD2 and LD4 binding with respective binding partners, inhibiting CD4 and CD8 binding with respective binding partners, inhibiting CD4 and CD8 binding with Lck, inhibiting Leupaxin binding with respective binding partners, inhibiting DCC binding with respective binding partners, inhibiting interactions of FAK-related molecules including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git½, Pax3, hic-5, and ARF.
84. The stapled peptide of claim 78, wherein the amino acids at one or more positions selected from the group consisting of i, i+3, i+4, i+7, i+8, i+10 and i+11 (where i is an integer) are connected to each other.
85. The stapled peptide of claim 78, wherein the stapled peptide comprises any one of SEQ ID Nos: 1-38.
86. The stapled peptide of claim 78, wherein the peptide is further conjugated with an imaging agent.
87. The stapled peptide of claim 86, wherein the imaging agent is (5-/6-) carboxytetramethylrhodamine (TAMRA).
88. A pharmaceutical composition comprising one or more stapled peptides recited in claim 78.
89. A composition comprising two or more stapled peptides as recited in claim 78, wherein the two or more stapled peptides are tethered together via a linker.
90. The composition of claim 89, wherein the linker is a PEG based linker.
91. A method of treating, ameliorating, or preventing a hyperproliferative disease or fibrosis (e.g., liver fibrosis, lung fibrosis, keloids, etc.) in a patient comprising administering to said patient a therapeutically effective amount of a compound as recited in claim 1, 24 or 48 or a stapled peptide of claim 78.
92. The method of claim 91, wherein said hyperproliferative disease is cancer.
93. The method of claim 91, wherein said cancer is a cancer characterized with FAK expression, FAK pathway activation, FAK dependence, FAK activity and/or FAK-paxillin related activity.
94. The method of claim 91 wherein said patient is a human patient.
95. The method of claim 91 further comprising administering to said patient one or more anticancer agents.
96. The method of claim 95 wherein said anticancer agent is a chemotherapeutic agent.
97. The method of claim 96 wherein said anticancer agent is radiation therapy.
98. A kit comprising one or more stapled peptides of claim 78, and/or compounds as recited in claim 1, 24 or 48, and instructions for administration to a patient having a hyperproliferative disease.
99. The kit of claim 98 wherein said hyperproliferative disease is cancer.
100. The kit of claim 99, wherein said cancer is characterized with FAK activity and/or FAK-paxillin related activity.
101. The kit of claim 98 further comprising one or more anticancer agents.
102. The kit of claim 101, wherein said stapled peptide is to be administered together with one or more anticancer agents.
103. A compound having Formula VII: ##STR00016## including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein Y.sub.C—Y.sub.T—SPA- and Y.sub.C—Y.sub.T—SPB— are independently a compound capable of binding with the FAT domain of FAK and inhibit FAK's interaction with a paxillin protein; wherein the C-termini of the two stapled peptides SPA and SPB are linked via tether D.sub.1T.sub.2D.sub.2, wherein D1 and D2 are independently selected from (optionally substituted) nitrogens, thereby being part of amide bonds with the C-termini; contain N, O or S as the atom of attachment; wherein SPA and SPB are synthesized on 2-chlorotrityl chloride resin, or some other resin that would allow for the cleavage from resin of a protected peptide.
104. A compound having Formula VIII: Y.sub.C—Y.sub.T—SPA-T.sub.2-SPB—NH2, including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof; wherein Y.sub.C—Y.sub.T—SPA- and Y.sub.C—Y.sub.T—SPB— are independently a compound capable of binding with the FAT domain of FAK and inhibit FAK's interaction with a paxillin protein.
105. A pharmaceutical composition comprising one or more compounds of claim 1, 24, 48, 72, 73 or 75 within a liposomal formulation.
106. The pharmaceutical composition of claim 105, wherein the liposomal formulation of the LD2 peptide may be consisting of HSPC, Cholesterol, PEG2000-DSPE, DSPC, DOPE, DOTAP, Triolein, EPC, DOPS, POPC, SM, DMPC, DMPG, DOPC, mPEG derivatives, MVL5, DOTMA, DDAB, DC-Cholesterol, GL67, DODMA, Soy phospholipids, cationic lipids, anionic lipids, neutral lipids in various combinations and/or ratios and/or buffer solutions.
107. The pharmaceutical composition of claim 105, wherein the liposomal formulation comprises a combination of one or any combination of sphingomyelin (SM), D-erythrose-sphingomyelin, D-erythrose dihydrosphingomyelin, palmitoylsphingomyelin, lysophospholipids, galactocerebroside, gangliosides, cerebrosides, glycerides, triglycerides, diglycerides, small alkyl chain phospholipids, phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), distearoylphosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerols such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, ceramides, a phosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride, aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipids, and cholesterol and its derivatives, lyso-phosphotydyl choline, lyso-sphingomyelin, dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], Lyso phoshphatidic acid, Lyso phosphatidylcholine, OA-NO.sub.2 (nitrated oleic acid 9- and 10-nitro-cis-octedecenolic acids), LNO.sub.2 (nitrated linoleic Acid 9-, 10-, 12- and 13-nitro-cis-octedecadienoic acids), AA-NO.sub.2 (nitrated Arachidonic Acid 5-, 6-, 8-, 9-, 11-, 12-, 14-, and 15-nitro-cis-eicosatetraenoic acids), CLNO.sub.2 (nitrated cholesteryl linoleate cholestaryl-9-, 10-, 12- and 13-nitro-cis-octedecadiencates), fatty acid, omega-3 polyunsaturated fatty acids, hexadecatrienoic acid (HTA; 16:3 (n-3); all-cis-7,10,13-hexadecatrienoic acid), a-Linolenic acid (ALA; 18:3 (n-3); all-cis-9,12,15-octadecatrienoic acid), stearidonic acid (SDA; 18:4 (n-3); all-cis-6,9,12,15-octadecatetraenoic acid), eicosatrienoic acid (ETE; 20:3 (n-3); all-cis-11,14,17-eicosatrienoic acid), eicosatetraenoic acid (ETA; 20:4 (n-3); all-cis-8,11,14,17-eicosatetraenoic acid), eicosapentaenoic acid (EPA; 20:5 (n-3); all-cis-5,8,11,14,17-eicosapentaenoic acid), heneicosapentaenoic acid (HPA; 21:5 (n-3); all-cis-6,9,12,15,18-heneicosapentaenoic acid); docosapentaenoic acid (DPA; clupanodonic acid; 22:5 (n-3); all-cis-7,10,13,16,19-docosapentaenoic acid), docosahexaenoic acid (DHA; 22:6 (n-3); all-cis-4,7,10,13,16,19-docosahexaenoic acid), tetracosapentaenoic acid; 24:5 (n-3); all-cis-9,12,15,18,21-tetracosapentaenoic acid), tetracosahexaenoic acid (Nisinic acid; 24:6 (n-3), all-cis-6,9,12,15,18,21-tetracosahexaenoic acid), sphingosine-1-phosphate analogs, sphingosine-1-phosphate antagonists, sphingosine-1-phosphate agonists, sphingosine-1-phosphate receptor agonists, sphingosine-1-phosphate receptor antagonists, and sphingosine-1-phosphate receptor analogs.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0218] Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in numerous tumors including melanoma, breast, colon, ovarian, pancreatic, glioblastoma, and others (1), and has been shown to be a necessary component for human cancer progression (2-4).
[0219] From a biological standpoint, FAK is involved in motility, invasion, angiocrine signaling, lymphangiogenesis, metastasis, and epithelial-mesenchymal transition (EMT) (5-8). FAK also sequesters and inactivates pro-apoptotic proteins like p53 and RIP to enhance the survival signals necessary for a cancer to invade and metastasize (9, 10). FAK's role in cancer has been confirmed by clinical prognostic studies, genetically-engineered mouse models, and knockout studies (11-15).
[0220] FAK knockdown results in robust activation of apoptosis and growth arrest in cancer cells with no effects in normal cells (16, 17). Conversely, FAK-kinase inhibitors have a partial effect on apoptosis/tumor growth (18-20) and it has been hypothesized that the FAK scaffold is the major modulator of FAK-dependent anti-apoptosis (21). FAK directly binds p53 to suppress p53-mediated apoptosis and disruption of the FAT domain by adenoviral FAK-CD was shown to induce apoptosis in cancer cells (22). It has been shown that FAK-kinase inhibitors do not drastically inhibit FAK phosphorylation at autophosphorylation residue Y397 and Receptor Tyrosine Kinases (RTKs) transphosphorylate FAK as a drug resistance mechanism (23). Furthermore, FAK-kinase inhibitors (e.g., FAK-kinase catalytic domain inhibitors) have shown limited efficacy in Phase I/II clinical trials (24-26).
[0221] The FAT domain is a four-helical bundle at the C-terminus of FAK that contains key residue Y925 and is involved in multiple protein-protein interactions at the focal adhesion site. Numerous data have emerged showing the importance of the Focal Adhesion Targeting (FAT) domain as the initiator of FAK activation through its multiple interactions with paxillin, Leupaxin, CD4, and DCC (27-29). The integrity of the FAT domain is essential for localization of FAK to focal adhesions, association with integrins/RTKs, and downstream FAK signaling (30-32). Mutation of the FAT domain demonstrated dramatic biological effects on metastasis, invasion, and apoptosis (33, 34). As such, experiments conducted during the course of developing embodiments for the present invention hypothesized that peptide inhibitors that target the FAT domain will be more efficacious than FAK-kinase inhibitors and less-prone to drug resistance mechanisms.
[0222] Paxillin is a major focal adhesion adaptor protein that integrates key cytoskeletal proteins and signaling molecules such as Vinculin, FAK, Actin, Src, and Crk (35). FAK localization to the focal adhesion is mediated by the FAK-paxillin interaction and mutation of the binding site was shown have drastic effects on FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and invasion (34, 36). Paxillin contains two alpha helical LD motifs (LD2 and LD4) that are required for binding to the FAT domain of FAK. The FAT-paxillin LD2/LD4 interaction is well-characterized, has a K.sub.D of 50-100 μM, and has been validated by multiple orthogonal assays (X-ray, SPR, ITC, NMR, FP, and mutagenesis). LD2 and LD4 interact at two separate hydrophobic patches on the FAT domain (Helix 1-4, Helix 2-3) and disruption of both sites is required for maximal biological effect (27, 37, 38).
[0223] Experiments conducted during the course of developing embodiments for the present invention synthesized and optimized peptides capable of effectively targeting FAK non-catalytic function through binding of the FAT domain and thereby inhibiting, for example, FAK-paxillin interaction (e.g., FAK-LD2 domain of paxillin). In particular, the present invention provides stapled LD2 domain peptides capable of inhibiting FAK-paxillin interaction. These LD2 peptides represent a significant advantage over existing FAK inhibitors due to the ability to disrupt FAK protein-protein interactions (PPIs) and therefore provide novel anti-cancer effects.
[0224] As such, the present invention provides a new class of peptides which function as inhibitors of focal adhesion kinase (FAK) activity through binding with its focal adhesion targeting (FAT) domain and thereby inhibiting FAK-paxillin interaction (e.g., LD2 peptides). Indeed, in certain embodiments, the present invention provides LD2 peptides capable of inhibiting FAK-paxillin interaction. In some embodiments, such LD2 peptides are amphipathic alpha-helical stapled peptides comprising hydrophobic amino acids and hydrophilic amino acids, wherein two or more amino acids of the peptide are connected to each other.
[0225] As used herein, the term “stapled peptide” means that peptide regions are connected to each other. In some embodiments, in order to increase the chemical stability and secondary structure of alpha-helices, the i position and i+ position of the alpha-helix can be stapled using various covalent bonding methods. Specifically, the amino acids at one or more positions selected from the group consisting of i, i+3, i+4, i+7, i+8, i+10 and i+11 (where i is an integer) may be stapled. Amino acids may be stapled by a covalent bond and linker moeity to thereby increase the cell-penetrating ability. In some cases, two or more amino acid positions selected from the group consisting of i, i+3, i+4, i+7, i+8, i+10 and i+11 (where i is an integer) may be stapled.
[0226] Typically, two amino acids may be connected to each other by a disulfide bond, a carbon-carbon double bond, an azide-alkyne cycloaddition, or an amide bond. Examples of the method for linking two amino acids to each other include introduction of disulfide between two amino acid positions, introduction of a carbon-carbon double bond by a metathesis reaction, introduction of an amide bond, introduction of a short linker by the Michael reaction, and the like. Such stapling makes it highly possible to prepare a cell-penetrating peptide having an improved cell-penetrating ability and desired chemical stability.
[0227] If a peptide is alpha-helical, two or more amino acids of the peptide can be connected to each other exclusive of the basic peptide backbone, creating a cyclic structure. The size of the cyclic ring may vary depending on the position of the amino acid and length of the linking moiety. One or more staples may be contained in the peptide.
[0228] In a particular embodiment of the present invention, one or more amino acids of the peptide may be functionalized with a double bond-containing compound. For example, amino acids can be connected to each other by a ring structure produced by a ring-closing metathesis between double bond-containing compounds. The functionalized amino acids may be amino acids substituted with an alkenyl side-chain. The alkenyl side-chain may be one or more selected from the group consisting of 2-propylenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 6-heptenyl, 7-octenyl, 8-nonenyl, 9-decenyl, 10-undecenyl and 11-dodecenyl groups.
[0229] The amino acids of the peptide are not specifically limited as long as they can maintain the α-helical structure while showing amphipathic properties. For example, the hydrophilic amino acid may be one or more selected from the group consisting of arginine, lysine, and histidine, and the hydrophobic amino acid may be one or more selected from the group consisting of leucine, valine, tryptophan, phenylalanine, tyrosine, and isoleucine. Non-natural amino acids may also be utilized in the peptide structure.
[0230] Specifically, in an example of the present invention, an amphipathic alpha-helical stapled peptide capable of inhibiting FAK-paxillin interaction was generated.
[0231] As noted, in certain embodiments, the present invention provides LD2 peptides capable of inhibiting FAK-paxillin interaction. In some embodiments, such LD2 peptides are amphipathic alpha-helical stapled peptides comprising hydrophobic amino acids and hydrophilic amino acids, wherein two or more amino acids of the peptide are connected to each other.
[0232] Specifically, such stapled LD2 peptides capable of inhibiting FAK-paxillin interaction may comprise any one of the following sequences, and are produced by introducing the carbon-carbon double bond through a metathesis reaction. In the following sequences, R.sub.8 denotes (R)-2-(7′-octenyl) alanine, S.sub.5 denotes (S)-2-(4′-pentenyl) alanine and R.sub.5 denotes (R)-2-(4′-pentenyl) alanine.
[0233] In some embodiments, two or more of the LD2 peptides are tethered together with a linker (e.g., a PEG based linker) that is constructed via chemistries that are compatible with the various functionalities and solvent and known to those skilled in the art (e.g., linked via “click” chemistry)).
[0234] In some embodiments, the LD2 peptides are further conjugated with an imaging agent (e.g., conjugated with (5-/6-)carboxytetramethylrhodamine (TAMRA)).
[0235] An important aspect of the present invention is that the LD2 peptides of the invention induce cell cycle arrest and/or apoptosis and also potentiate the induction of cell cycle arrest and/or apoptosis either alone or in response to additional apoptosis induction signals (e.g., through inhibiting FAK non-catalytic activity) (e.g., through inhibiting FAK-paxillin interaction). Therefore, it is contemplated that such LD2 peptides sensitize cells to induction of cell cycle arrest and/or apoptosis, including cells that are resistant to such inducing stimuli. The LD2 peptides of the present invention can be used to induce apoptosis in any disorder that can be treated, ameliorated, or prevented by the induction of apoptosis. In one embodiment, the LD2 peptides can be used to induce apoptosis in cells comprising functional FAK activity. In one embodiment, the LD2 peptides can be used to inhibit cancer metastasis. In one embodiment, the LD2 peptides can be used to inhibit angiogenesis.
[0236] In some embodiments, the compositions and methods of the present invention are used to treat diseased cells, tissues, organs, or pathological conditions and/or disease states in an animal (e.g., a mammalian patient including, but not limited to, humans and veterinary animals). In this regard, various diseases and pathologies are amenable to treatment or prophylaxis using the present methods and compositions. A non-limiting exemplary list of these diseases and conditions includes, but is not limited to, pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like, T and B cell mediated autoimmune diseases; inflammatory diseases; infections; hyperproliferative diseases; AIDS; degenerative conditions, vascular diseases, and the like. In some embodiments, the cancer cells being treated are metastatic. In other embodiments, the cancer cells being treated are resistant to anticancer agents. In other embodiments, the disorder is any disorder having cells having FAK activity and/or FAK-paxillin related activity.
[0237] Some embodiments of the present invention provide methods for administering an effective amount of an LD2 peptide of the invention and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g., surgical intervention, and/or radiotherapies). In a particular embodiment, the additional therapeutic agent(s) is an anticancer agent.
[0238] A number of suitable anticancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis; polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for co-administration with the disclosed stapled peptides (LD2 peptides) are known to those skilled in the art.
[0239] In certain embodiments, anticancer agents comprise agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitor, vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); BCL-2 family inhibitors (VENCLEXTA); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs (e.g., Butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.
[0240] In still other embodiments, the compositions and methods of the present invention provide an LD2 peptide of the invention and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).
[0241] Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenoimid-azolecarboxamide).
[0242] In some embodiments, antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2′-deoxycoformycin)).
[0243] In still further embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).
[0244] Any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies. Table 4 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the “product labels” required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.
TABLE-US-00002 TABLE 4 Aldesleukin Proleukin Chiron Corp., (des-alanyl-1, serine-125 human interleukin-2) Emeryville, CA Alemtuzumab Campath Millennium and ILEX (IgG1κ anti CD52 antibody) Partners, LP, Cambridge, MA Alitretinoin Panretin Ligand Pharmaceuticals, (9-cis-retinoic acid) Inc., San Diego CA Allopurinol Zyloprim GlaxoSmithKline, (1,5-dihydro-4 H -pyrazolo[3,4-d]pyrimidin-4- Research Triangle Park, one monosodium salt) NC Altretamine Hexalen US Bioscience, West (N,N,N′,N′,N″,N″,- hexamethyl-1,3,5-triazine- Conshohocken, PA 2, 4, 6-triamine) Amifostine Ethyol US Bioscience (ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester)) Anastrozole Arimidex AstraZeneca (1,3-Benzenediacetonitrile, a, a, a′, a′- Pharmaceuticals, LP, tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl)) Wilmington, DE Arsenic trioxide Trisenox Cell Therapeutic, Inc., Seattle, WA Asparaginase Elspar Merck & Co., Inc., (L-asparagine amidohydrolase, type EC-2) Whitehouse Station, NJ BCG Live TICE BCG Organon Teknika, Corp., (lyophilized preparation of an attenuated strain Durham, NC of Mycobacterium bovis (Bacillus Calmette- Gukin [BCG], substrain Montreal) bexarotene capsules Targretin Ligand Pharmaceuticals (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8- pentamethyl-2-napthalenyl) ethenyl] benzoic acid) bexarotene gel Targretin Ligand Pharmaceuticals Bleomycin Blenoxane Bristol-Myers Squibb (cytotoxic glycopeptide antibiotics produced by Co., NY, NY Streptomyces verticillus; bleomycin A.sub.2 and bleomycin B.sub.2) Capecitabine Xeloda Roche (5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]- cytidine) Carboplatin Paraplatin Bristol-Myers Squibb (platinum, diammine [1,1- cyclobutanedicarboxylato(2-)-O, O′]-,(SP-4-2)) Carmustine BCNU, BiCNU Bristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea) Carmustine with Polifeprosan 20 Implant Gliadel Wafer Guilford Pharmaceuticals, Inc., Baltimore, MD Celecoxib Celebrex Searle Pharmaceuticals, (as 4-[5-(4-methylphenyl)-3- (trifluoromethyl)- England 1H-pyrazol-1-yl] benzenesulfonamide) Chlorambucil Leukeran GlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin Platinol Bristol-Myers Squibb (PtCl.sub.2H.sub.6N.sub.2) Cladribine Leustatin, 2- R. W. Johnson (2-chloro-2′-deoxy-b-D-adenosine) CdA Pharmaceutical Research Institute, Raritan, NJ Cyclophosphamide Cytoxan, Neosar Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino] tetrahydro-2H- 13,2-oxazaphosphorine 2-oxide monohydrate) Cytarabine Cytosar-U Pharmacia & Upjohn (1-b-D-Arabinofuranosylcytosine, C.sub.9H.sub.13N.sub.3O.sub.5) Company cytarabine liposomal DepoCyt Skye Pharmaceuticals, Inc., San Diego, CA Dacarbazine DTIC-Dome Bayer AG, Leverkusen, (5-(3,3-dimethyl-1-triazeno)-imidazole-4- Germany carboxamide (DTIC)) Dactinomycin, actinomycin D Cosmegen Merck (actinomycin produced by Streptomyces parvullus, C.sub.62H.sub.86N.sub.12O.sub.16) Darbepoetin alfa (recombinant peptide) Aranesp Amgen, Inc., Thousand Oaks, CA daunorubicin liposomal DanuoXome Nexstar ((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy- Pharmaceuticals, Inc., á-L-lyxo-hexopyranosyl)oxy]-7,8,9,10- Boulder, CO tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedione hydrochloride) Daunorubicin HCl, daunomycin Cerubidine Wyeth Ayerst, Madison, ((1 S ,3 S)-3-Acetyl-1,2,3,4,6,11-hexahydro- NJ 3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1- naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)- L- lyxo -hexopyranoside hydrochloride) Denileukin diftitox Ontak Seragen, Inc., (recombinant peptide) Hopkinton, MA Dexrazoxane Zinecard Pharmacia & Upjohn ((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- Company piperazinedione) Docetaxel Taxotere Aventis ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert- Pharmaceuticals, Inc., butyl ester, 13-ester with 5b-20-epoxy- Bridgewater, NJ 12a,4,7b,10b,13a-hexahydroxytax- 11-en-9-one 4-acetate 2-benzoate, trihydrate) Doxorubicin HCl Adriamycin, Pharmacia & Upjohn (8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L- Rubex Company lyxo-hexopyranosyl)oxy] -8-glycolyl-7,8,9,10- tetrahydro-6,8,11- trihydroxy-1-methoxy-5,12- naphthacenedione hydrochloride) doxorubicin Adriamycin PFS Pharmacia & Upjohn Intravenous Company injection doxorubicin liposomal Doxil Sequus Pharmaceuticals, Inc., Menlo park, CA dromostanolone propionate Dromostanolone Eli Lilly & Company, (17b-Hydroxy-2a-methyl-5a-androstan-3-one Indianapolis, IN propionate) dromostanolone propionate Masterone Syntex, Corp., Palo injection Alto, CA Elliott′s B Solution Elliott's B Orphan Medical, Inc Solution Epirubicin Ellence Pharmacia & Upjohn ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L- Company arabino- hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl)-1-methoxy-5,12- naphthacenedione hydrochloride) Epoetin alfa Epogen Amgen, Inc (recombinant peptide) Estramustine Emcyt Pharmacia & Upjohn (estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3- Company [bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17- (dihydrogen phosphate), disodium salt, monohydrate) Etoposide phosphate Etopophos Bristol-Myers Squibb (4′-Demethylepipodophyllotoxin 9-[4,6-O-(R)- ethylidene-(beta)-D-glucopyranoside], 4′- (dihydrogen phosphate)) etoposide, VP-16 Vepesid Bristol-Myers Squibb (4′-demethylepipodophyllotoxin 9-[4,6-0-(R)- ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia & Upjohn (6-methylenandrosta-1,4-diene-3, 17-dione) Company Filgrastim Neupogen Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDR Roche (2′-deoxy-5-fluorouridine) Fludarabine Fludara Berlex Laboratories, (fluorinated nucleotide analog of the antiviral Inc., Cedar Knolls, NJ agent vidarabine, 9-b -D- arabinofuranosyladenine (ara-A)) Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals, (5-fluoro-2,4(1H,3H)-pyrimidinedione) Inc., Humacao, Puerto Rico Fulvestrant Faslodex IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-penta Guayama, Puerto Rico fluoropentylsulphinyl) nonyl]estra-1,3,5-(10)- triene-3,17-beta-diol) Gemcitabine Gemzar Eli Lilly (2′-deoxy-2′, 2′-difluorocytidine monohydrochloride (b-isomer)) Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33 hP67.6) Goserelin acetate Zoladex Implant AstraZeneca Pharmaceuticals Hydroxyurea Hydrea Bristol-Myers Squibb Ibritumomab Tiuxetan Zevalin Biogen IDEC, Inc., (immunoconjugate resulting from a thiourea Cambridge MA covalent bond between the monoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)- propyl]-[N-[2- bis(carboxymethyl)amino]-2-(methyl) - ethyl]glycine) Idarubicin Idamycin Pharmacia & Upjohn (5, 12-Naphthacenedione, 9-acetyl-7-[(3- Company amino-2,3,6-trideoxy-(alpha)-L- lyxo - hexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,9,11-trihydroxyhydrochloride, (7S- cis )) Ifosfamide IFEX Bristol-Myers Squibb (3-(2-chloroethyl)-2-[(2- chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide) Imatinib Mesyilate Gleevec Novartis AG, Basel, (4-[(4-Methyl-1-piperazinyl)methyl]-N-[4- Switzerland methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-phenyl]benzamide methanesulfonate) Interferon alfa-2a Roferon-A Hoffmann-La Roche, (recombinant peptide) Inc., Nutley, NJ Interferon alfa-2b Intron A Schering AG, Berlin, (recombinant peptide) (Lyophilized Germany Betaseron) Irinotecan HCl Camptosar Pharmacia & Upjohn ((4S)-4,11-diethyl-4-hydroxy-9-[(4- piperi- Company dinopiperidino)carbonyloxy]-1H-pyrano[3′, 4′: 6,7] indolizino[1,2-b] quinoline-3,14(4H, 12H) dione hydrochloride trihydrate) Letrozole Femara Novartis (4,4′-(1H-1,2,4 -Triazol-1-ylmethylene) dibenzonitrile) Leucovorin Wellcovorin, Immunex, Corp., Seattle, Leucovorin WA (L-Glutamic acid, N[4[[(2amino-5-formyl- 1,4,5,6,7,8 hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium salt (1:1)) Levamisole HCl Ergamisol Janssen Research ((−)-(S)-2,3,5, 6-tetrahydro-6-phenylimidazo Foundation, Titusville, [2,1-b] thiazole monohydrochloride NJ C.sub.11H.sub.12N.sub.2S•HCl) Lomustine CeeNU Bristol-Myers Squibb (1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Meclorethamine, nitrogen mustard Mustargen Merck (2-chloro-N-(2-chloroethyl)-N- methylethanamine hydrochloride) Megestrol acetate Megace Bristol-Myers Squibb 17α(acetyloxy)- 6 -methylpregna- 4,6- diene- 3,20- dione Melphalan, L-PAM Alkeran GlaxoSmithKline (4-[bis(2-chloroethyl) amino]-L-phenylalanine) Mercaptopurine, 6-MP Purinethol GlaxoSmithKline (1,7-dihydro-6 H -purine-6-thione monohydrate) Mesna Mesnex Asta Medica (sodium 2-mercaptoethane sulfonate) Methotrexate Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid) Methoxsalen Uvadex Therakos, Inc., Way (9-methoxy-7H-furo[3,2-g][1]-benzopyran-7- Exton, Pa one) Mitomycin C Mutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc., Dublin, CA Mitotane Lysodren Bristol-Myers Squibb (1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane) Mitoxantrone Novantrone Immunex Corporation (1,4-dihydroxy-5,8-bis[[2- [(2- hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione dihydrochloride) Nandrolone phenpropionate Durabolin-50 Organon, Inc., West Orange, NJ Nofetumomab Verluma Boehringer Ingelheim Pharma KG, Germany Oprelvekin Neumega Genetics Institute, Inc., (IL-11) Alexandria, VA Oxaliplatin Eloxatin Sanofi Synthelabo, Inc., (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] NY, NY [oxalato(2-)-O,O′] platinum) Paclitaxel TAXOL Bristol-Myers Squibb (5β, 20-Epoxy-1,2a, 4,7β, 10β, 13a- hexahydroxytax-11-en-9-one 4,10-diacetate 2- benzoate 13-ester with (2R, 3 S)- N-benzoyl-3- phenylisoserine) Pamidronate Aredia Novartis (phosphonic acid (3-amino-1- hydroxypropylidene) bis-, disodium salt, pentahydrate, (APD)) Pegademase Adagen Enzon Pharmaceuticals, ((monomethoxypolyethylene glycol (Pegademase Inc., Bridgewater, NJ succinimidyl) 11 - 17 -adenosine deaminase) Bovine) Pegaspargase Oncaspar Enzon (monomethoxypolyethylene glycol succinimidyl L-asparaginase) Pegfilgrastim Neulasta Amgen, Inc (covalent conjugate of recombinant methionyl human G-CSF (Filgrastim) and monomethoxypolyethylene glycol) Pentostatin Nipent Parke-Davis Pharmaceutical Co., Rockville, MD Pipobroman Vercyte Abbott Laboratories, Abbott Park, IL Plicamycin, Mithramycin Mithracin Pfizer, Inc., NY, NY (antibiotic produced by Streptomyces plicatus) Porfimer sodium Photofrin QLT Phototherapeutics, Inc., Vancouver, Canada Procarbazine Matulane Sigma Tau (N-isopropyl-μ-(2-methylhydrazino)-p- Pharmaceuticals, Inc., toluamide monohydrochloride) Gaithersburg, MD Quinacrine Atabrine Abbott Labs (6-chloro-9-(1 -methyl-4-diethyl-amine) butylamino-2-methoxyacridine) Rasburicase Elitek Sanofi-Synthelabo, Inc., (recombinant peptide) Rituximab Rituxan Genentech, Inc., South (recombinant anti-CD20 antibody) San Francisco, CA Sargramostim Prokine Immunex Corp (recombinant peptide) Streptozocin Zanosar Pharmacia & Upjohn (streptozocin 2 -deoxy - 2 - Company [[(methylnitrosoamino)carbonyl]amino] - a(and b ) - D - glucopyranose and 220 mg citric acid anhydrous) Talc Sclerosol Bryan, Corp., Woburn, (Mg.sub.3Si.sub.4O.sub.10 (OH).sub.2) MA Tamoxifen Nolvadex AstraZeneca ((Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N, Pharmaceuticals N-dimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1)) Temozolomide Temodar Schering (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as- tetrazine-8-carboxamide) teniposide, VM-26 Vumon Bristol-Myers Squibb (4′-demethylepipodophyllotoxin 9-[4,6-0-(R)- 2- thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac Bristol-Myers Squibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4- dien-17-oic acid [dgr]-lactone) Thioguanine, 6-TG Thioguanine GlaxoSmithKline (2-amino-1,7-dihydro-6 H - purine-6-thione) Thiotepa Thioplex Immunex Corporation (Aziridine, 1,1′,1″-phosphinothioylidynetris-, or Tris (1-aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin GlaxoSmithKline ((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9- dihydroxy-1H-pyrano[3′, 4′: 6,7] indolizino [1,2-b] quinoline-3,14-(4H,12H)-dione monohydrochloride) Toremifene Fareston Roberts Pharmaceutical (2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]- Corp., Eatontown, NJ phenoxy)-N,N-dimethylethylamine citrate (1:1)) Tositumomab, I 131 Tositumomab Bexxar Corixa Corp., Seattle, (recombinant murine immunotherapeutic WA monoclonal IgG.sub.2a lambda anti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody)) Trastuzumab Herceptin Genentech, Inc (recombinant monoclonal IgG.sub.1 kappa anti- HER2 antibody) Tretinoin, ATRA Vesanoid Roche (all-trans retinoic acid) Uracil Mustard Uracil Mustard Roberts Labs Capsules Valrubicin, N-trifluoroacetyladriamycin-14- Valstar Anthra --> Medeva valerate ((2S-cis)-2- [1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7 methoxy-6,11-dioxo-[[4 2,3,6- trideoxy-3- [(trifluoroacetyl)-amino-α-L-lyxo- hexopyranosyl]oxyl]-2-naphthacenyl]-2- oxoethyl pentanoate) Vinblastine, Leurocristine Velban Eli Lilly (C.sub.46H.sub.56N.sub.4O.sub.10•H.sub.2SO.sub.4) Vincristine Oncovin Eli Lilly (C.sub.46H.sub.56N.sub.4O.sub.10•H.sub.2SO.sub.4) Vinorelbine Navelbine GlaxoSmithKline (3′ ,4′-didehydro-4′-deoxy-C′- norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronic acid Zometa Novartis ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid monohydrate)
[0245] Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, eflornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hu14.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab, mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS-9, 06-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB—, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.
[0246] For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's “Pharmaceutical Basis of Therapeutics” tenth edition, Eds. Hardman et al., 2002.
[0247] The present invention provides methods for administering an LD2 peptide of the invention with radiation therapy. The invention is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal. For example, the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.
[0248] Antimicrobial therapeutic agents may also be used as therapeutic agents in the present invention. Any agent that can kill, inhibit, or otherwise attenuate the function of microbial organisms may be used, as well as any agent contemplated to have such activities. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptive agents and the like, used alone or in combination. Indeed, any type of antibiotic may be used including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like.
[0249] In some embodiments of the present invention, an LD2 peptide of the invention and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, the LD2 peptide is administered prior to the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of the therapeutic or anticancer agent. In some embodiments, the LD2 peptide is administered after the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of the anticancer agent. In some embodiments, the LD2 peptide and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., the LD2 peptide is administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the LD2 peptide is administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.
[0250] Compositions within the scope of this invention include all compositions wherein the LD2 peptides of the present invention are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
[0251] In addition to administering the stapled peptide (e.g., LD2 peptide) as a raw peptide, the LD2 peptides of the invention may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the LD2 peptides into preparations which can be used pharmaceutically. The preparations, particularly those preparations which can be administered orally or topically and which can be used for one type of administration, such as tablets, dragees, slow release lozenges and capsules, mouth rinses and mouth washes, gels, liquid suspensions, hair rinses, hair gels, shampoos and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by intravenous infusion, injection, topically or orally, contain from about 0.01 to 99 percent, in one embodiment from about 0.25 to 75 percent of active peptide(s), together with the excipient. In some embodiments, the formulation of the LD2 peptide can also be liposomal in nature. In some embodiments, the liposomal formulation of the peptides (e.g., LD2, LD4) may be consisting of HSPC, Cholesterol, PEG2000-DSPE, DSPC, DOPE, DOTAP, Triolein, EPC, DOPS, POPC, SM, DMPC, DMPG, DOPC, mPEG derivatives, MVL5, DOTMA, DDAB, DC-Cholesterol, GL67, DODMA, Soy phospholipids, cationic lipids, anionic lipids, neutral lipids in various combinations and/or ratios and/or buffer solutions. In some embodiments, the liposomal formulation of the peptides (e.g., LD2, LD4) may comprise a combination of one or any combination of sphingomyelin (SM), D-erythrose-sphingomyelin, D-erythrose dihydrosphingomyelin, palmitoylsphingomyelin, lysophospholipids, galactocerebroside, gangliosides, cerebrosides, glycerides, triglycerides, diglycerides, small alkyl chain phospholipids, phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), distearoylphosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerols such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, ceramides, a phosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride, aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipids, and cholesterol and its derivatives, lyso-phosphotydyl choline, lyso-sphingomyelin, dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyObutyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], Lyso phoshphatidic acid, Lyso phosphatidylcholine, OA-NO2 (nitrated oleic acid 9- and 10-nitro-cis-octedecenolic acids), LNO.sub.2 (nitrated linoleic Acid 9-, 10-, 12- and 13-nitro-cis-octedecadienoic acids), AA-NO.sub.2 (nitrated Arachidonic Acid 5-, 6-, 8-, 9-, 11-, 12-, 14-, and 15-nitro-cis-eicosatetraenoic acids), CLNO.sub.2 (nitrated cholesteryl linoleate cholestaryl-9-, 10-, 12- and 13-nitro-cis-octedecadiencates), fatty acid, omega-3 polyunsaturated fatty acids, hexadecatrienoic acid (HTA; 16:3 (n-3); all-cis-7,10,13-hexadecatrienoic acid), a-Linolenic acid (ALA; 18:3 (n-3); all-cis-9,12,15-octadecatrienoic acid), stearidonic acid (SDA; 18:4 (n-3); all-cis-6,9,12,15-octadecatetraenoic acid), eicosatrienoic acid (ETE; 20:3 (n-3); all-cis-11,14,17-eicosatrienoic acid), eicosatetraenoic acid (ETA; 20:4 (n-3); all-cis-8,11,14,17-eicosatetraenoic acid), eicosapentaenoic acid (EPA; 20:5 (n-3); all-cis-5,8,11,14,17-eicosapentaenoic acid), heneicosapentaenoic acid (HPA; 21:5 (n-3); all-cis-6,9,12,15,18-heneicosapentaenoic acid); docosapentaenoic acid (DPA; clupanodonic acid; 22:5 (n-3); all-cis-7,10,13,16,19-docosapentaenoic acid), docosahexaenoic acid (DHA; 22:6 (n-3); all-cis-4,7,10,13,16,19-docosahexaenoic acid), tetracosapentaenoic acid; 24:5 (n-3); all-cis-9,12,15,18,21-tetracosapentaenoic acid), tetracosahexaenoic acid (Nisinic acid; 24:6 (n-3), all-cis-6,9,12,15,18,21-tetracosahexaenoic acid), sphingosine-1-phosphate analogs, sphingosine-1-phosphate antagonists, sphingosine-1-phosphate agonists, sphingosine-1-phosphate receptor agonists, sphingosine-1-phosphate receptor antagonists, and sphingosine-1-phosphate receptor analogs.
[0252] The pharmaceutical compositions of the invention may be administered to any patient which may experience the beneficial effects of the stapled peptides (LD2 peptides) of the invention. Foremost among such patients are mammals, e.g., humans, although the invention is not intended to be so limited. Other patients include veterinary animals (cows, sheep, pigs, horses, dogs, cats and the like).
[0253] The LD2 peptides and pharmaceutical compositions thereof may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
[0254] The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active LD2 peptides with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
[0255] Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
[0256] Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active stapled peptides (e.g., LD2 peptides) in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active stapled peptides (e.g., LD2 peptides) are in one embodiment dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be added.
[0257] Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active peptides with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active stapled peptides (e.g., LD2 peptides) with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
[0258] Suitable formulations for parenteral administration include aqueous solutions of the active peptides in water-soluble form, for example, water-soluble salts and alkaline solutions. In addition, suspensions of the active stapled peptides (e.g., LD2 peptides) as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
[0259] The topical compositions of this invention are formulated in one embodiment as oils, creams, lotions, ointments and the like by choice of appropriate carriers. Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C12). The carriers may be those in which the active ingredient is soluble. Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired. Additionally, transdermal penetration enhancers can be employed in these topical formulations. Examples of such enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762; each herein incorporated by reference in its entirety.
[0260] Ointments may be formulated by mixing a solution of the active ingredient in a vegetable oil such as almond oil with warm soft paraffin and allowing the mixture to cool. A typical example of such an ointment is one which includes about 30% almond oil and about 70% white soft paraffin by weight. Lotions may be conveniently prepared by dissolving the active ingredient, in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.
[0261] One of ordinary skill in the art will readily recognize that the foregoing represents merely a detailed description of certain preferred embodiments of the present invention. Various modifications and alterations of the compositions and methods described above can readily be achieved using expertise available in the art and are within the scope of the invention.
Examples
[0262] The following examples are illustrative, but not limiting, of the stapled peptides (e.g., LD2 peptides), compositions, and methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.
Example I
[0263] This example describes the generation of peptides which have an affinity for the FAT domain of FAK, and which are capable of blocking the interaction of paxillin with focal adhesion kinase (FAK), thereby inhibiting FAK activity related to FAK-paxillin interaction.
[0264] To develop inhibitors of the FAK-paxillin interaction experiments were conducted that took a stapled peptide approach which has the following advantages: (1) using the native peptide as a starting point, (2) spanning the entire interaction interface, (3) having a chemical moiety to enhance alpha helicity, cell permeability, and proteolytic stability, and (4) a workable molecule for straight-forward SAR analysis (amide chemistry).
[0265] Experiments were conducted that first started with synthesis of a series of cyclic peptides based on the paxillin LD2 motif and all-hydrocarbon stapling (
[0266] There are three major approaches for hydrocarbon stapling of alpha helical peptides: (1) i, i+3, (2) i, i+4, and (3) i, i+7, each having the potential for different biological effects. Experiments were conducted that synthesized various iterations of these i motifs (staple scanning) at different positions (shifting N or C terminal), stapling at residues present at both the hydrophobic interface and solvent-accessible area. Experiments were conducted that extended residues on the N- and C-terminus of the peptide, as these amino acids can have a beneficial effect on both binding affinity and cell permeability. Finally, to enhance protein contacts, enhance permeability, and further inhibit recognition by proteases, experiments were conducted that substituted natural amino acids with hydrophobic/polar/charged substitutions, non-natural amino acids, or D-amino acids (39) (
[0267] To understand the structure-activity relationships of FAK stapled peptides, experiments were designed and 36 peptides were synthesized (Table 2) that varied based on staple strategy ((i, i+3), (i, i+4), and (i, i+7)), staple position, sequence length, amino acid composition, and homologous sequences (LD2, LD4, CD4, DCC, Leupaxin). Stapled peptide UACC-1907
##STR00008##
was identified which gave a K.sub.D of 3.4 μM in SPR affinity analysis, showed very high selectivity for WT/mutant protein in SPR, and competitively inhibited paxillin-FAT binding in fluorescence polarization (FP) assays (K.sub.i=5.8 μM) (
[0268] The specific staple position of 1907 was essential for biochemical/biophysical activity, as 1905, with the same i+7 stapling motif shifted just one amino acid away, had no binding and inhibitory properties. Other LD2 peptides with the same i+7 stapling motif in different positions (1914, 2017) also showed limited activity compared to 1907. LD2 peptides with different stapling motifs (1919—(i, i+4), 1921—(i, i+3), 2015—(i, i+4), 2022—(i, i+3), 2024—(i, i+3)) did not show much activity compared to 1907. Also, a peptide with the Aib (2-Aminoisobutyric acid)-based alpha-helical stabilization strategy, 1912, had lower activity compared to 1907. Amino acid sequence was also very important for activity, as 1910, with two glutamic acid to glutamine conversions, was not as active as 1907. Leucines 145, 149, and 152 (based off paxillin sequence) were very important for activity, as peptides with conversions to tryptophan (1933, 2007) had worse binding and inhibitory properties compared to 1907. Peptide 2014, with a L.sub.145E and L.sub.152E substitution, showed completely no binding and inhibitory properties. Overall, stapled peptides based off homologous protein sequences (1929, 1916) did not have comparable activity to LD2-derived 1907. Paxillin LD4-derived peptide 1917 had similar, albeit lower activity than LD2-derived 1907. The length of stapled peptide 1907 was also crucial for activity, as 2011, a truncated stapled peptide at the n- and c-terminus, had no binding and inhibitory properties. An extended peptide, 1920, had a modest binding affinity (K.sub.D=7.0 μM), however worse inhibitory properties (K.sub.i=25.0 μM) compared to 1907. These data showed that an empirically derived optimal stapling chemistry and amino acid sequence was required for best FAK binding and inhibition.
[0269] To further evaluate the activity of 1907, experiments were conducted that tested the peptide in a flow cytometry-based cell permeability assay. In this assay, Rhodamine-tagged 1907 successfully crossed the cell membrane in MDA-MB-453 breast cancer cells compared to Rhodamine only control (
[0270] Further structural biology experiments were conducted to test 1907 for binding to the paxillin binding site of the FAK FAT domain. In HSQC NMR experiments with .sup.15N labeled FAT domain protein, 1907 showed specific binding at both the FAT Helix 2-3 (L959, R962, K955) and FAT Helix 1-4 (V928, 1936) binding sites for the native paxillin LD2 peptide (
[0271] To improve the cellular potency of 1907, peptide analogs were designed and synthesized with a myristoyl (2012, 2029) or dodecyl (2025) modification for increased hydrophobicity and cellular uptake (
[0272] As evidenced by SPR, mutagenesis, and NMR data, peptide 1907 has the ability to bind to both the Helix 2-3 and Helix 1-4 binding sites on the FAT domain. To create a multivalent peptide with the ability to bind both sites simultaneously and therefore with nanomolar binding affinity, we designed and synthesized a series of linked peptides (2018, 2021, 2023) via the coupling of two azido-modified 1907 molecules (2006) to a DBCO-[PEG].sub.n-DBCO or an alkyne-[PEG].sub.n-alkyne linker (Table 2,
[0273] To facilitate membrane permeability of peptides 2023 and 1907, in vitro experiments were conducted with liposomal formulation Saint-Protein (Synvolux). A 3:1 volume:volume ratio of Saint:Peptide was used to prepare formulations and peptide concentration was titrated in PBS PH 7.4+1% DMSO. As shown in
[0274] To create bifunctional peptides with the dual-ability to inhibit both the FAK FAT domain and the FAK kinase domain, we designed hybrid peptides based on FAT stapled peptide 1907 and FAK kinase domain inhibitor PF-562271 (PF-271) (
[0275] To create FAT peptides with the ability to degrade FAK protein in cells through the PROteoylsis TArgeting Chimera (PROTAC) approach, we designed hybrid peptides based on FAT stapled peptide 1907 and E3-ligase targeting ligand thalidomide (
[0276] Experiments were conducted to test the protease resistance of synthesized peptides 1967 (native LD2), 1907, and 2012 (
[0277] Experiments were conducted to test the anti-fibrotic effects of stapled peptides targeting the FAK FAT domain (
[0278] Additional stapled peptides were generated. Table 2 provides additional stapled peptides with sequence/structural information, SPR and FP results.
TABLE-US-00003 TABLE 2 Stapled Peptide SAR Analysis. SEQ ID SPR K.sub.d FP K.sub.i NO: Name Sequence (μM) (μM) 1 1967 Ac-NLSELDRLLLELN-NH.sub.2 (Ac-LD.sub.2-NH.sub.2) 156 70.6 2 1905 Ac-NLSR.sub.8LDRLLLS.sub.5LN-NH.sub.2 >200 >1250 3 1906 H-NLSR.sub.5LDRLLNS.sub.5LN-NH.sub.2 >200 >1250 4 1907 Ac-NLR.sub.8ELDRLLS.sub.5ELN-NH.sub.2 3.43 5.80 5 1910 Ac-NLR.sub.8QLDRLLS.sub.5QLN-NH.sub.2 24.8 46.8 6 1912 Ac-NLAibELDRLLAibELN-NH.sub.2 38.7 28.1 7 1913 Ac-DLR.sub.8ELDRLLS.sub.5ELD-NH.sub.2 13.7 21.8 8 1914 Ac-NLSER.sub.8DRLLLES.sub.5N-NH.sub.2 64.3 >1250 9 1919 Ac-NLSES.sub.5DRLS.sub.5LELN-NH.sub.2 68.9 38.6 10 1920 Ac-SLGSNLR.sub.8ELDRLLS.sub.5ELNAVQH-NH.sub.2 6.95 25.0 11 1921 Ac-NLSELDRLR.sub.5LES.sub.5N-NH.sub.2 >200 492 12 1925 Ac-NLSER.sub.8DRAALES.sub.5N-NH.sub.2 >200 492 13 1929 Ac-RRR.sub.8ARLRFMS.sub.5QFY-NH.sub.2 77.6 >1250 14 1931 Ac-NLRES.sub.5DRLS.sub.5RELN-NH.sub.2 13.7 14.5 15 1933 Ac-NLR.sub.8EWDRLLS.sub.5EWN-NH.sub.2 155 281 16 2007 Ac-NLR.sub.8ELDRLWS.sub.5ELN-NH.sub.2 73.1 190 17 2009 Ac-NLR.sub.8ALDRLLS.sub.5ELN-NH.sub.2 6.96 26.7 18 2010 Ac-NLR.sub.8ALDALLS.sub.5ELN-NH.sub.2 12.6 68.5 19 2011 Ac-R.sub.8ELDRLLS.sub.5-NH.sub.2 >200 >333 20 2013 Ac-NLR.sub.8ELDRLLS.sub.5ELN-NH.sub.2 (unstapled) 37.2 20.3 21 2014 Ac-NLR.sub.8EEDRLLS.sub.5EEN-NH.sub.2 >200 >1250 22 2015 Ac-NLSELDRS.sub.5LLES.sub.5N-NH.sub.2 47.5 114 23 2017 Ac-NR.sub.8SELDRLS.sub.5LELN-NH.sub.2 20.3 80.6 24 2022 Ac-NLSER.sub.5DRS.sub.5LLELN-NH.sub.2 911 341 25 2024 Ac-NR.sub.5SES.sub.5DRLLLELN-NH.sub.2 >200 412 26 2028 Ac-NLR.sub.8ELDKLLS.sub.5ELN-NH.sub.2 1.36 (1) NC 10.9 (2)* 27 2006 N.sub.3(CH.sub.2CH.sub.2O).sub.3CH.sub.2CO-NLR.sub.8ELDRLLS.sub.5ELN-NH.sub.2 (Az-1907) 4.58 51.4 28 2012 Myristoyl-NLR.sub.8ELDRLLS.sub.5ELN-NH.sub.2 >200 83.0 29 2020 Myristoyl-NLR.sub.8EEDRLLS.sub.5EEN-NH.sub.2 >200 >1250 30 2029 Ac-NLR.sub.8ELDK(myristoyl)LLS.sub.5ELN-NH.sub.2 19.5 NC 31 2025 4-Dodecyl-(Az-1907) 190 257 32 2018 (Az-1907)-[DBCO-PEG-DBCO]-(Az-1907) 24.2 51.3 33 2021 (Az-1907)-PEG18-(Az-1907) 0.237 NA 34 2023 (Az-1907)-PEG10-(Az-1907) 0.0214 NA 35 2019 Thalidomide-(Az-1907) 10.2 29.1 36 1916 Ac-SATR.sub.8ELDELMS.sub.5SLSD-NH.sub.2 32.3 47.5 37 1917 Ac-SATRES.sub.5DELS.sub.5ASLSD-NH.sub.2 4.90 6.72 Synthesized hydrocarbon stapled peptides were varied based on staple strategy ((i, i + 3); (i, i + 4); and (i, i + 7)), staple position, sequence length, amino acid composition, and homologous sequences. MW, SPR data, and FP data are shown. Note: 1907 and 2023 were identified as top candidates based on data. Abbreviations: R.sub.8: (R)-2-(7-octenyl)alanine; S.sub.5: (S)-2-(4-pentenyl)alanine; R.sub.5: (R)-2-(4-pentenyl)alanine; Az: 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetyl; Aib: 2-aminoisobutyric acid; DBCO: 3-amino-1-(2-azatricyclo[10.4.0.0.sup.4,9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)propan-1-one; *two-site binding model; NC: not yet calculated; NA: Not applicable due to K.sub.i outside range of assay; or derivatives thereof. Unless otherwise stated, the peptides are the product(s) of an intramolecular ring-closing methasis reaction; double bond geometry has not been established or quantified. These sequences are abbreviated and may refer to “click” chemistry products. FIG. 14 shows the structures of peptides P29-P34.
Example II
[0279] This example describes the materials and methods for Example I.
Synthesis
[0280] The compounds described herein were synthesized using Rink amide resin and various combinations of Fmoc-(S)-2-(4-pentenyl)alanine, Fmoc-(R)-2-(4-pentenyl)alanine, Fmoc-(R)-2-(7-octenyl)alanine, N-α-Fmoc-α-aminoisobutyric acid (Fmoc-Aib-OH), Nα-Fmoc-Nε-(azido-PEG4)-L-Lysine, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, and Fmoc-Val-OH. Except for glycine, or as otherwise noted, these amino acids are in the “L” configuration. Standard Fmoc-based solid phase peptide synthesis chemistry, as known by those skilled in the art, was employed. Peptides were synthesized on a Biotage Initiator+ Alstra automated microwave-assisted peptide synthesizer using Rink amide MBHA ChemMatrix resin (typical loading of 0.45 meq/g). N-Fmoc-protected amino acids were used with standard side chain protecting groups. The alkenyl amino acids Fmoc-(R)-2-(7-octenyl)alanine, Fmoc-(S)-2-(4-pentenyl)alanine and Fmoc-(R)-2-(4-pentenyl)alanine were purchased from Advanced ChemTech. Reactions were carried out on a 0.1 mmol scale. Typical conditions follow. Concentrations of 0.5 M for the protected amino acids, HCTU, DIC and Oxyma Pure; 1.0 M for DIPEA and 5.0 M for acetic anhydride in DMF were used. To ensure efficient mixing, reaction volumes of 4 mL were used and DMF was added as needed. A solution of 20% 4-methylpiperidine in DMF was used for Fmoc-deprotection and cleavage/global deprotection was effected using 95:2.5:2.5 (v/v) FA: water:triisopropylsilane. Couplings used 5 eq of amino acid, 5 eq of HCTU and 10 eq of DIPEA except in the cases where the unnatural alkene amino acids were used, where 3:3:6 mole equivalents of amino acid:HCTU:DIPEA were used.
[0281] The resin, provided as the free amine, was first swelled with DMF (70° C. for 20 min). The Fmoc-protected amino acid was coupled at 75° C. for 4 min, then washed with DMF. Double couplings were used for all residues. Arginine was coupled at 50° C. for 6 minutes. A cycle of Fmoc-deprotection followed which consisted of a 3 min reaction, then a 10 min reaction with fresh reagent, at ambient temperature, followed by a DMF wash. Double Fmoc-deprotection cycles were used for N-terminal residues and olefinic amino acids.
[0282] Ring closing metathesis was performed on-machine in a manual injection mode on the Fmoc-protected peptide, using Grubbs' first-generation catalyst (benzylidene-bis(tricyclohexylphosphine)dichlororuthenium). Prior to the reaction, the resin was washed well with cycles of DCM followed by Et.sub.2O, dried briefly in vacuo, then washed and swelled with DCE. Grubbs' I (10 mM) was added then reacted at 40° C. for 1 h, with venting every 15 min. This was performed a total of three times, with DCE used to wash between reactions. Upon completion, the resin was washed with DCE then DCM, then swelled with DMF.
[0283] The Fmoc group was deprotected as above. When N-capped as the acetamide, 50 eq of acetic anhydride and 10 eq of DIPEA were used, and this reacted at ambient temperature for 45 min. Rhodamine B was coupled with 5 eq each of DIC and Oxyma Pure, at 75° C. for 4 min. The resin was washed with three cycles of DCM followed by Et2O, then dried in vacuo.
[0284] Cleavage using 95:2.5:2.5 TFA:water:triisopropylsilane was performed for 2 h at ambient temperature. The reaction solution was then added dropwise to 35 mL of cold Et2O. This was mixed, chilled at −80° C. for 30 min, then centrifuged at 6000×G for 6 min. After decanting the supernatant, the pellet was suspended in 20 mL of Et2O, then chilled, centrifuged and decanted as above. The crude peptide was dried in vacuo. Preparative HPLC was performed on an Agilent 1260 II quaternary HPLC, with a Variable Wavelength detector, using a Zorbax SB-C18 column (Agilent 880975-202; 9.4×250 mm; 80 Å pore size; 5 μm particle size) using a gradient of acetonitrile (0.1% AcOH) in water (0.1% AcOH).
[0285] Analytical HPLC was performed on an Agilent 1200 HPLC, with 1200 DAD and Infinity 6125 LCMSD detectors, using a Zorbax SB-C18 column (Agilent 830990-902; 2.1×150 mm; 80 Å pore size; 3.5 μm particle size) using a gradient of acetonitrile (0.1% AcOH) in water (0.1% AcOH).
##STR00009##
Surface Plasmon Resonance (SPR)
[0286] SPR binding studies were performed on a ForteBio Pioneer FE SPR system. In brief, a SADH Streptavidin in Dextran Hydrogel biosensor (ForteBio) was docked onto the flow cell and preconditioned with two injections of 10 mM NaOH, 1 M NaCl for 1 min at 504/min. Subsequently, biotinylated Avitag-FAT protein was diluted in running buffer (100 mM Tris-HCl, 200 mM NaCl, 0.05% Tween-20) and injected at 10 μL/min to achieve approximately 1,000 RU of immobilized protein on channel 1. Empty channel 2 served as the reference control. After achieving a stable baseline, a concentration series (200 μM-0.01 μM) of peptide was prepared in final running buffer (100 mM Tris-HCl, 200 mM NaCl, 0.05% Tween-20, 5% DMSO) and injected using the OneStep gradient injection method at a flow rate of 75 μL/min. 3% sucrose was utilized as a bulk standard control for OneStep injection and a DMSO calibration curve was performed using a concentration range of 3.5% to 6.5% DMSO. Raw SPR data were appropriately processed in Qdat software (ForteBio) by normalizing the baseline prior to injection, aligning the channels, subtraction of the reference channel, and blank subtraction. Kinetic data were fitted to a pseudo-first order 1:1 interaction binding model using to calculate K.sub.D. In addition, a steady-state model and Req data points were used to validate the binding affinity. Visual inspection of the SPR sensograms was performed to verify appropriate model fitting, lack of mass transport effects, return to baseline, and lack of irregular kinetics.
Fluorescence Polarization Assay
[0287] The FP assay buffer used was 20 mM Tris, 200 mM NaCl, 0.05% β-me, 0.1% Triton X-100, 5% glycerol, and 1× Halt protease inhibitor cocktail. All final FP reactions were placed into a 384-well plate (NUNC 267461) at 30 μL and shaken for 3 hours at room temperature to reach equilibrium. The plates were read on a PerkinElmer EnVision plate reader with software Envision Manager 1.13. Bodipy TMR FP optical module (2100-4100) was used as the mirror. The excitation filter (2100-5830) utilized wavelength at 531 nm and both emission filters (2100-5800 and 2100-5810) were at wavelengths of 579 nm. The baseline mP of TAMRA-LD2-L10D only was set to 15 mP through the assay optimization wizard on the Envision Manager software. The assay optimization set the measurement height to 6.5 mm, excitation light to 100%, G-factor to 1.01, detector gain to 300, and the number of flashes per well at 25.
[0288] For IC.sub.50 determination of peptide inhibitors, inhibitor was titrated from 325 nM to 667 μM into FP Buffer containing 20 μM FAT and 0.1 μM TAMRA-LD2. Wells with no FAT were used as a baseline value which was subtracted from the raw values to produce ΔmP values. The plate was read at every hour for four hours to test for time differences in IC.sub.50. The titration data was processed through GraphPad Prism to produce a dose response curve and a calculated IC.sub.50 with standard error (SE). A four-parameter dose-response inhibition model was utilized and the bottom fit was constrained to the lower plateau of the curve. K.sub.i was determined from the following calculation:
[0289] where I.sub.50 is the concentration of free inhibitor at 50% inhibition. L.sub.50 is the concentration of free ligand at 50% inhibition. P.sub.0 is the concentration of free protein and K.sub.D is calculated from the saturation curve. Iso is calculated by the following equation:
where P.sub.T is the total protein concentration, L.sub.T is the total labeled-ligand concentration, P.sub.0 is the positive root of P.sub.0.sup.2+(K.sub.D+L.sub.T)*P.sub.0−P.sub.T, PL.sub.0=P−P.sub.0, PL.sub.50=PL.sub.0/2, L.sub.0=L.sub.T−PL.sub.0, and L.sub.50=L.sub.T−PL.sub.50.
2D HSQC NMR
[0290] 2D HSQC-NMR samples were prepared at a .sup.15N FAT concentration of 100 μM. Peptides were screened at a concentration of 500 μM to 250 nM with a DMSO concentration of 5%. TROSY-based HSQC were collected on a Bruker Avance III-HD console equipped with a 5 mm TCI Prodigy cryoprobe operating at Larmor frequencies of 600.133 and 60.817 MHz for 1H and 15N respectively. Each 2D experiment was collected with sweep widths of 14.03 and 30.0 ppm, 2048 and 256 points respectively, and 16 signal averages [29-33]. All final data was processed through Bruker TopSpin. Chemical shift perturbations (CSPs) were mapped onto the structure of the FAT-LD2 complex (PDB 1OW8) using PyMOL software to examine the binding site and structural changes upon binding.
Flow Cytometry
[0291] Flow cytometry assays performed on the Canto II flow cytometer (BD Biosciences) were utilized to measure cellular uptake of TAMRA-tagged peptides. In brief, the instrument was properly gated using cells treated with TAMRA-only as a positive control. Also, cells treated with untagged peptide were used as a negative control to rule out non-specific signal. Cells were treated with TAMRA-tagged peptides for a duration of 2h-48h to measure cellular uptake. Permeability of peptide was reported as % positivity relative to TAMRA positive control and mean fluorescence intensity (MFI).
Immunofluorescence
[0292] Immunofluorescent staining was performed to measure the effect of stapled peptide on FAK localization. In brief, cells were seeded onto a coverslip and were fixed in 4% paraformaldehyde in 1×PBS PH 7.4 for 10 min and permeabilized with 0.2% Triton X-100 for 5 min on ice. Cells were blocked with 25% normal goat serum in 1×PBS PH 7.4 for 30 min, washed with 1×PBS PH 7.4, and incubated with primary FAK 4.47 antibody (Millipore) diluted 1:200 in 25% goat serum in 1×PBS PH 7.4. Cells were washed three times with 1×PBS PH 7.4, and a FITC-conjugated secondary antibody (1:400 dilution in 25% goat serum) was applied to the coverslip. Cells were imaged using a Zeiss AXIO Imager M2 Upright Widefield Fluorescent Microscope. Over six fields of view were captured per sample.
X-ray Crystallography
[0293] Co-crystallization of lead peptide SP3 with the human FAK FAT domain (AA 919-1052), in which peptide was mixed and incubated with protein (1:1 molar ratio) prior to crystallization, was performed using a Rigaku Phoenix-HT crystallization robot using commercially available crystallization kits (Hampton Research, Molecular Dimensions, Qiagen) and focused buffer plates around the initial crystallization condition (altering pH, salt, glycerol, and other precipitants). Data collection was performed at synchrotron radiation sources such as the Advanced Photon Source (APS) at the Argonne National Laboratory (Chicago, Ill.), and the Advanced Light Source (ALS) at the Lawrence Berkeley National Laboratory (Berkeley, Calif.). Structural determination was done by molecular replacement using the crystallographic structure PDB 1K05 as a reference model. Extra electron density in FAK-FAT domain binding pockets (Fo-Fc) was modeled to peptide structures and progressively refined over multiple cycles to build a high quality model.
[0294] Table 3 provides mass spectrometry data for peptide inhibitors of the present invention.
TABLE-US-00004 TABLE 3 Mass Spectrometry Data for Peptide Inhibitors. Mass spectra were by ESI+. SEQ ID NO: Name Exact mass Observed mass Calc mass Assignment 1 1967 1581.87 792.1 791.9 ½*[M + 2H].sup.2+ 2 1905 1616.00 809.1 809.0 ½*[M + 2H].sup.2+ 3 1906 1574.95 788.6 788.5 ½*[M + 2H].sup.2+ 4 1907 1673.97 838.1 838.0 ½*[M + 2H].sup.2+ 5 1910 1672.00 837.1 837.0 ½*[M + 2H].sup.2+ 6 1912 1551.86 777.1 776.9 ½*[M + 2H].sup.2+ 7 1913 1675.94 839.5 839.0 ½*[M + 2H].sup.2+ 8 1914 1647.92 825.1 825.0 ½*[M + 2H].sup.2+ 9 1919 1605.87 804.0 803.9 ½*[M + 2H].sup.2+ 10 1920 2453.36 1227.9 1227.7 ½*[M + 2H].sup.2+ 11 1921 1605.87 804.0 803.9 ½*[M + 2H].sup.2+ 12 1925 1563.83 783.0 782.9 ½*[M + 2H].sup.2+ 13 1929 1876.08 626.6 626.4 ⅓*[M + 3H].sup.3+ 14 1931 1717.96 860.5 860.0 ½*[M + 2H].sup.2+ 15 1933 1819.96 911.1 911.0 ½*[M + 2H].sup.2+ 16 2007 1746.97 874.6 874.5 ½*[M + 2H].sup.2+ 17 2009 1615.97 809.1 809.0 ½*[M + 2H].sup.2+ 18 2010 1530.90 766.6 766.5 ½*[M + 2H].sup.2+ 19 2011 1090.68 1091.6 1091.7 [M + H].sup.+ 20 2013 1702.00 852.1 852.0 ½*[M + 2H].sup.2+ 21 2014 1705.89 854.1 854.0 ½*[M + 2H].sup.2+ 22 2015 1605.87 804.1 803.9 ½*[M + 2H].sup.2+ 23 2017 1648.90 825.1 825.5 ½*[M + 2H].sup.2+ 24 2022 1605.87 803.9 803.9 ½*[M + 2H].sup.2+ 25 2024 1605.87 803.9 803.9 ½*[M + 2H].sup.2+ 26 2028 1645.97 823.3 824.0 ½*[M + 2H].sup.2+ 27 2006 1847.05 935.7 935.5 ½*[M + 2H].sup.2+ 28 2012 1842.16 933.3 933.1 ½*[M + H + Na].sup.2+ 29 2020 1874.08 938.2 938.0 ½*[M + 2H].sup.2+ 30 2029 1856.16 929.0 929.1 ½*[M + 2H].sup.2+ 31 2025 2041.26 681.0 681.4 ⅓*[M + 3H].sup.3+ 32 2018 4548.49 1138.7 1138.1 ¼*[M + 4H].sup.4+ 33 2021 4536.59 1135.7 1135.8 ¼*[M + 4H].sup.4+ 34 2023 4184.38 1047.3 1047.1 ¼*[M + 4H].sup.4+ 35 2019 2158.14 1079.4 1080.1 ½*[M + 2H].sup.2+ 36 1916 1742.86 872.5 872.4 ½*[M + 2H].sup.2+ 37 1917 1683.83 843.0 842.9 ½*[M + 2H].sup.2+ 38 2030 2,366.18 1184.3 1184.1 ½*[M + 2H].sup.2+ 39 2016 2,128.22 717.7 717.1 .sup. ⅓*[M + 2H + Na].sup.3+
[0295] Having now fully described the invention, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.
INCORPORATION BY REFERENCE
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EQUIVALENTS
[0337] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.