Peptide inhibitors of focal adhesion kinase activity and uses thereof

Abstract

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).

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, such as migration, invasion, metastasis, apoptosis, proliferation, angiogenesis, and immune cell suppression. However, previous FAK inhibitors generally target only the kinase function and ignore the role of FAK as a scaffold protein. Importantly, FAK scaffold interactions control many key functions of FAK, such as apoptosis, proliferation, invasion, and metastasis. FAK localization to focal adhesions is mediated by the FAK-paxillin interaction, and mutations in the binding site have been shown to have a great impact on FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and invasion.

[0005] There is a need for improved medicaments 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 via binding of the FAT domain, thereby inhibiting, for example, the FAK-paxillin interaction.

[0007] (Summary of the Invention) Experiments conducted during the development of aspects of the present invention have 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 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) and thus provide a new anti-cancer effect.

[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 certain embodiments of the present invention, the combined treatment of an animal with a therapeutically effective amount of the described peptides of the present invention (e.g., LD2 peptide, LD4 peptide) and an anti-cancer agent produces a superior tumor response and clinical benefit in the animal compared to an animal treated with the peptide or the anti-cancer agent / radiation alone. Since all approved dosages of anti-cancer agents and radiation treatments are known, the present invention contemplates various combinations 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 dosage of the anti-cancer agent can be less than the standard dosage.

[0012] In certain embodiments of the present invention, the combined treatment of an animal with a therapeutically effective amount of the described peptides 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 disorders) (e.g., fibrosis (e.g., IPF, liver fibrosis, keloid))) produces a superior clinical benefit in the animal compared to an animal treated with the peptide or the therapeutic agent alone. Since all approved dosages of therapeutic agents are known, the present invention contemplates various combinations 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 dosage of the therapeutic agent can be less than the standard dosage.

[0013] In one embodiment, the present invention provides formula I as follows:

[0014] [Chemical formula]

[0015] (wherein Y C 、Y T, Z1, R1, L1, Q1, R2, and Z2 each independently contain any chemical moiety that enables the resulting compound to bind to the FAT domain of FAK and inhibit the interaction between FAK and paxillin.) To provide a compound (including its pharmaceutically acceptable salts, solvates, and / or prodrugs) contained therein.)

[0016] In some embodiments, the compound can bind to one or more of helices 1-4 and helix 2-3 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 of the FAK protein. In some embodiments, the compound can inhibit the interaction between FAK and paxillin.)

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

[0018] In some embodiments, the compound enables one or more of the following: Disrupting FAK non-catalytic activity by inhibiting the interaction between FAK and paxillin, Disrupting FAK catalytic activity through direct binding of the FAT domain, Inhibiting FAK-related scaffolding function, Inhibiting FAK protein-protein interactions mediated by the FAT domain, Inhibiting 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-related apoptosis, proliferation, invasion, and / or metastasis, Inhibiting FAK-paxillin interaction, which results 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 each binding partner. Inhibiting the binding of CD4 and CD8 to each binding partner. Inhibiting the binding of CD4 and CD8 to Lck. Inhibiting the binding of Leupaxin to each binding partner. 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 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]-) that occur in the motifs of (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 the radionuclide moiety. In some embodiments, Y C is a covalent derivative of the drug transporter ligand. In some embodiments, Y C is a covalent derivative of the cell penetrating moiety / sequence (e.g., TAT, etc.). In some embodiments, Y C is a covalent derivative of the chemotherapeutic agent. In some embodiments, Y C is a covalent derivative of the lipid moiety. In some embodiments, Y C is a covalent derivative of the prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics. In some embodiments, Y C is a covalent derivative of the electrophilic moiety for covalent attachment to the target protein.

[0023] In some embodiments, Y T is C an optional chemical chain between Y T and Z1. In some embodiments, Y T , when present, forms an alkyl or amide (e.g., carbamide, sulfonamide or phosphoramide) group. In some embodiments, Y 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 poly(ethylene glycol) (PEG) monomers, oligomers or polymers.

[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 that, except for the unique peptide backbone, includes R1 of Formula II and can covalently bond with an amino acid that includes R2 of Formula II. In one embodiment, L2 is a hydrocarbon chain that includes 8 atoms and a single double bond of a cis or trans structure, or a mixture thereof. In another embodiment, L2 is a hydrocarbon chain that includes 11 atoms and a single double bond of a cis or trans structure, or a mixture thereof. In another embodiment, L2 is a hydrocarbon chain that includes neither 8 atoms nor 11 atoms and includes a single double bond of a cis or trans structure, or a mixture thereof. In another embodiment, L2 includes two sulfur atoms covalently bonded in a manner different from a disulfide bond. In another embodiment, L2 includes 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 can include or not include internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and can include or not include an internal structure such as a carbocyclic or heterocyclic ring that can or cannot function as a dye or chromophore, and can or cannot branch into a pendant moiety such as biotin or a dye or a chemical probe or a reactive group, and is selected from an ether (including polyethers such as poly(ethylene glycol)), an ester, an amide, a thioether, a thioester, or a hydrocarbon chain. L1 can include or not include one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms. This list is not meant to be exhaustive, and one of ordinary skill in the art can readily envision 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 that, except for its unique peptide backbone, includes R1 of Formula II and can covalently bond with an amino acid whose amino acid includes R2 of Formula II. In one embodiment, L3 is a hydrocarbon chain containing 8 atoms and a single double bond of cis or trans structure, or a mixture thereof. In another embodiment, L3 is a hydrocarbon chain containing 11 atoms and a single double bond of cis or trans structure, or a mixture thereof. In another embodiment, L3 is a hydrocarbon chain that contains neither 8 atoms nor 11 atoms and contains a single double bond of cis or trans structure, or a mixture thereof. In another embodiment, L3 contains two sulfur atoms covalently bonded in a manner different from a disulfide bond. In another embodiment, L3 includes 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 can include internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), or not, and can include an internal structure such as a carbocyclic or heterocyclic ring that can function as a dye or chromophore, or not, and can branch to a pendant moiety such as biotin or a dye or a chemical probe or a reactive group, or not, and is selected from ethers (including polyethers such as poly(ethylene glycol)), esters, amides, thioethers, thioesters, or hydrocarbon chains. L1 can include one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms, or not. This list is not meant to be exhaustive, and one of ordinary skill in the art can readily envision 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 compound 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 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 of the FAK protein. In some embodiments, the compound can inhibit the interaction between FAK and paxillin.

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

[0051] In some embodiments, the compound enables one or more of the following: Disrupting FAK non-catalytic activity by inhibiting the interaction between FAK and paxillin, Disrupting FAK catalytic activity through direct binding of the FAT domain, Inhibiting the FAK-related scaffolding 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-related apoptosis, proliferation, invasion, and / or metastasis, Inhibiting the FAK-paxillin interaction that results in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion, Inhibiting the FAK-Leupaxin interaction through binding to the FAT domain of FAK, Inhibiting the FAK-CD4 interaction through binding to the FAT domain of FAK, Inhibiting the FAK-CD8 interaction through binding to the FAT domain of FAK, Inhibiting FAK-DCC interaction through binding to the FAT domain of FAK, inhibiting the binding of paxillin LD2 and LD4 to each binding partner, inhibiting the binding of CD4 and CD8 to each binding partner, inhibiting the binding of CD4 and CD8 to Lck, inhibiting the binding of Leupaxin to each binding partner, 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, respectively, an amino acid chain 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 of (i, i + 3); (i, i + 4); (i, i + 7) and (i, i + 11), Q4 is, respectively, an amino acid chain of two (-[Aa11]-[Aa12]-), three (-[Aa11]-[Aa12]-[Aa13]-), six (-[Aa11]-[Aa12]-[Aa13]-[Aa14]-[Aa15]-[Aa16]-) or ten (-[Aa11]-[Aa12]-[Aa13]-[Aa14]-[Aa15]-[Aa16]-[Aa17]-[Aa18]-[Aa19]-[Aa20]-) occurring in the motifs of (i, i + 3); (i, i + 4); (i, i + 7) and (i, i + 11): (In the formula, "i" means an α - amino acid skeleton residue with respect to the N - terminus of the macrocycle, and "number" in "i + [number]" means the number of amino acid residues away from i with respect to the C - terminus. In the formula, Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8, Aa9, Aa10 are each, independently of one another, a natural or non - natural α - amino acid, and Aa11, Aa12, Aa13, Aa14, Aa15, Aa16, Aa17, Aa18, Aa19, Aa20 are each, independently of one another, a natural or non - natural α - amino acid.).

[0053] In some embodiments, Z1 and Z2 are, independently of one another, each a natural or non - natural amino acid chain with a unit length of 0 - 200.

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

[0055] In some embodiments, Y T is Y C and an optional chemical chain between Z1. In some embodiments, Y T when present, forms an alkyl or amide (e.g., carbamide, sulfonamide or phosphoramide) group. In some embodiments, Y 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 amine and acid functional groups are separated by poly(ethylene glycol) (PEG) monomers, oligomers or polymers.

[0056] In some embodiments, Y C is a covalent derivative of an inhibitor of FAK kinase catalytic activity. In some embodiments, Y C is a covalent derivative of an inhibitor of other kinases (e.g., EGFR, HER2, etc.). In some embodiments, Y C is a covalent derivative of a GPCR compound. In some embodiments, Y C is a covalent derivative of a nuclear receptor compound. In some embodiments, Y 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. In some embodiments, Y C is a covalent derivative of a radionuclide moiety. In some embodiments, Y C is a covalent derivative of a drug transporter ligand. In some embodiments, Y 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. In some embodiments, Y C is a covalent derivative of a lipid moiety. In some embodiments, Y C is a covalent derivative of a prodrug moiety that promotes favorable bioavailability and / or pharmacokinetics. In some embodiments, Y C is a covalent derivative of an electrophilic moiety for covalent bonding 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 that, except for its unique peptide backbone, includes R1 of Formula III and in which the amino acid can covalently bond with an amino acid that includes R2 of Formula III. In one embodiment, L5 is a hydrocarbon chain that includes 8 atoms and a single double bond in a cis or trans configuration, or a mixture thereof. In another embodiment, L5 is a hydrocarbon chain that includes 11 atoms and a single double bond in a cis or trans configuration, or a mixture thereof. In another embodiment, L5 is a hydrocarbon chain that includes neither 8 atoms nor 11 atoms and includes a single double bond in a cis or trans configuration, or a mixture thereof. In another embodiment, L5 includes two sulfur atoms covalently bonded in a manner different from a disulfide bond. In another embodiment, L5 includes 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 may or may not include internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not include an internal structure such as a carbocyclic or heterocyclic ring that can or cannot function as a dye or chromophore, and may or may not branch to a pendant moiety such as biotin or a dye or a chemical probe or a reactive group, and is selected from an ether (including polyethers such as poly(ethylene glycol)), an ester, an amide, a thioether, a thioester, or a hydrocarbon chain. L1 may or may not include one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms. This list is not meant to be exhaustive, and one of ordinary skill in the art can readily envision 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; which may or may not include an internal structure such as a carbocyclic or heterocyclic ring that can function as a pigment or chromophore, and which may or may not branch to a pendant moiety such as biotin or a pigment or a chemical probe or a reactive group or a reactive E3 ligase ligand.) To provide a compound (including its pharmaceutically acceptable salts, solvates, and / or prodrugs) contained therein. In some embodiments, the chain (T1) may or may not include internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and may or may not include an internal structure such as a carbocyclic or heterocyclic ring that can function as a pigment or chromophore, and may or may not branch to a pendant moiety such as biotin or a pigment or a chemical probe or a reactive group or a reactive E3 ligase ligand, and is selected from ethers (including polyethers such as poly(ethylene glycol)), esters, amides, thioethers, thioesters, or hydrocarbon chains. In some embodiments, T1 may or may not include 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 a 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 functional group. The products of these two species are substituted 1H-1,2,3-triazoles. In some examples, a catalyst is used in the reaction.

[0066] In one embodiment, the present invention provides the following formula V:

[0067]

Chemical formula

[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 functional group. 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 In the formula, the N-terminus of SPA-NH2 is linked via the linker L6 to the side chain represented by YC-YT-SPA-NH2, which is derived from an internal amino acid ([Aa1], [Aa2], [Aa3], [Aa4], [Aa5], [Aa6], [Aa7], [Aa8], [Aa9], [Aa10][Aa11], [Aa12], [Aa13], [Aa14], [Aa15], [Aa16], [Aa17], [Aa18], [Aa19] or [Aa20] individual members; 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 is derived from an internal amino acid ([Aa1], [Aa2], [Aa3], [Aa4], [Aa5], [Aa6], [Aa7], [Aa8], [Aa9], [Aa10][Aa11], [Aa12], [Aa13], [Aa14], [Aa15], [Aa16], [Aa17], [Aa18], [Aa19] or [Aa20] individual members; 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). In the formula, the linker is a hydrocarbon chain containing cis or trans alkene, or a mixture thereof; or the linker may or may not contain internal unsaturation (e.g., a single bond or multiple double bonds, or one or more alkynes), and is an ether (including polyethers such as poly(ethylene glycol)), ester, amide, thioether, thioester, or hydrocarbon chain. To provide a compound (including its pharmaceutically acceptable salts, solvates, and / or prodrugs) contained therein.

[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 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 an internal structure such as a carbocyclic or heterocyclic ring that can function as a dye or chromophore, and may or may not branch into a pendant moiety such as biotin or a dye or a chemical probe or a reactive group or a reactive E3 ligase ligand, and is selected from ethers (including polyethers such as poly(ethylene glycol)), esters, amides, thioethers, thioesters, or hydrocarbon chains. L1 may or may not contain one or more sulfur atoms, or one or more oxygen atoms, or one or more nitrogen atoms. This list is not meant to be exhaustive, and one of ordinary skill in the art can readily envision other covalent bonding strategies. L6 may be branched or may be a chemical derivative not encompassed above. Non-limiting examples of such derivatives include epoxidation or aziridination or cyclopropanation or dihydroxylation of a double bond.

[0075] In one embodiment, the invention provides Formula VII below:

[0076]

Chemical formula

[0077] (wherein Y C -Y T -SPA- and Y C -Y T -SPB- are 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 of Formulae I, II, III, IV, V, VI, VII, and VIII are selected from standard amino acids, L-α-tert-butylglycine, D-α-tert-butylglycine, β-(2-thienyl)-L-alanine, L-alloisoleucine, 4,5-dehydro-L-leucine, D-homoleucine, L-homoleucine, 1-aminocyclopentane-1-carboxylic acid, D-alloisoleucine, 3-(4-thiazolyl)-L-alanine, L-homoarginine, 5,5,5-trifluoro-DL-leucine, γ-carboxyγ-(di-tert-butyl ester)-L-glutamic acid, γ-carboxyγ-(di-tert-butyl ester)-D-glutamic acid, L-α-aminobutyric acid, D-α-aminobutyric acid, α,β-dehydro-2-aminobutyric acid, 4-nitro-L-phenylalanine, 4-chloro-L-phenylalanine, 4-chloro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-fluoro-D-phenylalanine, L-homophenylalanine, D-homophenylalanine, 3,4-dichloro-D-phenylalanine, 3-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, p-phenyl-L-phenylalanine, p-phenyl-D-phenylalanine, 4-bromo-L-phenylalanine, 4-bromo-D-phenylalanine, 2-chloro-L-phenylalanine, 2-chloro-D-phenylalanine, 3-cyano-L-phenylalanine, and 3-cyano-D-phenylalanine.

[0080] In some embodiments, further, the compounds of Formulae I, II, III, IV, V, VI, VII, and VIII are bound to additional therapeutic agents (e.g., thalidomide) (e.g., bifunctional compounds (e.g., PROTACs)).

[0081] In some embodiments, the compounds of 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 α-carbon flanked on both sides by complementary (in terms of spacing and stereochemistry) monoalkenyl residues. The geminal bis(linked alkene) is typically symmetrically substituted, but this is not a requirement.

[0082] In any exemplification of a compound of Formula I, or a compound of Formula II, or a compound of Formula III, or a compound of Formula IV, or a compound of Formula V, or a compound of Formula VI, or a compound of Formula VII, or a compound of 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 bonded to a chemical moiety other than a hydrogen atom. In one embodiment, this chemical moiety may further participate in, or may not participate in, chemical reactions including, but not limited to, ring-closing metathesis reactions, esterification, amide formation, Diels - Alder reactions, etc., and contains a hydrocarbon chain or a substituted hydrocarbon chain that is a reactive species.

[0083] In certain embodiments, the present invention provides an α - helical staple peptide comprising hydrophobic and hydrophilic amino acids, wherein two or more amino acids of the peptide are linked to each other and 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 helices 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 of the FAK protein. In some embodiments, the compounds can inhibit the interaction between FAK and paxillin.

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

[0086] In some embodiments, the peptide enables one or more of the following: Disrupting FAK non-catalytic activity by inhibiting the interaction between FAK and paxillin; Disrupting FAK catalytic activity via direct binding of the FAT domain; Inhibiting FAK-related scaffolding functions; Inhibiting FAK protein-protein interactions mediated by the FAT domain; Inhibiting the binding of paxillin to the helix 1-4 region of the FAK FAT domain; Inhibiting the binding of paxillin to the helix 2-3 region of the FAK FAT domain; Inhibiting FAK-related apoptosis, proliferation, invasion, and / or metastasis; Inhibiting the FAK-paxillin interaction that results in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion; Inhibiting the FAK-Leupaxin interaction via binding to the FAT domain of FAK; Inhibiting the FAK-CD4 interaction via binding to the FAT domain of FAK; Inhibiting the FAK-CD8 interaction via binding to the FAT domain of FAK; Inhibiting the FAK-DCC interaction via binding to the FAT domain of FAK; Inhibiting the binding of paxillin LD2 and LD4 to each binding partner; Inhibiting the binding of CD4 and CD8 to each binding partner; Inhibiting the binding of CD4 and CD8 to Lck; Inhibiting the binding of Leupaxin to each binding partner; 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 one 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 4,9 hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)propan-1-one; RhoB: rhodamine B; or a derivative thereof. Unless otherwise specified, the peptide is the product of an intramolecular ring-closing metathesis reaction; the double bond geometry has not been established or quantified. These sequences are omitted and may mean "click" chemical 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 are useful for treating, ameliorating, or preventing disorders, for example, 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 accumulate endogenous proteins on E3 ubiquitin ligases for degradation, and methods using such compounds. In particular, the present invention provides bifunctional or proteolysis-targeting chimera (PROTAC) compounds that have been found to be useful as regulators of the targeted ubiquitination (and subsequent degradation and / or other inhibition) of various polypeptides and other proteins. An exemplary advantage of the compounds provided by the present invention is that they enable a broad range of pharmacological activities, consistent with the degradation / inhibition of targeted polypeptides from virtually any protein class or family. Further, the present invention provides methods for treating or ameliorating a disease state, such as cancer (e.g., cancer characterized by FAK expression, FAK pathway activation, FAK-dependence, FAK activity and / or FAK-paxillin-related activity), using an effective amount of the compounds described herein.

[0108] In a further aspect, the present invention provides a bifunctional or PROTAC compound comprising 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), such that the target protein / polypeptide is placed in proximity to the ubiquitin ligase, resulting in degradation (and inhibition) of the protein (e.g., inhibiting the interaction between paxillin and FAK). In one embodiment, the PTM is any of the peptides described herein that have an affinity for the FAT domain of FAK (e.g., thereby inhibiting the interaction between FAK and paxillin) (e.g., any of the peptides included in Formulas I-VI) (e.g., any of the peptides listed in Table 1). In some embodiments, the ULM is an inhibitor of a 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 the bifunctional compound can be represented as PTM-ULM.

[0109] The position of each of the PTM and ULM moieties, and the numbers thereof exemplified herein are provided only as an example and are not intended to limit the peptide in any way. As understood by those skilled in the art, the bifunctional compounds described herein can be synthesized, and the number and position of each functional moiety can be varied according to the purpose.

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

[0111] The above embodiments are not limited to a particular 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, 2 to 4 ethylene glycol units, or an optionally substituted alkyl group interspersed with O, N, S, P or Si atoms as desired. In certain embodiments, the linker is substituted with aryl, phenyl, benzyl, alkyl, alkylene, azide, or heterocyclic groups. In certain embodiments, the linker is a dye compound. In certain embodiments, the linker is a photoreactive compound. In certain embodiments, the linker can be asymmetric or symmetric. In certain embodiments, the linker is a substituted or unsubstituted polyethylene glycol group in the size range of about 1 to about 12 ethylene glycol units, 1 to about 10 ethylene glycol units, about 2 to about 6 ethylene glycol units, about 2 to 5 ethylene glycol units, 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. Zoomed-in inset of the helix 2-3 binding interface of FAT and paxillin LD2. C. Helix wheel analysis of intermolecular and intramolecular interactions of LD2-FAT. 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 the stapled peptide UACC-1907.

[0118] Figure 4: Biochemical, biophysical, and cytological data of the stapled peptide UACC-1907 (1907). A. 3D model of peptide 1907 with the staple highlighted in red. B. Competitive FP experiment using 1907 showing inhibition of TAMRA-LD2 binding to FAT. C. SPR binding and selectivity analysis using wild-type FAT and mutants FAT (L994E, I936A) at the helix-protein interface. D. Flow cytometry analysis of rhodamine-1907 (10 μM) cell 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: HSQC NMR data of the N-labeled FAT domain protein in complex with peptide 1907. A. 1H / 15 FAT domain 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: Anti-cancer effect data of staple peptides 2023 and 1907 in liposomal formulations. A. 2D proliferation data of peptide 2023 combined with the cationic lipid reagent Saint-Protein (Synvolux). B. 2D proliferation data of peptide 1907 combined with the cationic lipid reagent Saint-Protein. Lipid:peptide formulations were prepared as 10-fold stocks using peptides at a 1:1 (v:v) ratio and titrated concentrations in PBS pH 7.4 + 1% DMSO.

[0124] Figure 10: Overview of the synthetic strategy of the FAT-kinase bifunctional inhibitor and the molecule UACC-2030 that demonstrates the concept. A. (Top) Adhesion site of the FAK kinase domain inhibitor PF-562271. (Bottom) Adhesion 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 synthetic strategy and the synthetic molecule UACC-2019 that demonstrates the concept.

[0126] Figure 12: In vitro trypsin digestion assay to measure the stability and protease resistance of peptides (1967, 1907, and 2012). The assay was 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 apoptosis activity and growth arrest in cancer cells, but has no effect on normal cells (16, 17). Conversely, FAK kinase inhibitors show a partial effect on apoptosis / tumor growth, and it is hypothesized that the FAK scaffold is a major modulator of FAK-dependent anti-apoptosis (21). FAK binds directly to p53 and suppresses p53-mediated apoptosis, and disruption of the FAT domain by adenoviral FAK-CD has been shown to induce apoptosis in cancer cells (22). FAK kinase inhibitors have been shown not to dramatically inhibit FAK phosphorylation at the autophosphorylated residue Y397 or FAK transphosphorylation by receptor tyrosine kinases (RTKs) as a drug resistance mechanism (23). Furthermore, FAK kinase inhibitors (e.g., FAK kinase catalytic domain inhibitors) have shown only limited effects in Phase I / II clinical trials (24 - 26).

[0132] The FAT domain is four helical bundles at the C-terminus of FAK that contain the important Y925 residue and are involved in multiple protein-protein interactions at focal adhesion sites. 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). Integration of the FAT domain is important for the localization of FAK to focal adhesions, its 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, in the experiments conducted during the development of the aspects of the present invention, a hypothesis was established that peptide inhibitors targeting the FAT domain are more effective than FAK kinase inhibitors, which tend to have less understood drug resistance mechanisms.

[0133] Paxillin is a major focal adhesion adapter protein that integrates important cytoskeletal proteins and signaling molecules such as vinculin, FAK, actin, Src, and Crk (35). FAK localization to focal adhesions is mediated by the FAK-paxillin interaction, 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 is well-characterized, has a KD of 50 - 100 μM, and has been confirmed by multiple assays (X-ray, SPR, ITC, NMR, FP, and mutagenesis). LD2 and LD4 interact with two separate hydrophobic patches (helix 1-4, helix 2-3) on the FAT domain, and disruption of both sites is required for maximal biological effects (27, 37, 38). In experiments conducted during the development of aspects of the present invention, peptides were synthesized and optimized that can efficiently target the non-catalytic function of FAK through 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), and thus provide a novel anti-cancer effect.

[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 present staple LD2 peptide that can inhibit the FAK-paxillin interaction can include any one of the following sequences and is generated by introducing a carbon-carbon double bond through 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 compatible with various functional groups and solvents and are linked together (e.g., linked via "click" chemistry properties) with a linker (e.g., a PEG-based linker) constructed through chemistry known to those skilled in the art.

[0144] In some embodiments, the LD2 peptide is further conjugated to a contrast agent (e.g., conjugated to (5- / 6-)carboxytetramethylrhodamine (TAMRA).).

[0145] An important aspect of the present invention is that the LD2 peptide of the present invention induces cell cycle arrest and / or apoptosis and enhances the induction of cell cycle arrest and / or apoptosis alone or in response to a further apoptosis-inducing signal (e.g., through inhibiting FAK non-catalytic activity) (e.g., through inhibiting the FAK-paxillin interaction). Therefore, the present LD2 peptide is contemplated to confer sensitivity to cells (including cells that are resistant to the inducing stimulus) with respect to the induction of cell cycle arrest and / or apoptosis. The LD2 peptide 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 present LD2 peptide can be used to induce apoptosis in cells containing functional FAK activity. In one embodiment, the present LD2 peptide can be used to inhibit cancer metastasis. In one embodiment, the present LD2 peptide 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 domestic animals). In this regard, various diseases and medical conditions are amenable to treatment or prevention using the methods and compositions of the present invention. A non-limiting 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 cancer tumor, breast cancer tumor, ovarian cancer tumor, lung cancer tumor, small cell lung cancer tumor, Wilms tumor, cervical cancer tumor, testicular cancer tumor, bladder cancer tumor, pancreatic cancer tumor, stomach cancer tumor, colon cancer tumor, prostate cancer tumor, urogenital cancer tumor, thyroid cancer tumor, esophageal cancer tumor, myeloma, multiple myeloma, adrenal cancer tumor, renal cell cancer tumor, endometrial cancer tumor, adrenocortical cancer tumor, malignant pancreatic insulinoma, malignant carcinoid cancer tumor, choriocarcinoma, fungating polypoma, hypercalcemia of malignancy, 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 thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, etc., autoimmune diseases mediated by T and B cells; 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 anti-cancer agents. In other embodiments, the disorder is any disorder having cells with FAK activity and / or FAK-paxillin related activity.

[0147] Some aspects of the present invention provide a method of administering an effective amount of the LD2 peptide of the present invention, and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic anti-neoplastic agents, apoptosis regulators, antibacterial agents, antiviral agents, antifungal agents, and anti-inflammatory agents) and / or therapeutic techniques (e.g., surgical intervention, and / or radiation therapy). In certain aspects, 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 administering, without limitation, many anti-cancer agents such as: agents that induce apoptosis; polynucleotides (e.g., antisense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated to anti-cancer agents, 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; proteasome inhibitors: NF-κB regulators; anti-CDK compounds; HDAC inhibitors, etc. Many examples of chemotherapeutic compounds and anti-cancer therapies suitable for co-administration with the staple peptide (LD2 peptide) are known to those of skill 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 yet a further aspect, chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mitomycin), 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-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 tumor lysing agent commonly used in the context of cancer treatment is found in the use of the compositions and methods of the present invention. For example, the US Food and Drug Administration maintains a regulatory system for tumor lysing agents approved for use in the United States. International agencies corresponding to the US FDA also maintain similar regulatory systems. Table 4 provides an exemplary list of anti-tumor agents approved for use in the United States. Those skilled in the art will understand that the "product label" required for all chemotherapy agents approved in the United States describes the approved indications, dosing information, toxicity data, etc. for the 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] The anticancer agent further includes compounds that have been identified to have anticancer activity. For example, it includes, but is 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, atrasentan, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, exisulind, enzastaurin, erlotinib, eribulin, fenretinide, flavopiridol, fludarabine, flutamide, fostmulin, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hu14.18-Interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprorelin, LMB-9 immunotoxin, lonafarnib, lulizumab, mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, norbendex, NS-9, O6-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC833, PXD101, pyrazoloacridine, R115777, RAD001, lampridine, levomycetin analog, 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, talapanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, timalphasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, ziloton, and zoscidartrihydrochloride.

[0165] For a more detailed description of anticancer agents and other therapeutic agents, one of ordinary skill in the art can refer to any number of reference manuals, including but not limited to the Physician’s Desk Reference and to Goodman and Gilman’s ”Pharmaceutical Basis of Terapeutics” tenth edition, Eds. Hardman et al., 2002.

[0166] The present invention provides a method for administering the LD2 peptide of the present invention, in combination 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 an animal. For example, the animal can receive photon radiation therapy, particle beam radiation therapy, other types of radiation therapy, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In other embodiments, the 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 microorganisms, inhibits their function, or otherwise attenuates them can be used, and any agent having 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, etc., used alone or in combination. In fact, any type of antibiotic can be used, including but not limited to antibacterial agents, antiviral agents, antifungal agents, etc. In some embodiments of the present invention, the LD2 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 periods, different concentrations, different administration routes, etc. In some embodiments, the LD2 peptide is administered prior to the therapeutic or anti-cancer agent, for example, 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 the administration of the therapeutic or anti-cancer agent. In some embodiments, the LD2 peptide is administered after the therapeutic or anti-cancer agent, for example, 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of the 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 the intended purpose. Although the needs of each individual vary, the determination of the optimal range of the effective amount of each component is within the scope of those skilled in the art.

[0169] In addition to administering the present staple peptide (e.g., LD2 peptide) as a raw peptide, the present LD2 peptide can be administered as part of a pharmaceutical preparation containing a suitable pharmaceutically acceptable carrier, including pharmaceutically useful excipients and adjuvants that facilitate the processing of the LD2 peptide into a formulation. The formulation, in particular, tablets, dragees, sustained-release lozenges and capsules, oral rinses, gels, liquid suspensions, hair rinses, hair gels, shampoos, etc., can be administered orally or topically and can be used for one type of administration. Preparations that can be administered rectally, such as suppositories, and solutions suitable for administration by intravenous injection, injection, topical or oral administration contain, together with the excipient, about 0.01 to 99%, in one embodiment about 0.25 to 75% of the active peptide. In some embodiments, the formulation of the present LD2 peptide can also be essentially liposomes. In some embodiments, the liposomal formulations of the present peptide (e.g., LD2, LD4) 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 in buffer solutions. In some embodiments, the liposomal formulations of the present peptide (e.g., LD2, LD4) can be sphingomyelin (SM), D-erythro-sphingomyelin, D-erythro-dihydrosphingomyelin, palmitoyl sphingomyelin, lysophospholipid, galactosylceramide, ganglioside, cerebroside, glyceride, triglyceride, diglyceride, small alkyl chain phospholipid, phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), distearoyl phosphatidylcholine 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine dioleoyl phosphatidylethanolamine, dilauroyl phosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol and other diphosphatidylglycerols, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, ceramide, phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, dipalmitoyl phosphatidylglycerol salt, phosphatidic acid, galactocerebroside, ganglioside, cerebroside, dilauryl phosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenyl glycoside, 3-cholesteryl-6'-(glycosylthio) hexyl ether glycolipid, as well as cholesterol and its derivatives, lysophosphatidylcholine, lysosphingomyelin, 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) butylamide], 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 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-octadecenoic acid), LNO2 (nitrate linolenic acid 9-, 10-, 12-, and 13-nitro-cis-octadecadienoic 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-octadecadiene ketone), Fatty acids, Omega-3 polyunsaturated fatty acids, Hexadecatrienoic acid (HTA; 16:3(n-3); all-cis-7,10,13-hexadecatrienoic acid), Alpha-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; clupanodonic acid; 22:5(n-3); all-cis-7,10,13,16,19-docosapentaenoic acid), Docosahexaenoic acid (DHA; 22:6(n-3); all-cis-4,7,10,13,16,19-docosahexaenoic acid), tetracosapentaenoic acid; 24:5(n-3); all-cis-9,12,15,18,21-tetracosapentaenoic acid), tetracosahexaenoic acid (nisinic acid; 24:6(n-3); all-cis-6,9,12,15,18,21-tetracosahexaenoic acid), sphingosine-1-phosphate analogs, sphingosine-1-phosphate antagonists, sphingosine-1-phosphate agonists, sphingosine-1-phosphate receptor agonists, sphingosine-1-phosphate receptor antagonists, and sphingosine-1-phosphate receptor analogs, 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, fillers such as saccharides (e.g., lactose or sucrose, mannitol or sorbitol), cellulose preparations and / or calcium phosphates (e.g., tricalcium phosphate or calcium hydrogen phosphate), and binders such as starch paste using, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrants such as the above-mentioned starches, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts (e.g., sodium alginate) can be added. Auxiliaries are, inter alia, flow regulators and lubricants such as silica, talc, stearic acid or its salts (e.g., magnesium stearate or calcium stearate), and / or polyethylene glycol. The tablet core is, if desired, provided with a suitable coating that is resistant to gastric juice. For this purpose, a concentrated sugar solution can be used, which can contain, if desired, gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. A solution of a suitable cellulose preparation (e.g., acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate) is used to produce a coating that is resistant to gastric juice. Dyes or pigments can be added to the tablets or the coating of the sugar-coated tablets, for example, to identify or characterize the combination of the active compound dosage.

[0174] Other pharmaceutical formulations that can be used orally include push-fit capsules formed of gelatin, and soft encapsulation capsules formed 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 the soft capsules, the active staple peptide (e.g., LD2 peptide) is dissolved or suspended in a suitable liquid such as fatty oil or liquid paraffin in one embodiment. Further, a stabilizer may be added.

[0175] Possible pharmaceutical formulations that can be used rectally include, for example, suppositories consisting of a combination of one or more active peptides and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffinic hydrocarbons. Further, gelatin rectal capsules consisting of a combination of an active staple peptide (e.g., LD2 peptide) and a base material can also be used. Possible base materials include, for example, liquid triglycerides, polyethylene glycol, or paraffinic 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. Further, as a suitable oily injection suspension, a suspension of the active staple peptide (e.g., LD2 peptide) can be administered. 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. The aqueous injection suspension can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. Optionally, the suspension can also contain a stabilizer.

[0177] In one aspect, the topical composition of the present invention can be formulated as an oil, cream, lotion, ointment, etc. by selecting a suitable carrier. Suitable carriers include vegetable oils or mineral oils, white petrolatum (white soft paraffin), branched-chain fats or oils, animal fats, and high molecular weight alcohols (C 12 greater than). The carrier can be one in which the active ingredient is soluble. Emulsifiers, stabilizers, wetting agents, and antioxidants, and, if desired, agents that impart colorants or fragrances may also be included. Additionally, transdermal penetration enhancers can 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 formulated by mixing the active ingredient in a vegetable oil such as almond oil with warmed soft paraffin and cooling the mixture. A typical example of such an ointment contains, by weight, about 30% almond oil and about 70% white soft paraffin. Lotions can be conventionally manufactured 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 understand that the above description merely illustrates the detailed description of certain preferred embodiments of the present invention. Various modifications and changes to the above compositions and methods can be easily achieved using the expertise available in the art and are within the scope of the present invention.

[0180] (Example) The following examples are illustrative of, but not limited to, the staple peptides (e.g., LD2 peptide), compositions, and methods of the present invention. Other suitable modifications and adaptations of various conditions and parameters are those typically encountered in clinical therapy and will be apparent to those skilled in the art and are within the spirit and scope of the present invention.

[0181] (Example I) This example describes the preparation of a peptide that has an affinity for the FAT domain of FAK and can interfere with the interaction between paxillin and focal adhesion kinase (FAK), and thus inhibits the FAK activity associated with the FAK-paxillin interaction.

[0182] To develop an inhibitor of the FAK-paxillin interaction, experiments were conducted using a stapled peptide approach that has the following advantages: (1) using a natural peptide as a starting point, (2) spanning the entire interaction interface, (3) having chemical moieties to enhance α-helicity, cell permeability, and proteolytic stability, and (4) being an executable molecule for pure SAR analysis (amide chemistry).

[0183] First, experiments were initiated starting from the synthesis of a series of cyclic peptides based on the paxillin LD2 motif and all-hydrocarbon stapling (Figure 1). 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 the i, i+7 positions (6 amino acids apart or two helical turns) were followed by ring-closing olefin metathesis and cleavage from the resin (see Figure 2.) (Kim Y et al., Nature Protocols. 2011;6:761-71). The stapled peptides were designed using molecular modeling and X-ray crystal structure analysis of the FAT-LD2 complex (PDB 10W8), and residues that are not part of the binding interface were selected for substitution with olefinic amino acids. Residue portions of the binding interface were also selected for SAR testing.

[0184] There are three main 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 perform various iterations (staple scanning) of these i-motifs at different positions (N- or C-terminal shifts) and to synthesize stapling at residues present in both the hydrophobic interface and the solvent-accessible surface area. Experiments were performed because the extended residues on the N- and C-termini of the peptide can have beneficial effects on both binding affinity and cell permeability. Finally, experiments were conducted to substitute natural amino acids with hydrophobic / polar / charged substitutions, unnatural amino acids, or D-amino acids (39) to enhance protein contact, enhance permeability, and further inhibit protease recognition (Figure 1).

[0185] To understand the structure-activity relationship of the FAK staple peptide, experiments were designed and 36 peptides were synthesized based on staple strategies ((i, i+3), (i, i+4), and (i, i+7)), staple positions, sequence length, amino acid composition, and homologous sequences (LD2, LD4, CD4, DCC, Leupaxin) (Table 2). The staple peptide UACC-1907 that gave a K D of 3.4 μM in SPR affinity analysis, showed a very high degree of selectivity for WT / mutant proteins in SPR, and competitively inhibited paxillin-FAT binding in a fluorescence polarization (FP) assay:

[0186]

Chemical Structure

[0187] was identified (K i = 5.8 μM) (Figure 4). The activity of UACC-1907 was significantly improved compared to the native 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] 1905, in which the same i+7 stapling motif was shifted by only 1 amino acid, had no binding and inhibitory properties, so the specific staple position of 1907 was essential for biochemical / biophysical activity. 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), 2024-(i,i+3)) showed little activity compared to 1907. Also, peptides with an Aib (2-aminoisobutyric acid)-based α-helix stabilization strategy (i.e., 1912) had lower activity compared to 1907. 1910, which involved the conversion of two glutamic acids to glutamine, was not as active as 1907, so the amino acid sequence was also very important for activity. The amino acid sequence was also very important for activity because peptides (1933, 2007) with the conversion of leucines 145, 149, and 152 (based on the paxillin sequence) to tryptophan had worse binding and inhibitory properties compared to 1907. Peptide 2014 with L145E and L152E substitutions showed no binding and inhibitory properties at all. Overall, staple peptides (1929, 1916) based on homologous protein sequences did not have activity comparable to 1907 derived from LD2. The paxillin LD4-derived peptide 1917 had similar activity, although lower than that of 1907 derived from LD2. The length of the staple peptide 1907 was also important for activity, and truncated staple peptides (i.e., 2011) at the n-terminus and c-terminus had no binding and inhibitory properties. The extended peptide 1920 had moderate binding affinity (K D = 7.0 μM), but worse inhibitory properties (K i = 25.0 μM) compared to 1907. These data showed that empirically derived optimal stapling chemistry and amino acid sequence were required for the best FAK binding and inhibition.

[0189] To further evaluate the activity of 1907, experiments were conducted to test the peptide in a cell permeability assay based on flow cytometry. In this assay, rhodamine-labeled 1907 passed well through the cell membrane in MDA-MB-453 breast cancer cells compared to the control with rhodamine only (Figure 4). Experiments were also conducted to test the cell efficacy in SK-MEL-103 melanoma cells. Peptide 1907 effectively inhibited SK-MEL-103 invasion and 3D growth at a lower relative concentration compared to the known FAK inhibitor defactinib (Compound K D compared to), as shown in Figure 4.

[0190] To examine 1907 for its binding to the paxillin binding site of the FAK FAT domain, additional structural biology experiments were performed. 15 In HSQC NMR experiments using N-labeled FAT domain protein, 1907 showed specific binding at both the helix 2-3 (L959, R962, K955) and helix 1-4 (V928, I936) binding sites to FAT relative to the native paxillin LD2 peptide (Figure 5). The FAT binding affinity derived from NMR showed that 1907 had a stronger affinity for the FAT helix 2-3 site (L959:K D =33.2 μM, K1032:K D =33.7 μM) compared to the FAT helix 1-4 site (I936:K D =1.0 μM, K955:K D =1.6 μM). Also, the X-ray co-crystal structure of 1907 complexed with the human FAK FAT domain was successfully determined (Figure 6). In this crystal structure, 1907 bound to the same FAT helix 2-3 site as paxillin LD2 and showed a prominent α-helix structure. Overall, these tests 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 (Figure 7, Table 2, Figure 14) with myristoyl (2012, 2029) or dodecyl (2025) modifications for increased hydrophobicity and cellular uptake. A negative control myristoylated peptide (2020) was designed and synthesized based on the inactive molecule 2014. Experiments were conducted to test the effects of these peptides in a 3D growth assay using SK-MEL-103 melanoma cells. Peptide 2012 (IC 50 = 5.5 μM) had improved cellular potency compared to 1907 (IC 50 = 60 μM), while the negative control 2020 had no effect (IC 50 = NA). Furthermore, 2012 did not affect the growth 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 create a multivalent peptide that can bind to both sites simultaneously and thus with nanomolar binding affinity, we designed and synthesized a series of conjugated peptides (2018, 2021, 2023) via the conjugation of two azide-modified 1907 molecules (2006) to a DBCO-[PEG] n -DBCO or alkyne-[PEG] n -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 a binding affinity for FAT comparable to that of the parent peptide 1907 (4.6 vs 2.8 μM). Multivalent peptides were ligated using azide-alkyne cycloaddition or “click” chemistry. Experiments were conducted to test the FAT binding affinities of the multivalent peptides 2018, 2021, and 2023 (Table 2). The DBCO-based multivalent peptide 2018 did not have an increased binding affinity (K D = 24.2 μM) compared to 1907, while 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 demonstrated the principle for the FAT multivalency approach and emphasized the empirical determination of the optimal linker length (PEG n ) and chemistry (DBCO vs alkyne) for maximal FAT binding.

[0193] To promote the membrane permeability of peptides 2023 and 1907, in vitro experiments were conducted using liposomal formulations Saint-Protein (Synvolux). Formulations were prepared using a 3:1 volume:volume ratio of Saint:Peptide and the peptide concentration was titrated in PBS pH 7.4 + 1% DMSO. As shown in Figure 9, the 2023 liposomal formulation showed nanomolar effects on the growth of various cancer cells (U87 IC 50 = 40 nM, MDA-MB-231 IC 50 = 27 nM, SK-MEL-103 IC 50 = 50 nM). The liposomal formulation of 1907 showed low micromolar effects on cancer cell growth (U87 IC 50 = 8.5 μM, MDA-MB-231 = 2.3 μM, SK-MEL-103 IC 50 = 2.4 μM). The cell IC50 calculated for both 2023 and 1907 was in line with the FAT domain K D determined by SPR and NMR.

[0194] To generate bifunctional peptides with the dual ability to inhibit both the FAK FAT domain and the FAK kinase domain, a hybrid peptide based on the FAT staple peptide 1907 and the FAK kinase domain inhibitor PF-562271 (PF-271) was designed (Figure 10). Using "click" chemistry, alkynylated PF-271 was conjugated to the azide-modified peptide 2006. A rational choice of 2006 as the conjugation partner was determined by X-ray crystallographic information and calculated biophysical data (Tables 2 and 6). The alkynylation site on PF-271 was selected based on the conventional X-ray co-crystal structure of PF-271:FAK kinase, demonstrating the feasibility of the PROTAC approach (40). The successful synthesis of the FAT-kinase bifunctional (UACC-2030) is shown in Table 3. The chemical structure of 2030 is shown in Figures 10 and 14.

[0195] To generate FAT peptides with the ability to degrade FAK protein in cells by the PROteoylsis TArgeting Chimera (PROTAC) approach, a hybrid peptide based on the FAT staple peptide 1907 and the E3 ligase targeting ligand thalidomide was designed (Figure 11). Using "click" chemistry, alkynylated thalidomide was coupled to the azide-modified peptide 2006. A rational choice of 2006 as the conjugation partner was determined by X-ray crystallographic information and calculated biophysical data (Tables 2 and 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. Peptide 2019 (K D = 10.2 μM) showed a FAT domain binding affinity comparable to that of the parent structure 1907 (Table 2).

[0196] Experiments were conducted 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, both stapled peptides 1907 (t 1 / 2 = 15.7 hours) and 2012 (t 1 / 2 > 48 hours) showed significantly improved stability and protease resistance compared to the native LD2 peptide 1967 (t 1 / 2 = 0.69 hours).

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

[0198] Additional stapled peptides were prepared. 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 staple strategy ((i, i+3); (i, i+4); and (i, i+7)), staple position, sequence length, amino acid composition, and homologous sequences. MW, SPR data, and FP data are shown. (Note) 1907 and 2023 were top candidates from 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-yl)propan-1-one; * : Two-site binding model; NC: Not calculated. NA: Not applicable for assays outside the range of K i to which it does not apply. Unless otherwise specified, peptides are products of intramolecular closed-loop mesasis reactions, and double bond geometry has not been established or quantified. These sequences are omitted and may refer to "click" chemical 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 in this specification were synthesized using various combinations of Rink amide resin and 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, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, and Fmoc-Val-OH. Except for glycine, or as otherwise noted, these amino acids are in the "L" configuration. As is known to those skilled in the art, standard Fmoc-based solid-phase peptide synthesis chemistry was used. The peptides were synthesized using a Biotage Initiator+ Alstra automated microwave-assisted peptide synthesizer with 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. The reactions were carried out on a 0.1 mmol scale. Typical conditions were as follows. Protected amino acids, HCTU, DIC, and Oxyma Pure were used at a concentration of 0.5 M; DIPEA was used at a concentration of 1.0 M, and acetic anhydride in DMF was used at a concentration of 5.0 M. 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 performed using 95:2.5:2.5 (v / v) FA:water:triisopropylsilane. Coupling was carried out using 5 equivalents of amino acid, 5 equivalents of HCTU and 10 equivalents of DIPEA, except when using non-natural alkene amino acids, for which 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 minutes). The Fmoc-protected amino acid was coupled at 75 °C for 4 minutes and then washed with DMF. Double coupling was used for all residues. Arginine was coupled at 50 °C for 6 minutes. Subsequently, a cycle of Fmoc-deprotection consisting of a 3-minute reaction, followed by a 10-minute reaction with fresh reagent at ambient temperature, followed by a DMF wash was performed. A double Fmoc-deprotection cycle was used for the N-terminal residue and the olefinic amino acid.

[0204] Ring-closing metathesis was performed on the Fmoc-protected peptide on a machine in manual injection mode using Grubbs' first-generation catalyst (benzylidene-bis(tricyclohexylphosphine)dichlororuthenium). Prior to the reaction, the resin was thoroughly washed with a cycle of DCM, followed by Et2O, dried briefly in vacuo, then washed and swollen in DCE. Then, Grubbs I (10 mM) was added and the reaction was carried out at 40 °C for 1 hour with evacuation every 15 minutes. This was performed a total of 3 times using the DCE used for washing during the reaction. After completion, the resin was washed with DCE, then DCM, and then swollen in DMF.

[0205] The Fmoc group was deprotected as described above. When N-capping as acetamide, 50 equivalents of acetic anhydride and 10 equivalents of DIPEA were used and reacted at ambient temperature for 45 minutes. Rhodamine B was coupled at 75 °C for 4 minutes with 5 equivalents each of DIC and Oxyma Pure. The resin was washed with 3 cycles of DCM, followed by Et2O, and then dried in vacuo.

[0206] Cleavage was performed using 95:2.5:2.5 TFA:water:triisopropylsilane for 2 hours at ambient temperature. The reaction solution was then added dropwise to 35 mL of cold water Et2O. This was mixed, cooled at -80 °C for 30 minutes, and then centrifuged at 6000×G for 6 minutes. After decanting the supernatant, the pellet was suspended in 20 mL of Et2O, then cooled, centrifuged, and decanted as above. The crude peptide was dried under vacuum.

[0207] Preparative HPLC was performed on an Agilent 1260 II quaternary HPLC equipped with a variable wavelength detector using a gradient of acetonitrile (0.1% AcOH) in water (0.1% AcOH) on a Zorbax SB-C18 column (Agilent 880975-202; 9.4×250 mm; pore size 80 Å; particle size 5 μm).

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

[0209]

Chemical formula

[0210] <Surface Plasmon Resonance (SPR)> The SPR binding assay was performed on a ForteBio Pioneer FE SPR system. Briefly, SADH streptavidin in a dextran hydrogel biosensor (ForteBio) was docked onto the flow cell and preconditioned with two injections of 10 mM NaOH and 1 M NaCl at 50 μL / min for 1 minute. 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. The empty channel 2 was used as a reference control. After achieving a stable baseline, a series of peptide concentrations (200 μM to 0.01 μM) were prepared in the 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 utilized as a bulk standard control for OneStep injection, and a DMSO calibration curve was performed using a concentration range of 3.5% to 6.5% DMSO. The raw SPR data was appropriately processed in Qdat software (ForteBio) by normalizing the baseline prior to injection, aligning the channels, subtracting the reference channel, and blank subtraction. K D To calculate KD, the kinetic data was fitted to a pseudo-first order 1:1 interaction binding model. Additionally, the steady state model and Req data points were used to verify the binding affinity. Visual inspection of the SPR sensogram was performed to verify proper model fitting, lack of mass transport effects, return to baseline, and lack of irregular kinetics.

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

[0212] IC of the peptide inhibitor 50 For measurement, the inhibitor was titrated from 325 nM to 667 μM in the FP buffer containing 20 μM FAT and 0.1 μM TAMRA - LD2. Wells without FAT were used as baseline values, and these were subtracted from the raw values to obtain ΔmP values. The plate was read every hour for 4 hours to confirm the time difference of IC 50 . The titration data was processed with GraphPad Prism to create a dose - response curve and a calculated IC 50 (SE) with standard error. A four - parameter dose - response inhibition model was utilized, and the bottom fit was constrained to the lower plateau of the curve. K i was determined from the following calculation formula:

[0213]

Equation

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

[0215]

Equation

[0216] where P T is the total protein concentration, L T is the total labeled conjugate concentration, P0 is 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 is the positive root of.

[0217] <2D HSQC NMR> 2D HSQC-NMR samples were prepared in 100 μM 15 N FAT. Peptides were screened at a concentration of 500 - 250 nM with a 5% DMSO concentration. 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. In each 2D experiment, 16 signals were co-registered with 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> Flow cytometry assays were performed using a Canto II flow cytometer (BD Biosciences) to measure the cellular uptake of TAMRA-tagged peptides. Briefly, the device was appropriately gated using cells treated with TAMRA only 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> Immunofluorescence staining was performed to measure the effect of stapled peptides on FAK localization. Briefly, cells were seeded on coverslips and fixed with 4% paraformaldehyde in 1×PBS pH 7.4 for 10 minutes and permeabilized with 0.2% Triton X-100 on ice for 5 minutes. Cells were blocked with 25% normal goat serum in 1×PBS pH 7.4 for 30 minutes, washed with 1×PBS pH 7.4, and incubated with FAK 4.47 primary antibody (Millipore) diluted 1:200 in 25% goat serum in 1×PBS pH 7.4. Cells were washed three times with 1×PBS pH 7.4 and FITC-conjugated secondary antibody (diluted 1:400 in 25% goat serum) was applied to the coverslips. Cells were imaged using a Zeiss AXIO Imager M2 Upright Widefield Fluorescent Microscope. For each sample, images were taken across 6 fields of view.

[0220] <X-ray crystallography> Co-crystallization of the lead peptide SP3 with the human FAK FAT domain (AA 919 - 1052), where the peptide was mixed with the protein (1:1 molar ratio) and incubated prior to crystallization, was performed using a Rigaku Phoenix-HT crystallization robot with focusing buffer plates around commercially available crystallization kits (Hampton Research, Molecular Dimensions, Qiagen) and initial crystallization conditions (varying pH, salts, 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. Extra electron density in the FAK-FAT domain binding pocket (Fo-Fc) was modeled into the peptide structure and refined progressively 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 were by ESI+.

[0224] Although the present invention has been fully described, it will be understood by those skilled in the art 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 of its embodiments. All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety as part of this specification.

[0225] (Incorporation by reference) The entire disclosure of each patent document and scientific paper referenced herein is hereby incorporated by reference into this specification for all purposes, including but not limited to the following references: 1. Golubovskaya VM, Kweh FA, Cance 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 analyses. 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: 22454420. 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 regulation of 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 can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are not intended to limit the invention described herein, but should be regarded as illustrative in all respects. Therefore, the scope of the present invention is indicated not by the foregoing description but by the appended claims, and all changes within the meaning and scope of the equivalence of the claims are intended to be included in the present invention.

Brief Description of the Drawings

[0227] [Figure 1] Overview of stapled α-helix peptides targeting the FAT domain of FAK. A. Crystal structure (PDB 1OW8) of FAT-paxillin interaction 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-in inset of the helix 2-3 binding interface of FAT and paxillin LD2. C. Helix wheel analysis of intermolecular and intramolecular interactions of LD2-FAT. D. 3D representation of the stapling strategy used for peptide optimization. E. Overview of amino acid modifications used in the peptide design strategy. [Figure 2] Synthetic scheme for stapled peptide synthesis. [Figure 3] Chemical structure of stapled peptide UACC-1907. [Figure 4]Biochemical, Biophysical, and Cytological Data of Stapled Peptide UACC-1907 (1907). A. 3D model of peptide 1907 with staples highlighted in red. B. Competitive FP experiment using 1907 showing inhibition of TAMRA-LD2 binding to FAT. C. SPR binding and selectivity analysis using wild-type FAT and mutants FAT (L994E, I936A) at the helix-protein interface. D. Flow cytometry analysis of rhodamine-1907 (10 μM) cell 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. 1H / 15N HSQC spectra of the FAT domain using 1% DMSO (maroon), 50 μM 1907 (red), 10 μM 1907 (green), and 5 μM 1907 (blue) on a 600 MHz NMR spectrometer. Note: The peptide causes peak intensity changes at both the helix 1-4 (K1032) and helix 2-3 (L959) binding sites. B. 1907 binding curves using HSQC NMR and four different residues (V932, L959, L994, and D1036) on the FAT domain. The 1907 concentration was titrated from 100 - 0.01 μM and the rate of change of peak integration was plotted against concentration to calculate KD. C. Mapping of the backbone perturbations caused by 1907 at the helix 2-3 site and D. at the 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 of FAK Peptide Inhibitor (i.e., 1907) in Complex with the Human FAK FAT Domain at 1.95 Å Resolution. The left panel shows the stapled peptide in blue and the FAT domain in green. The right panel shows the electron density map of the peptide in the binding pocket. [Figure 7]Anticancer effect of stapled 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 of stapled peptide UACC-2012 in HUVEC "normal" cells. [Figure 8] FAT bivalent stapled peptide strategy and SPR data of synthetic peptide UACC-2023. A. Overview of the bivalent stapled peptide strategy to enable FAT domain double-site binding using click chemistry and linker approach. B. Structure of UACC-2023. C. SPR sensogram of bivalent peptide UACC-2023 and PEG10 linker binding to FAT. [Figure 9] Anticancer effect data of stapled peptides 2023 and 1907 in liposome formulations. A. 2D proliferation data of peptide 2023 combined with cationic lipid reagent Saint-Protein (Synvolux). B. 2D proliferation data of peptide 1907 combined with cationic lipid reagent Saint-Protein. Lipid:peptide formulations were prepared as 10-fold stocks using peptides at a 1:1 (v:v) ratio and titrated concentrations in PBS pH 7.4 + 1% DMSO. [Figure 10] Overview of the synthetic strategy of FAT-kinase bifunctional inhibitor and proof-of-concept molecule UACC-2030. A. (Top) Adhesion site of FAK kinase domain inhibitor PF-562271. (Bottom) Adhesion site of FAT domain inhibitor 1907. B. Synthetic plan of FAT-kinase bifunctional group and chemical structure of synthetic molecule UACC-2030. [Figure 11] Overview of FAT-PROTAC synthetic strategy and proof-of-concept synthetic molecule UACC-2019. [Figure 12]In vitro trypsin digestion assay to measure the stability and protease resistance of peptides (1967, 1907, and 2012). The assay was performed using trypsin-agarose beads (ThermoFisher), and peptide concentration was measured using LC-MS. Peptide half-life was calculated using GraphPad Prism software. [Figure 13] Antifibrotic activity of FAT stapled peptide UACC-2012 in LX2 human hepatic stellate cells. A. Western blot assay of LX2 cells treated with 2 ng / mL TGF-β for 17 h to induce a pro-fibrotic phenotype. Cells were pretreated with either 10 μM, 5 μM, and 2 μM DMSO or UACC-2012 (mSP3) for 1 h. Quantification of the fibronectin results is shown in the lower panel. B. Morphological changes of LX2 cells after 17 h 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

**Claim 1**: A composition comprising a compound (including its pharmaceutically acceptable salts and / or solvates) having the sequence number 4 (Ac-NLR8ELDLLS5ELN-NH2) or sequence number 28 (Myristoyl-NLR8ELDLLS5ELN-NH2), where Ac means acetyl, R8 means (R)-2-(7-octenyl)alanine, S5 means (S)-2-(4-pentenyl)alanine, Myristoyl means a myristoyl group, and S5 and R8 are cross-linked to each other, and where the standard one-letter amino acid code is used, wherein the compound can bind to the FAT domain of FAK; wherein the compound enables inhibition of the interaction between the FAT domain of FAK and paxillin protein. **Claim 2** The composition according to claim 1, further comprising the compound bound to a further therapeutic agent. [[ID=*6*]]**Claim 3**: The composition according to claim 1, wherein the compound is further bound to a contrast agent. **Claim 4** The composition according to claim 1, wherein the contrast agent is (5- / 6-)carboxytetramethylrhodamine (TAMRA). **Claim 5** A pharmaceutical composition comprising the composition according to claim 1, wherein the pharmaceutical composition comprises one or more of the above compounds within a liposomal formulation. **Claim 6** The pharmaceutical composition according to claim 5, 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, MVL5, DOTMA, DDAB, DC-cholesterol, GL67, DODMA, soy phospholipids, cationic lipids, anionic lipids, neutral lipids in various combinations and / or ratios and / or in buffer solutions. **Claim*7** 16:3 (n-3); all-cis-7,10,13-hexadecatrienoic acid), alpha-linolenic acid (ALA; The liposome preparation is sphingomyelin (SM), D-erythro-sphingomyelin, D-erythro-dihydrosphingomyelin, palmitoyl sphingomyelin, lysophospholipid, galactocerebroside, ganglioside, cerebroside, glyceride, triglyceride, diglyceride, small alkyl chain phospholipid, phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC), 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dilauroyl phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol and other diphosphatidylglycerols, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, ceramide, phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, dipalmitoyl phosphatidylglycerol salt, phosphatidic acid, galactocerebroside, ganglioside, cerebroside, dilauryl phosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenyl glycoside, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipid, and cholesterol, lysophosphatidylcholine, lysosphingomyelin, 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)butylamide], 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 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 (Oleic Acid Nitrate 9- and 10-nitro-cis-octadecenoic acid), LNO ２ (Nitrate Linolenic Acid 9-, 10-, 12-, and 13-nitro-cis-octadecadienoic acid), AA-NO ２ (Nitrate Arachidonic Acid 5-, 6-, 8-, 9-, 11-, 12-, 14-, and 15-nitro-cis-eicosatetraenoic acid), CLNO ２ (Nitrate Cholesteryl Linoleate Cholesteryl-9-, 10-, 12-, and 13-nitro-cis-octadecadiene Cato), Fatty Acid, Omega-3-Polyunsaturated Fatty Acid, Hexadecatrienoic Acid (HTA; 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; It should be noted that there are some parts in the original text that seem to be a bit disordered in terms of the claim numbering and presentation. This translation attempts to make sense of it based on the overall context. If there are specific requirements or corrections needed regarding the claim structure, it may be necessary to further clarify the source text. 20:3(n - 3); all-cis-11,14,17-eicosatrienoic acid), eicosatetraenoic acid (ETA; 20:4(n - 3); all-cis-8,11,14,17-eicosatetraenoic acid), eicosapentaenoic acid (EPA; 20:5(n - 3); all-cis-5,8,11,14,17-eicosapentaenoic acid), heneicosapentaenoic acid (HPA; 21:5(n - 3); all-cis-6,9,12,15,18-heneicosapentaenoic acid); docosapentaenoic acid (DPA; clupanodonic acid; 22:5(n - 3); all-cis-7,10,13,16,19-docosapentaenoic acid), docosahexaenoic acid (DHA; 22:6(n - 3); all-cis-4,7,10,13,16,19-docosahexaenoic acid), tetracosapentaenoic acid; 24:5(n - 3); all-cis-9,12,15,18,21-tetracosapentaenoic acid), tetracosahexaenoic acid (nisinic acid; 24:6(n - 3); all-cis-6,9,12,15,18,21-tetracosahexaenoic acid), a sphingosine-1-phosphate analog, a sphingosine-1-phosphate antagonist, a sphingosine-1-phosphate agonist, a sphingosine-1-phosphate receptor agonist, a sphingosine-1-phosphate receptor antagonist, and a combination of one or any combination of sphingosine-1-phosphate receptor analogs, the pharmaceutical composition according to claim 5.

8. The composition according to claim 1, wherein the compound enables one or more of the following: Binding to one or more of the helix 1 - 4 region and the helix 2 - 3 region of the FAT domain of the FAK, Inhibiting the interaction between FAK and paxillin, Destroying the non-catalytic activity of FAK by inhibiting the interaction between FAK and paxillin, Destroying the catalytic activity of FAK through direct binding of the FAT domain, Inhibiting the FAK-related scaffolding function, Inhibiting the FAK protein-protein interaction mediated by the FAT domain, Inhibiting the binding between paxillin and the helix 1 - 4 region of the FAT domain of FAK, Inhibiting the binding between paxillin and the helix 2 - 3 region of the FAT domain of FAK, Inhibiting FAK-related apoptosis, proliferation, invasion, and / or metastasis Inhibiting the FAK-paxillin interaction, which results in inhibition of FAK phosphorylation, paxillin phosphorylation, focal adhesion turnover, cell adhesion, migration, and / or invasion Inhibiting the FAK-Leupaxin interaction via binding to the FAT domain of FAK Inhibiting the FAK-CD4 interaction via binding to the FAT domain of FAK Inhibiting the FAK-CD8 interaction via binding to the FAT domain of FAK Inhibiting the FAK-DCC interaction via binding to the FAT domain of FAK Inhibiting the binding of the LD motifs of paxillin LD2 and LD4 to each binding partner Inhibiting the binding of CD4 and CD8 to each binding partner Inhibiting the binding of Leupaxin to each binding partner Inhibiting the binding of DCC to each binding partner Inhibiting the binding of the paxillin protein to one or more of PyK2, Vinculin, ILK, Actopaxin, PKL, Git1 / 2, Pax3, hic-5, and ARF

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