Peptide inhibitors of focal adhesion kinase activity and uses thereof

Abstract

To provide peptides having an affinity for the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) for the treatment of cancer and other diseases characterized by FAK activity and / or expression (for example, fibrosis).SOLUTION: A compound having the following formula (where YC, YT, Z1, R1, L1, Q1, R2, and Z2 independently include any chemical moiety that permits the resulting compound to bind with the FAT domain of FAK and inhibit the interaction between FAK and a paxillin protein), and pharmaceutically acceptable salts, solvates, and / or prodrugs thereof are provided.SELECTED DRAWING: Figure 1

Description

Detailed Description of the Invention

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 894,726, filed August 31, 2019, which is incorporated herein by reference in its entirety.

[0002] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with government support under Grant No. R01 CA065910 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present application provides peptides having affinity for the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). In particular, the peptides are modified and derived from the sequences of the LD2 alpha helix domain of paxillin (e.g., LD2 peptide), the LD4 domain of paxillin (e.g., LD4 peptide), and the CD8 peptide. These peptides interfere with the interaction between paxillin and FAK, thereby inhibiting FAK activity associated with FAK-paxillin interaction. The present invention further provides the use of the peptides as therapeutic agents for the treatment of cancer and other diseases characterized by FAK activity and / or expression (e.g., fibrosis).

[0004] (introduction) Focal adhesion kinase (FAK) is an extremely attractive cancer drug target due to its overexpression in 80% of solid tumors and its involvement in multiple hallmarks of cancer, including migration, invasion, metastasis, apoptosis, proliferation, angiogenesis, and immune cell suppression. However, previous FAK inhibitors have generally targeted only the kinase function, ignoring FAK's role as a scaffold protein. Importantly, FAK-scaffold interactions regulate many key functions of FAK, such as apoptosis, proliferation, invasion, and metastasis. FAK localization to focal adhesions is mediated by FAK-paxillin interactions, and mutations in the binding site have been shown to have profound effects on FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and invasion.

[0005] There is a need for improved pharmaceuticals and related methods for treating diseases and conditions characterized by FAK activity.

[0006] The present invention addresses this need by providing a novel class of molecules (e.g., polypeptides, compounds) that can efficiently target FAK non-catalytic functions through binding of the FAT domain, thereby inhibiting, for example, FAK-paxillin interactions.

[0007] (Summary of the Invention) Experiments conducted during the development of embodiments of the present invention have led to the synthesis and optimization of peptides that efficiently target FAK non-catalytic functions via binding of the FAT domain, thereby inhibiting, for example, the FAK-paxillin interaction (e.g., the LD2 domain of FAK-paxillin). In particular, the present invention provides stapled peptides (e.g., LD2 peptide, LD4 peptide) that can inhibit the FAK-paxillin interaction. These peptides exhibit significant advantages over existing FAK inhibitors due to their ability to disrupt FAK protein-protein interactions (PPIs), thus providing novel anti-cancer effects.

[0008] Thus, the present invention provides a novel class of LD2 peptides that function as inhibitors of focal adhesion kinase (FAK) activity through binding to the focal adhesion targeting (FAT) domain, thereby inhibiting the FAK-paxillin interaction. The present invention further provides the use of such LD2 peptides as therapeutic agents for the treatment of cancer, fibrotic diseases, and other diseases characterized by FAK activity.

[0009] Thus, the present invention contemplates that exposure of an animal (e.g., a human) suffering from a disorder characterized by FAK activity and / or expression (e.g., cancer (e.g., and / or a cancer-related disorder) (e.g., fibrosis (e.g., IPF, liver fibrosis, keloids)) to a therapeutically effective amount of a peptide capable of binding to the FAT domain of the FAK protein will completely inhibit the growth and / or metastasis of cancer cells or supporting cells and / or render the cells more susceptible to the cell death-inducing activity of cancer therapeutics or radiation therapy. In some embodiments, inhibition of FAK activity occurs, for example, via inhibiting FAK-paxillin binding (e.g., via binding to the FAT domain of FAK).

[0010] The present invention contemplates that inhibitors of FAK activity will satisfy an unmet need for the treatment of multiple cancer types when administered as monotherapy to induce cell growth inhibition, apoptosis and / or cell cycle arrest in cancer cells, or when administered in a temporal relationship with additional agents, e.g., other cell death-inducing or cell cycle-disrupting cancer therapeutic agents, or targeted therapeutic agents, or tumor immunotherapeutic agents, or radiation therapy (combination therapy), so that a greater proportion of cancer cells or supporting cells are prone to the apoptotic program compared to the corresponding proportion of cells in animals treated only with cancer therapeutic agents or radiation therapy alone.

[0011] In some embodiments of the present invention, combined treatment of animals with a therapeutically effective amount of the described peptides of the present invention (e.g., LD2 peptide, LD4 peptide) and an anti-cancer drug produces superior tumor responses and clinical benefits in the animals compared to animals treated with the peptide or the anti-cancer drug / radiation alone. Because the doses of all approved anti-cancer drugs and radiation treatments are known, the present invention contemplates various combinations of them with the described peptides (e.g., LD2 peptide, LD4 peptide). In some embodiments, due to the synergistic effect with the described peptides of the present invention, the dose of the anti-cancer drug can be lower than the standard dose.

[0012] In certain embodiments of the present invention, combination treatment of an animal with a therapeutically effective amount of a described peptide of the present invention (e.g., LD2 peptide, LD4 peptide) and any therapeutic agent for treating a disorder characterized by FAK activity and / or expression (e.g., cancer (e.g., and / or cancer-related disorder) (e.g., fibrosis (e.g., IPF, liver fibrosis, keloid)) results in a greater clinical benefit in the animal compared to an animal treated with the peptide or the therapeutic agent alone. Because the doses of all approved therapeutic agents are known, the present invention contemplates various combinations of them with the described peptides (e.g., LD2 peptide, LD4 peptide). In some embodiments, due to a synergistic effect with the described peptide of the present invention, the dose of the therapeutic agent may be lower than the standard dose.

[0013] In one embodiment, the present invention provides a compound of formula I:

[0014] [ka]

[0015] (In the formula, Y C , Y T, Z1, R1, L1, Q1, R2, and Z2 independently comprise any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction of FAK with the paxillin protein. The present invention provides compounds comprised within (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

[0016] In some embodiments, the compounds can bind to one or more of the helix 1-4 and helix 2-3 portions of the FAT domain of FAK. In some embodiments, the compounds can bind to one or more of the following amino acid residues in the wild-type FAK protein: V928, I936, R962, and K955. However, the compounds can also bind to additional amino acid residues in the FAK protein. In some embodiments, the compounds can inhibit the interaction of FAK with paxillin.

[0017] In some embodiments, the compound is an isolated polypeptide.

[0018] In some embodiments, the compounds are capable of one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity. disruption of FAK catalytic activity via direct binding of the FAT domain; Inhibiting FAK-associated scaffold function, Inhibiting FAK protein-protein interaction mediated by the FAT domain; Inhibiting the binding of paxillin to helix 1-4 of the FAT domain of FAK Inhibiting the binding of paxillin to helix 2-3 of the FAT domain of FAK inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; Inhibition of FAK-Leupaxin interaction via binding to the FAT domain of FAK Inhibition of FAK-CD4 interaction via binding to the FAT domain of FAK; Inhibition of FAK-CD8 interaction via binding to the FAT domain of FAK; Inhibition of FAK-DCC interaction via binding to the FAT domain of FAK inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; Inhibiting the binding of Leupaxin to each of its binding partners inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL Git1 / 2, Pax3, hic-5, and ARF.

[0019] In some embodiments, Q1 is an amino acid chain of 2 amino acids (-[Aa1]-[Aa2]-), 3 amino acids (-[Aa1]-[Aa2]-[Aa3]-), 6 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively: (In the formula, "i" means an α-amino acid backbone residue relative to the N-terminus of the macrocycle, and the "number" in "i + [number]" means the number of amino acid residues away from i relative to the C-terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, and Aa10 are each, independently of one another, a natural or unnatural α-amino acid.)

[0020] In some embodiments, Z1 and Z2, independently of each other, are each a natural or unnatural amino acid chain of 0-200 units in length.

[0021] In some embodiments, Y C is a desired moiety such as an affinity tag (e.g., biotin), a molecular probe or dye (fluorescent or otherwise), or a chemically reactive moiety including, but not limited to, an azide, alkyne, or photoreactive species. Examples are found in Molecular Probes Handbook, Eleventh edition, Iain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), or FAK kinase inhibitors, but are not limited to that selection.

[0022] In some embodiments, Y C is a covalent derivative of an inhibitor of FAK kinase catalytic activity. C is a covalent derivative of another kinase (e.g., Src, EGFR, HER2, etc.) inhibitor. In some embodiments, Y C is a covalent derivative of the GPCR compound. C is a covalent derivative of a nuclear acceptor compound. C is a covalent derivative of an E3 ubiquitin ligase targeting ligand. In some embodiments, Y C is a covalent derivative of a protein-protein interaction inhibitor. Cis a covalent derivative of a radionuclide moiety. C is a covalent derivative of a drug transporter ligand. C is a covalent derivative of a cell-penetrating moiety / sequence (e.g., TAT, etc.). In some embodiments, Y C is a covalent derivative of a chemotherapeutic agent. C is a covalent derivative of a lipid moiety. C is a covalent derivative of a prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics. C is a covalent derivative of an electrophilic moiety for covalent attachment to a target protein.

[0023] In some embodiments, Y T is Y C and Z1. In some embodiments, Y T When present, Y forms an alkyl or amide (e.g., carbamide, sulfonamide, or phosphoramide) group. T may or may not contain one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or one or more carbocyclic or heterocyclic rings. In some embodiments, the chemical chain is selected from β-alanine, 6-aminohexanoic acid, and amino acids in which the amine and acid functional groups are separated by a poly(ethylene glycol) (PEG) monomer, oligomer, or polymer.

[0024] In some embodiments, R1 is hydrogen or a lower alkyl or substituted methyl group.

[0025] In some embodiments, R2 is hydrogen or a lower alkyl or substituted methyl group.

[0026] In some embodiments, L1 is a hydrocarbon containing a single double bond, typically in a cis or trans configuration, or a mixture thereof, and typically contains 8 or 11 atoms, although it can be a different integer.

[0027] In some embodiments, L1 is any combination of atoms and molecules, excluding the inherent peptide backbone, that allows an amino acid comprising R1 of Formula I to be covalently bonded to an amino acid comprising R2 of Formula I. In some embodiments, L1 is a hydrocarbon chain comprising 8 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In some embodiments, L1 is a hydrocarbon chain comprising 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In some embodiments, L1 is a hydrocarbon chain comprising neither 8 nor 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In some embodiments, L1 comprises two sulfur atoms covalently bonded in a manner other than a disulfide bond. In some embodiments, L1 comprises a hydrocarbon chain having an internal polysubstituted triazole, or a substituted 4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole, or a substituted 8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole, or a substituted 2,3,8,9-tetrahydrodibenzo[3,4:7,8]cycloocta[1,2-d]isoxazole, or a substituted 4a,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazine. In some embodiments, the linker is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and may or may not branch to pendant moieties such as biotin, dyes, chemical probes, or reactive groups. L1 may or may not contain one or more sulfur atoms, one or more oxygen atoms, or one or more nitrogen atoms. This list is not intended to be exhaustive, and those skilled in the art can easily imagine other covalent bonding strategies.

[0028] In some embodiments, the compound is at least 60% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 75% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 80% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 85% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 90% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 95% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 98% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound is at least 99% identical to one of SEQ ID NOs: 1-38. In some embodiments, the compound comprises, consists of, or consists essentially of one of SEQ ID NOs: 1-38.

[0029] In one embodiment, the present invention provides a compound of formula II:

[0030] [ka]

[0031] (In the formula, Y C , Y T , Z1, R1, Q1, L2, R2, Z3, R3, Q2, L3, R4, and Z2 independently comprise any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction of FAK with the paxillin protein. The present invention provides compounds comprised within (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

[0032] In some embodiments, the compound can bind to one or more of helix 1-4 and helix 2-3 portions of the FAT domain of FAK. In some embodiments, the compound can bind to one or more of the following amino acid residues in the wild-type FAK protein: V928, I936, R962, and K955. However, the compound can also bind to additional amino acid residues in the FAK protein. In some embodiments, the compound can inhibit the interaction of FAK with paxillin. In some embodiments, the compound is an isolated polypeptide.

[0033] In some embodiments, the compounds are capable of one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity. disruption of FAK catalytic activity via direct binding of the FAT domain; Inhibiting FAK-associated scaffold function, Inhibiting FAK protein-protein interaction mediated by the FAT domain; Inhibiting the binding of paxillin to helix 1-4 of the FAT domain of FAK Inhibiting the binding of paxillin to helix 2-3 of the FAT domain of FAK inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; Inhibition of FAK-Leupaxin interaction via binding to the FAT domain of FAK Inhibition of FAK-CD4 interaction via binding to the FAT domain of FAK; Inhibition of FAK-CD8 interaction via binding to the FAT domain of FAK; Inhibition of FAK-DCC interaction via binding to the FAT domain of FAK inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; Inhibiting the binding of Leupaxin to each of its binding partners inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

[0034] In some embodiments, Q1 is an amino acid chain of 2 amino acids (-[Aa1]-[Aa2]-), 3 amino acids (-[Aa1]-[Aa2]-[Aa3]-), 6 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively; Q2 is a 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 acid chain occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively: (In the formula, "i" refers to an α-amino acid backbone residue relative to the N-terminus of the macrocycle, and the "number" in "i + [number]" refers to the number of amino acid residues away from i relative to the C-terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, and Aa10 are each independently a natural or unnatural α-amino acid, and Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, and Aa20 are each independently a natural or unnatural α-amino acid.)

[0035] In some embodiments, Z1, Z2 and Z3, independently of one another, are each a natural or unnatural amino acid chain of 0-200 units in length.

[0036] In some embodiments, Y C is a desired moiety such as an affinity tag (e.g., biotin), a molecular probe or dye (fluorescent or otherwise), or a chemically reactive moiety including, but not limited to, an azide, alkyne, or photoreactive species. Examples are found in Molecular Probes Handbook, Eleventh edition, Iain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), or FAK kinase inhibitors, but are not limited to that selection.

[0037] In some embodiments, Y C is a covalent derivative of an inhibitor of FAK kinase catalytic activity. C is a covalent derivative of another kinase (e.g., EGFR, HER2, etc.) inhibitor. In some embodiments, Y C is a covalent derivative of the GPCR compound. C is a covalent derivative of a nuclear acceptor compound. C is a covalent derivative of an E3 ubiquitin ligase targeting ligand. In some embodiments, YC is a covalent derivative of a protein-protein interaction inhibitor. C is a covalent derivative of a radionuclide moiety. C is a covalent derivative of a drug transporter ligand. C is a covalent derivative of a cell-penetrating moiety / sequence (e.g., TAT, etc.). In some embodiments, Y C is a covalent derivative of a chemotherapeutic agent. C is a covalent derivative of a lipid moiety. C is a covalent derivative of a prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics. C is a covalent derivative of an electrophilic moiety for covalent attachment to a target protein.

[0038] In some embodiments, Y T is Y C and Z1. In some embodiments, Y T When present, Y forms an alkyl or amide (e.g., carbamide, sulfonamide, or phosphoramide) group. T may or may not contain one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or one or more carbocyclic or heterocyclic rings. In some embodiments, the chemical chain is selected from β-alanine, 6-aminohexanoic acid, and amino acids in which the amine and acid functional groups are separated by a poly(ethylene glycol) (PEG) monomer, oligomer, or polymer.

[0039] In some embodiments, R1 and R2 are independently hydrogen or a lower alkyl or substituted methyl group.

[0040] In some embodiments, R3 and R4 are independently hydrogen or a lower alkyl or substituted methyl group.

[0041] In some embodiments, L2 is a hydrocarbon containing a single double bond, typically in a cis or trans configuration, or a mixture thereof, and typically contains 8 or 11 atoms, although it can be a different integer.

[0042] In some embodiments, L2 is any combination of atoms and molecules, excluding the inherent peptide backbone, that can covalently bond an amino acid comprising R1 of Formula II to an amino acid comprising R2 of Formula II. In one embodiment, L2 is a hydrocarbon chain comprising 8 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L2 is a hydrocarbon chain comprising 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L2 is a hydrocarbon chain comprising neither 8 nor 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L2 comprises two sulfur atoms covalently bonded in a manner other than a disulfide bond. In another embodiment, L2 comprises a hydrocarbon chain having an internal polysubstituted triazole, or a substituted 4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole, or a substituted 8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole, or a substituted 2,3,8,9-tetrahydrodibenzo[3,4:7,8]cycloocta[1,2-d]isoxazole, or a substituted 4a,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazine. In some embodiments, the linker is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and may or may not branch to pendant moieties such as biotin, dyes, chemical probes, or reactive groups. L1 may or may not contain one or more sulfur atoms, one or more oxygen atoms, or one or more nitrogen atoms. This list is not intended to be exhaustive, and those skilled in the art can easily imagine other covalent bonding strategies.

[0043] In some embodiments, L3 is a hydrocarbon containing a single double bond, typically in a cis or trans configuration, or a mixture thereof, and typically contains 8 or 11 atoms, although it can be a different integer.

[0044] In some embodiments, L3 is any combination of atoms and molecules, excluding the inherent peptide backbone, that allow an amino acid comprising R1 of Formula II to be covalently bonded to an amino acid comprising R2 of Formula II. In one embodiment, L3 is a hydrocarbon chain comprising 8 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L3 is a hydrocarbon chain comprising 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L3 is a hydrocarbon chain comprising neither 8 nor 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L3 comprises two sulfur atoms covalently bonded in a manner other than a disulfide bond. In another embodiment, L3 comprises a hydrocarbon chain having an internal polysubstituted triazole, or a substituted 4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole, or a substituted 8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole, or a substituted 2,3,8,9-tetrahydrodibenzo[3,4:7,8]cycloocta[1,2-d]isoxazole, or a substituted 4a,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazine. In some embodiments, the linker is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and may or may not branch to pendant moieties such as biotin, dyes, chemical probes, or reactive groups. L1 may or may not contain one or more sulfur atoms, one or more oxygen atoms, or one or more nitrogen atoms. This list is not intended to be exhaustive, and those skilled in the art can easily imagine other covalent bonding strategies.

[0045] In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 60% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 75% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 85% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 98% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 99% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises one of SEQ ID NOs: 1-38.

[0046] In one embodiment, the present invention provides a compound of formula III:

[0047] [ka]

[0048] (In the formula, Y C , Y T , Z1, R1, Q1, L4, Q4, L5, R5, and Z2 independently comprise any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction of FAK with the paxillin protein. The present invention provides compounds comprised within (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

[0049] In some embodiments, the compounds can bind to one or more of the helix 1-4 and helix 2-3 portions of the FAT domain of FAK. In some embodiments, the compounds can bind to one or more of the following amino acid residues in the wild-type FAK protein: V928, I936, R962, and K955. However, the compounds can also bind to additional amino acid residues in the FAK protein. In some embodiments, the compounds can inhibit the interaction of FAK with paxillin.

[0050] In some embodiments, the compound is an isolated polypeptide.

[0051] In some embodiments, the compounds are capable of one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity. disruption of FAK catalytic activity via direct binding of the FAT domain; Inhibiting FAK-associated scaffold function, Inhibiting FAK protein-protein interaction mediated by the FAT domain; Inhibiting the binding of paxillin to helix 1-4 of the FAT domain of FAK Inhibiting the binding of paxillin to helix 2-3 of the FAT domain of FAK inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; Inhibition of FAK-Leupaxin interaction via binding to the FAT domain of FAK Inhibition of FAK-CD4 interaction via binding to the FAT domain of FAK; Inhibition of FAK-CD8 interaction via binding to the FAT domain of FAK; Inhibition of FAK-DCC interaction via binding to the FAT domain of FAK inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; Inhibiting the binding of Leupaxin to each of its binding partners inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

[0052] In some embodiments, Q1 is an amino acid chain of 2 amino acids (-[Aa1]-[Aa2]-), 3 amino acids (-[Aa1]-[Aa2]-[Aa3]-), 6 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively; Q4 is a 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 acid chain occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively: (In the formula, "i" refers to an α-amino acid backbone residue relative to the N-terminus of the macrocycle, and the "number" in "i + [number]" refers to the number of amino acid residues away from i relative to the C-terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, and Aa10 are each independently a natural or unnatural α-amino acid, and Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, and Aa20 are each independently a natural or unnatural α-amino acid.)

[0053] In some embodiments, Z1 and Z2, independently of each other, are each a natural or unnatural amino acid chain of 0-200 units in length.

[0054] In some embodiments, Y C is a desired moiety such as an affinity tag (e.g., biotin), a molecular probe or dye (fluorescent or otherwise), or a chemically reactive moiety including, but not limited to, an azide, alkyne, or photoreactive species. Examples are found in Molecular Probes Handbook, Eleventh edition, Iain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), or FAK kinase inhibitors, but are not limited to that selection.

[0055] In some embodiments, Y T is Y C and Z1. In some embodiments, Y T When present, Y forms an alkyl or amide (e.g., carbamide, sulfonamide, or phosphoramide) group. Tmay or may not contain one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or one or more carbocyclic or heterocyclic rings. In some embodiments, the chemical chain is selected from β-alanine, 6-aminohexanoic acid, and amino acids in which the amine and acid functional groups are separated by a poly(ethylene glycol) (PEG) monomer, oligomer, or polymer.

[0056] In some embodiments, Y C is a covalent derivative of an inhibitor of FAK kinase catalytic activity. C is a covalent derivative of another kinase (e.g., EGFR, HER2, etc.) inhibitor. In some embodiments, Y C is a covalent derivative of the GPCR compound. C is a covalent derivative of a nuclear acceptor compound. C is a covalent derivative of an E3 ubiquitin ligase targeting ligand. In some embodiments, Y C is a covalent derivative of a protein-protein interaction inhibitor. C is a covalent derivative of a radionuclide moiety. C is a covalent derivative of a drug transporter ligand. C is a covalent derivative of a cell-penetrating moiety / sequence (e.g., TAT, etc.). In some embodiments, Y C is a covalent derivative of a chemotherapeutic agent. C is a covalent derivative of a lipid moiety. C is a covalent derivative of a prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics. C is a covalent derivative of an electrophilic moiety for covalent attachment to a target protein.

[0057] In some embodiments, R1 and R5 are independently selected from hydrogen or a lower alkyl or substituted methyl group.

[0058] In some embodiments, L4 and L5 are independently selected from hydrocarbons containing a single double bond in a cis or trans configuration, or a mixture thereof, the hydrocarbon chain typically containing 8 or 11 atoms, but may be a different integer.

[0059] In some embodiments, L4 is any combination of atoms and molecules, excluding the inherent peptide backbone, that allow an amino acid comprising R1 of Formula III to be covalently bonded to an amino acid comprising R2 of Formula III. In one embodiment, L4 is a hydrocarbon chain comprising 8 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L4 is a hydrocarbon chain comprising 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L4 is a hydrocarbon chain comprising neither 8 nor 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L4 comprises two sulfur atoms covalently bonded in a manner other than a disulfide bond. In another embodiment, L4 comprises a hydrocarbon chain having an internal polysubstituted triazole, or a substituted 4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole, or a substituted 8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole, or a substituted 2,3,8,9-tetrahydrodibenzo[3,4:7,8]cycloocta[1,2-d]isoxazole, or a substituted 4a,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazine. In some embodiments, the linker is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and may or may not branch to pendant moieties such as biotin, dyes, chemical probes, or reactive groups. L1 may or may not contain one or more sulfur atoms, one or more oxygen atoms, or one or more nitrogen atoms. This list is not intended to be exhaustive, and those skilled in the art can easily imagine other covalent bonding strategies.

[0060] In some embodiments, L5 is any combination of atoms and molecules, excluding the inherent peptide backbone, that allow an amino acid comprising R1 of Formula III to be covalently bonded to an amino acid comprising R2 of Formula III. In one embodiment, L5 is a hydrocarbon chain comprising 8 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L5 is a hydrocarbon chain comprising 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L5 is a hydrocarbon chain comprising neither 8 nor 11 atoms and a single double bond in either cis or trans configuration, or a mixture thereof. In another embodiment, L5 comprises two sulfur atoms covalently bonded in a manner other than a disulfide bond. In another embodiment, L5 comprises a hydrocarbon chain having an internal polysubstituted triazole, or a substituted 4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole, or a substituted 8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole, or a substituted 2,3,8,9-tetrahydrodibenzo[3,4:7,8]cycloocta[1,2-d]isoxazole, or a substituted 4a,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazine. In some embodiments, the linker is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and may or may not branch to pendant moieties such as biotin, dyes, chemical probes, or reactive groups. L1 may or may not contain one or more sulfur atoms, one or more oxygen atoms, or one or more nitrogen atoms. This list is not intended to be exhaustive, and those skilled in the art can easily imagine other covalent bonding strategies.

[0061] In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 60% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 75% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 85% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 98% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises an amino acid sequence that is at least 99% identical to one of SEQ ID NOs: 1-38. In some embodiments, at least a portion of the compound comprises one of SEQ ID NOs: 1-38.

[0062] In one embodiment, the present invention provides a compound of formula IV:

[0063] [ka]

[0064] wherein SPA-NH2 and SPB-NH2 are independently a compound of formula I (as defined in claim 1), excluding YC-YT-, or a compound of formula II (as defined in claim 24), excluding YC-YT-, or a compound of formula III (as defined in claim 48), excluding YC-YT-; wherein T1 is a chain or 0-400 atoms in length, typically but not limited to, comprising a poly(ethylene glycol) chain, typically but not limited to, linked to SPA-NH2 and SPB-NH2 as an amide functional group; may or may not contain internal structures such as carbon or heterocyclic rings that may or may not function as pigments or chromophores, and The pendant moiety may be branched or unbranched, such as biotin or a dye or a chemical probe or a reactive group or a reactive E3 ligase ligand. In some embodiments, the chain (T1) is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and which may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and which may or may not branch to pendant moieties such as biotin, dyes, chemical probes, reactive groups, or reactive E3 ligase ligands. In some embodiments, T1 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.

[0065] In some embodiments, T1 is the product of a "click chemistry" reaction. As a non-limiting example, SPA-NH2 can have an N-terminal alkyne, and SPB-NH2 can have an N-terminal azide functionality. The product of these two species is a substituted 1H-1,2,3-triazole. In some instances, a catalyst is used in the reaction.

[0066] In one embodiment, the present invention provides a compound of formula V:

[0067] [ka]

[0068] (In the formula, Y C -Y T -SPA-NH2 and Y C -Y T -SPB-NH2 is independently a compound of formula I, or a compound of formula II, or a compound of formula III; wherein the side chain represented by YC-YT-SPA-NH2 is derived 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 an amino acid that is part of Z1; or an amino acid that is part of Z2; or an amino acid that is part of Z3; or an amino acid that is part of Z1; or an amino acid that is part of Z2; or an amino acid that is part of Z3; and a side chain represented by YC-YT-SPB-NH2 is linked via a chain (T2) by chemical ligation to an individual member of [Aa3], [Aa4], [Aa5], [Aa6], [Aa7], [Aa8], [Aa9], [Aa10], [Aa11], [Aa12], [Aa13], [Aa14], [Aa15], [Aa16], [Aa17], [Aa18], [Aa19] or [Aa20]; or an amino acid that is part of Z1; or an amino acid that is part of Z2; or an amino acid that is part of Z3. The present invention provides compounds comprised within (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

[0069] In some embodiments, chain (T2) is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and which may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and which may or may not branch to pendant moieties such as biotin, dyes, chemical probes, reactive groups, or reactive E3 ligase ligands. In some embodiments, T2 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.

[0070] In some embodiments, T2 is the product of a "click chemistry" reaction. As a non-limiting example, SPA-NH2 may have an N-terminal alkyne, and SPB-NH2 may have an N-terminal azide functionality. The product of these two species is a substituted 1H-1,2,3-triazole. In some instances, a catalyst is used in the reaction.

[0071] In one embodiment, the present invention provides a compound of formula VI:

[0072] [ka]

[0073] (Wherein, SPA-NH2 is Y C -Y T - except for compounds of formula I (as defined in claim 1), or Y C -Y T - except for compounds of formula II (as defined in claim 24), or Y C -Y T a compound of formula III (as defined in claim 48), except for wherein the N-terminus of SPA-NH2 is connected via linker L6 to an internal amino acid from SPA-NH2 (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 an amino acid that is part of Z1; or an amino acid that is part of Z2; or an amino acid that is part of Z3). the side chain, designated YC-YT-SPA-NH2, derived from an amino acid, is linked to 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 an amino acid that is part of Z1; or an amino acid that is part of Z2; or an amino acid that is part of Z3; wherein the linker is a hydrocarbon chain comprising a cis or trans alkene, or a mixture thereof; or the linker is an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes). The present invention provides compounds comprised within (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

[0074] In some embodiments, L6 is any combination of atoms and molecules that allows for covalent bonding between the N-terminus of SPA-NH2 and any other amino acid, including SPA-NH2, except for direct bonding via the peptide backbone. Typically, the bond between L6 and the N-terminus of SPA-NH2 is via an amide bond. In one embodiment, L6 comprises a hydrocarbon chain containing eight atoms and a single double bond in a cis or trans configuration, or a mixture thereof. In another embodiment, L6 comprises a hydrocarbon chain containing a single double bond in a cis or trans configuration, or a mixture thereof. In another embodiment, L6 comprises one oxygen atom within the hydrocarbon chain. In another embodiment, L6 comprises multiple oxygen atoms between the carbon atoms, as in the poly(ethylene glycol) system. In another embodiment, L6 comprises one sulfur atom within the hydrocarbon chain. In another embodiment, L6 comprises two sulfur atoms covalently bonded by a method other than a disulfide bond. In another embodiment, L6 comprises an ester functional group within the hydrocarbon chain. In another embodiment, L6 comprises a thioester functional group within the hydrocarbon chain. In another embodiment, L6 comprises a hydrocarbon chain having an internal polysubstituted triazole, or a substituted 4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole, or a substituted 8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole, or a substituted 2,3,8,9-tetrahydrodibenzo[3,4:7,8]cycloocta[1,2-d]isoxazole, or a substituted 4a,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazine.In some embodiments, the linker is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and may or may not branch to pendant moieties such as biotin, dyes, chemical probes, reactive groups, or reactive E3 ligase ligands. L1 may or may not contain one or more sulfur atoms, one or more oxygen atoms, or one or more nitrogen atoms. This list is not intended to be exhaustive, and those skilled in the art can easily imagine other covalent bonding strategies. L6 may be branched or may be a chemical derivative not encompassed by the above. Non-limiting examples of these derivatives include epoxidation, aziridination, cyclopropanation, or dihydroxylation of the double bond.

[0075] In one embodiment, the present invention provides a compound of formula VII:

[0076] [ka]

[0077] (In the formula, Y C -Y T -SPA- and Y C -Y T -SPB- is independently a compound of formula I, or a compound of formula II, or a compound of formula III. The present invention provides compounds (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof) encompassed within the scope of the present invention. The C-termini of two staple peptides, SPA and SPB, are linked via the chain D1T2D2. In some examples, D1 and D2 are (optionally substituted) nitrogen atoms, thereby forming an amide bond with the C-terminus. D1 and D2, independently of each other, can contain N, O, or S as the linking atom. SPA and SPB are synthesized on a 2-chlorotrityl chloride resin or some other resin that allows for cleavage of the protected peptide from the resin. An example of this chemistry can be found in Alhassan et al. Green Chem. 2020, 22, 2840-2845. Protected staple peptides as free carboxylic acids can also be synthesized by conventional solution-phase methods. The protected peptide acid can be reacted with a polyfunctional molecule, such as a polyamine or amino alcohol, to generate a protected form of the compound of Formula VII. Global deprotection can provide a compound of formula VII. In some instances, SPA and SPB can be independently reacted with complementary click chemistry partners, for example, SPA can be reacted with an aminoalkyne, and SPB can be reacted with an aminoazide. These novel derivatives can generate the protected form of the compound of formula VII under appropriate reaction conditions (e.g., copper(I) species). Global deprotection can provide a compound of formula VII.

[0078] In one embodiment, the present invention provides a compound of formula VIII: Y C -Y T -SPA-T2-SPB-NH2 (In the formula, Y C -Y T -SPA- and Y C -Y T -SPB- is independently a compound of formula I, or a compound of formula II, or a compound of formula III. The compounds of Formula VIII can be synthesized in a similar manner to the compounds of Formula I or Formula II or Formula III, and the compounds of Formula VIII can be synthesized in a similar manner to the compounds of Formula I or Formula II or Formula III, and the compounds of Formula VIII can be synthesized in a similar manner to the compounds of Formula VIII ... VIII, and the compounds of Formula VIII can be synthesized in a similar manner to the C

[0049] This involves the displacement of the SPB, thereby extending the SPB via a tether, to produce an SPA, etc. In another scenario, a protected peptide carboxylic acid SPA, such as described for Formula VII, or a protected click chemistry partner, such as described for Formula VII, is added to the SPB via chemistry known to those skilled in the art (e.g., amide bond formation or CuAAC reaction, as appropriate). Global deprotection can provide a compound of Formula VIII.

[0079] In some embodiments, the compounds within Formula I, II, III, IV, V, VI, VII, and VIII are selected from the group consisting of standard amino acids, 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, ... , 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.

[0080] In some embodiments, the compounds within Formulas I, II, III, IV, V, VI, VII, and VIII are further conjugated to an additional therapeutic agent (e.g., thalidomide) (e.g., a bifunctional compound (e.g., a PROTAC)).

[0081] In some embodiments, the compounds within Formulas I, II, III, IV, V, VI, VII, and VIII have a central disubstituted amino acid with a geminal bis(linked alkene) pattern at the alpha carbon flanked on either side by complementary (in terms of spacing and stereochemistry) monoalkenyl residues. The geminal bis(linked alkene) is typically symmetrically substituted, although this is not a requirement.

[0082] In any of the exemplary compounds of Formula I, or Formula II, or Formula III, or Formula IV, or Formula V, or Formula VI, or Formula VII, or Formula VIII, a nitrogen atom that is part of the peptide backbone or part of the side chain of an amino acid may be covalently attached to a chemical moiety that is not a hydrogen atom. In one embodiment, the chemical moiety is a hydrocarbon or substituted hydrocarbon chain containing a reactive species that may or may not further participate in chemical reactions, including, but not limited to, ring-closing metathesis reactions, esterification, amide formation, Diels-Alder reactions, etc.

[0083] In one aspect, the present invention provides an α-helical stapled peptide comprising hydrophobic and hydrophilic amino acids, wherein two or more amino acids of the peptide are linked to each other, and wherein the peptide is capable of binding to the FAT domain of FAK.

[0084] In some embodiments, the compounds can bind to one or more of the helix 1-4 and helix 2-3 portions of the FAT domain of FAK. In some embodiments, the compounds can bind to one or more of the following amino acid residues in the wild-type FAK protein: V928, I936, R962, and K955. However, the compounds can also bind to additional amino acid residues in the FAK protein. In some embodiments, the compounds can inhibit the interaction of FAK with paxillin.

[0085] In some embodiments, the compound is an isolated polypeptide.

[0086] In some embodiments, the peptides enable one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity. disruption of FAK catalytic activity via direct binding of the FAT domain; Inhibiting FAK-associated scaffold function, Inhibiting FAK protein-protein interaction mediated by the FAT domain; Inhibiting the binding of paxillin to helix 1-4 of the FAT domain of FAK Inhibiting the binding of paxillin to helix 2-3 of the FAT domain of FAK inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; Inhibition of FAK-Leupaxin interaction via binding to the FAT domain of FAK Inhibition of FAK-CD4 interaction via binding to the FAT domain of FAK; Inhibition of FAK-CD8 interaction via binding to the FAT domain of FAK; Inhibition of FAK-DCC interaction via binding to the FAT domain of FAK inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; Inhibiting the binding of Leupaxin to each of its binding partners inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

[0087] In some embodiments, the amino acids at two 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 linked to each other.

[0088] In some embodiments, the staple peptide comprises any of SEQ ID NOs: 1-38, as set forth in Table 1.

[0089] [Table 1]

[0090] Table 1: Peptides and identifiers. Abbreviations: R8: (R)-2-(7-octenyl)alanine; S5: (S)-2-(4-pentenyl)alanine; R5: (R)-2-(4-pentenyl)alanine; Az: 2-(2-(2-(2-azidoethoxy)ethoxy)acetyl); Aib: 2-aminoisobutyric acid; DBCO: 3-amino-1-(2-azatricyclo[10.4.0.0])-1H-pyridin-1H-pyridin-2 ... 4,9 ]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)propan-1-one; RhoB: rhodamine B; or its derivatives. Unless otherwise noted, peptides are the product of intramolecular ring-closing metathesis reactions; double bond geometries have not been established or quantified. These sequences may be omitted to denote "click" chemistry products. Figure 14 shows the structures of peptides P29-P34, P37, and P38.

[0091] As described in Table 1, R8 means (R)-2-(7-octenyl)alanine, S5 means (S)-2-(4-pentenyl)alanine, R5 means (R)-2-(4-pentenyl)alanine, and Aib means 2-aminoisobutyric acid; Az means 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetyl, Ac means acetyl, and DBCO means 3-amino-1-(2-azatricyclo[10.4.0.04,9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)propan-1-one, where standard one-letter amino acid codes are used.

[0092] In some embodiments, the peptide is further conjugated to an imaging agent, hi some embodiments, the imaging agent is 5- or 6-carboxytetramethylrhodamine (TAMRA), or a mixture of isomers.

[0093] In one aspect, the present invention provides pharmaceutical compositions comprising one or more of the present staple peptides in a pharmaceutically acceptable carrier.

[0094] In certain embodiments, the present invention provides pharmaceutical compositions comprising two or more of the present stapled peptides linked together by a linker (e.g., a PEG-based linker).

[0095] In one embodiment, the present invention provides a method for treating, ameliorating, or preventing a hyperproliferative disorder in a patient, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising one or more of the present staple peptides.

[0096] In some embodiments, the hyperproliferative disease is cancer (eg, a cancer characterized by FAK expression, FAK pathway activation, FAK dependence, FAK activity and / or FAK-paxillin associated activity).

[0097] In some embodiments, the patient is a human patient.

[0098] In some embodiments, the present invention further comprises administering to the patient one or more anti-cancer agents. In some embodiments, the anti-cancer agent is a chemotherapy agent. In some embodiments, the anti-cancer agent is radiation therapy.

[0099] In one embodiment, the present invention provides one or more of the stapled peptides and instructions for administering the stapled peptides to a patient with a hyperproliferative disease.

[0100] In some embodiments, the stapled peptides are capable of binding to other proteins with similar FAT domain-type structures (e.g., Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF).

[0101] The present invention further provides methods for producing the present stapled peptides, at least in part through the following techniques described in Example I:

[0102] The present invention also provides the use of the stapled peptides to induce cell cycle arrest and / or apoptosis in cells containing a FAK protein (e.g., a functional FAK protein, a non-functional FAK protein, an aberrantly regulated FAK protein, or a mutant FAK protein). The present invention also relates to the use of the stapled peptides to sensitize cells to additional agents, e.g., inducers of apoptosis and / or cell cycle arrest, and to provide chemoprotection of normal cells through the induction of cell cycle arrest prior to treatment with a chemotherapeutic agent.

[0103] As described above, the stapled peptides of the present invention are useful for treating, ameliorating, or preventing disorders, e.g., disorders responsive to the induction of apoptotic cell death, e.g., disorders characterized by aberrant apoptosis (including hyperproliferative diseases such as cancer). In some embodiments, the stapled peptides can be used to treat, ameliorate, or prevent cancers characterized by resistance to cancer therapy (e.g., cancer cells that are chemotherapy-resistant, radiation-resistant, hormone-resistant, etc.). In some embodiments, the cancer is multiple myeloma, acute myeloid leukemia, melanoma, breast cancer, head or neck cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, and / or pancreatic cancer. In other embodiments, the stapled peptides can be used to treat hyperproliferative diseases characterized by the expression of functional FAK protein activity, particularly functional FAK-paxillin-associated activity.

[0104] The stapled peptides of the present invention can be used to treat disorders, e.g., disorders associated with FAK expression and / or activity (e.g., bone disease, bone density regulation, fibrotic disorders, rheumatoid arthritis, osteoarthritis, neuropathy, Alzheimer's disease, pro-inflammatory gene expression, chronic inflammatory diseases, vascular inflammation, vitiligo, psoriasis, acute lung injury (ALI), cardiovascular disease, diabetic nephropathy, HHV-8 (Kaposi's sarcoma-associated herpesvirus (KSHV)), ventilator-induced lung injury, and / or AIDS). & HIV-CD4 associated).

[0105] The stapled peptides of the present invention are useful for treating, ameliorating, or preventing disorders, such as disorders associated with FAK and Pyk2 expression and / or activity (e.g., chronic diseases such as cardiovascular disease, bone disease, fibrosis (e.g., liver fibrosis, pulmonary fibrosis, keloids, etc.), rheumatoid arthritis, and neurological disorders).

[0106] The present invention also provides kits comprising the stapled peptides of the present invention and instructions for administration to an animal, which may optionally contain other therapeutic agents, such as anti-cancer agents or apoptosis-modulating agents.

[0107] The present invention further provides bifunctional compounds that recruit endogenous proteins to E3 ubiquitin ligases for degradation, and methods using the compounds. In particular, the present invention provides bifunctional or proteolysis-targeting chimeric (PROTAC) compounds that find use as regulators of targeted ubiquitination of various polypeptides and other proteins, which are then degraded and / or otherwise inhibited. An exemplary advantage of the compounds provided herein is that they are capable of a broad range of pharmacological activity, consistent with the degradation / inhibition of targeted polypeptides from virtually any protein class or family. Furthermore, the present invention provides methods using an effective amount of the compounds described herein for the treatment or amelioration of disease conditions, such as cancer (e.g., cancers characterized by FAK expression, FAK pathway activation, FAK dependency, FAK activity, and / or FAK-paxillin-associated activity).

[0108] In a further aspect, the present invention provides bifunctional or PROTAC compounds that include an E3 ubiquitin ligase binding moiety (e.g., a ligand of an E3 ubiquitin ligase, i.e., a "ULM" group) and a moiety that binds to a target protein (e.g., a protein / polypeptide targeting ligand, i.e., a "PTM" group) (e.g., the FAT domain of FAK), thereby bringing the target protein / polypeptide into close proximity with the ubiquitin ligase, resulting in degradation (and inhibition) of the protein (e.g., inhibiting the interaction of paxillin with FAK). In some embodiments, the PTM is any of the peptides described herein (e.g., any of the peptides included in Formulas I-VI) (e.g., any of the peptides described in Table 1) that have affinity for the FAT domain of FAK (e.g., thereby inhibiting the interaction of FAK with paxillin). In some embodiments, the ULM is an inhibitor of von-Hippel-Lindau (VHL) ligase, cereblon, mouse double minute 2 (MDM2), and / or an apoptosis protein (IAP) E3 ligase binding moiety. For example, the structure of a bifunctional compound can be represented as PTM-ULM.

[0109] The locations of each of the PTM and ULM moieties, and their numbers exemplified herein, are provided by way of example only and are not intended to limit the peptides in any way. As will be understood by those of skill in the art, the bifunctional compounds described herein can be synthesized, and the number and location of each functional moiety can be varied as desired.

[0110] In some embodiments, the bifunctional compound further comprises a chemical linker ("L"). As an example of this, the structure of the bifunctional compound can be depicted as PTM-L-ULM, where PTM is a protein / polypeptide targeting moiety (e.g., any of the compounds described herein that exhibit modulatory activity against FAK), L is a linker, and ULM is a VHL, cereblon, MDM2, or IAPE3 ligase binding moiety.

[0111] The above embodiments are not limited to a specific type of linker. In some embodiments, the linker group is an optionally substituted (poly)ethylene glycol having 1 to about 100 ethylene glycol units, about 1 to about 50 ethylene glycol units, 1 to about 25 ethylene glycol units, about 1 to 10 ethylene glycol units, 1 to about 8 ethylene glycol units, 1 to about 6 ethylene glycol units, or 2 to 4 ethylene glycol units, or an optionally substituted alkyl group interdispersed with O, N, S, P, or Si atoms. In some embodiments, the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, azido, or heterocyclic group. In some embodiments, the linker is a dye compound. In some embodiments, the linker is a photoreactive compound. In some embodiments, the linker can be asymmetric or symmetric. In certain embodiments, the linker is a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, from 1 to about 10 ethylene glycol units, from about 2 to about 6 ethylene glycol units, from about 2 to 5 ethylene glycol units, or from about 2 to 4 ethylene glycol units.

[0112] The ULM group and the PTM group may also be covalently linked to the linker group via any chemically suitable and stable group. In exemplary aspects of the invention, the linker is independently covalently linked to the ULM group and the PTM group, in some embodiments, via an amide, ester, thioester, keto group, carbamate (urethane), carbon, or ether, and each of these groups may be inserted anywhere in the ULM group and the PTM group to provide maximal binding of the ULM group on the ubiquitin ligase and the PTM group on the target protein to be degraded. In certain aspects where the PTM group is a ULM group, the target protein for degradation may be the ubiquitin ligase itself. In certain exemplary aspects, the linker may optionally be attached to a substituted alkyl, alkylene, alkene or alkyne group, an aryl group, or a heterocyclic group on the ULM and / or PTM group.

[0113] In some embodiments, the compounds described herein comprise multiple ULMs, multiple PTMs, multiple chemical linkers, or any combination thereof.

[0114] In some embodiments, the invention provides one or more of the following methods, comprising administering a bifunctional compound described herein comprising ULM and a PTM (in some embodiments, the ULM and PTM are linked via a linker moiety, as described elsewhere herein), wherein ULM binds to the PTM, the ULM recognizes a ubiquitin pathway protein, and the PTM recognizes a target protein, such that degradation of the target protein occurs when the target protein is brought into proximity with a ubiquitin ligase (resulting in degradation of the target protein / inhibition of its effect and control of protein levels): Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity. disruption of FAK catalytic activity via direct binding of the FAT domain; Inhibiting FAK-associated scaffold function, Inhibiting FAK protein-protein interaction mediated by the FAT domain; Inhibiting the binding of paxillin to helix 1-4 of the FAT domain of FAK Inhibiting the binding of paxillin to helix 2-3 of the FAT domain of FAK inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; Inhibition of FAK-Leupaxin interaction via binding to the FAT domain of FAK Inhibition of FAK-CD4 interaction via binding to the FAT domain of FAK; Inhibition of FAK-CD8 interaction via binding to the FAT domain of FAK; Inhibition of FAK-DCC interaction via binding to the FAT domain of FAK inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; Inhibiting the binding of Leupaxin to each of its binding partners inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF. The control of protein levels provided by the present invention provides for the treatment of disease states or conditions, which are regulated through target proteins, by lowering the levels of said proteins in the patient's cells.

[0115] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Overview of stapled α-helical peptides targeting the FAT domain of FAK. A. Crystal structure of the FAT-paxillin interaction (PDB 1OW8), containing two paxillin LD2 motifs (shown in green and magenta). The stapled peptide is based on the structure of the paxillin LD2 motif. B. Zoom inset of the FAT helix 2-3 binding interface with paxillin LD2. C. Helix wheel structural analysis of the LD2-FAT inter- and intramolecular interactions. D. 3D representation of the stapling strategy used for peptide optimization. E. Overview of amino acid modifications used in the peptide design strategy.

[0116] Figure 2: Synthetic scheme for stapled peptide synthesis.

[0117] Figure 3: Chemical structure of stapled peptide UACC-1907.

[0118] Figure 4: Biochemical, biophysical, and cytological data for the stapled peptide UACC-1907 (1907). A. 3D model of peptide 1907 with the staples highlighted in red. B. Competition FP experiment with 1907 showing inhibition of TAMRA-LD2 binding to FAT. C. SPR binding and selectivity analysis with wild-type FAT and mutant FAT (L994E, I936A) at the helix-protein interface. D. Flow cytometry analysis of rhodamine-1907 (10 μM) cellular uptake in MDA-MB-453 breast cancer cells. E. Boyden chamber invasion assay in SK-MEL-103 melanoma cells. F. 3D Matrigel-on-top proliferation assay in SK-MEL-103 melanoma cells.

[0119] Figure 5: In complex with peptide 1907 15 A. HSQC NMR data of N-labeled FAT domain proteins. B. HSQC NMR data of N-labeled FAT domain proteins using a 600 MHz NMR spectrometer with 1% DMSO (maroon), 50 μM 1907 (red), 10 μM 1907 (green), and 5 μM 1907 (blue). 1 H / 15N HSQC spectrum. NOTE: The peptide induces peak intensity changes at both the helix 1-4 (K1032) and helix 2-3 (L959) binding sites. B. HSQC NMR and 1907 binding curves using four different residues on the FAT domain (V932, L959, L994, and D1036). The 1907 concentration was titrated from 100 to 0.01 µM, and the rate of change in peak integration was plotted against the concentration to determine K. D was calculated. Mapping of key perturbations caused by 1907 at C. helix 2-3 site and D. helix 1-4 site. Residues with large shifts are highlighted in green. Note that 1907 binds to the same site as native paxillin LD2 (yellow and cyan).

[0120] Figure 6: X-ray crystal structure at 1.95 Å resolution of a FAK peptide inhibitor (i.e., 1907) in complex with the human FAK FAT domain. The left panel shows the stapled peptide in blue and the FAT domain in green. The right panel shows an electron density map of the peptide in the binding pocket.

[0121] Figure 7: Anti-cancer effect of myristoylated peptide 1907 (UACC-2012). A. 3D structure of UACC-2012. B. 3D structure of negative control molecule (UACC-2014). C. 3D Matrigel-on-top cell proliferation data of stapled peptide in SK-MEL-103 melanoma cells. D. 3D Matrigel-on-top cell proliferation data of stapled peptide UACC-2012 in HUVEC "normal" cells.

[0122] Figure 8: FAT bivalent stapled peptide strategy and SPR data for synthetic peptide UACC-2023. A. Overview of the bivalent stapled peptide strategy to enable dual-site FAT domain conjugation using click chemistry and linker approaches. B. Structure of UACC-2023. C. PEG conjugated to the bivalent peptide UACC-2023 and FAT. 10 SPR sensogram of the linker.

[0123] Figure 9: Anticancer efficacy data for stapled peptides 2023 and 1907 in liposomal formulations. A. 2D proliferation data for peptide 2023 in combination with the cationic lipid reagent Saint-Protein (Synvolux). B. 2D proliferation data for peptide 1907 in combination with the cationic lipid reagent Saint-Protein. Lipid:peptide formulations were prepared as 10x stocks using a 1:1 (v:v) ratio and titrated concentrations of peptide in PBS pH 7.4 + 1% DMSO.

[0124] Figure 10: Overview of the synthetic strategy for FAT-kinase bifunctional inhibitors and the proof-of-concept molecule UACC-2030. A. (Top) Attachment site of the FAK kinase domain inhibitor PF-562271. (Bottom) Attachment site of the FAT domain inhibitor 1907. B. Synthetic plan of the FAT-kinase bifunctional group and chemical structure of the synthetic molecule UACC-2030.

[0125] Figure 11: Overview of the FAT-PROTAC synthesis strategy and the proof-of-concept synthetic molecule UACC-2019.

[0126] Figure 12: In vitro trypsin digestion assay to measure the stability and protease resistance of peptides (1967, 1907, and 2012). Assays were performed using trypsin-agarose beads (ThermoFisher), and peptide concentrations were measured using LC-MS. Peptide half-lives were calculated using GraphPad Prism software.

[0127] Figure 13: Antifibrotic activity of the FAT-stapled peptide UACC-2012 in LX2 human hepatic stellate cells. A. Western blot study of LX2 cells treated with 2 ng / mL TGF-β for 17 hours to induce a profibrotic phenotype. Cells were pretreated for 1 hour with either DMSO or UACC-2012 (mSP3) at 10 μM, 5 μM, and 2 μM. Quantification of fibronectin results is shown in the bottom panel. B. Morphological changes in LX2 cells after 17 hours of TGF-β and peptide treatment. Images were captured using bright-field microscopy. Abbreviations: mSP3 = UACC-2012; SP3 = UACC-1907.

[0128] Figure 14: Structures of compounds P29-P34, P37 and P38.

[0129] (Detailed Description of the Invention) Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many tumors, including melanoma, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, and glioblastoma (1), and has been shown to be an essential component of human cancer progression (2-4).

[0130] From a biological perspective, FAK is involved in motility, invasion, angiocrine signaling, lymphangiogenesis, metastasis, and epithelial-mesenchymal transition (EMT) (5-8). FAK also sequester and inactivate proapoptotic proteins such as p53 and RIP, enhancing survival signals necessary for cancer invasion and metastasis (9, 10). The role of FAK in cancer has been confirmed by clinical prognostic studies, genetically modified mouse models, and knockout studies (11-15).

[0131] Knockdown of FAK results in strong apoptotic activity and growth arrest in cancer cells but not in normal cells (16, 17). Conversely, FAK kinase inhibitors show only partial effects on apoptosis / tumor growth, and the FAK scaffold is hypothesized to be a key modulator of FAK-dependent anti-apoptosis (21). FAK directly binds to p53 and suppresses p53-mediated apoptosis, and disruption of the FAT domain with adenoviral FAK-CD has been shown to induce apoptosis in cancer cells (22). FAK kinase inhibitors have been shown to not dramatically inhibit FAK phosphorylation at the autophosphorylation residue Y397 or transphosphorylation of FAK by receptor tyrosine kinases (RTKs) 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).

[0132] The FAT domain, a four-helix bundle at the C-terminus of FAK containing the critical Y925 residue, is involved in multiple protein-protein interactions at focal adhesions. Multiple data have emerged demonstrating the importance of the focal adhesion targeting (FAT) domain as an initiator of FAK activity through multiple interactions with paxillin, leupaxin, CD4, and DCC (27-29). The integrity of the FAT domain is crucial for FAK localization to focal adhesions, association with integrins / RTKs, and downstream FAK signaling (30-32). Mutations in the FAT domain have demonstrated dramatic biological effects on metastasis, invasion, and apoptosis (33, 34). Therefore, experiments conducted during the development of embodiments of the present invention led us to hypothesize that peptide inhibitors targeting the FAT domain would be more effective than FAK kinase inhibitors, which tend to have less understood drug resistance mechanisms.

[0133] 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 focal adhesions is mediated by FAK-paxillin interactions, and mutations in its binding site have been shown to have dramatic effects on FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and invasion (34, 36). Paxillin contains two α-helical LD ​​motifs (LD2 and LD4) required for binding to the FAT domain of FAK. The FAT-paxillin LD2 / LD4 interaction has been well characterized, with a KD of 50–100 μM and confirmed by multiple assays (X-ray, SPR, ITC, NMR, FP, and mutagenesis). LD2 and LD4 interact at two separate hydrophobic patches on the FAT domain (helices 1-4 and helices 2-3), and disruption of both sites is required for maximal biological effect (27, 37, 38). Experiments conducted during the development of embodiments of the present invention synthesized and optimized peptides that can efficiently target FAK non-catalytic functions via binding of the FAT domain, thereby inhibiting, for example, the FAK-paxillin interaction (e.g., the FAK-LD2 domain of paxillin). In particular, the present invention provides stapled LD2 domain peptides capable of inhibiting the FAK-paxillin interaction. These LD2 peptides exhibit significant advantages over existing FAK inhibitors due to their ability to disrupt FAK protein-protein interactions (PPIs), thus providing novel anti-cancer effects.

[0134] Thus, the present invention provides a novel class of peptides (e.g., LD2 peptides) that function as inhibitors of focal adhesion kinase (FAK) activity through binding to the focal adhesion targeting (FAT) domain, thereby inhibiting FAK-paxillin interaction. Indeed, in some embodiments, the present invention provides LD2 peptides that can inhibit FAK-paxillin interaction. In some embodiments, the LD2 peptides are amphipathic α-helical stapled peptides containing hydrophobic and hydrophilic amino acids, where two or more amino acids of the peptide are linked to each other.

[0135] As used herein, the term "stapled peptide" refers to peptide regions linked to one another. In some embodiments, to increase the chemical stability and secondary structure of an α-helix, positions i and i+ of the α-helix can be stapled using various covalent bonding methods. Specifically, 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) can be stapled. The amino acids can be stapled via a covalent bond and a linker moiety, thereby increasing cell penetration 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) can be stapled.

[0136] Typically, two amino acids can be bonded to each other via a disulfide bond, a carbon-carbon bond, an azide-alkyne cycloaddition, or an amide bond. Examples of methods for bonding two amino acids to each other include introducing a disulfide between the two amino acid positions, introducing a carbon-carbon double bond by a metathesis reaction, introducing an amide bond, or introducing a short linker by a Michael reaction. Such stapling significantly enables the production of cell-penetrating peptides with improved cell-penetrating ability and desired chemical stability.

[0137] When a peptide is an α-helical peptide, two or more amino acids of the peptide can be linked together, excluding the basic peptide backbone, to form a cyclic structure. The size of the cyclic ring can vary depending on the position of the amino acids and the length of the linking moiety. One or more staples can be included in the peptide.

[0138] In certain embodiments of the present invention, one or more amino acids of a peptide can be functionalized with a double bond-containing compound. For example, the amino acids can be linked to each other through a ring structure generated by ring-closing metathesis between double bond-containing compounds. The functionalized amino acids can be amino acids substituted with an alkenyl side chain. The alkenyl side chain can 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.

[0139] The amino acids of the peptide are not particularly limited as long as they maintain an α-helical structure and exhibit amphipathic properties. For example, the hydrophilic amino acids may be one or more selected from the group consisting of arginine, lysine, and histidine, and the hydrophobic amino acids may be one or more selected from the group consisting of leucine, valine, tryptophan, phenylalanine, tyrosine, and isoleucine. Unnatural amino acids may also be used in the peptide structure.

[0140] In particular, in the present examples, amphipathic α-helical stapled peptides were generated that are capable of inhibiting the FAK-paxillin interaction.

[0141] As described above, in some embodiments, the present invention provides LD2 peptides capable of inhibiting FAK-paxillin interaction. In some embodiments, the LD2 peptides are amphipathic α-helical stapled peptides comprising hydrophobic and hydrophilic amino acids, wherein two or more amino acids of the peptide are linked to each other.

[0142] Specifically, the stapled LD2 peptide capable of inhibiting FAK-paxillin interaction can include any one of the following sequences and is generated by introducing a carbon-carbon double bond via a metathesis reaction: In the following sequences, R8 represents (R)-2-(7'-octenyl)alanine, S5 represents (S)-2-(4'-pentenyl)alanine, and R5 represents (R)-2-(4'-pentenyl)alanine.

[0143] In some embodiments, two or more LD2 peptides are joined together (e.g., linked via "click" chemistry) with a linker (e.g., a PEG-based linker) that is compatible with various functional groups and solvents and constructed via chemistry known to those of skill in the art.

[0144] In some embodiments, the LD2 peptide is further conjugated to an imaging agent (eg, conjugated to (5- / 6-)carboxytetramethylrhodamine (TAMRA)).

[0145] An important aspect of the present invention is that the LD2 peptides of the present invention induce cell cycle arrest and / or apoptosis, and enhance the induction of cell cycle arrest and / or apoptosis, either alone or in response to additional apoptosis-inducing signals (e.g., via inhibiting FAK non-catalytic activity) (e.g., via inhibiting FAK-paxillin interaction). Thus, it is contemplated that the LD2 peptides sensitize cells (including cells resistant to the inductive stimuli) to the induction of cell cycle arrest and / or apoptosis. 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 containing 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.

[0146] In some embodiments, the compositions and methods of the present invention are used to treat diseased cells, tissues, organs, or pathological and / or disease states in animals (e.g., mammalian patients, including, but not limited to, humans and livestock animals). In this regard, a variety of diseases and conditions are amenable to treatment or prevention using the methods and compositions of the present invention. A non-exhaustive 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 cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and 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, prostate carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex T- and B-cell mediated autoimmune diseases such as carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid 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; inflammatory diseases; infectious diseases; hyperproliferative diseases; AIDS; degenerative conditions, vascular diseases, etc. In some embodiments, the cancer being treated is metastatic. In other embodiments, the cancer being treated is resistant to anticancer drugs. In other embodiments, the disorder is any disorder having cells with FAK activity and / or FAK-paxillin associated activity.

[0147] Some embodiments of the present invention provide methods for administering an effective amount of an LD2 peptide of the present invention and at least one additional therapeutic agent (including, but not limited to, a chemotherapeutic anti-neoplastic agent, an apoptosis-modulating agent, an antibacterial agent, an antiviral agent, an antifungal agent, and an anti-inflammatory agent) and / or a therapeutic technique (e.g., surgical intervention and / or radiation therapy). In certain embodiments, the additional therapeutic agent is an anti-cancer agent.

[0148] Many suitable anti-cancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates the administration of many anti-cancer agents, including, but not limited to, agents that induce apoptosis; polynucleotides (e.g., antisense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biomimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated to anti-cancer drugs, toxins, defensins); toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g., IL-2)); adoptive immunotherapeutic agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans retinoic acid); gene therapy agents (e.g., antisense therapeutic agents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors; NF-κB modulators; anti-CDK compounds; HDAC inhibitors, and the like. Many examples of chemotherapeutic compounds and anti-cancer treatments suitable for co-administration with the present stapled peptide (LD2 peptide) are known to those skilled in the art.

[0149] In some embodiments, the anti-cancer agent comprises an agent that induces or stimulates 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., antibodies against TNF family receptor proteins, TNF family ligands, TRAIL, TRAIL-R1, or TRAIL-R2); kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitors, vascular growth factor receptor (VGFR) kinase inhibitors, fibroblast growth factor receptor (FGFR) kinase inhibitors, platelet-derived growth factor receptor ( PDGFR) kinase inhibitors, and Bcr-Abl kinase inhibitors (e.g., GLEEVEC); BCL-2 family inhibitors (VENCLEXTA); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); antiestrogens (e.g., raloxifene and tamoxifen); antiandrogens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (C OX-2 inhibitors (e.g., celecoxib, meloxicam, NS-398, and nonsteroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory agents (e.g., butazolidine, DECADRON, DELTASONE, dexamethasone, intenzol, 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); cell signaling molecules; ceramides and cytokines; staurosporine, etc.

[0150] In yet another aspect, the compositions and methods of the present invention provide an LD2 peptide of the present invention and at least one anti-hyperproliferative or anti-tumor agent selected from the group consisting of alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant- and / or animal-derived compounds).

[0151] Alkylating agents suitable for use in the compositions and methods of the present invention include, but are not limited to, 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil); 2) ethyleneimines and methylmelamines (e.g., hexamethylamine and thiotepa); 3) alkylsulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozotocin (streptozotocin); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenoimide-azolecarboxamide).

[0152] In some embodiments, antimetabolites suitable for use in the compositions and methods of the present invention 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 (fluorodeoxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin)).

[0153] In still further embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the present invention include: 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-alpha); 6) platinum coordination complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., procarbazine (N-methylpropional); methylhydrazine); 9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) adrenocortical steroids (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).

[0154] Any oncolytic agent commonly used in the context of cancer therapy finds use in the compositions and methods of the present invention. For example, the U.S. Food and Drug Administration maintains a regulatory system for oncolytic agents approved for use in the United States. International counterparts to the U.S. FDA maintain similar regulatory systems. Table 4 provides an exemplary list of antitumor agents approved for use in the United States. Those skilled in the art will understand that the "product labeling" required for all U.S.-approved chemotherapeutic agents describes the approved indications, dosing information, toxicity data, etc., for exemplary agents.

[0155] [Table 2]

[0156] [Table 3]

[0157] [Table 4]

[0158] [Table 5]

[0159] [Table 6]

[0160] [Table 7]

[0161] [Table 8]

[0162] [Table 9]

[0163]

Table 10

[0164] Anti-cancer agents further include compounds identified as having anti-cancer activity. For example, including, but not limited to, 3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG20, AE-941, AG-013736, AGRO100, alanosine, AMG706, antibody G250, antineoplaston, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cephalosporin ... Fixim, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, exceincidin 743, efaproxiral, eflornithine, EKB-569, enzaurin, erotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histoline, HKI-272, homoharringtonine, HSPPC-96, hu14.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12, IPI-504, irofenib, 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, nitrilamide Rocamptothecin, nolatrexed dihydrochloride, nolvadex, NS-9, O6-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, Pixatron, PS-341, PSC833, PXD101, pyrazoloacridine, R115777, RAD001, lamprinase, 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, talamostat, talampanel, tariquidar, temsirolimus, TGFα-PE38 Immunotoxins, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosquidar trihydrochloride.

[0165] For a more detailed description of anti-cancer and other therapeutic agents, those skilled in the art can refer to any number of instruction 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.

[0166] The present invention provides a method for administering the LD2 peptide of the present invention together with radiation therapy. The present invention is not limited by the type, amount, or delivery and administration system used to deliver a therapeutic dose of radiation to animals. For example, animals can receive photon radiation therapy, particle beam radiation therapy, other types of radiation therapy, and combinations thereof. In some embodiments, radiation is delivered to animals using a linear accelerator. In other embodiments, radiation is delivered using a gamma knife.

[0167] Antimicrobial therapeutic agents can also be used as therapeutic agents in the present invention. Any agent that kills, inhibits the function of, or otherwise attenuates microorganisms can be used, and any agent with such activity is contemplated. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disrupting agents, and the like, used alone or in combination. Indeed, any type of antibiotic can be used, including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like. In some embodiments of the present invention, the LD2 peptide of the present invention and one or more therapeutic or anti-cancer agents are administered to an animal under one or more of the following conditions: different cycles, different durations, different concentrations, different routes of administration, etc. In some embodiments, the LD2 peptide is administered before the therapeutic or anti-cancer 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 before administration of the therapeutic or anti-cancer agent. In some embodiments, the LD2 peptide is administered after the therapeutic or anti-cancer 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 administration of the anti-cancer agent. In some embodiments, the LD2 peptide and the therapeutic or anti-cancer agent are administered simultaneously but on different schedules, for example, the LD2 peptide is administered daily while the therapeutic or anti-cancer 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 anti-cancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.

[0168] Compositions within the scope of the present invention include all compositions containing the LD2 peptide of the present invention in an amount effective to achieve its intended purpose. While individual needs vary, determining the optimal range of effective amounts of each component is within the skill of the art.

[0169] In addition to administering the stapled peptides (e.g., LD2 peptides) as raw peptides, the LD2 peptides can be administered as part of a pharmaceutical formulation containing a suitable pharmaceutically acceptable carrier, including pharmaceutically acceptable excipients and auxiliary agents that facilitate processing of the LD2 peptide into a formulation. These formulations, particularly oral or topical formulations and formulations that can be used for one type of administration, such as tablets, dragees, sustained-release lozenges and capsules, mouthwashes, gels, liquid suspensions, hair rinses, hair gels, shampoos, and other formulations that can be administered rectally, such as suppositories, and solutions suitable for intravenous infusion, injection, topical, or oral administration, contain about 0.01-99%, and in one embodiment, about 0.25-75%, of the active peptide, together with the excipients. In some embodiments, the LD2 peptide formulations can also be liposomal in nature. In some embodiments, liposomal formulations of the peptides (e.g., LD2, LD4) may consist 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, soybean 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) is / are comprised of sphingomyelin (SM), D-erythrose-sphingomyelin, D-erythrose-dihydrosphingomyelin, palmitoylsphingomyelin, lysophospholipids, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, diglycerides, small alkyl chain phospholipids, phosphatidylcholine, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-Distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), Distearoylphosphatidylcholine, 1-Myristoyl-2-Palmitoylphosphatidylcholine, 1-Palmitoyl-2-myristoylphosphatidylcholine, 1-Palmitoyl-2-stearoylphosphatidylcholine, 1-Stearoyl-2-Palmitoylphosphatidylcholine, Dioleoylphosphatidylcholine, Dioleoylphosphatidylethanolamine, Dilauroylphosphatidylglycerol Phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, ceramide, phosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoylsphingomyelin, phytosphingomyelin, and dipalmitoylsphingomyelin Phosphorus, distearoyl sphingomyelin, dipalmitoyl phosphatidylglycerol salts, phosphatidic acid, galactocerebroside, ganglioside, cerebroside, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenylglycosides, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipid, and cholesterol and its derivatives, lyso- Phosphatidylcholine, 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-maleimidophenyl)cyclohexane-carboxamide], 1,2-Di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], Lysophosphatidic acid, Lysophosphatidylcholine, OA-NO2( Linoleic acid nitrate 9- and 10-nitro-cis-octedecenoic acid), LNO2 (nitrate linolenic acid 9-, 10-, 12-, and 13-nitro-cis-octedecadienoic acid), AA-NO2 (nitrate arachidonic acid 5-, 6-, 8-, 9-, 11-, 12-, 14-, and 15-nitro-cis-eicosatetraenoic acid), CLNO2 (nitrate cholesteryl linoleate cholesteryl-9-, 10-, 12-, and 13-nitro-cis-octedecadienoic acid), fatty acids, omega-3 polyunsaturated fatty acids, hexadeca trienoic acid (HTA; 16:3(n-3); all-cis-7,10,13-hexadecatrienoic acid), α-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-octadecatrienoic 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; clupanodonioic 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, or any combination thereof.

[0170] The pharmaceutical compositions of the present invention can be administered to any patient who can benefit from the beneficial effects of the staple peptide (LD2 peptide) of the present invention. While not intending to limit the present invention, the most important of such patients are mammals, such as humans. Other patients include livestock (cattle, sheep, pigs, horses, dogs, cats, etc.).

[0171] LD2 peptide and its pharmaceutical compositions can be administered by any means that achieves their intended purpose. For example, administration can be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal, or topical. Alternatively, or concurrently, administration can be oral. The dosage depends on the age, health, and weight of the recipient, concurrent treatments (if any), frequency of treatment, and the nature of the desired effect.

[0172] The pharmaceutical preparations of the present invention are produced in a manner known per se, for example, by conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by mixing the active LD2 peptide with solid excipients, optionally milling the resulting mixture, and, if desired or necessary, adding suitable auxiliaries and then processing the granular mixture to obtain tablets or dragee cores.

[0173] Suitable excipients are, in particular, sugars (e.g., lactose or sucrose, mannitol or sorbitol), cellulose preparations and / or fillers such as calcium phosphates (e.g., tricalcium phosphate or calcium hydrogen phosphate), and binders such as starch pastes using, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrants can be added, such as the above-mentioned starches, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate). Auxiliaries are, inter alia, flow regulators and lubricants, such as silica, talc, stearic acid or a salt thereof (e.g., magnesium stearate or calcium stearate), and / or polyethylene glycol. Dragee cores are, if desired, provided with a suitable coating that is resistant to gastric juices. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. To produce coatings that are resistant to gastric juices, solutions of suitable cellulose preparations (e.g., acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate) are used. Dyes or pigments can be added to tablets or dragee coatings, for example, to identify or characterize the active compound dosage combination.

[0174] Other pharmaceutical formulations that can be used orally include push-fit capsules made of gelatin and encapsulated soft capsules made of gelatin and a plasticizer such as glycerin or sorbitol. The push-fit capsules can contain the active staple peptide (e.g., LD2 peptide) in the form of granules that can be mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, a stabilizer. In one embodiment, the active staple peptide (e.g., LD2 peptide) in the soft capsule is dissolved or suspended in a suitable liquid such as fatty oil or liquid paraffin. Additionally, stabilizers may be added.

[0175] Possible pharmaceutical preparations for rectal administration include, for example, suppositories, which consist of a combination of one or more active peptides with a suppository base. Suitable suppository bases include, for example, natural or synthetic triglycerides or paraffin hydrocarbons. In addition, gelatin rectal capsules, which consist of a combination of an active staple peptide (e.g., LD2 peptide) with a base, can also be used. Possible bases include, for example, liquid triglycerides, polyethylene glycol, or paraffin hydrocarbons.

[0176] Formulations suitable for parenteral administration include aqueous solutions of the active peptide in water-soluble form, such as water-soluble salts and alkaline solutions. In addition, suspensions of the active staple peptide (e.g., LD2 peptide) can be administered as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or polyethylene glycol-400. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. Optionally, the suspension can also contain stabilizers.

[0177] The topical compositions of the present invention may, in one embodiment, be formulated as oils, creams, lotions, ointments, etc., by selection of an appropriate carrier. Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats, and high molecular weight alcohols (C 12 The carrier may be one in which the active ingredient is soluble. Emulsifiers, stabilizers, humectants, and antioxidants, as well as agents that impart color or fragrance, if desired, may also be included. In addition, transdermal penetration enhancers may be used in these topical formulations. Examples of such enhancers can be found in U.S. Patent No. 3,989,816 and U.S. Patent No. 4,444,762, each of which is incorporated herein by reference in its entirety.

[0178] Ointments can be prepared by mixing the active ingredient in a vegetable oil such as almond oil with warm soft paraffin and then cooling the mixture. A typical example of such an ointment contains about 30% almond oil and about 70% white soft paraffin by weight. Lotions can be conventionally prepared by dissolving the active ingredient in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.

[0179] Those skilled in the art will readily appreciate that the foregoing description merely represents a detailed description of certain preferred embodiments of the invention. Various modifications and variations of the compositions and methods described above can be readily accomplished using the expertise available in the art and are within the scope of the invention.

[0180] (Example) The following examples are illustrative, but not limiting, of the staple 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 that are obvious to those skilled in the art are within the spirit and scope of the present invention.

[0181] Example I This example describes the generation of peptides that have affinity for the FAT domain of FAK and can disrupt the interaction between paxillin and focal adhesion kinase (FAK), thus inhibiting FAK activity associated with FAK-paxillin interaction.

[0182] To develop inhibitors of the FAK-paxillin interaction, we conducted experiments taking a stapled peptide approach, which has the following advantages: (1) using a native peptide as a starting point, (2) spanning the entire interaction interface, (3) possessing chemical moieties to enhance α-helicity, cell permeability, and proteolytic stability, and (4) viable molecules for pure SAR analysis (amide chemistry).

[0183] We first performed experiments starting with the synthesis of a series of cyclic peptides based on the paxillin LD2 motif and all-hydrocarbon stapling (Figure 1). We used solid-phase peptide synthesis and incorporation of olefinic amino acids ((R)-N-Fmoc-2-(4'-pentenyl)alanine and (S)-N-Fmoc-2-(7'-octenyl)alanine) at positions i and i+7 (six amino acids apart, or two helical turns), followed by ring-closing olefin metathesis and cleavage from the resin (see Figure 2) (Kim Y et al., Nature Protocols. 2011;6:761-71). Stapled peptides were designed using molecular modeling and X-ray crystallography of the FAT-LD2 complex (PDB 10W8). Residues not part of the binding interface were selected for substitution with olefinic amino acids. Residues in the binding interface were also selected for SAR testing.

[0184] There are three major approaches to hydrocarbon stapling of α-helical peptides: (1) i, i+3, (2) i, i+4, and (3) i, i+7, each with the potential for different biological effects. Experiments were conducted to synthesize various iterations (staple scans) of these i motifs at different positions (N- or C-terminal shifts) and to staple residues present in both the hydrophobic interface and the solvent-accessible area. Extended residues on the N- and C-termini of peptides can have beneficial effects on both binding affinity and cell permeability, so experiments were conducted. Finally, experiments were conducted to replace natural amino acids with hydrophobic / polar / charged substitutions, unnatural amino acids, or D-amino acids (39) to enhance protein contacts, enhance permeability, and further inhibit recognition by proteases (Figure 1).

[0185] To understand the structure-activity relationship of FAK stapled peptides, an experiment was designed and 36 peptides were synthesized (Table 2), varying based on stapling strategy ((i, i+3), (i, i+4), and (i, i+7)), staple position, sequence length, amino acid composition, and homologous sequence (LD2, LD4, CD4, DCC, Leupaxin). A K of 3.4 μM was obtained in SPR affinity analysis. D The stapled peptide UACC-1907, which exhibits a very high degree of selectivity for WT / mutant proteins in SPR and competitively inhibits paxillin-FAT binding in fluorescence polarization (FP) assays:

[0186] [ka]

[0187] was identified (K i = 5.8 μM) (Figure 4). The activity of UACC-1907 was significantly higher than that of the natural paxillin LD2 peptide UACC-1967 (K D = 156 μM, K i = 70.6 μM). The chemical structure of UACC-1907 (1907) is also shown in Figure 3.

[0188] The specific staple position of 1907 was essential for its biochemical / biophysical activity, as 1905, which had the same i+7 stapling motif shifted by one amino acid, had no binding or inhibitory properties. Other LD2 peptides (1914, 2017) with the same i+7 stapling motif at different positions 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), and 2024-(i,i+3)) showed less activity compared to 1907. Furthermore, a peptide with an Aib (2-aminoisobutyric acid)-based α-helix stabilization strategy (i.e., 1912) had lower activity compared to 1907. The amino acid sequence was also crucial for activity, as peptide 1910, which contains two glutamic acid to glutamine conversions, was not as active as peptide 1907. The amino acid sequence was also crucial for activity, as peptides with leucines 145, 149, and 152 (based on the paxillin sequence) converted to tryptophan (1933, 2007) had poorer binding and inhibitory properties compared to peptide 1907. Peptide 2014, which contains the L145E and L152E substitutions, showed no binding or inhibitory properties. Overall, stapled peptides based on homologous protein sequences (1929, 1916) did not have comparable activity to peptide 1907 derived from LD2. The paxillin LD4-derived peptide 1917 had similar, albeit lower, activity than peptide 1907 derived from LD2. The length of stapled peptide 1907 was also important for activity, as truncated stapled peptides at the N- and C-termini (i.e., 2011) had no binding or inhibitory properties. The extended peptide 1920 had a moderate binding affinity (K D =7.0 μM), but had poorer inhibitory properties (K i = 25.0 μM). These data indicated that empirically derived optimal stapling chemistry and amino acid sequence were required for best FAK binding and inhibition.

[0189] To further evaluate the activity of 1907, experiments were performed to test the peptide in a flow cytometry-based cell permeability assay. In this assay, rhodamine-labeled 1907 successfully crossed the cell membrane in MDA-MB-453 breast cancer cells compared to the rhodamine-only control (Figure 4). Experiments were also performed to test cellular efficacy in SK-MEL-103 melanoma cells. Peptide 1907 exhibited significantly higher activity than the known FAK inhibitor defactinib (compound K). D (compared to ) effectively inhibited SK-MEL-103 invasion and 3D proliferation at lower relative concentrations (Figure 4).

[0190] Further structural biology experiments were performed to examine 1907 for binding to the paxillin-binding site of the FAK FAT domain. 15 In HSQC NMR experiments using N-labeled FAT domain proteins, 1907 showed specific binding to the native paxillin LD2 peptide at both the FAT helix 2-3 (L959, R962, K955) and FAT helix 1-4 (V928, I936) binding sites (Figure 5). NMR-derived FAT binding affinity indicated that 1907 binds to the helix 1-4 site (I936:K). D =33.2μM, K1032:K D = 33.7 μM) compared with the FAT helix 2-3 site (L959:K D =1.0 μM, K955:K D The 1907 showed a stronger affinity for the FAK domain (p = 1.6 μM). We also successfully determined the X-ray co-crystal structure of 1907 in complex with the human FAK FAT domain (Figure 6). In this crystal structure, 1907 binds to the same FAT helix 2-3 site as paxillin LD2 and exhibits a pronounced α-helical structure. Overall, these studies supported the feasibility of the stapled peptide approach and provided 1907 as a peptide lead for further chemical optimization.

[0191] To improve the cellular potency of 1907, peptide analogs were designed and synthesized with myristoyl (2012, 2029) or dodecyl (2025) modifications for increased hydrophobicity and cellular uptake (Figure 7, Table 2, Figure 14). A negative control myristoylated peptide (2020) was designed and synthesized based on the inactive molecule 2014. Experiments were performed to test the effects of these peptides in a 3D proliferation assay using SK-MEL-103 melanoma cells. Peptide 2012 (IC 50 = 5.5 μM) is 1907 (IC 50 = 60 μM), whereas the negative control 2020 had no effect (IC 50 = NA). Furthermore, 2012 did not affect the proliferation of normal HUVEC cells (IC 50 =NA) (Figure 7).

[0192] As shown 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 generate a multivalent peptide capable of binding to both sites simultaneously, and thus with nanomolar binding affinity, we synthesized DBCO-[PEG] n -DBCO or alkyne-[PEG] n A series of binding peptides (2018, 2021, and 2023) were designed and synthesized via the attachment of two azide-modified 1907 molecules (2006) to a -alkyne linker (Table 2, Figure 8). The chemical structures of 2018, 2021, and 2023 are shown in Figure 14. The synthesized azide-containing stapled peptide (2006) showed binding affinity for FAT comparable to that of the parent peptide 1907 (4.6 vs. 2.8 μM). Azide-alkyne cycloaddition, or "click," chemistry, was used to link the multivalent peptides. Experiments were performed to test the FAT binding affinity of the multivalent peptides 2018, 2021, and 2023 (Table 2). The DBCO-based multivalent peptide 2018 showed increased binding affinity (K) compared to 1907. D = 24.2 μM), but not the alkyne-based peptide 2021 (K D= 237 nM) and 2023 (K D =21 nM) showed a dramatic improvement in FAT binding affinity (Figure 8). These data demonstrate the proof of principle for the FAT polyvalent method and identify the optimal linker length (PEG 1.5) for maximal FAT binding. n ) and empirical determination of chemistry (DBCO vs. alkyne).

[0193] To facilitate membrane permeability of peptides 2023 and 1907, in vitro experiments were performed using the liposomal formulation Saint-Protein (Synvolux). Formulations were prepared using a 3:1 volume:volume ratio of Saint:Peptide, and peptide concentrations were titrated in PBS pH 7.4 + 1% DMSO. As shown in Figure 9, the 2023 liposomal formulation demonstrated nanomolar efficacy against the proliferation of various cancer cells (U87 IC 50 =40 nM, MDAMB231 IC 50 =27nM, SKMEL103 IC 50 =50 nM). Liposomal formulations of 1907 showed low micromolar effects on cancer cell proliferation (U87 IC 50 =8.5μM, MDAMB231=2.3μM, SKMEL103 IC 50 = 2.4 μM). The calculated cellular IC50 for both 2023 and 1907 was significantly higher than the FAT domain K determined by SPR and NMR. D It was a straight line.

[0194] To create a bifunctional peptide with dual capabilities to inhibit both the FAK FAT domain and the FAK kinase domain, we designed a hybrid peptide based on the FAT-stapled peptide 1907 and the FAK kinase domain inhibitor PF-562271 (PF-271) (Figure 10). Using "click" chemistry, alkyne-derivatized PF-271 was linked to the azide-modified peptide 2006. The rational selection of 2006 as the conjugation partner was determined by X-ray crystallography information and calculated biophysical data (Table 2 and Figure 6). The alkyne functionalization site on PF-271 was selected based on the previous X-ray cocrystal structure of PF-271:FAK kinase, demonstrating the feasibility of the PROTAC approach (40). The successful synthesis of the FAT-kinase bifunctional peptide (UACC-2030) is shown in Table 3. The chemical structure of 2030 is shown in Figure 10 and Figure 14.

[0195] To generate a FAT peptide capable of degrading FAK protein in cells via a PROteolysis TArgeting Chimera (PROTAC) approach, we designed a hybrid peptide based on the FAT staple peptide 1907 and the E3 ligand targeting ligand thalidomide (Figure 11). Using "click" chemistry, alkyne-derivatized thalidomide was attached to the azide-modified peptide 2006. The rational selection of 2006 as the conjugation partner was determined by X-ray crystallographic information and calculated biophysical data (Table 2 and Figure 6). The successful synthesis of the FAT-PROTAC molecule (UACC-2019) is shown in Table 3. The chemical structure of 2019 is shown in Figures 11 and 14. The peptide 2019 (K D = 10.2 μM) exhibited FAT domain binding affinity comparable to the parent structure 1907 (Table 2).

[0196] Experiments were performed to test the protease resistance of synthetic peptides 1967 (native LD2), 1907, and 2012 (Figure 12). Trypsin-agarose beads were used to test the potential cleavage of the peptides in physiological buffer, and LC-MS was used as the analytical method to determine the peptide concentration. As shown in Figure 12, stapled peptide 1907 (t 1 / 2 =15.7 hours) and 2012(t 1 / 2 >48 hours) both expressed the natural LD2 peptide 1967 (t 1 / 2 =0.69 hours), demonstrating greatly improved stability and protease resistance.

[0197] Experiments were conducted to test the antifibrotic effects of stapled peptides targeting the FAK FAT domain (Figure 13). TGF-β induction was performed in LX-2 human hepatic stellate cells, promoting a profibrotic phenotype and differentiation into myofibroblast-like cells. 2012 treatment dose-dependently reduced the TGF-β-induced expression of fibronectin, a marker of fibrosis. 2012 treatment also induced morphological changes in TGF-β-induced LX-2 cells, resulting in cell dedifferentiation and phenotypic reversion.

[0198] Additional stapled peptides were generated. Table 2 provides additional stapled peptides with sequence / structure information, SPR and FP results.

[0199] [Table 11]

[0200] Table 2: Stapled peptide SAR analysis. Synthetic hydrocarbon stapled peptides were varied based on stapling strategy ((i, i+3); (i, i+4); and (i, i+7)), staple position, sequence length, amino acid composition, and homologous sequence. MW, SPR data, and FP data are shown. Note: 1907 and 2023 were selected as top candidates based on the data. Abbreviations: R8: (R)-2-(7-octenyl)alanine; S5: (S)-2-(4-pentenyl)alanine; R5: (R)-2-(4-2-pentenyl)alanine; Az: 2-(2-(2-azidoethoxy)ethoxy)acetyl; Aib: 2-aminoisobutyric acid; DBCO: 3-amino-1-(2-azatricyclo[10.4.0.0 4,9]hexadeca-1(16),4,6,8,12,14-hexen-10-yn-2-yl)propan-1-one; * : two-site binding model; NC: not calculated. NA: for applications outside the scope of the assay, K i Unless otherwise noted, peptides are the product of an intramolecular ring-closing methanogenesis reaction, and double bond geometries have not been established or quantified. These sequences may be omitted to refer to "click" chemistry products. Figure 14 shows the structures of peptides P29-P34.

[0201] Example II This example describes the materials and methods for Example I.

[0202] <Synthesis> The compounds described herein can be prepared by cleaving Rink amide resin with Fmoc-(S)-2-(4-pentenyl)alanine, Fmoc-(R)-2-(4-pentenyl)alanine, Fmoc-(R)-2-(7-octenyl)alanine, N-α-aminoisobutyric acid (Fmoc-Aib-OH), Nα-Fmoc-Nε-(azido-PEG4)-L-lysine, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, FmocAsp(Ot-Bu)-OH, Fmoc-G Synthesis was performed using various combinations of In(Trt)-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Gly-OH, FmocHis(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. With the exception of glycine, or where otherwise noted, these amino acids are in the "L" configuration. Standard Fmoc-based solid-phase peptide synthesis chemistry was used, as known to those skilled in the art. Peptides were synthesized on a Biotage Initiator+ Alstra automated microwave-assisted peptide synthesizer using Rink Amide MBHA ChemMatrix resin (typical loading 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 performed on a 0.1 mmol scale. Typical conditions were as follows: 0.5 M concentrations of protected amino acids, HCTU, DIC, and Oxyma Pure; 1.0 M concentrations of DIPEA; and 5.0 M concentrations of acetic anhydride in DMF were used. A 4 mL reaction volume was used to ensure efficient mixing, and DMF was added as needed.A solution of 20% 4-methylpiperidine in DMF was used for Fmoc-deprotection, and cleavage / global deprotection was carried out using 95:2.5:2.5 (v / v) FA:water:triisopropylsilane. Couplings were performed using 5 equivalents of amino acid, 5 equivalents of HCTU, and 10 equivalents of DIPEA, except when using unnatural alkene amino acids, where 3:3:6 molar equivalents of amino acid:HCTU:DIPEA were used.

[0203] The resin, provided as a free amine, was first swollen in DMF (70°C, 20 min). Fmoc-protected amino acids were coupled at 75°C for 4 min, followed by a DMF wash. Double coupling was used for all residues. Arginine was coupled at 50°C for 6 min. This was followed by a cycle of Fmoc-deprotection consisting of a 3 min reaction, followed by a 10 min reaction with fresh reagents at ambient temperature, followed by a DMF wash. Double Fmoc-deprotection cycles were used for N-terminal residues and olefinic amino acids.

[0204] Grubbs' first generation catalyst (benzylidene-bis(tricyclohexylphosphine)dichlororuthenium) was used to perform ring-closing metathesis on an Fmoc-protected peptide in manual injection mode on the machine. Prior to the reaction, the resin was thoroughly washed with cycles of DCM followed by EtO, briefly dried in vacuo, then washed and swollen with DCE. Grubbs I (10 mM) was then added and the reaction was run for 1 h at 40 °C with evacuation every 15 min. This was done a total of three times, with DCE used for washing between reactions. After completion, the resin was washed with DCE, then DCM, and then swollen with DMF.

[0205] The Fmoc group was deprotected as described above. When N-capped as acetamide, 50 equivalents of acetic anhydride and 10 equivalents of DIPEA were used, which was allowed to react for 45 minutes at ambient temperature. Rhodamine B was coupled with 5 equivalents each of DIC and Oxyma Pure at 75°C for 4 minutes. The resin was washed with three cycles of DCM followed by EtO and then dried in vacuo.

[0206] Cleavage with 95:2.5:2.5 TFA:water:triisopropylsilane was carried out at ambient temperature for 2 hours. The reaction solution was then added dropwise to 35 mL of cold EtO. This was mixed, cooled to -80°C for 30 minutes, and then centrifuged at 6000 x G for 6 minutes. After decanting the supernatant, the pellet was suspended in 20 mL of EtO, then cooled, centrifuged, and decanted as above. The crude peptide was dried under vacuum.

[0207] Preparative HPLC was performed on an Agilent 1260 II quatemary HPLC equipped 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) with a gradient of acetonitrile (0.1% AcOH) in water (0.1% AcOH).

[0208] Analytical HPLC was performed using a Zorbax SB-C18 column (Agilent 830990-902; 2.1 × 150 mm; 80 Å pore size; 3.5 μm particle size) with a gradient of acetonitrile (0.1% AcOH) in water (0.1% AcOH) at 1200°C. Analysis was performed on an Agilent 1200 HPLC using a DAD and an Infinity 6125 LCMSD detector.

[0209] [ka]

[0210] <Surface Plasmon Resonance (SPR)> SPR binding studies were performed on a ForteBio Pioneer FE SPR system. Briefly, SADH streptavidin in a dextran hydrogel biosensor (ForteBio) was docked onto a flow cell and preconditioned with two injections of 10 mM NaOH, 1 M NaCl at 50 μL / min for 1 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 1000 RU of immobilized protein on channel 1. Empty channel 2 served as a reference control. After achieving a stable baseline, a series of peptide concentrations (200 μM–0.01 μM) was prepared in final running buffer (100 mM Tris-HCl, 200 mM NaCl, 0.05% Tween-20, 5% DMSO) and injected at a flow rate of 75 μL / min using the OneStep gradient injection method. 3% sucrose was used as the bulk standard control for OneStep injection, and a DMSO calibration curve was performed using a concentration range of 3.5%–6.5% DMSO. The original SPR data were appropriately processed in Qdat software (ForteBio) by baseline normalizing, channel alignment, reference channel subtraction, and blank subtraction prior to injection. D The kinetic data were fitted to a pseudo-first-order 1:1 interaction binding model to calculate the . Furthermore, the steady-state model and Req data points were used to verify the binding affinity. Visual inspection of the SPR sensograms was performed to verify proper model fitting, the absence of mass transport effects, return to baseline, and the absence of erratic kinetics.

[0211] Fluorescence polarization assay The FP assay buffer used was 20 mM Tris, 200 mM NaCl, 0.05% β-methylcellulose, 0.1% Triton X-100, 5% glycerol, and 1X Halt protease inhibitor cocktail. All final FP reactions were placed in 30 μL into a 384-well plate (NUNC 267461) and allowed to equilibrate for 3 hours with shaking at room temperature. Plates were read on a PerkinElmer EnVision plate reader with Envision Manager 1.13 software. A Bodipy TMR FP optical module (2100-4100) was used as the mirror. The excitation filter (2100-5830) utilized a wavelength of 531 nm, and both emission filters (2100-5800 and 2100-5810) were at 579 nm. The baseline mP for the TAMRA-LD2-L10D alone was set to 15 mP through the assay optimization wizard in Envision Manager software. Assay optimization included a measurement height of 6.5 mm, excitation light at 100%, G factor of 1.01, detector gain of 300, and flashes per well of 25.

[0212] IC of peptide inhibitors 50 For measurements, inhibitors were titrated from 325 nM to 667 μM in FP buffer containing 20 μM FAT and 0.1 μM TAMRA-LD2. Wells without FAT were used as baseline values, which were subtracted from the raw values ​​to obtain ΔmP values. Plates were read every hour for 4 hours, and IC values ​​were calculated. 50 The titration data were processed in GraphPad Prism to generate dose-response curves and calculated IC with standard errors. 50 (SE) was generated. A four-parameter dose-response inhibition model was used, with the bottom fit constrained to the lower plateau of the curve. i was calculated using the following formula:

[0213]

number

[0214] In the formula, I 50 is the concentration of free inhibitor at 50% inhibition. 50 is the concentration of free ligand at 50% inhibition, P0 is the concentration of free protein, and K D is calculated from the saturation curve. 50 is calculated as follows:

[0215]

number

[0216] In the formula, P T is the total protein concentration, and L T is the total labeled ligature cardinality, and P0 is the P0 2 +(K D +L T ) * P0-P T ,PL0=P-P0,PL 50 =PL0 / 2, L0=L T -PL0, and L 50 =L T -PL 50 This is the correct route.

[0217] <2D HSQC NMR> 2D HSQC-NMR samples were prepared at 100 μM 15 N FAT was prepared. Peptides were screened at concentrations ranging from 500 to 250 nM in 5% DMSO. TROSHSQC was 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 included 16 overlapping signals collected at sweep widths of 14.03 and 30.0 ppm, and 2048 and 256 points, respectively [29-33]. All final data were processed with Bruker TopSpin. Chemical shift perturbations (CSPs) were mapped onto the structure of the FAT-LD2 complex (PDB 1OW8) using PyMOL software to investigate the binding site and conformational changes upon binding.

[0218] <Flow cytometry> Canto II Flow cytometry assays were performed using a flow cytometer (BD Biosciences) to measure the cellular uptake of TAMRA-tagged peptides. Briefly, the device was appropriately gated using cells treated with only TAMRA as a positive control. Also, cells treated with untagged peptides were used as a negative control to exclude non-specific signals. To measure cellular uptake, cells were treated with TAMRA-tagged peptides for 2 - 48 hours. Peptide permeability was reported as the percentage positive and mean fluorescence intensity (MFI) relative to the TAMRA positive control.

[0219] <Immunofluorescence> To measure the effect of stapled peptides on FAK localization, immunofluorescence staining was performed. Briefly, cells were seeded on coverslips and fixed with 4% paraformaldehyde in 1×PBS PH7.4 for 10 minutes and permeabilized with 0.2% Triton X-100 for 5 minutes on ice. Cells were blocked with 25% normal goat serum in 1×PBS PH7.4 for 30 minutes, washed with 1×PBS PH7.4, and incubated with the FAK 4.47 primary antibody (Millipore) diluted 1:200 in 25% goat serum in 1×PBS PH7.4. Cells were washed three times with 1×PBS PH7.4 and the FITC-conjugated secondary antibody (diluted 1:400 in 25% goat serum) was applied to the coverslip. Cells were imaged using a Zeiss AXIO Imager MII Upright Widefield Fluorescent Microscope. Six fields were photographed for each sample.

[0220] <X-ray crystallography> Co-crystallization of the lead peptide SP3 with the human FAK FAT domain (AA 919-1052), in which the peptide was mixed and incubated with the protein (1:1 molar ratio) prior to crystallization, was performed using a Rigaku Phoenix-HT crystallization robot, using a buffer plate focused around a commercial crystallization kit (Hampton Research, Molecular Dimensions, Qiagen) and varying initial crystallization conditions (pH, salt, glycerol, and other precipitants). Data collection was performed at synchrotron radiation sources such as the Advanced Photon Source (APS) at Argonne National Laboratory (Chicago, IL) and the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory (Berkeley, CA). Structure determination was performed by molecular replacement using the crystal structure PDB 1K05 as a reference model. The extra electron density in the FAK-FAT domain binding pocket (Fo-Fc) was modeled onto the peptide structure and progressively refined over multiple cycles to build a high-quality model.

[0221] Table 3 provides mass spectrometry data for the peptide inhibitors of the present invention.

[0222] [Table 12]

[0223] Table 3. Mass spectrometry data for peptide inhibitors. Mass spectra are from ESI+.

[0224] Although the present invention has been fully described, those skilled in the art will recognize that the same can be practiced 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 incorporated by reference in their entirety.

[0225] (Incorporated by reference) The entire disclosure of each patent document and scientific article referenced herein is incorporated by reference for all purposes, including, but not limited to, the following references: 1. Golubovskaya VM, Kweh FA, Cancer WG. Focal adhesion kinase and cancer. Histol Histopathol. 2009;24(4):503-10. Epub 2009 / 02 / 19. PubMed PMID: 19224453. 2. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of focal adhesion kinase gene and invasive cancer. Lancet. 1993;342(8878):1024-5. 3. Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, Liu ET, Cance WG. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Research. 1995;55(13):2752-5. 4. Lark AL, Livasy CA, Calvo B, Caskey L, Moore DT, Yang X, Cance WG. Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyzes. Clin Cancer Res. 2003;9(1):215-22. PubMed PMID: 12538472. 5. Siesser PMF, Hanks SK. The Signaling and Biological Implications of FAK Overexpression in Cancer. Clin Cancer Res %R 101158 / 1078-0432CCR-06-0456. 2006;12(11):3233-7. 6. McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat Rev Cancer. 2005;5(7):505-15. PubMed PMID: 16069815. 7. Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. pp125fak a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proceedings of the National Academy of Sciences of the United States of America. 1992;89(11):5192-6. 8. Zhao X, Guan JL. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Advanced drug delivery reviews. 2011;63(8):610-5. Epub 2010 / 12 / 02. doi: 10.1016 / j.addr.2010.11.001. PubMed PMID: 21118706; PMCID: 3132829. 9. Golubovskaya VM, Finch R, Cance WG. Direct Interaction of the N-terminal domain of Focal Adhesion Kinase with the N-terminal transactivation domain of p53. J Biol Chem. 2005;280(26):25008-21. doi: 10.1074 / jbc.M414172200. 10. Kurenova E, Xu L-H, Yang X, Baldwin AS, Jr., Craven RJ, Hanks SK, Liu Z-g, Cance WG. Focal Adhesion Kinase Suppresses Apoptosis by Binding to the Death Domain of Receptor-Interacting Protein. Mol Cell Biol. 2004;24(10):4361-71. 11. Lahlou H, Sanguin-Gendreau V, Zuo D, Cardiff RD, McLean GW, Frame MC, Muller WJ. Mammary epithelial-specific disruption of the focal adhesion kinase blocks mammary tumor progression. Proc Natl Acad Sci U S A. 2007;104(51):20302-7. PubMed PMID: 18056629. 12. Pylayeva Y, Gillen KM, Gerald W, Beggs HE, Reichardt LF, Giancotti FG. Ras- and PI3K-dependent breast tumorigenesis in mice and humans requires focal adhesion kinase signaling. The Journal of Clinical Investigation. 2009;119(2):252-66. 13. Nishimura M, Machida K, Imaizumi M, Abe T, Umeda T, Takeshima E, Watanabe T, Ohnishi Y, Takagi K, Hamaguchi M. Tyrosine phosphorylation of 100-130 kDa proteins in lung cancer correlates with poor prognosis. British Journal of Cancer. 1996;74(5):780-7. 14. Recher C, Ysebaert L, Beyne-Rauzy O, Mansat-De Mas V, Ruidavets J-B, Cariven P, Demur C, Payrastre B, Laurent G, Racaud-Sultan C. Expression of Focal Adhesion Kinase in Acute Myeloid Leukemia Is Associated with Enhanced Blast Migration, Increased Cellularity, and Poor Prognosis. Cancer Res. 2004;64(9):3191-7. doi: 10.1158 / 0008-5472.can-03-3005. 15. Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995;377(6549):539-44. doi: 10.1038 / 377539a0. PubMed PMID: 7566154. 16. Xu L-h, Yang X-h, Bradham CA, Brenner DA, Baldwin AS, Craven RJ, Cance WG. The focal adhesion kinase suppresses transformation-associated, anchorage-Independent apoptosis in human breast cancer cells. J Biol Chem. 2000;275:30597-604. 17. Sonoda Y, Matsumoto Y, Funakoshi M, Yamamoto D, Hanks SK, Kasahara T. Anti-apoptotic Role of Focal Adhesion Kinase (FAK). INDUCTION OF INHIBITOR-OF-APOPTOSIS PROTEINS AND APOPTOSIS SUPPRESSION BY THE OVEREXPRESSION OF FAK IN A HUMAN LEUKEMIC CELL LINE, HL-60. J Biol Chem. 2000;275(21):16309-15. PubMed PMID: 0010821872. 18. Kessler BE, Sharma V, Zhou Q, Jing X, Pike LA, Kerege AA, Sams SB, Schweppe RE. FAK Expression, Not Kinase Activity, Is a Key Mediator of Thyroid Tumorigenesis and Protumorigenic Processes. Mol Cancer Res. 2016;14(9):869-82. Epub 2016 / 06 / 05. doi: 10.1158 / 1541-7786.MCR-16-0007. PubMed PMID: 27259715; PMCID: PMC5025360. 19. Walsh C, Tanjoni I, Uryu S, Tomar A, Nam JO, Luo H, Phillips A, Patel N, Kwok C, McMahon G, Stupack DG, Schlaepfer DD. Oral delivery of PND-1186 FAK inhibitor decreases tumor growth and spontaneous breast to lung metastasis in pre-clinical models. Cancer Biol Ther. 2010;9(10):778-90. PubMed PMID: 20234193; PMCID: PMC2933309. 20. Stokes JB, Adair SJ, Slack-Davis J, Walters DM, Tilghman RW, Hershey ED, Lowrey B, Thomas KS, Bouton AH, Hwang RF, Stelow EB, Parsons JT, Bauer TW. Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment. Molecular Cancer Therapeutics. 2011. doi: 10.1158 / 1535-7163.mct-11-0261. 21. Cance WG, Golubovskaya VM. Focal adhesion kinase versus p53: apoptosis or survival? Sci Signal. 2008;1(20):pe22. PubMed PMID: 18493017. 22. Xu LH, Yang X, Craven RJ, Cance WG. The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth Differ. 1998;9(12):999-1005. 23. Marlowe TA, Lenzo FL, Figel SA, Grapes AT, Cance WG. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors. Mol Cancer Ther. 2016;15(12):3028-39. Epub 2016 / 09 / 18. doi: 10.1158 / 1535-7163.MCT-16-0366. PubMed PMID: 27638858; PMCID: PMC5136315. 24. Infante JR, Camidge DR, Mileshkin LR, Chen EX, Hicks RJ, Rischin D, Fingert H, Pierce KJ, Xu H, Roberts WG, Shreeve SM, Burris HA, Siu LL. Safety, pharmacokinetic, and pharmacodynamic phase I dose-escalation trial of PF-00562271, an inhibitor of focal adhesion kinase, in advanced solid tumors. J Clin Oncol. 2012;30 (13):1527-33. Epub 2012 / 03 / 29. doi: 10.1200 / JCO.2011.38.9346. PubMed PMID: 22454 420. 25. Jones SF, Siu LL, Bendell JC, Cleary JM, Razak AR, Infante JR, Pandya SS, Bedard PL, Pierce KJ, Houk B, Roberts WG, Shreeve SM, Shapiro GI. A phase I study of VS-6063, a second-generation focal adhesion kinase inhibitor, in patients with advanced solid tumors. Invest New Drugs. 2015;33(5):1100-7. doi: 10.1007 / s10637-015-0282-y. PubMed PMID: 26334219. 26. Verastem I. Placebo Controlled Study of VS-6063 in Subjects With Malignant Pleural Mesothelioma (COMMAND 2015 [updated October 5, 2015; cited 2016 April 24, 2016]. Available from: https: / / clinicaltrials.gov / ct2 / show / study / NCT01870609. 27. Hoellerer MK, Noble ME, Labesse G, Campbell ID, Werner JM, Arold ST. Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain. Structure. 2003;11(10):1207-17. Epub 2003 / 10 / 07. PubMed PMID: 14527389. 28. Garron ML, Arthos J, Guichou JF, McNally J, Cicala C, Arold ST. Structural basis for the interaction between focal adhesion kinase and CD4. J Mol Biol. 2008;375(5):1320-8. Epub 2007 / 12 / 15. doi: 10.1016 / j.jmb.2007.11.040. PubMed PMID: 18078954. 29. Xu S, Liu Y, Li X, Liu Y, Meijers R, Zhang Y, Wang JH. The binding of DCC-P3 motif and FAK-FAT domain mediates the initial step of netrin-1 / DCC signaling for axon attraction. Cell Discov. 2018;4:8. Epub 2018 / 02 / 27. doi: 10.1038 / s41421-017-0008-8. PubMed PMID: 29479476; PMCID: PMC5818605. 30. Sieg DJ, Hauck CR, Schlaepfer DD. Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci. 1999;112(Pt):2677-91. 31. Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH, Schlaepfer DD. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol. 2000;2(5):249-56. 32. Thomas JW, Cooley MA, Broome JM, Salgia R, Griffin JD, Lombardo CR, Schaller MD. The role of FAK binding in the regulationof tyrosine phosphorylation of paxillin. J Biol Chem. 1999;in press. 33. Kaneda T, Sonoda Y, Ando K, Suzuki T, Sasaki Y, Oshio T, Tago M, Kasahara T. Mutation of Y925F in focal adhesion kinase (FAK) suppresses melanoma cell proliferation and metastasis. Cancer Lett. 2008;270(2):354-61. Epub 2008 / 07 / 09. doi: 10.1016 / j.canlet.2008.05.042. PubMed PMID: 18606490. 34. Deramaudt TB, Dujardin D, Noulet F, Martin S, Vauchelles R, Takeda K, Ronde P. Altering FAK-paxillin interactions reduces adhesion, migration and invasion processes. PLoS One. 2014;9(3):e92059. Epub 2014 / 03 / 20. doi: 10.1371 / journal.pone.0092059. PubMed PMID: 24642576; PMCID: PMC3958421. 35. Kanteti R, Batra SK, Lennon FE, Salgia R. FAK and paxillin, two potential targets in pancreatic cancer. Oncotarget. 2016;7(21):31586-601. Epub 2016 / 03 / 17. doi: 10.18632 / oncotarget.8040. PubMed PMID: 26980710; PMCID: PMC5058780. 36. Scheswohl DM, Harrell JR, Rajfur Z, Gao G, Campbell SL, Schaller MD. Multiple paxillin binding sites regulate FAK function. J Mol Signal. 2008;3:1. Epub 2008 / 01 / 04. doi: 10.1186 / 1750-2187-3-1. PubMed PMID: 18171471; PMCID: PMC2246129. 37. Gao G, Prutzman KC, King ML, Scheswohl DM, DeRose EF, London RE, Schaller MD, Campbell SL. NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model. J Biol Chem. 2004;279(9):8441-51. PubMed PMID: 14662767. 38. Bertolucci CM, Guibao CD, Zheng J. Structural features of the focal adhesion kinase-paxillin complex give insight into the dynamics of focal adhesion assembly. Protein Sci. 2005;14(3):644-52. Epub 2005 / 02 / 04. doi: 10.1110 / ps.041107205. PubMed PMID: 15689512; PMCID: PMC2279287. 39. Chang YS, Graves B, Guerlavais V, Tovar C, Packman K, To KH, Olson KA, Kesavan K, Gangurde P, Mukherjee A, Baker T, Darlak K, Elkin C, Filipovic Z, Qureshi FZ, Cai H, Berry P, Feyfant E, Shi XE, Horstick J, Annis DA, Manning AM, Fotouhi N, Nash H, Vassilev LT, Sawyer TK. Stapled alpha-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc Natl Acad Sci U S A. 2013;110(36):E3445-54. Epub 2013 / 08 / 16. doi: 10.1073 / pnas.1303002110. PubMed PMID: 23946421; PMCID: PMC3767549. 40. Gao H, Wu Y, Sun Y, Yang Y, Zhou G, Rao Y. Design, Synthesis, and Evaluation of Highly Potent FAK-Targeting PROTACs. ACS Med Chem Lett. Article ASAP. DOI: 10.1021 / acsmedchemlett.9b00372。

[0226] (equivalent) The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is therefore 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. [Brief explanation of the drawings]

[0227] [Figure 1] Overview of stapled α-helical peptides targeting the FAT domain of FAK. A. Crystal structure of the FAT-paxillin interaction (PDB 1OW8) containing two paxillin LD2 motifs (shown in green and magenta). The stapled peptide is based on the structure of the paxillin LD2 motif. B. Zoomed inset of the FAT helix 2-3 binding interface with paxillin LD2. C. Helix wheel structural analysis of the LD2-FAT inter- and intramolecular interactions. D. 3D representation of the stapling strategy used for peptide optimization. E. Overview of amino acid modifications used in peptide design strategies. [Figure 2] Synthetic scheme for stapled peptide synthesis. [Figure 3] Chemical structure of stapled peptide UACC-1907. [Figure 4]Biochemical, biophysical, and cytological data for the stapled peptide UACC-1907 (1907). A. 3D model of peptide 1907 with the staples highlighted in red. B. Competition FP experiment with 1907 showing inhibition of TAMRA-LD2 binding to FAT. C. SPR binding and selectivity analysis with wild-type FAT and mutant FAT (L994E, I936A) at the helix-protein interface. D. Flow cytometry analysis of rhodamine-1907 (10 μM) cellular uptake in MDA-MB-453 breast cancer cells. E. Boyden chamber invasion assay in SK-MEL-103 melanoma cells. F. 3D Matrigel-on-top proliferation assay in SK-MEL-103 melanoma cells. [Figure 5] HSQC NMR data of 15N-labeled FAT domain protein in complex with peptide 1907. A. H / 15N HSQC spectra of the FAT domain using a 600 MHz NMR analyzer with 1% DMSO (maroon), 50 μM 1907 (red), 10 μM 1907 (green), and 5 μM 1907 (blue). Note: The peptide induces peak intensity changes at both the helix 1-4 (K1032) and helix 2-3 (L959) binding sites. B. HSQC NMR and 1907 binding curves using four different residues on the FAT domain (V932, L959, L994, and D1036). 1907 concentrations were titrated from 100 to 0.01 μM, and the KD was calculated by plotting the percentage change in peak integration versus concentration. Mapping of key perturbations caused by 1907 at C. helix 2-3 site and D. helix 1-4 site. Residues with large shifts are highlighted in green. Note that 1907 binds to the same site as native paxillin LD2 (yellow and cyan). [Figure 6] X-ray crystal structure at 1.95 Å resolution of a FAK peptide inhibitor (i.e., 1907) in complex with the human FAK FAT domain. The left panel shows the staple peptide in blue and the FAT domain in green. The right panel shows an electron density map of the peptide in the binding pocket. [Figure 7]Anti-cancer effect of myristoylated peptide 1907 (UACC-2012). A. 3D structure of UACC-2012. B. 3D structure of negative control molecule (UACC-2014). C. 3D Matrigel-on-top cell proliferation data of stapled peptide in SK-MEL-103 melanoma cells. D. 3D Matrigel-on-top cell proliferation data of stapled peptide UACC-2012 in HUVEC "normal" cells. [Figure 8] FAT bivalent stapled peptide strategy and SPR data for the synthetic peptide UACC-2023. A. Overview of the bivalent stapled peptide strategy to enable dual-site conjugation of the FAT domain using click chemistry and linker approaches. B. Structure of UACC-2023. C. SPR sensogram of the bivalent peptide UACC-2023 and the PEG10 linker bound to FAT. [Figure 9] Anticancer efficacy data for stapled peptides 2023 and 1907 in liposomal formulations. A. 2D growth data for peptide 2023 in combination with the cationic lipid reagent Saint-Protein (Synvolux). B. 2D growth data for peptide 1907 in combination with the cationic lipid reagent Saint-Protein. Lipid:peptide formulations were prepared as 10x stocks using a 1:1 (v:v) ratio and titrated concentrations of peptide in PBS pH 7.4 + 1% DMSO. [Figure 10] Overview of the synthetic strategy for FAT-kinase bifunctional inhibitors and the proof-of-concept molecule UACC-2030. A. (Top) Attachment site of the FAK kinase domain inhibitor PF-562271. (Bottom) Attachment site of the FAT domain inhibitor 1907. B. Synthetic plan of the FAT-kinase bifunctional group and chemical structure of the synthetic molecule UACC-2030. [Figure 11] Overview of the FAT-PROTAC synthesis strategy and the proof-of-concept synthetic molecule UACC-2019. [Figure 12]In vitro trypsin digestion assays were performed to measure the stability and protease resistance of peptides (1967, 1907, and 2012). Assays were performed using trypsin-agarose beads (ThermoFisher), and peptide concentrations were measured using LC-MS. Peptide half-lives were calculated using GraphPad Prism software. [Figure 13] Antifibrotic activity of the FAT-stapled peptide UACC-2012 in LX2 human hepatic stellate cells. A. Western blot analysis of LX2 cells treated with 2 ng / mL TGF-β for 17 hours to induce a profibrotic phenotype. Cells were pretreated for 1 hour with either DMSO or UACC-2012 (mSP3) at 10 μM, 5 μM, and 2 μM. Quantification of fibronectin results is shown in the bottom panel. B. Morphological changes in LX2 cells after 17 hours of TGF-β and peptide treatment. Images were captured using bright-field microscopy. Abbreviations: mSP3 = UACC-2012; SP3 = UACC-1907. [Figure 14] Structures of compounds P29–P34, P37, and P38.

Claims

1. Formula I below: 【Chemical 1】 (In the formula, Y Ｃ , Y Ｔ , Z １ , R １ , L １ , Q １ , R ２ , and Z ２ independently includes any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction of FAK with the paxillin protein. (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

2. The compound of claim 1, which is capable of binding to one or more of the helix 1-4 and helix 2-3 portions of the FAT domain of the FAK protein.

3. 2. The compound of claim 1, which is capable of binding to one or more of the following amino acid residues in a wild-type FAK protein: V928, I936, R962, and K955.

4. The compound of claim 1, which is capable of inhibiting the interaction between FAK and paxillin.

5. The compound of claim 1 which is an isolated polypeptide.

6. The compound of claim 1, which enables one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity; disrupting FAK catalytic activity via direct binding of the FAT domain; inhibiting FAK-associated scaffold function; inhibiting FAK protein-protein interactions mediated by the FAT domain; inhibiting the binding of paxillin to the helix 1-4 region of the FAT domain of FAK; inhibiting the binding of paxillin to the helix 2-3 region of the FAT domain of FAK; inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; inhibiting FAK-Leupaxin interaction via binding to the FAT domain of FAK; inhibiting FAK-CD4 interaction via binding to the FAT domain of FAK; inhibiting FAK-CD8 interaction via binding to the FAT domain of FAK; inhibiting FAK-DCC interaction via binding to the FAT domain of FAK; Inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; inhibiting the binding of Leupaxin to each of its binding partners; inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

7. Q １ are amino acid chains of two amino acids (-[Aa1]-[Aa2]-), three amino acids (-[Aa1]-[Aa2]-[Aa3]-), six amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or ten amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively: (In the formula, "i" means an α-amino acid backbone residue relative to the N-terminus of the macrocycle, and the "number" in "i + [number]" means the number of amino acid residues away from i relative to the C-terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, and Aa10 are each independently a natural or unnatural α-amino acid.)

8. Z １ and Z ２ and each independently represent a natural or unnatural amino acid chain of 0 to 200 units in length.

9. Y Ｃ a desired moiety such as biotin or a dye, molecular probe, fluorescent or other, or chemically reactive moiety including, but not limited to, an azide, alkyne, or photoreactive species (examples are found in Molecular Probes Handbook, Eleventh edition, Iain D. Johnson, Life Technologies Corporation, 2010 (ISBN 978-0-9829279-0-8), but is not limited to the selection thereof.), 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 covalent derivative of a FAK kinase inhibitor, an inhibitor of another kinase (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 radionuclide moiety, a covalent derivative of a drug transporter 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 prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics, and a covalent derivative of an electrophilic moiety for covalent attachment to a target protein.

10. Y Ｔ But Y Ｃ and Z １ 2. The compound of claim 1, wherein the optional chemical chain between the groups, if present, can form an alkyl or amide (e.g., carbamide, sulfonamide, or phosphoramide) group.

11. the chemical chain is selected from β-alanine, 6-aminohexanoic acid, and amino acids in which the amine and acid functional groups are separated by a poly(ethylene glycol) (PEG) monomer, oligomer, or polymer; and / or Y Ｔ The compound of claim 10, wherein 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 carbocyclic or heterocyclic rings.

12. R １ 2. The compound of claim 1, wherein is hydrogen or a lower alkyl or substituted methyl group.

13. R ２ 2. The compound of claim 1, wherein is hydrogen or a lower alkyl or substituted methyl group.

14. L １ is a hydrocarbon containing a single double bond, typically in a cis or trans configuration, or a mixture thereof, typically containing 8 or 11 atoms, but which may be a different integer, and / or may be a disulfide staple or a triazole staple.

15. The compound of claim 1, which is at least 60% identical to one of SEQ ID NOs: 1-38.

16. The compound of claim 1, which is at least 75% identical to one of SEQ ID NOs: 1-38.

17. The compound of claim 1, which is at least 80% identical to one of SEQ ID NOs: 1-38.

18. The compound of claim 1, which is at least 85% identical to one of SEQ ID NOs: 1-38.

19. The compound of claim 1, which is at least 90% identical to one of SEQ ID NOs: 1-38.

20. The compound of claim 1, which is at least 95% identical to one of SEQ ID NOs: 1-38.

21. The compound of claim 1, which is at least 98% identical to one of SEQ ID NOs: 1-38.

22. The compound of claim 1, which is at least 99% identical to one of SEQ ID NOs: 1-38.

23. 2. The compound of claim 1, comprising, consisting of, or consisting essentially of one of SEQ ID NOs: 1-38.

24. Formula II below: 【Chemistry 2】 (In the formula, Y Ｃ , Y Ｔ , Z １ , R １ , Q １ , L ２ , R ２ , Z ３ , R ３ , Q ２ , L ３ , R ４ , and Z ２ independently includes any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction of FAK with the paxillin protein. (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

25. The compound of claim 24, which is capable of binding to one or more of the helix 1-4 and helix 2-3 portions of the LD2 domain of a paxillin protein.

26. 25. The compound of claim 24, which is capable of binding to one or more of the following amino acid residues in a wild-type FAK protein: V928, I936, R962, and K955.

27. 25. The compound of claim 24, which is capable of inhibiting the interaction of FAK with paxillin.

28. 25. The compound of claim 24, which is an isolated polypeptide.

29. 25. The compound of claim 24, which enables one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity; disrupting FAK catalytic activity via direct binding of the FAT domain; inhibiting FAK-associated scaffold function; inhibiting FAK protein-protein interactions mediated by the FAT domain; inhibiting the binding of paxillin to the helix 1-4 region of the FAT domain of FAK; inhibiting the binding of paxillin to the helix 2-3 region of the FAT domain of FAK; inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; inhibiting FAK-Leupaxin interaction via binding to the FAT domain of FAK; inhibiting FAK-CD4 interaction via binding to the FAT domain of FAK; inhibiting FAK-CD8 interaction via binding to the FAT domain of FAK; inhibiting FAK-DCC interaction via binding to the FAT domain of FAK; Inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; inhibiting the binding of Leupaxin to each of its binding partners; inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

30. Q １ are amino acid chains of 2 amino acids (-[Aa1]-[Aa2]-), 3 amino acids (-[Aa1]-[Aa2]-[Aa3]-), 6 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively; Q ２ are 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 acid chains occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively: (In the formula, "i" means an α-amino acid backbone residue relative to the N-terminus of the macrocycle, and the "number" in "i + [number]" means the number of amino acid residues away from i relative to the C-terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, and Aa10 are each independently a natural or unnatural α-amino acid, and Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, and Aa20 are each independently a natural or unnatural α-amino acid.)

31. Z １ and Z ２ and Z ３ and each independently represent a natural or unnatural amino acid chain of 0 to 200 units in length.

32. Y Ｃ a desired moiety such as biotin or a dye, molecular probe, fluorescent or other, or chemically reactive moiety including, but not limited to, an azide, alkyne, or photoreactive species (examples are found in Molecular Probes Handbook, Eleventh edition, Iain 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 covalent derivative of a FAK kinase inhibitor, an inhibitor of another kinase (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 radionuclide moiety, a covalent derivative of a drug transporter 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 prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics, and a covalent derivative of an electrophilic moiety for covalent attachment to a target protein.

33. Y Ｔ But Y Ｃ and Z １ 25. The compound of claim 24, wherein the optional chemical chain between the groups, if present, can form an alkyl or amide (e.g., carbamide, sulfonamide, or phosphoramide) group.

34. the chemical chain is selected from β-alanine, 6-aminohexanoic acid, and amino acids in which the amine and acid functional groups are separated by a poly(ethylene glycol) (PEG) monomer, oligomer, or polymer; and / or Y Ｔ 25. The compound of claim 24, wherein 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 carbocyclic or heterocyclic rings.

35. R １ and R ２ 25. The compound of claim 24, wherein is independently hydrogen or a lower alkyl or substituted methyl group.

36. R ３ and R ４ 25. The compound of claim 24, wherein is independently hydrogen or a lower alkyl or substituted methyl group.

37. L ２ is a hydrocarbon containing a single double bond, typically in a cis or trans configuration, 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. L ３ is a hydrocarbon containing a single double bond, typically in a cis or trans configuration, or a mixture thereof, and typically contains 8 or 11 atoms, but can be a different integer.

39. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 60% identical to one of SEQ ID NOs: 1-38.

40. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 75% identical to one of SEQ ID NOs: 1-38.

41. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 1-38.

42. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 85% identical to one of SEQ ID NOs: 1-38.

43. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 1-38.

44. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 1-38.

45. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 98% identical to one of SEQ ID NOs: 1-38.

46. 25. The compound of claim 24, wherein at least a portion of the compound comprises an amino acid sequence that is at least 99% identical to one of SEQ ID NOs: 1-38.

47. 25. The compound of claim 24, wherein at least a portion of the compound comprises one of SEQ ID NOs: 1-38.

48. Formula III below: 【Chemistry 3】 (In the formula, Y Ｃ , Y Ｔ , Z １ , R １ , Q １ , L ４ , Q ４ , L ５ , R ５ , and Z ２ independently includes any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction of FAK with the paxillin protein. (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

49. 49. The compound of claim 48, capable of binding to one or more of the helix 1-4 and helix 2-3 portions of the LD2 domain of a paxillin protein.

50. 49. The compound of claim 48, which is capable of binding to one or more of the following amino acid residues in a wild-type FAK protein: V928, I936, R962, and K955.

51. 49. The compound of claim 48, which is capable of inhibiting the interaction of FAK with paxillin.

52. 49. The compound of claim 48, which is an isolated polypeptide.

53. 49. The compound of claim 48, which enables one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity; disrupting FAK catalytic activity via direct binding of the FAT domain; inhibiting FAK-associated scaffold function; inhibiting FAK protein-protein interactions mediated by the FAT domain; inhibiting the binding of paxillin to the helix 1-4 region of the FAT domain of FAK; inhibiting the binding of paxillin to the helix 2-3 region of the FAT domain of FAK; inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; inhibiting FAK-Leupaxin interaction via binding to the FAT domain of FAK; inhibiting FAK-CD4 interaction via binding to the FAT domain of FAK; inhibiting FAK-CD8 interaction via binding to the FAT domain of FAK; inhibiting FAK-DCC interaction via binding to the FAT domain of FAK; Inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; inhibiting the binding of Leupaxin to each of its binding partners; inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

54. Q １ are amino acid chains of 2 amino acids (-[Aa1]-[Aa2]-), 3 amino acids (-[Aa1]-[Aa2]-[Aa3]-), 6 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-) or 10 amino acids (-[Aa1]-[Aa2]-[Aa3]-[Aa4]-[Aa5]-[Aa6]-[Aa7]-[Aa8]-[Aa9]-[Aa10]-) occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively; Q ４ are 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 acid chains occurring in the motifs (i, i+3); (i, i+4); (i, i+7) and (i, i+11), respectively: (In the formula, "i" means an α-amino acid backbone residue relative to the N-terminus of the macrocycle, and the "number" in "i + [number]" means the number of amino acid residues away from i relative to the C-terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, and Aa10 are each independently a natural or unnatural α-amino acid, and Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, and Aa20 are each independently a natural or unnatural α-amino acid.)

55. Z １ and Z ２ and each independently represent a natural or unnatural amino acid chain of 0 to 200 units in length.

56. Y Ｃ a desired moiety such as biotin or a dye, molecular probe, fluorescent or other, or chemically reactive moiety including, but not limited to, an azide, alkyne, or photoreactive species (examples are found in Molecular Probes Handbook, Eleventh edition, Iain 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 covalent derivative of a FAK kinase inhibitor, an inhibitor of another kinase (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 radionuclide moiety, a covalent derivative of a drug transporter 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 prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics, and a covalent derivative of an electrophilic moiety for covalent attachment to a target protein.

57. Y Ｔ But Y Ｃ and Z １ 49. The compound of claim 48, wherein the optional chemical chain between the groups, if present, can form an alkyl or amide (e.g., carbamide, sulfonamide, or phosphoramide) group.

58. the chemical chain is selected from β-alanine, 6-aminohexanoic acid, and amino acids in which the amine and acid functional groups are separated by a poly(ethylene glycol) (PEG) monomer, oligomer, or polymer; and / or Y Ｔ 58. The compound of claim 57, wherein 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 carbocyclic or heterocyclic rings.

59. R １ and R ５ is independently selected from hydrogen or a lower alkyl or substituted methyl group.

60. L ４ and L ５ are independently selected from hydrocarbons containing a single double bond, typically in a cis or trans configuration, or a mixture thereof, said hydrocarbons typically containing 8 or 11 atoms, but may be different integers, and / or disulfide or triazole staples.

61. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 60% identical to one of SEQ ID NOs: 1-38.

62. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 75% identical to one of SEQ ID NOs: 1-38.

63. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 1-38.

64. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 85% identical to one of SEQ ID NOs: 1-38.

65. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 1-38.

66. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 1-38.

67. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 98% identical to one of SEQ ID NOs: 1-38.

68. 49. The compound of claim 48, wherein at least a portion of the compound comprises an amino acid sequence that is at least 99% identical to one of SEQ ID NOs: 1-38.

69. 49. The compound of claim 48, wherein at least a portion of the compound comprises one of SEQ ID NOs: 1-38.

70. Formula IV below: 【Chemistry 4】 (In the formula, SPA-NH ２ and SPB-NH ２ are independently compounds of formula I, excluding YC-YT- (as defined in claim 1), or compounds of formula II, excluding YC-YT- (as defined in claim 24), or compounds of formula III, excluding YC-YT- (as defined in claim 48), In the ceremony, T １ is a chain or 0-400 atoms in length, typically but not limited to, comprising a poly(ethylene glycol) chain, typically but not limited to, as an amide functional group, SPA-NH ２ and SPB-NH ２ Connected to may or may not contain internal structures such as carbon or heterocyclic rings that may or may not function as pigments or chromophores; The pendant moiety, such as biotin or a dye or a chemical probe or a reactive group, may be branched or unbranched, and It 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. (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

71. Chain (T １ 71. The compound of claim 70, wherein the aryl group is selected from an ether (including polyethers such as poly(ethylene glycol)), an ester, an amide, a thioether, a thioester, or a hydrocarbon chain that may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and that may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and that may or may not branch to pendant moieties such as biotin or dyes or chemical probes or reactive groups or reactive E3 ligase ligands.

72. Formula V below: 【Chemistry 5】 (In the formula, YC-YT-SPA-NH ２ and YC-YT-SPB-NH ２ are independently a compound of formula I (as defined in claim 1), or a compound of formula II (as defined in claim 24), or a compound of formula III (as defined in claim 48), wherein 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 Z １ or Z ２ or Z ３ YC-YT-SPA-NH ２ is 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 Z １ or Z ２ or Z ３ YC-YT-SPB-NH ２ and a side chain represented by ２ ) are connected via (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

73. Chain (T ２ 73. The compound of claim 72, wherein the aryl group is selected from an ether (including polyethers such as poly(ethylene glycol)), an ester, an amide, a thioether, a thioester, or a hydrocarbon chain that may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and that may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, and that may or may not branch to pendant moieties such as biotin or dyes or chemical probes or reactive groups or reactive E3 ligase ligands.

74. 74. The compound of claim 73, wherein the chain is selected from an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), which may or may not contain internal structures such as carbon or heterocycles that may or may not function as dyes or chromophores, which may or may not contain internal structures such as biotin or dyes or chemical probes or reactive groups that have pendant moieties, and which 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. Formula VI below: 【Chemistry 6】 (In the formula, SPA-NH ２ Is Y Ｃ -Y Ｔ -, except for compounds of formula I (as defined in claim 1), or Y Ｃ -Y Ｔ -, except for compounds of formula II (as defined in claim 24), or Y Ｃ -Y Ｔ - except for compounds of formula III (as defined in claim 48), In the formula, SPA-NH ２ The N-terminus of ６ Through SPA-NH ２ internal amino acids 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 Z １ or Z ２ or Z ３ YC-YT-SPA-NH ２ is 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 Z １ or Z ２ or Z ３ It is derived from the amino acid that is part of wherein the linker is a hydrocarbon chain comprising a cis or trans alkene, or a mixture thereof; or the linker is an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain, which may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes). (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

76. The amino acids may be selected from the group consisting of standard amino acids, 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 49. The compound of claim 1, 24, or 48, wherein the compound is selected from the group consisting of 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. 100. The compound of claim 1, 24 or 48, further comprising a compound conjugated to an additional therapeutic agent.

78. an α-helical stapled peptide comprising hydrophobic and hydrophilic amino acids; two or more amino acids of the peptide are linked to each other; An α-helical stapled peptide, which is capable of binding to the FAT domain of FAK and inhibits the interaction of FAK with xylin protein.

79. The staple peptide of claim 78, which is capable of binding to one or more of the helix 1-4 and helix 2-3 portions of the LD2 domain of a paxillin protein.

80. 79. The staple peptide of claim 78, wherein the compound is capable of binding to one or more of the following amino acid residues in a wild-type FAK protein: V928, I936, R962, and K955.

81. The staple peptide of claim 78, which is capable of inhibiting the interaction between FAK and paxillin.

82. The staple peptide of claim 78, wherein inhibition of the interaction between FAK and paxillin can disrupt FAK non-catalytic activity.

83. 79. The staple peptide of claim 78, which enables one or more of the following: Inhibition of the interaction between FAK and paxillin disrupts FAK non-catalytic activity; disrupting FAK catalytic activity via direct binding of the FAT domain; inhibiting FAK-associated scaffold function; inhibiting FAK protein-protein interactions mediated by the FAT domain; inhibiting the binding of paxillin to the helix 1-4 region of the FAT domain of FAK; inhibiting the binding of paxillin to the helix 2-3 region of the FAT domain of FAK; inhibiting FAK-associated apoptosis, proliferation, invasion, and / or metastasis; inhibiting FAK-paxillin interaction, resulting in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; inhibiting FAK-Leupaxin interaction via binding to the FAT domain of FAK; inhibiting FAK-CD4 interaction via binding to the FAT domain of FAK; inhibiting FAK-CD8 interaction via binding to the FAT domain of FAK; inhibiting FAK-DCC interaction via binding to the FAT domain of FAK; Inhibiting the binding of paxillin LD2 and LD4 to their respective binding partners; inhibiting the binding of CD4 and CD8 to their respective binding partners; inhibiting the binding of CD4 and CD8 to Lck; inhibiting the binding of Leupaxin to each of its binding partners; inhibiting the binding of DCC to each binding partner; Inhibiting the interaction of FAK-related molecules, including Pyk2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF.

84. The staple peptide of claim 78, wherein 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 linked to each other.

85. The staple peptide of claim 78, wherein the staple peptide comprises any one of SEQ ID NOs: 1 to 38.

86. 79. The staple peptide of claim 78, wherein the peptide is further conjugated to a contrast agent.

87. The staple peptide of claim 86, wherein the imaging agent is (5- / 6-) carboxytetramethylrhodamine (TAMRA).

88. 79. A pharmaceutical composition comprising one or more stapled peptides of claim 78.

89. 89. The pharmaceutical composition of claim 88, comprising two or more stapled peptides of claim 78, wherein the two or more stapled peptides are linked via a linker.

90. 90. The composition of claim 89, wherein the linker is a PEG-based linker.

91. A method for treating, ameliorating, or preventing a hyperproliferative disease or fibrosis (e.g., liver fibrosis, pulmonary fibrosis, keloid, etc.) in a patient, comprising administering to the patient a therapeutically effective amount of a compound described in claim 1, 24, or 48, or a stapled peptide described in claim 78.

92. 92. The method of claim 91, wherein the hyperproliferative disease is cancer.

93. 92. The method of claim 91, wherein the hyperproliferative disease is a cancer characterized by FAK expression, FAK pathway activation, FAK dependence, FAK activity and / or FAK-paxillin associated activity.

94. 92. The method of claim 91, wherein the patient is a human patient.

95. 92. The method of claim 91, further comprising administering to the patient one or more anti-cancer agents.

96. 96. The method of claim 95, wherein the anti-cancer agent is a chemotherapeutic agent.

97. 97. The method of claim 96, wherein the anti-cancer agent is radiation therapy.

98. A kit comprising the stapled peptide of claim 78 and / or the compound of claim 1, 24 or 48, and instructions for administration to a patient with a hyperproliferative disease.

99. 99. The kit of claim 98, wherein the hyperproliferative disease is cancer.

100. 100. The kit of claim 99, wherein the cancer is characterized by FAK activity and / or FAK-paxillin associated activity.

101. 99. The kit of claim 98, further comprising one or more anti-cancer drugs.

102. 102. The kit of claim 101, wherein the stapled peptide is administered in conjunction with one or more anti-cancer drugs.

103. Formula VII below: 【Chemistry 7】 (In the formula, Y Ｃ -Y Ｔ -SPA- and Y Ｃ -Y Ｔ -SPB- is a compound that independently binds to the FAT domain of FAK and is capable of inhibiting the interaction of FAK with the paxillin protein, wherein the C-termini of the two staple peptides SPA and SPB are linked via a chain D1T2D2, wherein D1 and D2 are independently an (optionally substituted) nitrogen atom, which can thereby be part of an amide bond to the C-terminus, and are selected from those containing N, O, or S as the linking atom; where SPA and SPB are synthesized on 2-chlorotrityl chloride resin or some other resin that allows cleavage of the protected peptide from the resin. (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

104. Formula VIII below: Y Ｃ -Y Ｔ -SPA-T ２ -SPB-NH ２ (In the formula, Y Ｃ -Y Ｔ -SPA- and Y Ｃ -Y Ｔ -SPB- is a compound that independently binds to the FAT domain of FAK and inhibits the interaction of FAK with the paxillin protein. (including pharmaceutically acceptable salts, solvates, and / or prodrugs thereof).

105. 80. A pharmaceutical composition comprising one or more compounds of claims 1, 24, 48, 72, 73 or 75 in a liposomal formulation.

106. The pharmaceutical composition of claim 105, wherein the liposomal formulation of the LD2 peptide can consist 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, soybean phospholipids, cationic lipids, anionic lipids, neutral lipids in various combinations and / or ratios and / or buffer solutions.

107. The liposome formulation may be selected from the group consisting of sphingomyelin (SM), D-erythrose-sphingomyelin, D-erythrose-dihydrosphingomyelin, palmitoylsphingomyelin, lysophospholipids, galactocerebrosides, 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-glycerol-3-phosphatidylcholine (DMPC), 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), distearoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerol Phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, ceramide, phosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoylsphingomyelin, phytosphingomyelin, and dipalmitoylsphingomyelin Phosphorus, distearoyl sphingomyelin, dipalmitoyl phosphatidylglycerol salts, phosphatidic acid, galactocerebroside, ganglioside, cerebroside, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenylglycoside, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipid, and cholesterol and its derivatives, lyso- Phosphatidylcholine, 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-maleimidophenyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], lysophosphatidic acid, lysophosphatidylcholine, OA-NO, ２ (Linoleic acid nitrate 9- and 10-nitro-cis-octedecenoic acid), LNO ２ (nitratolinolenic acid 9-, 10-, 12-, and 13-nitro-cis-octedadecadienoic acid), AA-NO ２ (nitrate arachidonic acid 5-, 6-, 8-, 9-, 11-, 12-, 14-, and 15-nitro-cis-eicosatetraenoic acid), CLNO ２ (Cholesteryl linoleate cholesteryl-9-, 10-, 12-, and 13-nitro-cis-octedadecadienoate), fatty acids, omega-3-polyunsaturated fatty acids, hexadecatrienoic acid (HTA; 16:3 (n-3); all-cis-7,10,13-hexadecatrienoic acid), α-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-octadecatrienoic 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; clupanodonioic 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 analog, sphingosine-1-phosphate antagonist, sphingosine-1-phosphate agonist, sphingosine-1-phosphate receptor agonist, sphingosine-1-phosphate receptor antagonist, and sphingosine-1-phosphate receptor analog, or any combination thereof.

Images