Bifunctional molecules for selective modification of target substrates

Abstract

The present disclosure relates to bifunctional chemical conjugation molecules, which find utility as target substrate modification agents. The present disclosure includes multifunctional compounds comprising an enzyme binding moiety, a chemical linker moiety, and a target binding moiety, which may further comprise an electrophilic reactive group. The molecules described herein find use in modifying substrates, e.g., post-translational modifications, on proteins that are not natural substrates of the enzyme. Diseases or disorders may be treated or prevented by the molecules of the present disclosure.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 291,942, filed December 20, 2021, U.S. Provisional Application No. 63 / 211,307, filed June 16, 2021, U.S. Provisional Application No. 63 / 173,351, filed April 9, 2021, and U.S. Provisional Application No. 63 / 173,357, filed April 9, 2021. The entire contents of the above-identified applications are hereby incorporated by reference herein in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. AI154099 awarded by the National Institutes of Health and Grant No. N66001-17-2-4055 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in this invention.

[0003] The subject matter disclosed herein is generally directed to chimeric small molecules that are utilized to induce modifications in target substrates. [Background technology]

[0004] Protein kinases regulate important cellular functions such as the cell cycle, metabolism, differentiation, proliferation and apoptosis. Kinase dysfunction is also associated with a variety of human diseases, including cancer, inflammatory conditions, autoimmune disorders and heart disease. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a continuing need in the art for effective treatments and modifications, such as post-translational modifications, for diseases associated with enzyme dysfunction. However, obstacles, such as non-specific effects, remain obstacles to the development of effective modifications and treatments. For example, there are no small molecules that induce phosphorylation of any given protein, and phosphorylation of any protein on demand using small molecules would be advantageous. Therefore, new small molecules that confer new functions to enzymes through proximity-mediated effects may be useful in the study and treatment of important cellular functions and diseases. [Means for solving the problem]

[0006] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

[0007] overview In one embodiment, a bifunctional small molecule is provided that includes a kinase-binding moiety and a target protein-binding moiety connected via a linker molecule, where the kinase-binding moiety brings the kinase into proximity with the target protein and induces phosphorylation of the target protein. A chimeric small molecule is provided that includes an enzyme-binding moiety and a target-binding moiety connected via one or more linker molecules, and optionally an electrophilic reactive group, where the enzyme-binding moiety brings the enzyme into proximity with a target substrate and induces modification of the target substrate, or where the enzyme-binding moiety facilitates labeling of the enzyme with the target-binding moiety via the electrophilic reactive group.

[0008] In one embodiment, the formula A-(L) n -B wherein A is an enzyme binding moiety, B is a target binding moiety, L is a linker, and n is 0 to 6, or AL-El-B or AL 1 -El-L 2 -B where A is an enzyme binding moiety, B is a target binding moiety, L is a linker, and EI is an electrophilic reactive group. In one embodiment, L1 and L2 are the same or different molecules selected from an alkane, alkene, alkyne, amine, ether, thiol, sulfone, carbonyl, acyl, ketone, carboxylic acid ester; amide, enone, anhydride, imide, PEG, or any combination thereof, and n is 0-6. In one embodiment, A and B can be the same molecule or different molecules that bind to the same type of enzyme (e.g., both are small molecule binders of Abl kinase).

[0009] In one embodiment, the enzyme binding moiety is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or transferase binding moiety. In some embodiments, the binding moiety is a kinase binding moiety, which can be a PK, e.g., a receptor tyrosine kinase, a non-receptor tyrosine kinase, a serine / threonine specific kinase, a PKC, an AMPK, a MAPK, an EGFR, a FGFR, a NGFR, a TrkA, an ABL, a BCKDK, a CDK, a PI3K, a VEGFR, a BRAF, a MEK, an AKT, an ALK, a BTK, a FLT3, a JAK2, a AURKA, a c-MET, a DDR, a FKBP, an INSR, an IKK, a JNK, a mTOR, a PAK, a PDK1, a PDK2, a PTK2 / FAK, a pyruvate kinase, a RAC-α, a RIPK, a TYK2, a SHP, aPKC, a NOP, a μ opioid receptor, a δ opioid receptor, a UMPK, a SphK, or a GSK-3 binding moiety.

[0010] In certain embodiments, the bifunctional molecule has the formula A-(L) n -B (wherein A is a kinase binding moiety, B is a target protein binding moiety, L is a linker, and n is 0 to 6). In some embodiments, the kinase binding moiety is an AMPK, ABL, or PKC binding moiety. In some embodiments, B is K-Ras, HSP90, BRD4, BTK, FKB12 F36VIn one embodiment, L is selected from an alkane, an alkene, an alkyne, an amine, an ether, a thiol, a sulfone, a carbonyl, an acyl, a ketone, a carboxylic acid ester; an amide, an enone, an anhydride, an imide, a PEG, or any combination thereof, and n is 0-6.

[0011] In some embodiments, the target binding moiety B is an enzyme binding moiety, and A and B each separately bind to the same type of enzyme. In some embodiments, the bound enzymes are oligomeric proteins, and the chimeric small molecule locks the oligomeric enzymes in an active or inactive state. In one embodiment, the enzymes are kinases, and optionally one of the bound kinases phosphorylates the other bound enzyme, thereby activating the other bound enzyme, and optionally the kinases are receptor tyrosine kinases, non-receptor tyrosine kinases, or serine-threonine kinases.

[0012] In one embodiment, the bifunctional molecule has the formula [ka] where W is independently selected from amine, O, S, NH, a bond, alkane, alkene, alkyne, amine, ether, thiol, sulfone, carbonyl, acyl, ketone, carboxylic ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, O, S, NH, or any combination thereof, and A and B are linked to their respective W via any functional group or ring position of A and B. In one embodiment, A and B bind to the same type of enzyme, and in some aspects, A and B are BCR-ABL binding agents. In some aspects, A and B are the same binding moiety.

[0013] In certain embodiments, the kinase binding moiety is selected from the group consisting of FKBP, PKC, AMPK, ABL, PK, MAPK, e.g., MAPK1, MAPK11, MAPK12, MAPK13, MAPK14, p38α The targeting moiety is a MAPK, EGFREGFR, FGFR, NGFR, TrkA, ABL, CDK, e.g., CDK2, CDK4, PI3K, VEGFR, BRAF, MEK, e.g., MEK1 / 2, MEK5, AKT, ALK, BTK, BCKDK, FLT3, JAK2, AURKA, c-MET, DDR, INSR, JNK, IκB, IKK, Lyn, mTOR, e.g., mTORC-1, PAK, PDK, e.g., PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, e.g., PKC-ζ, NOP, μ opioid receptor, δ opioid receptor, UMPK, SphK, or GSK-3 binding moiety. In one embodiment, the targeting moiety binds to the same type of kinase as the kinase binding moiety. In some embodiments, B is K-Ras, HSP90, BRD4, BTK, FKB12 F36V It is the binding part.

[0014] In certain embodiments, the kinase binding moiety has the formula [ka] (Wherein, R is [ka] , a carbohydrate mimetic, a heterocycle, a diahydrohexitol, a pyranose or a furanose; and Q is selected from the group consisting of B, C, N, O, S, where H is N. A Or N B Located on either X 1 and X 2is independently selected from the group consisting of C, N and O; Y is selected from the group consisting of H, OH, halogen, CN or a hydrogen bond donating substituent; Z is H, an alkane, an alkene, an alkyne, an amine, a nitrile, nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2 Cl, and optionally further substituted). In one embodiment, Z comprises an AMPK binding moiety according to the formula: Z a -Z b (In the formula, Z a teeth [ka] is selected from the group consisting of Z b teeth [ka] and n is 0 to 6.

[0015] In one embodiment, the AMPK binding moiety [ka] was selected from the group consisting of:

[0016] In one embodiment, A is [ka] And B is [ka] It is.

[0017] In one embodiment, L is [ka] (wherein n is 1, 2, 3, 4 or 5). is selected from the group consisting of:

[0018] In one embodiment, the small chimeric molecule [ka] It is.

[0019] In one embodiment, A is of the formula [ka] or a PKC binding portion of an analog thereof.

[0020] In one embodiment, the small chimeric molecule [ka] (Wherein, R is tBuC(O)) It is.

[0021] In one embodiment, the small chimeric molecule [ka] It is.

[0022] In one embodiment, the small chimeric molecule [ka] It is.

[0023] In one embodiment, A is of the formula [ka] (wherein R1 to R5 are independently H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more condensed rings thereof, or an aliphatic halogen compound, for example, -OCF 2 Cl; Z is independently selected from B, C, N, O, S, preferably one or two atoms of Z are N, O, S, or a combination thereof; Ra, Rb, and Rc are independently selected from alkanes, alkenes, alkynes, ethers, alcohols, amines, nitriles, nitro, thiols, sulfones, sulfonates, halogens, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, acid anhydrides, imides, aliphatic halogen compounds, such as -OCF 2 Cl, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, one or more fused rings including any combination of any of the above listed rings, and Re is an alkane, alkene, alkyne, ether, alcohol, amine, nitrile, nitro, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, acid anhydride, imide, aliphatic halogen compound, e.g., OCF 2 Cl, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof at one or more positions, or R 1 Or R 5 and R a , R b , R c One or more of [ka] and an amide further attached to a molecule selected from the group consisting of:

[0024] In one embodiment, A is according to formula II(b) and Re is [ka] wherein Rf and Rg are selected at one or more positions from alkanes, alkenes, alkynes, ethers, alcohols, amines, nitriles, nitro, thiols, sulfones, sulfonates, halogens, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, anhydrides, imides, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, cyclic hydrocarbons optionally substituted with one or more fused rings comprising any combination of any of the previously listed rings, unsaturated cyclic hydrocarbons, heterocycles, one or more fused rings comprising any combination of any of the previously listed rings; and Rf and Rg are independently selected from the group consisting of: [ka] is selected from the group consisting of:

[0025] In one embodiment, the ABL kinase binding moiety has the formula: [ka] R is selected from H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, an aliphatic halogen compound, e.g., -OCF2Cl, or any combination thereof, preferably [ka] (selected from It is described in.

[0026] In one embodiment, the ABL kinase binding moiety [ka] R is selected from H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, an aliphatic halogen compound, e.g., -OCF 2 Cl, or any combination thereof, preferably [ka] (wherein X and Y are CH or N, and R is H, D, F, Me, CF 3 is) is selected from the group consisting of:

[0027] In one embodiment, the ABl kinase binding moiety [ka] is selected from.

[0028] In one embodiment, the ABl kinase binding moiety has the formula: [ka] wherein X is selected from C, N, O and S; R 2is H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more condensed rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof, preferably [ka] (selected from the group consisting of It is described in.

[0029] In one embodiment, the ABl kinase binding moiety has the formula: [ka] X is a halogen, Y is selected from C, N, O and S, and R 1 , R 2 , and R 3 are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof) is selected from.

[0030] In one embodiment, the ABl kinase binding moiety has the formula: [ka] (Y 1 is selected from C, N, O and S; R 4 , R 6 and R 7are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof) is selected from.

[0031] In one embodiment, the ABl kinase binding moiety has the formula: [ka] TIFF2024513566000030.tif218170(Y 1 is selected from C, N, O and S; R 3 , R 4 , R 6 and R 7 are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof) is selected from.

[0032] In one embodiment, the ABl kinase binding moiety has the formula: [ka] (Y 1 is selected from C, N, O and S; R 3 , R 4 , R 6 and R 7are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof) is selected from.

[0033] In one embodiment, the Abl kinase binding moiety has the formula: [ka] is selected from.

[0034] In one embodiment, the Abl kinase binding moiety [ka] is selected from the group consisting of:

[0035] In one embodiment, the Abl kinase binding moiety [ka] is selected from the group consisting of:

[0036] In one embodiment, R a , R b , R c One or more of [ka] and an amide further attached to a molecule selected from the group consisting of:

[0037] In one embodiment, A is according to formula II(b), where Re is [ka] wherein Rf and Rg are selected at one or more positions from alkanes, alkenes, alkynes, ethers, alcohols, amines, nitriles, nitros, thiols, sulfones, sulfonates, halogens, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, anhydrides, imides, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, cyclic hydrocarbons optionally substituted with one or more fused rings containing any combination of any of the previously listed rings, unsaturated cyclic hydrocarbons, heterocycles, one or more fused rings containing any combination of any of the previously listed rings. is selected from the group consisting of:

[0038] In one embodiment, Rf and Rg are independently [ka] is selected from the group consisting of:

[0039] In one embodiment, the small chimeric molecule [ka] (wherein n is 0 to 3). It is described in.

[0040] In one embodiment, the small chimeric molecule [ka] It is.

[0041] In one embodiment, the small chimeric molecule [ka] It is.

[0042] In one embodiment, the small chimeric molecule [ka] It is.

[0043] In certain embodiments, the enzyme-binding moiety is [ka] or analogs thereof.

[0044] In one embodiment, B is [ka] It is.

[0045] In one embodiment, B is [ka] is selected from the group consisting of:

[0046] In one embodiment, B is [ka] (wherein R is an electrophilic reactive group; X is the formula [ka] and Y is H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2 Cl) In one embodiment, the electrophilic reactive group is selected from the group consisting of: [ka] or analogs thereof.

[0047] In one embodiment, B is of the formula [ka] or an analogue thereof.

[0048] In one embodiment, B is [ka] or analogs thereof.

[0049] In one embodiment, B is [ka] or analogs thereof.

[0050] In one embodiment, B is [ka] Selected from FKBP12 F36V binding molecules or analogs thereof.

[0051] In one example, the small chimeric molecule [ka] It is.

[0052] In one embodiment, B is of the formula [ka] or an analog thereof.

[0053] In one embodiment, A is an AMPK-binding moiety and B is a KRAS-binding moiety as described herein. In some embodiments, A is an ABL kinase-binding moiety and B is a BRD4-binding moiety as described herein.

[0054] In one embodiment, L is [ka] and n is 0 to 3, or L is [ka] and n is 0 to 6. In one embodiment, L is [ka] wherein n is 1, 2, 3, 4 or 5.

[0055] In one embodiment, L is a rigid linker, which is [ka] or any combination thereof, wherein any atom in the ring may be optionally substituted with C, N, O, S, the linker may be attached to one or more PEG molecules prior to attachment to A and optionally B, and m and n may be independently selected from 0 to 6. may be selected from the group consisting of:

[0056] In an example embodiment, the linker L has one covalent attachment point to the kinase binding molecule and two covalent attachment points to the other kinase binding molecule. The covalent attachment points may be any single, double, triple or quadruple bond between one component of the BFM and another. In an example embodiment, the formula [ka] According to the present invention, the linker binds one kinase binding molecule, say A, and another, say B.

[0057] In one embodiment, the PEG compound in the above-listed linker can be replaced with any linker listed herein. In one embodiment, the above-listed linker is optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. In one embodiment, the electrophilic reactive group in the formula is selected from N-acyl-N-alkylsulfonamide (NASA), dibromophenylbenzoate, or N-sulfonylpyridone. In one aspect, the electrophilic reactive group is [ka] where EWG is any electron withdrawing group known in the art. In one embodiment, the electrophilic reactive group reacts with a nucleophilic reactive group. The enzyme binder may further comprise a bioorthogonal group. In one embodiment, the bioorthogonal group is selected from tetrazine, triazine, cyclooctene, cyclopropene, and diazo; [ka] may be selected from the group consisting of:

[0058] In certain embodiments, the enzyme binding agent has a half-life that is shorter than the half-life of the target to which the targeted binding agent can bind, which may be 2, 3, 4, 5 or less times shorter than the half-life of the target bound to the targeted binding agent.

[0059] In some embodiments, the target is a protein. In some embodiments, the molecule may comprise an enzyme binding agent that is a kinase binding agent, which may be a kinase inhibitor or activator. In one embodiment, the kinase inhibitor is a promiscuous kinase inhibitor.

[0060] In one embodiment, the molecule is [ka] It is.

[0061] In one embodiment, the molecule is [ka] It is.

[0062] In one embodiment, the molecule is [ka] It is.

[0063] In one embodiment, the molecule is [ka] It is.

[0064] In one embodiment, the molecule is [ka] It is.

[0065] In one embodiment, the molecule is [ka] It is.

[0066] In one embodiment, the molecule is [ka] It is.

[0067] In one embodiment, the molecule is [ka] It is.

[0068] In one embodiment, the molecule is [ka] It is.

[0069] In one embodiment, the molecule is [ka] It is.

[0070] In one embodiment, the molecule is [ka] It is.

[0071] In one embodiment, the molecule is [ka] It is.

[0072] In one embodiment, the molecule is [ka] It is.

[0073] In one embodiment, the molecule is [ka] It is.

[0074] In one embodiment, the molecule is [ka] It is.

[0075] In one embodiment, the molecule is [ka] It is.

[0076] In one embodiment, the molecule is [ka] It is.

[0077] In one embodiment, the molecule is [ka] It is.

[0078] In one embodiment, the molecule is [ka] It is.

[0079] In one embodiment, the molecule is [ka] It is.

[0080] In one embodiment, the molecule is [ka] It is.

[0081] In one embodiment, the molecule is [ka] It is.

[0082] In one embodiment, the molecule is [ka] It is.

[0083] In one embodiment, the molecule is [ka] It is.

[0084] A method of inducing phosphorylation of a target protein is provided, the method comprising administering to a cell or population of cells a chimeric small molecule.

[0085] A method for modifying a target substrate in a cell is provided. In one embodiment, the method comprises: 1 -EL 2-B, where A is an enzyme binding moiety specific for the cellular enzyme to be reprogrammed, B is a target binding moiety specific for the target substrate to be modified, L is a linker, and EI is an electrophilic reactive group, whereby the chimeric molecule labels the cellular enzyme with a target binding moiety for the target substrate, and modifies the target substrate by binding the reprogrammed enzyme to the target substrate via the target binding agent, whereby the reprogrammed cellular enzyme introduces one or more modifications to the target substrate. In one embodiment, the enzyme binding moiety has a half-life that is about 2, 3, 4, 5, 6, or 7 times shorter than the half-life of the reprogrammed enzyme. In one embodiment, the reprogrammed enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In one embodiment, the inhibitor is an enzyme binding moiety. In one embodiment, the enzyme to be repurposed / reprogrammed is a kinase and the enzyme-binding moiety is a kinase inhibitor. In one embodiment, the kinase inhibitor is a "promiscuous" kinase inhibitor. In one embodiment, the method includes administering a quenching molecule, thereby quenching or reducing the inhibitory activity of the enzyme inhibitor. In one embodiment, the quenching molecule is one or more of an aldehyde, an alkene, an alkyne, a strained alkyne, a cyclooctyne, a trans-cyclooctene, a cyclopropene, an oxanorbornadiene, a norbornene, a phosphine, an electron-rich dienophile, an isonitrile, an isocyanopropionate, a tetrazole, a 2-acylboronic acid, or any derivative thereof. In one embodiment, the cyclooctyne derivative includes a dibenzocyclooctyne, a biarylazacyclooctyne, or a dimethoxyazacyclooctyne. In one embodiment, the method includes a strained alkyne including a bicyclononyne or a dioxabiaryldecyne.

[0086] A method for modifying a substrate is provided. In one embodiment, a chimeric small molecule as described herein is introduced. In one embodiment, modifying comprises inducing post-translational modification of a target protein. In one embodiment, the post-translational modification is phosphorylation.

[0087] Methods of treating cancer are provided. The methods of treating cancer include administering to a patient a compound of the formula: ALEB, AL 1 -EL 2 -B or A-(L) n The present invention includes creating an initialized cellular enzyme by administering to a subject in need thereof a chimeric molecule of formula (I)-B, where A is an enzyme binding moiety, L is a linker, n is 0-6, E is an electrophilic reactive group, and B is an oncogenic protein to be modified, whereby the chimeric molecule labels the cellular enzyme with a targeted binding agent for the target substrate, and modifying the oncogenic protein by binding the repurposed / initialized enzyme to the target substrate via the targeted binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. In one embodiment, the targeted binding agents are KRAS, RAS, FKPB, 12F36V , EGFR, HSP90, BTK, MDM2, BRD4, BCR-ABL, NF-kB, LDH-A, p53, GP73, MUC1, MUC16, CD44, GPCR, HMGB1, RIOK1, CHK1, UBE2F, HuR, PTEN, STAT-3, osteopontin, EGFR, AKT, DAPK1, Rho, Ubc9, FOXK2, HIC1, HER2, BRAF, BCL-2, CD117, (KIT), ALK, PI3K, Delta, DNMT1 or SMO.

[0088] In one embodiment, the cellular enzyme to be reprogrammed is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In one embodiment, the enzyme binding agent is an enzyme inhibitor, preferably a kinase inhibitor. In one embodiment, the kinase inhibitor is specific for PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, NOP, μ-opioid receptor, δ-opioid receptor, UMPK, SphK, or GSK-3. In one embodiment, a quenching molecule is administered, thereby quenching the inhibitory activity of the enzyme inhibitor.

[0089] A method for treating a disease associated with abnormal KRAS signaling is provided, comprising administering a composition comprising a bifunctional molecule, the bifunctional molecule comprising a KRAS binding molecule and a kinase binding molecule as described herein. In one embodiment, the enzyme binding molecule is a target of an enzyme selected from the group consisting of PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, NOP, μ opioid receptor, δ opioid receptor, UMPK, SphK, or GSK-3. In one embodiment, the kinase binding molecule is an AMPK binding moiety. In one embodiment, the target binding moiety is a KRAS binding moiety. G12CIn one embodiment, the bifunctional molecule phosphorylates one or more residues on KRAS selected from the group consisting of Ser17, Ser39, Ser65, Ser106, Ser122, Ser136, Ser2, Thr2, Thr35, Thr50, Thr74, Thr87, Thr124, Thr127, and Thr148.

[0090] In one embodiment, the target substrate comprises a nuclear localization sequence (NLS), also known as a nuclear localization signal, or a nuclear export signal sequence (NES), also known as a nuclear export signal. Upon binding of the small chimeric molecule and recruitment of the target enzyme, the target enzyme can modify the target substrate, e.g., protein and / or NLS or NES. The result of targeting the NLS-bound protein with the small chimeric molecule will be either introduction of the enzyme-modified protein into the nucleus or prevention of the enzyme-modified protein into the nucleus, depending on the enzyme that is recruited for modification. In one embodiment, an FKBP containing an NLS is included on the Cas9 system, which is targeted by the kinase. Upon recruitment / initialization of the kinase via the chimeric small molecule, the NLS is phosphorylated. In one aspect, the modification disrupts signal transduction and prevents localization to the nucleus. The phosphorylated NLS can no longer facilitate the transport of the Cas9 system into the nucleus, thus localizing it to the cytoplasm.

[0091] In some embodiments where the target substrate comprises a NES, modification of the NES-functionalized target substrate can result in modification of the target substrate and / or the NES. Targeting a protein with an NES-linked small chimeric molecule results in either the introduction of the enzyme-modified protein from the nucleus to the cytoplasm, or the prevention of the enzyme-modified protein from the nucleus to the cytoplasm, depending on the enzyme (e.g., kinase, phosphatase) that is recruited for modification. In some embodiments, an FKBP containing an NES is included on the Cas9 system, which is targeted by the kinase. Upon initial recruitment of the kinase via the chimeric small molecule, the NES is phosphorylated. In some embodiments, the modification disrupts signal transduction and prevents localization to the cytoplasm. The phosphorylated NES can no longer facilitate the transport of the Cas9 system to the cytoplasm, thus localizing it to the nucleus.

[0092] A method for treating an infection caused by a pathogen is provided, the method comprising administering to said patient an antibody of formula: ALEB or AL 1 -EL 2The present invention relates to a method for producing an initialized cellular enzyme by administering a chimeric molecule of -B, where A is an enzyme binding moiety, L is a linker, E is an electrophilic reactive group, and B is a pathogen protein to be modified, to a subject in need thereof, whereby the chimeric molecule labels the cellular enzyme with a target binding agent for the target substrate, and modifies the pathogen protein by binding the repurposed / initialized enzyme to the target substrate via the target binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. In one embodiment, the initialized cellular enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, translocase. In one embodiment, the pathogen is a virus, a bacterium, a fungus, or a protozoan. In one embodiment, the bacterium is Mycobacterium tuberculosis (Mtb) or Pseudomonas aeruginosa (PsA). In one embodiment, the pathogen is Mtb and the pathogen protein is one or more of PtpA, PtpB, SapM, ESAT-6 and Rv2966c. In one embodiment, the pathogen is (PsA) and the targeted binding agent is colistin. In one embodiment, the enzyme binding agent is a kinase inhibitor. In one embodiment, the kinase inhibitor is a promiscuous inhibitor. In one embodiment, a quenching molecule is administered, thereby quenching or reducing the inhibitory activity of the enzyme inhibitor.

[0093] In one aspect, the invention provides a method of treating cancer, comprising: a) administering to a subject in need thereof a therapeutically effective amount of a composition comprising any of the preceding molecules. In one embodiment, the cancer is characterized by aberrant kinase signaling, a cancerous fusion. In one embodiment, the cancer is characterized by aberrant BCR-ABL kinase signaling. In one embodiment, the cancer is characterized by a cancerous fusion of ABL kinase. In one embodiment, the cancerous fusion is a TEL-ABL or NUP214-ABL fusion. In one embodiment, the method may further comprise administering a therapeutically effective amount of a monomer of A or B to reverse the activity of the chimeric molecule.

[0094] These and other aspects, objects, features and advantages of the example embodiments will become apparent to those of ordinary skill in the art upon consideration of the following detailed description of the example embodiments.

[0095] BRIEF DESCRIPTION OF THE DRAWINGS A general understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention may be utilized. [Brief description of the drawings]

[0096] [Figure 1] FIG. 1 includes a schematic diagram providing a depiction of a bifunctional molecule, according to certain example embodiments.

[0097] [Figure 2A-B] 2A-2B provide additional schematics of possible mechanisms of action for bifunctional molecules, according to certain example embodiments.

[0098] [Diagram 3] FIG. 3 provides a Western blot assay of the Abl-BRD4 bifunctional molecule, according to certain example embodiments.

[0099] [Figure 4]FIG. 4 provides a Western blot assay of an Abl-BRD4 bifunctional molecule, according to certain example embodiments.

[0100] [Diagram 5] FIG. 5 provides a Western blot assay of the Abl-BRD4 bifunctional molecule, according to certain example embodiments.

[0101] [Figure 6] FIG. 6 provides a Western blot assay of the Abl-BRD4 bifunctional molecule, according to certain example embodiments.

[0102] [Figure 7] FIG. 7 provides a Western blot assay of the Abl-BRD4 bifunctional molecule, according to certain example embodiments.

[0103] [Figure 8] FIG. 8 provides a Western blot assay of the Abl-BRD4 bifunctional molecule, according to certain example embodiments.

[0104] [Figure 9] FIG. 9 provides a Western blot of Abl-BRD4 bifunctional molecule activity in HEK293 cells, according to certain example embodiments.

[0105] [Figure 10] FIG. 10 provides a Western blot of Abl-BRD4 bifunctional molecule activity in HEK293 cells, according to certain example embodiments.

[0106] [Figure 11] FIG. 11 provides a Western blot demonstrating that Abl-BRD4 bifunctional molecules induce ternary complexes of Abl-flag (BRD4-HA), according to certain example embodiments.

[0107] [Figure 12] FIG. 12 provides a Western blot of an Abl-Hsp90 bifunctional molecule, according to certain example embodiments.

[0108] [Figure 13] FIG. 13 provides a Western blot of an Abl-AMPK bifunctional molecule, according to certain example embodiments.

[0109] [Figure 14] FIG. 14 is a heat map demonstrating that, according to certain example embodiments, the Abl-FGR1 bifunctional molecule can activate FGFR / mTOR / G-CSF signaling in cells with endogenous Abl.

[0110] [Figure 15] FIG. 15 provides a heat map demonstrating that tyrosine PHICS VS1043 activates FGFR1 / mTOR / G-CSF signaling in a dose-dependent manner, according to certain example embodiments.

[0111] [Figure 16] Exemplary chimeras for recruitment of P. aeruginosa to antibodies, complement or macrophages, and recruitment of M.tb proteins to host kinases.

[0112] [Figure 17] A binder discovery platform for microbial and host targets.

[0113] [Figure 18] Selected inhibitors for covalent labeling of kinases with their residence times. The sites of linker and bioorthogonal group attachment are indicated by an arrow and a star (*), respectively.

[0114] [Figures 19A-19C](A) Representative example of a chimeric small molecule designed for proximity induced labeling of MAPK p38α based on its inhibitor SB203580 and the mechanism of covalent modification. (B) Exemplary inactivation of an inhibitor via click reaction with bulky cyclooctyne, the bulky group prevents the inhibitor from binding to the kinase. (C) Exemplary embodiment of a sorafenib-based chimeric small molecule designed for proximity induced labeling of MAPK p38α with a binder of PtpA.

[0115] [Figure 20] Modular components for an exemplary chimera: known binders to microbial targets (blue), and known binders to host targets (green). Applicants used these to optimize the assay. Applicants also used these building blocks to create pseudochimeras, consisting of known binders (shown here) and binders found that were identified from screening.

[0116] [Figure 21A-21B] (A) Improved synthesis of the benzolactam core (binder for PKC). (B) Schematic representation of the modular synthesis of exemplary PHICS molecules from acid / amine-containing binders and commercially available bifunctional linkers.

[0117] [Fig. 22A-22B] (A) Structures of the exemplary small molecule PHICS4 and the inactive analogue iPHICS4. (B) PHICS4-induced phosphorylation of cytoplasmic BRD4-HA by PKC-HA in HEK293T cells detected with a PKC motif antibody after immunoprecipitation (IP).

[0118] [Figure 23A-23B] (A) Structure of an exemplary small molecule PHICS5. (B) PHICS5-induced phosphorylation of endogenous BCR-ABL in K562 cells by endogenous PKC detected with phospho-c-ABL (Thr735) antibody.

[0119] [Fig. 24A-24B](A) Structures of an exemplary small molecule PHICS6 and an inactive analogue of PHICS6. (B) PHICS6-induced phosphorylation of BTK-S180A-flag by PKC-HA in HEK293T cells, probed with a PKC motif antibody after IP.

[0120] [Diagram 25] Structure of an exemplary small molecule, PHICS7.

[0121] [Figure 26] Design of the C1B-ABL-flag construct for PHICS4-mediated induction of proximity between tyrosine kinases and BRD4 allows PHICS4-induced phosphorylation of cytoplasmic BRD4-HA by C1B-ABL-flag in HEK293 cells, probed with a pan-phosphotyrosine antibody after IP.

[0122] [Figure 27A-27B] General mechanisms of (A) heterobifunctional PHICS and (B) homobifunctional PHICS.

[0123] [Fig. 28A-28B] (A) The phosphorylation cascade induced by autophosphorylation of BCR-ABL. (B) Mechanism of oligomerization and PHICS-induced cell death, hypothetical mechanism of inactivation. Known binding sites for molecules at the active (ATP) site, and allosteric sites are listed.

[0124] [Figures 29A-29E] (A) Structure of PHICS8 and the inactive analog iPHICS8. (B) PHICS8-induced ternary complex formation between BRD4 and ABL observed by AlphaScreen assay. (C) PHICS8-induced phosphorylation of BRD4 by ABL in vitro. ADPGlo assay for BRD4 phosphorylation by ABL in the presence of PHICS8 and iPHICS8. (E) Co-immunoprecipitation of ABL and BRD4 in the presence of PHICS8.

[0125] [Fig. 30A-30H] Medicinal chemistry optimization. Optimization of Abl binders: Structure (A) and corresponding EC50 values ​​in K563 (B). Linker optimization: Structure (C) and corresponding EC50 values ​​in BaF3 and K562 cells (D). (E) Best structure of bifunctional and corresponding monomer. Best compound VS1161 vs. imatinib in (F) KCL-22S cells, (G) K562 cells. (H) VS1161 is non-toxic in Ba / F3 and HEK293T cells.

[0126] [Fig. 31A-31H] Preliminary mechanistic studies of bifunctional and monomeric forms. (A) Competition of VS1150 bifunctional with VS1148 monomeric form at various ratios. (B,C) Phosphorylation of downstream targets (pSTAT5, pERK) is reduced by the same bifunctional but not monomeric forms. (D,E) Nanobit data showing dose-dependent complex formation of BCR-ABL in cells in the presence of bifunctional forms. (F) Known autophosphorylation sites (pY177, pY245, Y412) are reduced by bifunctional VS1161, whereas monomeric VS1171 remains the same. (G) The level of total phosphorylation of BCR-ABL remains the same in the presence of homo-PHICS, thus suggesting neophosphorylation, which remains to be confirmed. (H) Non-cancerous fusion of ABL with SH1-3 domains is inhibited by homo-PHICS but not by monomeric forms.

[0127] [Fig. 32A-32F] The best compound (VS1161) is shown in green for all experiments. (A-C) VS1161 works on active site mutations T315I, E225V and Y253H, but imatinib does not. (D) VS1161 works better than imatinib in TEL-ABL fusion cell lines. (E) Asciminib cannot compete with VS1161 in TEL-ABL fusion cell lines. (F) Reduced cell viability in the presence of VS1161 compared to known drugs asciminib, ponatinib and imatinib in PEER cells harboring NUP214-ABL.

[0128] [Fig. 33A-33B] Methods for detecting complex formation in cells: (A) SmBiT / LgBiT assay constructs to optimize the NanoBiT assay (see FIG. 31D), (B) in vivo XL mass spectrometry, (C) PRISM barcoding.

[0129] [Fig. 34A-34B] (A) Workflow for determining generalizability. (B-C) Examples of methods and compounds described herein.

[0130] [Fig. 35A-35B] (A) CRISPR Scanning: A protein of interest (BCR-ABL) is expressed in K562 cells and CRISPR is used to induce mutations in the POI and the efficacy of our homo-PHICS to bind and subsequently kill cells is assessed in the presence of these mutations to identify drug resistant (aka escape) mutants. (B) PRISM workflow for determining selectivity.

[0131] [Fig. 36A-36E] (A) Proposed fragments of VS1161 to optimize ABL dimerization, (B) fragment 1, (C) fragment 2, (D) fragment 3 exit vector, and (E) fragment 4 linker options.

[0132] [Figure 37] An example of the effect of bifunctional molecules that activate phosphorylation of FGFR.

[0133] [Fig. 38A-38C] (A) Exemplary structure for ABL PHICS targeting BRD4 and FGFR, (B) BRD4 phosphorylation and ternary complex formation, (C) gene expression levels downstream of FGFR induced by ABL recruitment.

[0134] [Fig. 39A-39F](A) Cell viability in BCR-ABL-dependent cell lines K562, KCL-22, Ba / F3, U2OS and HEK293 harboring p210 BCR-ABL. (B) Western blot analysis of 14 h treatment of KCL-22s cells with dimeric VS1161 (2 μM), monomeric VS1171 (4 μM), associated pY and downstream targets of BCR-ABL. (C) pSTAT5, (D) pCRKL, (E) pERK, (F) pAKT.

[0135] [Diagram 40] Cell viability data for VS1161 in the cell line NUP214-ABL.

[0136] [Fig. 41A-41D] Exemplary methods for detecting complex formation in cells: (A) SmBit / LgBit assay, (B) constructs for A, (C) XL mass spectrometry, (D) PRISM barcoding.

[0137] [Fig. 42A-42B] A and B: Proposed structure of ABL.

[0138] [Diagram 43] Dimers of ABL activators reduced BCR-ABL autophosphorylation, as well as phosphorylation of its downstream targets STAT5 and ERK, while monomers had no effect.

[0139] [Diagram 44] ABL activator dimers selectively killed K562 cells.

[0140] [Diagram 45] Optimization of chemicals: effect of enantiomeric purity.

[0141] [Figure 46] Correlation between dimer toxicity towards K562 and in vitro binding or activation of ABL kinase.

[0142] [Figure 47] Effect of linker type: Removal of the diamide further improved efficacy.

[0143] [Figure 48] Effect of linker length: The PEG2 linker is the most effective linker.

[0144] [Figure 49] Effect of linker length: PEG2 linkers are most effective when the C2 linker is too short for any effect.

[0145] [Figure 50] The combination of a chiral dihydropyrazole, a pyridine exit vector, and a PEG2 linker gives the most potent molecule.

[0146]

[0147] [Figure 51] Rescue experiments: Co-treatment with activators reduces the effect of the bifunctional molecules in a dose-dependent manner.

[0148]

[0149] [Figure 52] Asciminib-resistant mutants are also resistant to VS1161.

[0150] [Figure 53] The active site mutant is efficiently inhibited by VS1161.

[0151] [Figure 54] Dimers of other tyrosine kinase inhibitors (TKIs) are less effective than their monomers.

[0152] [Figure 55] VS1161 inhibits TEL-ABL fusion in BaF3 cells.

[0153] [Figure 56] Asciminib does not inhibit TEL-ABL fusion but rescues cells treated with VS1161.

[0154] [Figure 57] Exemplary ABL activator dimers are more potent than approved TKIs.

[0155] [Figure 58] An illustrative hook effect.

[0156] [Figure 59] Time dependence in phosphorylation levels.

[0157] [Figure 60] Exemplary linker attachment sites in Abl kinase activator molecules.

[0158] [Figure 61] Toxicology research.

[0159] [Figure 62] Additional general workflow for resistance evolution of homo-PHICS: A protein of interest (BCR-ABL) is expressed in K562 cells, CRISPR is used to induce mutations in the POI, and the efficacy of our homo-PHICS to bind and subsequently kill cells is evaluated in the presence of these mutations to identify drug-resistant (aka escape) mutants.

[0160] [Fig. 63A-63B] Exemplary embodiment in which phosphorylation of a transcription factor disrupts its protein-DNA (A) and protein-protein (B) binding.

[0161] [Figure 64]FIG. 64 depicts an exemplary phosphorylation-inducing chimeric small molecule (PHICS), which is formed by joining two small molecules - a kinase binder (triangle) and a target protein ligand (circle) - to increase the effective molar concentration of the target protein around the kinase, resulting in phosphorylation.

[0162] [Figure 65] Exemplary modular synthesis of PHICS molecules for kinase evaluation.

[0163] [Fig. 66A-66D] Exemplary binders of transcription factors targeted by PHICS.

[0164] [Figure 67] Viability study of Z138 cell line between the two bifunctional molecules and ibrutinib.

[0165] [Figure 68] A bifunctional molecule containing a BTK-binding agent, a variable linker, and a variable functional group.

[0166] [Figure 69] Z-138 cell viability studies of molecules from Figure 68.

[0167] [Figure 70] Z-138 cell viability studies of molecules from Figure 68 focusing on linker dependency.

[0168] [Figure 71] Z-138 cell viability study of molecules from Figure 68 focusing on variable linkers and PROTAC MT802.

[0169] [Fig. 72A-72D](A) Structure of PHICS5. (B-C) PHICS5-induced phosphorylation of endogenous BCR-ABL (B) and c-ABL (C) in K562 cells by endogenous PKC detected with phospho-c-ABL (Thr735) antibody. (D) Effect of PHICS5 on K562 cell viability by Cell-Titer Glo assay. VS1088-ABL kinase binder.

[0170] [Fig. 73A-73D] (A) Structure of PHICS6 and the inactive analog iPHICS6. (B) PHICS6-induced phosphorylation of BTK(S108A) by PKC in HEK293T. (C) Effect of Ser to Asp / Ala mutations on BTK autophosphorylation. (D) Effect of PHICS6 on the viability of the ibrutinib-resistant, BTK-dependent cell line Z-138.

[0171] [Fig. 74A-74C] (A) Design of the RTK-FKBP construct and predicted mechanism of RTK phosphorylation by ABL in the presence of PHICS molecules. (B) Structure of VS1043, a PHICS molecule designed to bind FKBP and ABL. (C) VS1043-induced phosphorylation of the HER2-FKBP construct by ABL detected by pY1221 HER2-specific antibody. VS772 and AD235-binders of ABL and FKBPF36V, respectively.

[0172] [Fig. 75A-75F] (A-F) Effect of VS1161 on the viability of BaF3 cells expressing K562 (A), KCL-22s (B), SUP-B15 (C), and various imatinib-resistant BCR-ABL mutants: T315I (D), E255V (E), and Y25 (F).

[0173] [Fig. 76A-76B] (A) Example of a BTK-BRD4 chimeric small molecule, (B) Western blot demonstrating induction of BTK phosphorylation by a BTK-BRD4 chimeric small molecule in pY1000-rabbit_HA-mouseBRD4.

[0174] [Fig. 77A-77B] (A) Example of an EGFR-BTK chimeric small molecule, (B) Western blot demonstrating induction of EGFR phosphorylation by BTK-EGFR chimeric small molecules in both pY1000-rabbit_FLAG-mouse EGFR and pY223-rabbit_FLAG-mouse BTK.

[0175] [Fig. 78A-78B] (A) Examples of EGFR-BTK chimeric small molecules, (B) Western blots demonstrating induction of EGFR phosphorylation by EGFR-BTK small molecules in probe_pY1000-rabbit_FLAG-mice, pY845-rabbit_FLAG-mice, and pERK-rabbit_ERK-mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0176] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0177] General definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th Edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (eds. FM Ausubel et al.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (eds. MJ MacPherson, BD Hames, and GR Taylor); Antibodies, A Laboratory Manual (1988) (eds. Harlow and Lane): Antibodies A Laboratory Manual, 2nd Edition 2013 (ed. EA Greenfield); Animal Cell Culture (1987) (ed. RI Freshney); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710);Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J.Wiley & Sons (New York, NY 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd ed. (2011).

[0178] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0179] The term "optionally" or "optionally" means that the subsequently described event, circumstance, or substituent may or may not be present, and that the description includes examples when the event or circumstance occurs as well as examples when it does not occur.

[0180] The limitations of numerical ranges by endpoints include all numbers and fractions subsumed within the individual ranges, as well as the recited endpoints.

[0181] The terms "about" and "approximately" as used herein when referring to measurements, e.g., parameters, amounts, time periods, and the like, are meant to encompass variations in and from the stated value, e.g., variations of + / - 10% or less, + / - 5% or less, + / - 1% or less, and + / - 0.1% or less, to the extent that such variations are appropriate for functioning in the disclosed invention. It should be understood that the values ​​to which the "about" or "approximately" modifier refers are themselves specifically, and preferably, disclosed.

[0182] As used herein, a "biological sample" may contain whole cells and / or live cells and / or cell debris. A biological sample may contain (or be derived from) a "body fluid". The invention encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax (ear wax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, and cell cultures derived from body fluids. Bodily fluids may be obtained from a mammalian organism, for example, by puncture or other collection or sampling procedures.

[0183] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0184] "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is defined according to the Cahn-Ingold-Prelog RS system when the compounds are enantiomers, and the stereochemistry of each chiral carbon can be defined as either R or S. Resolved compounds of unknown absolute configuration can be designated as (+) or (-) depending on the way they rotate plane polarized light at the wavelength of the sodium D line (dextrorotatory or levorotatory). Certain compounds described herein contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers and other stereoisomeric forms that can be defined in terms of the absolute stereochemistry at each asymmetric atom as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optionally in substantially pure form and intermediate mixtures. In some chemical structures, stereocenters may be identified with a "wavy" bond indicating that the stereocenter may be in the R or S configuration, unless otherwise specified. However, stereocenters without a wavy line (i.e., a "straight" bond) may also be in the (R) or (S) configuration, unless otherwise specified. Compositions that include compounds may contain stereocenters, each of which may independently be in the (R) configuration, the (S) configuration, or a racemic mixture.

[0185] Optically active (R)- and (S)-isomers can be prepared, for example, using chiral synthons or chiral reagents or resolved using conventional techniques. Enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high performance liquid chromatography (HPLC), the formation and crystallization of chiral salts, or can be prepared by asymmetric synthesis.

[0186] Optical isomers can be obtained by resolution of racemic mixtures according to conventional processes, for example, by formation of diastereoisomeric salts, by treatment with optically active acids or bases. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, and camphorsulfonic acid. Separation of the diastereoisomeric mixtures by crystallization followed by liberation of optically active bases from these salts provides separation of the isomers. Another method involves the synthesis of covalent diastereoisomeric molecules by reacting the disclosed compounds with optically pure acids or optically pure isocyanates in active form. The synthesized diastereoisomers can be separated by conventional means, for example, chromatography, distillation, crystallization, or sublimation, and then hydrolyzed to enantiomerically enriched compounds.

[0187] Optically active compounds can also be obtained by using active starting materials, hi some embodiments, these isomers can be in the form of a free acid, a free base, an ester or a salt.

[0188] In certain embodiments, the disclosed compounds may be tautomers. As used herein, the term "tautomer" refers to a type of isomer that includes two or more interconvertible compounds that result in the formal migration of at least one hydrogen atom and at least one change in valence (e.g., from a single bond to a double bond, a triple bond to a single bond, or vice versa). Tautomerization includes protonation or proton shift tautomerization, which is considered a subset of acid-base science. Protonation tautomerization or proton shift tautomerization involves the migration of a proton accompanied by a change in bond order. The abundance of tautomers depends on several factors, including temperature, solvent, and pH. When tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. Tautomerization (i.e., the reaction that provides the tautomer pair) can be catalyzed by acid or base, or can occur without the action or presence of an external agent. Exemplary tautomerizations include, but are not limited to, keto-enol, amide-imide, lactam-lactim, enamine-imine, and enamine-(different)enamine tautomerizations. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

[0189] All chiral, diastereomeric, racemic and geometric isomers of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare the compounds and intermediates carried out herein are encompassed by this disclosure. All tautomers of the compounds shown or described are also encompassed by this disclosure.

[0190] As used herein, out-of-ring bond substitutions, e.g. [ka] means that the substitution can be at any available position on the ring.

[0191] As used herein, a derivative of a compound is used interchangeably with a structural or chemical analogue. A derivative of a compound may contain modifications or changes to one or more functional groups, atoms, or substructures compared to the compound.

[0192] Various embodiments are described herein below. It should be noted that specific embodiments are not intended as exhaustive descriptions or limitations to the broader aspects discussed herein. An aspect described in conjunction with a particular embodiment does not necessarily limit that embodiment and can be practiced with any other embodiment. References throughout this specification to "one embodiment," "an embodiment," or "an example embodiment" mean that the particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the invention. Thus, the appearance of the phrases "in one embodiment," "in an embodiment," or "an example embodiment" in various places throughout this specification may, but do not necessarily, all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features but not other features included in other embodiments, meaning that combinations of features of different embodiments are within the scope of the invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0193] "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is defined according to the Cahn-Ingold-Prelog RS system when the compounds are enantiomers, and the stereochemistry of each chiral carbon can be defined as either R or S. Resolved compounds of unknown absolute configuration can be designated as (+) or (-) depending on the way they rotate plane polarized light at the wavelength of the sodium D line (dextrorotatory or levorotatory). Certain compounds described herein contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers and other stereoisomeric forms that can be defined in terms of the absolute stereochemistry at each asymmetric atom as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optionally in substantially pure form and intermediate mixtures. In some chemical structures, stereocenters may be identified with a "wavy" bond indicating that the stereocenter may be in the R or S configuration, unless otherwise specified. However, stereocenters without a wavy bond (i.e., a "straight" bond) may also be in the (R) or (S) configuration, unless otherwise specified. Compositions that include compounds may contain stereocenters, each of which may independently be in the (R) configuration, the (S) configuration, or a racemic mixture.

[0194] Optically active (R)- and (S)-isomers can be prepared, for example, using chiral synthons or chiral reagents or resolved using conventional techniques. Enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high performance liquid chromatography (HPLC), the formation and crystallization of chiral salts, or can be prepared by asymmetric synthesis.

[0195] Optical isomers can be obtained by resolution of racemic mixtures according to conventional processes, for example, by formation of diastereoisomeric salts, by treatment with optically active acids or bases. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, and camphorsulfonic acid. Separation of the diastereoisomeric mixtures by crystallization followed by liberation of optically active bases from these salts provides separation of the isomers. Another method involves the synthesis of covalent diastereoisomeric molecules by reacting the disclosed compounds with optically pure acids or optically pure isocyanates in active form. The synthesized diastereoisomers can be separated by conventional means, for example, chromatography, distillation, crystallization, or sublimation, and then hydrolyzed to enantiomerically enriched compounds.

[0196] Optically active compounds can also be obtained by using active starting materials, hi some embodiments, these isomers can be in the form of a free acid, a free base, an ester or a salt.

[0197] In one embodiment, the disclosed compounds may be tautomers. As used herein, the term "tautomer" refers to a type of isomer that includes two or more interconvertible compounds that undergo a formal shift of at least one hydrogen atom and at least one change in valence (e.g., from a single bond to a double bond, from a triple bond to a single bond, or vice versa). Tautomerization includes protonation or proton shift tautomerization, which is considered a subset of acid-base science. Protonation tautomerization or proton shift tautomerization involves the shift of a proton accompanied by a change in bond order. The ratio of tautomers present depends on several factors, including temperature, solvent, and pH. When tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. Tautomerization (i.e., the reaction that provides a tautomer pair) can be catalyzed by acid or base, or can occur without the action or presence of an external agent. Exemplary tautomerizations include, but are not limited to, keto-enol, amide-imide, lactam-lactim, enamine-imine, and enamine-(different)enamine tautomerizations. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

[0198] All chiral, diastereomeric, racemic and geometric isomers of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare the compounds and intermediates carried out herein are encompassed by this disclosure. All tautomers of the compounds shown or described are also encompassed by this disclosure.

[0199] As used herein, out-of-ring bond substitutions, e.g. [ka] means that the substitution can be at any available position on the ring.

[0200] Alkyl generally refers to a linear or branched aliphatic group. The alkyl group may be unsubstituted or substituted with halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, cycloalkyl, aryl, aryloxy, heteroaryl or heteroaryloxy groups, among others. The linear or branched carbon chain of an alkenyl has one or more double bonds. Alkynyl includes linear or branched carbon chains with at least one triple bond. Alkenyl and alkynyl groups can each have one or more double bonds or triple bonds, or a combination of double and triple bonds. Alkenyl and alkynyl groups can be unsubstituted or substituted with functional groups as described herein.

[0201] As used herein, a hydrocarbon substituent means any group consisting exclusively of hydrogen and carbon atoms, including alkyl, alkylene, alkyne, and saturated and unsaturated rings and fused rings.

[0202] As used herein, nitrogen-based substituent refers to any group containing one or more nitrogens. Non-limiting examples of nitrogen-based substituents may include aminyl, quaternary ammonium cation, amidyl, iminyl, imidyl, azidyl, azo group, cyano, nitrate, nitrile group, nitrite group, nitryl, nitrosyl, oxime, carbamoyl.

[0203] As used herein, sulfur-based substituent means any group containing one or more sulfurs. Non-limiting examples of sulfur-based substituents may include H or R sulfanyl, disulfanyl, sulfinyl, sulfino group, sulfo group, alkosulfonyl, thiocyanato group, isothiocyanato group, thioyl, sulfanylidene, methanethioyl, mercaptocarbonyl, hydroxy(thiocarbonyl), thioester group, thionoester group, dithiocarboxy group, dithiocarboxylate group, dithiocarbamate group.

[0204] As used herein, oxygen-based substituent refers to any group that contains one or more oxygens. Non-limiting examples of oxygen-based substituents may include hydroxyl, carbonyl, formyl, haloformyl, (alkoxycarbonyl)oxy, carboxyl, carboxylate, carboalkoxyl, hydroperoxyl, peroxyl, alkoxyl, dialkoxyl, trialkoxyl, methylenedioxyl, tetraalkoxyl, and carboxylic anhydride groups.

[0205] As used herein, boron-based substituent refers to any group that contains one or more boron atoms. Non-limiting examples of boron-based substituents may include boronyl, borono group, O-[bis(alkoxy)alkylboronyl], hydroxyborino group, O-[alkoxydialkylboronyl].

[0206] As used herein, a halogen-based substituent means any group that contains one or more halogens.

[0207] As used herein, heterocycle means any molecule that forms adjacent covalent bonds and contains an element that is not hydrogen or carbon. Non-limiting examples of heterocycles include oxetane, thietane, azetidine, β-lactam, oxirane, thiirane, aziridine, azirine, diaziridine, diazirine, epoxide, tetrahydrofuran, furan, thiolane, thiophene, pyrrolidine, pyrrole, 3-pyrroline, 2-H-pyrrole, benzofuran, coumaran, isobenzofuran, benzothiophene, dibenzothiophene, indoline, indole, indolinine, oxindole, indoxyl, isatin, isoindole, isoisopropyl, iso ... indole, indolizine, pyrrolidine, carbazole, dioxolane, dithiolane, oxazolidine, oxazolidinone, oxazole, isoxazole, thiazole, isothiazole, imidazolidine, 2-imidazoline, imidazole, pyrazolidine, 2-pyrazoline, pyrazole, benzodioxole, benzoxazole, indoxazine, benzothiazole, benzimidazole, 1H-indazole, purine, azaindole, 1,2,3-oxadiazo , 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, benzotriazole, quinuclidine, diazabicyclooctane, diazabicycloundecane, 4H-pyran, tetrahydropyran, dihydropyran, 2H-pyran, piperidine, pyridine, picoline, lutidine, collidine, pyridone, acridine, chromene, coumarin, isocoumarin, xanthene, tetrahydroquinoline, quinoline, isoquinoline, quinolone, These may include 4H-quinolizine, quinolizinium, 1,4-dioxane, morpholine, paraformaldehyde, 1,4-dithiane, 1,3-dithiane, thiomorpholine, trithiane, piperazine, pyrazine, pyrimidine, pyridazine, 1,3,5-triazine, tetrazine, cinnoline, phthalazine, 1,8-naphthyridine, quinoxaline, quinazoline, and combinations thereof including fused or covalently linked linkages, optionally further substituted with any of the previously listed substituents.

[0208] The additional substituents may include any combination of the above substituents.

[0209] Throughout this description, the molecule [ka] However, further optimization of the binding locations of the molecules can be performed, including by methods of screening and computational approaches detailed herein, and are further explored in the examples of this application. [ka] The bond locations specified on the molecule as depicted by are not intended to be limiting, but are merely exemplary, and it is implicitly recognized that further optimization and bond site locations can be specified using the methods and guidance described herein, including any position on the rings within the structure and any other substituent on the molecule.

[0210] Carbocycle or cycloalkyl means a monocyclic or bicyclic carbocyclic functional group and includes both substituted and unsubstituted cycloalkyl groups. Cycloalkyl groups can optionally contain double bonds and are intended to encompass cycloalkenyl groups. Unless otherwise indicated, (C 3 ~C 8 ) References to cycloalkyl refer to cycloalkyl groups containing from 3 to 8 carbons and are intended to include monocyclic cycloalkyl groups containing from 3 to 8 carbons and bicyclic cycloalkyl groups containing from 6 to 8 carbons.

[0211] Heterocycloalkyl generally refers to a ring functional group having carbon atoms and one or more heteroatoms independently selected from S, N, or. Heterocycloalkyl is intended to include one or more double bonds that may be between two carbons or between a carbon and a heteroatom. For example, an exemplary 5-membered heterocycloalkyl can have one carbon-carbon double bond or one carbon-nitrogen bond in the ring, such as dihydropyrazole, pyrrolinyl.

[0212] As used herein, aryl refers to aromatic hydrocarbon groups, including cyclic and polycyclic, e.g., bicyclic, tricyclic, aromatic ring moieties. Exemplary aryl groups include phenyl and naphthyl. Phenyl may be unsubstituted or substituted at one or more positions with substituents, including but not limited to those listed above for alkyl groups.

[0213] Heteroaryl groups as used herein refer to aromatic moieties having carbon atoms and one or more selected from O, S or N, including cyclic and polycyclic, e.g., bicyclic, tricyclic, moieties.

[0214] Reference is made to International Patent Application No. PCT / US21 / 12816, filed January 8, 2021, which is specifically incorporated by reference in its entirety into this specification.

[0215] All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0216] Summary Small molecules are classically developed to inhibit enzyme activity. The embodiments disclosed herein define a new class of bifunctional small molecules that confer new functions to enzymes through proximity-mediated effects. In general, bifunctional molecules include an effector-binding moiety connected to a substrate-binding moiety via a linker. Bifunctional molecules can be used to improve the kinetics of native enzyme modification by bringing substrate molecules into proximity with the enzyme, including using chimeric small molecule configurations that may be energetically or otherwise more favorable. In addition, chimeric small molecules may be used to retarget enzymes to modify non-native or neo-substrates.

[0217] In one aspect, the embodiments disclosed herein provide targeted chimeric molecules, also referred to herein as chimeric small molecules, that can covalently label a target enzyme with a targeted binding agent, e.g., a target protein binding agent. Reprogramming native enzymes to recognize new substrates then expands the range of therapeutic targets that can be exploited in the treatment of disease, including making previously undruggable targets accessible. Targeted chimeras disclosed herein generally include an enzyme binding agent and a targeted binding agent connected via one or more linkers. The molecule may include an exit vector at one or both ends of the linker, with the linker at one end connected to a first moiety via an exit vector and the linker at the opposite end connected to a second moiety via an exit vector.

[0218] In another aspect, the chimeric small molecule can be configured to facilitate the covalent labeling of an enzyme with a target binding moiety. This labeling of an enzyme with a target binding agent can be used to define new substrates that are not normally targeted or modified by such enzymes. In such an embodiment, the chimeric molecule comprises an enzyme binding moiety that further comprises an electrophilic reactive group linked to the target binding moiety. The enzyme binding agent non-covalently binds to the enzyme of interest, resulting in an electrophilic reactive group that "tags" the enzyme by covalently linking the target binding agent to a nucleophile located on the enzyme via a proximity-driven reaction. In such an embodiment, the enzyme binding moiety is then released from the labeled enzyme, either by the inherent kinetics of the molecule or by application of a quencher. The target binding agent can then home to the labeled enzyme and bind and modify new target substrates. This approach also expands the number of enzyme binding agents that can be used. For example, although there are some high-quality enzyme inhibitors that exist, such inhibitors are not suitable for use in targeting chimeras that remain bound to the enzyme and thus may normally inhibit the enzymatic activity of the bound enzyme. As discussed in more detail below, the selection of appropriate inhibitors and optimal use of quenching molecules allows these inhibitors to be used in the labeling process described below without affecting downstream modification reactions.The embodiments disclosed herein advantageously provide for labeling of enzymes at one or more locations to nucleophiles located on the enzyme by targeted binding agents.

[0219] Exemplary embodiments provide targeting chimeras having a target binding moiety and an enzyme binding moiety that are configured to bind to the same enzyme. The binding moieties may be chemically identical or chemically different from each other, but are configured to target multimeric enzymes. In some aspects, the molecules are designed to target abnormal protein dimerization. Since abnormal dimerization can create constitutively active or inactive dimers that are shown in disorders, in exemplary embodiments, the compositions can be utilized to bind and lock multimeric enzymes in an inactive state, or to generate multimeric enzymes in an active state.

[0220] Exemplary embodiments provide targeted chimeras designed for oncogenic targets. In one embodiment, the method provides for the modification, e.g., neophosphorylation, of oncogenic targets. Methods of use can include eliciting an immune response, creating autoantigens, and inactivating targets. In one exemplary embodiment, hyperphosphorylation or neophosphorylation of a target protein can result in immune recruitment to the target, e.g., via triggering presentation of neoepitopes and T cell attack on cells presenting the epitope. Modification of kinases and key regulator proteins involved in cancer is also within the scope of the methods disclosed herein.

[0221] In certain exemplary embodiments, a method of modifying a target substrate in a cell is provided. For example, when the enzyme binding agent is a kinase and the target is a substrate located in a cell, the labeled kinase can bind to the substrate. The bound kinase can then neophosphorylate the target substrate, thereby modifying it.

[0222] In certain exemplary embodiments, a method of recruiting the host's immune system against cancer is provided. For example, if the enzyme binder is a kinase and the target protein is an oncogenic protein, the labeled kinase can bind to the cancer through the oncogenic protein. The bound kinase can then neophosphorylate the target protein, thereby signaling the host's immune system to attack the cancer.

[0223] In certain exemplary embodiments, a method is provided for recruiting the host's immune system against a pathogen. For example, if the enzyme-binding agent is a kinase and the target protein is located on the surface of a pathogenic bacterium, the labeled kinase can bind to the bacterium through the target protein. The bound kinase can then neophosphorylate the target protein, thereby signaling the host's immune system to attack the bacterium.

[0224] Also provided is a method for modifying a protein of interest comprising contacting the protein of interest with a compound disclosed herein in an environment comprising one or more kinases. A method for treating a disease, disorder or condition in a subject in need thereof can comprise administering to the subject a therapeutically effective amount of a molecule disclosed herein.

[0225] Chimeric small molecules In one embodiment, the chimeric small molecule has the general formula A-(L) n -B wherein A is an enzyme binding moiety, B is a target binding moiety, and L is a linker, n being 0-6. It is described in.

[0226] Exemplary linkers may be selected from alkanes, alkenes, alkynes, amines, ethers, thiols, sulfones, carbonyls, acyl, ketones, carboxylic acid esters; amides, enones, anhydrides, imides, PEG, heterocycles, or any combination thereof, and are discussed in more detail below.

[0227] In one example embodiment, A and B may both bind to the same type of enzyme. In one example embodiment, A and B may be the same molecule or binding moiety, or A and B may be different molecules that bind to the same enzyme. Small molecules that contain binding moieties that bind to the same type of enzyme may be particularly useful when the enzyme oligomerizes and the molecule acts as a type of molecular glue, allowing the oligomer to lock into a particular conformation that is desired to inhibit or activate a particular activity of the oligomer.

[0228] In one embodiment, the chimeric small molecule has the formula [ka] where W is an exit vector. W can be independently selected from amine, O, S, NH, a bond, alkane, alkene, alkyne, amine, ether, thiol, sulfone, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, O, S, NH, or any combination thereof, and A and B are linked to their respective Ws through any functional group or ring position of A and B.

[0229] The embodiments of Formula I, II-A, and II-B are useful when the enzyme-binding moiety does not inactivate or inhibit the enzyme upon binding. Formula I, II-A, and II-B are further useful when it is desired to bring an enzyme bound to the enzyme-binding moiety and a target substrate bound to the target-binding moiety into close proximity via the chimeric small molecule. This may be desirable with certain enzyme-activator binding moieties, particularly for additional reasons related to the chimeric small molecule and / or reaction kinetics.

[0230] In another exemplary embodiment, the chimeric small molecule has the general formula AL-El-B where A is an enzyme binding moiety, L is a linker, E is an electrophilic reactive group, and B is a target binding moiety; or AL 1 -EL 2 -B where A is an enzyme binding moiety, L1 and L2 are first and second linkers, E is an electrophilic reactive group, and B is a target binding moiety. These formula embodiments, while not necessarily limiting, may be useful in situations where the enzyme binding moiety would otherwise inhibit or interact with the ability of an enzyme bound to the enzyme binding moiety to modify a target substrate bound to the target binding moiety.

[0231] The enzyme binding moiety binds to an enzyme and may be specific for one or more enzymes. The electrophilic reactive group may be designed to react with a moiety on the enzyme to which the enzyme binding agent binds. In certain embodiments, the enzyme binding agent may further comprise a bioorthogonal group. The enzyme binding agent, as further detailed herein, may in one embodiment be selected to have a half-life that is shorter than the half-life of the target bound to the target binding agent.

[0232] The electrophilic reactive group of the chimeric small molecule may be designed to react with a moiety on the enzyme, for example, on an amino acid of the enzyme. The electrophilic reactive group can advantageously be designed to react with a moiety on the enzyme that is in close proximity to the binding site of the enzyme binder. Reaction of the electrophilic reactive group with a moiety on the enzyme, for example, a nucleophilic group located on the enzyme, can allow for labeling or binding of the enzyme with a targeted binder. Binding of such a targeted binder to the enzyme can create an initialized enzyme that can modify the target substrate.

[0233] The targeted binding agent may be specific for one or more targets of interest. In one embodiment, the target of interest is a macromolecule, such as a protein. The targeted binding agent binds to the target of interest (target substrate), thereby bringing the enzyme into proximity with the target of interest. In some embodiments, the target of interest may advantageously be a non-cognate substrate of the enzyme. The target of interest may be a pathogenic or oncogenic target.

[0234] In some embodiments, the moiety specified herein as enzyme binding moiety can be used as target binding moiety.For example, enzyme is a desirable target because overexpression of enzyme can lead to disease.Therefore, small chimeric molecules can be designed such that the target binding moiety is any enzyme binding moiety specified herein that targets enzyme.For example, overexpression of EGFR can lead to tumor growth, and therefore the EGFR enzyme binding moiety specified herein can be used as target binding moiety.

[0235] Similarly, target binding moiety can be used as enzyme binding moiety.For example, FKBP can be used to phosphorylate substrate.Thus, FKBP target binding moiety can be used as enzyme binding moiety to make FKBP enzyme phosphorylate substrate.

[0236] The use of an enzyme binding moiety for a target binding moiety, or vice versa, may be due to increased or decreased half-life or binding kinetics. For example, in order to successfully modify a target substrate, the target binding moiety must remain bound to the enzyme binding moiety long enough to modify it. In some embodiments, an enzyme binding moiety may be used for a target binding moiety because of the desired binding kinetics or longer binding half-life. In some embodiments, a target binding moiety may be used as an enzyme binding moiety because the target binding moiety has decreased binding kinetics or decreased half-life. Selecting a target binding moiety with decreased binding kinetics or decreased half-life for the enzyme binding moiety may be advantageous, for example, when the target binding moiety is an inhibitor and a longer binding time leads to decreased target substrate modification.

[0237] Enzyme binding part The enzyme moiety can be selected based on the target substrate and the modification to the substrate that is desired. The enzyme binding moiety may be a small molecule that binds to an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. Examples of oxidoreductases include dehydrogenases and oxidases. Examples of transferases include transaminases, kinases, and methyltransferases. Examples of hydrolases may include lipases, amylases, peptidases, and phosphatases. Examples of lyases may include decarboxylases. Examples of isomerases may include isomerases and mutases. Examples of ligases may include synthetases. Examples of translocases may include transporters.

[0238] The enzyme-binding moiety may be selected based on high enzyme abundance in the target cell, binder molecules with activity at lower concentrations, e.g., nanomolar activity, available crystal structures and low residence times, ability to accommodate bioorthogonal groups without affecting binding potency and residence time, e.g., small bioorthogonal handles, high density of amino acids with nucleophilic side chains, e.g., serine / threonine / tyrosine / lysine, close to the binding pocket, and / or whether labeling of the kinase interferes with its enzyme activity, which may be based on experimental data and / or modeling. The length of the linker may be adjusted to allow for modifications, e.g., phosphorylation, with increased distance from the binding pocket, and modifications targeted to locations further away from the binding pocket, e.g., amino acid residues. In certain exemplary embodiments, the enzyme-binding moiety is an allosteric modulator. Considerations in selecting the enzyme-binding moiety may include allosteric signaling, which may include a network of non-covalently interacting protein residues, conformational preferences, and changes associated with induced fit in both spatial and temporal aspects. In one embodiment, the enzyme binding moiety may be an allosteric activator or inhibitor of the enzyme. The allosteric activator or inhibitor may be computationally discovered. In one method, a high-quality drug target is obtained. Then, prediction of the allosteric site is performed using methods such as perturbation response scanning (PRS) combined with all-atom molecular dynamics (MD) and dynamic residue network (DRN). The allosteric modulator is then identified using methods such as homology modeling, docking or essential dynamics. A description of this process can be found in Figures 2 and 3 of Amamuddy S. et al. "Integrated Computational Approaches and Tools for Allosteric Drug Discovery," IJMS 2020, 21(3), pp. 847, which is incorporated herein by reference.

[0239] The enzyme binding moiety may be selected based on the type of desired modification, for example, post-translational modification of the target substrate. In one embodiment, the enzyme binding moiety can bind to an enzyme that phosphorylates the target, and therefore the type of enzyme can be selected for this desired modification of the target substrate. A post-translational modification (PTM) is one type of modification that is performed. This may include cleavage of peptide bonds, formation of disulfide bonds, acylation, prenylation, lipoylation, acetylation, deacetylation, formylation, alkylation, carbonylation, phosphorylation, dephosphorylation, glycosylation, lipidation, hydroxylation, S-nitrosylation, S-sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, or malonylation (Taherzadeh et al., 2018). Thus, post-translational modification enzymes are one set of enzyme binding agents envisioned for use in the present invention.

[0240] In one embodiment, the enzyme binding agent provides modifications to amino acids, see, for example, Table 1 in Karve et al., Journal of Amino Acids Vol. 2011, Article ID 207691, p. 13, DOI: 10.4061 / 2011 / 207691, incorporated herein by reference. Karve et al. summarize some post-translational modifications and their importance in various diseases, as well as normal occurrence. Karve et al. evaluate potential functional modifications, including acetylation, carbonylation, glycosylation and glycation, hydroxylation, methylation, nitration, palmitoylation, phosphorylation, sulfation, and ubiquitination, for each amino acid, which are specifically incorporated for the modifications detailed therein.

[0241] Reaction of an electrophilic reactive group with a moiety on an enzyme, such as a nucleophilic reactive group located on an enzyme, can allow for labeling or binding of the enzyme with a targeted binding agent. Binding of such a targeted binding agent to an enzyme can create an initialized enzyme that can modify a target substrate. Thus, in certain exemplary embodiments, an enzyme binding moiety is bound to the enzyme and is selected based on the availability of a binding pocket and amino acid side chains proximate to the binding pocket that can be used in reaction with the electrophilic reactive group of the targeting chimera.

[0242] Half-life In one embodiment, when the chimeric molecule is used to label the surface of an enzyme with a target binding moiety, the enzyme binding moiety may be selected in part based on its half-life. In one embodiment, the enzyme binding moiety may be selected in part based on its half-life compared to the half-life of the target substrate. In some embodiments, the half-life of the enzyme binding moiety is 2, 3, 4 or 5 times shorter than the half-life of the target substrate. Without being bound by theory, designing a targeting chimera with a half-life of the enzyme binding agent shorter than the half-life of the target substrate may allow for desirable reaction kinetics in the case of an enzyme being labeled with a target binding agent via an electrophilic reactive group, followed by enzymatic modification of the target substrate when the target binding agent binds to the target substrate. The half-life of the enzyme binding agent and the target substrate generally relates to the time required for the concentration of the enzyme binding agent or the target substrate to decrease to half of its initial concentration. In one embodiment, the half-life may measure, for example, the time it takes for half of the molecules initially measured in a sample, which may include a cell, multiple cells, a tissue, an organ, or a mammal, to degrade. In one embodiment, the half-lives of the target substrate and the enzyme-binding agent are measured under the same or similar conditions, e.g., in the same cell type, tissue or organ, hi one embodiment, the half-life measurements can be measured in the same sample or system having the particular phenotype, genotype, disease or condition being studied, treated and / or evaluated.

[0243] The half-life of an enzyme-binding agent can be measured, for example, using the dissociation time t, which describes the average time required to liberate half of the initially occupied receptors under conditions in which the association or rebinding of the enzyme-binding moiety occurs. 1 / 2 or receptor occupancy 1 / 2Dissociation requiring a conformational change in the receptor or binding pocket size may be a factor in residence time and can be considered when selecting an enzyme-binding moiety. See, e.g., Roskoski R Jr. Classification of small molecule protein kinase inhibitors based upon the structures of their drug-enzyme complexes. Pharmacol Res. 2016;103:26-48. doi:10.1016 / j.phrs.2015.10.021.

[0244] The time that a compound resides on its target, e.g., residence time, may be used. See Willemsen-Seegers N, Uitdehaag JCM, Prinsen MBW et al. Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance. J Mol Biol. 2017;429(4):574-586. doi:10.1016 / j.jmb.2016.12.019 for a discussion and identification of residence time and kinetic parameters of exemplary kinase binders, which is incorporated herein in its entirety, in particular Tables 1, 3A-3B, 4A-4C, S3 and S4 for teachings on tyrosine kinase inhibitors, EGFR inhibitors, ponatinib for various kinases, certain kinases and their related inhibitors, Aurora A and B kinase inhibitors, and P13k lipid kinase inhibitors. Elimination half-life may also be utilized alone or in conjunction with residence time assessment. Additional pharmacodynamics and pharmacokinetics may also be considered in the assessment of half-life for enzyme binders. Half-life may be modeled. See, for example, Callegari D, Lodola A, Pala D et al. Metadynamics Simulations Distinguish Short- and Long-Residence-Time Inhibitors of Cyclin-Dependent Kinase 8 [published revision found in J Chem Inf Model. 2017 Feb. 27;57(2):386]. J Chem Inf Model. 2017;57(2):159-169. doi:10.1021 / acs.jcim.6b00679, incorporated herein by reference.

[0245] The measurement of the half-life of the target substrate depends on the type of target substrate being measured. In an example embodiment, when the target is a protein, an approach to measure half-life, such as mass spectrometry-based proteomics, such as SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture)-based proteomics, may be used, see, for example, Matheison et al., Nature Communications Vol. 9, Paper No.: 689 (2018). High-throughput proteomics may be used to estimate the half-life of a protein in a particular tissue and / or cell, or even a predictive model may be used to predict the half-life of such a protein in a tissue from the properties of the cell, see, for example, Rahman M, Sadygov RG (2017) Predicting the protein half-life in tissue from its cellular properties. PLoS ONE 12(7): e0180428. https: / / doi.org / 10.1371 / journal.pone.0180428.

[0246] By way of example, further embodiments are discussed in the context of kinase binding moieties, which may be used to guide the selection of other allosteric binders for other classes of enzymes.

[0247] Kinase-binding moiety In one embodiment, the enzyme binding moiety is a kinase binding moiety. Kinases belong to the family of phosphotransferases and phosphorylate substrates by transferring the gamma phosphate of ATP at the hydroxyl group of the substrate. The substrate may include lipids, sugars, or amino acids. The kinase binding moiety may be any molecule capable of binding to a kinase. Some kinase binding molecules are known to activate kinases upon binding, while others are known to inhibit kinases upon binding. In one embodiment, the kinase binding moiety is a kinase activator. In one embodiment, the kinase binding moiety is a kinase inhibitor. However, since the design of the targeting chimera creates a kinase labeled with a targeted binding agent, the binding of the kinase is the primary objective, rather than the inhibitory or activating behavior of the kinase binding agent, the kinase binding moiety utilized for initial binding to the kinase allows for the generation of a repurposed enzyme labeled with a targeted binding agent, rather than the use of the kinase binding agent for kinase activation or inhibition.

[0248] In some embodiments, the enzyme binder is a kinase activator moiety. The kinase activator moiety can be a small molecule or compound that activates the kinase. As used herein, a kinase is an enzyme that adds a phosphate group to an amino acid of another molecule, typically a protein substrate. An activator of a kinase enhances phosphorylation. In one embodiment, the kinase activator moiety promotes the active conformation of the enzyme, in one aspect, through a binding interaction with a regulatory subunit. See, e.g., Zorn et al., Nat. Chem. Biol. (2010), doi:10.1038 / NCHEMBIO.318. The kinase can act on the amino acids serine, threonine, tyrosine, or combinations thereof.

[0249] The activator moieties can be identified from activators known in the art. Activators may be derivatives of activators known in the art and may contain fewer or additional functional groups that still permit their use as activators, but may enhance or facilitate the desired formation, conformation or binding site for the multifunctional molecules described herein. Exemplary modifications may include derivatives for increased solubility, charge, functionality for use with orientation adaptors or linkers, as detailed elsewhere herein.

[0250] In one embodiment, the enzyme-binding moiety is a kinase inhibitor. Kinase inhibitors (KIs) are generally designed to bind to the highly conserved Asp-Phe-Gly (DFG) motif of kinases. KIs can be classified by the conformation of the DFG-binding site. Type I inhibitors bind to the active DFG-Asp-in conformation, while type II inhibitors bind to the inactive DFG-Asp-out conformation. Further considerations for kinase inhibitors include mimicking the hydrogen-bonding interactions normally formed by the adenosine ring of ATP, or the mechanism of inhibition, e.g., competing with ATP binding, which may involve reversible or irreversible covalent binding. See, e.g., (Gross et al. J Clin Invest. 2015;125(5):1780-1789).

[0251] A consideration in kinase inhibitor design has been the degree of specificity for a particular kinase. A hypothesized advantage has been for higher specificity, whereas kinase inhibitors with a low degree of specificity for a particular kinase promote the recruitment of many types of kinases. Promiscuous kinase inhibitors are advantageous because the kinase is a vehicle for modification of the target substrate.

[0252] In one embodiment, the enzyme-binding moiety is a promiscuous kinase inhibitor (PKI). Promiscuous kinase inhibitors refer to molecules that bind to two or more kinases. Promiscuous kinase inhibitors are molecules that have binding specificity to a binding pocket that is highly conserved across kinases. Promiscuous kinase inhibitors may bind to two, three, four, five or more different kinases. In one embodiment, the promiscuous kinase inhibitor is an ATP-competitive kinase inhibitor. In one embodiment, the PKI targets one or more kinases selected from PDGFRA, PDGFRB, KIT, CSF1R, DDR1, DDR2, MEK5 and YSK4. See, e.g., Seeliger, MA et al., "What Makes a Kinase Promiscuous for Inhibitors?" Cell Chem. Biol., 26(3), 2019; pp. 390-399. For example, the kinase inhibitor imatinib can inhibit c-KIT, PDGFR-alpha, and BCR-ABL kinases (see, e.g., Iqbal N, Iqbal N. Imatinib: a breakthrough of targeted therapy in cancer. Chemother Res Pract. 2014;2014:357027. doi: 10.1155 / 2014 / 357027. Epub 19 May 2014), as well as sunitinib, sorafenib, and cabozantinib, which are known for their promiscuous activity, and are provided as non-limiting examples of promiscuous kinase inhibitors. In one embodiment, the PKI is modified to contain a bioorthogonal group.

[0253] In one embodiment, the enzyme-binding moiety is a kinase-binding moiety. Examples of kinases that may bind to the chimeric small molecules of the invention include, but are not limited to, PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, NOP, μ (mu) opioid receptor, δ (delta) opioid receptor, UMPK, SphK, or GSK-3.

[0254] ABL joint part In one embodiment, the enzyme-binding moiety is an ABL kinase-binding moiety. Abelson kinase (ABL) is a ubiquitously expressed non-receptor tyrosine kinase that plays a key role in cell differentiation and survival. Simpson et al., J. Med. Chem. 2019 62, 2154-2171 (Simpson et al., 2019). ABL tyrosine kinase can be found in the nucleus, cytoplasm and mitochondria. ABL protein is normally under well-organized regulation. However, chromosomal translocations joining the ABL gene with genes encoding other proteins result in various fusion proteins that tend to dimerize (or oligomerize) and autophosphorylate. Hence, ABL kinase becomes constitutively active, resulting in myeloproliferative disorders. In one embodiment, one of the ABL kinase-binding moieties detailed herein is used together with the target-binding moiety described herein in a chimeric small molecule.

[0255] In one embodiment, the ABL kinase binding moiety is an ABL kinase activator. In one embodiment, the c-Abl kinase activator is (5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,4-imidazolidinedione or 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin, as described in Yang et al., "Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site," Chem. & Biol., 18, pp. 177-186, Feb. 25, 2011; DOI: 10.1016 / j.chembiol.2010.12.013): [ka] (DPH).

[0256] In one embodiment, the c-Abl kinase activator is a kinase inhibitor that has been shown to activate c-Abl in vivo in Simpson et al., 2019. [ka] The novel aminopyrazoline small molecule activators described in Table 6 of Simpson et al., 2019, are specifically incorporated herein by reference.

[0257] In one embodiment, the c-Abl kinase activator moiety is [ka] It is.

[0258] In one embodiment, the ABL kinase activator is [ka] where the dashed circle identifies a bond for orienting an adaptor / exit vector and / or linker. The functional groups represented by the dashed circle on the ABL kinase activator can be utilized in methods to attach linkers and orient the adaptor prior to attachment to the protein binding moiety. The ABL kinase binding moiety can be further modified, for example, at any moiety around the ring of the moiety, as detailed below.

[0259] In one embodiment, the c-Abl binding moiety has the formula [ka] (Wherein, R is [ka] (It is described in the above).

[0260] In one embodiment, the DPH is, e.g. [ka] It is functionalized with.

[0261] The binding moiety, e.g., activator moiety, may be functionalized for a method of attachment that directs the adaptor and / or linker. The discussion herein is of an exemplary ABL kinase binding moiety, but can be applied to other binding moieties in a similar manner. The parent molecule of the ABL kinase activator, DPH, can be functionalized for a method of attachment that directs the adaptor and linker. In one example embodiment, the binding moiety is [ka] Exemplary functionalized molecules of the binding moiety can be [ka] It is.

[0262] Once functionalized, the directing adaptor, the linker, or both, can be added, either sequentially or at once, with the directing adaptor and linker added as one molecule. Exemplary molecules are provided below, where the R group represents the target binding moiety. [ka] In some cases, more than one activator moiety can be attached to the protein binding moiety. In each instance, the activator moieties can be attached to a linker and functionalized as described herein for methods to orient the adaptor prior to attachment to the protein binding moiety, e.g., utilizing functional groups represented by dashed circles.

[0263] In certain embodiments, the Abl kinase activator is a DHP or dihydropyrazole activator. Exemplary molecules include [ka] wherein X is (CH2)n, which may be substituted with, for example, one or more of amide, acetal, aminal, amine, alkyl, ether, hydrocarbyl and derivatives thereof, or other groups described elsewhere herein. In one embodiment, n is 0-20, more preferably n is 1-10 or 2-7, and R is [ka] In one embodiment, the ABL kinase activator is linked to the dihydropyrazole via various types of linkers, see, for example, (PHICS 10.1-10.5, Figure 64A of PCT / US2021 / 012816).

[0264] In one embodiment, one of the ABL kinase binding moieties detailed herein is used in a chimeric small molecule together with a BRD4 binding moiety described herein. In one embodiment, when the target binding moiety is for BRD4, the exemplary molecule is [ka] teeth [ka] may include.

[0265] In one embodiment, the enzyme binding moiety has the formula [ka] (wherein R1 to R5 are independently H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more condensed rings thereof, or an aliphatic halogen compound, for example, -OCF 2 Cl; Z is independently selected from B, C, N, O, S, preferably one or two atoms of Z are N, O, S, or a combination thereof; Ra, Rb, and Rc are independently selected from alkanes, alkenes, alkynes, ethers, alcohols, amines, nitriles, nitro, thiols, sulfones, sulfonates, halogens, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, acid anhydrides, imides, aliphatic halogen compounds, such as -OCF 2 Re is selected from one or more fused rings including Cl, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, any combination of any of the above-listed rings; Re is an alkane, alkene, alkyne, ether, alcohol, amine, nitrile, nitro, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, acid anhydride, imide, aliphatic halogen compounds, e.g., OCF 2 Cl, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof at one or more positions, or R 1 Or R 5 can form a ring with or any combination thereof.

[0266] In one embodiment, R a , R b , R c One or more of [ka] and an amide further attached to a molecule selected from the group consisting of:

[0267] In one embodiment, the ABL binding moiety is according to formula II(b), where Re is [ka] wherein Rf and Rg are selected at one or more positions from the group consisting of: alkanes, alkenes, alkynes, ethers, alcohols, amines, nitriles, nitros, thiols, sulfones, sulfonates, halogens, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, anhydrides, imides, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, cyclic hydrocarbons optionally substituted with one or more fused rings comprising any combination of any of the previously listed rings, unsaturated cyclic hydrocarbons, heterocycles, one or more fused rings comprising any combination of any of the previously listed rings. In one embodiment, Rf and Rg are independently selected from the group consisting of: [ka] is selected from the group consisting of:

[0268] In one embodiment, the kinase binding moiety [ka] In the formula, R is H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more condensed rings thereof, an aliphatic halogen compound, e.g., -OCF 2 Cl, or any combination thereof; [ka] may be selected from is selected from the group consisting of:

[0269] In one embodiment, the enzyme-binding moiety is an ABL kinase inhibitor. In one embodiment, the ABL inhibitor has the formula: [ka] Imatinib has the following structure:

[0270] In one exemplary embodiment, the ABL inhibitor is nilotinib, dasatinib, bosutinib, ponatinib, or any derivative thereof. In an exemplary embodiment, the kinase binding molecule is [ka] Selected from.

[0271] In one embodiment, the ABL kinase binding molecule [ka] is selected from the group consisting of:

[0272] In one embodiment, the ABL kinase binding molecule [ka] is selected from the group consisting of:

[0273] In one embodiment, the ABL kinase binding molecule [ka] is selected from the group consisting of:

[0274] In one example embodiment, the ABL kinase binding moiety has the formula: [ka] and asciminib, also known as ABL-001, described in US Pat. [Table 1] Asciminib is a negative allosteric modulator of BCR-ABL1, which induces the kinase to adopt a form of autoinhibition, thereby inactivating the conformation. A PROTAC based on asciminib has been granted fast track designation. In the UK, asciminib is available for compassionate use for named organ dysfunction and has been shown to have minimal effects on platelet function. Asciminib has been shown to inhibit GI markers in wild-type ABL1 cell lines. 50 1.5nM and ABL1 T315I It has inhibitory activity against cell proliferation in vitro at 35 nM for cell lines. Asciminib has a pIC of 8.6-9.5 against the non-receptor tyrosine kinase ABL proto-oncogene 1. 50 See Schoepfer, J. et al., "Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1," J. Med. Chem. 2018, 61(18), which is incorporated by reference in its entirety. In some embodiments, the compound is selected from the group consisting of Schoepfer et al. (2018) [ka] [Table 2] or a derivative thereof.

[0275] In one example embodiment, the ABL kinase binding moiety has the formula: [ka] BO1 is a non-ATP-competitive negative allosteric modulator of mutant BCR-ABL kinase protein. BO1 interaction with wild-type protein shows an ATP-competitive / mixed mechanism of action. BO1 has a pK of 7.0-7.4 for the non-receptor tyrosine kinase ABL proto-oncogene 1. i In addition, 1,3,4 thiadiazole derivatives as Abl tyrosine kinase inhibitors can be used, for example, Radi, M. et al., "Discovery and SAR of 1,3,4-Thiadiazole Derivatives as Potent Abl Tyrosine Kinase Inhibitors and Cytodifferentiating Agents," Bioorganic & Medicinal Chemistry Letters 2008, 18(3), pp. 1207-1211, which is incorporated herein by reference in its entirety, in particular their cSrc and Abl inhibitory activity and the following formula: [ka] See compounds 6a-6u in Table 1 of Radi et al., reproduced below, which have the following structure: [Table 3]

[0276] In one embodiment, the ABL kinase binding moiety has the formula: [ka] GNF-2 is a highly selective, non-ATP-competitive inhibitor of Bcr-Abl. It acts as a negative allosteric modulator that binds to a site distant from the ATP pocket. GNF-2 has an IC of 267 nM. 50 The present invention inhibits Bcr / Abl fusion proteins at low levels. See, for example, Zhang, J. et al., "Targeting Bcr-Abl by Combining Allosteric with ATP-Binding-Site Inhibitors," Nature 2010, 463(7280), 501-506, which is incorporated herein by reference in its entirety.

[0277] In one embodiment, the ABL kinase binding moiety has the formula: [ka] GNF-5 is a selective and allosteric BCR-ABL inhibitor. GNF-5 can largely overcome resistance patterns associated with imatinib or nilotinib treatment (except for the gatekeeper mutation T315I). Co-treatment with GNF-2 (the original structural embodiment of GNF-5) and imatinib significantly reduces the emergence of resistant clones in vitro. GNF-5 down-regulates BCR-ABL kinase activity by mimicking the effect of myristate binding, which directs the protein towards adopting an inactive conformational state. GNF-5 has a pIC of 6.7 for the non-receptor tyrosine kinase ABL proto-oncogene 1. 50 See, for example, Deng, X. et al., "Expanding the Diversity of Allosteric Bcr-Abl Inhibitors," J. Med. Chem. 2010, 53(19), pp. 6934-6946, and Zhang Nature 2010, which are incorporated by reference in their entireties. In some embodiments, SAR can be performed around the GNF-2 scaffold with functionalities modified at specific positions. [ka] The crystal structure of GNF-2 bound to the Abl myristoyl pocket can also be used for further optimization, see for example Figure 2 in Zhang, Nature, 2010 463, 501-506, which is incorporated herein by reference. A co-crystal structure of the complex imatinib and GNF-2 with c-Abl is also available (PDB ID: 3K5V). Additional targeting moieties can be designed as described in Figure 3 in Zhang Nature (Nature, 2010 463, 501-506), which is incorporated by reference and presented below. [ka]

[0278] In one example embodiment, the ABL kinase binding moiety has the formula: [ka] The DPH is listed in ICW EC 6.1. 50 See, e.g., Simpson, GL et al., “Identification and Optimization of Novel Small C-Abl Kinase Activators Using Fragment and HTS Methodologies,” J. Med. Chem. 2019, 62(4), 2154-2171, which is incorporated by reference in its entirety, and which includes the following: [ka] and those described in DPH and compounds identified in Simpson et al.: [Table 4] In another example embodiment, the ABL targeted binding agent is any c-ABL kinase activator from Simpson, GL et al., Identification and Optimization of Novel Small c-Abl Kinase Activators Using Fragment and HTS Methodologies. J Med Chem, 2019. 62(4):2154-2171.

[0279] In one example embodiment, the ABL kinase binding moiety has the formula: [ka] It is a dihydropyrazole according to the above.

[0280] In one embodiment, the ABL kinase binding moiety is [ka] is selected from the group consisting of:

[0281] In one embodiment, the ABL kinase binding moiety is [ka] It is.

[0282] R in the formula of the ABL inhibitor is optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. In an exemplary embodiment, R is selected from any boron-based, carbon-based, nitrogen-based, oxygen-based, sulfur-based, halogen-based substituent, heterocycle, fused ring, or any combination thereof. In an exemplary embodiment, R is H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, such as -OCF 2 In an example embodiment, R is selected from [ka] is selected from.

[0283] In one embodiment, the kinase binding molecule of the ABL inhibitor is [ka] is selected from.

[0284] In another exemplary embodiment, the kinase binding molecule of the ABL inhibitor has the formula [ka] is selected from.

[0285] In one embodiment, X and R 2 is optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. In one embodiment, X is any viable boron-, carbon-, nitrogen-, oxygen- or sulfur-based element or compound. In one embodiment, X is selected from C, N, O and S. In one embodiment, R 2is selected from any boron-based, carbon-based, nitrogen-based, oxygen-based, sulfur-based, halogen-based substituent, heterocyclic ring, fused ring, or any combination thereof. 2 is H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more condensed rings thereof, aliphatic halogen compounds, e.g., -OCF 2 In an example embodiment, R 2 teeth [ka] is selected from.

[0286] In one embodiment, the ABL kinase binding molecule is [ka] is selected from.

[0287] In one embodiment, the X, Y and R groups are optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. In one embodiment, X and Y are independently selected from any boron-based, carbon-based, nitrogen-based, oxygen-based, sulfur-based, halogen-based substituent, heterocycle, fused ring, or any combination thereof. In one embodiment, X is a halogen. In one embodiment, Y is selected from C, N, O and S. In one embodiment, R 1 , R 2 and R 3 is independently selected from any boron-based, carbon-based, nitrogen-based, oxygen-based, sulfur-based, halogen-based substituent, heterocyclic ring, fused ring, or any combination thereof. 1 , R 2 , and R 3are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof.

[0288] In another exemplary embodiment, the kinase binding molecule of the ABL inhibitor is [ka] is selected from.

[0289] In one embodiment, the Y and R groups are optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. 1 is any viable boron-, carbon-, nitrogen-, oxygen-, or sulfur-based element or compound. In an example embodiment, Y and Y 1 is selected from C, N, O and S. In one embodiment, R 3、 R 4 , R 6 and R 7 is independently selected from any boron-based, carbon-based, nitrogen-based, oxygen-based, sulfur-based, halogen-based substituent, heterocyclic ring, fused ring, or any combination thereof. 3、 R 4 , R 6 and R 7 are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2Cl, or any combination thereof.

[0290] In another exemplary embodiment, the ABL kinase inhibitor binding molecule is [ka] is selected from.

[0291] In one embodiment, Y 1 The Y and R groups are optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. 1 is any viable boron-, carbon-, nitrogen-, oxygen-, or sulfur-based element or compound. In an example embodiment, Y 1 is selected from C, N, O and S. In one embodiment, R 4 , R 6 and R 7 is independently selected from any boron-based, carbon-based, nitrogen-based, oxygen-based, sulfur-based, halogen-based substituent, heterocyclic ring, fused ring, or any combination thereof. 4 , R 6 and R 7 are independently H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof.

[0292] In one embodiment, the target binding moiety is an ABL inhibitor. In one embodiment, the ABL inhibitor is DCC-2036, which has the formula: [ka] It is a dual anchoring inhibitor that binds to both the E282 / R386 pair of the switch control pocket and the Met318 ATP hinge, with an IC50 value of 0.8 nM as described in

[0293] In one embodiment, the kinase binding moiety is a c-ABL tyrosine kinase inhibitor or any derivative thereof from International Patent Application No. WO2019173761, which is incorporated herein by reference.

[0294] AMPK binding part In one embodiment, the enzyme binding moiety is an AMPK kinase binding moiety. AMPK is a serine / threonine kinase that assembles into a heterotrimeric complex composed of a catalytic α-subunit and two regulatory β and γ subunits. See, for example, Wells et al. (2012). It is believed that small molecules that mimic AMP binding to the γ subunit can directly activate AMPK.

[0295] In one embodiment, the AMPK kinase binding moiety has the formula: [ka] (Wherein, R is [ka] , a carbohydrate mimetic, a heterocycle, a dihydrohexitol, a pyranose or a furanose; Q is selected from the group consisting of B, C, N, O, and S; where H is N A Or N B Located on either X 1 and X 2 is independently selected from the group consisting of C, N, and O; Y is selected from the group consisting of H, OH, halogen, CN, or a hydrogen bond donor substituent; Z is H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2 Cl, which is optionally further substituted). It is described in.

[0296] In one embodiment, Z has the formula: Z a -Z b (In the formula, Z a teeth [ka] is selected from the group consisting of Z b teeth [ka] is selected from the group consisting of n is 0 to 6. The present invention can be as described in

[0297] In one embodiment, the AMPK binding moiety [ka] The AMPK binding moiety is selected from the group consisting of:

[0298] Additional AMPK kinase binding moieties that can be used in accordance with the present invention include other AMPK activators, including A769662 (Cool et al., Cell Metab. 3, 403-416 (2006)) and PT1 (Pang et al., J. Biol. Chem. 283, 16051-16060 (2008)), and derivatives thereof, as further modified in accordance with the teachings detailed herein for use and optimization in the bifunctional molecules of the present invention.

[0299] The AMPK binding moiety can be, for example, as described in U.S. Patent Publication No. 20050038068, which is incorporated herein by reference; [ka] or derivatives thereof, as further modified in accordance with the teachings detailed herein for use and optimization in the bifunctional molecules of the present invention.

[0300] In one embodiment, the kinase binding moiety is an AMPK activator. [ka] Other AMPK activators include A769662, which has a pEC 50 see, e.g., Cool, B. et al., "Identification and Characterization of a Small Molecule AMPK Activator That Treats Key Components of Type 2 Diabetes and the Metabolic Syndrome," Cell Metabolism 2006, 3(6), 403-416, which is incorporated herein by reference in its entirety.

[0301] The AMPK activator can be, for example, as described in U.S. Patent Publication No. 20050038068, which is incorporated herein by reference; [ka] The AMPK activator can be as described in International Patent Publication Nos. WO2007019914, WO2009124636, WO2009135580, WO2008006432, or WO2009152909, which are incorporated herein by reference. In one embodiment, the activator is [ka] The present invention can be as described in

[0302] The AMPK activator may be as described in International Patent Publication No. WO2009100130, which is incorporated herein by reference. In one embodiment, the AMPK activator is [ka] It is described in.

[0303] The AMPK activator can be as described in International Patent Publication Nos. WO2010036613, WO2010047982, WO2010051176, WO2010051206, WO2011106273, or WO2012116145. In one embodiment, the AMPK activator is [ka] It is described in.

[0304] In an exemplary embodiment, the AMPK activator may be as described in International Patent Publication Nos. WO2011029855, WO2011138307, WO2012119979, and WO2012119978, which are incorporated herein by reference. [ka] You can choose from.

[0305] In an example embodiment, the AMPK activator can be as described in International Patent Publication Nos. WO2011032320, WO2011033099, WO2011069298, WO2011070039, WO2011128251, WO2012001020, which are incorporated by reference herein. [ka] You can choose from.

[0306] In one embodiment, the AMPK activator can be as described in International Patent Publication No. WO2011080277, which is incorporated herein by reference. In one aspect, the AMPK activator is [ka] It could be.

[0307] In one embodiment, the AMPK activator can be as described in International Patent Publication No. WO2012033149, which is incorporated herein by reference. In one aspect, the AMPK activator is [ka] You can choose from.

[0308] In one embodiment, the AMPK activator is MT47-100 and has the formula: [ka] MT47-100 modulates the activity of AMPK, but not the direction of modulation, depending on the subunit composition of the enzyme. MT47-100 acts as a direct activator of β1 subunit-containing AMPK and as an allosteric inhibitor of β2 subunit-containing AMPK. pK as an activator i The value is 5.4, but the pK iThe value is 4.6 as an allosteric inhibitor. See, for example, Scott, JW et al., "Inhibition of AMP-Activated Protein Kinase at the Allosteric Drug-Binding Site Promotes Islet Insulin Release," Chemistry & Biology 2015, 22(6), pp. 705-711, the entire contents of which are incorporated herein by reference.

[0309] Additional AMPK binding moieties for use in the present invention are disclosed in International Patent Publication Nos. WO2007019914, WO2009124636, WO2009135580, WO2008006432, WO2009152909, WO2011029855, WO2011138307, WO2012119979, WO2012119978, WO20110 32320, WO2011033099, WO2011069298, WO2011070039, WO2011128251, WO2012001020, WO2011080277, WO2012033149, and derivatives thereof, and may be further modified in accordance with the teachings detailed herein for use and optimization in the bifunctional molecules of the present invention.

[0310] PKC binding part In one embodiment, the enzyme-binding moiety is a PKC kinase-binding moiety. In one embodiment, the enzyme-binding moiety is a PKC activator or inhibitor. Protein kinase C (PKC) is composed of multiple isozymes, plays a role in signal transduction pathways, exhibits tissue-specific expression, and plays a variety of biological roles. In one embodiment, the PKC kinase-binding moiety can be utilized in small chimeric molecules disclosed herein that are selective for PKC isoforms, such as traditional (cPKCs-α, βI, βII, γ), novel (nPKCs-δ, ε, η, θ), atypical (aPKCs-ζ, ι / λ), and PKCμ (a form between the novel and atypical isoforms). In one embodiment, the PKC binding moiety has the formula [ka] or a PKC binding portion of an analog thereof.

[0311] Additional PKC binding moieties that can be configured for use in the molecules described herein are found, for example, in

[0179] to

[0194] of International Patent Application No. PCT / US21 / 12816, which is specifically incorporated herein.

[0312] In one embodiment, the enzyme-binding molecule can be designed as an activator of diacylglycerol (DAG) responsive to C1 domain-containing proteins, such as protein kinase C. Protein kinase C (PKC) is composed of multiple isozymes, plays a role in signal transduction pathways, exhibits tissue-specific expression, and plays a variety of biological roles. Activators of PKC can be utilized in the small chimeric molecules disclosed herein, where the activating moiety is selective for PKC isoforms.

[0313] In one embodiment, the kinase binding moiety is a DAG activator. Activators of DAG-responsive proteins may include DAG-indolactones as described in LC Garcia et al., Bioorg. Med. Chem., 22 (2014) 3123-3140. Exemplary DAG-indolactones have the formula: [ka] wherein R is indole. R can be, for example, 1-methyl, 1H-indol-5-yl, 1-methyl, 1H-indol-6-yl, 1-methyl, 1H-indol-4-yl, or 1-methyl, 1H-indol-7-yl. In one embodiment, the compound is selective for PKCα or PKCε.

[0314] The DAG lactone, e.g., AJH-836, is as described in Cooke et al., J. Biol. Chem. (2018) 293(22) pp. 8330-8341. In one embodiment, the DAG lactone has the formula [ka] As provided in Cooke, the formula for AJH-836 is: [ka] and is selective for PKCδ and PKC.

[0315] Teleocidins, such as (-)-indolactam-V (ILV), and benzolactam-V8, such as 7-substituted benzolactam-V8, can be utilized as PKC activators. The PKC activator can be as described in Ma et al., Org. Lett. 4:14 (2002) DOI:10.1021 / ol026125l.

[0316] In one embodiment, the PKC activator has the formula [ka] (Wherein, R1, R3, and R4 are each independently an alkyl, alkenyl, or alkynyl; R2 is a saturated or unsaturated alkylene (e.g., branched alkylene, linear alkylene, cycloalkylene, C 1 ~C 22 Branched alkylene, C 1 ~C 22 Linear alkylene, C 3 ~C 22 Cycloalkylene, C 1 ~C 10 Branched alkylene, C 1 ~C 10 Linear alkylene, C 3 -C 10 Cycloalkylene, C 1 -C 8 Branched alkylene, C 1 ~C 8 Linear alkylene, C 3 ~C 8 Cycloalkylene), C 1 ~C 22 Saturated or unsaturated heteroalkylene (e.g., branched heteroalkylene, linear heteroalkylene, heterocycloalkylene, C 1 ~C 22 Branched Heteroalkylene, C 1 -C 22 Linear heteroalkylene, C 3 ~C 22 Heterocycloalkylene, C 1 ~C 10 Branched Heteroalkylene, C 1 ~C 10 Linear heteroalkylene, C 3 ~C 10 Heterocycloalkylene, C 1 ~C 8 Branched Heteroalkylene, C 1 ~C 8 Linear heteroalkylene, C 3 ~C 8 heterocycloalkylene), arylene (e.g. C 5 ~C 22 arylene), heteroarylene (e.g., C 5 ~C22 R2 is -(C(R a )(R a )) 1~8 -, -(OC(R a )(R a )) 1~8 -, -(OC(R a )(R a )-C(R a )(R a )) 1~8 -, -N(R a )-, -O-, -C(O)-, optionally substituted C 6 Arylene, optionally substituted C 5~12 Heteroarylene, Hydroxy-Substituted C 3~ 6 Cycloalkylene or hydroxy-substituted C 4 heterocycloalkylene, each of the foregoing may have one or more (e.g., 2, 3, 4, 5) points of substitution; R a is independently, at each occurrence, hydrogen or alkyl (e.g., C 1 ~C 7 Alkyl, C 1 ~C 3 alkyl).

[0317] In one embodiment, the formula is [ka] (Wherein, R1, R3, and R4 are each independently an alkyl, alkenyl, or alkynyl; R2 is a saturated or unsaturated alkylene (e.g., branched alkylene, linear alkylene, cycloalkylene, C 1 ~C 22 Branched alkylene, C1 ~C 22 Linear alkylene, C 3 ~C 22 Cycloalkylene, C 1 ~C 10 Branched alkylene, C 1 ~C 10 Linear alkylene, C 3 -C 10 Cycloalkylene, C 1 -C 8 Branched alkylene, C 1 ~C 8 Linear alkylene, C 3 ~C 8 Cycloalkylene), C 1 ~C 22 Saturated or unsaturated heteroalkylene (e.g., branched heteroalkylene, linear heteroalkylene, heterocycloalkylene, C 1 ~C 22 Branched Heteroalkylene, C 1 -C 22 Linear heteroalkylene, C 3 ~C 22 Heterocycloalkylene, C 1 ~C 10 Branched Heteroalkylene, C 1 ~C 10 Linear heteroalkylene, C 3 ~C 10 Heterocycloalkylene, C 1 ~C 8 Branched Heteroalkylene, C 1 ~C 8 Linear heteroalkylene, C 3 ~C 8 heterocycloalkylene), arylene (e.g. C 5 ~C 22 arylene), heteroarylene (e.g., C 5 ~C 22heteroarylene), or a divalent hydrocarbon selected from combinations thereof (wherein each of the foregoing may have one or more (e.g., 2, 3, 4, 5) points of substitution, which may be selected from substituted amides including those selected from those set forth in Table 1 of Kozikowski et al. J. Med. Chem, 2003, 46:3, pp. 364-373, Table 1 on page 366, which is specifically incorporated herein by reference).

[0318] R2 is -(C(R a )(R a )) 1~8 -, -(OC(R a )(R a )) 1~8 -, -(OC(R a )(R a )-C(R a )(R a )) 1~8 -, -N(R a )-, -O-, -C(O)-, optionally substituted C 6 Arylene, optionally substituted C 5~12 Heteroarylene, Hydroxy-Substituted C 3~ 6 Cycloalkylene or hydroxy-substituted C 4 heterocycloalkylene, each of the foregoing may have one or more (e.g., 2, 3, 4, 5) points of substitution; R a is independently, at each occurrence, hydrogen or alkyl (e.g., C 1 ~C 7 Alkyl, C 1 ~C 3 alkyl).

[0319] In one embodiment, the formula is [ka] (wherein R1, R3, and R4 are independently alkyl, alkenyl, alkynyl, and R2 may be selected from the following: In one embodiment, the PKC activator is a benzolactam analog of ILV, as described in Kozikowski et al., J. Med. Chem., 1997, 40:9 1316-1326, where R is CC(CH 2 ) 7 CH 3 or (CH 2 ) 9 CH 3 It could be.

[0320] In one embodiment, R1, R3 and R4 are alkyl, and in some embodiments, R1, R3 and R4 are methyl. In one embodiment, the formula is [ka] It is described in.

[0321] In one embodiment, the PKC activator is a natural product activator, e.g., DPP, prostratin, mezerein, octahydromezerein, thimeleatoxin, (-)-octylindolactam V, OAG, or resiniferatoxin, as described in Kazanietz et al., Mol. Pharma. 44:296-307 (1993).

[0322] In one embodiment, the PKC binding moiety has the formula [ka] or a PKC binding portion of an analog thereof.

[0323] In one embodiment, the PKC activator is selective for PKC delta.In one embodiment, the PKC activator is 7α-acetoxy-6β-benzoyloxy-12-Obenzoylroylanone (Roy-Bz), as described in Bessa et al., Cell Death and Disease (2018) 9:23.

[0324] The PKC activator may be an ILV derivative, such as n-hexyl ILV or 10-membered 1-hexyl indolakulam-V10, or a derivative thereof, as described in Yanagita et al. J. Med. Chem., 2008, 51:1, pp. 46-56, which is incorporated herein by reference. ... [ka] (wherein R1 and R2=H, R1=H and R2=Cl, or R1=Br and R2=H), and in some instances may be PKCδ, PKCε, or PKCη.

[0325] In one embodiment, the activator moiety is 6-chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid (PF-06409577), benzolactam, DPP, prostratin, mezerein, octahydromezerein, thimeleatoxin, (-)-indolactam V, (-)-octylindolactam V, OAG, or a derivative thereof.

[0326] In one embodiment, the activator moiety is thieno[2,3-b]pyridine, thienopyridone, quinoxalinedione, imidazo[4,5-b]pyridine, [2,3-d]pyridine, benzimidazole, pyrrolo[2,3-d]pyrimidine, spirocyclic indolinone, tetrahydroquinoline, thieno[2,3-b]pyridinedione, and derivatives thereof. See Expert Opin Ther. Patents (2012) 22(12), which is incorporated herein by reference.

[0327] In other example embodiments, the PKC activator may be selected from Table 1 of PCT / US2021 / 012816, which is incorporated by reference herein.

[0328] FKBP binding part In one embodiment, the enzyme-binding moiety is an FKBP kinase-binding moiety. In one embodiment, the enzyme-binding moiety can be designed as an activator or inhibitor of FK506-binding protein (FKBP). FKBPs belong to the immunophilin family. FKBPs are present in all eukaryotic organisms ranging from yeast to humans and are expressed in most tissues. Mammalian FKBPs can be subdivided into four groups: cytoplasmic, endoplasmic reticulum, nuclear, and TPR (tetratricopeptide repeat)-containing FKBPs. In one embodiment, the FKBP is FKBP12, which binds to intracellular calcium release channels and TGF-β type I receptors. In one embodiment, the FKBP activator moiety has the formula: [ka] and any derivatives thereof. See, for example, (Kolos et al. FKBP Ligands-Where We Are and Where to Go? Front. (2018) FKBP Ligands-Where We Are and Where to Go? Front. Pharmacol. 9:1425).

[0329] IRTK binding part

[0330] In one embodiment, the enzyme-binding moiety is an IRTK kinase-binding moiety. The insulin receptor (IR) is a heterotetrameric protein consisting of two extracellular α subunits and two transmembrane β subunits. Binding of a ligand to the α subunit of IR induces a conformational change in the receptor. As a result, the tyrosine kinase activity intrinsic to the β subunit of IR is stimulated (Salituro GM et al. Discovery of a small molecule insulin receptor activator. Recent Prog Horm Res. 2001;56:107-26). In one embodiment, the enzyme-binding moiety is an IR activator or inhibitor. In one embodiment, the activator for IRTK is kojic acid or a derivative thereof.

[0331] In one embodiment, the target is the androgen receptor. In one embodiment, the localization moiety may include enzalutamide. In one embodiment, enzalutamide is attached via an ether bond to a linker that includes an azide terminus. Thus, in one embodiment, the addition of an alkyne functionality in the activator moiety allows for connection via bioorthogonal click chemistry. In one embodiment, the insulin receptor is represented by the formula: [ka] wherein C, N, O, S or P. In another exemplary embodiment, the IRTK activator is of the formula: [ka] and any derivatives thereof.

[0332] In one embodiment, the IRTK activator is XMetA, also known as XOMA-159, which is a monoclonal antibody and an allosteric partial agonist of the insulin receptor. See, e.g., Bedinger DH et al., "Differential Pathway Coupling of the Activated Insulin Receptor Drives Signaling Selectivity by XMetA, an Allosteric Partial Agonist Antibody," J Pharmacol Exp Ther 2015, 353(1), pp. 35-43.

[0333] Lyn bond part In one embodiment, the enzyme-binding moiety is a Lyn kinase-binding moiety. Lyn kinase belongs to the Src family of kinases and is the predominant Src kinase in B cells. The regulatory properties of Lyn play a role in immune system function. See, for example, Xu Y. "Lyn Tyrosine Kinase," Immunity 2005, 22 (1), 9-18. In one embodiment, the Lyn-binding moiety is an activator or inhibitor. In one embodiment, a Lyn activator has the formula: [ka] Trimidone, also known as MLR-1023, described in , is a selective allosteric activator of Lyn kinase and was developed for the treatment of type 2 diabetes. Experiments with knockout mice revealed that in the absence of Lyn kinase, trimidone did not lower glucose. Currently, trimidone is in phase 2 studies in patients with uncontrolled type 2 diabetes. Trimidone has a pEC of 7.2. 50 See, e.g., Saporito, MS et al., "MLR-1023 Is a Potent and Selective Allosteric Activator of Lyn Kinase In Vitro That Improves Glucose Tolerance In Vivo," J Pharmacol Exp Ther 2012, 342(1), pp. 15-22, with the following comparisons in cellular and enzyme assays referenced below, which are incorporated herein by reference. [Table 5]

[0334] PK binding part In one embodiment, the enzyme binding moiety is a PK kinase binding moiety. Pyruvate kinase (PK) catalyzes the transphosphorylation of phosphoenolpyruvate (PEP) to ADP to generate ATP in glycolysis. PK is expressed in four different isoenzyme forms: L, R, M1 and M2 in mammalian tissues depending on metabolic requirements and their regulatory properties. The M2, L and R isoenzymes have homotropic coactivation with PEP and heterotropic coactivation with FBP. See, for example, Gupta V. et al., "Human Pyruvate Kinase M2: A Multifunctional Protein," Protein Science 2010, 19(11), pp. 2031-2044.

[0335] In one embodiment, the PK kinase binding moiety is a PK activator. In one embodiment, the PK activator has the following properties: [ka] and mitapivat, also known as AG-348, described in US Pat. [Table 6] Mitapivat is a small molecule allosteric activator of pyruvate kinase. It activates the PK isoforms found in red blood cells, the PKR protein expressed from the PKLR gene, and the embryonic PKM2 isoform expressed from the PKM gene. Mitapivat was developed as a novel therapy for disorders of the red blood cells associated with inherited PKR deficiency and for cancer therapy via activation of PKM2. Activation of PK in red cells increases hemoglobin levels. The active drug is the sulfate hydrate. Mitapivat has a pEC of >7.0 for PKM2. 50In one embodiment, the PK activator is any compound from U.S. Patent No. 8,785,450B2, which is incorporated herein by reference, or any derivative thereof. In one embodiment, the PK activator is any compound from International Patent Publication No. WO2013056153A1, which is incorporated herein by reference, or any derivative thereof.

[0336] In one embodiment, the kinase binding moiety is a PK inhibitor, see above for more information regarding PK kinases. In one embodiment, the PK inhibitor is any of those identified in U.S. Patent No. 6,534,501, which is incorporated herein by reference, or any derivative thereof.

[0337] NOP connection part The nociceptin opioid peptide (NOP) receptor is part of the opioid receptor family of GPCRs, which couples to Gi / Go and inhibits adenylate cyclase activity. In one embodiment, the enzyme binding moiety or target binding moiety binds to the GPCR opioid receptor. In one embodiment, the enzyme binding moiety is a NOP activator. In one embodiment, the NOP activator has the formula: [ka] or any derivative thereof.

[0338] In some embodiments, the NOP activator is the NOP agonist Ser100 according to the formula: Ac-RYYRWKKKKKKK-NH2. In some embodiments, the NOP activator is the NOP agonist N / OFQ according to the formula: FGGFTGARKSARKLANQ. In some embodiments, the NOP activator is JNJ-19385899, see, e.g., Zaveri, NT, "Nociceptin Opioid Receptor (NOP) as a Therapeutic Target: Progress in Translation from Preclinical Research to Clinical Utility," J. Med. Chem. 2016, 59(15), pp. 7011-7028, which is incorporated herein by reference in its entirety.

[0339] Several proteins, such as G protein-coupled receptor kinases, β-arrestins and G proteins, specifically regulate NOP receptor function. It has also been shown that sodium and guanyl nucleotides can modify functional NOP complexes and G protein interactions. Other G protein-coupled receptors, such as the mu-opioid receptor, appear to be able to form heterodimers with the NOP receptor, potentially modifying the receptor protein; see, for example, Wang, H.-L. et al., "Heterodimerization of Opioid Receptor-like 1 and μ-Opioid Receptors Impairs the Potency of μ Receptor Agonist," Journal of Neurochemistry 2005, 92(6), pp. 1285-1294.

[0340] In certain embodiments, the binding agent is an allosteric modulator of a delta opioid receptor. [ka] BMS-986187, 3,3,6,6-tetramethyl-9-[4-[(2-methylphenyl)methoxy]phenyl]-4,5,7,9-tetrahydro-2H-xanthene-1,8-dione.

[0341] In certain embodiments, the binding agent is an allosteric modulator of the mu opioid receptor. [ka] These are BMS-986121 [(4-{2-[(2,6-dichlorophenyl)amino]-1,3-thiazol-4-yl}phenyl)(hydroxy)imino]-λ 1 -Oxidaniyl; BMS-986122 2-(3-bromo-4-methoxyphenyl)-3-(4-chlorophenyl)sulfonyl-1,3-thiazolidine; BMS-986123 [Hydroxy({2-methoxy-5-[3-(4-methylbenzenesulfonyl)-1,3-thiazolidine-2-yl]phenyl})imino]-λ 1 -oxidanyl; BMS-986124 2-(4-bromo-2-methoxyphenyl)-3-(4-chlorobenzenesulfonyl)-1,3-thiazolidine; or BMS-986187 3,3,6,6-tetramethyl-9-[4-[(2-methylphenyl)methoxy]phenyl]-4,5,7,9-tetrahydro-2H-xanthene-1,8-dione.

[0342] In one embodiment, the NOP binding agent is a NOP antagonist. In one embodiment, the NOP antagonist has the formula: [ka] or any of the following, see Zaveri J. Med. Chem. 2016.

[0343] MAPK binding part In one embodiment, the enzyme-binding moiety is a mitogen-activated protein kinase (MAPK)-binding moiety. In one embodiment, the MAPK-binding moiety is an inhibitor or activator. MAPKs are involved in signal transduction pathways. A common feature of MAPKs is their ability to phosphorylate the transactivation domains of transcription factors, thereby modulating transcriptional activity. In one embodiment, the kinase-binding moiety is a MAPK inhibitor.

[0344] In certain embodiments, the MAPK inhibitor is [ka] p38α MAPK inhibitors and their derivatives, including, which can be utilized as an activating moiety in the chimeric small molecules of the present invention. Inhibitor B96 may also be known as doramapimod, which is an allosteric inhibitor. Doramapimod exhibits moderate selectivity for p38 alpha, beta, and gamma isozymes compared to p38 delta. It exhibits moderate selectivity for p38 alpha, beta, and gamma isozymes compared to p38 delta. A Kd value of 0.1 nM has been reported, and in a screening panel of kinases, doramapimod inhibited many kinases with IC50 values ​​of <100 nM. Doramapimod has been shown to block TNFα release in LPS-stimulated THP-1 cells with an IC50 value of 18 nM. Dramapimod inhibits MAPK14 with a pKd of 9.4 and a pIC50 of 7.7, MAPK11 with a pIC50 of 8.1, MAPK12 with a pIC50 of 7.5, and MAPK13 with a pIC50 of 6.5, see Moffett et al., Bioorg. Med. Chem. Lett. 2011, 21, 7155-7165. Further areas for modification when tailoring molecules for use in chimeric small molecules are described in Moffett, which is incorporated by reference, particularly Figure 3 and the associated teachings thereof. In some embodiments, the molecule incorporates a non-aromatic fragment that makes a productive hydrogen bonding interaction with Arg 70 on the αC-helix.

[0345] In one embodiment, the MAPK inhibitor is an allosteric inhibitor of p38 as described in compound 10, which is discussed in more detail in the context of Jnk-1.

[0346] In one embodiment, the MAPK inhibitor is SB203580 (SB6). In one embodiment, the MAPK inhibitor is represented by the formula [ka] In one embodiment, the MAPK inhibitor is skepinone-L having the formula [ka] and sorafenib and its derivatives.

[0347] In one embodiment, the MAPK inhibitor is the small molecule KC-706.

[0348] EGFR binding part In one embodiment, the enzyme-binding moiety is an EGFR-binding moiety. In one embodiment, the EGFR-binding moiety is an inhibitor or activator. EGFR is a tyrosine kinase receptor that belongs to the ErbB family of receptors that mediate cell growth, differentiation and repair in cells, particularly non-cancer cells.

[0349] In one embodiment, the EGFR binding molecule has the formula [ka] Or an analog thereof.

[0350] In one embodiment, the EGFR binding molecule is gefitinib. Gefitinib selectively binds to the ATP binding site of EGFR, thereby causing inhibition. In one embodiment, the EGFR binding molecule can be any one selected from the group consisting of erlotinib, afatinib, osimertinib, lapatinib, neratinib, or any derivative thereof.

[0351] In some embodiments, the kinase is an EGFR mutant. In some embodiments, the EGFR mutant comprises L858R, C797S, T790M, V984R, or a combination thereof.

[0352] In one embodiment, the EGFR inhibitor is EAI001, designed to overcome clinically acquired EGFR T790M / C797S mutant resistance in NSCLC by binding extrinsic ATP. EAI001 binds to the allosteric MT3 site of EGFR with a carboxamide forming a hydrogen bond with Asp 855, a phenyl group forming a hydrophobic interaction with the DFG-in pocket, and a 1-oxoisoindolinyl spanning the solvent accessible region. EAI001 has the formula: [ka] It is described in.

[0353] In certain embodiments, the EGFR inhibitor is an EAII001 analog, e.g., a compound of the formula: [ka] The compound is EAI045 described in

[0354] Both EAI001 and its analog EAI045 show potent inhibitory activity against EGFR L858R / T790M with IC50 values ​​of 24 and 3 nM, respectively. EAI045 is an allosteric inhibitor of a mutant form of EGFR found in lung cancer that leaves the wild-type receptor intact, with an IC50 of 3 nM. 50 and inhibits the L858R / T790M mutant EGFR and is >1000-fold selective for this mutant compared to the wild-type receptor. The IC50s for additional EGFR and its mutants, as well as EAI045, are: [Table 7]

[0355] In screening panels, EAI045 does not inhibit any other kinases by >20% (at 1000 nM EAI045) or show any liability to non-kinase targets, and in xenograft models, EAI045 is effective against EGFR (L858R / T790M / C797S) tumors with a mutant profile that is resistant to all currently available ATP-competitive EGFR tyrosine kinase inhibitors. See Angew. Chem. Int. Ed. 2020, 59, 13764-13776, incorporated herein by reference. EAI1045 exhibits the following properties: [Table 8]

[0356] In certain embodiments, EGFR inhibitors are designed to overcome acquired resistance to current EGFR tyrosine kinase inhibitors that bind to the ATP pocket of the enzyme, the site of many of the identified resistance mutations.

[0357] In an exemplary embodiment, the EGFR inhibitor is an analog that includes the addition of a phenylpiperazine substitution on the isoindolinone ring of EAI045. The analog has the formula: [ka] It may also be JBJ-04-125-02 described in

[0358] In certain embodiments, JBJ-04-125-02 exhibits subnanomolar potency against EGFR L858R / T790M kinase with a biochemical IC50 value of 0.26 nM. Notably, it potently inhibits cell proliferation and EGFR L858R / T790M / C797S signaling in vitro and in vivo as a single agent. X-ray crystal structures of JBJ-04-125-02 and EGFR T790M demonstrate that it binds to an allosteric site of EGFR in a manner similar to EAI001. In certain embodiments, JBJ-04-125-02 inhibits EGFR phosphorylation at (0.01-10 uM) and has demonstrated mutant selectivity by inhibiting mutant EGFR and downstream AKT and ERK1 / 2 phosphorylation. Angew. Chem. Int. Ed. 2020, 59, 13764-13776, which are incorporated by reference in their entireties; see, e.g., To et al., Single and dual targeting of mutant EGFR with an allosteric inhibitor, Cancer Discov. 2019 July; 9(7): 926-943. Doi:10.1158 / 2159-8290.CD-18-0903.

[0359] In certain embodiments, the EGFR inhibitor is an inhibitor identified in US Pat. No. 8,242,080, which is incorporated herein by reference, or a derivative thereof.

[0360] BCKDK joint part In one embodiment, the enzyme-binding moiety is a branched chain alpha-keto acid dehydrogenase kinase (BCKDK) binding moiety, also referred to as the 3-methyl-2-oxobutanoate dehydrogenase kinase binding site. In one embodiment, the BCKDK binding moiety is an inhibitor or activator. BCKDK has been targeted to address conditions such as obesity, maple syrup urine disease, and diabetes. In one embodiment, the binding moiety is ADR000362, which has the formula [ka] or a derivative thereof.

[0361] In one embodiment, the allosteric inhibitor has the formula [ka] The S-enantiomer of α-chlorophenylpropionate [(S)-CPP] described in Tso SC, Qi X, Gui WJ et al. Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase. Proc Natl Acad Sci US A. 2013;110(24):9728-9733. doi:10.1073 / pnas.1303220110, which is incorporated herein by reference, in particular, BCKDK inhibitor compounds and their IC 50 and K d See Table 1 which contains the values.

[0362] In one embodiment, the BCKDK inhibitor is a benzothiophene carboxylate derivative. In one embodiment, the binding moiety has the formula [ka] or derivatives thereof. See Tso et al., Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase. J Biol Chem. 2014 Jul. 25;289(30):20583-93. doi: 10.1074 / jbc.M114.569251.

[0363] FGFR binding part In one embodiment, the enzyme-binding moiety is an FGFR kinase-binding moiety. In one embodiment, the FGFR-binding moiety is an inhibitor or activator. Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases expressed on cell membranes, consisting of four members: FGFR1-FGFR4. All four FGFR members contain, from N-terminus to C-terminus, a large extracellular ligand-binding domain that includes an immunoglobulin (Ig)-like subunit (D1, D2, and D3) followed by a single transmembrane helix and an intracellular tyrosine kinase domain. The native ligands of FGFRs are fibroblast growth factors. FGFRs play important roles in both developmental and adult cells. See, for example, Dai S. et al., "Fibroblast Growth Factor Receptors (FGFRs): Structures and Small Molecule Inhibitors," Cells 2019, 8(6), 614.

[0364] In certain embodiments, the FGFR inhibitor has the formula: [ka] The SSR is SSR128129 described in.

[0365] Characteristics of SSR128129 include: [Table 9] In some embodiments, SSR128129E is used as the sodium salt. SSR128129E is a negative allosteric modulator of the FGF receptor. The compound has an IC of <100 nM. 50SSR128129E inhibits FGF1-induced ERK phosphorylation through FGFR2. SSR128129E inhibits FGF ligand-induced receptor dimerization in an allosteric manner, without affecting EGF binding, by interactions at Lys279, Thr320, Thr319, Cys278, Trp290, Phe 276, Wal 274, Tyr 340, Ile329, Tyr328, Leu327, Leu312. See Cancer Cell, 2013, 23, 477-488, which is incorporated by reference. For the effect of SSRs on cellular responses to different FGFRs, see Table 1 in Cancer Cell, 2013, 23, 4774-88, which is specifically incorporated by reference herein and shows the SSR concentrations that result in at least 50% inhibition at concentrations between 10 nM and 100 nM. Also see generally Herbert et al., Molecular Mechanism of SSR128129E, an Extracellularly Acting, Small-Molecule, Allosteric Inhibitor of FGF Receptor Signaling. Cancer Cell July 11, 2016; doi: 10.1016 / j.ccr.2013.02.018, which is incorporated by reference, for the chemical structure of SR128129E, the predicted binding detailed in Figure 727, and the effect of SSR on cellular responses to different FGFRs provided in Table 1, each of which is specifically incorporated by reference herein.

[0366] HA-NGFR binding part In one embodiment, the enzyme binding moiety is an allosteric tropomyosin receptor kinase A (TrkA) or high affinity nerve growth factor (HA-NGFR) kinase binding moiety. In one embodiment, the TrkA or HA-NGFR binding moiety is an inhibitor or activator. High affinity nerve growth factor receptor (HA-NGFR) is a family of receptor tyrosines that regulate the proliferation, differentiation and survival of sympathetic and neural neurons in the central and peripheral nervous systems. The native ligand of HA-NGFR is nerve growth factor. Absence of the ligand resulting in lack of activation can promote cell death, making neurons dependent on trophic factors for survival. See, e.g., National Center for Biotechnology Information, 2021. PubChem Protein Summary for NCBI Protein P04629, High affinity nerve growth factor receptor.

[0367] In certain embodiments, the pan-Trk inhibitor is GZ389988, AR786 (an allosteric selective TrkA inhibitor), ASP7962 (a TrkA receptor agonist), ONO-4474 (a pan-Trk inhibitor), or VM902A (an allosteric TrkA selective inhibitor). Additional Trk inhibitors are described in Bailey et al., Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016, doi: 10.1080 / 13543776.2017.1297797 and Bailey et al. (2020) Tropomyosin receptor kinase inhibitors: an updated patent review for 2016-2019, Expert Opinion on Therapeutic Patents, 30:5, pp. 325-339, DOI: 10.1080 / 13543776.2020, both of which are incorporated by reference in their entirety.

[0368] In certain embodiments, the HA-NGFR is VM-902A or a related compound or analog thereof. [ka] or an analog thereof.

[0369] IkappaB binding part In one embodiment, the enzyme binding moiety is an IkappaB kinase binding moiety. In one embodiment, the IkappaB binding moiety is an inhibitor or activator. In one aspect, the IkappaB kinase binding moiety inhibits one or both of the IKK-alpha and IKK-beta subunits of IkappaB kinase. In one embodiment, the binding moiety is the selective allosteric inhibitor BMS-345541, which has the following properties: [ka] It is described in. [Table 10] BMS-345541 has been shown to block NF-kB-dependent transcription in mice and is active against LPS-induced NF-kB activation in mice. In one embodiment, the negative allosteric modulator BMS-345541 has a pK of 6.9 as an inhibitor of nuclear factor kappaB kinase subunit beta. d , 6.5 pIC 50 , as well as a pIC of 5.4 for the inhibitor components of the nuclear factor kappaB kinase complex. 50 has.

[0370] CDK binding part In one embodiment, the enzyme-binding moiety is a CDK kinase-binding moiety. In one embodiment, the enzyme-binding moiety is a CDK inhibitor or activator. Cyclin-dependent kinases (CDKs) are characterized by the requirement of separate subunits, cyclins, which provide domains for enzymatic activity. CDKs control cell division and modulate transcription. The CKD family is divided into three cell cycle-related subfamilies: CDK1, CDK 2, and CDK 3; CDK4 and CDK6; and CDK5 and CDK14-CDK18, as well as five transcription subfamilies: CDK7; CDK8 and CDK19; CDK9; CDK10 and CDK11; CDK12 and CDK13; and CDK20. In one embodiment, the CDK inhibitor comprises palbociclib, ribociclib, abemaciclib, or any derivative thereof. In one embodiment, the CDK8 inhibitor is represented by the formula: [ka] Compound 5 has the formula:

[0371] In some embodiments, the CDK2 inhibitor is a flavopiridol analog. In some embodiments, the CDK2 inhibitor is 8-amido flavone, 8-sulfonamido flavone, 8-amido-7-hydroxyflavone, or a heterocyclic analog of flavopiridol. See Ahn et al., Design, synthesis, and antiproliferative and CDK2-cyclin a inhibitory activity of novel flavopiridol analogues, Bioorganic & Medicinal Chemistry, Vol. 15, No. 2, 2007, pp. 702-713, doi: 10.1016 / j.bmc.2006.10.063, which is incorporated herein by reference. In one embodiment, the compound is selected from the 8-amino flavopiridol analogues in Table 1 of Ahn, which may be selected based on the antiproliferative and inhibitory activities of Table 1, which is specifically incorporated herein by reference. Modifications to the Ahn molecule may be made based in part on the desired interaction between the analog and the CDK, with exemplary modifications made based on Figures 2A-2B.

[0372] In certain exemplary embodiments, the CDK inhibitor has the formula: [ka] and alvocidib, an inhibitor that causes cell cycle arrest and is currently undergoing Phase 2 clinical evaluation for its anti-cancer potential.

[0373] In one embodiment, alvocidib has a CDK4 pK of 7.2. i and CDK2 pIC of 6.4 to 7.0 50 and is utilized as a CDK2 and / or CDK4 kinase binding moiety having the following properties: [Table 11]

[0374] In certain embodiments, properties can be optimized for use in chimeric small molecules based on sites for modification, which can be identified and optimized according to the formula and as discussed in Bioorg. Med. Chem. 2007, 15, 702-713: [ka] (wherein R can be an alkane, alkene, alkyne, ether, alcohol, amine, nitrile, nitro, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, acid anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more fused rings including any combination of any of the above-listed rings, optionally substituted at one or more positions with any of the above-listed rings, preferably piperidine, pyrrolidine, thiane, or morpholine rings, which may be further substituted at any position on the ring). In particular, the 8 amino flavopiridol analogue is as detailed in Table 1 in Bioorg. Med. Chem. 2007, 15, pp. 702-713, as shown below. [Table 12]

[0375] PI3K binding part In one embodiment, the enzyme-binding moiety is a PI3K kinase-binding moiety. In one embodiment, the enzyme-binding moiety is a PI3K inhibitor or activator. Phosphoinositide 3-kinases (PI3Ks) are a superfamily of lipid kinases central to human cancer, diabetes, and aging. There are three different PI3K classes (I, II, and III) as well as different isoforms (e.g., class I has four isoforms: α, β, γ, δ), and within each class, there are distinct roles for each of the PI3Ks. Class I has been implicated in many cancers, especially those with pathogenic mutations. PI3K acts downstream on many growth factors and upstream on AKT and mTOR (Kannaiyan et al. Expert Rev Anticancer Ther. 2018; 18(12): pp. 1249-1270).

[0376] In one embodiment, the PI3K inhibitor has the formula: [ka] It is an idelalisib having the formula:

[0377] Idelalisib is a small molecule inhibitor of the delta isoform of PI3K. In certain embodiments, the PI3K inhibitor has the formula: [ka] PIK-108 is an allosteric inhibitor of the lipid-modifying kinase phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunits β and δ (PI3Kβ / δ). In addition to binding at the ATP-binding pocket, the compound binds at an allosteric site close to the H1047R mutation hotspot in the mouse PI3Kα C-lobe. See, e.g., Tables 2 and 3, and Certal, V. et al., "Discovery and Optimization of New Benzimidazole- and Benzoxazole-Pyrimidone Selective PI3Kβ Inhibitors for the Treatment of Phosphatase and TENsin Homologue (PTEN)-Deficient Cancers," J. Med. Chem. 2012, 55(10), pp. 4788-4805, which is incorporated by reference in its entirety, with specific reference to the biochemical and cellular activities of pyrimidone benzimidazoles and their substitutes.

[0378] VEGFR binding part In one embodiment, the enzyme-binding moiety is a VEGFR-binding moiety. In one embodiment, the VEGFR-binding moiety is an inhibitor or activator. Vascular endothelial growth factors (VEGFs) are a family of polypeptides with a conserved receptor-binding domain composed of a disulfide-knot structure. There are two VEGFs, VEGF-A and VEGF-B, which bind to VEGFRM, a receptor tyrosine kinase located on vascular endothelial cells. In one embodiment, the kinase-binding moiety is a VEGFR inhibitor. In one embodiment, the VEGFR inhibitor is sorafenib, sunitinib, pazopanib, axitinib, cabozantinib, lenvatinib, vandetanib, or regorafenib.

[0379] BRAF binding part In one embodiment, the kinase binding moiety is a BRAF binding moiety. In one embodiment, the binding moiety is a BRAF inhibitor or activator. BRAF is a member of the aggressive fibrosarcoma family of serine / threonine kinases and is frequently activated in patients with cancer due to genetic abnormalities. BRAF has three conserved regions: conserved region 1 (CR1) is a Ras-GTP binding autoregulatory domain; conserved region 2 (CR2) is a serine-rich region that functions as a hinge on the molecule; and conserved region 3 (CR3) is a catalytic protein kinase domain. In one embodiment, the kinase binding moiety is a BRAF inhibitor. In one embodiment, the BRAF inhibitor comprises vemurafenib or dabrafenib.

[0380] MEK joint part In one embodiment, the enzyme binding moiety is a MEK binding moiety. In one embodiment, the MEK binding moiety is an inhibitor or activator. MEK is a kinase enzyme that phosphorylates mitogen-activated protein kinases (MAPKs). Seven MEK subtypes have been identified, all mediating cellular responses to distinct growth signals. In one embodiment, the kinase binding moiety is a MEK inhibitor. In one embodiment, the binding moiety is a type 3 kinase inhibitor. In one embodiment, the MEK inhibitor binding moiety has the formula: [ka] Trametinib is used as an adjunct treatment for patients with BRAF V600E or V600K mutant melanoma, inhibiting MAP2K1 and MAP2K2 (also known as MEK1 and 2) in the p42 / p44 MAPK pathway. The absorption / distribution of an oral dose of trametinib tablets is 72%. Trametinib is 97.4% bound to human plasma proteins, which can be utilized when determining dosages for the small molecules detailed herein. See Gilmartin et al., GSK1120212 (JTP-74057) Is An Inhibitor of MEK Activity and Activation with Favorable Pharmacokinetic Properties for Sustained In Vivo Pathway Inhibition. Clin Cancer Res. 2011 Mar 1;17(5):989-1000. doi: 10.1158 / 1078-0432.CCR-10-2200. Published online Jan 18, 2011. Trametinib has a MAPK1 inhibition pIC of 9 / 0-9.1. 50 and MAPK2 pIC of 8.7 50 Trametinib has been shown to sustain inhibition of p-ERK1 / 2 for more than 24 hours with high potency, selectivity and a long circulating half-life.

[0381] In one embodiment, the MEK inhibitor binding moiety comprises cobimetinib, an allosteric inhibitor of MEK serine / threonine protein kinases with selectivity for MEK1 and MEK2. Cobimetinib selectively inhibits the activity of MEK serine / threonine protein kinases and has the following properties: [ka] It is described in. [Table 13] Additional in vitro activity of cobimetinib and related analogs is reviewed in Rice et al., "Novel Carboxamide-Based Allosteric MEK Inhibitors: Discovery and Optimization Efforts toward XL518 (GDC-0973)," ACS Med. Chem. Lett. 2012, 3, 5, 416-421, particularly Tables 1 and 3, which are incorporated herein by reference.

[0382] In certain embodiments, the MEK inhibitor binding moiety has the formula: [ka] The present invention also includes pimasertib, which is described in Pimasertib is an orally bioavailable small molecule inhibitor of the mitogen-activated protein kinases MEK1 and MEK2 (MEK1 / 2) with potential antineoplastic activity. It binds to an allosteric site distinct from the ATP-binding site, e.g., preventing activity rather than inhibiting catalytic activity. Pimasertib (AS703026) is cytotoxic to CD138 purified multiple myeloma (MM) cells from patients with relapsed and refractory MM with IC50 values ​​ranging from 2 to 200 nM. MEK1 / 2 (MAP2K1 / K2) are dual-specificity threonine / tyrosine kinases that play a key role in the activation of the RAS / RAF / MEK / ERK pathway and are frequently upregulated in various tumor cell types. It selectively binds to and inhibits the activity of MEK1 / 2, preventing the activation of MEK1 / 2-dependent effector proteins and transcription factors, which may result in the inhibition of growth factor-mediated cell signaling and tumor cell proliferation. Yoon J, Koo KH, Choi KY. MEK1 / 2 inhibitors AS703026 and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy. Cancer Res. 2011 Jan 15;71(2):445-53. See doi: 10.1158 / 0008-5472.

[0383] In certain embodiments, the MEK inhibitor has the formula: [ka] This includes CI-1040, which is described in

[0384] In certain embodiments, the MEK1 and MEK2 inhibitor binding moiety has the following properties: [ka] The first is selumetinib (AZD6244, ARRY-142886), which is described in [Table 14] Selumetinib is an orally bioavailable, non-ATP-competitive inhibitor that is highly specific for MEK1 / 2. It is a negative allosteric modulator of MEK1 with a pIC50 of 7.8-7.9. Sensitivity to selumetinib in a panel of NSCLC and CRC cell lines showed sensitivity to specific mutations of KRAS in GEO cells with the amino acid change p.G12A, SW480 cells with the amino acid change G12V, SW620 cells with the amino acid change p.G12V, and in HCT116 cells with the amino acid change G13D and the PIK3CA amino acid change p.H1047R, and in H1299 cells with the NRAS amino acid change p.Q61K.

[0385] In certain embodiments, the allosteric MEK inhibitor is a compound of the formula: [ka] (Wherein, R and R5 are as shown in the table below.) [Table 15] The compound is 3,4-difluoro-2-(2-halo-4-iodo-phenylamino)-N-2-hydroxy-ethoxy)-benzamide according to the present invention.

[0386] In one embodiment, MEK inhibitors have been investigated in advanced KRAS mutant colorectal cancer, non-small cell lung cancer, melanoma, colon neoplasia and breast cancer and have the formula: [ka] and mirdametinib (PD 0325901), a selective and non-ATP-competitive MEK inhibitor, described in . Mirdametinib has an EK1 inhibition pIC of 8.1. 50 It has a value and has the following properties: [Table 16]

[0387] In one embodiment, the MEK binding moiety is the allosteric inhibitor refametinib: [ka] or its analogues, e.g. [ka] or derivatives thereof.

[0388] In certain exemplary embodiments, the MEK binding moiety inhibitor has the following properties: [ka] and binimetinib as described in [Table 17] Binimetinib is FDA-approved for treatment of advanced BRAF-mutant melanoma in conjunction with the BRAF-mutant kinase inhibitor encorafenib. See Dummer et al., Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-mutant melanoma (COLUMBUS): a multicenter, open-label, randomized phase 3 trial. Lancet Oncol. 2018 May;19(5):603-615. doi: 10.1016 / S1470-2045(18)30142-6.

[0389] Additional studies in other solid tumor types, neoplasms, and hematological cancers are under investigation. See, e.g., Woodfield SE, Zhang L, Scorsone KA, Liu Y, Zage PE. Binimetinib inhibits MEK and is effective against neuroblastoma tumor cells with low NF1 expression. BMC Cancer. 2016 Mar 1;16:172. doi: 10.1186 / s12885-016-2199-z. Binimetinib is a negative allosteric modulator with MEK1 and MEK2 pIC50 of 7.9.

[0390] AKT joint part In one embodiment, the enzyme-binding moiety is an AKT-binding moiety. In one embodiment, the AKT-binding moiety is an inhibitor or activator. RAC-alpha serine / threonine-protein kinase (AKT) in humans has three isozymes (AKT1, 2 and 3, also known as PKB-α, -β and -γ). Each isozyme contains an amino (N)-terminal PH domain, an internal domain linker, a kinase domain, and a 21-residue carboxy (C)-terminal hydrophobic motif. In one embodiment, the ATK inhibitor has the formula: [ka] and borsertib, which has the formula:

[0391] In certain exemplary embodiments, the kinase inhibitor has the following properties: [ka] The compound MK-2206 has the following structure: [Table 18] MK-2206 is an orally bioavailable allosteric inhibitor of the serine / threonine protein kinase AKT (protein kinase B) with potential antineoplastic activity. MK-2206 has pIC values ​​of 8.3, 7.9 and 7.2 for AKT1, 2 and 3, respectively. 50 value. MK-2206 can enhance the antitumor efficacy of standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. As of 2018, ClinicalTrials.gov had 50 registered clinical trials of MK-2206. Many have been withdrawn, terminated, or completed. The ring fused to the pyridine may be modified to a monocyclic, bicyclic, tricyclic linear fused ring, or an angular tricyclic ring. The pyridine may be modified to a pyrazine. The substituted benzene moiety may also be modified. The strained cyclobutene may be substituted with any substituent known in the art. Furthermore, the hydrogen on the amine in the moiety may be substituted with any substituent known in the art. For further design guidance, see Kettle, JG et al., "Diverse Heterocyclic Scaffolds as Allosteric Inhibitors of AKT," J. Med. Chem. 2012, 55(3), 1261-1273, which is incorporated by reference in its entirety.

[0392] In one embodiment, the AKT inhibitor is any inhibitor from International Patent Application No. WO2008070016A2, which is incorporated herein by reference in its entirety, and any derivatives thereof. In addition, see, for example, Wu, W.-I. et al., "Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition," PLoS ONE 2010, 5(9), e12913, which is incorporated herein by reference in its entirety. In one embodiment, the inhibitor of AKT or a derivative thereof is [ka] (Wherein, R1=H and R2= [ka] and Any N in the ring may be replaced by C, N, O, S, B or P, or [ka] (Wherein R=NHMe) See Bioorg. Med. Chem. Let. 2008, 18, 4191-4194; doi:10.1371 / journal.pone.0012913, which is incorporated herein by reference. Optimization of allosteric inhibition of AKT can be performed based on the following guidance. [ka] Strategies for reducing the combined potency and hERG affinity for AKT binders may be based in whole or in part on the following. [ka]

[0393] In certain embodiments, the kinase inhibitor has the formula: [ka] Compound 16h is an AKT inhibitor VIII, also known as compound 16h, having the formula: AKT inhibitor VII is a cell-permeable quinoxaline compound that has been shown to potently, selectively, allosterically, and reversibly inhibit AKT (protein kinase B), with selectivity for AKT1 and 2 over AKT3. 50The values ​​are 7.2, 6.7 and 5.7 for AKT1, 2 and 3, respectively. See Lindsley, CW et al., "Allosteric Akt (PKB) Inhibitors: Discovery and SAR of Isozyme Selective Inhibitors," Bioorganic & Medicinal Chemistry Letters 2005, 15(3), pp. 761-764, which is incorporated herein by reference in its entirety, with specific reference to Lindsley's Tables 1 and 2, reproduced below. [Table 19] [Table 20]

[0394] In certain embodiments, the kinase inhibitor has the formula: [ka] and milansertib, also known as ARQ-092, described in US Pat. No. 6,339,663. milansertib is an orally active, selective, potent allosteric AKT inhibitor. milansertib has pIC values ​​of 8.3, 8.4, and 7.8 against AKT1, 2, and 3. 50Miransertib has progressed into Phase 1 and 2 development in solid and liquid tumors. See, e.g., "Discovery of 3-(3-(4-(1-Aminocyclobutyl)Phenyl)-5-Phenyl-3H-Imidazo[4,5-b]Pyridin-2-Yl)Pyridin-2-Amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor," J. Med. Chem. 2016, 59(13), pp. 6455-6469, which is incorporated by reference in its entirety, with Tables 2 and 4 in J. Med. Chem. 2016, 59(13), pp. 6455-6469, reproduced below, with specific reference to Tables 1, 2, 3, 4, 6, and 9. [Table 21] [Table 22]

[0395] In one embodiment, the kinase inhibitor is ARQ 751. In one embodiment, the kinase inhibitor is selected from the group consisting of those described in Ashwell, MA et al., "Discovery and Optimization of a Series of 3-(3-Phenyl-3H-Imidazo[4,5-b]Pyridin-2-Yl)Pyridin-2-Amines: Orally Bioavailable, Selective, and Potent ATP-Independent Akt Inhibitors," J. Med. Chem. 2012, 55, with specific reference to Tables 3, 4, 6, and 8 of Ashwell et al., reproduced below by reference. (11), pages 5291-5310, or any derivative thereof. [Table 23] [Table 24] [Table 25] [Table 26]

[0396] In certain embodiments, the kinase inhibitor has the formula: [ka] and borsertib, which has the formula:

[0397] Borsertib inhibits the AKT wt For IC of 0.8m or less 50 and K of 2.2 nM i It is a covalent allosteric inhibitor of AKT with the EC 50 The values ​​are 191±90 nM, 48±15 nM, 5±1 nM, 277±90 nM, 373±54 nM, 7770±641 nM in AN3CA (endometrial), T47D (breast), ZR-75-1 (breast), MCF-7 (breast), BT-474 (breast), and KU-19-19 (bladder) cell lines, respectively. In certain embodiments, the allosteric inhibitor can be one described in the table from Uhlenbrock et al., "Structural and chemical insights into the covalent-allosteric inhibition of the protein kinase Akt," Chem Sci. 2019 Mar. 28; 10(12): 3573-3585. doi: 10.1039 / c8sc05212c, reproduced below. [Table 27] The scaffolding changes are as follows: [ka] The present invention can be as described in

[0398] In certain embodiments, the AKT inhibitor has the formula: [ka] or any derivative thereof, see, for example, "Lactoquinomycin C and D, Two New Medermycin Derivatives from the Marine-Derived Streptomyces Sp. SS17A," Natural Product Research 2019, 34(9), pp. 1213-1218. In an exemplary embodiment, the lactoquinomycin is medermycin.

[0399] In certain embodiments, the AKT activator has the following properties: [ka] and BIND-2206, also known as MK-2206 or NSC-749607, described in US Pat. [Table 28] The AKT portion can be synthesized following the guidance and design provided herein, taking into account the AKT binding moiety, as disclosed, for example, in Panicker et al. Adv Exp Med Biol 1163:253-278 (2019); Botello-Smith et al. PLoS Comp Biol 13(8):e1005711 (2017); Mou et al. Chem Biol Drug Des 89(5):723-731 (2017); Ruiz-Carillo et al. Sci Rep 8:7365 (2018), and Budas et al. Biochem Soc Trans 35:1021-1026 (2007). Further information on AKT allosteric inhibitors can be found in Wu, W.-I. et al. "Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition" PLoS ONE 2010, 5(9), e12913 and guidance on the crystal structure of human AKT1 with an allosteric inhibitor to help identify interactions in the binding pocket for allosteric changes.

[0400] ALK joint part In one embodiment, the enzyme-binding moiety is an ALK kinase-binding moiety. In one embodiment, the ALK-binding moiety is an inhibitor or activator. Anaplastic lymphoma kinase is also known as ALK tyrosine kinase receptor or CD246. ALK participates in cell communication and the development and function of the nervous system. Upon ligand binding, the full-length receptor ALK dimerizes, changes conformation, and autoactivates its own kinase domain. The autoactivated ALK dimer phosphorylates other ALK receptors at specific tyrosine amino acid residues. The ALK phosphorylation residues are binding sites for the recruitment of several adaptors. In one embodiment, the ALK inhibitor includes crizotinib, ceritinib, alectinib, brigatinib, or lorlatinib.

[0401] In one embodiment, the ALK inhibitor has the formula: [ka] or a derivative thereof.

[0402] BTK joint part In one embodiment, the enzyme-binding moiety is a BTK kinase-binding moiety. In one embodiment, the BTK-binding moiety is an inhibitor or activator. Bruton's tyrosine kinase (Btk) is involved in multiple signaling cascades and plays a role in B cell developmental signaling and oncogenic signaling. See, e.g., Singh et al., 2018; Pal et al., 2018. In one embodiment, the BTK inhibitor is ibrutinib, acalabrutinib, or a derivative thereof. [ka] Exemplary derivatives are detailed in Liclican et al., Biochimica et Biophysica Acta (BBA) 1864(4): 129531, DOI: 10.1016 / j.bbagen.2020.129531, [ka] Includes.

[0403] In one embodiment, the BTK activator is [ka] is selected from.

[0404] In one embodiment, the BTK activator moiety [ka] is provided in the targeting portion.

[0405] FLT3 binding part In one embodiment, the enzyme-binding moiety is a FLT3 kinase-binding moiety. In one embodiment, the FLT3-binding moiety is an inhibitor or activator. FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase belonging to the subclass III family. FLT3 contains five immunoglobulin-related domains in the extracellular region and an intracellular tyrosine kinase domain that is separated into two by a specific hydrophilic insertion. In one embodiment, the FLT3 inhibitor comprises midostaurin.

[0406] JAK2 binding part In one embodiment, the enzyme binding moiety is a JAK2 kinase binding moiety. In one embodiment, the JAK2 binding moiety is an inhibitor or activator. Janus kinase 2 (JAK2) is a non-receptor tyrosine kinase and belongs to the Janus kinase family. JAK2 lacks the Src homology binding domains SH2 and SH3 but contains seven JAK homology domains JH1-JH7. In one embodiment, the JAK2 inhibitor has the formula: [ka] and ruxolitinib, also known as INCB018424, which is described in

[0407] In another exemplary embodiment, the JAK2 inhibitor has the formula: [ka] and tasocitinib, also known as CP6905502, described in

[0408] AURKA joint part In one embodiment, the enzyme-binding moiety is an AURKA kinase-binding moiety. In one embodiment, the AURKA-binding moiety is an inhibitor or activator. Aurora A kinase (AURKA) is a member of the Setr / Thr kinases whose orthologues control progression through mitotic cell death. Other members of the Aurora family are Aurora B and C, which all share a relatively conserved kinase catalytic domain at the carboxy (C) terminus. In one embodiment, an Aurora A inhibitor has the formula: [ka] Orkin A has an IC of 12.7 μM. 50 and K of 2.7 μM i has.

[0409] In certain embodiments, the Aurora A inhibitor has the formula: [ka] AA29 has an IC of 34.4 μM. 50 and K of 7.4 μM i has.

[0410] In certain embodiments, the Aurora A inhibitor has the formula: [ka] AA30 has an IC of 25.6 μM. 50 and K of 5.5 μM i In one embodiment, the compound has the formula: [Table 29] Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2. Sci Rep 6, 28528 (2016). Doi: 10.1038 / srep28528, which is incorporated herein by reference. In one embodiment, the kinase binding moiety is a monobody targeting Aurora A, as described in Zorba A. et al. "Allosteric Modulation of a Human Protein Kinase with Monobodies," Proc Natl Acad Sci USA 2019, 116(28), 13937-13942, which is incorporated herein by reference.

[0411] In one embodiment, the Aurora inhibitor is an Aurora inhibitor identified in US Patent Application No. 20080051327, which is incorporated herein by reference, or any derivative thereof.

[0412] c-MET binding part In one embodiment, the enzyme-binding moiety is a c-MET kinase binding moiety. In one embodiment, the c-MET binding moiety is an inhibitor or activator. c-MET is a receptor tyrosine kinase involved in cell signaling pathways. After binding to hepatocyte growth factor, it activates signaling pathways such as proliferation, motility, migration and invasion, among others, see e.g., Organ, SL et al., An Overview of the C-MET Signaling Pathway, Ther Adv Med Oncol 2011, 3, S7-S19. In one embodiment, the c-MET inhibitor is a compound of the formula [ka] and tivantinib, also known as ARQ-197, described in US Pat.

[0413] In one embodiment, the tivantinib binding agent or a derivative thereof targets the receptor tyrosine kinase of the MET proto-oncogene, is an allosteric inhibitor, and has one or more of the following properties: tivantinib or a derivative thereof has an enzyme IC of 50 nM or less in NCI-H441 cells, respectively. 50 , 100 nM phospho-MET IC 50 , Viability IC of 100 nM 50 , and 80 nM invasion IC 50 Tivantinib is a non-ATP-competitive MET-specific inhibitor that is 10-100-fold more selective for c-Met than other kinases tested (see Munshi et al., Moll. Can. Ther. doi:10.1158 / 1535-7163.MCT-09-1173). Tivantinib has not only demonstrated growth inhibition in breast, prostate, colon, and pancreatic cancer xenografts, but also inhibits metastasis formation in an experimental metastasis model of orthotopic colon cancer xenografts. In addition, the tivantinib inhibitor has a pKi value of 6.4. These features allow for appropriate selection and modification for the design of chimeric small molecules, as detailed elsewhere herein.

[0414] DDR binding site In one embodiment, the enzyme-binding moiety is a DDR kinase-binding moiety. In one embodiment, the DDR-binding moiety is an inhibitor or activator. Discoidin domain receptors (DDRs) belong to the receptor tyrosine kinase family and are identified by their activating ligand, fibrillar collagen. Furthermore, their activation and inactivation kinetics are slow and they exist as dimers at the cell surface in the absence of their ligand. See, for example, Grither, WR et al., "Inhibition of Tumor-Microenvironment Interaction and Tumor Invasion by Small-Molecule Allosteric Inhibitor of DDR2 Extracellular Domain," Proc Natl Acad Sci USA 2018, 115(33), pp. E7786-E7794.

[0415] In one embodiment, the DDR inhibitor is [ka] or derivatives thereof.

[0416] In one embodiment, the DDR inhibitor has the formula: [ka] IC of 230 nM 50 The WRG-28 has the following features.

[0417] In one embodiment, WRG-28 or a derivative thereof is an extracellular acting allosteric inhibitor that inhibits receptor-ligand interactions via allosteric modulation of the receptor. WRG-28 has been shown to inhibit tumor invasion and migration, as well as the tumor-supportive role of the stroma, and inhibits establishment of metastatic breast tumor cells in the lung by targeting DDR2.

[0418] INSR joint part In one embodiment, the enzyme-binding moiety is an INSR kinase-binding moiety. In one embodiment, the INSR-binding moiety is an inhibitor or activator. The insulin receptor (INSR) is a member of the receptor tyrosine kinase (RTK) family that is located on a cell membrane glycoprotein and modulates insulin. The INSR family is composed of RTKs including the insulin-like growth factor-1 receptor (IGF1R) and insulin receptor-related receptors. See, for example, Hubbard, SR, "The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase," Cold Spring Harbor Perspectives in Biology 2013, 5(3). In one embodiment, the kinase-binding moiety is an INSR inhibitor. In one embodiment, the INSR inhibitor is XMetD, also known as RZ-358 or XOMA358, which is a human anti-INSR IgG2 monoclonal antibody. XMetD is a negative allosteric modulator of INSR. See, for example, Patel P. et al., "A Unique Allosteric Insulin Receptor Monoclonal Antibody That Prevents Hypoglycemia in the SUR-1- / - Mouse Model of KATP Hyperinsulinism," mAbs 2018, 10(5), pp. 796-802.

[0419] In one embodiment, the enzyme is the insulin receptor and the binding moiety is RZ-358, also known as XOMA-358, a fully human negative allosteric modulating insulin receptor antibody. RZ358 can be administered intravenously and binds to a site on the insulin receptor present in the liver, fat and muscle. The RZ358 molecule has high selectivity for the insulin receptor, without IGF-1 interaction, and still allows insulin to bind and signal attenuating the insulin signal only if insulin is elevated. Clinical trials have been conducted at doses ranging from 0.1 to 9 mg / kg and have been studied in congenital hyperinsulinism and hypoglycemia after gastric bypass.

[0420] Additional selective allosteric antibodies against the insulin receptor, including XMetD, have been identified using the research platform and can be utilized in the small molecules disclosed herein. See J Journal of Diabetes Science and Technology 2014, 8, 865-873, doi:10.4161 / mabs.26871. In one embodiment, the binding moiety is an allosteric insulin receptor antibody, such as XOMA358. Phase 2 clinical trials show that ZOMA358 exhibits inhibition against insulin signaling in patients with inappropriate insulin signaling, including congenital hyperinsulinism. Treatment with the antibody in volunteers ranges from 01.mg / kg to 9mg / kg. See Johnson et al., Attenuation of Insulin Action by an Allosteric Insulin Receptor Antibody in Healthy Volunteers. J Clin Endocrinol Metab. 2017 Aug 1;102(8):3021-3028. doi: 10.1210 / jc.2017-00822.

[0421] IKK joint part In one embodiment, the enzyme-binding moiety is an IKK kinase-binding moiety. In one embodiment, the IKK-binding moiety is an inhibitor or activator. The IκB kinase (IKK) complex is composed of three subunits: IKKα, IKKβ, and IKKγ / NEMO. The subunits IKKα and IKKβ are catalytic, and IKKγ / NEMO is regulatory. See, for example, Karin, M. "The IκB Kinase - a Bridge between Inflammation and Cancer," Cell Res 2008, 18(3), pp. 334-342. In one embodiment, the IKK inhibitor has the formula: [ka] The compound is BMS-345541 described in

[0422] mTOR binding site In one embodiment, the enzyme-binding moiety is an mTOR kinase-binding moiety. In one embodiment, the mTOR-binding moiety is an inhibitor or activator. The mammalian target of rapamycin (mTOR) is a serine / threonine protein kinase of the PI3K-related protein kinase family. mTOR is large, approximately 300-500 kDa, and contains a conserved kinase catalytic domain. mTOR also contains HEAT repeats, a FAT domain, a FATC domain, and an FRB (FKBP12 / rapamycin-binding) domain that binds the drug rapamycin in complex with its intracellular receptor protein FKBP12. See, for example, Ballou LM et al., "Rapamycin and MTOR Kinase Inhibitors," J Chem Biol 2008, 1(1-4), pp. 27-36.

[0423] In certain embodiments, the mTOR activator has the following properties: [ka] and sirolimus, also known as rapamycin, described in US Pat. [Table 30]

[0424] Sirolimus is a macrolide produced by the bacterium Streptomyces hygroscopicus. It has potent immunosuppressive and antiproliferative properties. Sirolimus binds to FK506 binding protein 12 (FKBP12), creating a complex that inhibits the mammalian target of rapamycin (mTOR). Sirolimus inhibition of FKBP prolyl isomerase 1A has a pK i has.

[0425] The FKBP12-sirolimus complex has been reported to bind to a site distinct from the kinase domain of mTOR and acts as a negative allosteric modulator of mTOR activity. This action reduces mTOR-induced proliferation of activated T cells, the cells normally involved in immunological attacks in transplanted tissues. See Am. J. Health-Syst. Pharm. 2000, 57, 437-448. In vitro studies have been performed and these show that sirolimus inhibits MERS-CoV infection of Huh7 cells. This mechanism may also be applicable to SAR-CoV-2 infection. Sirolimus has been used in kidney transplantation.

[0426] In certain exemplary embodiments, the mTOR inhibitor is any inhibitor or derivative thereof encompassed in International Patent Application No. WO2014177123, which is incorporated herein by reference.

[0427] PAK binding part In one embodiment, the enzyme-binding moiety is a p21 kinase-binding moiety. In one embodiment, the PAK-binding moiety is an inhibitor or activator. p21-activated kinases (PAKs) are serine / threonine protein kinases. PAKs can be divided into two groups: group I consisting of PAK1-3 and group II consisting of PAK-6. They are effectors of Rac / Cdc42 GTPases and play important roles in cell proliferation, survival, motility and angiogenesis. See, for example, Karpov AS et al., "Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor," ACS Med. Chem. Lett. 2015, 6(7), pp. 776-781. In one embodiment, the PAK inhibitor has the formula: [ka] Compound 3, PMID 26191365, is a highly selective negative allosteric regulator of the protein kinase p21 (Cdc42 / Rac)-activated kinase 1 with favorable physicochemical properties. Compound 3 binds to a site adjacent to the ATP-binding site of the kinase. Compound 3 has pK values ​​of 8.1 and 6.4 for PAK1 (RAC1) and PAK2 (RAC1), respectively. d For example, see Karpov, AS et al. "Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor," ACS Med. Chem. Lett. 2015, 6(7), pp. 776-781, the entire contents of which are incorporated herein by reference. In one embodiment, the PAK inhibitor is the PAK inhibitor from Karpov ACS Med. Chem. Lett. 2015. The selectivity of PAK1 inhibitors versus the SAR of other PAK inhibitors is detailed in Table 3 of ACS Med. Chem. Lett, 2015, 6, 776-781, reproduced below. [Table 31]

[0428] In certain exemplary embodiments, the PAK inhibitor has the following properties: [ka] The IPA-3 described in [Table 32] IPA-3 is a cell-permeable, non-ATP-competitive, allosteric, selective inhibitor of p21 protein (Cdc42 / Rac)-activated kinase 1 (PAK1). IPA-3 binds covalently to the autoregulatory domain of PAK1 and prevents its activation by Cdc42. IPA-3 has a pIC of 5.6 for PAK1 (RAC1). 50 See, e.g., Viaud, J.; Peterson, JR, "An Allosteric Kinase Inhibitor Binds the P21-Activated Kinase Autoregulatory Domain Covalently," Mol Cancer Ther 2009, 8(9), 2559-2565, and Deacon, SW et al., "An Isoform-Selective, Small-Molecule Inhibitor Targets the Autoregulatory Mechanism of P21-Activated Kinase," Chemistry & Biology 2008, 15(4), 322-331, both of which are incorporated herein by reference in their entireties.

[0429] In certain embodiments, the PAK inhibitor has the formula: [ka] KPT-9274 is a small molecule that inhibits PAK4 and NAMPT. In contrast to the PAK kinase inhibitor PF-3758309, KPT-9274 acts as an allosteric modulator of PAK4 that does not interfere with the kinase activity of the enzyme. KPT-9274 has begun phase 1 clinical evaluation for non-Hodgkin's lymphoma and solid tumors. KPT-9274 has an IC of 120 nM in a cell-free assay. 50 KPT-9274 inhibits the proliferation of MS751 cervical carcinoma and Z138 B-cell acute lymphoblastic leukemia cell lines in vitro with an IC of <100 nM. 50 values ​​and induces the shrinkage of Molt-4 (T-cell acute lymphoblastic leukemia) xenografts in SCID mice. In addition, KPT-9274 has IC values ​​of 2.4, 5.6, 14.3, 16.7, 18.0, 19.0, 22.6, and >10,000 nM, respectively. 50 KPT-9274 inhibits the following B-ALL cell lines: KOPN-8, RS4, REH, 697 cells, OP-1, Nalm6, SupB15, SEM with IC values ​​of 19.4, 32.7, and 25.9 nM. 50 At low values, PDX B-ALL: inhibits LAX2, LAX7R and ICN13.

[0430] PDK1 binding part In one embodiment, the enzyme-binding moiety is a PDK1 kinase-binding moiety. In one embodiment, the PDK1-binding moiety is an inhibitor or activator. Phosphoinositide-dependent protein kinase-1 (PDK1) regulates the AGC family of kinases. PDK1 contains three ligand-binding sites: a substrate-binding site, a catalytic ATP-binding site, and a PDK1-interacting fragment (PIF)-binding site. The PIF-binding site, which is hydrophobic, has two functions: recruitment of downstream substrate kinases that carry a hydrophobic motif (HM), and stimulation of the intrinsic activity of PDK1. See, for example, "The Chemical Diversity and Structure-Based Discovery of Allosteric Modulators for the PIF-Pocket of Protein Kinase PDK1," Journal of Enzyme Inhibition and Medicinal Chemistry 2019, 34(1), pp. 361-374. In one embodiment, the kinase-binding moiety is a PDK1 inhibitor. In one embodiment, the kinase-binding moiety is a PDK1 inhibitor. In certain embodiments, the PDK1 inhibitor has the formula: [ka] The PS48 described in.

[0431] PS48 has an AC of 8.0 μM 50 See, for example, Hindie, V. et al., "Structure and Allosteric Effects of Low-Molecular-Weight Activators on the Protein Kinase PDK1," Nat Chem Biol 2009, 5(10), pp. 758-764, which is incorporated by reference in its entirety, with specific reference to FIG. 3 and Table 1, which depict the binding pocket, reproduced below. [Table 33]

[0432] See also Stroba, A. et al., "3,5-Diphenylpent-2-Enoic Acids as Allosteric Activators of the Protein Kinase PDK1: Structure-Activity Relationships and Thermodynamic Characterization of Binding as Paradigms for PIF-Binding Pocket-Targeting Compounds," J. Med. Chem. 2009, 52(15), 4683-4693, both of which are incorporated by reference in their entireties. Table 1 from Stroba, entitled Effect of Compound son Catalytic Activity of PDK1 and Thermodynamic Characterization of Binding, is specifically incorporated by reference and reproduced below. [Table 34]

[0433] In certain embodiments, the PDK1 inhibitor has the formula: [ka] RS1 is of the formula: [ka] and RS2 as described in 1999. RS1 and RS2 bind to PDK1 with Kds of 1.5 μM and 9 μM, respectively.

[0434] In some embodiments, the PDK1 inhibitor is a peptide docking motif (piftide). The piftide is a synthetic peptide. In one embodiment, the piftide is REPRILSEEEQEMFRDFDYIADW. In some embodiments, the piftide is a small molecule mimic of a peptide. See, for example, Rettenmaier TJ et al., "A Small-Molecule Mimic of a Peptide Docking Motif Inhibits the Protein Kinase PDK1." Proc Natl Acad Sci USA 2014, 111(52), pp. 18590-18595, the entire contents of which are incorporated herein by reference.

[0435] In certain embodiments, the PDK1 inhibitor has the formula: [ka] The PS210 described in

[0436] PTK2 / FAK binding part In one embodiment, the enzyme-binding moiety is a PTK2 / FAK kinase-binding moiety. In one embodiment, the PTK2 / FAK-binding moiety is an inhibitor or activator. Protein tyrosine kinase 2 (PTK2), also known as focal adhesion kinase (FAK), is a non-receptor tyrosine kinase, but is only distantly related to other tyrosine kinases. PTK2 / FAK plays an essential role in mammalian development and many physiological functions, most notably cell migration, by integrating signals from integrins and growth factor receptors. See, for example, Hirt UA et al., "Efficacy of the Highly Selective Focal Adhesion Kinase Inhibitor BI 853520 in Adenocarcinoma Xenograft Models Is Linked to a Mesenchymal Tumor Phenotype," Oncogenesis 2018, 7(2).

[0437] In certain embodiments, the PTK2 / FAK inhibitor has the formula: [ka] Compound 30, PMID 23414845, is described in. Compound 30 is a selective inhibitor of the tyrosine kinase PTK2 (also known as FAK). It is a type III inhibitor in that it binds to an allosteric site but not to the ATP active site of the kinase. In vitro, compound 30 inhibits the autophosphorylation of PTK2 with an IC50 of 7.1 μM in prostate cancer cells. PTK2 plays an important role in the control of cell proliferation, migration and invasion and helps regulate resistance to apoptosis. The enzyme is overexpressed in some cancer cells and reduction of PTK2 activity has growth inhibitory effects in vitro and in vivo. These factors make inhibition of PTK2 a novel mechanism for diseases of cell hyperproliferation. In a separate experiment, compound 30 inhibited the autophosphorylation of PTK2 with a pIC50 of 6.2 for inhibition of PAK2. 50 For example, see Tomita, N. et al., "Structure-Based Discovery of Cellular-Active Allosteric Inhibitors of FAK," Bioorganic & Medicinal Chemistry Letters 2013, 23(6), pp. 1779-1785, which is incorporated herein by reference in its entirety, with specific reference to Tables 1, 3 and 5, which represent the evaluation of the SAR of the substituents in Tables 1 and 5, reproduced below, for binding agents that may be used herein. [Table 35] [Table 36]

[0438] RIPK binding part In one embodiment, the enzyme-binding moiety is a RIPK kinase-binding moiety. In one embodiment, the RIPK-binding moiety is an inhibitor or activator. Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways that lead to cell death and / or inflammation. See, e.g., Degterev A. et al., "Targeting RIPK1 for the Treatment of Human Diseases," Proc Natl Acad Sci USA 2019, 116(20), pp. 9714-9722. In one embodiment, the RIPK inhibitor is of the formula [ka] RIPA-56 is a highly potent, selective, metabolically stable type III (allosteric) inhibitor of RIPK1. RIPA-56 is also known as compound 92 in patent WO2016101885, which is incorporated herein by reference. RIPA-56 is a drug candidate for the treatment of systemic inflammatory response syndrome (SIRS). RIPA-56 is active against human and mouse RIPK1 and is effective in animal models. It lacks off-target IDO inhibitory activity. RIPA-56 has a pIC of 7.9 against RIPK-1. 50 For example, see Ren, Y. et al., "Discovery of a Highly Potent, Selective, and Metabolically Stable Inhibitor of Receptor-Interacting Protein 1 (RIP1) for the Treatment of Systemic Inflammatory Response Syndrome," J. Med. Chem. 2017, 60(3), 972-986, the entire contents of which are incorporated herein by reference, with specific references in Table 5.

[0439] TYK2 binding part In one embodiment, the enzyme-binding moiety is a TYK2 kinase-binding moiety. In one embodiment, the TYK2-binding moiety is an inhibitor or activator. Tyrosine kinase (TYK2) is a member of the JAK kinase family that regulates signaling downstream of the receptor. TYK2 pairs with JAK2 to regulate the IL-23 / IL-12 pathway and JAK1 to regulate the type I interferon family. In one embodiment, the TYK2 inhibitor has the formula: [ka] The compound is Compound 29 described in, for example, Moslin R. et al., "Identification of Imidazo[1,2-b]Pyridazine TYK2 Pseudokinase Ligands as Potent and Selective Allosteric Inhibitors of TYK2 Signalling," Med. Chem. Commun. 2017, 8(4), pp. 700-712.

[0440] In certain exemplary embodiments, the TYK2 inhibitor has the formula: [ka] where D is deuterium. The deuterated methylamide group confers selectivity by binding to the pocket of the TYK2 JH2 ligand binding domain. Deuclavacitinib is a selective, orally active allosteric inhibitor of TYK2 that binds to the JH2 (pseudokinase) domain. Deuclavacitinib is quinone selective and does not bind to the JAK1-3 or TYK2 JH1 (ATP) binding domains. Deuclavacitinib has an in vitro IC of 5 nM. 50 In particular, duravacitinib has a pK of 10.7 for TYK2, inhibiting IFNγ production in TYK2 cells. i values, and pIC values ​​of 9.7 and 9.01 for TYK2 and JAK1, respectively. 50Currently, duravacitinib is undergoing evaluation in clinical studies in patients with systemic lupus erythematosus and ulcerative colitis (both in Phase 2) and moderate to severe psoriasis (in Phase 3). See, for example, Wrobleski, ST et al., "Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165," J. Med. Chem. 2019, 62(20), 8973-8995, and "Tyrosine Kinase 2 (TYK2) Allosteric Inhibitors To Treat Autoimmune Diseases," J. Med. Chem. 2019, 62(20), 8951-8952, which are incorporated herein by reference in their entireties. Tables 1 and 3 from Wrobleski et al. are specifically incorporated herein by reference. Table 1 shows the biochemical availability of the JAK family for clinical inhibitors, and Table 3, reproduced below, provides an expansion of the C3' amide SAR. [Table 37]

[0441] SHP joint part In one embodiment, the enzyme-binding moiety is a Scr kinase-binding moiety. In one embodiment, the Scr kinase-binding moiety is an inhibitor or activator. Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) belongs to the protein tyrosine phosphatase (PTP) family and is a positive transducer of proliferation and anti-apoptotic signals from receptor tyrosine kinases. SHP2 is composed of three folded domains and a C-terminal tail. SHP2 modulates phosphatase activity by binding to phosphopeptides at the N-terminal SH2 and C-terminal SH2 domains. The PTP domain retains catalytic functionality in the conserved signature motif HCX5R. The aberrant C-terminal tail contains a putative regulatory function. See, for example, Marasco M. et al., "Molecular Mechanism of SHP2 Activation by PD-1 Stimulation." Sci. Adv. 2020, 6(5), eaay4458. In certain exemplary embodiments, the SHP3 inhibitor is any from International Patent Application No. WO2020076723, which is incorporated herein by reference.

[0442] aPKC binding part In one embodiment, the enzyme-binding moiety is an atypical PKC kinase-binding moiety. In one embodiment, the aPKC-binding moiety is an inhibitor or activator. Atypical protein kinase C (aPKC) belongs to the protein kinase C family, which is divided into three groups based on their structure and cofactor regulation. The aPKC isozymes: ζ and λ, are at least understood and are significantly different in structure from the other two classes. First, the C1 domain contains one Cys-rich motif instead of two. Second, the aPKC isozymes do not appear to contain the critical residues that maintain the C2 fold. In addition, a distinctive feature of aPKC is that they are reportedly unresponsive to phorbol esters in vivo or in vitro. See, e.g., Newton, A.C., "Protein Kinase C: Structure, Function, and Regulation," Journal of Biological Chemistry 1995, 270(48), pp. 28495-28498.

[0443] In certain exemplary embodiments, the aPKC inhibitor is an inhibitor identified in International Patent Application No. WO2015075051, which is incorporated herein by reference, or any derivative thereof.

[0444] In certain embodiments, the PKC inhibitor is a PKC-zeta (PKCζ) inhibitor. See Abdel-Halim, Discovery and Optimization of 1,3,5-Trisubstituted Pyrazolines as Potent and Highly Selective Allosteric Inhibitors of Protein Kinase C-ζ, Journal of Medicinal Chemistry 2014 57(15), pp. 6513-6530, DOI: 10.1021 / jm500521n, which is incorporated herein by reference in its entirety, with specific references in Tables 1 and 2 reproduced below. [Table 38] [Table 39] In some embodiments, the binding moiety has the formula: [ka] is a 1,3,5-trisubstituted pyrazoline according to More preferably the binding molecule is [ka] or any derivative thereof. 1,3,5-trisubstituted pyrazolines are potent and selective allosteric PKCζ inhibitors. A phenolic group on the 5-phenyl is essential for inhibitory activity, with catechol providing the best activity. The presence of a lipophilic (halogen or alkyl) substituent on the 1-phenyl has been shown to be essential for high potency.

[0445] SphK binding part In one embodiment, the enzyme binding moiety is a SphK kinase binding moiety. In one embodiment, the SphK binding moiety is an inhibitor or activator. Sphingosine kinase (SphK) is a biological lipid kinase that regulates the sphingolipid metabolic pathway and controls multiple important cellular processes. SphK is the only enzyme that catalyzes the ATP-dependent phosphorylation of sphingosine to sphingosine-1-phosphate. SphK has five conserved domains, C1-C5. The C4 domain appears to be unique to SphKs, while the C1-C3 domains are also found in ceramide kinase (CERK) and diacylglycerol kinase (DAGK). The two SphK isoforms are SphK1 and SphK2. SphK2 has about 240 more amino acids than SphK1. See, for example, Cao M. "Sphingosine Kinase Inhibitors: A Patent Review" Int J Mol Med 2018.

[0446] In certain exemplary embodiments, the SphK inhibitor is an inhibitor identified in International Patent Application No. WO2014118556, which is incorporated herein by reference, or a derivative thereof.

[0447] GSK-3 binding part In one embodiment, the enzyme-binding moiety is a GSK-3 kinase-binding moiety. In one embodiment, the GSK-3-binding moiety is an inhibitor or activator. Glycogen synthase kinase 3 (GSK3) includes two isoforms, GSK3α and GSK3β, which regulate many interactions, such as intracellular receptor-coupled signaling proteins, insulin receptor, and some ionotropic receptors. GSK3 can be found in the cytosol, mitochondria, and nucleus, as well as other intracellular compartments. Two important functional domains of GSK3 are the stimulatory substrate-binding domain, which recruits substrates to GSK3, and the kinase domain, which phosphorylates substrates. See, for example, Glycogen Synthase Kinase-3 (GSK3): Regulation, Actions, and Diseases. Pharmacology & Therapeutics 2015, 148, pp. 114-131. In one embodiment, the GSK3 inhibitor is an inhibitor identified in U.S. Pat. No. 9,757,369, which is incorporated herein by reference, or a derivative thereof.

[0448] JNK joint part c-Jun N-Terminal Kinases (JNKs) are involved in stress signaling pathways involved in the regulation of gene expression, neuroplasticity, regeneration, cell death, and cellular aging. JNK is one of three families of MAP kinases. JNK has three isoforms: JNK1 and JNK2, which are found throughout tissues, and JNK3, which is found in nerves, heart, and testes. See, for example, Yarza, R. et al., "C-Jun N-Terminal Kinase (JNK) Signaling as a Therapeutic Target for Alzheimer's Disease," Front. Pharmacol. 2016, p. 6. In one embodiment, the enzyme-binding moiety is a JNK-binding moiety. In one embodiment, the JNK-binding moiety is an inhibitor or activator. In one embodiment, the JNK inhibitor has the formula: [ka] Compound 10 has the following IC in μM: 50 1.2 in 0.1 mM p38α assay; 0.8 in 0.1 mM MKK6 / p38α cascade assay; 1.4 in 0.01 mM p38α / MK2 cascade assay; >100 in 0.1 mM MKK6 assay; >40 in 0.01 mM MK2 assay; >40 in 0.1 mM p38β assay; >40 in 0.1 mM p38β assay; >40 in 0.1 mM p38γ assay; and >40 in 0.1 mM p28δ assay, see e.g., Comess, KM et al., "Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases," ACS Chem. Biol. 2011, 6(3), pp. 234-244, incorporated herein by reference. In certain embodiments, compound 10 binds to a lipid binding pocket. Additional JNK1 non-ATP site inhibitors can also be used in the small molecules disclosed herein, including biaryl-tetrazole Jnk-1 activation inhibitors. In some embodiments, the biaryl-tetrazole binding site for Jnk-1 is selected from Table 2 in ACS Chem. Biol. 2011, 6, pp. 234-244, reproduced below. [Table 40] Further details regarding biaryl-tetrazole affinity and coupling assay data for JNK isoforms and related MAP kinase proteins from Table 1 of ACS Chem. Biol. 2011, 6, pp. 234-244 are also provided and incorporated by reference below. [Table 41]

[0449] In certain examples of embodiments, the binding moiety is adapted from FIG. 1B, available in Lombard et al. Allosteric modulation of JNK docking-site interactions with ATP-competitive inhibitors. Biochemistry. Author manuscript; PMC October 9, 2019, incorporated herein by reference: [ka] is selected from.

[0450] TRK binding part The neurotrophic tyrosine kinase receptor 1 gene (NTRK1) encodes the tropomyosin-related kinase A (TRKA) receptor tyrosine kinase. TRKA is a high affinity receptor for nerve growth factor (NGF) and a member of the neurotrophin receptor family of receptor tyrosine kinases. TRKA is important for the development and maturation of the central and peripheral nervous systems during embryonic development. It is involved in pain and temperature sensing in sympathetic and sensory neurons, as well as memory processes in adults where it is expressed in the basal forebrain. NGF-mediated dimerization activates TRKA, which induces autophosphorylation of specific tyrosine residues and transphosphorylation of additional substrates, leading to activation of the PI3K / AKT, Ras / MAPK and PLC-γ pathways. See, e.g., Ardini, E. et al., "The TPM3-NTRK1 Rearrangement Is a Recurring Event in Colorectal Carcinoma and Is Associated with Tumor Sensitivity to TRKA Kinase Inhibition," Molecular Oncology 2014, 8(8), 1495-1507.

[0451] In one embodiment, the enzyme-binding moiety is a TRK-binding moiety. In one embodiment, the TRK-binding moiety is an inhibitor or an activator. In one embodiment, the TRK inhibitor has the formula: [ka] Compound 13, an allosteric inhibitor of TRKA, has an IC of 99 nM. 50 , and has better selectivity than TRKB and TRKC, each of which has an IC of 81 mM and 25 mM, respectively. 50In addition, compound 15 also demonstrated better selectivity for TRKA than TRKB. The crystal structures of TRKA and compounds 13, 15 and 16 from Chinese Patent Application No. 103649076, especially Figures 10 and 11, are incorporated herein by reference (PDB code: 5KMI, 5H3Q). In another example of the embodiment, the TRK inhibitor is any pyrrolidinyl urea or pyrrolidinyl thiourea, and any derivative thereof, as described in International Patent Application No. WO2012158413A2, which is incorporated herein by reference.

[0452] Additional Trk inhibitors are described in Bailey et al., Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016, Expert Opinion on Therapeutic Patents, doi: 10.1080 / 13543776.2017.1297797 and Bailey et al. (2020) Tropomyosin receptor kinase inhibitors: an updated patent review for 2016-2019, Expert Opinion on Therapeutic Patents, 30:5, pp. 325-339, DOI: 10.1080 / 13543776.2020, both of which are incorporated by reference in their entireties.

[0453] PDGFR binding part The Platelet-Derived Growth Factor (PDGF) system includes two receptors: PDGFRA and PDGFRB, and four ligands: PDGFA, PDGFB, PDGFC, and PDGFD. Ligand binding induces receptor dimerization, allowing autophosphorylation of specific tyrosine residues and subsequent recruitment of various signaling molecules. PDGFR regulates normal cell growth and differentiation, and expression of activated PDGFR promotes oncogenic transformation, see, e.g., McDermott, U. et al. Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-αActivation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors. Cancer Res 2009, 69(9), pp. 3937-3946. In one embodiment, the enzyme-binding moiety is a PDGFR targeting molecule. In one embodiment, the PDGFR-binding moiety is an inhibitor or activator. In certain embodiments, the PDGFR targeting molecule is imatinib, nilotinib, or dasatinib.

[0454] target substrate The target substrate may be a natural substrate of the enzyme bound to the kinase binding moiety described above. However, the target binding moieties discussed below may also be used to direct the enzyme to modify a non-natural or neo-substrate for that enzyme. Target substrates are polypeptides, nucleic acids, polynucleotides, lipids and oligosaccharides. The target binding moiety may be selected for a specific substrate of interest, which may be located in different localized sites of the cell, e.g., nucleus, cytoplasm, mitochondria, cell surface.

[0455] target binding moiety The target binding moiety comprises a chimeric small molecule by a mechanism that binds or associates with a target, including the target substrate described above. The target binding moiety of the chimeric molecule binds to the target substrate and brings the target substrate into proximity with the enzyme via the enzyme binding moiety or by virtue of the target binding moiety attached or labeled on the enzyme. The reaction may allow the enzyme to modify multiple substrates, non-natural target substrates of the enzyme, and increase the kinetics / efficiency of modification of such substrates. For that purpose, the target binding moiety must be capable of binding to the desired substrate of interest and be linked to the enzyme binding moiety via a linker to allow modification of the substrate.

[0456] Polynucleotide-binding moiety In one embodiment, the target binding moiety binds to a polynucleotide. Examples of polynucleotide binding moieties include small molecules. Small molecules that target polynucleotides include groove binders and intercalators, see, for example, Wang M. et al., "Recent Advances in Developing Small Molecules Targeting Nucleic Acid," IJMS 2016, 17(6), 779, and Warner KD et al., "Principles for Targeting RNA with Drug-like Small Molecules," Nat Rev Drug Discov 2018, 17(8), 547-558, which are incorporated herein by reference. Additional examples of polynucleotide binding moieties include protein-binding proteins. Polynucleotide binding proteins can be identified from nucleotide binding folds in proteins, such as Rossman type (see, e.g., Kleiger et al., J. of Mol. Biol. 323: 69-76) and P-loop-containing nucleotidyl hydrolase folds (see, e.g., Saraste et al., Trends in Bio Sci, 15: 430-434). Chauhan et al. developed a method for identifying ATP and GTP binding residues, and Ansari et al. designed a specific method for NAD. Parca et al. (2012) identified nucleotide binding sites in protein structures, including nucleotides bound to proteins, protein names, and organism names in Table 1 of DOI: 10.1371 / journal.pone.0050240, which is incorporated herein by reference. Thus, nucleotide binding sites are known in the art and can be identified by the skilled artisan for use as target binding moieties in the present compositions.

[0457] Oligosaccharide binding site In one embodiment, the target binding moiety is an oligosaccharide binding moiety. The oligosaccharide binding moiety includes a small molecule. For example, small molecules containing boronic acid are typically used to bind to oligosaccharides. See, for example, Jin S. et al., "Carbohydrate Recognition by Boronolectins, Small Molecules, and Lectins. Med. Res. Rev." 2009, incorporated herein by reference. Other oligosaccharide binding moieties include carbohydrate binding proteins, which are important targets when considering antiviral and anticancer drugs. The localization moiety can be, for example, lecithin, promoting an interaction site for carbohydrates. Exemplary molecules include small molecule boronolectins, nucleic acid-based boronolectins, and peptide boronolectins. See, e.g., Jin et al., Med. Res Rev. 2010 Mar;30(2):171-257; doi: 10.1002 / med.20155, which is incorporated herein by reference, specifically Figures 1-50 for the binding molecules and complexes formed. Publicly available computational methods are available using bioinformatics developed to select small molecules capable of binding carbohydrates, see, e.g., Zhao et al., Current Protocols in Protein Science 94: 1 10.1002 / cpps.75; Shionyu-Mitsuyama C, Shirai T, Ishida H, Yamane T (2003) Protein Eng 16: 467-478; and Kulharia M, Bridgett SJ, Goody RS, Jackson RM (2009) InCa-SiteFinder: a method for structure-based prediction of inositol and carbohydrate binding sites on proteins. J Mol Graph Model 28: 297-303.

[0458] Analysis of binding site residues in conjunction with stabilizing residues in protein-carbohydrate complexes may allow identification of folding and binding of the complex to understand interactions in addition to non-covalent hydrogen bonding and non-polar interactions. See, e.g., Shanmugam et al., doi.:10.2174 / 0929866525666180221122529. Using publicly available tools, carbohydrate binding moieties containing binding sites and predicted folds can be used for the design of multifunctional molecules containing such carbohydrate binding moieties.

[0459] Lipid binding moiety In one embodiment, the target-binding moiety is a lipid-binding moiety. The lipid-binding moiety can be utilized as a target-binding moiety in the chimeric small molecules disclosed herein. As a regulator of cell stabilization and signal transduction, their modification, distribution or tracking in compositions will be useful in treating, regulating and / or modifying pathways, processes and conditions. Lipids include charged lipids such as phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), and PI-phosphate, -diphosphate and -triphosphate (PIP-a family of seven negatively charged lipids) and gangliosides (GM), zwitterionic lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) lipids, ceramide (CER), diacylglycerol (DAG) and lysophosphatidylcholine (LPC) lipids, sphingolipids, glycerophospholipids, cholesterol, phosphatidylglycerol.

[0460] Lipid binding moieties may be incorporated into chimeric small molecules. For example, certain steroids can target and bind to lipids. Other lipid binding moieties, such as proteins, can bind lipids either specifically, where a distinct binding site for a given lipid can be identified, or non-specifically, where the lipid acts as a medium and physical properties such as thickness, fluidity, or curvature regulate protein function. Phosphoinositide binding domains, such as FYVE or PX, or the FRRG motif in the β-propeller of PROPPIN, are more common domains that can be used to identify lipid-binding proteins. The FYVE domain, named after the first four proteins containing the motif (Fab1, YOTB, Vac1 EEA1), contains several conserved regions that can also be utilized to identify related domains. See, for example, AH Lystad, A. Simonsen Phosphoinositide-binding proteins in autophagy, FEBS Lett., 590 (2016), pp. 2454-2468, 10.1002 / 1873-3468.12286. Additional FYVE domain-containing proteins include SARA, FRABIN, DFCP1 FGD1, ANKFY1, EEA1 FGD1, FGD2, FGD3, FGD4, FGD5, FGD6, FYCO1, HGS MTMR3, MTMR4, PIKFYVE, PLEKHF1, PLEKHF2, RUFY1, RUFY2, WDF3, WDFY1, WDFY2, WDFY3, ZFYVE1, ZFYVE16, ZFYVE19, ZFYVE20, ZFYVE21, ZFYVE26, ZFYVE27, ZFYVE28, ZFYVE9.

[0461] Eukaryotic cells can degrade intracellular components through a lysosomal degradation pathway called macroautophagy, where dysfunction of the pathway is associated with several diseases. Dikic et al., Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol., 19 (2018), pp. 349-364, doi: 10.1038 / s41580-018-0003-4. Thus, autophagy-related (ATG) proteins may be utilized as lipid-binding moieties in the present invention, including LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2. De la Ballina (2019), doi.org / 10.1016 / j.jmb.2019.05.051. Lipid-binding proteins include the protein HCLS1-binding protein 3 (HS1BP3), which can negatively regulate the activity of phospholipase D1 (PLD1).

[0462] Protein-binding moiety In one embodiment, the target binding moiety is a protein binding moiety.Protein binding moieties are provided for exemplary proteins of interest that are targeted for modification, as detailed herein.Protein binding moieties are selected based on desired association and modification.Therefore, the desired modification, which can be tailored based on a particular condition, disease, treatment, or other desired effect, is a design consideration when selecting a protein binding moiety.

[0463] Target protein binding moieties can be selected for specific proteins of interest, which can be located in different localized sites of a cell, e.g., the nucleus, cytoplasm, mitochondria, cell surface. Examples of target protein binding moieties are disclosed, for example, in Sun et al., Signal Transduction and Targeted Therapy, 4:64 (2019), which provides exemplary proteins and corresponding ligands (i.e., target polypeptide binding moieties, see in particular Figures 5-48, which are incorporated herein by reference). The target protein binding moiety may bind to a protein that undergoes a conformational change upon binding.

[0464] The target protein binding moiety may bind to a protein that undergoes a conformational change upon binding, such as the androgen receptor (AR). In one embodiment, activation of the enzyme results in modification of the target substrate by the enzyme at one or more new modification sites that would otherwise remain unmodified by the enzyme if not activated by the chimeric molecule. The target substrate need not be the natural substrate of the enzyme. The target substrate may be a protein, and discussion herein of a gene includes the product of gene expression.

[0465] In one embodiment, the target protein binding moiety can bind to a protein that is an ATPase or a GTPase. Exemplary GTPases can be from the Ras, Rho, Rab, Arf or Ran family, see, for example, Yoshimi Takai, Takuya Sasaki and Takashi Matozaki, Small GTP-Binding Proteins, Physiological Reviews 2001 81:1, pp. 153-208; doi: 10.1152 / ohysrev.2001.81.1.153. Exemplary targeting molecules can include molecules such as ibrutinib (BTK), dasatinib (BCR-ABL), MRTX (KRAS), MI-1061 (MDM2), gefitinib (EGFR), palbociclib (CDK4 / 6) and foretinib (C-MET), or analogs thereof.

[0466] In one embodiment, the target protein binding moiety is a fusion protein. In one embodiment, the target protein binding moiety is any of the previously identified enzyme binding moieties whose targets include fusion proteins.

[0467] In one embodiment, the target substrate comprises an orthogonal tag, e.g., FKBP12. F36V Modified with SNAP-, CLIP-, ACP- and MCP-tags, the target binding moiety is a binder of an orthogonal tag, see, e.g., neb.com / tools-and-resources / feature-articles / snap-tag-technologies-novel-tools-to-study-protein-function, incorporated herein by reference.

[0468] Provided below are further non-limiting examples of target protein binding moieties for various target proteins of interest in the context of oncology and infectious disease, as well as examples of uses that are further discussed in the Methods and Uses section below.

[0469] KRAS In one embodiment, the target protein binding moiety is a KRAS binding moiety. [ka] wherein R is an electrophilic reactive group and X is a group of formula [ka] and Y is H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2Cl).

[0470] In one embodiment, the electrophilic reactive group is [ka] or analogs thereof.

[0471] In one embodiment, the target binding moiety is a hydrogen bond surrogate (HBS) son of sevenless (SOS) peptidomimetic (PM). In one embodiment, HBS-SOS-PM has the sequence: * GIYRTDILRTEEGN-NH2, XFE * GIYRTELLKAEEAN-NH2, XFE * GIYRLELLKAEEAN-NH2, XFE * GIYRLELLK-NH2, XFE * AIYRLELLKAEEAN-NH2, XFE * GIYRLELLKAibEEAibN-NH2 and XAE * GIYRLELLKAEAAA-NH2 (where X represents a 4-pentenoic acid residue and the asterisk ( * ) is an N-allyl residue ( * In one embodiment, the target binding moiety is of the formula: XFE, wherein XFE represents an N-allyl glycine. * In one embodiment, the target binding moiety has the formula: * The KRAS binding molecule HB7 is described as GIYRLELLKAEAAA-NH2. See Nickerson et al., An Orthosteric Inhibitor of the RAS-SOS Interaction, doi: 10.1016 / B978-0-12-420146-0.00002-0, which is incorporated by reference in its entirety, with specific references in Table 2.1.

[0472] In one embodiment, the target binding moiety has the formula: [ka] In one embodiment, the target binding moiety has the formula: [ka] wherein R can be H, Gly, Ala, β-Ala, Val, Ile, Pro, or any other viable substituent known in the art. In one embodiment, the target binding moiety is a KRAS binding moiety that is an indole, phenol, sulfonamide, or any modified version thereof. See Sun et al., Angew Chem Int Ed Engl. 2012 Jun. 18; 51(25): 6140-6143. doi: 10.1002 / anie.201201358, which is incorporated herein by reference in its entirety.

[0473] In one embodiment, the target binding moiety has the formula: [ka] JPEG2024513566000290.jpg99170JPEG2024513566000291.jpg103170JPEG2024513566000292.jpg86170JPEG2024513566000293.jpg85170JPEG2024513566000294.jpg86170JPEG2024513566000295.jpg98170JPEG2024513566000296.jp g101170JPEG2024513566000297.jpg107170JPEG2024513566000298.jpg86170JPEG2024513566000299.jpg88170JPEG2024513566000300.jpg90170JPEG2024513566000301.jpg88170JPEG2024513566000302.jpg102170 is a KRAS binding molecule. In one embodiment, the target binding moiety has the formula: Ac-FIGRLCTEILKLREGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-AIGRLCTEILRLRNGA-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; Ac-LAWALRELERELARLC-NH2; Ac-WIGRLCTEIR HRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEIRRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-FIGRLCTEILKLREGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-AIGRLCTEILRLRNGA-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; FITC-AβLAWALRELERELARLC-NH2; Ac-WIGRLCTEIR H RLRNGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEIRRLRNGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEIR H RLRNGN-NH2; DZ-GLAWRLRELERELARLC-NH2; Ac-WIGRLCTEIK(DZ)RLRNGN-NH2 or Ac-LAWRLRELERELARLC-NH2 (in the formula, R H is L-homoarginine, Aβ is L-β-alanine, DZ is a diazirine photocrosslinker, and FITC is 5-fluorescein isothiocyanate linked via a thiourea bond to the N-terminal amine. See Hong et al., PNAS 2021 May 4 118(18) e2101027118; doi:10.1073 / pnas.2101027118, which is incorporated by reference in its entirety with specific references in Table S2.

[0474] In one embodiment, the target binding moiety has the formula: [ka] wherein R can be any substituent known in the art. In one embodiment, R is an electrophilic group. In one embodiment, R is [ka] (Wherein R is H, [ka] See Yoo et al., ACS Chem. Biol. 2020, 15, 6, 1604-1612, which are incorporated by reference in their entirety.

[0475] FKBP12 F36V In another exemplary embodiment, the target protein binding moiety can be designed to bind to FK506 binding protein (FKBP). The FKBP can be FKBP12, which binds to the intracellular calcium release channel and the TGF-β type I receptor. In one exemplary embodiment, the FKBP protein binding moiety is FKBP12 F36V In another exemplary embodiment, the binding molecule is [ka] or analogs thereof.

[0476] Tyrosine phosphorylation in FGFR1 can trigger a signaling cascade that induces PI3K / AKT / mTOR signaling and increased transcription of the blood growth factor G-CSF. See, e.g., Turner et al., Nature Reviews Cancer 2010.

[0477] In one embodiment, ABL kinase is used to F36V In one embodiment, the bifunctional small chimeric molecule is [ka] TIFF2024513566000308.tif49170TIFF2024513566000309.tif53170TIFF2024513566000310.ti f48170TIFF2024513566000311.tif51170TIFF2024513566000312.tif59170TIFF20245135660003 Selected from 13.tif68170TIFF2024513566000314.tif61170TIFF2024513566000315.tif59170TIFF2024513566000316.tif49170TIFF2024513566000317.tif42146TIFF2024513566000318.tif65170.

[0478] In one embodiment, the bifunctional small chimeric molecule is [ka] In one embodiment, the molecule is capable of activating FGFR1 / mTOR / G-CSF signaling in a dose-dependent manner.

[0479] EGFR In one embodiment, the target protein binding moiety is an EGFR binding moiety. EGFR is a tyrosine kinase receptor that belongs to the ErbB family of receptors that mediate cell growth, differentiation and repair of cells, particularly non-cancerous cells, whereas EGF is overexpressed in certain cancer cells, e.g., many solid tumors, including colorectal cancer, non-small cell lung cancer, ovarian, renal, head, pancreatic, cervical and prostate squamous cell carcinomas, and particularly breast cancer.

[0480] In one embodiment, the target protein binding moiety has the formula [ka] or an analog thereof.

[0481] HSP90 Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone whose co-chaperones modulate proteins involved in cell cycle control and signal transduction. Like many ATP-dependent proteins, the protein undergoes a functional cycle associated with its ATPase cycle.

[0482] In one embodiment, the HSP90 binding molecule is [ka] Or an analog thereof.

[0483] Additional HSP90 binding agents have the formula: [ka] Tanespimycin (IC of 5 nM in cell-free assay) 50 ), formula: [ka] Alvespimycin as described in (IC50 of 62 nM in cell-free assay), formula: [ka] EC141 described in formula [ka] Novobiocin according to Novobiocin analogs can also be utilized and can be used as MAPK signaling disrupting agents, as described in Hall et al., J Med Chem. 2016 Feb. 11; 59(3): 925-933; doi: 10.1021 / acs.jmedchem.5b01354, which is incorporated by reference.

[0484] BTK Bruton's tyrosine kinase (BTK) is a protein involved in multiple signal transduction cascades and is widely expressed in B cells. First, BTK is a cytoplasmic protein and is therefore available for interaction with cytoplasmic kinases such as AMPK.

[0485] In one embodiment, the BTK binding molecule [ka] or analogs thereof.

[0486] MDM2 In certain embodiments, the protein binding moiety is [ka] or a derivative or analogue thereof.

[0487] BRD4 In certain embodiments, the protein binding moiety is [ka] or analogs thereof.

[0488] FGFR1 In one embodiment, the protein binding moiety inhibits the FGFR1 fusion protein. In one embodiment, the FGFR1 fusion protein inhibitor has the formula [ka] and dovitinib, also known as TKI258, described in

[0489] PtpA, PtpB In one embodiment, the protein binding moiety has the formula [ka] or any derivative thereof.

[0490] In an exemplary embodiment, the PtpB binding moiety has the formula [ka] or any derivative thereof.

[0491] SapM In one embodiment, the protein binding moiety is a SapM binding moiety. In some embodiments, the SapM binding moiety contains a trihydroxybenzene group. In some embodiments, the SapM binding moiety is composed of a benzylidenemalononitrile scaffold. In one embodiment, the SapM binding moiety has the formula: [ka] JPEG2024513566000333.jpg150170 or any derivative thereof. In one embodiment, the SapM binding moieties are L-ascorbic acid (L-AC) and 2-phospho-L-ascorbic acid (2P-AC).

[0492] UMPK In one embodiment, the targeted binding agent is an M.tb kinase inhibitor. In one embodiment, the M.tb kinase inhibitor is an UMPK inhibitor identified in U.S. Patent Application Publication No. 20090209022, which is incorporated herein by reference, and any derivatives thereof.

[0493] Colistin In an exemplary embodiment, the PsA-associated target protein binding moiety is colistin, which has the formula: [ka] has.

[0494] Linker part The linker or linking moiety is a bifunctional or multifunctional moiety that can be used to link one or more of the target binding moieties, enzyme binding moieties. In some embodiments, the linker has a functionality that can react with moieties for covalent binding. The linker moiety is preferably a chemical linker moiety, represented as L in the formula of the present invention. In some embodiments, the linker moiety may preferably include one or more repeats, for example, 1, 2, 3, 4, 5, 6, 7, 8 or more repeats, which may be utilized to promote or improve the spacing, conformation and / or performance of the molecule. The linker described herein may refer to both L1 and L2, or L1 and L2 are different linkers described herein.

[0495] A linker or linking moiety can be used to link the enzyme binding agent to the target binding agent and / or the electrophilic reactive group to either the enzyme binding agent, the target binding agent, or both. When more than one linker molecule is used in a molecule, the linkers may be the same as one another or may be different.

[0496] In some embodiments, the linker moiety is polyethylene glycol (PEG). In some embodiments, the linker moiety is two or more PEGs, such as 2, 3, 4, 5, 6, 7, 8 or more PEGs.

[0497] In certain embodiments, the linker is [ka] (wherein n is 0 to 3 or more).

[0498] In certain embodiments, the linker is [ka] (wherein n is 0 to 6 or 7 or more). In an exemplary embodiment, the linker [ka] And the methyl may be substituted with ethyl, propyl, butyl, hexyl, or larger alkyl groups.

[0499] In one embodiment, the linker is [ka] where n is 1, 2, 3, 4 or 5.

[0500] In certain exemplary embodiments, the linker may be a reversible linker. In exemplary embodiments, the reversible linker is [ka] Includes.

[0501] In some embodiments, the linker is rigid. A rigid linker is a non-flexible linker. A rigid linker reduces or prevents rotation of bonds in the linker. The linker may use a higher bond order to increase rigidity. In some embodiments, the rigid linker may contain bond orders of 2, 3 or more, for example double or triple bonds. The linker may be composed of one or more rings or cycles of atoms and bonds to increase rigidity. The rings may be composed of 3, 4, 5, 6, 7 or more atoms connected via bonds. In some embodiments, the rings are homocyclic rings, for example, cycloalkanes or cycloalkenes. In some embodiments, the rings are heterocyclic rings, for example, piperidine or pyridine. In some embodiments, the rigid linker may be composed of one or more homocyclic rings, one or more heterocyclic rings, or a combination of homocyclic and heterocyclic rings. The one or more homocyclic or heterocyclic rings may be directly bonded together, e.g., naphthalene or 3,9-diazaspiro[5.5]undecane, or may be linked via one or more bonds, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 bonds. Homocyclic and heterocyclic rings are well known in the art and although not specifically listed, variations and combinations thereof are contemplated herein.

[0502] In one embodiment, L is a rigid linker, which is [ka] or any combination thereof, wherein any atom in the ring may be optionally substituted with C, N, O, S; the linker may be attached to one or more PEG molecules prior to attachment to A and optionally B; and m and n may be independently selected from 0 to 6.

[0503] In an example embodiment, the linker L has one covalent point of attachment to the kinase binding molecule and two covalent points of attachment to the other kinase binding molecule. The covalent points of attachment may be any single, double, triple, or quadruple bond between one component of the BFM / small chimera molecule and another. In an example embodiment, the formula [ka] According to the present invention, the linker binds one kinase binding molecule, say A, and another, say B.

[0504] In one embodiment, the PEG compound in the above-mentioned linker can be replaced with any linker mentioned herein. In one embodiment, the above-mentioned linker is optimized for physicochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding.

[0505] Exit Vector In one embodiment, the linker may include an exit vector. In one embodiment, the exit vector may be expressed independently of the linker. The exit vector parameters may be determined based in part on the average orientation of the substituents attached to the variable point, which may be generated using cheminformatics software. The exit vector may include a bond emanating from the chemical moiety. In one embodiment, the exit vector is provided as a bond on the linker or from the binding moiety, providing a conformation of the bond between the linker and the enzyme binding moiety. The exit vector may also be expressed independently of the linker in the formula detailed herein. In one embodiment, the exit vector is included in W.

[0506] One or more exit vectors may be utilized with the molecules described herein. In certain embodiments, the linker or enzyme binding moiety may be represented by an exit vector included in the linker or enzyme binding moiety. In embodiments, the exit vector may be represented independently of the linker or enzyme binding moiety.

[0507] In certain embodiments, the reactive bond of the exit vector is selected to be energetically favorable, preferably to increase binding affinity. The exit vector may be adjusted depending on the linker used in the molecule. In embodiments, the exit vector is a chemical moiety or bond that facilitates stereochemical protrusion, which may further facilitate subsequent coupling, binding and / or accessibility.

[0508] In one embodiment, the enzyme binding moiety comprises an exit vector. The exit vector comprises a group on the enzyme binding moiety that is attached to a linker. In one embodiment, the exit vector is capable of click chemistry, amide coupling chemistry, crosslinking chemistry, alkylation or sulfonation chemistry. In an embodiment, the exit vector is provided as a bond on the linker or from the Abl binding moiety, providing a conformation of the bond between the linker and the Abl binding moiety and / or the second Abl binding moiety. In one embodiment, the exit vector provided as a bond in the Abl binding moiety comprises a pyridine exit vector, as depicted in FIG. 50.

[0509] covalent warhead In one embodiment, the molecules or binding moieties disclosed herein may be modified to include or remove a covalent warhead. As used herein, a covalent warhead is typically an electrophilic functional group that can form a reversible or irreversible bond with a nucleophilic functional group. In one embodiment, the molecules or binding moieties may be modified in the covalent warhead to reduce or decrease the strength of the covalent binding ability of the covalent warhead, or to increase the binding affinity or strength of the binding of the covalent warhead as desired according to the application. In one exemplary embodiment, a binding molecule that creates an irreversible covalent bond with the target may be selected. When used in bifunctional molecules, such tight binding may be less desirable, and therefore such covalent warhead modifications may be desirable and may be modified to reduce interactions, see, for example, sciencedirect.com / science / article / pii / S0968089618320807. In particular, the reactivity of the warhead may be designed to allow covalent binding with the target with reversible or irreversible properties, depending on the desired functionality. An exemplary database that can be useful in identifying protein-ligand interactions around the binding site is described in Du et al., Nucleic Acids Research, Vol. 49, No. D1, Jan. 8, 2021, pp. D1122-D1129, incorporated herein by reference, with the database CovalentInDB accessible at cadd.zju.edu.cn / cidb / . This approach can be used with the design of any covalent warhead for the molecules disclosed herein.

[0510] Electrophilic reactive groups In one embodiment, the molecules or binding moieties disclosed herein may be modified to include an electrophilic reactive group. In one embodiment, the electrophilic reactive group is located between the linker and the target binding agent. In an embodiment, the electrophilic reactive group is located between two linkers, a first linker attached to the enzyme binding agent and a second linker attached to the target binding agent. As used herein, an electrophilic reactive group is typically a functional group that can form a reversible or irreversible bond with a nucleophilic functional group. The electrophilic reactive group allows the target binding moiety to directly bind to the target enzyme. Upon binding to the electrophilic reactive group, the enzyme is immediately tagged with the target binding moiety. In one embodiment, the molecules or binding moieties may be modified in the electrophilic reactive group to reduce or decrease the strength of the covalent binding ability of the electrophilic reactive group, or to increase the binding affinity or strength of the binding of the electrophilic reactive group as desired according to the application. In an embodiment, a binding molecule that creates an irreversible covalent bond with the target may be selected. When bifunctional molecules are used, such tight binding may be less desirable. Therefore, modification of such electrophilic reactive groups is desirable and can be modified to reduce interactions, see, for example, sciencedirect.com / science / article / pii / S0968089618320807. In particular, the reactivity can be designed to allow covalent binding at the target with reversible or irreversible properties depending on the desired functionality. In some embodiments, the electrophilic reactive group is designed to react with an amino acid side chain reactive group. The amino acid side chain reactive group may be nucleophilic. Nucleophilic amino acid side chain reactive groups may include arginine, lysine, histidine, cysteine, aspartic acid, glutamic acid, and tyrosine. In one embodiment, the electrophilic reactive group reacts with lysine.Exemplary databases that may be useful in identifying protein-ligand interactions around the binding site are described in Du et al., Nucleic Acids Research, Vol. 49, No. D1, Jan. 8, 2021, pp. D1122-D1129, incorporated herein by reference, with the database CovalentInDB accessible at cadd.zju.edu.cn / cidb / . This approach can be used with any of the designs of electrophilic reactive groups for the molecules disclosed herein.

[0511] N-Acyl-N-alkylsulfonamide (NASA) The design of electrophilic reactive groups may be accomplished using NASA chemistry by forming reversible or irreversible bonds with nucleophilic reactive groups located on the enzyme. NASA chemistry is generally described in Nat Commun 9, 1870 (2018), which is incorporated herein by reference. In example embodiments, the enzyme binding moiety can be attached to a linker utilizing an N-acyl N-alkylsulfonamide (NASA) electrophilic reactive group further attached to the protein binding moiety. The M&M containing NASA is covalently attached to the enzyme as NASA chemically reacts with adjacent lysines upon non-covalent attachment to the target enzyme. The NASA-modified M&M then dissociates from the enzyme leaving behind the protein target binder covalently attached to the enzyme. This modified enzyme then binds to the target protein through the newly attached binder, further modifying the protein. In certain example embodiments, the kinase binding moiety is labeled using NASA chemistry. Thus, embodiments include methods of making the compositions disclosed herein using NASA chemistry, as further described in the examples.

[0512] Dibromophenyl benzoate In an example embodiment, the electrophilic reactive group is dibromophenyl benzoate (DB). DB can be used to functionalize the linker by reacting with a nucleophile located on the enzyme. The dibromophenyl group acts as a leaving group to facilitate the reaction, while the benzoate immediately stabilizes the attached moiety. In an example embodiment, the linker connecting the enzyme binding moiety and the protein binding moiety is functionalized with DB to label the target enzyme with the protein binding moiety. DB chemistry is generally described in Takaoka et al. Chem. Sci., (2015), 6, pp. 3217-3224, which is incorporated herein by reference.

[0513] N-Sulfonylpyridone In an example embodiment, the electrophilic reactive group is N-sulfonylpyridone (SP). SP can be used to functionalize the linker by undergoing sulfonylation with a nucleophile located on the enzyme. In an example embodiment, the linker connecting the enzyme binding moiety and the protein binding moiety is functionalized with SP to label the target enzyme with the protein binding moiety. SP chemistry is generally described in K. Matsuo et al. Angew. Chem. Int. Ed. 2018, 57, 659, which is incorporated herein by reference.

[0514] In one embodiment, the electrophilic reactive group is [ka] Includes one of the following.

[0515] Photoreactive groups In one embodiment, the electrophilic reactive group is a photoreactive group. In one embodiment, the photoreactive group is a photoactivated cell surface reactive group. In another embodiment, the photoactivated cell surface reactive group is a benzophenone, azide, or diazirine, where the group is activated to a carbon-centered radical, a nitrene, or a carbene, respectively. In another embodiment, the photoreactive group is a thienyl-substituted alpha-ketoamide, see, e.g., Ota, E. et al., "Thienyl-Substituted α-Ketoamide: A Less Hydrophobic Reactive Group for Photo-Affinity Labeling," ACS Chem. Biol. 2018, 13(4), pp. 876-880.

[0516] Bioorthogonal Group The chimeric molecules disclosed herein may further comprise a bioorthogonal group. The chimeric molecules may be configured to comprise a bioorthogonal group as a device for removing the enzyme-binding moiety from the target enzyme. This occurs when a coupling molecule selected to react with the bioorthogonal molecule is introduced into a system containing the enzyme-linked chimeric molecule and binds to the bioorthogonal group. As a result, the enzyme-linked molecule is no longer operational and cannot bind to the target enzyme. In one embodiment, the enzyme-linking agent comprises a bioorthogonal group. In one embodiment, the enzyme-linking agent is modified to contain a bioorthogonal group. Bioorthogonal chemistry includes chemical reactions that take place in a biological environment without reacting with an endogenous system, e.g., a functional group. Bioorthogonal groups include moieties capable of bioorthogonal chemistry. Non-limiting examples of bioorthogonal groups include tetrazine, triazine, cyclooctene, cyclopropene, and diazo groups.

[0517] In one embodiment, the bioorthogonal group is [ka] Includes one of the following.

[0518] Examples of chimeric molecules Chimeric molecules may be assembled using any combination of the enzyme binding moieties, linkers, electrophilic activating groups and target binding moieties described above. The following description provides certain chimeric molecules that can be made according to the design principles and moiety examples provided above for reference only. In one embodiment, A may be an AMPK binding moiety, an ABL binding moiety or a PKC binding moiety. In another embodiment, B may be a KRAS binding moiety, HSP90, BRD4, BTK, FKB12 F36V In one embodiment, A is an AMPK binding moiety and B is a KRAS binding moiety. In one embodiment, the AMPK binding moiety is [ka] or

[0519] or each of which is as disclosed in Yoo et al., ACS Chem. Biol. 2020, 15, 6, 1604-1612, each of which is incorporated by reference in its entirety.

[0520] In one embodiment, the AMPK binding moieties each have the sequence: * GIYRTDILRTEEGN-NH2, XFE * GIYRTELLKAEEAN-NH2, XFE * GIYRLELLKAEEAN-NH2, XFE * GIYRLELLK-NH2, XFE * AIYRLELLKAEEAN-NH2, XFE * GIYRLELLKAibEEAibN-NH2 and XAE * GIYRLELLKAEAAA-NH2 (where X represents a 4-pentenoic acid residue and the asterisk ( * ) is an N-allyl residue ( *In one embodiment, the AMPK binding moiety is utilized with a KRAS binding molecule selected from the group consisting of HBS 1-7 according to the formula: XFE, wherein XFE represents an N-arylglycine, N-arylglycine, and X-arylglycine. * In one embodiment, the AMPK binding moiety is of the formula: * GIYRLELLKAEAAA-NH2 is utilized with the KRAS binding molecule HB3. See Nickerson et al., An Orthosteric Inhibitor of the RAS-SOS Interaction, doi: 10.1016 / B978-0-12-420146-0.00002-0, which is incorporated by reference in its entirety, with specific references in Table 2.1.

[0521] In one embodiment, the AMPK binding moiety has the formula: [ka] In one embodiment, the AMPK binding moiety has the formula: [ka] where R can be H, Gly, Ala, β-Ala, Val, Ile, Pro, or any other viable substituent known in the art. In one embodiment, the AMPK binding moiety is used with a KRAS binding molecule that is an indole, phenol, sulfonamide, or any modified version thereof. See Sun et al., Angew Chem Int Ed Engl. 2012 Jun. 18; 51(25): 6140-6143. doi: 10.1002 / anie.201201358, which is incorporated herein by reference in its entirety.

[0522] In one embodiment, the AMPK binding moiety has the formula: [ka] JPEG2024513566000348.jpg99170JPEG2024513566000349.jpg103170JPEG2024513566000350.jpg85170JPEG202451356 6000351.jpg84170JPEG2024513566000352.jpg86170JPEG2024513566000353.jpg97170JPEG2024513566000354.jpg1011 70JPEG2024513566000355.jpg103170JPEG2024513566000356.jpg85170JPEG2024513566000357.jpg86170JPEG2024513566000358.jpg91170JPEG2024513566000359.jpg88170JPEG2024513566000360.jpg104170. In one embodiment, the AMPK binding moiety has the formula: Ac-FIGRLCTEILKLREGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-AIGRLCTEILRLRNGA-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; Ac-LAWALRELERELARLC-NH2; Ac-WIGRLCTEIR HRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEIRRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; Ac-LAWRLRELERELARLC-NH2; Ac-FIGRLCTEILKLREGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-AIGRLCTEILRLRNGA-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEILRLRNGN-NH2; FITC-AβLAWALRELERELARLC-NH2; Ac-WIGRLCTEIR H RLRNGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEIRRLRNGN-NH2; FITC-AβLAWRLRELERELARLC-NH2; Ac-WIGRLCTEIR H RLRNGN-NH2; DZ-GLAWRLRELERELARLC-NH2; Ac-WIGRLCTEIK(DZ)RLRNGN-NH2 or Ac-LAWRLRELERELARLC-NH2 (in the formula, R H is L-homoarginine, Aβ is L-β-alanine, DZ is a diazirine photocrosslinker, and FITC is 5-fluorescein isothiocyanate linked via a thiourea bond to the N-terminal amine) are utilized with the SOS peptidomimetic of KRAS binding molecules described in Hong et al., PNAS 2021 May 4 118(18) e2101027118; doi:10.1073 / pnas.2101027118, which is incorporated by reference in its entirety with specific references in Table S2.

[0523] In one embodiment, the AMPK binding moiety has the formula: [ka] wherein the R group can be any substituent known in the art. In one embodiment, R4 is an electrophilic group. In one embodiment, R4 is [ka] (Wherein R is H, [ka] See Yoo et al., ACS Chem. Biol. 2020, 15, 6, 1604-1612, which are incorporated by reference in their entirety.

[0524] In another exemplary embodiment, A is an ABL binding moiety and B is a BRD4 binding moiety.

[0525] In one embodiment, the small molecule comprises an enzyme-binding portion and a target-binding portion that binds to the same kinase and comprises a BCRC-ABL-binding portion. In some embodiments, the BCR-ABL-binding agents are the same. In some embodiments, the BCR-ABL-binding agents are different. In some embodiments, the two BCR-ABL-binding agents are independently selected from the Abl-binding agents detailed elsewhere herein. The Abl-binding agents according to the formulas detailed herein can be further optimized for physicochemical properties, e.g., solubility and / or permeability, and / or pharmacokinetic properties, e.g., microsomal stability or target binding. In one aspect, the small chimeric molecule has the formula: [ka] In one embodiment, the small chimeric molecule has the formula: [ka] In one embodiment, the small chimeric molecule has the formula: [ka] In other example embodiments, the molecule can be any of those depicted in the figures.

[0526] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0527] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0528] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0529] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0530] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0531] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0532] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0533] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0534] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0535] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0536] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0537] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0538] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0539] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0540] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0541] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0542] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0543] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0544] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0545] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0546] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0547] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0548] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0549] In one embodiment, the small chimeric molecule has the formula: [ka] It is described in.

[0550] How to use In another aspect, the chimeric small molecules described above may be used in methods to confer new functions to cellular enzymes or modulate the activity of cellular enzymes. The chimeric molecules find use for treatment in a variety of diseases and disorders. In certain examples of embodiments, the protein binding moiety may bind to a target of interest and preferably localize to the region of the target of interest, allowing the kinase to bind and modify the target. Exemplary applications include use in rewiring cellular signaling. See Lim et al., Nat Rev Mol Cell Biol 2010, 11(6), pp. 393-403. Cell signaling can be addressed by adding phosphoryl groups to specific signaling proteins of interest with dose and temporal control that allows rewiring in kinase signaling pathways in disease or health. The multifunctional system herein may allow targeted degradation of proteins whose phosphorylation sites are targets for recruiting ubiquitin ligases and signal degradation. See Toure et al., Angewandte Chemie (Inter'l ed. In English) 2016, 55(6), 1966-73. Similarly, preventing protein aggregation can be useful in cancer treatment approaches. As described herein, the addition of negatively charged phosphoryl groups using bifunctional molecules on proteins prone to aggregation can increase solubility and reduce self-aggregation. Guo et al., FEBS Letters, 2005, 579(17), 3574-3578; Zhang et al., Protein Expression and Purification 2004 36(2) 207-216. Exemplary embodiments are also provided that include methods of treatment of kinasopathies. Exemplary embodiments further include modulation of nucleotide binding proteins, which can include the use of orthogonally tagged nucleases, e.g., Cas, and phosphorylation of transcription factors to affect binding. In one embodiment, the invention described herein relates to a method for therapy in which cells are modified ex vivo with multifunctional molecules that modify at least one target substrate, followed by administration of the edited cells to a patient in need thereof.

[0551] Methods for modifying target substrates The chimeric small molecules disclosed herein can be utilized in methods of modifying target substrates. Methods of modifying target substrates can include creating repurposed / initialized cellular enzymes by delivering chimeric molecules as described herein. In some examples of embodiments, chimeric small molecules can be used to inhibit nucleotide binding proteins, inhibit oncogenic kinases, create neoantigens, and elicit immune responses such as molecular prostheses of kinasopathies, treatment of pathogens, and induction of receptor tyrosine kinase signaling.

[0552] A method of modifying a substrate is provided, which may be in a cell. In one embodiment, a chimeric small molecule as described herein is introduced. In one embodiment, modifying includes inducing a post-translational modification of the target protein. In one embodiment, the post-translational modification is phosphorylation. The method includes administering the chimeric small molecule to a cell or cell population. The method of modifying a target substrate may include contacting the target substrate with a chimeric small molecule of the present invention, e.g., a bifunctional molecule. The contacting may allow binding to or association with the target substrate or a molecule in proximity to the target substrate. In some embodiments, the bifunctional molecule may act as a molecular adhesive by binding to a specific protein, e.g., TKR or ABL, and modifying aberrant function in the cell. The modification may be by inducing a conformational change through binding, by changing the stability of the structure, by phosphorylation of the target, or through another mechanism that affects the behavior of the target substrate. By way of example, activation or inactivation of a target substrate via binding of a bifunctional molecule results in modification of the target substrate at one or more new modification sites that would otherwise remain unmodified if the bifunctional molecule were not bound to the target substrate. In some embodiments, the method includes inducing phosphorylation of a protein in a cell. The method can include contacting the target substrate with a chimeric small molecule.

[0553] In one embodiment, the chimeric molecule can label a cellular enzyme with a target binder for a target substrate via an electrophilic reactive group moiety. The electrophilic reactive group reacts with and binds to a nucleophilic side chain on the cellular enzyme. Labeling of the cellular enzyme can allow binding or association with the target substrate or a molecule proximal to the target substrate, facilitating modification of the target substrate. In one embodiment, this approach allows the use of enzyme inhibitor moieties, as well as activators and neutral binding molecules that induce target modification. Such enzyme binders tethered with electrophilic reactive groups, e.g., chemoselective electrophilic warheads, exhibit site-specific labeling of side chain nucleophilic residues, e.g., nucleophilic side chain amino acids, proximal to the inhibitor binding site. In general, labeling proximal to the inhibitor binding site refers to a reactive group at, within, or at a distance from the binding site of the binding moiety, which allows the electrophilic reactive group to react at or near the binding site of the binding moiety in time and / or space. The tethering of the electrophilic warhead can include a linker, bond and / or an exit vector or adapter, where in some instances, in one embodiment, when the target substrate is not a natural substrate for the enzyme or activation of the enzyme by the binding moiety is not activated by binding to the activator moiety, results in modification of the target substrate by the enzyme at one or more new modification sites that would otherwise remain unmodified by the enzyme. The modification can be by inducing a conformational change via binding, altering the stability of the structure, by phosphorylation of the target, or through other mechanisms that affect the behavior of the target substrate, such as removal of groups, such as, for example, phosphatases, methyltransferases. The modifying can include post-translational modifications disclosed herein, including, for example, phosphorylation, hydroxylation, acetylation, methylation, glycosylation, prenylation, amidation, elimination, lipidation, acylation, lipoylation, deacetylation, formylation, S-nitrosylation, S-sulfenylation, sulfonylation, sulfinylation, succinylation, sulfation, carbonylation, or alkylation. In one embodiment, the method comprises inducing phosphorylation of a protein in or on a cell.The method can include contacting a target substrate with a chimeric small molecule.In one embodiment, the target substrate is in proximity to a kinase specific for the enzyme-binding portion of the molecule. The chimeric small molecule that induces phosphorylation can optionally be provided with adenosine monophosphate (AMP) or another molecule that provides an additional phosphate group. Without being bound by theory, the addition of AMP or another phosphate-providing molecule can enhance phosphorylation.

[0554] In certain examples of embodiments, inhibition of nucleotide binding proteins may include inhibition of binding of CRISPR-Cas proteins to nucleic acids or transcription factors that bind to DNA. Thus, proteins that have been modified to include a binding domain that can be targeted by Cas9, including an orthogonal tag, such as an FKBP binding domain, may be inhibited by the use of a small molecule that includes an orthogonal tag, such as dTAG. Sequence-specific modular adapters consisting of DNA binding proteins and self-ligating protein tags may be utilized. See, for example, Nguyen et al., Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics, Chem Sci., 40(2019) doi: 10.1039 / C9SC02990G. Similarly, nucleotide binding may be modified through modification of transcription factors with chimeric small molecules. Methods of transcription factor modification are provided, as post-translational phosphorylation of transcription factors may be required for direct binding interactions or conformational changes in transcription factors that may result in, activate, or inhibit gene transcription. In certain exemplary embodiments, the method of use can include inducing an immune response, creating autoantigens, and inactivating targets. In certain exemplary embodiments, hyperphosphorylation or neophosphorylation of target proteins can result in immune recruitment to the target, for example, via triggering presentation of neoepitopes and T cell attack on cells presenting the epitope. In one application, the small molecules disclosed herein are utilized in human leukocyte antigen (HLA) presentation and immune responses. Neophosphorylation to induce an immune response can find use in cancer immunotherapy approaches. In an exemplary approach, a kinase is selected for phosphorylation of p53, for example, Ser33, Ser315, and / or Thr82. This phosphorylation results in subsequent binding and conformational changes that result in activation as a transcription factor. See, for example, Ryan and Vousden, Nature, 419 (2002).Therefore, designing molecules containing binding moieties for enzymes that phosphorylate or dephosphorylate with a target for p53 may allow control of nucleotide binding based on the desired conformation of the transcription factor. See also, e.g., Mattiske T, Tan MH, Dearsley O, Cloosterman D, Hii ​​CS, Gecz J et al. (2018) Regulating transcriptional activity by phosphorylation: A new mechanism for the ARX homeodomain transcription factor. PLoS ONE 13(11): e0206914. Doi:10.1371 / journal.pone.0206914.

[0555] Quinasopathy Treatment of kinasopathies may also be contemplated, see generally Lahiry et al., Nature Reviews Genetics, 2011, with disclosure of Table 1 of hereditary kinasopathies, incorporated herein by reference. Thus, for kinasopathies with loss of function, the chimeric small molecules described in the present invention can recruit a functional kinase to provide the lost function. See, for example, Lahiry et al., Nature Reviews Genetics vol. 11, pp. 60-74 (2010) (discussing various germline disorders and cancers associated with kinase dysfunction), particularly Supplementary Table 1 for intrinsic kinasopathies and Supplementary Table 2 for kinases associated with cancer, incorporated herein by reference. In one exemplary embodiment, Src family protein tyrosine kinases (SFKs) bind to the conserved Y in an active A-loop conformation. ASFK is stabilized in an active conformation by phosphorylation. By targeting SFK for modification, e.g., phosphorylation, in the A-loop, treatment of abnormal SFK can address kinasopathies associated with SKF, e.g., ALL, CML. See, e.g., Mechanism of Drug-Resistance in Kinases, Expert Opin Investig Drugs. 2011 February; 20(2): 153-208; doi: 10.1517 / 13543784.2011.546344.

[0556] Methods of Use with Kinase Inhibitor Binding Moieties In one embodiment, the method comprises the step of: 1 -EL 2-B, where A is an enzyme binding moiety specific for the cellular enzyme to be repurposed / initialized, B is a target binding moiety specific for the target substrate to be modified, L is a linker, and EI is an electrophilic reactive group, whereby the chimeric molecule labels the cellular enzyme with a target binding moiety for the target substrate, and modifies the target substrate by binding the repurposed / initialized enzyme to the target substrate via the target binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. In one embodiment, the enzyme binding moiety has a half-life that is about 2, 3, 4, 5, 6, or 7 times shorter than the half-life of the repurposed / initialized enzyme. In one embodiment, the initialized enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In one embodiment, the inhibitor is an enzyme binding moiety. In one embodiment, the enzyme to be repurposed / reprogrammed is a kinase and the enzyme-binding moiety is a kinase inhibitor. In one embodiment, the kinase inhibitor is a "promiscuous" kinase inhibitor. In one embodiment, the method includes administering a coupling molecule, thereby quenching the inhibitory activity of the enzyme inhibitor. In one embodiment, the coupling molecule is one or more of an aldehyde, an alkene, an alkyne, a strained alkyne, a cyclooctyne, a trans-cyclooctene, a cyclopropene, an oxanorbornadiene, a norbornene, a phosphine, an electron-rich dienophile, an isonitrile, an isocyanopropionate, a tetrazole, a 2-acylboronic acid, or any derivative thereof. In one embodiment, the cyclooctyne derivative includes a dibenzocyclooctyne, a biarylazacyclooctyne, or a dimethoxyazacyclooctyne. In one embodiment, the method includes a strained alkyne including a bicyclononyne or a dioxabiaryldecyne.

[0557] In one embodiment, the method includes a chimeric small molecule in which the enzyme-binding moiety has a half-life that is about 2, 3, 4, 5, or 6 times shorter than the half-life of the initialized enzyme. In some embodiments, the initialized enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In some embodiments, the enzyme-binding agent is an inhibitor. In some embodiments, the initialized enzyme is a kinase and the enzyme-binding agent is a kinase inhibitor. In some embodiments, the kinase inhibitor is a promiscuous kinase inhibitor.

[0558] In one embodiment, the method includes a small chimeric molecule containing a bioorthogonal group that allows the enzyme linkage to react and bind with a coupling molecule. In one embodiment, the method further includes administering a coupling molecule. The coupling small molecule is administered to react with the bioorthogonal group on the chimeric small molecule, thereby quenching the kinase inhibitor from binding to the kinase. In one embodiment, the coupling molecule is one or more of an aldehyde, alkene, alkyne, strained alkyne, cyclooctyne, trans-cyclooctene, cyclopropene, oxanorbornadiene, norbornene, phosphine, electron rich dienophile, isonitrile, isocyanopropionate, tetrazole, 2-acylboronic acid, or any derivative thereof. In one embodiment, the cyclooctyne derivative includes dibenzocyclooctyne, biarylazacyclooctyne, or dimethoxyazacyclooctyne. In one embodiment, the strained alkyne includes bicyclononyne or dioxabiaryldecyne.

[0559] In one embodiment, the coupling molecule is co-administered with the chimeric small molecule. In one embodiment, the coupling molecule is administered after administration of the chimeric small molecule. In some embodiments, the coupling molecule is administered within 24 hours, or within 12 hours, or within 11 hours, or within 10 hours, or within 9 hours, or within 8 hours, or within 7 hours, or within 6 hours, or within 5 hours, or within 4 hours, or within 3 hours, or within 2 hours, or within 1 hour, or within 30 minutes or less of administration of the small chimeric molecule.

[0560] Delivery of coupling molecules In one aspect, the method includes the additional step of administering or delivering a coupling molecule. The coupling molecule reacts with the bioorthogonal group on the enzyme targeting moiety as described above. This reaction inhibits the binding of the binding moiety to the enzyme. When used with a chimeric small molecule that contains an electrophilic reactive group, the enzyme binding moiety may be released from the chimeric small molecule and the coupling molecule may bind to the bioorthogonal group of the enzyme binding moiety, thereby preventing the enzyme binding agent from further binding of the enzyme. In one embodiment, the coupling molecule is used with an enzyme binding moiety that is an inhibitor of the enzyme.

[0561] The coupling molecule may be administered in any pharmaceutical formulation, effective amount, and dosage form as previously described. The coupling molecule may be delivered using any previously described method, or may be administered as described herein with any co-therapeutic agent or combination. The chimeric small molecule and the coupling molecule may be delivered or administered simultaneously or sequentially. The simultaneous delivery of the coupling molecule and the chimeric small molecule may be within the same delivery method or using separate delivery methods. The simultaneous but separate delivery of the coupling small molecule may be the same type of delivery method or different types of delivery methods as previously described. The sequential delivery of the coupling molecule may be using the same type of delivery method or different types of delivery methods. The sequential delivery of the coupling molecule may be within 24 hours, or within 12 hours, or within 11 hours, or within 10 hours, or within 9 hours, or within 8 hours, or within 7 hours, or within 6 hours, or within 5 hours, or within 4 hours, or within 3 hours, or within 2 hours, or within 1 hour, or within 30 minutes or less of administration of the chimeric small molecule.

[0562] Coupling Molecules In one embodiment, a coupling molecule is introduced into a system containing the small chimeric molecule. As the coupling molecule comes into contact with the chimeric small molecule bound to the target enzyme, it quenches the binding between the enzyme-binding moiety and the target enzyme. In one embodiment, the coupling molecule is a molecule that can undergo a reaction with a bioorthogonal that is a substituent of the enzyme-binding moiety. The reaction results in the coupling molecule being attached to the enzyme-binding moiety, and as a result, the enzyme-binding moiety is no longer bound to the enzyme. In one embodiment, the coupling molecule can react with the bioorthogonal moiety by aldehyde / ketone nucleophilic reaction, dipolar cycloaddition, phosphine ligation, Diels-Alder cycloaddition, [4+1] cycloaddition, nitrile imine-alkene reaction, or 2-acylboronic acid condensation, or any other bioorthogonal reaction.

[0563] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled by aldehyde / ketone nucleophilic condensation. Conveniently, for example, an aldehyde is coupled with an amine group, such as an alkoxyamine or hydrazine. This approach is effective on the cell surface, although intracellular metabolites contain aldehydes and ketones. In one embodiment, the coupling molecule is an aldehyde.

[0564] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled by a dipolar cycloaddition. The dipolar cycloaddition typically occurs between an azide and an alkyne, either in the presence or absence of copper. In the case of copper-free dipolar cycloaddition, the alkyne is strained to facilitate the reaction. In most cases, the strained alkyne is a cyclooctyne or any derivative thereof. Non-limiting examples of cyclooctynes ​​include dibenzocyclooctyne, biarylazacyclooctynone, and dimethoxyazacyclooctyne. In one embodiment, the coupling molecule is an alkyne. In one embodiment, the coupling molecule is a strained alkyne. In one embodiment, the coupling molecule is a cyclooctyne. It is understood that any strained alkyne may be used, but other non-limiting examples include bicyclononyne, dioxabiaryldecyne, and any derivatives thereof.

[0565] The dipolar cycloaddition may also involve the reaction between an oxanorbornadiene and an azide. In this case, the cycloaddition between an oxanorbornadiene and an azide is followed by a spontaneous retro-Diels-Alder reaction to produce a triazole and a furan. In one embodiment, the coupling molecule is an oxanorbornadiene or any derivative thereof.

[0566] The dipolar cycloaddition may also include the reaction between norbornene and a nitrile oxide. In one embodiment, the coupling molecule is norbornene. The coupling molecule may also undergo a dipolar cycloaddition with another dipolar molecule, such as a nitrone, (imino)synthon, or 1,3-dithiolium-4-oleate, and may be constructed from the corresponding unsaturated hydrocarbon.

[0567] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled by phosphine ligation, or interchangeably referred to as Staudinger ligation. Phosphine ligation typically occurs between an azide and a phosphine, typically forming a phosphine oxide and a stable amide linkage, or, if an electron-deficient aromatic azide is used, forming an iminophosphorane. In one embodiment, the coupling molecule is a phosphine or any derivative thereof. Phosphine ligation may also involve a cyclopropane in place of the azide. Non-limiting examples of cyclopropanes include cyclopropenone, cyclopropenethione, and cyclopropenium ion.

[0568] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled by a Diels-Alder cycloaddition. The reaction is an inverse electron demand Diels-Alder, classically occurring between an electron-deficient diene and an electron-rich dienophile. In one embodiment, the coupling molecule is an electron-rich dienophile. The Diels-Alder cycloaddition may include a tetrazine ligation, in which a strained unsaturated hydrocarbon and a tetrazine or triazene are coupled to form a pyridazine. In one embodiment, the coupling molecule is a strained unsaturated hydrocarbon. The unsaturated hydrocarbon may also be cyclic. Non-limiting examples of strained cyclic unsaturated hydrocarbons include cyclooctyne, trans-cyclooctene, norbornene, cyclopropene, and azetine. In one embodiment, the coupling molecule is cyclooctyne, trans-cyclooctene, or a derivative thereof.

[0569] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled by a [4+1] cycloaddition. This reaction classically involves coupling of an isonitrile with a tetrazole followed by spontaneous retro Diels-Alder elimination. The conjugate of the reaction is more stable when the isonitrile is tertiary. However, when the isonitrile is primary or secondary, less stable conjugates are formed. In one embodiment, the coupling molecule is an isonitrile or any derivative thereof. In one embodiment, the isonitrile is tertiary. In one embodiment, the coupling molecule is an isocyanopropanoate or any derivative thereof.

[0570] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled via nitrile imine-alkene cycloaddition. Classically, tetrazole is photolyzed to produce a nitrile imine that readily couples with unsaturated hydrocarbons. The wavelength required for photolysis depends on the substituents on the tetrazine. However, photolysis is not required when hydrazonoyl chloride is present, which spontaneously produces the nitrile imine from the tetrazole at neutral pH. In one embodiment, the coupling molecule is an unsaturated hydrocarbon, optionally introduced using hydrazonoyl chloride.

[0571] In one embodiment, the coupling molecule and the bioorthogonal moiety are coupled by 2-acylboronic acid condensation. In this reaction, a boronic acid is coupled with an amine to form a stable diazoborine. In one embodiment, the coupling molecule is a 2-acylboronic acid or any derivative thereof. See, for example, Shieh P, Bertozzi CR, Design strategies for bioorthogonal smart probes. Org Biomol Chem. 2014;12(46):9307-9320. doi:10.1039 / c4ob01632g and Mike LWJ et al., Recent developments in bioorthogonal chemistry and the orthogonality within, Curr. Opin. Chem. Biol., 2021, 60, 79-88, which are incorporated herein by reference.

[0572] Carcinogenicity Application In one embodiment, the disease is associated with cancer. In particular, the disease is oncogenesis. Many oncogenic targets are known and can be regulated by post-translational modifications. See, for example, Chen, L., Liu, S., and Tao, Y. Regulating tumor suppressor genes: post-translational modifications. Sig Transduct Target Ther 5, 90(2020); doi:10.1038 / s41392-020-0196-9. Exemplary types of post-translational modifications of proteins involved in oncogenesis and their expression patterns can be found in Table 1 of Sharma et al. (2019). Post-Translational Modifications (PTMs), from a Cancer Perspective: An Overview. Oncogen 2(3): p. 12, specifically incorporated herein by reference.

[0573] The chimeric small molecules disclosed herein can be utilized in methods of treating cancer. The methods of treating cancer can include creating repurposed / initialized cellular enzymes by administering chimeric molecules as described herein. The chimeric molecules label the cellular enzymes with oncogenic protein binding agents via electrophilic reactive group moieties. The electrophilic reactive groups react and bind with nucleophilic side chains on the cellular enzymes. Labeling the cellular enzymes can allow binding or association with oncogenic proteins or molecules in close proximity to oncogenic proteins, facilitating modification of target substrates. In one aspect, the method includes inducing phosphorylation of oncogenic proteins in or on cells. The method can include contacting the oncogenic protein with the chimeric small molecule. In one embodiment, the oncogenic protein is in close proximity to a kinase specific for the enzyme-binding portion of the molecule. The chimeric small molecule that induces phosphorylation can optionally be provided with adenosine monophosphate (AMP) or another molecule that provides an additional phosphate group. Without being bound by theory, the addition of AMP or other phosphate-providing molecules can enhance phosphorylation.

[0574] Methods of treating cancer are provided. The methods of treating cancer include administering to a patient a compound of the formula: ALEB, AL 1 -EL 2 -B or A-(L) n The present invention includes creating an initialized cellular enzyme by administering to a subject in need thereof a chimeric molecule of formula (I)-B, where A is an enzyme binding moiety, L is a linker, n is 0-6, E is an electrophilic reactive group, and B is an oncogenic protein to be modified, whereby the chimeric molecule labels the cellular enzyme with a targeted binding agent for the target substrate, and modifying the oncogenic protein by binding the repurposed / initialized enzyme to the target substrate via the targeted binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. In one embodiment, the targeted binding agents are KRAS, RAS, FKPB, 12F36V, EGFR, HSP90, BTK, MDM2, BRD4, BCR-ABL, NF-kB, LDH-A, p53, GP73, MUC1, MUC16, CD44, GPCR, HMGB1, RIOK1, CHK1, UBE2F, HuR, PTEN, STAT-3, osteopontin, EGFR, AKT, DAPK1, Rho, Ubc9, FOXK2, HIC1, HER2, BRAF, BCL-2, CD117, (KIT), ALK, PI3K, Delta, DNMT1 or SMO. In one embodiment, the cellular enzyme to be reprogrammed is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, translocase. In one embodiment, the enzyme binding agent is an enzyme inhibitor, preferably a kinase inhibitor. In one embodiment, the kinase inhibitor is specific for PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, NOP, μ opioid receptor, δ opioid receptor, UMPK, SphK, or GSK-3. In one embodiment, a coupling molecule is administered, thereby quenching the inhibitory activity of the enzyme inhibitor.

[0575] A method for treating a disease associated with abnormal KRAS signaling is provided, comprising administering a composition comprising a bifunctional molecule, the bifunctional molecule comprising a KRAS binding molecule and a kinase binding molecule as described herein. In one embodiment, the enzyme binding molecule is a target of an enzyme selected from the group consisting of PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, NOP, μ opioid receptor, δ opioid receptor, UMPK, SphK, or GSK-3. In one embodiment, the kinase binding molecule is an AMPK binding moiety. In one embodiment, the KRAS is G12C In one embodiment, the bifunctional molecule phosphorylates one or more residues on KRAS selected from the group consisting of Ser17, Ser39, Ser65, Ser106, Ser122, Ser136, Ser2, Thr2, Thr35, Thr50, Thr74, Thr87, Thr124, Thr127, and Thr148.

[0576] In one embodiment, a method of treating cancer in a cell is provided, comprising administering a chimeric small molecule of the present invention. In one embodiment, the small molecule comprises a P13K kinase binding agent, a linker, an electrophilic reactive group, and a p53 target binding agent, such as a p53 target binding agent based on idasanutlin. In one embodiment, the molecule comprises a P13K binding agent based on the inhibitor PIK108, which optionally further comprises a bioorthogonal group, such as cyclopropenyl. The binding moiety PIK108 comprises a linker connected to an electrophilic reactive group, such as dibromophenyl benzoate. The electrophilic reactive group is then connected to a p53 protein target binding agent, optionally via a linker. Upon binding to PI3K kinase via PIK108, the adjacent lysine of the binding pocket of PI3K reacts with the lysine reactive group (e.g., dibromophenyl benzoate), releasing the kinase inhibitor and leaving P13K tagged with the p53 binding agent. Kinases that are covalently labeled with the targeted binding agent can then hyperphosphorylate and / or neophosphorylate p53. Administration of a tetrazine coupling molecule, when presented on the PI3K binding molecule, can quench the cyclopropenyl biorthogonal group and inactivate the released kinase binding moiety.

[0577] An enzyme and a target binding moiety that binds to the same enzyme In one embodiment, both the enzyme and the target are the same, e.g., two of the same type of enzyme. Advantageously, the binding of two enzymes, e.g., two kinases, may be utilized to provide a molecular glue, with the chimeric molecule configured to bind the two enzymes in a manner that allows them to assume a desired configuration, e.g., resulting in a dimeric or multimeric enzyme that is adapted to an active or inactive conformation. Thus, there is also provided a method of modifying a target substrate in a subject in need thereof, comprising administering a molecule disclosed herein. The binding of the two kinases by the bifunctional molecule may lock the kinases in an inactive state, or may "flip" the state of the kinases, e.g., from inactive to active or from active to inactive. In some embodiments, the subject has a condition to be treated, which may be cancer, and the cancer is associated with an oncogenic fusion.

[0578] In one embodiment, cancer is treated using molecules that contain binding moieties that each bind to the same type of enzyme (e.g., kinase), where a therapeutically effective amount of the composition is administered to the cancer patient. In one embodiment, a therapeutically effective amount of a monomer capable of binding to an enzyme, e.g., kinase, as described herein is administered to the cancer patient after the chimeric small molecule is administered. The purpose of the monomer is to reveal the effect of the chimeric small molecule. Upon introduction into the affected cell, the monomer competitively binds to the same kinase as the chimeric small molecule, where dissociation of the chimeric small molecule stops the effect that the chimeric small molecule had on the enzyme, e.g., an effect that may change the conformation of the active or inactive state. In one embodiment, the disease is characterized by aberrant kinase signaling, in one embodiment, by aberrant BCR-ABL kinase signaling. Additional cancerous fusions (e.g., via chromosomal translocations resulting in gene fusions encoding cancerous fusion proteins) and cancers characterized by aberrant kinase signaling are further detailed herein.

[0579] Exemplary oncogenic fusion proteins that can be treated by the binding of multimeric enzymes include fusions related to the ABL protein. The ABL protein is a non-receptor tyrosine kinase that is normally under well-organized regulation. However, chromosomal translocations that join the ABL gene with genes encoding other proteins result in various oncogenic fusion proteins (BCR-ABL, TEL-ABL, NUP214-ABL, etc.) that are prone to dimerization (or oligomerization) and subsequent autophosphorylation. Thus, the ABL kinase becomes constitutively active, resulting in diseases such as chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and other myeloproliferative disorders. One example of an oncogenic fusion is BCR-ABL. The binding of two kinases by a bifunctional molecule can lock the kinase in an inactive state. This is particularly true for kinases such as Abl, which must form a complex to become active.

[0580] In an exemplary embodiment, the cancer is characterized by a cancerous fusion of a kinase, for example, ABL kinase. Oncogenic ABL fusion proteins are known in the art and are involved in a variety of proliferative disorders. Chromosomal translocations occur joining the gene for ABL with genes encoding other proteins, resulting in various proteins prone to dimerization (or oligomerization) and autophosphorylation, making ABL kinase constitutively active and resulting in myeloproliferative disorders. In an exemplary embodiment, the cancerous fusion is TEL-ABL or NUP214-ABL.

[0581] In one embodiment, the targeted binding agent and the enzyme binding moiety target BCR-ABL. In one embodiment, the chimeric small molecule has the formula: A-(L) n -B

[0582] where A and B are first and second kinase binding moieties, L is a linker, and n is 0-6. Both A and B may be chemically identical or chemically distinct, but in both scenarios bind to the same kinase type, e.g., both A and B bind to Abl kinase, such that two copies of the same complex are linked in close proximity to each other by a chimeric small molecule. The Abl binding moiety can be selected from any of the Abl binding moieties disclosed herein.

[0583] In one embodiment, the ABL kinase binding moiety and B are [ka] is selected from the group consisting of:

[0584] Although the discussion of the BCR-ABL method has been discussed with respect to small molecule-mediated conformational locking, e.g., molecular adhesives, described herein, a similar approach is contemplated with other oncogenic fusions. In one embodiment, neophosphorylation in oncogenic targets is used as an autoantigen. A notable class of oncogenic targets for neophosphorylation are kinases activated by gene fusions. They are generated by translocations or other chromosomal rearrangements and are associated with both hematopoietic malignancies and solid tumors. Kinase fusions vary considerably between cancer types, reflecting the differences in the etiology of these tumors.

[0585] Translocation events in cancer have been shown to be associated with fusions involving ALK, BRAF, EGFR, FGFR1, 2 and 3, NTRK1, 2 and 3, PDGFRA, PRKCA and B, RAF1, RET, ROS1, FGR, MET, PIK3CA and PKN1. Chimeric small molecules were designed as molecular adhesives for targeting fusions using the small molecule design considerations described herein. In particular, druggable kinases involved in fusions including AKT3, ALK, BRAF, BRD4, CD74, EGFR, EML4, ERBB4, ESR1, FGFR2, FGFR3, JAK2, MET, NOTCH1, NRG1, NTRK1, NTRK3, NUP214-ABL1, PDGFRA, PDGFRB, PML-RARA, RAF1, RET, ROS1, TMPRSS2 and TRIM33-RET were also identified.

[0586] Additional fusions that may be triggered by the chimeric small molecules taught herein include, but are not limited to, ACSM2B--NOTCH2, ACTG2-ALK, ACVR2A--AKT3, AFF3--TMPRSS2, AGGF1--RAF1, AGK-BRAF, AKAP13--NRG1, AKAP13--NTRK3, AKAP13-RET, AKAP7--ESR1, AKT3-ADSS, AKT3--CDC14A, AKT3--HEATR1, AKT3--PPP2R2A, AKT3-PTPRR, ALK--GALNT14, AL K-SCEL, ALK--STK39, AP3B1-BRAF, ARHGEF25--NTRK1, ARID2--TMPRSS2, ATAD2--ERBB4, ATF7IP--TMPRSS2, ATG7-BRAF, ATP1A1--NOTCH2, ATP1B1--NRG 1, ATP2B4--ERBB4, B4GALT1--RAF1, BACE2--TMPRSS2, BAIAP2L1-MET, BCL2L11-BRAF, BCR--ABL1, BRAF--AP3B1, BRAF--ATG7, BRAF--CUL1, BRAF--DENN D2A, BRAF--FAM114A2, BRAF-HIBADH, BRAF--MACF1, BRAF--MED4, BRAF--SND1, BRAF-SUGCT, BRD4--AKAP8L, BRD4--CC2D1A, BRD4--CSE1L, BRD4--CSN2, BRD4--CYP4F22, BRD4--GNAT1, BRD4--MFSD12, BRD4--NOTCH3, BRD4--PGLYRP1, BRD4--PGLYRP2, BRD4--SLC1A6, BRD4--ZC3H15, C8orf34-MET, CBR4--E RBB4, CCAR2--FGFR2, CCDC6-RET, CD74--ROS1, CDC27-BRAF, CDK12--JAK2, CDK2-ALK, CEL--NTRK1, CEP170--AKT3, CEP85L--ROS1, CHIC2-PDGFRA, CLCN 6--RAF1, CLOCK-PDGFRA, CLTC--ROS1, CMTM8--RAF1, CUX1-BRAF, DANCR-PDGFRA, DLG5-RET, DLG5--TMPRSS2, DNM1--FGFR2, DOCK8--JAK2, DSTYK-BRAF,EGFR-ACADM、EGFR--C7orf72、EGFR-CHODL、EGFR-DYM、EGFR--GRB10、EGFR--GYG1、EGFR--INSL4、EGFR-LYST、EGFR--RCL1、EGFR--SEPT14、EGFR--SEPT 14P24、EGFR--TEAD3、EGFR--VSTM2A、EIF5--NOTCH2、EML4-ALK、EML4--NTRK3、EPHB2--NTRK1、EPS15L1--BRD4、ERBB4--RBM33、ERBB4--SDCCAG8、ERBB4 --SLC25A10、ERC1-RET、ERG--TMPRSS2、ESR1-ASPH、ESR1--BNC2、ESR1-GNAS、ESR1--MYCT1、ESR1--、DE7B、ESR1-POLH、ESR1--POLR2E、ESR1--SIM1、ESR1--SYNE1、ESR1--TFB1M、ESR1-UTRN、ETV6--NTRK3、EZR--ROS1、FAM114A2-BRAF、FAM193A--FGFR3、FAT1--NTRK3、FBXL20--NOTCH2、FGFR2--AP1M1、FGF R2--BICC1、FGFR2--CASP7、FGFR2--CCAR2、FGFR2--CCDC186、FGFR2--CCDC6、FGFR2--CTNNA3、FGFR2--EIF4A2、FGFR2--ENPP2、FGFR2--FRK、FGFR2--OFD 1、FGFR2--SHTN1、FGFR2--SMN1、FGFR2--TACC2、FGFR2--USP10、FGFR3-AES、FGFR3--AMBRA1、FGFR3--ELAVL3、FGFR3--FBXO28、FGFR3--MLLT10、FGFR3-- -TACC3、FKBP15-RET、FOXO1-PDGFRB、FRMD3--BRD4、GPRC5A--NRG1、GTF2IRD1-ALK、HDLBP--TMPRSS2、HIBADH-BRAF、HMGN2P46--TMPRSS2、IGHGP--NOTC H1、IRF2BP2--NTRK1、JAK2--CSTF3、JAK2--DOCK8、JAK2-GLDC、JAK2--RCL1、KANSL1L--ERBB4、KCNQ5-ALK、KDM7A-BRAF、KIAA1211-PDGFRA、KIF5B-MET、KLHL7-BRAF、LMNA--NTRK1、LMNA--RAF1、LYN--NTRK3、MACF1-BRAF、MAGI3--NOTCH2、MALAT1-ALK、MAP3K7-PDGFRB、MAPK1--NOTCH1、MESDC2--TMPRSS2 、MET--C8orf34、MET--CNTNAP5、MET--DYNC1I1、MET--ST7-AS2、MET-TFG、MET--WNT2、MGP--ESR1、MKRN1-BRAF、MPRIP--RAF1、NCOA4-RET、NDUFS4--TM PRSS2、NOTCH1--CHST9、NOTCH1--EXD3、NOTCH1--LCN15、NOTCH1--MAPK1、NOTCH1--SDCCAG3、NOTCH1--SPTAN1、NOTCH1--TMEM117、NOTCH2--ADAM30、 NOTCH2--CWH43、NOTCH2-MNDA、NOTCH2--PSMA5、NOTCH2--REG4、NOTCH2--SEC22B、NOTCH2--SPAG17、NRG1--PMEPA1、NRG1--STMN2、NTRK1--DYNC2H1、NT RK3--ETV6、NTRK3--LOXL2、NTRK3--PEAK1、NTRK3-RBPMS、NUP214--ABL1、OXR1-MET、PAICS-PDGFRA、PAPD7--RAF1、PCM1--NRG1、PDE7A--NRG1、PDE9A- -TMPRSS2、PDGFRA--FIP1L1、PDGFRA--GRID2、PDGFRA--SCFD2、PDGFRA--USP8、PKHD1--ESR1、PLGRKT--JAK2、PML-RARA、PPP4R3B-ALK、PTGFRN--NOTCH2 、PTPRZ1-MET、RAB3IL1--NRG1、RAB5B-ALK、RAC1P2-EGFR、RAF1--AGGF1、RAF1--C9orf153、RAF1--EIF3L、RAF1--GXYLT2、RAF1--IQSEC1、RAF1--NXPH1 、RAF1--PHC3、RAF1--RPL32、RAF1--SSUH2、RAF1--TRAK1、RBPMS--NTRK3、RET--CCDC6、RET-MRLN、RET--NCOA4、RHBDD2-EGFR、ROS1--CD74、ROS1-CLTC、Including ROS1--FBXO9, SCP2--TMPRSS2, SDC4--NRG1, SEC61G-EGFR, SIK3--TMPRSS2, SLC34A2--ROS1, SLC45A3--TMPRSS2, SMAD4--NRG1, SMARCA4--BRD4, SMN1--FGFR2, SND1-BRAF, SPECC1L-RET, SQSTM1--NTRK1, SSBP2--NTRK1, STRN-ALK, SYNE1--ESR1, TACC3--FGFR3, TAX1BP1-BRAF, TBL1XR1-RET, TCEA1-EGFR, TFG-MET, TFG--NTRK1, THAP7--NRG1, THBS1--NRG1, TMEFF2--TMPRSS2, TMEM165-PDGFRA, TMPRSS2--ATF7IP, TMPRSS2-BRAF, TMPRSS2--CALB1, TMPRSS2-DGKG, TMPRSS2--DIAPH1, TMPRSS2--EML4, TMPRSS2-ERG, TMPRSS2--ETV4, TMPRSS2--ETV5, TMPRSS2--GUCA2A, TMPRSS2-HDLBP, TMPRSS2--HSF2BP, TMPRSS2--INPP4B, TMPRSS2--IRS2, TMPRSS2--KLF4, TMPRSS2--MORC3, TMPRSS2--RPS6, TMPRSS2--MX1, TMPRSS2--PDE9A, TMPRSS2--PHF12, TMPRSS2-SARS, TMPRSS2--TMEFF2, TMPRSS2--TMEM109, TPM1-ALK, TPM3--NTRK1, TRAK1--RAF1, TRIM24-BRAF, TRIM27-RET, TTC13--JAK2, TULP4--ESR1, UBXN8--NRG1, USP28--TMPRSS2, USP46-PDGFRA, VCL--FGFR2, VPS18--NTRK3, WRN--NRG1, ZBTB7B--NTRK1, ZC3HAV1-BRAF, ZEB2--AKT3 and ZNF430--BRD4.,

[0587] Examples of targetable fusions include ALK fusions, such as TFG-ALK. ALK fusions have been identified in multiple cancer types, such as lung adenocarcinoma, bladder cancer, colorectal cancer, breast cancer, renal cell carcinoma, renal medullary carcinoma and thyroid cancer. In particular, EML4-ALK fusions have been found in lung adenocarcinoma, STRN-ALK fusions have been found in thyroid cancer and papillary renal cell carcinoma, TPM1-ALK fusions have been found in bladder cancer, SMEK2-ALK fusions have been found in rectal adenocarcinoma, and GTF2IRD1-ALK fusions have been found in thyroid cancer. Another targetable fusion includes BRAF fusions, which are associated with prostate cancer, melanoma, radiation-induced thyroid cancer and pediatric low-grade glioma. In particular, TRIM-BRAF fusions have been found in renal adenocarcinomas, ATG7-BRAF in melanomas, and ZC3HAV1-BRAF and FAM114A2-BRAF in thyroid cancers. Other examples of fusions include AGK-BRAF, SND1-BRAF, MACF1-BRAF, TAX1BP1-BRAF, and CDC27-BRAF. It is known in the art that BRAF dimers are not sensitive to RAF inhibitors, but are instead treated for downstream inhibition, for example, by MEK inhibition.

[0588] Another targetable fusion includes FGFR fusion, which has been identified in the following tumor types: glioblastoma multiforme, bladder urothelial carcinoma, lung squamous cell carcinoma, renal papillary cell carcinoma, brain low-grade glioma, prostate adenocarcinoma, head and neck squamous cell carcinoma, invasive breast carcinoma, and gastric adenocarcinoma. In particular, FGFR3-TACC3 fusion has been found in papillary renal carcinoma, FGFR3-ELAVL3 has been found in low-grade glioma, and FGFR3-BAIAP2L1 has been found in bladder cancer. Another targetable fusion includes WASF2-FGR fusion, which has been found in lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, and skin melanoma. Another targetable fusion includes MET fusion, which has been found in low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, and thyroid carcinoma. In particular, BAIAP2L1-MET and C8orf34-MET have been found in papillary renal carcinoma, KIF5B-MET in lung adenocarcinoma, and TFG-MET in papillary thyroid carcinoma. Another notable fusion is TPR-MET.

[0589] Another targetable fusion includes NTRK fusion, which is associated with congenital fibrosarcoma, human secretory breast cancer, and papillary thyroid cancer, including glioblastoma, cholangiocarcinoma, and pediatric high-grade glioma. In particular, PAN3-NTRK2 is found in head and neck squamous cell carcinoma, AFAP1-NTRK2 is found in low-grade carcinoma, TRIM24-NTRK2 is found in lung adenocarcinoma, and TPM3-NTRK1 is found in sarcoma and thyroid cancer. Another targetable fusion includes PIK3CA fusion, which is found in endometrial cancer, invasive breast cancer, and colorectal cancer and head and neck cancer. In particular, TBL1XR1-PIK3CA fusion is found in breast cancer and prostate adenocarcinoma, FNDC3B-PIK3CA fusion is found in uterine endometrial cancer, and TBL1XR1-PIK3CA fusion is found in invasive breast cancer and prostate cancer. Another targetable fusion is PKC fusion, which is found in papillary glioneuronal tumors and benign fibrous histiocytoma.PRKCA fusion is found in lung squamous cell carcinoma, and PRKCB fusion is found in lung squamous cell carcinoma, lung adenocarcinoma and low-grade glioma.An example of a fusion includes PRKCA fused to IGF2BP3, TANC2-PRKCA. 【0...

Claims

1. A chimeric small molecule comprising an enzyme-binding moiety and a target-binding moiety connected via one or more linker molecules, which may contain an electrophilic reactive group, where the enzyme-binding moiety brings the enzyme into proximity with the target substrate and induces modification of the target substrate, or where the enzyme-binding moiety facilitates labeling of the enzyme with the target-binding moiety via the electrophilic reactive group.

2. formula A-(L) n -B where A is an enzyme-binding moiety, B is a target binding moiety, L is a linker and n is 0 to 6; or formula AL-El-B or AL 1 -El-L 2 -B where A is an enzyme-binding moiety, B is a target binding moiety, L is a linker, E is an electrophilic reactive group. The chimeric small molecule of claim 1 , represented by:

3. A and B bind separately to the same kind of enzyme, The enzyme may be an oligomeric enzyme and the chimeric small molecule may lock the oligomeric enzyme in an active or inactive state; The enzymes may be kinases, and one of the bound kinases may phosphorylate the other bound kinase, thereby activating it, and the kinases may be receptor tyrosine kinases, non-receptor tyrosine kinases, or serine tyrosine kinases; formula 【Chemistry 1】 wherein W is independently selected from an amine, O, S, NH, a bond, an alkane, an alkene, an alkyne, an amine, an ether, a thiol, a sulfone, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, O, S, NH, or any combination thereof; and A and B are linked to their respective Ws through any functional group or ring position of A and B. The chimeric small molecule of claim 2, wherein the chimeric small molecule is

4. The chimeric small molecule of any one of the preceding claims, wherein L, L1, and / or L2 are individually selected from an alkane, an alkene, an alkyne, an amine, an ether, a thiol, a sulfone, a carbonyl, an acyl, a ketone, a carboxylic acid ester; an amide, an enone, an anhydride, an imide, PEG, or any combination thereof, and n is 0-6.

5. The chimeric small molecule of claim 1 , wherein the enzyme binding moiety is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or transferase binding moiety.

6. the enzyme-binding moiety is a kinase-binding moiety; The kinase binding moiety is selected from FKBP, PKC, AMPK, ABL, PK, MAPK, e.g., MAPK1, MAPK11, MAPK12, MAPK13, MAPK14, p38a a MAPK, EGFREGFR, FGFR, NGFR, TrkA, ABL, CDK, e.g., CDK2, CDK4, PI3K, VEGFR, BRAF, MEK, e.g., MEK1 / 2, MEK5, AKT, ALK, BTK, BCKDK, FLT3, JAK2, AURKA, c-MET, DDR, INSR, JNK, IkB, IKK, Lyn, mTOR, e.g., mTORC-1, PAK, PDK, e.g., PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-a, RIPK, TYK2, SHP, aPKC, e.g., PKC-zeta, NOP, GPC family, e.g., μ opioid receptor or δ opioid receptor, UMPK, SphK or GSK-3 binding moiety; The chimeric small molecule of claim 1 , wherein the targeting moiety may bind to the same type of kinase as the kinase binding moiety.

7. B is K-Ras, HSP90, BRD4, BTK, and FKB12 F36V The chimeric small molecule of claim 2 which is a binding moiety.

8. The kinase binding moiety has the formula 【Chemistry 2】 (Wherein, R is 【Chemistry 3】 , a carbohydrate mimetic, a heterocycle, a dihydrohexitol, a pyranose or a furanose; Q is selected from the group consisting of B, C, N, O, and S; where H is N A Or N B Located on either X 1 and X 2 is independently selected from the group consisting of C, N, and O; Y is selected from the group consisting of H, OH, halogen, CN, or a hydrogen bond donor substituent; Z is H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2 Cl, and optionally further substituted).

20. The AMPK binding moiety of claim 19, Z has the formula: Z a -Z b (In the formula, Z a is 【Chemistry 4】 is selected from the group consisting of Z b is 【Chemistry 5】 is selected from the group consisting of (n is 0 to 6) The chimeric small molecule of claim 1 ,

9. The AMPK binding site 【Chemistry 6】 The chimeric small molecule of claim 8, selected from the group consisting of:

10. A 【Chemistry 7】 and B 【Chemistry 8】 and L 【Chemistry 9】 (wherein n is 1, 2, 3, 4 or 5). The chimeric small molecule of claim 9, which may be selected from the group consisting of:

11. 【Catalog 10】 The chimeric small molecule of claim 1 ,

12. A is the formula 【Chemistry 11】 or an analog thereof.

13. 【Chemical 12】 13. The chimeric small molecule of claim 12, wherein

14. A is the formula 【Chemistry 13】 (wherein R1 to R5 are independently H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2 Selected from Cl; Z is independently selected from B, C, N, O, S, preferably one or two atoms of Z are N, O, S, or a combination thereof; Ra, Rb, and Rc each independently represent an alkane, alkene, alkyne, ether, alcohol, amine, nitrile, nitro, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, acid anhydride, imide, or an aliphatic halogen compound, e.g., -OCF 2 selected from one or more fused rings including Cl, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, any combination of any of the preceding listed rings; Re is an alkane, alkene, alkyne, ether, alcohol, amine, nitrile, nitro, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, acid anhydride, imide, aliphatic halogen compound, e.g., OCF 2 Cl, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof at one or more positions, or R 1 Or R 5 can form a ring with or any combination thereof, The formula is 【Chemistry 14】 wherein R is selected from H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, an aliphatic halogen compound, e.g., -OCF2Cl, or any combination thereof; 【Chemistry 15】 (selected from may be selected from The formula is 【Chemistry 16】 wherein R is selected from H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, an aliphatic halogen compound, e.g., -OCF2Cl, or any combination thereof; 【Chemistry 17】 (wherein X and Y are CH or N, and R is H, D, F, Me, or CF 3 ). (selected from the group consisting of may be selected from The kinase binding site 【Chemistry 18】 The chimeric small molecule of claim 1, which may be selected from:

15. The enzyme-binding moiety and / or the kinase-binding moiety of claim 1, 【Chemistry 19】 wherein X is selected from C, N, O, and S; R 2 is H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, one or more condensed rings thereof, aliphatic halogen compounds, e.g., -OCF 2 Cl, or any combination thereof, preferably 【Chemistry 20】 (selected from the group consisting of The molecule of claim 14 , 16. The enzyme-binding moiety and / or the kinase-binding moiety of claim 1, 【Chemistry 21】 【change】 【change】 (X is a halogen; Y and Y1 are independently selected from C, N, O and S; R 1 , R 2 , R 3 R 4 , R 6 , and R 7 are independently selected from H, an alkane, an alkene, an alkyne, an amine, a nitrile, nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, an aliphatic halogen compound, e.g., -OCF 2 Cl, or any combination thereof) The molecule of claim 14, selected from:

17. The enzyme-binding moiety and / or the kinase-binding moiety of claim 16, 【Chemical 22】 The molecule of claim 14, selected from:

18. R a , R b , R c One or more of 【Chemistry 23】 15. The molecule of claim 14, further attached to a molecule selected from the group consisting of:

19. A is according to formula II(b), where Re is 【Chemistry 24】 wherein Rf and Rg are selected at one or more positions from alkanes, alkenes, alkynes, ethers, alcohols, amines, nitriles, nitros, thiols, sulfones, sulfonates, halogens, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, anhydrides, imides, cyclic hydrocarbons, unsaturated cyclic hydrocarbons, heterocycles, cyclic hydrocarbons optionally substituted with one or more fused rings containing any combination of any of the previously listed rings, unsaturated cyclic hydrocarbons, heterocycles, one or more fused rings containing any combination of any of the previously listed rings. is selected from the group consisting of Rf and Rg are independently 【Chemistry 25】 15. The molecule of claim 14, which may be selected from the group consisting of:

20. B, 【Chemistry 26】 15. The molecule of claim 14, selected from the group consisting of:

21. The enzyme-binding moiety and / or the kinase-binding moiety, 【Chemistry 27】 or derivatives thereof.

22. Formula IV 【Chemistry 28】 (wherein n is 0 to 3); 【Chemical 29】 The molecule of claim 14, which is

23. B 【Chemistry 30】 (wherein R is an electrophilic reactive group; X is the formula 【Chemistry 31】 and Y is H, an alkane, an alkene, an alkyne, an amine, a nitrile, a nitro, an ether, an alcohol, a thiol, a sulfone, a sulfonate, a halogen, a carbonyl, an acyl, a ketone, a carboxylic acid ester, an amide, an enone, an anhydride, an imide, a cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof, or an aliphatic halogen compound, e.g., -OCF 2 Cl) is a KRAS binding molecule selected from the group consisting of Electrophilic reactive groups are 【Chemistry 32】 or derivatives thereof.

24. B is the formula 【Chemical 33】 or a derivative thereof, Or B is 【Chemical 34】 or analogs thereof; Or B is 【Chemistry 35】 or derivatives thereof; Or B is 【Chemical 36】 or derivatives thereof; Or B is of the formula 【Chemical 37】 or an analog thereof.

25. The chimeric small molecule of claim 1, wherein A is an AMPK binding moiety described in claim 8 and B is a KRAS binding moiety described in claim 23, or A is an ABL kinase binding moiety described in claim 14 and B is a BRD4 binding moiety described in claim 24.

26. L 【Chemical 38】 where n is 0 to 3; or L 【Chemical 39】 where n is 0 to 6, or L 【Chemistry 40】 (wherein n is 1, 2, 3, 4 or 5). or The formula in which L connects A and B: 【Chemistry 41】 The molecule of claim 1 ,

27. L is a rigid linker, 【Chemistry 42】 or any combination thereof (wherein any atom in the ring may be substituted with C, N, O, or S; The linker may be attached to one or more PEG molecules prior to attachment to A and, optionally, B; m and n may be independently selected from 0 to 6. The molecule of claim 1, which may be selected from the group consisting of:

28. The electrophilic reactive groups are N-acyl-N-alkylsulfonamides (NASA), dibromophenyl benzoates, N-sulfonylpyridones, 【Chemistry 43】 is selected from The chimeric small molecule of claim 2 , wherein the electrophilic reactive group is optionally reacted with a nucleophilic reactive group.

29. the enzyme binding agent further comprises a bioorthogonal group; The bioorthogonal group may be selected from tetrazine, triazine, cyclooctene, cyclopropene and diazo; Bioorthogonal groups are 【Chemistry 44】 29. The molecule of claim 28, which may be selected from the group consisting of:

30. the enzyme binding agent has a half-life that is shorter than the half-life of the target to which the target binding agent can bind; The half-life of the enzyme binding agent may be at least 2, 3, 4, 5 or less times shorter than the half-life of the target bound to the target binding agent; The molecule of claim 1 , wherein the target may be a protein.

31. the enzyme-binding agent is a kinase-binding agent; The kinase-binding agent may be a kinase inhibitor or a kinase activator; The molecule of claim 1 , wherein the kinase inhibitor is a promiscuous kinase inhibitor.

32. 【Catalogue 45】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 The molecule of claim 1, selected from the group consisting of:

33. 13. A method for inducing modification of a target substrate, comprising administering to an isolated cell or population of cells a chimeric small molecule described in claim 1.

34. A method for modifying a target substrate in an isolated cell, comprising: AL-El-B and AL 1 -The-L 2 -B where A is an enzyme-binding moiety specific for the cellular enzyme being repurposed / reprogrammed; B is a target binding moiety specific for the target substrate to be modified; L is a linker, EI is an electrophilic reactive group) creating an initialized cellular enzyme by delivering a chimeric molecule of the above formula, whereby the chimeric molecule labels the cellular enzyme with a target binding moiety for the target substrate; and Modifying the target substrate by binding the repurposed / initialized enzyme to the target substrate via a targeted binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. Including, The enzyme-binding moiety may have a half-life that is about 2, 3, 4 or 5 times the half-life of the initiated enzyme; The enzyme to be initialized may be an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase; The enzyme-binding moiety may be an inhibitor; The enzyme to be initialized may be a kinase and the enzyme-binding moiety may be a kinase inhibitor; The kinase inhibitor may be a "promiscuous" kinase inhibitor; and and optionally further comprising administering a coupling molecule to thereby quench the inhibitory activity of the enzyme inhibitor; and the coupling molecule may be one or more of an aldehyde, alkene, alkyne, strained alkyne, cyclooctyne, trans-cyclooctene, cyclopropene, oxanorbornadiene, norbornene, phosphine, electron rich dienophile, isonitrile, isocyanopropionate, tetrazole, 2-acylboronic acid, or any derivative thereof; The cyclooctyne derivative may include dibenzocyclooctyne, biarylazacyclooctyne, or dimethoxyazacyclooctyne; and The method wherein the strained alkyne comprises a bicyclononyne or a dioxabiaryldecyne.

35. A method for modifying a substrate, comprising introducing into a cell a molecule according to claim 1, The modification may include inducing a post-translational modification of the target protein; and The method, wherein the post-translational modification is phosphorylation.

36. 1. Use of a chimeric molecule in the manufacture of a medicament for treating cancer to generate an initialized cellular enzyme, wherein the chimeric molecule has the formula: A-L-E-B, A-L 1 -E-L 2 -B or A-(L) n -B and where A is an enzyme binding moiety, L is a linker, n is 0-6, E is an electrophilic reactive group, and B is an oncogenic protein to be modified, whereby the chimeric molecule labels a cellular enzyme with a targeted binding agent for the target substrate; and Modifying an oncogenic protein by binding a repurposed / initialized enzyme to a target substrate via a targeted binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. Including, The targeted binding agent may be specific for KRAS, RAS, FKPB 12F36V, EGFR, HSP90, BTK, MDM2, BRD4, NF-kB, LDH-A, p53, GP73, MUC1, MUC16, CD44, GPCR, HMGB1, RIOK1, CHK1, UBE2F, HuR, PTEN, STAT-3, osteopontin, EGFR, AKT, DAPK1, Rho, Ubc9, FOXK2, HIC1, HER2, BRAF, BCL-2, CD117, (KIT), ALK, PI3K, Delta, DNMT1 or SMO; The cellular enzyme to be initialized may be an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase; and The enzyme binding agent may be an enzyme inhibitor, preferably a kinase inhibitor; The kinase inhibitor may be a promiscuous inhibitor; The kinase inhibitor may be specific for PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-a, RIPK, TYK2, SHP, aPKC, NOP, GPC family, e.g., μ or δ opioid receptor, UMPK, SphK, GSK-3; and A quenching molecule may be administered to thereby quench the inhibitory activity of the enzyme inhibitor.

37. 23. Use of a chimeric small molecule comprising a KRAS binding molecule of claim 21 and an enzyme-binding molecule of claim 1 in the manufacture of a medicament for treating a disease associated with aberrant KRAS signaling, comprising administering a composition comprising the chimeric functional small molecule, The enzyme-binding molecule may be a target of an enzyme selected from the group consisting of PK, PKC, AMPK, MAPK, EGFR, FGFR, NGFR, TrkA, ABL, BCKDK, CDK, PI3K, VEGFR, BRAF, MEK, AKT, ALK, BTK, FLT3, JAK2, AURKA, c-MET, DDR, FKBP, INSR, IKK, JNK, mTOR, PAK, PDK1, PDK2, PTK2 / FAK, pyruvate kinase, RAC-a, RIPK, TYK2, SHP, aPKC, NOP, GPC family, e.g., μ-opioid receptor or δ-opioid receptor, UMPK, SphK or GSK-3; The AMPK may be an AMPK binding moiety according to claim 8; KRAS may be KRAS G12C ; The chimeric small molecule may phosphorylate one or more residues on KRAS selected from the group consisting of Ser17, Ser39, Ser65, Ser106, Ser122, Ser136, Ser2, Thr2, Thr35, Thr50, Thr74, Thr87, Thr124, Thr127, Thr148.

38. 2. Use of a chimeric molecule in the manufacture of a medicament for treating an infection by a pathogen, comprising: and producing an initialized cellular enzyme by administering to a subject in need thereof, wherein the chimeric molecule has the formula: A-L-E-B or A-L 1 -E-L 2 -B and where A is an enzyme binding moiety, L is a linker, E is an electrophilic reactive group, and B is a pathogen protein to be modified, whereby the chimeric molecule labels a cellular enzyme with a targeted binding agent for the target substrate; and Modifying a pathogen protein by binding a repurposed / initialized enzyme to a target substrate via a target binding agent, whereby the repurposed / initialized cellular enzyme introduces one or more modifications to the target substrate. Including, The cellular enzyme to be initialized may be an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, translocase; The pathogen may be a virus, a bacterium, a fungus or a protozoan; The bacterium may be Mycobacterium tuberculosis (Mtb) or Pseudomonas aeruginosa (PsA); The pathogen may be Mtb and the pathogen protein may be one or more of PtpA, PtpB, SapM, ESAT-6, and Rv2966c; The pathogen may be (PsA) and the targeted binding agent may be colistin; The enzyme binding agent may be a kinase inhibitor; The kinase inhibitor may be a promiscuous inhibitor; and A quenching molecule may be administered to thereby quench the inhibitory activity of the enzyme inhibitor.

39. 13. Use of the chimeric small molecule of claim 3 in the manufacture of a medicament for treating cancer, comprising: The cancer may be characterized by aberrant kinase signaling; The aberrant kinase signaling may be characterized by a cancerous fusion of ABL kinase; The cancerous fusion may be a TEL-ABL or NUP214-ABL fusion; The cancer may be characterized by aberrant BCR-ABL kinase signaling; The medicament may be for administering a monomer of A or B in a therapeutically effective amount to reverse the effect of the chimeric small molecule.

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