Bifunctional chimeric molecules for labeling kinases with target binding moieties and methods of use thereof

Abstract

The present disclosure relates to chimeric small molecules that find utility as modulators of target substrates according to the formula A-L1-EB or A-L1-E-L2-B, where A is a kinase-binding moiety; B is a target-binding moiety; L1 and L2 are each a linker; and E is an electrophilic reactive group. The molecules of the present invention find use in effecting substrate modifications, such as post-translational modifications, on targets that are not natural substrates of kinases, such that diseases or disorders can be treated or prevented with the molecules of the present disclosure.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 412,837, filed October 3, 2022; U.S. Provisional Patent Application No. 63 / 414,827, filed October 10, 2022; U.S. Provisional Patent Application No. 63 / 457,339, filed April 5, 2023; and U.S. Provisional Patent Application No. 63 / 535,010, filed August 28, 2023. The entire contents of the above-identified applications are hereby incorporated by reference in their entirety.

[0002] The subject matter disclosed herein relates generally to multifunctional chemical conjugation molecules that are utilized to induce modifications in target substrates.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with federal support under Grant Nos. N66001-17-2-4055 and HR00112120010 awarded by the Department of Defense and Grant No. GM137606 awarded by the National Institutes of Health. The federal government has certain rights in this invention.

[0004] Sequence Listing This application contains a Sequence Listing submitted electronically as an xml file entitled BROD-5705WP_ST26.xml, created on October 2, 2023, and 56,444 bytes in size, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0005] There is a continuing need in the art for effective treatments for diseases involving enzymatic and other dysfunctions, as well as a need to perform modifications, such as post-translational modifications. However, obstacles such as non-specific effects remain an impediment to the development of effective modifications and treatments. Small molecules that confer novel functions to enzymes through proximity-mediated effects could be useful in the study and treatment of important cellular functions and diseases.

[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. Summary of the Invention [Means for solving the problem]

[0007] In certain exemplary embodiments, the formula: A-L1-EB or A-L1-E-L2-B where A is a kinase-binding moiety; B is a target-binding moiety; L1 and L2 are each a linker; and E is an electrophilic reactive group. Chimeric small molecules according to the invention are provided.

[0008] 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 kinase-binding moiety may be a MAPK, EGFR, FGFR, NGFR, TrkA, ABL, CDK, such as CDK2, CDK4, CDK8, PI3K, VEGFR, BRAF, MEK, such as MEK1 / 2, MEK5, AKT, ALK, BTK, BCKDK, FLT3, JAK2, AURKA, c-MET, DDR, INSR, JNK, IB, IKK, Lyn, mTOR, such as mTORC-1, PAK, PDK, such as PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-, RIPK, TYK2, SHP, aPKC, such as PKC-ζ, NOP, GPC family, such as μ-opioid receptor or δ-opioid receptor, UMPK, SphK or GSK-3, IRTK, PDGFR, IDH, ITK, TAK, BMX, LIMK or IRE binding moiety. The kinase-binding moiety may comprise a kinase inhibitor or a kinase activator. In certain embodiments, the kinase inhibitor is a broad spectrum kinase inhibitor.

[0009] In some embodiments, the kinase inhibitor is sorafenib, SB2035890, or skepinone B, or an analog or derivative thereof. In some embodiments, the kinase inhibitor is gefitinib, or an analog or derivative thereof, imatinib, or an analog or derivative thereof, or idelalisib, or an analog or derivative thereof.

[0010] A chimeric small molecule can comprise a kinase-binding moiety that has a half-life that is shorter than the half-life of the target to which the target-binding moiety is capable of binding, hi certain embodiments, the half-life of the kinase-binding moiety is at least 2, 3, 4, or 5 times shorter than the half-life of the target bound by the target-binding moiety.

[0011] The chimeric small molecule can include an electrophilic reactive group selected from N-acyl-N-alkylsulfonamide (NASA), dibromophenylbenzoate, or N-sulfonylpyridone. The chimeric small molecule of any preceding claim, wherein the electrophilic reactive group is: [ka] is selected from the group consisting of:

[0012] In some embodiments, the electrophilic reactive group has the formula: [ka] wherein R1 is selected from CO, SO2, Me-CO or Me-SO2, R2 is selected from 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 halide such as -OCF2Cl, or any combination thereof, and the benzene ring is optionally substituted at any position. It has.

[0013] In certain embodiments, the electrophilic reactive group is [ka] JPEG2025533072000005.jpg112170 (wherein the electrophilic reactive group is attached to the chimeric small molecule via a terminal carbon group; a carbon on the cyclic moiety and / or a nitrogen on the cyclic moiety) is selected from the group consisting of:

[0014] The chimeric small molecule may comprise a linker, as represented by L1 and optionally L2, 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. The L1 and L2 linkers may be 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.

[0015] The chimeric small molecule may comprise a kinase-binding agent further comprising a bioorthogonal group. In certain embodiments, the bioorthogonal group is selected from tetrazine, triazine, cyclooctene, cyclopropene, and diazo. The bioorthogonal group is [ka] may be selected from the group consisting of:

[0016] The target of the target binding moiety of the chimeric small molecule is a protein. In some embodiments, the target protein is from a pathogen. The pathogen can be a virus, bacterium, fungus, or protozoan. In some embodiments, the microbial protein is an intracellular or extracellular pathogen protein. The intracellular pathogen can be Mycobacterium tuberculosis, or the extracellular pathogen is Pseudomonas aeruginosa. The chimeric small molecule can include a target binding moiety that is a phosphatase A (PtpA) binder, a PtpB binder, a SapM binder, an ESAT-6 binder, and an Rv2966c binder. The chimeric small molecule can include a target binding moiety that is colistin.

[0017] The chimeric small molecule may be capable of covalently labeling a kinase with a kinase-binding agent. In some embodiments, the label is a label of a nucleophile disposed on the kinase.

[0018] The chimeric small molecule may comprise a target binding moiety for a target that has the ability to bind an oncogenic target.

[0019] In an exemplary embodiment, the kinase inhibitor is SB sorafenib, 2035890, or Skepinone B, or an analog or derivative thereof. In an exemplary embodiment, the kinase inhibitor is gefitinib, or an analog or derivative thereof. In an exemplary embodiment, the kinase inhibitor is imatinib, or an analog or derivative thereof. In an exemplary embodiment, the kinase inhibitor is idelalisib, or an analog or derivative thereof. In an exemplary embodiment, the kinase inhibitor is enasidenib, erdafitinib, ivosidenib, pemigatinib, pralsetinib, infigratinib, dacomitinib, capmatinib, mobocertinib, gilteritinib, or an analog or derivative thereof.

[0020] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0021] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0022] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0023] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0024] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0025] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0026] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0027] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0028] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0029] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0030] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0031] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0032] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0033] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0034] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0035] In certain exemplary embodiments, the kinase inhibitor is: [ka] or an analogue or derivative thereof.

[0036] In certain exemplary embodiments, the binding moiety is: [ka] or an analogue or derivative thereof.

[0037] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0038] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0039] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0040] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0041] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0042] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0043] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0044] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0045] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0046] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0047] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0048] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0049] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0050] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0051] In an exemplary embodiment, A-L1-E has the formula: [ka] relates to.

[0052] A-L1-E is the formula [ka] or an analog or derivative thereof.

[0053] The chimeric small molecule is [ka] wherein m=0 or 1; n=1, 2, 3, 4, or 5; and X=CH or (CH)O. It may be related to.

[0054] The chimeric small molecule is [ka] wherein X and Y are independently selected from CH or (CH)O, and n and m are independently selected from 1, 2, 3, 4, 5, or 6. It may be related to.

[0055] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] (Wherein JQ1 is a compound represented by the formula: [ka] or its analogues or derivatives) relates to.

[0056] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0057] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0058] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0059] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0060] In an exemplary embodiment, R1 is [ka] or an analogue or derivative thereof, R2 is [ka] or an analogue or derivative thereof.

[0061] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0062] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0063] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0064] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0065] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0066] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0067] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0068] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0069] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0070] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0071] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0072] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0073]

[0010] Embodiments herein include methods of inducing modification of a target substrate, comprising administering to a cell or cell population a chimeric small molecule as described herein.

[0011] Methods of modifying a substrate, comprising introducing into a cell a molecule as described herein. In some embodiments, the electrophilic reactive group of the chimeric small molecule can react with a nucleophilic reactive group. In some embodiments, the nucleophilic reactive group is optionally contained in an amino acid selected from cysteine, serine, threonine, tyrosine, glutamic acid, aspartic acid, lysine, arginine, and histidine.

[0074] In one embodiment, a method of modifying a target substrate in a cell includes: generating a reprogrammed cellular kinase by delivering a chimeric small molecule described herein, where A is a kinase binding moiety specific for the cellular kinase to be repurposed / reprogrammed, whereby the chimeric small molecule labels the cellular kinase with a target binding moiety for the target substrate; and modifying the target substrate by binding the repurposed / reprogrammed kinase to the target substrate via the target binding moiety, whereby the repurposed / reprogrammed cellular kinase introduces one or more modifications to the target substrate.

[0075] The methods described herein can further include administering a coupling molecule, thereby quenching the inhibitory activity of the kinase-binding moiety. The method of modifying can include inducing a post-translational modification of the target protein. In some aspects, the post-translational modification is phosphorylation.

[0076] A method of treating cancer is provided, comprising: administering to a subject in need thereof a chimeric small molecule as detailed herein to generate a reprogrammed cellular kinase, wherein target binding moiety B binds to the oncogenic protein to be modified, thereby the chimeric small molecule tags the cellular kinase with the target binding moiety for the target substrate; and modifying the oncogenic protein by binding the repurposed / reprogrammed kinase to the target substrate via the target binding moiety, thereby the repurposed / reprogrammed cellular kinase introduces one or more modifications to the target substrate. The method may further comprise administering a coupling molecule, thereby quenching the activity of the kinase binding moiety. In some embodiments, the modification includes inducing a post-translational modification of the target protein; in some embodiments, the post-translational modification is phosphorylation.

[0077]

[0003] A method for treating a pathogen infection is provided, comprising: administering to a subject in need thereof a chimeric small molecule described herein to generate a reprogrammed cellular kinase, where B is the pathogen protein to be modified, whereby the chimeric small molecule tags the cellular kinase with a target binding moiety for a target substrate; and modifying the pathogen protein by binding the repurposed / reprogrammed kinase to the pathogen protein via the target binding moiety, whereby the repurposed / reprogrammed cellular kinase introduces one or more modifications to the target substrate. The method may further include administering a coupling molecule, thereby quenching the inhibitory activity of the kinase binding moiety. In embodiments, the modification includes inducing a post-translational modification of the target protein, optionally the post-translational modification being phosphorylation.

[0078] In one aspect, the present disclosure provides electrophilically reactive linkers with enhanced stability, reactivity, and tunability according to the formula L1-El, E1-L1, or L1-El-L2, where L1 and L2 are independently selected from an alkane, alkene, amine, ether, thiol, sulfone, carbonyl, acyl, ketone, carboxylic acid ester, amide, enone, anhydride, imide, and PEG, and E1 is an electrophilic reactive group. In exemplary embodiments, E1 is configured to facilitate cleavage of the linker and attachment of all or a portion of L1 or L2 to a cysteine, lysine, methionine, or tyrosine amino acid on a target polypeptide.

[0079] In exemplary embodiments, the linker further comprises an attachment moiety linked to L1 or L2 and configured to covalently attach to the target polypeptide via El. In exemplary embodiments, the linker further comprises a binding moiety located on the opposite side of the linker from the attachment moiety and capable of binding to the target polypeptide. In exemplary embodiments, El is configured to facilitate attachment of all or a portion of L1 or L2 to a cysteine, and El is [ka] is selected from the group consisting of:

[0080] In an exemplary embodiment, El is configured to facilitate attachment of all or a portion of L1 or L2 to a lysine, and El is [ka] (Wherein R1 is [ka] selected from the group consisting of: R2 is [ka] selected from the group consisting of is.

[0081] In an exemplary embodiment, El is configured to facilitate attachment of all or a portion of L1 or L2 to a lysine, and El is [ka] is selected from the group consisting of:

[0082] In an exemplary embodiment, El is configured to facilitate attachment of all or a portion of L1 or L2 to a methionine, and El is [ka] is selected from the group consisting of:

[0083] In one aspect, the present disclosure provides heterobifunctional molecules comprising an electrophilically reactive linker as described herein, a target polypeptide binding moiety attached to the linker at one end, and a modifying moiety attached to the linker at the opposite end. In exemplary embodiments, the target polypeptide moiety binds to a kinase, phosphatase, ubiquitinase, deubiquitinase, acetyltransferase, deacetylase, methyltransferase, demethylase, or glycosyltransferase, and the modifying moiety binds to a neo-substrate of the kinase, phosphatase, ubiquitinase, deubiquitinase, acetyltransferase, deacetylase, methyltransferase, demethylase, or glycosyltransferase. In exemplary embodiments, the modifying moiety is an immunogenic moiety, and the target polypeptide binding moiety is a polypeptide to which the immunogenic moiety is attached.

[0084] These and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the exemplary embodiments.

[0085] A better 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 and the accompanying drawings of which: [Brief explanation of the drawings]

[0086] [Figure 1] Exemplary chimeras for recruiting Pseudomonas aeruginosa to antibodies, complement, or macrophages, and Mycobacterium tuberculosis (M. tb) proteins to host kinases.

[0087] [Figure 2] A binder drug discovery platform for microbial and host targets.

[0088] [Figure 3]Inhibitors selected for covalent labeling of kinases and their residence times. The sites where the linker and bioorthogonal group are attached are indicated by an arrow and an asterisk (*), respectively.

[0089] [Figures 4A-4C] (4A) Representative example of a chimeric small molecule designed to label MAPK p38α with its inhibitor SB203580 and induce proximity based on a covalent modification mechanism. (4B) Exemplary deactivation of the inhibitor by click reaction with bulky cyclooctyne; the bulky group prevents the inhibitor from binding to the kinase. 4C. Exemplary embodiment of a sorafenib-based chimeric small molecule designed to label MAPK p38α with a binder of PtpA via induce proximity.

[0090] [Figure 5] Modular components of an exemplary chimera: a known binder to a microbial target (blue) and a known binder to a host target (green). Applicants used these to optimize the assay. Applicants also use these building blocks to create pseudochimeras consisting of known binders (shown herein) and binders identified from screening.

[0091] [Figure 6] Exemplary chimeric platforms for target labeling.

[0092] [Figure 7] General preparation strategies for NASA and its analogues / substitutes.

[0093] [Figure 8] Additional exemplary NASA analogs / alternatives according to and for use in embodiments of the present invention described herein.

[0094] [Figure 9] Exemplary kinase binding moieties according to and for use in embodiments of the invention described herein.

[0095] [Figures 10A-10B] 10A) An exemplary scheme of an embodiment described herein. 10B) An exemplary chimeric molecule as described herein.

[0096] [Figure 11] Further exemplary kinase binding moieties with exemplary linkers / electrophilic reactive groups according to and for use in embodiments of the invention described herein.

[0097] [Figure 12] Experimental validation of electrophilic reactive group addition-elimination reactions with glutathione as described in embodiments herein.

[0098] [Figure 13] Electrophilic reactive group design considerations, including exemplary electrophilic reactive groups according to and for use in embodiments of the invention described herein.

[0099] [Figure 14] Experimental reactivity validation of aryl and alkyl design considerations for electrophilic reactive groups.

[0100] [Figure 15] Experimental reactivity / hydrolysis validation of nitrile and ketone design considerations for electrophilic reactive groups.

[0101] [Figure 16] Experimental reactivity of exemplary electrophilic reactive groups. These exemplary results demonstrate the tunability of electrophilic reactive groups according to and for use in embodiments of the invention described herein.

[0102] [Figure 17] Exemplary designs of chimeric small molecules bearing a kinase-binding moiety and an electrophilic reactive group.

[0103] [Figure 18]Experimental validation of exemplary chimeric small molecules demonstrating phosphorylation of BTK by ABL and reduced EC50 of exemplary chimeric small molecules compared to ibrutinib.

[0104] [Figure 19] Proof-of-concept phosphorylation of BRD4 by EGFR PHICS (HEK293T cells).

[0105] [Figure 20] Cell target engagement: Biotin click / Cy5 azide click for compound 1, HEK293T.

[0106] [Figure 21] Cell target engagement: Biotin click / Cy5 azide click for compound 2, HEK293T.

[0107] [Figure 22] Cell target engagement: Biotin click / Cy5 azide click for compound 3, HEK293T.

[0108] [Figure 23] Induction of BRD4 phosphorylation by JAK3 PHICS (HEK293T cells).

[0109] [Figures 24A-24C] FGFR4 / pan-FGFR biochemical target engagement: 24A) LC-MS with in vitro labeling (intact mass for addition and elimination on the kinase), 24B) partial chimeric small molecules, 24C) in-gel fluorescence (fluorescence via Cu2+ click reaction of azido-Cy-5).

[0110] [Figures 25A-25C]ITK biochemical target engagement: 24A) LC-MS by in vitro labeling (intact mass for addition and elimination on the kinase), 24B) partial chimeric small molecules, 24C) in-gel fluorescence (fluorescence by Cu2+ click reaction of azido-Cy-5).

[0111] [Figures 26A-26B] EGFR biochemical target engagement: 24A) Partial chimeric small molecules, 24B) In-gel fluorescence (fluorescence via Cu2+ click reaction of azido-Cy-5).

[0112] [Figures 27A-27B] FGFR and EGFR biochemical target engagement: 24A) Partial FGFR chimeric small molecule and in-gel fluorescence (fluorescence via Cu2+ click reaction of azide-Cy-5), 24B) Partial EGFR chimeric small molecule and in-gel fluorescence (fluorescence via Cu2+ click reaction of azide-Cy-5).

[0113] [Figure 28] JAK3 and ITK biochemical target engagement: 24A) Partial JAK3 chimeric small molecule and in-gel fluorescence (fluorescence via Cu2+ click reaction of azide-Cy-5), 24B) Partial ITK chimeric small molecule and in-gel fluorescence (fluorescence via Cu2+ click reaction of azide-Cy-5).

[0114] [Figure 29] FGFR4 cellular target engagement: Nano Bret-based luminescence with various partially chimeric small molecules at various concentrations.

[0115] [Figure 30] Proof-of-concept: BRD4 phosphorylation by various FGFR4 small chimeric molecules.

[0116] [Figure 31] Experimental results for the ITK-BRD4 small chimeric molecule.

[0117] [Figure 32] Exemplary lysine reactive electrophilic groups for use in exemplary electrophilic reactive linkers disclosed herein and the tunability of the linker molecule by modification of the electrophilic reactive group are provided. Reactivity: 4 mL of 10 mM compound, 32 mL 1×PBS + 4 mL 50 mM lysine.

[0118] [Figure 33A-33B] 33A) First generation PHICS using an allosteric, non-inhibitory, rare kinase binder. 33B) Second generation PHICS using an inhibitory, allosteric / active site-specific, abundantly available kinase binder. AH can be Cys, Lys, Tyr, or Met.

[0119] [Figures 34A-34D] 34A) ABL PHICS induces phosphorylation of the EGFR receptor, which initiates signal transduction. 34B) Structures of PHICS1.1 and ABL kinase binder 1.2. 34C-34D) PHICS-induced EGFR phosphorylation 34C) and induction of downstream signaling as observed using a luciferase-based reporter assay 34D).

[0120] [Figures 35A-35F] 35A) Homo-PHICS-induced neophosphorylation inhibits BCR-ABL by disrupting the ATP pocket. 35B-C) Tyr253 phosphorylation (located in the P-loop of the ATP pocket) is observed in cells (or in vitro) treated with homo-PHICS, but not in control treatments. 35C-35D) Homo-PHICS induced death of CML cells and cells harboring gatekeeper mutations resistant to imatinib with an EC50 of 52 nM. 35E) KRAS neophosphorylation disrupts membrane / GTP binding, potentially rendering them neoantigen-bearing. 35F) An example of neophosphorylation interfering with KRAS GTP binding / membrane translocation.

[0121] [Figures 36A-36G]36A) PHICS from BTK inhibitors against the target protein BRD4. 36B) Structures of BTK inhibitors 1 and 2, the corresponding alkyne-containing analogs 3 and 4, and BTK-BRD4 PHICS 5 and 7, along with the corresponding inactive controls 6 and 8. 36C) Demonstration of covalent labeling of BTK in HEK293T cells with 1 μM 3 and 4 using in-gel fluorescence. 36D) NanoBRET assay assessing ATP pocket occupancy and BTK inhibition. 36E) ATP pocket occupancy by NanoBRET for known BTK inhibitors 1 and 2 and their derivatives 3, 5, 4, and 7. 36F-36G) PHICS-induced phosphorylation of BRD4 by BTK observed in cells in the presence of 5 36F) or 7 36G), but not their inactive controls, 6 36F) or 8 36G), respectively.

[0122] [Figures 37A-37E] 37A) Click chemistry-based platform for rapid PHICS assembly. 37B-37C) Kinase inhibitors where the leaving group is an aliphatic amine 37B) or an aryl amine 37C). 37D) Targeted binder ligands for BRD4 (b), KRAS (c), and Myc (d). 37E) PHICS-mediated BRD4 phosphorylation by FGFR4, ITK, and JAK3.

[0123] [Figure 38] Scheme 1. Cooperativity in three-body equilibria.

[0124] [Figures 39A-39C] 39A) N-Acyl-N-alkylsulfonamides (NASAs) follow a characteristic structural motif with R1 and R2 serving as suitable positions for modification to achieve high atom economy and tunable reactivity with respect to hydrolysis or aminolysis. 39B) Synthesis of NASA derivatives with various R1 and R2 groups allows tuning of reactivity. 39C) -CH2CF3 is identified as a superior alternative because it exhibits attenuated hydrolysis while retaining sufficient aminolysis.

[0125] [Figures 40A-40D] 40A) Structure of a NASA-alkyne designed and synthesized based on a previously described high-affinity kinase inhibitor. The targeted kinase is provided in parentheses. 40B) Convergent synthesis of Lys-targeted PHICS. A NASA-alkyne is combined with the target binder azide via highly chemoselective CuAAC. 40C) In vitro fluorescent labeling (with Cy5 dye) of purified kinase with the corresponding NASA-alkyne probe. 40D) An ideal PHICS would exhibit fine-tuned retention times. Molecules with long retention times would primarily act as inhibitors, while molecules with short retention times would facilitate phosphorylation of the target protein by allowing partial activation of the kinase.

[0126] [Figure 41] Challenges Associated with the Development of Traditional Bifunctional Molecules. Similar PTM induction / removal chimeras are currently being developed by various laboratories, and they involve the identification of non-inhibitory binders of various PTM induction / removal enzymes through screening (e.g., mass spectrometry (ABPP), DNA-encoded libraries, or small molecule screening). These approaches pose several challenges: 1) de novo ligand discovery and optimization is involved, which is 32-pyridinone and very time-consuming; 2) identifying non-inhibitory pockets on enzymes is not straightforward, and for some enzymes, such pockets do not exist; 3) the resulting ligands have unknown pharmacology (e.g., specificity, off-targets, PK / PD); and 4) mass spectrometry primarily uses small molecule fragments with poor binding affinity. Applicants repurpose existing high-quality PTM induction / removal enzyme inhibitors. For nearly all PTM induction / removal enzymes, such inhibitors are abundantly available.

[0127] [Figure 42] Examples of kinase inhibitors abundantly available for PHICS development.

[0128] [Figures 43A-43C]PHICS can be developed using kinase inhibitors. PHICS can be designed from kinase inhibitors, where a nucleophile (e.g., cysteine) near the inhibitor binding pocket initiates an addition-elimination reaction, resulting in the addition of a target protein binder to the kinase and simultaneous release of the inhibitor from the kinase active site (43A). Such cysteine-driven PHICS kinase binders can be derived from abundantly available acrylamide-based inhibitors, where the amide nitrogen is often an aryl-amine (43C) or, less commonly, an aliphatic amine (43B). Using such BTK inhibitor scaffolds, we synthesized BRD4 PHICS with an arylamine scaffold (43C) or an aliphatic amine scaffold (43B). Here, the inhibitor is linked to the BRD4 binder JQ1 via a cleavable methacrylamide linker. We also synthesized iPHICS (an inactive control using I-JQ1, which does not bind to BRD4) to test whether PHICS could rewire BTK specificity and induce BRD4 phosphorylation. Indeed, we observed significantly higher BRD4 phosphorylation in the presence of PHICS than in the presence of iPHICS. We also observed significantly higher levels of co-immunoprecipitated BTK-FLAG and higher levels of BRD4 phosphorylation in the presence of PHICS than in the presence of iPHICS. Beyond BTK, we have successfully developed recruitment ligands for EGFR, FGFR (pan), FGFR4, ITK, JAK3, and CDK2.

[0129] [Figures 44A-44C]Data demonstrating functional group transfer reactivity in cells. Applicants performed target engagement studies using compounds bearing an alkyne handle instead of JQ1 (41A) and confirmed covalent association of the scaffold with BTK in cells (41B). Briefly, HEK293T cells transiently expressing BTK were treated with alkyne-containing compounds for 4 hours. After washing and lysis with PBS, the cell lysate was subjected to a Cu-catalyzed click reaction with sulfo-Cy5.5 azide, and labeling of BTK was confirmed by in-gel fluorescence (41B).

[0130] [Figures 45A-45E] Data demonstrating inhibitor dissociation from the ATP pocket. To demonstrate release of the BTK inhibitor scaffold from the ATP-binding pocket, we used a previously reported assay based on bioluminescence resonance energy transfer (BRET) between nanoluciferase (nanoLuc) and a fluorophore probe that binds to the ATP pocket. Higher inhibitor occupancy of this ATP pocket prevents tracer binding, resulting in a reduced BRET signal (42A). As expected, acrylamide-based BTK inhibitors (42B, 42C) dramatically reduced the BRET signal, indicating blockage of the ATP pocket, while their derivatives with cleavable methacrylamide linkers (alkynes or PHICS, 42D and 42E) did not affect tracer binding. Consistent with these findings, we observed higher autophosphorylation of BTK with PHICS compounds compared to the parent acrylamide inhibitor. We also extended this approach to EGFR, FGFR, ITK, JAK3, and CDK2 kinases.

[0131] [Figure 46] Challenges surrounding the development of Cys-reactive linkers for BTK, EGFR, FGFR (pan), FGFR4, ITK, JAK3, CDK2, BMX, AKT, and JNK1.

[0132] [Figure 47]A still unmet need: on-demand phosphorylation of any protein using small molecules.

[0133] [Figure 48] Phosphorylation converts a neutral residue into a negatively charged residue.

[0134] [Figure 49] Kinase number 1. AMP-activated protein kinase (AMPK).

[0135] [Figure 50] Kinase number 2. Protein kinase C required for specificity and MoA rewiring.

[0136] [Figure 51A-51B] Negishi coupling route to benzolactams: 9 steps, 22% yield.

[0137] [Figure 52A-52B] Gain-of-function: PHICS can rewire kinase specificity.

[0138] [Figure 53] PHICS is "catalytic" and exhibits turnover.

[0139] [Figure 54] PHICS is "catalytic" and exhibits turnover.

[0140] [Figure 55] The Hooke effect: A comparison of three-body and two-body equilibria.

[0141] [Figure 56] PHICS exhibits greater specificity than the sum of the two parts.

[0142] [Figures 57A-57E] Kinase number 3. Abelson kinase for tyrosine phosphorylation.

[0143] [Figures 58A-58C] Evidence of addition and elimination reactivity in cells.

[0144] [Figure 59] Evidence of addition and elimination reactivity in cells.

[0145] [Figure 60] Challenges surrounding the development of Cys-reactive linkers.

[0146] [Figure 61] Substitution of the nitrile with a fluoro group reduces the rate of hydrolysis by 5-fold.

[0147] [Figure 62] A platform for pharmacological protein editing using writer and eraser inhibitors. (De) denotes modification in both directions, e.g., (de)phosphorylation can mean phosphorylation and / or dephosphorylation.

[0148] [Figure 63] C1 domain: a tiny chemical genetic tag of human origin.

[0149] [Figure 64] C1 domain: a tiny chemical genetic tag of human origin.

[0150] [Figure 65] (SEQ ID NOs: 1-5)-PHICS is capable of inducing naturally occurring phosphorylation and signal transduction.

[0151] [Figure 66] PHICS is capable of inducing naturally occurring phosphorylation and signal transduction.

[0152] [Figure 67] PHICS-mediated phosphorylation can induce liprin phase separation.

[0153] [Figure 68] PHICS can recruit the atypical kinase BRD4 to induce Myc degradation.

[0154] [Figures 69A-69C] PHICS induced BCR-ABL phosphorylation and sequestration.

[0155] [Figure 70] Homo-PHICS increases pY253, which is located in the ATP-binding loop.

[0156] [Figure 71] Homo-PHICS: Neophosphorylation-mediated inhibition of tumor fusion kinase. Homo-PHICS kills tumor fusion cancer lines at approximately 6 nM; kills resistant mutant cancer lines better than known drugs; is more effective than known drugs in other tumor fusion cell lines; MOA is neophosphorylation and potentially can be extended to other fusions.

[0157] [Figure 72A-72B] Homo-PHICS is an allosteric binder but not an inhibitor of ABL.

[0158] [Figure 73] Homo-PHICS dimerizes ABL.

[0159] [Figure 74] Homo-PHICS blocks downstream signaling of BCR-ABL.

[0160] [Figure 75] Homo-PHICS induces apoptotic cell death.

[0161] [Figure 76]PRISM profile of compound in 1,000 cancer cell lines of diverse lineage / dependency. Homo-PHICS PRISM data demonstrates selectivity.

[0162] [Figure 77] Homo-PHICS is functional in the presence of active site mutants.

[0163] [Figure 78] Drug resistance profile of asciminib and VS1150. Treatment doses increased over time (days 0–7: 1 nM ABL001, 20 nM VS1150; days 7–14: 2 nM ABL001, 40 nM VS1150; days 14–21: 4 nM ABL001, 80 nM VS1150; days 21–63: 100 nM ABL001, 500 nM VS1150).

[0164] [Figure 79] Statistically significant gRNA enrichment for asciminib. Red underlines in the enrichment plot correspond to gRNAs that will be followed up in subsequent single-guide experiments.

[0165] [Figure 80] Statistically significant gRNA enrichment for asciminib but not for VS1150. Red underlines in the enrichment plot correspond to gRNAs that will be followed up in subsequent single-guide experiments.

[0166] [Figure 81] PHICS can induce inhibitory neophosphorylation of known substrates.

[0167] [Figures 82A-82C] PHICS allows for the control of overactive BTK mutants.

[0168] [Figure 83]PHICS enables control of hyperactive BTK mutants: targeted polypharmacology.

[0169] [Figure 84] ABL / PKC / AMPK PHICS induces death of ibrutinib-resistant cells.

[0170] [Figure 85] PHICS can hyperphosphorylate and inhibit K-Ras signaling.

[0171] [Figure 86] PHICS can hyperphosphorylate and inhibit K-Ras signaling.

[0172] [Figure 87] Generalization to other enzymes for PTM addition or removal.

[0173] [Figure 88] Approximately 10 analogs were synthesized to achieve nM potency.

[0174] [Figure 89] PHICS kinase.

[0175] [Figure 90] AKT phosphorylation on BRD4.

[0176] [Figures 91A-91C] (91A) Current design of a dumbbell-shaped bifunctional molecule. (91B) Ternary complex formation with a dumbbell-shaped bifunctionality requires an allosteric non-inhibitory binder (green triangle). The natural substrate (blue triangle) can bind to the active site, facilitating PTM. (91C) Our novel design can be extended to active site inhibitors. Michael addition followed by retro-azaMichael allows the natural substrate to bind to the POI and facilitate PTM.

[0177] [Figures 92A-92C] (92A) Methacrylamides 4–6 bearing alkyne handles from acrylamides 1–3 for Cys labeling. (92B) Reaction scheme for determining the reactivity of methacrylamides 4–6 with N-Ac-Cys-Ome in PBS buffer at room temperature. (92C) Kinetic data of methacrylamides along with rate constants.

[0178] [Figures 93A-93H] (93A) Reaction scheme for the design of CGT-PHICS for FGFR targeting BRD4. (93B, 93C, 93D) Nano-BRET assay of binding pocket occupancy for known FGFR inhibitors fisogatinib, futibatinib, PRN1317, and their non-covalent derivatives, and CGT-PHICS. (93E, 93F, 93G, 93H) CGT-PHICS induced phosphorylation of BRD4 by FGFR4 and FGFR2 in HEK293T cells.

[0179] [Figures 94A-94C] (94A) Schematic of dPHICS-induced FGFR receptor dimerization and signaling. (94B) dPHICS design for FGFR dimerization. (94C) MAPK pathway activation in the presence of dPHICS.

[0180] [Figure 95A-95J]Development of PHICS using cysteine ​​group transfer chemistry. (95A) Structures of noncovalent binders (12A–16A), covalent inhibitors (12–16), methacrylamides (17–21), and PHICS (22–26) for each kinase (JAK3, ITK, and EGFR). (95B) Kinetic data for methacrylamides (17–21) along with rate constants. (95C, 95D, and 95E) Nano-BRET assays comparing occupancy of covalent (12, 13, and 14), noncovalent (12A, 13A, and 14A), and CGT-PHICS (22, 23, and 24) inhibitors for JAK3 (95C) and ITK (95D and 95E). (95F, 95G, 95H, 95I, and 95J) PHICS-induced phosphorylation of BRD4 by the kinases JAK3, ITK, and EGFR, respectively, expressed in HEK293T cells.

[0181] [Figure 96] Phosphorylation-inducing chimeric small molecules (PHICS).

[0182] [Figure 97] KRAS phosphorylation: Thinking beyond ABL and AMPK. Experimental results showing tyrosine phosphorylation by various kinases.

[0183] [Figures 98A-98C] Reduction of pERK signaling in KRAS G12D cells (AGS cell line) by chimeric molecules. 98A) Exemplary chimeric small molecules of the invention. 98B) Immunoblotting of phosphorylated ERK (pERK) using exemplary chimeric molecules. 98C) Alpha SureFire pERK assay using exemplary chimeric molecules.

[0184] [Figure 99] Reduction of viability of AGS cells (KRAS G12D) by chimeric molecules from Figure 98.

[0185] [Figure 100]Chimeras exhibit linker length dependence: optimal length is required. Chimeric molecules reduce (KRAS) viability and normalized BRET.

[0186] [Figure 101] Cell killing activity is not entirely dependent on the affinity of the KRAS binding agent. Reduction of the viability and BRET ratio of (KRAS) by the chimeric molecule.

[0187] [Figure 102] Is the RIPTAC mechanism being neglected? Viability and BRET ratio of (KRAS) by chimeric molecules.

[0188] [Figure 103] SNT-243 kills futibatinib-resistant cancer cells (H929). The chimeric molecule reduces viability.

[0189] [Figure 104] Chimeras from the fuchibatinib scaffold are less potent than those from the ibrutinib scaffold. Reduced viability with chimeric molecules.

[0190] [Figure 105] Bifunctional example with sulfonamide-based leaving group.

[0191] [Figure 106] Kinetics of thiol addition for the FDA-approved drugs osimertinib, dacomitinib, and pyrotinib after derivatization of their aniline groups to sulfonamides. JK4 consists of an example of R1 to a trifluoroethyl group. JF70 and JF74 represent Ar-thio leaving groups in two different oxidation states. DETAILED DESCRIPTION OF THE INVENTION

[0192] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0193] general definition Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For definitions of common terms and techniques in molecular biology, see Molecular Cloning: A Laboratory Manual, 2002. nd edition(1989)(Sambrook,Fritsch,and Maniatis);Molecular Cloning:A Laboratory Manual,4 th edition (2012) (Green and Sambrook);Current Protocols in Molecular Biology (1987) (FMAusubel et al. eds.); Lane, eds.): Antibodies A Laboratory Manual, 2 ndedition 2013(EAGreenfield ed.);Animal Cell Culture(1987)(RIFreshney,ed.);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, NY1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd You can refer to the 2011 edition.

[0194] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0195] The terms "optional" or "optionally" mean that the subsequently described event, circumstance, or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0196] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0197] The terms "about" or "approximately," when used herein in reference to a measurable value, such as a parameter, amount, time duration, etc., are intended to encompass variations at and from the specified value, such as variations of ±10% or less, ±5% or less, ±1% or less, and ±0.1% or less from and to the specified value, where such variations are appropriate for the practice of the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically and preferably disclosed.

[0198] As used herein, a "biological sample" may include whole cells and / or viable cells and / or cell debris. A biological sample may include (or be derived from) a "body fluid." The present 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 semen, gastric acid, gastric juice, lymph, mucus (including nasal discharge and phlegm), pericardial fluid, peritoneal fluid, pleural effusion, 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 culture media, body fluids, and cell culture media from body fluids. Body fluids may be obtained from a mammalian organism, for example, by paracentesis or other collection or sampling procedures.

[0199] 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, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0200] Various embodiments are described below. Note that these specific embodiments are not intended to be exhaustive or limiting of the broad aspects discussed herein. An aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any one or more other embodiments. Throughout this specification, the reference to "one embodiment," "an embodiment," or "an exemplary embodiment" means that the particular features, structures, or characteristics described in connection with that embodiment are included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," or "an exemplary embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one skilled in the art from this disclosure, in one or more embodiments. Furthermore, some embodiments described herein may include some features included in other embodiments but not others, meaning that combinations of features from different embodiments are within the scope of the present invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0201] All publications, published patent documents, and patent applications cited in this specification are hereby 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.

[0202] overview Small molecules are classically developed to inhibit enzymatic activity. The embodiments disclosed herein define a novel class of chimeric small molecules that confer novel functions to kinases through proximity-mediated effects. Generally, the chimeric small molecules comprise an effector-binding moiety connected to a substrate-binding moiety via a linker. The chimeric small molecules can be used to improve the kinetics of modification of native proteins by bringing the substrate molecule into proximity with the kinase, including by using energetically or otherwise more favorable chimeric small molecule configurations. Additionally, the chimeric small molecules can be used to retarget proteins to modify non-natural or neo-substrates.

[0203] In one aspect, chimeric small molecules can be constructed to facilitate covalent labeling of proteins with target-binding moieties. Labeling kinases with target-binding moieties can be used to define novel substrates not normally targeted or modified by such proteins. In such embodiments, the chimeric small molecule comprises a kinase-binding moiety linked to the target-binding moiety, but also comprises an electrophilic reactive group. The kinase-binding moiety non-covalently binds to the kinase of interest, resulting in the electrophilic reactive group "labeling" the protein by covalently binding the target-binding moiety to a nucleophile located on the protein—via a proximity-driven reaction. In such embodiments, the kinase-binding moiety is then released from the labeled kinase either by the intrinsic kinetics of the molecule or by application of a quencher. The target-binding moiety can then direct the labeled kinase to bind and modify novel target substrates. This approach also expands the number of available kinase-binding agents. For example, although several high-quality kinase inhibitors exist, such inhibitors remain bound to the kinase and are therefore unsuitable for use in targeted chimeras where they may otherwise inhibit the enzymatic activity of the bound kinase. As discussed in more detail below, the selection of appropriate inhibitors and the optional use of quenching molecules allows these inhibitors to be used in the labeling process described above without affecting downstream modification reactions.

[0204] Embodiments disclosed herein provide targeted chimeras designed for oncogenic targets. In one exemplary embodiment, a method for modifying, e.g., neophosphorylating, oncogenic targets is provided. Methods of use may include eliciting an immune response, generating autoantigens, and target inactivation. In exemplary embodiments, hyperphosphorylation or neophosphorylation of a target protein can result in immune recruitment to the target, e.g., by presenting a neoepitope and inducing T cells to attack cells presenting that epitope. Modification of kinases and key regulator proteins implicated in cancer is also within the scope of the methods disclosed herein.

[0205]

[0003] Embodiments disclosed herein provide methods for modifying a target substrate in a cell. For example, if the kinase-binding moiety is a kinase and the target is a substrate located in the cell, then the kinase labeled with the target-binding moiety can bind to the target substrate. The bound kinase can then neophosphorylate, thereby modifying, the target substrate, even if it is a non-cognate substrate of the kinase.

[0206]

[0003] Embodiments disclosed herein provide methods for mobilizing the host's immune system against cancer. For example, if the target substrate is an oncogenic protein, then a kinase labeled with an oncogenic target binding moiety using a chimeric small molecule can bind to the cancer through the oncogenic protein binding moiety. The bound kinase can then neophosphorylate the target oncogenic protein, thereby signaling the host's immune system to attack the cancer.

[0207]

[0003] Embodiments disclosed herein provide methods for mobilizing the host's immune system against pathogens. For example, if a target substrate is located on the surface of a pathogenic bacterium, a kinase that has been tagged with a pathogenic protein target binding moiety using a chimeric small molecule can bind to the bacterium through the target protein binding moiety. The bound kinase can then neophosphorylate the target pathogenic protein, thereby signaling the host's immune system to attack the bacterium.

[0208] Chimeric small molecules In one exemplary embodiment, the chimeric small molecule has the general formula A-L1-EB or A-L1-E-L2-B wherein A is a kinase-binding moiety, B is a target-binding moiety, L1 and L2 are each a linker, and E is an electrophilic reactive group. relates to.

[0209] These formula embodiments may be useful in, but are not necessarily limited to, situations in which the kinase binding moiety might otherwise inhibit or interfere with the ability of a kinase to which the kinase binding moiety is bound to modify a target substrate to which the target binding moiety is bound.

[0210] The electrophilic reactive group of the chimeric small molecule can be designed to react with a moiety on the kinase, e.g., on an amino acid of the kinase. The electrophilic reactive group can advantageously be designed to react with a moiety on the kinase that is proximal to the binding site of the kinase-binding moiety. When the electrophilic reactive group reacts with a moiety on the kinase, e.g., a nucleophilic group on an amino acid located on the protein, it can enable labeling or binding of the kinase with a target-binding moiety. Attaching a target-binding moiety to a kinase in this manner can create a reprogrammed kinase that is capable of modifying target substrates, including target substrates other than naturally occurring substrates of that kinase. Thus, in exemplary embodiments, the kinase-binding moiety binds to the kinase and is selected based on the availability of a binding pocket and an amino acid side chain proximal to the binding pocket that can be used to react with the electrophilic reactive group of the target chimera.

[0211] The kinase-binding moiety can bind to a kinase and can be specific for one or more kinases. In certain embodiments, the kinase-binding moiety can further comprise a bioorthogonal group. The kinase-binding moiety can be selected to have a half-life that is shorter than the half-life of the kinase, as further detailed herein.

[0212] The target binding moiety can be specific for one or more desired targets. In one exemplary embodiment, the desired target is a macromolecule, such as a protein. The target binding moiety can bind to the desired target (target substrate), thereby bringing the kinase into proximity with the desired target. In certain embodiments, the desired target can advantageously be a non-cognate substrate of the kinase. The desired target can be a pathogenic or oncogenic target.

[0213] Kinase-binding moiety Chimeric small molecules according to the general formula A-L1-EB or A-L1-E-L2-B comprise a kinase-binding moiety, A, which may include any molecule capable of non-covalently binding to a kinase. The kinase-binding moieties of the chimeric small molecule, also referred to interchangeably herein as kinase-binding agents, may target one or more different kinases or one or more locations on a kinase.

[0214] Some kinases belong to the phosphotransferase family and phosphorylate substrates by transferring the gamma phosphate of ATP to a hydroxyl group of the substrate. The substrate may include a lipid, sugar, or amino acid. 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 exemplary embodiment, the kinase-binding moiety is a kinase activator. In an exemplary embodiment, the kinase-binding moiety is a kinase inhibitor. However, as the design of the targeting chimera is to generate a kinase labeled with a target-binding moiety, binding of the kinase is the primary objective, rather than its inhibitory or activating behavior, which, when initially utilized to bind to a kinase, allows for the generation of repurposed kinases labeled with a target-binding moiety, rather than using the kinase-binding moiety for its kinase-activating or inhibitory properties.

[0215] Chimeric small molecules are preferably designed so that one or more nucleophilic groups located on a protein, e.g., a kinase, readily react with an electrophilic reactive group on the chimeric small molecule. The nucleophilic group-containing portion of the protein can be an amino acid side chain. Thus, kinase-binding moieties can be selected based on the availability of a binding pocket and amino acid side chains proximal to the binding pocket that are available for reaction with the electrophilic reactive group on the target chimera. Further design of exit vectors contained on the kinase-binding agent or linker of the chimeric small molecule can further enable desired configuration of the kinase-binding moiety at the protein binding site.

[0216] Half-life In one exemplary embodiment, when a chimeric small molecule is used to label the surface of a kinase with a target-binding moiety, the kinase-binding moiety can be selected, in part, based on its half-life. In one exemplary embodiment, the kinase-binding moiety can be selected, in part, based on its half-life relative to the half-life of the kinase. In some embodiments, the half-life of the kinase-binding moiety is two-, three-, four-, or five-fold shorter than that of the kinase. Without being bound by any particular theory, designing a chimeric small molecule such that the half-life of the kinase-binding moiety is shorter than that of the kinase may enable desirable reaction rates when the kinase is labeled with the kinase-binding moiety via an electrophilic reactive group. The half-life of the kinase-binding moiety and the kinase generally relates to the time required for the concentration of the kinase-binding moiety or kinase to decrease to half of its initial concentration. In one exemplary embodiment, the half-life can be measured, for example, as the time it takes for a molecule to degrade half of the molecule initially measured in a sample that may include a cell, a plurality of cells, a tissue, an organoid, or a mammal. In an exemplary embodiment, the half-lives of the kinase and kinase-binding moiety are measured under the same or similar conditions, e.g., in the same cell type, tissue, or organism. In an exemplary embodiment, 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.

[0217] Measurement of the half-life of a kinase binding moiety can be, for example, the dissociation time t, which describes the average time required for half of the initially occupied receptor to become vacant under conditions that allow association or rebinding of the protein binding moiety. 1 / 2 or receptor occupancy t 1 / 2 The size of the dissociation or binding pocket, which requires a conformational change in the receptor, can play a factor in residence time and can be considered when selecting a protein-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.

[0218] The time a compound resides at its target, e.g., residence time, can be used. For a discussion and identification of residence times and kinetic parameters of exemplary kinase-binding moieties, 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, which is incorporated herein by reference in its entirety, and in particular, see Table 1, Table 3A-3B, Table 4A-4C, Table S3, and Table S4 for teachings regarding tyrosine kinase inhibitors, EGFR inhibitors, ponatinib against various kinases, specific kinases and their related inhibitors, Aurora A and B kinase inhibitors, and P13k lipid kinase inhibitors. Elimination half-life can also be used alone or in conjunction with assessing residence time. Additional pharmacodynamics and pharmacokinetics can also be considered in assessing the half-life of kinase-binding moieties. Half-life can be modeled. See, e.g., Callegari D, Lodola A, Pala D, et al. Metadynamics Simulations Distinguish Short- and Long-Residence-Time Inhibitors of Cyclin-Dependent Kinase 8 [published correction appears 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).

[0219] Kinase half-life measurements, mass spectrometry-based proteomics such as SILAC (stable isotope labeling with amino acids in cell culture)-based proteomics, and other half-life measurement techniques (see Matheison et al., Nature Communications volume 9, Article number: 689 (2018)) can be used. High-throughput proteomics can be used to estimate kinase half-lives in specific tissues and / or cells, or further predictive modeling can be used to predict such kinase half-lives in tissues from cellular properties (see, e.g., Rahman M, Sadygov RG Predicting the protein half-life in tissue from its cellular properties. PLoS ONE 12(7):e0180428.doi.org / 10.1371 / journal.pone.0180428 (2017)).

[0220] The kinase-binding moiety can be selected based on the desired target substrate and the modifications to that substrate. Advantageously, the kinase-binding moiety can be an activator or inhibitor of the kinase. The kinase-binding moiety can be selected based on the abundance of the kinase in the target cell; kinases with high activity at low concentrations, e.g., nanomolar activity; available crystal structures and characterization of kinase activity; kinase-binding moieties with short residence times; the ability of the kinase-binding moiety of the chimeric small molecule to accommodate bioorthogonal groups, e.g., small bioorthogonal handles, without affecting binding capacity and / or residence time; kinases with a high density of amino acids with nucleophilic side chains, e.g., serine / threonine / tyrosine / lysine, near the binding pocket; and / or whether labeling the kinase may interfere with its enzymatic activity (which can be based on experimental data and / or modeling). The linker length on the chimeric small molecule can be adjusted to allow for modifications, e.g., phosphorylation, and increasing the distance from the binding pocket allows for targeting modifications to positions, e.g., amino acid residues, further away from the binding pocket. For example, when binding to a target substrate in close proximity to the binding pocket of a kinase, a longer linker length can be utilized when the bioconjugation reaction is optimized to be further away from the binding pocket but still allow for the target binding moiety. Adjusting the linker length can also include the level of flexibility or rigidity depending on the configuration of the target binding moiety desired for amino acid residue modification. Shorter linker lengths can allow for modifications within the binding pocket, which may be desirable for some applications.

[0221] In an exemplary embodiment, the kinase-binding moiety is an allosteric modulator. Considerations for selecting a kinase-binding moiety include allosteric signaling, which may involve changes related to a network of non-covalently interacting protein residues, conformational selection, and induced fit in both spatial and temporal aspects. In one exemplary embodiment, the kinase-binding moiety can be an allosteric activator or inhibitor of the kinase. Allosteric activators or inhibitors can be discovered computationally. In one exemplary method, a high-quality drug target is obtained. Allosteric site prediction is then performed using a method such as perturbation response scanning (PRS) combined with all-atom molecular dynamics (MD) and dynamic residue networks (DRN). Allosteric modulators are then identified using methods such as homology modeling, docking, or essential dynamics. For an illustration of this process, see Figures 2 and 3 of Amamuddy S., et al. Integrated Computational Approaches and Tools for Allosteric Drug Discovery. 21 IJMS, 847 (2020) (incorporated herein by reference).

[0222] Kinase-binding moieties can be selected based on the type of desired modification performed by the kinase, for example, post-translational modification of the target substrate. In one exemplary embodiment, the kinase-binding moiety has the ability to bind to a kinase that phosphorylates the target, and therefore the type of kinase can be selected based on the desired modification of the target substrate. Post-translational modification (PTM) is one type of modification that can be performed. Thus, post-translationally modified kinases are a set of kinase-binding moieties contemplated for use in the present invention.

[0223] In one exemplary embodiment, the kinase binding moiety provides modifications to amino acids, see, e.g., Table 1 in Karve et al., Journal of Amino Acids Volume 2011, Article ID 207691, 13 pages, DOI: 10.4061 / 2011 / 207691, incorporated herein by reference. Karve et al. summarizes some post-translational modifications and their importance in various diseases and normal development. Karve et al. assesses phosphorylation of amino acids and is specifically incorporated herein with reference to the phosphorylation modifications detailed therein.

[0224] The electrophilic reactive group can react with a moiety on the kinase, e.g., a nucleophilic group located on the kinase, allowing for labeling or binding of the kinase by the target binding moiety. Binding of the target binding moiety to the kinase in this manner can generate a reprogrammed kinase capable of modifying the target substrate. Thus, in exemplary embodiments, the kinase binding moiety binds to the kinase and is selected based on the availability of a binding pocket and amino acid side chains proximal to the binding pocket that can be used to react with the electrophilic reactive group of the target chimera.

[0225] Exemplary Kinase Binding Moieties In an exemplary embodiment, the kinase-binding moiety 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 such phosphorylation activity. In one exemplary embodiment, the kinase activator moiety promotes the active conformation of the enzyme, in one aspect through binding interactions with a regulatory subunit. See, e.g., Zorn et al. Nat Chem Biol. 2010 Mar;6(3):179-188; doi:10.1038 / nchembio.318. Kinases can act on the amino acids serine, threonine, tyrosine, or combinations thereof.

[0226] Activator moieties can be identified from activators known in the art. Activators can be derivatives of activators known in the art and can contain fewer or additional functional groups that still enable activator activity, but that may enhance or facilitate the desired formation, conformation, or binding site of a chimeric small molecule described herein. Exemplary modifications can include derivatives to increase solubility, charge, functionality for use with orientation-determining adaptors or linkers, as detailed elsewhere herein.

[0227] In one embodiment, the kinase-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 according to 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. Additional considerations for kinase inhibitors include competition with ATP binding, which may include inhibition mechanisms such as mimicking the hydrogen-bonding interactions normally formed by the adenosine ring of ATP or reversible or irreversible covalent binding. See, for example, (Gross et al. J Clin Invest. 2015;125(5):1780-1789).

[0228] A key consideration in the design of kinase inhibitors is the degree of specificity for a particular kinase. While higher specificity is considered advantageous, kinase inhibitors with lower specificity for a particular kinase facilitate the recruitment of a broad range of kinases. Because the kinase is a vehicle for modifying target substrates, broad-spectrum kinase inhibitors are advantageous.

[0229] In one embodiment, the protein-binding moiety is a broad-spectrum kinase inhibitor (PKI). A broad-spectrum kinase inhibitor refers to a molecule that binds to two or more kinases. A broad-spectrum kinase inhibitor is a molecule that has binding specificity for a binding pocket that is highly conserved among kinases. A broad-spectrum kinase inhibitor may bind to two, three, four, five, or more different kinases. In one exemplary embodiment, the broad-spectrum kinase inhibitor is an ATP-competitive kinase inhibitor. In one exemplary 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; 390-399. For example, the kinase inhibitor imatinib can inhibit c-KIT, PDGFR-α, 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 2014 May 19); similarly, sunitinib, sorafenib, and cabozantinib are known for their broad spectrum activity and are provided as non-limiting examples of broad spectrum kinase inhibitors. In one exemplary embodiment, the PKI is modified to include a bioorthogonal group.

[0230] In one exemplary embodiment, the protein-binding moiety is a kinase-binding moiety. Exemplary kinases to which the chimeric small molecules of the invention may bind 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.

[0231] ABL joint part In one exemplary embodiment, the protein-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. ABL tyrosine kinase can be found in the nucleus, cytoplasm, and mitochondria. ABL protein is normally under well-orchestrated regulation. However, chromosomal translocations that join the ABL gene with genes encoding other proteins result in various fusion proteins that are prone to dimerization (or oligomerization) and autophosphorylation. As a result, ABL kinase becomes constitutively active, leading to myeloproliferative disorders. In one exemplary embodiment, a chimeric small molecule utilizes one of the ABL kinase-binding moieties described herein in conjunction with a target-binding moiety described herein.

[0232] In one exemplary embodiment, the ABL kinase binding moiety is an ABL kinase activator. In one exemplary 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, 177-186, February 25, 2011: [ka] (DPH).

[0233] In one exemplary embodiment, the c-Abl kinase activator is [ka] These can be selected from the following, which demonstrated in vivo activation of c-Abl in 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. The novel aminopyrazoline small molecule activators listed in Table 6 of Simpson et al. are specifically incorporated herein by reference.

[0234] In one exemplary embodiment, the c-Abl kinase activator is [ka] is.

[0235] In one exemplary embodiment, the c-AB1 kinase binding moiety has the formula [ka] (Wherein R is [ka] is) The compound is a (5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,4-imidazolidinedione or 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin) (DPH) derivative according to the present invention.

[0236] In an exemplary embodiment, the DPH is functionalized. [ka]

[0237] In one exemplary embodiment, the ABL kinase activator is: [ka] (where the dashed circle identifies the attachment of an orientation-determining adaptor and / or linker) The method of attaching the linker and orientation adaptor prior to attachment to the protein-binding moiety can utilize the functional group depicted in the dashed circle on the ABL kinase activator.

[0238] The activator moiety can be functionalized for methods of attaching an orientation-determining adaptor and a linker. The ABL kinase activator parent molecule DPH can be functionalized for methods of attaching an orientation-determining adaptor and a linker. Exemplary molecules include: [ka] It could be.

[0239] Once functionalized, the orientation adaptor and linker can be added either sequentially, or the orientation adaptor and linker can be added at once as one molecule. Exemplary molecules are provided below, where the R group represents the protein-binding moiety. [ka] Optionally, more than one activator moiety can be attached to a protein binding moiety. In either case, the identified activator moieties can be functionalized as described herein for methods of attaching linkers and orienting adaptors prior to attachment to the protein binding moiety, e.g., utilizing the functional groups depicted in the dashed circles.

[0240] In an exemplary embodiment, the Abl kinase activator is DPH or a dihydropyrazole activator. [ka] wherein X is (CH), which may be substituted with, for example, one or more of amide, acetal, aminal, amine, alkyl, ether, hydrocarbyl, and derivatives thereof, or other groups as described elsewhere herein. In one exemplary embodiment, n is 0 to 20, more preferably n is 1 to 10 or 2 to 7, and R is [ka] In one exemplary embodiment, the ABL kinase activator dihydropyrazole is linked via a variety of linkers, see, e.g., (PCT / US2021 / 012816, PHICS 10.1-10.5, Figure 64A).

[0241] In a preferred embodiment, one of the ABL kinase binding moieties as detailed herein is used in a chimeric small molecule together with a BRD4 binding moiety as described herein. In an exemplary embodiment, when the protein binding moiety is for BRD4: [ka] Exemplary molecules of the formula are: [ka] may include:

[0242] In one embodiment, the kinase binding moiety has the formula [ka] (wherein R1 to R5 are independently selected from 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; or aliphatic halide such as -OCF2Cl; Z is independently selected from B, C, N, O, S (preferably one or two atoms of Z = N, O, S), or a combination thereof; Ra, Rb, and Rc are independently selected from alkane, alkene, alkyne, ether, alcohol, amine, nitrile, nitro, thiol, sulfone, sulfonate, halogen, carbonyl; acyl; ketone; carboxylic acid ester and Re is independently selected from 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 halide such as -OCF2Cl; cyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle; one or more fused rings comprising any combination of any of the foregoing rings; and Re is one or more fused rings at one or more positions, or can be joined together with R1 or R5 to form a ring, or any combination thereof. is an ABL kinase binding moiety according to

[0243] In one embodiment, R a , R b , R c One or more of the [ka] and an amide further attached to a molecule selected from the group consisting of: (which may optionally be further substituted at one or more positions with 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; or any combination of these groups).

[0244] In one embodiment, the ABL binding moiety has formula II(b), where Re is [ka] wherein Rf and Rg are selected from the group consisting of: cyclic hydrocarbons; unsaturated cyclic hydrocarbons; heterocycles; one or more fused rings comprising any combination of any of the foregoing rings (optionally substituted at one or more positions with 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 comprising any combination of any of the foregoing rings). In an exemplary embodiment, Rf and Rg are [ka] are independently selected from the group consisting of:

[0245] In one embodiment, the kinase binding moiety is [ka] wherein R is selected from 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 halide such as -OCF2Cl, or any combination thereof; and optionally [ka] is selected from.

[0246] In an exemplary embodiment, the kinase binding moiety is an ABL kinase inhibitor. In an exemplary embodiment, the ABL inhibitor has the formula: [ka] The most effective treatment is imatinib.

[0247] In one exemplary embodiment, the ABL inhibitor is nilotinib, dasatinib, bosutinib, ponatinib, or any derivative thereof. [ka] The kinase-binding molecule is selected from:

[0248] In one embodiment, the ABL kinase binding molecule is [ka] is selected from the group consisting of:

[0249] In one embodiment, the ABL kinase binding molecule is [ka] is selected from the group consisting of:

[0250] In one embodiment, the ABL kinase binding molecule is [ka] is selected from the group consisting of:

[0251] In an exemplary embodiment, the ABL kinase binding moiety has the formula: [ka] [Table 1] The drug is asciminib, also known as ABL-001. Asciminib is a negative allosteric modulator of BCR-ABL1, inducing the kinase to adopt an autoinhibitory, and therefore inactive, conformation. Asciminib-based PROTACs have received fast-track designation. In the UK, asciminib is available for compassionate use for designated organ dysfunction and has been shown to have minimal effects on platelet function. Asciminib inhibits cell proliferation in vitro and in wild-type ABL1 cell lines using GI agonists. 50 1.5nM and ABL1 T315I Asciminib has an inhibitory effect of 35 nM against ABL proto-oncogene 1, a non-receptor tyrosine kinase, with a pIC of 8.6-9.5. 50 See, e.g., 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), 8120-8135 (hereby incorporated by reference in its entirety). In some embodiments, the compound is a compound according to the method of Schoepfer et al. (2018). [Table 2] relates to.

[0252] In an exemplary 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. The interaction of BO1 with wild-type protein shows an ATP-competitive / mixed mechanism of action. BO1 has a pK of 7.0-7.4 for ABL proto-oncogene 1, a non-receptor tyrosine kinase. i See, 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), 1207-1211 (hereby incorporated by reference in its entirety), specifically compounds 6a-6u. [Table 3]

[0253] In one exemplary embodiment, the ABL kinase binding moiety has the formula: [ka] GNF-2 is a highly selective, non-ATP-competitive inhibitor of Bcr-Abl. It binds to a site distant from the ATP pocket and acts as a negative allosteric modulator. GNF-2 inhibits Bcr / Abl fusion proteins with an IC of 267 nM. 50 See, for example, Zhang, J., et al. "Targeting Bcr-Abl by Combining Allosteric with ATP-Binding-Site Inhibitors." Nature 2010, 463(7280), 501-506 (incorporated herein by reference in its entirety).

[0254] In one exemplary 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 the resistance patterns associated with imatinib or nilotinib treatment (except for the gatekeeper mutation T315I). Co-treatment with GNF-2 (the original structural reincarnation of GNF-5) plus imatinib significantly reduces the emergence of resistant clones in vitro. GNF-5 downregulates BCR-ABL kinase activity by mimicking the effect of myristic acid binding, which forces the protein to adopt an inactive conformational state. GNF-5 has a pIC of 6.7 against ABL proto-oncogene 1, a non-receptor tyrosine kinase. 50 See, for example, Deng, X., et al. "Expanding the Diversity of Allosteric Bcr-Abl Inhibitors", J. Med. Chem. 2010, 53(19), 6934-6946 (hereby incorporated by reference in its entirety) and Zhang Nature 2010. In some embodiments, functional groups can be modified at specific positions to achieve SAR around the GNF-2 scaffold. [ka] The crystal structure of GNF-2 bound to the Abl myristoyl pocket is also available for further optimization; see Figure 2 in Zhang, Nature, 2010 463, 501-506, incorporated herein by reference. A co-crystal structure of imatinib and GNF-2 complexed with c-Abl is also available (PDB ID: 3K5V). Additional targeting moieties can be designed as described in Figure 3 in Zhang, Nature (2010:, Figure 3, incorporated by reference and illustrated below). [ka]

[0255] In an exemplary embodiment, the ABL kinase binding moiety has the formula: [ka] DPH is the ICW EC 50 is 6.1, 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 (incorporated herein by reference in its entirety), and DPH and the compounds as specified below: [ka] and compounds 45 and 32 from Simpson et al.: [Table 4] In another exemplary embodiment, the ABL target binding moiety is any c-ABL kinase activator from Simpson J. Med. Chem. 2019.

[0256] In an exemplary embodiment, the ABL kinase binding moiety has the formula: [ka] It is a dihydropyrazole according to the formula:

[0257] In one exemplary embodiment, the ABL kinase binding moiety is: [ka] is selected from the group consisting of:

[0258] In one embodiment, the ABL kinase binding moiety [ka]

[0259] R in the formula of the ABL inhibitor is optimized for physiochemical 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, or halogen-based substituent, heterocycle, fused ring, or any combination thereof. In a preferred exemplary embodiment, R is selected from 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 halide such as -OCF2Cl, or any combination thereof. In a preferred exemplary embodiment, R is [ka] is selected from.

[0260] In an exemplary embodiment, the ABL inhibitor kinase binding molecule is [ka] is selected from.

[0261] In another exemplary embodiment, the ABL inhibitor kinase binding molecule has the formula [ka] is selected from.

[0262] In an exemplary embodiment, X and R2 are optimized with respect to physicochemical properties such as solubility and / or permeability and / or pharmacokinetic properties such as microsomal stability or target binding. In an exemplary embodiment, X is any feasible boron-, carbon-, nitrogen-, oxygen-, or sulfur-based element or compound. In a preferred exemplary embodiment, X is selected from C, N, O, and S. In an exemplary embodiment, R2 is selected from any boron-, carbon-, nitrogen-, oxygen-, sulfur-, or halogen-based substituent, heterocycle, fused ring, or any combination thereof. In a preferred exemplary embodiment, R2 is selected from 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 halide such as -OCF2Cl, or any combination thereof. In a preferred exemplary embodiment, R2 is [ka] is selected from.

[0263] In one exemplary embodiment, the ABL kinase binding molecule is [ka] is selected from.

[0264] In an exemplary embodiment, the X, Y, and R groups are optimized for physiochemical properties such as solubility and / or permeability and / or pharmacokinetic properties such as microsomal stability or target binding. In an exemplary embodiment, X and Y are independently selected from any boron-, carbon-, nitrogen-, oxygen-, sulfur-, or halogen-based substituent, heterocyclic ring, fused ring, or any combination thereof. In a preferred exemplary embodiment, X is a halogen. In a preferred exemplary embodiment, Y is selected from C, N, O, and S. In an exemplary embodiment, R1, R2, and R3 are independently selected from any boron-, carbon-, nitrogen-, oxygen-, sulfur-, or halogen-based substituent, heterocyclic ring, fused ring, or any combination thereof. In preferred exemplary embodiments, R1, R2, and R3 are independently selected from 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 halide such as -OCF2Cl, or any combination thereof.

[0265] In another exemplary embodiment, the ABL inhibitor kinase binding molecule is [ka] is selected from.

[0266] In an exemplary embodiment, the Y and R groups are optimized for physiochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding. In an exemplary embodiment, Y and Y1 in the above formula are any feasible boron-, carbon-, nitrogen-, oxygen-, or sulfur-based element or compound. In a preferred exemplary embodiment, Y and Y1 are selected from C, N, O, and S. In an exemplary embodiment, R3, R4, R6, and R7 in the above formula are independently selected from any boron-, carbon-, nitrogen-, oxygen-, sulfur-, or halogen-based substituent, heterocycle, fused ring, or any combination thereof. In preferred exemplary embodiments, R3, R4, R6, and R7 are independently selected from 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 halide such as -OCF2Cl, or any combination thereof.

[0267] In another exemplary embodiment, the ABL kinase inhibitor binding molecule is [ka] is selected from.

[0268] In an exemplary embodiment, Y and R groups are optimized for physiochemical properties such as solubility and / or permeability and / or pharmacokinetic properties such as microsomal stability or target binding. In an exemplary embodiment, Y in the above formula is any viable boron-, carbon-, nitrogen-, oxygen-, or sulfur-based element or compound. In a preferred exemplary embodiment, Y is selected from C, N, O, and S. In an exemplary embodiment, R, R, and R in the above formula are independently selected from any boron-, carbon-, nitrogen-, oxygen-, sulfur-, or halogen-based substituent, heterocycle, fused ring, or any combination thereof. In preferred exemplary embodiments, R4, R6, and R7 are independently selected from 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 halide such as -OCF2Cl, or any combination thereof.

[0269] In an exemplary embodiment, the protein-binding moiety is an ABL inhibitor. In an exemplary embodiment, the ABL inhibitor is DCC-2036, which has the formula: [ka] It is a dual anchor inhibitor that binds to both the switch control pocket E282 / R386 pair and the Met318 ATP hinge with an IC50 value of 0.8 nM.

[0270] In one exemplary embodiment, the kinase binding moiety is a c-ABL tyrosine kinase inhibitor from WO2019173761 (hereby incorporated by reference) or any derivative thereof.

[0271] AMPK binding part In one exemplary embodiment, the kinase binding moiety is an AMPK kinase binding moiety. AMPK is a serine / threonine kinase that assembles into a heterotrimeric complex consisting of a catalytic α-subunit and two regulatory β- and γ-subunits. See, for example, Wells et al. (2012). If a small molecule mimics AMP binding to the γ-subunit, it may be able to directly activate AMPK.

[0272] In one embodiment, the AMPK kinase binding moiety has the formula: [ka] (Wherein R is [ka] , carbohydrate mimetics, heterocycles, dianhydrohexitols, pyranoses, or furanoses; Q is selected from the group consisting of B, C, N, O, and S; H is N A or N B and X1 and X2 are 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; and Z is selected from the group consisting of 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; or an aliphatic halide such as -OCF2Cl, which may be optionally further substituted. relates to.

[0273] In an exemplary embodiment, Z has the formula: Z a -Z b (In the formula, Z a teeth, [ka] selected from the group consisting of: Z b teeth, [ka] and n is 0 to 6. It may be related to.

[0274] In one embodiment, the AMPK binding moiety is [ka] The AMPK binding moiety is selected from the group consisting of:

[0275] Additional AMPK kinase binding moieties that can be used in 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 their use and optimization in the chimeric small molecules of the present invention.

[0276] The AMPK binding moiety can be, for example, as described in U.S. Patent Application Publication No. 20050038068, which is incorporated herein by reference; [ka] or derivatives thereof as further modified in accordance with the teachings detailed herein for their use and optimization in the chimeric small molecules of the present invention.

[0277] In one exemplary embodiment, the kinase-binding moiety is an AMPK activator. In an exemplary embodiment, the AMPK activator is [ka] Other AMPK activators include those with a pEC50 of 6.0 50 Examples include A769662, which has a value similar to that of the AMPK activator A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A30, A31, A32, A33, A44, A45, A46, A47, A48, A49, A50, A51, A52, A53, A54, A55, A56, A57, A58, A59, A60, A61, A62, A63, A64, A65, A66, A67, A68, A69, A769662, which has a value similar to the AMPK activator

[0278] The AMPK activator can be, for example, as described in U.S. Patent Application Publication No. 20050038068, which is incorporated herein by reference; [ka] The AMPK activator can be as described in WO 2007019914, WO 2009124636, WO 2009135580, WO 2008006432, or WO 2009152909, which are incorporated by reference herein. In an exemplary embodiment, the activator is [ka] It may be related to.

[0279] The AMPK activator can be as described in WO2009100130, which is incorporated herein by reference. In one aspect, the AMPK activator is [ka] relates to.

[0280] The AMPK activator can be as described in WO 2010036613, WO 2010047982, WO 2010051176, WO 2010051206, WO 2011106273, or WO 2012116145. In one exemplary embodiment, the AMPK activator is: [ka] relates to.

[0281] In an exemplary embodiment, the AMPK activator can be as described in International Publication Nos. 2011029855, 2011138307, 2012119979, and 2012119978, which are incorporated herein by reference. [ka] You can choose from:

[0282] In an exemplary embodiment, the AMPK activator can be as described in International Publication Nos. 2011032320, 2011033099, 2011069298, 2011070039, 2011128251, and 2012001020, which are incorporated by reference herein. [ka] You can choose from:

[0283] In one exemplary embodiment, the AMPK activator can be as described in WO2011080277, which is incorporated herein by reference. In one aspect, the AMPK activator is: [ka] It could be.

[0284] In one exemplary embodiment, the AMPK activator can be as described in WO2012033149, which is incorporated herein by reference. In one aspect, the AMPK activator is: [ka] You can choose from:

[0285] In an exemplary embodiment, the AMPK activator is MT47-100, having the formula: [ka] MT47-100 regulates AMPK activity, but the direction of regulation depends 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. i The value is 5.4, while the pK i The value is 4.6 for allosteric inhibitors. 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), 705-711 (incorporated herein by reference in its entirety).

[0286] Additional AMPK binding moieties for use in the present invention are disclosed in WO 2007019914, WO 2009124636, WO 2009135580, WO 2008006432, WO 2009152909, WO 2011029855, WO 2011138307, WO 2012119979, WO 2012119978, WO 2011032320, WO 2011032321, WO 2011032322, WO 2011032323, WO 2011032324, WO 2011032325, WO 2011032326, WO 2011032327, WO 2011032328, WO 2011032329 ... Nos. 2011033099, 2011069298, 2011070039, 2011128251, 2012001020, 2011080277, 2012033149 (incorporated herein by reference), and may be derivatives thereof as further modified in accordance with the teachings detailed herein for their use and optimization in the chimeric small molecules of the invention.

[0287] PKC binding part In one exemplary embodiment, the kinase-binding moiety is a PKC kinase-binding moiety. In one exemplary embodiment, the kinase-binding moiety is a PKC activator or inhibitor. Protein kinase C (PKC) is composed of multiple isozymes, which play roles in signal transduction pathways, exhibit tissue-specific expression, and fulfill various biological roles. In certain embodiments, the chimeric small molecules disclosed herein can utilize PKC kinase-binding moieties selective for PKC isoforms, such as classical (cPKC-α, βI, βII, γ), novel (nPKC-δ, ε, η, θ), atypical (αPKC-ζ, ι / λ), and PKCμ (a form between the novel and atypical isoforms). In one exemplary embodiment, the PKC-binding moiety has the formula [ka] or an analog thereof.

[0288] For additional PKC binding moieties that can be constructed for use in the molecules described herein, see, e.g., PCT / US21 / 12816, paragraphs

[0179] to

[0194] (specifically incorporated herein by reference).

[0289] In one exemplary embodiment, the kinase-binding molecule can be designed as an activator of diacylglycerol (DAG)-reactive C1 domain-containing proteins, such as protein kinase C. Protein kinase C (PKC) is composed of multiple isozymes, which play roles in signal transduction pathways, exhibit tissue-specific expression, and fulfill various biological roles. The chimeric small molecules disclosed herein can utilize activators of PKC, where the activating moiety is selective for PKC isoforms.

[0290] In one exemplary embodiment, the kinase-binding moiety is a DAG activator. Activators of DAG-reactive proteins may include DAG-indolactones as described in L.C. 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 an exemplary embodiment, the compound is selective for PKCα or PKCε.

[0291] DAG lactones, such as AJH-836, as described in Cooke, et al., J. Biol. Chem. (2018) 293(22) 8330-8341. In one exemplary embodiment, the DAG lactone is represented by the formula [ka] As provided in Cooke, the formula in AJH-836 is: [ka] and is selective for PKCδ and PKC.

[0292] Teleocidins such as (-)-indolactam-V (ILV) and benzolactam-V8, such as 7-substituted benzolactam-V8, can be used as PKC activators. The PKC activator can be as described in Ma, et al., Org. Lett. 4:14 (2002) DOI:10.1021 / ol0261251.

[0293] In an exemplary embodiment, the PKC activator has the formula [ka] (wherein R1, R3, and R4 are each independently alkyl, alkenyl, or alkynyl, and R2 is a saturated or unsaturated alkylene (e.g., branched alkylene, straight chain alkylene, cycloalkylene, C1-C 22 Branched alkylene, C1-C 22 Straight chain alkylene, C3-C 22 Cycloalkylene, C1-C 10 Branched alkylene, C1-C 10 Straight chain alkylene, C3-C 10 Cycloalkylene, C1-C8 branched alkylene, C1-C8 straight chain alkylene, C3-C8 cycloalkylene), C1-C 22 Saturated or unsaturated heteroalkylene (e.g., branched heteroalkylene, straight-chain heteroalkylene, heterocycloalkylene, C1-C 22 Branched heteroalkylene, C1-C 22 Straight chain heteroalkylene, C3-C 22 Heterocycloalkylene, C1-C 10 Branched heteroalkylene, C1-C 10 Straight chain heteroalkylene, C3-C 10heterocycloalkylene, C1-C8 branched heteroalkylene, C1-C8 straight chain heteroalkylene, C3-C8 heterocycloalkylene), arylene (e.g., C5-C 22 arylene), heteroarylene (e.g., C5-C 22 divalent hydrocarbons selected from heteroarylene, substituted amides ... 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 C6 arylene, optionally substituted C 5~12 Heteroarylene, hydroxy-substituted C 3~6 cycloalkylene or C heterocycloalkylene substituted with hydroxy; each of the foregoing may have one or more (e.g., 2, 3, 4, 5) points of substitution; and R a is independently selected at each occurrence from hydrogen or alkyl (eg, C1-C7 alkyl, C1-C3 alkyl).

[0294] In one exemplary embodiment, the formula is: [ka] (wherein R1, R3, and R4 are each independently alkyl, alkenyl, or alkynyl, and R2 is a saturated or unsaturated alkylene (e.g., branched alkylene, straight chain alkylene, cycloalkylene, C1-C 22 Branched alkylene, C1-C 22 Straight chain alkylene, C3-C 22 Cycloalkylene, C1-C 10 Branched alkylene, C1-C 10 Straight chain alkylene, C3-C 10 Cycloalkylene, C1-C8 branched alkylene, C1-C8 straight chain alkylene, C3-C8 cycloalkylene), C1-C 22 Saturated or unsaturated heteroalkylene (e.g., branched heteroalkylene, straight-chain heteroalkylene, heterocycloalkylene, C1-C 22 Branched heteroalkylene, C1-C 22 Straight chain heteroalkylene, C3-C 22 Heterocycloalkylene, C1-C 10 Branched heteroalkylene, C1-C 10 Straight chain heteroalkylene, C3-C 10 heterocycloalkylene, C1-C8 branched heteroalkylene, C1-C8 straight chain heteroalkylene, C3-C8 heterocycloalkylene), arylene (e.g., C5-C 22 arylene), heteroarylene (e.g., C5-C 22 divalent hydrocarbons selected from heteroarylene, substituted amides ... relates to.

[0295] 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 C6 arylene, optionally substituted C 5~12 Heteroarylene, hydroxy-substituted C 3~6 cycloalkylene or C heterocycloalkylene substituted with hydroxy; each of the foregoing may have one or more (e.g., 2, 3, 4, 5) points of substitution; and R a is independently selected at each occurrence from hydrogen or alkyl (eg, C1-C7 alkyl, C1-C3 alkyl).

[0296] In one exemplary embodiment, the formula is: [ka] wherein R1, R3, and R4 are independently alkyl, alkenyl, alkynyl, and R2 can be selected. In one exemplary embodiment, the PKC activator is a benzolactam analog of ILV, and R can be CC(CH2)7CH3 or (CH2)9CH3, as described in Kozikowski et al., J. Med. Chem., 1997, 40:9 1316-1326.

[0297] In an exemplary embodiment, R1, R3, and R4 are alkyl, and in some embodiments, R1, R3, and R4 are methyl. In an exemplary embodiment, the formula is: [ka] relates to.

[0298] In an exemplary embodiment, the PKC activator is a natural product activator as described in Kazanietz et al., Mol. Pharma. 44:296-307 (1993), such as DPP, prostratin, mezerein, octahydromezerein, thimeleatoxin, (-)-octylindolactam V, OAG, or resiniferatoxin.

[0299] In an exemplary embodiment, the PCK binding moiety has the formula [ka] or an analog thereof.

[0300] In one exemplary embodiment, the PKC activator is selective for PKCδ. In one exemplary embodiment, the PKC activator is 7α-acetoxy-6β-benzoyloxy-12-Obenzoylroyleanone (Roy-Bz) as described in Bessa et al., Cell Death and Disease (2018) 9:23.

[0301] The PKC activator can be an ILV derivative, such as n-hexyl ILV, or the 10-membered ring 1-hexylindolactam-V10, or a derivative thereof as described in Yanagita, et al., J. Med. Chem., 2008, 51:1, 46-56 (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.

[0302] In an exemplary embodiment, the activator moiety is 6-chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid (PF-06409577), a benzolactam, DPP, prostratin, mezerein, octahydromezerein, thimeleatoxin, (-)-indolactam V, (-)-octylindolactam V, OAG, or a derivative thereof.

[0303] In an exemplary 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), incorporated herein by reference.

[0304] In other exemplary embodiments, the PKC activator may be selected from Table 1 of PCT / US2021 / 012816, which is incorporated herein by reference.

[0305] FKBP binding part In one exemplary embodiment, the kinase-binding moiety is an FKBP kinase-binding moiety. In one exemplary embodiment, the kinase-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, 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 exemplary embodiment, the FKBP is FKBP12, which binds to intracellular calcium release channels and TGF-β type I receptors. In one exemplary 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).

[0306] IRTK binding part In one exemplary embodiment, the kinase-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. Ligand binding to the α subunit of the IR induces a conformational change in the receptor. As a result, the intrinsic tyrosine kinase activity of the β subunit of the 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 exemplary embodiment, the kinase-binding moiety is an IR activator or inhibitor. In one example, the activator for IRTK is kojic acid or a derivative thereof.

[0307] In one exemplary embodiment, the target is the androgen receptor. In one exemplary embodiment, the localization moiety may comprise enzalutamide. In one exemplary embodiment, enzalutamide is attached via an ether bond to a linker containing an azide terminus. Thus, in one exemplary embodiment, the addition of an alkyne functional group to the activator moiety allows for linkage via bioorthogonal click chemistry. In one exemplary embodiment, the insulin receptor is represented by the formula: [ka] (wherein X is C, N, O, S or P) In another exemplary embodiment, the IRTK activator has the formula: [ka] and any derivatives thereof.

[0308] In one exemplary embodiment, the IRTK activator is a monoclonal antibody and allosteric partial agonist of the insulin receptor, XMetA, also known as XOMA-159. 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), 35-43.

[0309] Lyn bond part In an exemplary embodiment, the kinase-binding moiety is a Lyn kinase-binding moiety. Lyn kinase belongs to the Src kinase family and is the predominant Src kinase in B cells. The regulatory properties of Lyn play a role in the function of the immune system. See, for example, Xu Y., "Lyn Tyrosine Kinase". Immunity 2005, 22(1), 9-18. In an exemplary embodiment, the Lyn-binding moiety is an activator or inhibitor. In an exemplary embodiment, a Lyn activator has the formula: [ka] Trimidone, also known as MLR-1023, is a selective allosteric activator of Lyn kinase and was developed for the treatment of type 2 diabetes. Experiments in knockout mice revealed that in the absence of Lyn kinase, trimidone does not lower glucose. Trimidone is currently undergoing phase 2 studies in patients with uncontrolled type 2 diabetes. Trimidone has a pEC50 of 7.2. 50See, 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), 15-22, the following activity comparisons in cellular and enzyme assays are cited below and incorporated by reference herein. [Table 5]

[0310] PK binding part In one exemplary embodiment, the kinase-binding moiety is a PK kinase-binding moiety. Pyruvate kinase (PK) catalyzes the transphosphorylation of phosphoenolpyruvate (PEP) to ADP to generate ATP during glycolysis. PK is expressed in four different isoenzyme forms in mammalian tissues: L, R, M1, and M2, depending on metabolic needs and its regulatory properties. The M2, L, and R isoenzymes exhibit homotropic coactivation with PEP and heterotropic coactivation with FBP. See, e.g., Gupta V., et al., "Human Pyruvate Kinase M2: A Multifunctional Protein," Protein Science 2010, 19(11), 2031-2044.

[0311] In one exemplary embodiment, the PK kinase binding moiety is a PK activator. In an exemplary embodiment, the PK activator is mitapivat, also known as AG-348, according to the formula: [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 fetal PKM2 isoform expressed from the PKM gene. Mitapivat was developed as a novel treatment for erythrocyte disorders associated with inherited PKR deficiency and for the therapy of cancers that involve activation of PKM2. PK activation in red blood cells increases hemoglobin levels. The active drug is the sulfate hydrate. Mitapivat has a pEC50 activity of greater than 7.0 for PKM2. 50 In an exemplary embodiment, the PK activator is any compound from U.S. Pat. No. 8,785,450 B2 (incorporated herein by reference) or any derivative thereof. In an exemplary embodiment, the PK activator is any compound from WO 2013056153 A1 (incorporated herein by reference) or any derivative thereof.

[0312] In an exemplary embodiment, the kinase binding moiety is a PK inhibitor (see above for more information regarding PK kinases). In an exemplary embodiment, the PK inhibitor is any one identified in U.S. Patent No. 6,534,501 (herein incorporated by reference), or any derivative thereof.

[0313] NOP connection part Nociceptin opioid peptide (NOP) receptors are part of the opioid receptor GPCR family that couple to Gi / Go and inhibit adenylate cyclase activity. In an exemplary embodiment, the kinase-binding moiety or target-binding moiety binds to a GPCR opioid receptor. In an exemplary embodiment, the kinase-binding moiety is a NOP activator. In an exemplary embodiment, the NOP activator has the following formula: [ka] or any derivative thereof.

[0314] In an exemplary embodiment, the NOP activator is the NOP agonist Ser100 according to the formula: Ac-RYYRWKKKKKKK-NH2 (SEQ ID NO: 6). In an exemplary embodiment, the NOP activator is the NOP agonist N / OFQ according to the formula: FGGFTGARKSARKLANQ (SEQ ID NO: 7). In an exemplary embodiment, 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), 7011-7028 (incorporated herein by reference in its entirety).

[0315] Several proteins, including G protein-coupled receptor kinases, β-arrestins, and G proteins, clearly regulate NOP receptor function. It has also been shown that sodium and guanyl nucleotides can modify the interaction of functional NOP complexes with G proteins. Other G protein-coupled receptors, such as the μ-opioid receptor, appear to be capable of heterodimerizing 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), 1285-1294.

[0316] In certain embodiments, the binding agent is an allosteric modulator of the δ 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.

[0317] 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 -oxidanyl;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-thiazolidin-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.

[0318] In an exemplary embodiment, the NOP binder is a NOP antagonist. In an exemplary embodiment, the NOP antagonist has one of the following formulas: [ka] See Zaveri J.Med.Chem.2016.

[0319] MAPK binding part In one exemplary embodiment, the kinase-binding moiety is a mitogen-activated protein kinase (MAPK)-binding moiety. In one exemplary 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 domain of transcription factors, thereby regulating transcriptional activity. In one exemplary embodiment, the kinase-binding moiety is a MAPK inhibitor.

[0320] In an exemplary embodiment, the MAPK inhibitor is: [ka] p38α MAPK inhibitors and derivatives thereof, including the compound (I), can be used as activating moieties in the chimeric small molecules of the present invention. Inhibitor B96 is also known as dramapimod, an allosteric inhibitor. Dramapimod exhibits moderate selectivity for p38α, p38β, and p38γ isozymes compared to p38δ. It exhibits moderate selectivity for p38α, p38β, and p38γ isozymes compared to p38δ. A Kd value of 0.1 nM has been reported, and in a kinase screening panel, dramapimod inhibited many kinases with IC50 values ​​of less than 100 nM. Dramapimod has been shown to block TNFα release in THP-1 cells after LPS stimulation 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 scope for modifications when tailoring the molecule for use in chimeric small molecules is described in Moffett, incorporated by reference, and in detail in Figure 3 and its associated teachings. In one embodiment, the molecule incorporates a non-aromatic fragment to form a productive hydrogen-bonding interaction with Arg 70 on the αC-helix.

[0321] In one embodiment, the MAPK inhibitor is an allosteric inhibitor of p38 according to compound 10, which is discussed in more detail in the context of Jnk-1.

[0322] In one embodiment, the MAPK inhibitor is SB203580 (SB6). In one embodiment, the MAPK inhibitor is a compound of the formula [ka] In one embodiment, the MAPK inhibitor is sorafenib and its derivatives of the formula:

[0323] In one exemplary embodiment, the MAPK inhibitor is the small molecule KC-706.

[0324] EGFR binding part In one exemplary embodiment, the kinase-binding moiety is an EGFR-binding moiety. In one exemplary embodiment, the EGFR-binding moiety is an inhibitor or activator. EGFR is a tyrosine kinase receptor belonging to the ErbB receptor family that mediates cell growth, differentiation, and repair of cells, particularly non-cancerous cells.

[0325] In one embodiment, the EGFR binding molecule has the formula: [ka] or its analogues.

[0326] In an exemplary embodiment, the EGFR binding molecule is gefitinib. Gefitinib selectively binds to the ATP binding site of EGFR, thereby causing inhibition. In one exemplary embodiment, the EGFR binding molecule can be any one from the group including erlotinib, afatinib, osimertinib, lapatinib, neratinib, dacomitinib, or any derivative thereof.

[0327] In some embodiments, the kinase is an EGFR mutant, hi some embodiments, the EGFR mutant comprises L858R, C797S, T790M, V984R, or a combination thereof.

[0328] In an exemplary embodiment, the EGFR inhibitor is EAI001, which was designed to overcome clinically acquired EGFR T790M / C797S mutation resistance in NSCLC by binding to ATP. When EAI001 binds to the allosteric MT3 site of EGFR, the carboxamide forms a hydrogen bond with Asp 855, the phenyl group forms a hydrophobic interaction with the DFG-in pocket, and the 1-oxoisoindolinyl spans the solvent-exposed region. EAI001 has the formula: [ka] relates to.

[0329] In an exemplary embodiment, the EGFR inhibitor has the formula: [ka] and EAII001 analogs according to the present invention, such as EAI045.

[0330] Both EAI001 and its analog EAI045 exhibit 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 but absent from the wild-type receptor, inhibiting the L858R / T790M mutant EGFR with an IC50 of 3 nM. 50 and is over 1000-fold selective for this mutant compared to the wild-type receptor. The IC50s for additional EGFR and its mutants and EAI045 are: [Table 7]

[0331] In screening panels, EAI045 did not inhibit any other kinases by more than 20% (at 1000 nM EAI045) or show any tendency toward non-kinase targets, and in xenograft models, EAI045 is effective against EGFR (L858R / T790M / C797S) tumors, a mutation profile that confers resistance 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]

[0332] 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 location of many of the identified resistance mutations.

[0333] In an exemplary embodiment, the EGFR inhibitor is an analog that comprises EAI045 by adding a phenylpiperazine substituent to the isoindolinone ring. The analog has the formula: [ka] JBJ-04-125-02.

[0334] In certain embodiments, JBJ-04-125-02 exhibits subnanomolar potency against the 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 as a single agent in vitro and in vivo. X-ray crystal structures of JBJ-04-125-02 and EGFR T790M demonstrate that it binds to an allosteric site on EGFR in a manner similar to EAI001. In certain embodiments, JBJ-04-125-02 inhibits EGFR phosphorylation (at 0.01-10 μM) and demonstrates mutant selectivity by inhibiting mutant EGFR and downstream AKT and ERK1 / 2 phosphorylation. Angew. Chem. Int. Ed. 2020, 59, 13764-13776 (incorporated herein by reference in its entirety); 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.

[0335] In exemplary embodiments, the EGFR inhibitor is an inhibitor identified in U.S. Patent No. 8,242,080 (hereby incorporated by reference) or a derivative thereof. In certain exemplary embodiments, the EGFR inhibitor is dacomitinib, mobocertinib, or any derivative thereof.

[0336] In certain exemplary embodiments, the EGFR inhibitor has the formula: [ka] or derivatives thereof, where the in group / hexagon represents the point of attachment to the remainder of the chimeric small molecule.

[0337] In an exemplary embodiment, the LIMK inhibitor has the formula: [ka] or derivatives thereof, where "linker" and "target binding agent" represent the remainder of the chimeric small molecule.

[0338] BCKDK joint part In one exemplary embodiment, the kinase binding moiety is a branched-chain α-keto acid dehydrogenase kinase (BCKDK) binding moiety, also known as a 3-methyl-2-oxobutyrate dehydrogenase kinase binding moiety. In one exemplary 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 derivatives thereof.

[0339] In one embodiment, the allosteric inhibitor is of the formula [ka] The S-enantiomer of α-chlorophenylpropionic acid [(S)-CPP] is the S-enantiomer of α-chlorophenylpropionic acid according to the present invention. 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 (incorporated herein by reference), specifically, BCKDK inhibitor compounds and their IC 50 and K. d See Table 1 for values.

[0340] In certain embodiments, the BCKDK inhibitor is a benzothiophene carboxylic acid derivative. In certain embodiments, the binding moiety has the formula [ka] and 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.

[0341] FGFR binding part In one exemplary embodiment, the protein-binding moiety is an FGFR kinase-binding moiety. In one exemplary embodiment, the FGFR-binding moiety is an inhibitor or activator. Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases expressed on the cell membrane, consisting of four members: FGFR1 to FGFR4. All four FGFR members have a large extracellular ligand-binding domain from the N-terminus to the C-terminus, containing three immunoglobulin (Ig)-like subunits (D1, D2, and D3), followed by a single transmembrane helix and an intracellular tyrosine kinase domain. The natural ligands of FGFRs are fibroblast growth factors. FGFRs play critical roles in both developing 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.

[0342] In an exemplary embodiment, the FGFR inhibitor has the formula: [ka] This is SSR128129.

[0343] SSR128129 characteristics include: [Table 9] In one embodiment, SSR128129E is used as a sodium salt. SSR128129E is a negative allosteric modulator of FGF receptors. This compound inhibits FGF1-induced ERK phosphorylation via FGFR2 with an IC of less than 100 nM. 50 SSR128129E inhibits FGF ligand-induced receptor dimerization in an allosteric manner by interacting with Lys279, Thr320, Thr319, Cys278, Trp290, Phe276, Wal274, Tyr340, Ile329, Tyr328, Leu327, and Leu312 without affecting FGF binding. See Cancer Cell, 2013, 23, 477-488 (incorporated by reference). For the effects of SSRs on cellular responses to various different FGFRs, see Table 1 in Cancer Cell, 2013, 23, 4774-88 (specifically incorporated by reference herein), which shows SSR concentrations at which at least 50% inhibition is achieved at concentrations between 10 nM and 100 nM. See also, for an overview, 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 (incorporated by reference) for the chemical structure of SSR128129E, binding predictions as detailed in FIG. 17, and the effect of SSR on cellular responses to a variety of different FGFRs as provided in Table 1 (each of which is specifically incorporated herein by reference).

[0344] In certain exemplary embodiments, the FGFR inhibitor is pemigatinib, infigratinib, fisogatinib, or any derivative thereof.

[0345] In certain exemplary embodiments, the FGFR binding moiety has the formula: [ka] or a derivative thereof, wherein the in group represents the point of attachment to the remainder of the chimeric small molecule.

[0346] In an exemplary embodiment, the kinase-binding moiety is a pan-FGFR kinase-binding moiety. In an exemplary embodiment, the pan-FGFR binding moiety is an inhibitor or activator. In an exemplary embodiment, the pan-FGFR inhibitor is erdafitinib, futibatinib, or any derivative thereof.

[0347] In an exemplary embodiment, the pan-FGFR binding moiety has the formula: [ka] or a derivative thereof, wherein the in group represents the point of attachment to the remainder of the chimeric small molecule.

[0348] HA-NGFR binding part In one exemplary embodiment, the protein-binding moiety is an allosteric tropomyosin receptor kinase A (TrkA) or high-affinity nerve growth factor receptor (HA-NGFR) kinase-binding moiety. In one exemplary embodiment, the TrkA or HA-NGFR binding moiety is an inhibitor or activator. The high-affinity nerve growth factor receptor (HA-NGFR) is a family of receptor tyrosine kinases that regulates the proliferation, differentiation, and survival of sympathetic and neural neurons in the central and peripheral nervous system. The natural ligand for HA-NGFR is nerve growth factor. Absence of the ligand, resulting in a loss of activation, promotes cell death and can make 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.

[0349] In certain embodiments, the pan-Trk inhibitor is GZ389988, AR786 (an allosteric selective TrkA inhibitor), ASP7962 (a TrkA receptor antagonist), 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 2016-2016, Expert Opinion on Therapeutic Patents, 30:5, 325-339, DOI:10.1080 / 13543776.2020 (both of which are incorporated by reference in their entirety).

[0350] In an exemplary embodiment, the HA-NGFR is VM-902A or a related compound or analog thereof. [ka] or an analog thereof.

[0351] IκB binding part In one embodiment, the protein-binding moiety is an IκB kinase binding moiety. In an exemplary embodiment, the IκB binding moiety is an inhibitor or activator. In certain aspects, the IκB kinase binding moiety inhibits one or both subunits of IκB kinase, IKK-α and IKK-β. In one embodiment, the binding moiety is the selective allosteric inhibitor BMS-345541, which has the formula: [ka] and has the following characteristics: [Table 10] BMS-345541 has been shown to block NF-kB-dependent transcription in mice and exhibits activity against LPS-induced NF-kB activation in mice. In one embodiment, the negative allosteric modulator BMS-345541 has a pK of 6.9. d , a pIC of 6.5 as an inhibitor of nuclear factor κB kinase subunit β 50 and a pIC of 5.4 for the inhibitor components of the nuclear factor-κB kinase complex 50 It has.

[0352] CDK binding part In an exemplary embodiment, the protein-binding moiety is a CDK kinase-binding moiety. In an exemplary embodiment, the protein-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 regulate transcription. The CKD family is divided into three cell cycle-related subfamilies: CDK1, CDK2, and CDK3; CDK4 and CDK6; and CDK5 and CDK14-CDK18, and five transcription subfamilies: CDK7; CDK8 and CDK19; CDK9; CDK10 and CDK11; CDK12 and CDK13; and CDK20. In an exemplary embodiment, the CDK inhibitor comprises palbociclib, ribociclib, abemaciclib, or any derivative thereof. In an exemplary embodiment, the CDK8 inhibitor is a compound of the formula: [ka] Compound 5 of the formula:

[0353] In an exemplary embodiment, the (CDK2 inhibitor) is a flavopiridol analog. In an exemplary embodiment, 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, Volume 15, Issue 2, 2007, Pages 702-713, doi:10.1016 / j.bmc.2006.10.063 (incorporated herein by reference). In one embodiment, the compound is selected from the 8-amino flavopiridol analogs of Table 1 of Ahn, which may be selected based on the antiproliferative and inhibitory activity of Table 1 (specifically incorporated herein by reference). Modifications to the Ahn molecule can be made based in part on the desired interaction between the analog and the CDK, with exemplary modifications made based on Figures 2A-2B.

[0354] In an exemplary embodiment, the CDK inhibitor is alvocidib, an inhibitor that causes cell cycle arrest, which is in Phase 2 clinical evaluation for its potential anti-cancer capabilities and has the formula: [ka] relates to.

[0355] In one embodiment, alvocidib is utilized as a CDK2 and / or CDK4 kinase binding moiety, which binds CDK4 pK i 7.2 and CDK2 pIC 50 is 6.4 to 7.0 and has the following characteristics: [Table 11]

[0356] In one embodiment, they can be identified and optimized according to the formula and discussion in Bioorg. Med. Chem. 2007, 15, 702-713: [ka] Properties can be optimized for use in chimeric small molecules based on the modification site as follows: (wherein R can be any cyclic hydrocarbon; unsaturated cyclic hydrocarbon; heterocycle; one or more fused rings containing any combination of any of the above rings (optionally substituted at one or more positions with 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 containing any combination of any of the above rings, preferably a piperidine ring, a pyrrolidine ring, a thiane ring, or a morpholine ring, which may be further substituted at any position on the ring). Specific 8-aminoflavopiridol analogs are detailed in Table 1 of Bioorg. Med. Chem. 2007, 15, 702-713, as illustrated below. [Table 12] In an exemplary embodiment, the CDK inhibitor has the formula: [ka] or derivatives thereof, and the oval represents the remainder of the chimeric small molecule.

[0357] In an exemplary embodiment, the CDK2 inhibitor has the formula: [ka] or derivatives thereof, where "linker" and "target binding agent" represent the remainder of the chimeric small molecule.

[0358] PI3K binding part In one exemplary embodiment, the protein-binding moiety is a PI3K kinase-binding moiety. In one exemplary embodiment, the protein-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 distinct PI3K classes (I, II, and III), with distinct isoforms (e.g., class I has four isoforms: α, β, γ, and δ), and within each class, there are distinct, individual roles for each PI3K. Class I has been implicated in many cancers, particularly those with pathogenic mutations. PI3K acts downstream of many growth factors and upstream of AKT and mTOR. (Kannaiyan et al. Expert Rev Anticancer Ther. 2018;18(12):1249-1270).

[0359] In an exemplary embodiment, the PI3K inhibitor has the formula: [ka] It is an idelalisib.

[0360] Idelalisib is a small molecule inhibitor of the delta isoform of PI3K. In an exemplary embodiment, 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 in the ATP-binding pocket, this compound binds at an allosteric site near the H1047R mutation hotspot in the mouse PI3Kα C-lobe. See, e.g., 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), 4788-4805 (herein incorporated by reference in its entirety), particularly Tables 2 and 3, and the biochemical and cellular activities of pyrimidone benzimidazoles and their replacements.

[0361] VEGFR binding part In one exemplary embodiment, the protein-binding moiety is a VEGFR-binding moiety. In one exemplary 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 containing a disulfide knot structure. There are two VEGFs, VEGF-A and VEGF-B, which bind to VEGFR, a receptor tyrosine kinase located in vascular endothelial cells. In one exemplary embodiment, the kinase-binding moiety is a VEGFR inhibitor. In an exemplary embodiment, the VEGFR inhibitor is sorafenib, sunitinib, pazopanib, axitinib, cabozantinib, lenvatinib, vandetanib, or regorafenib.

[0362] BRAF binding part In one exemplary embodiment, the kinase-binding moiety is a BRAF-binding moiety. In one exemplary embodiment, the binding moiety is a BRAF inhibitor or activator. BRAF is a member of the rapidly accelerated fibrosarcoma serine / threonine kinase family and is frequently activated by genetic abnormalities in cancer patients. 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 molecular hinge; and conserved region 3 (CR3) is a catalytic protein kinase domain. In one exemplary embodiment, the kinase-binding moiety is a BRAF inhibitor. In one embodiment, the BRAF inhibitor comprises vemurafenib or dabrafenib.

[0363] MEK joint part In one exemplary embodiment, the protein-binding moiety is a MEK-binding moiety. In one exemplary embodiment, the MEK-binding moiety is an inhibitor or activator. MEK is a kinase enzyme that phosphorylates mitogen-activated protein kinase (MAPK). Seven MEK subtypes have been identified, all mediating cellular responses to different growth signals. In one exemplary 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 comprises trametinib according to the formula: Trametinib has been used for the adjuvant treatment of patients with BRAF V600E or V600K mutant melanoma, inhibiting MAP2K1 and MAP2K2 (also known as MEK1 and 2) of 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 in determining the dosage of 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. Epub 2011 Jan 18. Trametinib has a MAPK1 inhibitory pIC of 9 / 0-9.1. 50 and MAPK2 pIC of 8.7 50 Trametinib has been shown to exhibit sustained inhibition of p-ERK1 / 2 for more than 24 hours, with high potency, selectivity, and a long circulating half-life.

[0364] In one exemplary embodiment, the MEK inhibitor comprises cobimetinib, an allosteric inhibitor of MEK serine / threonine protein kinases with selectivity over MEK1 and MEK2. Cobimetinib selectively inhibits the activity of MEK serine / threonine protein kinases and has the formula: [Table 13] Additional in vitro activity of cobimetinib and related analogs has been 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 (incorporated herein by reference), specifically Tables 1 and 3.

[0365] In an exemplary embodiment, the RIP1 inhibitor has the formula: [ka] or derivatives thereof, where "linker" and "target binding agent" represent the remainder of the chimeric small molecule.

[0366] In an exemplary embodiment, the MEK inhibitor has the formula: [ka] 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, thus preventing activation 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 activating the RAS / RAF / MEK / ERK pathway and are frequently upregulated in various tumor cell types. By selectively binding to and inhibiting MEK1 / 2 activity, it prevents the activation of MEK1 / 2-dependent effector proteins and transcription factors, potentially inhibiting growth factor-mediated cell signaling and tumor cell proliferation. See 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. doi:10.1158 / 0008-5472.

[0367] In an exemplary embodiment, the MEK inhibitor has the formula: [ka] Including CI-1040 relating to

[0368] In an exemplary embodiment, the MEK1 and MEK2 inhibitor is selumetinib (AZD6244, ARRY-142886) according to the formula: and has the following properties: [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 demonstrated sensitivity to specific KRAS mutations 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, HCT116 cells with the amino acid change G13D, and H1299 cells with the amino acid change p.H1047R in PIK3CA and the amino acid change p.Q61K in NRAS.

[0369] In an exemplary embodiment, the allosteric MEK inhibitor has the formula [ka] (wherein R and R5 are selected from the table below) 3,4-Difluoro-2-(2-halo-4-iodo-phenylamino)-N-2-hydroxy-ethoxy)-benzamide according to the present invention. [Table 15] (As described in Hartung et al., Optimization of allosteric MEK inhibitors, Part 1: Venturing into underexplored SAR territories, Bioorganic and Medicinal Chemistry Letters 23 (2013) 2384-2390, incorporated herein by reference).

[0370] In one exemplary embodiment, MEK inhibitors are investigated in advanced KRAS-mutated colorectal cancer, non-small cell lung cancer, melanoma, colon neoplasia, and breast cancer, and have the formula: [ka] The present study is directed at mirdametinib (PD 0325901), a selective non-ATP-competitive MEK inhibitor. Mirdametinib has an EK1 inhibitory pIC of 8.1. 50 It has a value and has the following properties: [Table 16]

[0371] In one exemplary embodiment, the MEK binding moiety is the allosteric inhibitor refametinib: [ka] or analogs thereof, e.g., [ka] or derivatives thereof.

[0372] In an exemplary embodiment, the MEK inhibitor is binimetinib according to the formula: and has the following properties: [Table 17] Binimetinib has received FDA approval for the treatment of advanced BRAF-mutant melanoma in combination 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.

[0373] Additional investigations in other solid tumor types, neuroblastoma, and hematological cancers are underway. 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 of MEK1 and MEK2 with a pIC50 of 7.9.

[0374] AKT joint part In one exemplary embodiment, the protein-binding moiety is an AKT-binding moiety. In one exemplary embodiment, the AKT-binding moiety is an inhibitor or activator. RAC-α 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 carboxyl-terminal hydrophobic motif. In an exemplary embodiment, the ATK inhibitor is represented by the formula: [ka] volsertib or a derivative thereof, and the oval represents the remaining chimeric small molecule.

[0375] In an exemplary embodiment, the kinase inhibitor has the formula: [ka] MK-2206, which has the following characteristics: [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 MK-2206 has potential therapeutic value. MK-2206 can enhance the antitumor efficacy of standard chemotherapeutic agents or molecularly targeted drugs in vitro and in vivo. As of 2018, there were 50 registered MK-2206 trials on ClinicalTrilas.gov. Many have been discontinued, terminated, or completed. The pyridine-fused ring can be modified to form a monocyclic, bicyclic, or tricyclic linear fused ring or an angular tricyclic ring. Pyridine can be modified to form a pyrazine. The substituted benzene moiety can also be modified. The strained cyclobutene can be substituted with any substituent known in the art. Furthermore, the hydrogen on the amine in the moiety can 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 (herein incorporated by reference in its entirety).

[0376] In one exemplary embodiment, the AKT inhibitor is any inhibitor from International Publication No. WO 2008070016A2 (herein incorporated by reference in its entirety) and any derivative 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 (herein incorporated 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 can be replaced with C, N, O, S, B or P. may relate to; or [ka] (wherein R=NHMe) See Bioorg.Med.Chem.Let.2008,18,4191-4194;doi:10.1371 / journal.pone.0012913 (incorporated herein by reference). Optimization of allosteric inhibition of AKT can be performed based on the following guidelines. [ka] Strategies for reducing hERG affinity in combination with potency for AKT binders may be based in whole or in part on the following. [ka]

[0377] In an exemplary embodiment, the kinase inhibitor has the formula: [ka] The AKT inhibitor VIII, also known as compound 16h, 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. The pIC of AKT inhibitor VIII is 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), 761-764 (herein incorporated by reference in its entirety), in particular Tables 1 and 2, reproduced below. [Table 19]

[0378] In an exemplary embodiment, the kinase inhibitor has the formula: [ka] Miransertib, also known as ARQ-092, is an orally active, selective, and potent allosteric AKT inhibitor. Miransertib has pIC values ​​of 8.3, 8.4, and 7.8 against AKT1, 2, and 3. 50 Miransertib has progressed into Phase 1 and Phase 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), 6455-6469 (herein incorporated by reference in its entirety), specifically Tables 1, 2, 3, 4, 6, and 9; Tables 2 and 4 are reproduced below. [Table 20] [Table 21]

[0379] In an exemplary embodiment, the kinase inhibitor is ARQ 751. In one exemplary embodiment, the kinase inhibitor is any inhibitor from 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(11), 5291-5310, or any derivative thereof, including in particular Table 3, Table 4, Table 6, and Table 8, which are reproduced below for reference. [Table 22] [Table 23] [Table 24] or [Table 25]

[0380] In an exemplary embodiment, the kinase inhibitor has the formula: [ka] Volsertib is a covalent allosteric inhibitor of AKT. wt For IC 50 is 0.8m< and K i The EC of volsertib is 2.2 nM. 50The values ​​are 191±90 nM, 48±15 nM, 5±1 nM, 277±90 nM, 373±54 nM, and 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 some embodiments, the allosteric inhibitor can be according to the table reproduced below. [Table 26] The variability of the scaffold can be according to the following scheme: [ka]

[0381] In an exemplary embodiment, 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), 1213-1218. In an exemplary embodiment, the lactoquinomycin is medermycin.

[0382] In an exemplary embodiment, the AKT inhibitor is BIND-2206, also known as MK-2206 or NSC-749607, according to the formula: and has the following properties: [Table 27] The AKT moiety can be synthesized following the guidance and design provided herein in light of the AKT binding moieties 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). For further information regarding AKT allosteric inhibitors, see 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.

[0383] ALK joint part In one exemplary embodiment, the protein-binding moiety is an ALK kinase-binding moiety. In one exemplary embodiment, the ALK-binding moiety is an inhibitor or activator. Anaplastic lymphoma kinase, also known as ALK tyrosine kinase receptor or CD246, is involved in intercellular communication and the development and function of the nervous system. Upon binding to a ligand, the full-length ALK receptor dimerizes, changes conformation, and autoactivates its own kinase domain. The autoactivated ALK dimer phosphorylates other ALK receptors on specific tyrosine amino acid residues. The ALK phosphorylated residues are binding sites for recruiting several adaptors. In one exemplary embodiment, the ALK inhibitor comprises crizotinib, ceritinib, alectinib, brigatinib, or lorlatinib.

[0384] In one embodiment, the ALK inhibitor has the formula: [ka] and CH5424802 or a derivative thereof.

[0385] BTK joint part In one exemplary embodiment, the protein-binding moiety is a BTK kinase-binding moiety. In one exemplary 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 development and oncogenic signaling. See, e.g., Singh et al., 2018; Pal et al., 2018. In an exemplary embodiment, the BTK inhibitor is ibrutinib, acalabrutinib, or a derivative thereof. [ka] Exemplary derivatives include: [ka] and as detailed in Liclican et al, Biochimica et Biophysica Acta (BBA) 1864(4):129531, DOI:10.1016 / j.bbagen.2020.129531.

[0386] In one exemplary embodiment, the BTK activator is [ka] is selected from.

[0387] In one exemplary embodiment, the BTK activator moiety includes: [ka] A targeting moiety of is provided.

[0388] In an exemplary embodiment, the BTK inhibitor has the formula: [ka] or a derivative thereof.

[0389] FLT3 binding part In one exemplary embodiment, the protein-binding moiety is a FLT3 kinase-binding moiety. In one exemplary 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 has five immunoglobulin-like domains in its extracellular region, and the intracellular tyrosine kinase domain is divided into two by a specific hydrophilic insertion. In one exemplary embodiment, the FLT3 inhibitor comprises midostaurin, gilteritinib, or a derivative thereof.

[0390] JAK joint part Janus kinases (JAKs) are a group of intracellular non-receptor tyrosine kinases. JAKs transduce cytokine-mediated signals through the JAK-STAT pathway. Adjacent to the plasma membrane, JAKs integrate with the proline-rich region of their intracellular domains. JAKs autophosphorylate each other, resulting in a conformational change in JAKs, which allows them to transduce intracellular signals through further phosphorylation and activation of transcription factors (i.e., STATs). The JAK family includes JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2).

[0391] In an exemplary embodiment, the protein-binding moiety is a JAK2 kinase-binding moiety. In an exemplary 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 an exemplary embodiment, the JAK2 inhibitor has the formula: [ka] This includes ruxolitinib, also known as INCB018424.

[0392] In another exemplary embodiment, the JAK2 inhibitor has the formula: [ka]

[0023] The chimeric small molecule of the present invention is tasocitinib, also known as CP690550, or a derivative thereof, wherein the carboxyl group represents the point of attachment to the remainder of the chimeric small molecule.

[0393] JAK3 functions in signal transduction by receptors containing the common gamma chain (γc) of the type I cytokine receptor family. JAK3 is generally expressed in hematopoietic and epithelial cells, such as T cells and NK cells. In an exemplary embodiment, the kinase-binding moiety is a JAK3-binding molecule. In an exemplary embodiment, the JAK3-binding moiety is an inhibitor or activator. In an exemplary embodiment, the JAK3 inhibitor has the formula: [ka] or derivatives thereof, where the oval represents the remainder of the chimeric small molecule. In an exemplary embodiment, the JAK3 inhibitor has the formula: [ka] or derivatives thereof, where the in group / hexagon represents the point of attachment to the remainder of the chimeric small molecule.

[0394] AURKA joint part In an exemplary embodiment, the protein-binding moiety is an AURKA kinase-binding moiety. In an exemplary embodiment, the AURKA-binding moiety is an inhibitor or activator. Aurora A kinase (AURKA) is a member of the Setr / Thr kinase family whose orthologues control the progression of mitotic cells through division. Other members of the Aurora family are Aurora B and C, which all share a relatively conserved kinase catalytic domain at the carboxyl terminus. In an exemplary embodiment, an Aurora A inhibitor has the formula: [ka] Olkin A has an IC50 of 12.7 in μM. 50and has a K in μM of 2.7 i It has.

[0395] In an exemplary embodiment, the Aurora A inhibitor has the formula: [ka] AA29 has an IC of 34.4 in μM. 50 and a K in μM of 7.4 i It has.

[0396] In certain exemplary embodiments, the Aurora A inhibitor has the formula: [ka] AA30 is in μM and has an IC of 25.6 50 and has a K of 5.5 in μM i In some embodiments, the compound has the formula: [Table 28] See Janechek, M., Rossmann, M., Sharma, P. et al. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2. Sci Rep 6, 28528 (2016). Doi:rep28528 (incorporated herein by reference). In one exemplary embodiment, the kinase-binding moiety is a monobody targeting Aurora A as described by Zorba A., et al. "Allosteric Modulation of a Human Protein Kinase with Monobodies". Proc Natl Acad Sci USA 2019, 116(28), 13937-13942 (incorporated herein by reference).

[0397] In an exemplary embodiment, the Aurora inhibitor is an Aurora inhibitor identified in US Patent Application Publication No. 20080051327, which is incorporated herein by reference, or any derivative thereof.

[0398] c-MET binding part In an exemplary embodiment, the protein-binding moiety is a c-MET kinase binding moiety. In an exemplary embodiment, the c-MET binding moiety is an inhibitor or activator. C-MET (mesenchymal-epithelial transition factor) is a receptor tyrosine kinase involved in cell signaling pathways. After binding to hepatocyte growth factor, it activates signaling pathways, including 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 an exemplary embodiment, the c-MET inhibitor is a compound of the formula [ka] The drug is tivantinib, also known as ARQ-197.

[0399] In one embodiment, the tivantinib binding agent or a derivative thereof targets the MET proto-oncogene, a receptor tyrosine kinase, is an allosteric inhibitor, and has one or more of the following properties: tivantinib or a derivative thereof is a non-ATP-competitive, MET-specific inhibitor, with 10- to 100-fold higher selectivity for c-Met over other kinases tested (see Munshi et al., Moll. Can. Ther. doi:10.1158 / 1535-7163.MCT-09-1173), with an enzyme IC of 50 nM in NCI-H441 cells, respectively. 50 , 100 nM fluorophore-MET IC 50 , 100 nM viability IC 50 and 80 nM invasion IC 50Tivantinib has demonstrated growth inhibition in breast, prostate, colon, and pancreatic cancer xenografts, as well as inhibiting metastasis formation in an experimental metastatic model of orthotopic colon cancer xenografts. In addition, the tivantinib inhibitor has a pKi value of 6.4. These characteristics allow for the appropriate selection and modification of chimeric small molecule designs, as detailed elsewhere herein.

[0400] In an exemplary embodiment, the c-MET inhibitor is capmatinib or any derivative thereof.

[0401] DDR joint part In one exemplary embodiment, the protein-binding moiety is a DDR kinase-binding moiety. In one exemplary embodiment, the DDR-binding moiety is an inhibitor or activator. Discoidin domain receptors (DDRs) belong to the receptor tyrosine kinase family and are distinguished by their activating ligand, fibrillar collagen. Furthermore, their activation and deactivation kinetics are slow, and they exist as dimers on the cell surface in the absence of their ligand. See, e.g., 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), E7786-E7794.

[0402] In an exemplary embodiment, the DDR inhibitor is: [ka] or a derivative thereof.

[0403] In an exemplary embodiment, the DDR inhibitor has the formula: [ka] IC related to50 is 230 nM WRG-28.

[0404] In one embodiment, WRG-28 or a derivative thereof is an extracellularly acting allosteric inhibitor that inhibits receptor-ligand interactions through allosteric modulation of the receptor. WRG-28 has been shown to inhibit tumor invasion and migration and the tumor-supportive role of the stroma, and inhibits metastatic breast tumor cell colonization in the lung by targeting DDR2.

[0405] INSR joint part In an exemplary embodiment, the protein-binding moiety is an INSR kinase-binding moiety. In an exemplary embodiment, the INSR-binding moiety is an inhibitor or activator. The insulin receptor (INSR) is located on a plasma membrane glycoprotein and is a member of the receptor tyrosine kinase (RTK) family that regulates insulin. The INSR family is composed of RTKs, including the insulin-like growth factor-1 receptor (IGF1R) and insulin receptor-related receptors. See, e.g., Hubbard, SR, "The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase," Cold Spring Harbor Perspectives in Biology 2013, 5(3). In an exemplary embodiment, the kinase-binding moiety is an INSR inhibitor. In an exemplary 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), 796-802.

[0406] In one embodiment, the protein is an insulin receptor and the binding moiety is RZ-358, also known as XOMA-358, a fully human negative allosterically modulating insulin receptor antibody. When administered intravenously, RZ358 can bind to sites on the insulin receptor present in liver, fat, and muscle. The RZ358 molecule is highly selective for insulin receptors that lack IGF-1 interaction, still allowing insulin to bind and signal, and attenuating insulin signaling only when insulin levels are elevated. Clinical trials have been conducted at doses ranging from 0.1 to 9 mg / kg and have been studied in congenital hyperinsulinism and post-gastric bypass hypoglycemia.

[0407] Additional selective allosteric antibodies against the insulin receptor, including XMetD, have been identified using research platforms and can be utilized with 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 have shown that XOMA358 inhibits insulin signaling in patients with inadequate insulin signaling, including congenital hyperinsulinism. Treatment with the antibody in volunteers ranges from 0.1 mg / kg to 9 mg / 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.

[0408] IKK joint part In an exemplary embodiment, the kinase-binding moiety is an IKK kinase-binding moiety. In an exemplary 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, e.g., Karin, M. "The IκB Kinase - a Bridge between Inflammation and Cancer". Cell Res 2008, 18(3), 334-342. In an exemplary embodiment, the IKK inhibitor is a compound of the formula: [ka] This is BMS-345541.

[0409] mTOR binding part In one exemplary embodiment, the protein-binding moiety is an mTOR kinase-binding moiety. In one exemplary 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 has 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, e.g., Ballou LM, et.al. "Rapamycin and mTOR Kinase Inhibitors," J Chem Biol 2008, 1(1-4), 27-36.

[0410] In an exemplary embodiment, the mTOR inhibitor has the formula: [ka] Sirolimus, also known as rapamycin, has the following properties: [Table 29]

[0411] 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 mammalian target of rapamycin (mTOR). Sirolimus inhibition of FKBP prolyl isomerase 1A has a pK of 9.7. i It has.

[0412] The FKBP12-sirolimus complex has been reported to bind to a site distinct from the kinase domain of mTOR, acting as a negative allosteric modulator of mTOR activity. This action reduces mTOR-induced proliferation of activated T cells, cells normally involved in immunological attack on transplant tissue. See Am. J. Health-Syst. Pharm. 2000, 57, 437-448. In vitro studies have been conducted, showing 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.

[0413] In an exemplary embodiment, the mTOR inhibitor is any inhibitor encompassed by WO2014177123 (hereby incorporated by reference) or a derivative thereof.

[0414] PAK binding part In an exemplary embodiment, the protein-binding moiety is a p21 kinase-binding moiety. In an exemplary 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, which includes PAKs 1-3, and Group II, which includes PAKs 4-6. These 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), 776-781. In an exemplary embodiment, the PAK inhibitor is a compound represented by the formula: [ka] Compound 3, PMID 26191365, is a highly selective negative allosteric modulator of the protein kinase p21 (Cdc42 / Rac)-activated kinase 1 with advantageous 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

[0013] The PAK inhibitors have a specific activity, which is shown in Table 3 of ACS Med. Chem. Lett. 2015, 6(7), 776-781, which is incorporated herein by reference in its entirety. In an exemplary embodiment, the PAK inhibitor is a PAK inhibitor from Karpov ACS Med. Chem. Lett. 2015. The SAR of PAK1 inhibitors, their selectivity relative to other PAK inhibitors, is detailed in Table 3 of ACS Med. Chem. Lett. 2015, 6, 776-781, and is reproduced below. [Table 30]

[0415] In an exemplary embodiment, the PAK inhibitor has the formula: [ka] It is an IPA-3 related to the above and has the following characteristics: [Table 31] IPA-3 is a cell-permeable, non-ATP-competitive, allosteric, and selective inhibitor of p21 protein (Cdc42 / Rac)-activated kinase 1 (PAK1). IPA-3 covalently binds to the autoregulatory domain of PAK1, preventing its activation by Cdc42. IPA-3 has a pIC of 5.6 for PAK1 (RAC1). 50 See, for example, 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).

[0416] In an exemplary embodiment, the PAK inhibitor has the formula: [ka] The present invention relates to KPT-9274, 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 without interfering with the enzyme's kinase activity. KPT-9274 has begun phase 1 clinical evaluation for non-Hodgkin's lymphoma and solid tumors. KPT-9274 inhibits recombinant human NAMPT with an IC50 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<100 nM. 50 and induces shrinkage of Molt-4 (T-cell acute lymphoblastic leukemia) xenografts in SCID mice. Additionally, KPT-9274 inhibits the proliferation of B-ALL cell lines: KOPN-8; RS4; REH; 697; OP-1; Nalm6; SupB15; with IC50 values ​​of 2.4; 5.6; 14.3; 16.7; 18.0; 19.0; 22.6; and >10,000 in nM, respectively. 50 KPT-9274 also inhibited PDX B-ALL: LAX2; LAX7R; and ICN13 with IC values ​​of 19.4; 32.7; and 25.9 in nM. 50 Inhibit by value.

[0417] PDK1 binding part In one exemplary embodiment, the protein-binding moiety is a PDK1 kinase-binding moiety. In one exemplary embodiment, the PDK1-binding moiety is an inhibitor or activator. Phosphoinositide-dependent protein kinase-1 (PDK1) regulates the AGC kinase family. 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 is hydrophobic and has two functions: recruiting downstream substrate kinases containing hydrophobic motifs (HMs) and stimulating the intrinsic activity of PDK1. See, e.g., "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), 361-374. In one exemplary embodiment, the kinase-binding moiety is a PDK1 inhibitor. In one exemplary embodiment, the kinase-binding moiety is a PDK1 inhibitor. In an exemplary embodiment, the PDK1 inhibitor has the formula: [ka] This is PS48.

[0418] PS48 was 8.0 μM AC 50 See, e.g., Hindie, V., et al. "Structure and Allosteric Effects of Low-Molecular-Weight Activators on the Protein Kinase PDK1", Nat Chem Biol 2009, 5(10), 758-764 (incorporated by reference in its entirety), with particular reference to Figure 3, which illustrates the binding pocket, and Table 1, which is illustrated below. [Table 32]

[0419] 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 herein by reference in their entireties. Table 1 from Stroba, entitled "Effect of Compounds on the Catalytic Activity of PDK1 and Thermodynamic Characterization of Binding," is specifically incorporated by reference and reproduced below. [Table 33]

[0420] In an exemplary embodiment, the PDK1 inhibitor has the formula: [ka] RS1 selectively binds to PDK1. In an exemplary embodiment, the PDK1 inhibitor is an RS1 according to the formula: [ka] RS1 and RS2 bind to PDK1 with Kd of 1.5 μM and 9 μM, respectively.

[0421] In an exemplary embodiment, the PDK1 inhibitor is a peptide docking motif (piftide). Piftide is a synthetic peptide. In an exemplary embodiment, piftide is REPRILSEEEQEMFRDFDYIADW (SEQ ID NO: 8). In an exemplary embodiment, piftide is a small molecule mimic of a peptide. See, e.g., 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), 18590-18595 (incorporated herein by reference in its entirety).

[0422] In an exemplary embodiment, the PDK1 inhibitor has the formula: [ka] This is PS210.

[0423] PTK2 / FAK binding part In one exemplary embodiment, the protein-binding moiety is a PTK2 / FAK kinase-binding moiety. In one exemplary 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 important role in mammalian development and numerous 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).

[0424] In an exemplary embodiment, the PTK2 / FAK inhibitor has the formula: [ka] Compound 30, PMID 23414845, 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 on the kinase, rather than the ATP active site. In vitro, compound 30 inhibited PTK2 autophosphorylation in prostate cancer cells with an IC50 of 7.1 μM. 50 PTK2 plays a key role in the control of cell proliferation, migration, and invasion, and helps regulate resistance to apoptosis. This enzyme is overexpressed in several cancers, and reduced PTK2 activity has growth inhibitory effects in vitro and in vivo. These factors make PTK2 inhibition a novel mechanism for treating hyperproliferative diseases. In a separate experiment, compound 30 exhibited a pIC of 6.2 for the inhibition of PAK2. 50 See, for example, Tomita, N., et al. "Structure-Based Discovery of Cellular-Active Allosteric Inhibitors of FAK", Bioorganic & Medicinal Chemistry Letters 2013, 23(6), 1779-1785 (hereby incorporated by reference in its entirety), in particular Tables 1, 3, and 5, which illustrate the evaluation of the SAR of substituents, and Tables 1 and 5 are reproduced below for binders that can be used herein. [Table 34] [Table 35]

[0425] RIPK binding part In an exemplary embodiment, the protein-binding moiety is a RIPK kinase-binding moiety. In an exemplary 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), 9714-9722. In an exemplary embodiment, the RIPK inhibitor is represented by the formula: [ka] RIPA-56 is a highly potent, selective, and metabolically stable type III (allosteric) RIPK1 inhibitor. RIPA-56 is also known as compound 92 in WO2016101885 (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 See, for example, 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 (herein incorporated by reference in its entirety), in particular Table 5.

[0426] In an exemplary embodiment, the RIP1 inhibitor has the formula: [ka] or derivatives thereof, where "linker" and "target binding agent" represent the remainder of the chimeric small molecule.

[0427] TYK2 binding part In an exemplary embodiment, the protein-binding moiety is a TYK2 kinase-binding moiety. In an exemplary embodiment, the TYK2-binding moiety is an inhibitor or activator. Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signaling downstream of receptors. TYK2 pairs with JAK2 to regulate the IL-23 / IL-12 pathway and with JAK1 to regulate the type I interferon family. In an exemplary embodiment, the TYK2 inhibitor has the formula: [ka] See, for example, Moslin R., et al. "Identification of Imidazo[1,2-b]Pyridazine TYK2 Pseudokinase Ligands as Potent and Selective Allosteric Inhibitors of TYK2 Signaling", Med. Chem. Commun. 2017, 8(4), 700-712.

[0428] In an exemplary embodiment, the TYK2 inhibitor has the formula: [ka] (wherein D is deuterium) Deuclavacitinib, also known as BMS-986165, is a selective, orally active, and allosteric TYK2 inhibitor that binds to the JH2 (pseudokinase) domain. Deuclavacitinib is kinome-selective and does not bind to JAK1-3 or the TYK2 JH1 (ATP) binding domain. Deuclavacitinib has an in vitro IC of 5 nM. 50 Specifically, deuclavacitinib has a pK of 10.7 for TYK2. i values ​​and pIC values ​​of 9.7 and 9.0 for TYK2 and JAK1, respectively 50 Currently, duravacitinib is undergoing clinical evaluation in patients with systemic lupus erythematosus and ulcerative colitis (both in Phase 2) and moderate to severe psoriasis (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 (incorporated herein by reference in their entirety). Tables 1 and 3 from Wrobleski et al. are specifically incorporated herein by reference. Table 1 shows the JAK family biochemical potency for clinical inhibitors, and Table 3, reproduced below, provides an extension of the C3' amide SAR. [Table 36]

[0429] SHP joint part In one exemplary embodiment, the protein-binding moiety is a Scr kinase-binding moiety. In one exemplary 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 growth and anti-apoptotic signals from receptor tyrosine kinases. SHP2 is composed of three folded domains and a C-terminal tail. SHP2 regulates phosphatase activity by binding to phosphopeptides in the N-terminal SH2 and C-terminal SH2 domains. The PTP domain harbors a catalytic function in the conserved signature motif HCX5R. The disordered C-terminal tail contains and has 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 an exemplary embodiment, the SHP3 inhibitor is any from WO2020076723, which is incorporated herein by reference.

[0430] aPKC binding part In one exemplary embodiment, the protein-binding moiety is an atypical PKC kinase-binding moiety. In one exemplary embodiment, the aPKC-binding moiety is an inhibitor or activator. Atypical protein kinase C (aPKC) belongs to the protein kinase C family and is classified into three groups based on its structure and cofactor regulation. The aPKC isozymes, ζ and λ, are the least understood and differ significantly in structure from the other two classes. First, they have one Cys-rich motif in the C1 domain instead of two. Second, aPKC isozymes appear to lack key residues that maintain the C2 fold. In a further characteristic of aPKC, aPKC has been reported to be 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), 28495-28498.

[0431] In an exemplary embodiment, the aPKC inhibitor is an inhibitor identified in WO2015075051 (hereby incorporated by reference) or any derivative thereof.

[0432] In an exemplary embodiment, 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), 6513-6530, DOI:10.1021 / jm500521n (incorporated herein by reference in its entirety), in particular Tables 1 and 2 reproduced below. [Table 37] [Table 38] In some embodiments, the binding moiety has the formula: [ka] and more preferably the linking molecule is a 1,3,5-trisubstituted pyrazoline according to [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 yielding the best activity. The presence of a lipophilic (halogen or alkyl) substituent on the 1-phenyl was found to be essential for producing high potency.

[0433] SphK binding part In one exemplary embodiment, the protein-binding moiety is a SphK kinase-binding moiety. In one exemplary 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. SphKs have 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). These two SphK isoforms are SphK1 and SphK2. SphK2 has approximately 240 more amino acids than SphK1. See, e.g., Cao M., "Sphingosine Kinase Inhibitors: A Patent Review." Int J Mol Med 2018.

[0434] In an exemplary embodiment, the SphK inhibitor is an inhibitor identified in WO2014118556 (hereby incorporated by reference) or a derivative thereof.

[0435] GSK-3 binding part In one exemplary embodiment, the protein-binding moiety is a GSK-3 kinase-binding moiety. In one exemplary 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 with intracellular receptor-coupled signaling proteins, such as insulin receptors and several ionotropic neurotransmitter receptors. GSK3 can be found in the cytosol, mitochondria, and nucleus, as well as other intracellular compartments. The two key functional domains of GSK3 are a priming substrate-binding domain, which recruits substrates to GSK3, and a kinase domain, which phosphorylates the substrate. See, e.g., Glycogen Synthase Kinase-3 (GSK3): Regulation, Actions, and Diseases. Pharmacology & Therapeutics 2015, 148, 114-131. In an exemplary embodiment, the GSK3 inhibitor is an inhibitor identified in US Pat. No. 9,757,369 (hereby incorporated by reference) or a derivative thereof.

[0436] JNK joint part c-Jun N-Terminal Kinase (JNK) is involved in stress signaling pathways that have been implicated in regulating gene expression, neuroplasticity, regeneration, cell death, and cellular senescence. JNK is one of three families of MAP kinases. JNK has three isoforms: JNK1 and JNK2, which are found throughout all tissues; and JNK3, which is found in neurons, the heart, and the 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, 6. In an exemplary embodiment, the kinase-binding moiety is a JNK-binding moiety. In an exemplary embodiment, the JNK-binding moiety is an inhibitor or activator. In an exemplary embodiment, the JNK inhibitor is a compound represented by the formula: [ka] Compound 10 has an IC of 1.2 in a 0.1 mM p38α assay; 0.8 in a 0.1 mM MKK6 / p38α cascade assay; 1.4 in a 0.01 mM p38α / MK2 cascade assay; greater than 100 in a 0.1 mM MKK6 assay; greater than 40 in a 0.01 mM MK2 assay; greater than 40 in a 0.1 mM p38β assay; greater than 40 in a 0.1 mM p38γ assay; and greater than 40 in a 0.1 mM p28δ assay. 50See, for example, 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), 234-244 (incorporated herein by reference). In one aspect, compound 10 binds to the lipid-binding pocket. Additional JNK1 non-ATP site inhibitors can also be used with the small molecules disclosed herein, including biaryl tetrazole-based Jnk-1 activation inhibitors. In one embodiment, the biaryl tetrazole-based binding moiety for Jnk-1 is selected from Table 2 of ACS Chem. Biol. 2011, 6, 234-244, reproduced below. [Table 39] Further details regarding biaryltetrazole affinity and coupling assay data for JNK isoforms and related MAP kinase proteins from Table 1 of ACS Chem. Biol. 2011, 6, 234-244 are also provided and incorporated by reference below. [Table 40]

[0437] In certain exemplary embodiments, the binding moiety is a nucleotide sequence as described in Lombard et al. Allosteric modulation of JNK docking-site interactions with ATP-competitive inhibitors. Biochemistry. Author manuscript; available in PMC 2019 Oct 9, Fig. 1B (incorporated herein by reference). [Table 41] is selected from.

[0438] In an exemplary embodiment, the JNK inhibitor has the formula: [ka] or derivatives thereof, and the oval represents the remainder of the chimeric small molecule.

[0439] 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 critical for the development and maturation of the central and peripheral nervous systems during embryogenesis. It has been implicated in pain and temperature sensation in sympathetic and sensory neurons and in memory processes in adults, and 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, for example, 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.

[0440] In an exemplary embodiment, the protein-binding moiety is a TRK-binding moiety. In an exemplary embodiment, the TRK-binding moiety is an inhibitor or activator. In an exemplary embodiment, the TRK inhibitor has the formula: [ka] The compound 13, an allosteric TRKA inhibitor, exhibited an IC value of 99 nM.50 TRKB and TRKC (each of which has an IC of 81 mM and 25 mM, respectively) 50 In addition, compound 15 also demonstrated good selectivity over TRKA over TRKB. The crystal structures of TRKA and compounds 13, 15, and 16 (PDB codes: 5KMI, 5H3Q) from Chinese Patent No. CN103649076, specifically Figures 10 and 11, are incorporated herein by reference. In another exemplary embodiment, the TRK inhibitor is any pyrrolidinyl urea or pyrrolidinyl thiourea and any derivative thereof as described in WO2012158413A2, incorporated herein by reference.

[0441] 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, 325-339, DOI:10.1080 / 13543776.2020 (both of which are incorporated by reference in their entirety).

[0442] 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, for example, McDermott, U., et al., "Ligand-Dependent Platelet-Derived Growth Factor Receptor (PDGFR)-α Activation Sensitizes Rare Lung Cancer and Sarcoma Cells to PDGFR Kinase Inhibitors," Cancer Res 2009, 69(9), 3937-3946. In an exemplary embodiment, the kinase-binding moiety is a PDGFR target molecule. In an exemplary embodiment, the PDGFR-binding moiety is an inhibitor or activator. In exemplary embodiments, the PDGFR targeting molecule is imatinib, nilotinib, or dasatinib.

[0443] IDH binding part Isocitrate dehydrogenase (IDH) converts isocitrate to α-ketoglutarate via NADP +IDH1 is involved in cellular metabolism by converting IDH1 in a cell-dependent manner. There are three isozymes—IDH1, IDH2, and IDH3—which function in different intracellular compartments. Mutations in IDH result in the production of 2-hydroxyglutarate instead of the normal product, which can lead to various cancers. See, for example, Jiang, B.; et al., “IDH1 Mutation Promotes Tumorigenesis by Inhibiting JNK Activation and Apoptosis Induced by Serum Starvation,” Cell Reports, 2017, 19, 389-400 and Malarz, K.; et al., “The Landscape of the Anti-Kinase Activity of the IDH1 Inhibitors,” Cancers, 2020, 12, 536.

[0444] In exemplary embodiments, the kinase-binding moiety is an IDH targeting molecule. In certain exemplary embodiments, the IDH targeting molecule is an inhibitor or activator. In certain exemplary embodiments, the IDH inhibitor is enasidenib, ivosidenib, or any derivative thereof.

[0445] RET joint part The rearranged during transfection (RET) oncogene fusion encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of ligands. RET has been shown to play a role in kidney development and enteric nervous system signal transduction. See, e.g., Arighi, E.; Borrello, MG; Sariola, H. RET Tyrosine Kinase Signaling in Development and Cancer. Cytokine & Growth Factor Reviews, 2005, 16, 441-467. In an exemplary embodiment, the kinase-binding moiety is a RET targeting molecule. In an exemplary embodiment, the RET targeting molecule is an inhibitor or activator. In an exemplary embodiment, the RET inhibitor is pralsetinib or any derivative thereof.

[0446] ITK joint part Interleukin-2-inducible T-cell kinase (ITK), also known as tyrosine-protein kinase (TSK), is a member of the TEC kinase family that is generally expressed in T cells and NK cells. ITK mediates signal transduction in T cells and NK cells triggered by T cell receptors and Fc receptors. See, for example, Zhong, Y.; et al., Targeting Interleukin-2 Inducible T-Cell Kinase (ITK) in T-Cell Related Diseases. Postdoc Journal, 2014, 2.

[0447] In an exemplary embodiment, the kinase-binding moiety is an ITK targeting molecule. In an exemplary embodiment, the ITK targeting molecule is an inhibitor or activator. In an exemplary embodiment, the ITK inhibitor has the formula: [ka] or a derivative thereof, where the oval represents the remainder of the chimeric small molecule and the in group represents the point of attachment to the remainder of the chimeric small molecule.

[0448] TAK binding part Transforming growth factor β-activated kinase 1 (TAK1) is a member of the MAPK family and functions as a signaling intermediate in tumor necrosis factor (TNF), interleukin-1, and Toll-like receptor (TLR) signaling pathways. TAK1 transmits receptor complex signals to downstream MAPK and NF-κB pathways. TAK1 initiates signaling cascades by associating with the TNF receptor complex via TNF receptor-associated factor 2 / 5 (TRAF2 / 5) and kinase receptor-interacting protein 1 (RIP1). See, for example, Broglie, P. et al., J. Transforming Growth Factor β-Activated Kinase 1 (TAK1) Kinase Adaptor, TAK1-Binding Protein 2, Plays Dual Roles in TAK1 Signaling by Recruiting Both an Activator and an Inhibitor of TAK1 Kinase in Tumor Necrosis Factor Signaling Pathway. Journal of Biological Chemistry, 2010, 285, 2333-2339.

[0449] In an exemplary embodiment, the kinase-binding moiety is a TAK1 targeting molecule. In an exemplary embodiment, the TAK1 targeting molecule is an inhibitor or activator. In an exemplary embodiment, the TAK1 inhibitor has the formula: [ka] or derivatives thereof, and the oval represents the remainder of the chimeric small molecule.

[0450] BMX joint part Bone marrow kinase on chromosome X (BMX) belongs to the non-receptor tyrosine kinase family of tyrosine kinases expressed in hepatocellular carcinoma (TEC). BMX is expressed in cardiac endocardial cells, cardiac endothelial cells, and hematopoietic cells of the myeloid lineage, such as granulocytes and monocytes. BMX may function in cell differentiation, motility, and survival. For example, Gottar-Guillier, M.; Dodeller, F.; Huesken, D.; Iourgenko, V.; Mickanin, C.; Labow, M.; Gaveriaux, S.; Kinzel, B.; Mueller, M.; Alitalo, K.; Littlewood-Evans, A.; Cenni, B. See Kinase-Independent Manner. The Journal of Immunology, 2011, 186, 6014-6023.

[0451] In an exemplary embodiment, the kinase-binding moiety is a BMX targeting molecule. In an exemplary embodiment, the BMX targeting molecule is an inhibitor or activator. In an exemplary embodiment, the BMX inhibitor has the formula: [ka] or derivatives thereof, and the oval represents the remainder of the chimeric small molecule.

[0452] LIMK joint part The LIM kinase (LIMK) family includes two closely related kinases, LIMK1 and LIMK2. LIMK family members are structurally composed of two amino-terminal LIM domains, a PDZ domain, a proline / serine (P / S)-rich region, and a kinase domain. These components form a signaling domain that regulates cytoskeletal dynamics through phosphorylation of cofilin family proteins, which are downstream effectors of several signaling pathways. See, for example, Prunier C, Prudent R, Kapur R, Sadoul K, Lafanechere L. LIM kinases: cofilin and beyond. Oncotarget. 2017 Jun 20;8(25):41749-41763.

[0453] In exemplary embodiments, the kinase-binding moiety is a LIMK targeting molecule. In certain exemplary embodiments, the LIMK targeting molecule is an inhibitor or activator. In certain exemplary embodiments, the LIMK inhibitor has the formula: [ka] or derivatives thereof, where "linker" and "target binding agent" represent the remainder of the chimeric small molecule.

[0454] IRE joint part The serine / threonine-protein kinase / endoribonuclease inositol-requiring enzyme 1α (IRE) is an ER transmembrane protein with two enzymatic activities: a kinase and an endoribonuclease (Rnase). During ER stress, the ER luminal domain becomes oligomerized, leading to ER protein unfolding. Oligomerization causes a rearrangement of these two enzymatic domains, resulting in trans-autophosphorylation. Rnase cleaves XBP1 mRNA, releasing the intron. Religation of the cleaved XBP1 mRNA then shifts the open reading frame. The translated, spliced ​​XBP1 mRNA produces a transcription factor called XBP1. ER protein folding, as well as capacity and quality control, are enhanced by the XBP1 protein. Thus, IRE promotes ER adaptation.

[0455] In an exemplary embodiment, the kinase binding moiety is an IRE targeting molecule. In an exemplary embodiment, the IRE targeting molecule is an inhibitor or activator. In an exemplary embodiment, the IRE inhibitor has the formula: [ka] or derivatives thereof, where "linker" and "target binding agent" represent the remainder of the chimeric small molecule.

[0456] Additional kinase-binding moieties In an exemplary embodiment, the kinase binding moiety has the formula: [ka] or a derivative thereof, with the remaining chimeric small molecule attached to the methyl group at the left end.

[0457] target substrate The target substrate can be a natural substrate of the enzyme to which the kinase-binding moiety binds. However, the target-binding moieties discussed below can also be used to direct an enzyme to modify a non-natural substrate or neo-substrate of that kinase. Target substrates are polypeptides, nucleic acids, polynucleotides, lipids, and oligosaccharides. The target-binding moiety can be selected for a specific substrate of interest that can be located in different cellular locations, such as the nucleus, cytoplasm, mitochondria, or cell surface.

[0458] target binding moiety The target binding moiety confers on the chimeric small molecule a mechanism for binding or associating with a target, including the target substrates noted above. The target binding moiety of the chimeric small molecule binds the target substrate and brings it into proximity with the kinase, either via the kinase binding moiety or due to the target binding moiety attached or labeled on the kinase. This reaction may allow the protein to modify a larger number of substrates, non-natural target substrates of the protein, and may increase the reaction rate / efficiency of such substrate modification. To this end, the target binding moiety must be capable of binding to the desired substrate of interest and of being linked via a linker to the kinase binding moiety, so as to allow for substrate modification.

[0459] Polynucleotide-binding moiety In one exemplary embodiment, the target-binding moiety binds to a polynucleotide. Exemplary 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 (incorporated herein by reference). Further exemplary polynucleotide-binding moieties include polynucleotide-binding proteins. Polynucleotide-binding proteins can be identified from nucleotide-binding folds in proteins, such as the Rossmann fold (see, e.g., Kleiger et al., J. of Mol. Biol. 323:69-76) and the P-loop-containing nucleotide hydrolase fold (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 method specifically for NAD. Parca et al. (2012) identified nucleotide-binding sites in protein structures and listed the nucleotides, protein names, and organism names to which these proteins bind in Table S1 of DOI: 10.1371 / journal.pone.0050240 (incorporated herein by reference). Thus, nucleotide-binding moieties are known in the art and can be identified by those skilled in the art for use as target-binding moieties in the present compositions.

[0460] Oligosaccharide binding moiety In one exemplary embodiment, the target-binding moiety is an oligosaccharide-binding moiety. Oligosaccharide-binding moieties include small molecules. 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 for antiviral and anticancer drugs. The localization moiety can be, for example, a lectin, which promotes an interaction site for the carbohydrate. Exemplary molecules include small molecule boronolectins, nucleic acid-based boronolectins, and peptide boronolectins. See, e.g., Jin et al., Med. Res Rev. 2010 March;30(2):171-257; doi:10.1002 / med.20155 (hereby incorporated by reference), particularly Figures 1-50 regarding the binding molecules and complexes formed. Publicly available computational methods are available for selecting small molecules with carbohydrate binding capabilities using developed bioinformatics, 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.

[0461] Analysis of binding site residues in protein-carbohydrate complexes, along with stabilizing residues, can identify complex folding and binding to understand interactions in addition to non-covalent hydrogen bonding and non-polar interactions. See, e.g., Shanmugam et al., doi.:10.2174 / 0929866525666180221122529. Publicly available tools can be used to design carbohydrate-binding moieties, including binding sites and predicted folds, for the design of chimeric small molecules containing such carbohydrate-binding moieties.

[0462] Lipid binding moiety In one exemplary embodiment, the target-binding moiety is a lipid-binding moiety. Lipid-binding moieties can be utilized as target-binding moieties for the chimeric small molecules disclosed herein. As regulators of cellular stability and signal transduction, modifying their composition, distribution, or trafficking can 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, PI-diphosphate, and PI-triphosphate (PIP—a family of seven anionic 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, and phosphatidylglycerol.

[0463] Lipid-binding moieties can be incorporated into chimeric small molecules. For example, certain steroids have the ability to target and bind lipids. Other lipid-binding moieties, such as proteins, can either bind lipids specifically (in which case a distinct binding site for a given lipid can be identified) or nonspecifically (in which case the lipid acts as a vehicle and physical properties such as thickness, fluidity, or curvature regulate protein function). Phosphoinositide-binding domains such as FYVE or PX or the FRRG motif of the β-propeller of PROPPIN may be more general domains that can be used to identify lipid-binding proteins. The FYVE domain, named after the first four proteins with the motif (Fab1, YOTB, Vac1 EEA1), contains several conserved regions that can also be used to identify related domains. See, for example, A.H. 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.

[0464] Eukaryotic cells can degrade intracellular components through a lysosomal degradation pathway called macroautophagy, and malfunction of this pathway has been linked to 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) Therefore, autophagy-related (ATG) proteins, including LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2, can be used as lipid-binding moieties in the present invention. (De la Ballina (2019), doi.org / 10.1016 / j.jmb.2019.05.051) Lipid-binding proteins include HCLS1-binding protein 3 (HS1BP3), a protein that negatively regulates the activity of phospholipase D1 (PLD1).

[0465] Target protein binding moiety As detailed herein, target protein binding moieties are provided for exemplary proteins of interest targeted for modification. Target protein binding moieties are selected based on the desired association and modification. Thus, the desired modification, which can be tailored to a particular pathology, disease, treatment, or other desired effect, can be a design consideration when selecting a protein binding moiety.

[0466] The target protein binding moiety can be selected for a specific protein of interest, which can be located in different cellular locations, e.g., the nucleus, cytoplasm, mitochondria, or the cell surface. Exemplary 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 Figures 5-48 for details, which are incorporated herein by reference). The target protein binding moiety can bind to a protein that undergoes a conformational change upon binding.

[0467] The target protein binding moiety can bind to a protein that undergoes a conformational change upon binding, such as the androgen receptor (AR). In one embodiment, upon activation of the protein, the target substrate is modified by the protein at one or more novel modification sites that would otherwise remain unmodified by the protein when not activated by the chimeric small molecule. The target substrate need not be a natural substrate of the protein. The target substrate can be a protein, and the discussion herein of a gene includes the product of gene expression.

[0468] In one embodiment, the target protein binding moiety is capable of binding a protein that is an ATPase or a GTPase. Exemplary GTPases can be from the Ras, Rho, Rab, Arf, or Ran families; see, e.g., Yoshimi Takai, Takuya Sasaki, and Takashi Matozaki, "Small GTP-Binding Proteins," Physiological Reviews 2001 81:1,153-208; doi:10.1152 / ohysrev.2001.81.1.153. Exemplary molecular targeting moieties 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.

[0469] In one exemplary embodiment, the target substrate is tagged with an orthogonal tag, e.g., FKBP12 F36V Modified with SNAP tags, CLIP tags, ACP tags, and MCP tags, and the target binding moiety is an orthogonal tag binder. See, e.g., neb.com / tools-and-resources / feature-articles / snap-tag-technologies-novel-tools-to-study-protein-function (incorporated herein by reference).

[0470] Below are provided further non-limiting examples of target protein binding moieties for various target proteins of interest in the oncology and infectious disease contexts, and the uses of which are further discussed in the Methods of Use section below.

[0471] KRAS In one exemplary embodiment, the target protein binding moiety is a KRAS binding moiety. [ka] wherein R is a covalent warhead; X is a group of formula [ka] and Y is selected from the group consisting of 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, or an aliphatic halide such as -OCF2Cl. The KRAS binding agent is selected from the group consisting of:

[0472] In one exemplary embodiment, the target-binding moiety is a hydrogen-bond surrogate (HBS) Son of Sevenless (SOS) peptidomimetic (PM). In one exemplary embodiment, HBS-SOS-PM is HBS1-7 according to the sequences: XFE*GIYRTDILRTEEGN-NH2 (SEQ ID NO: 9); XFE*GIYRTELLKAEEAN-NH2 (SEQ ID NO: 10); XFE*GIYRLELLKAEEAN-NH2 (SEQ ID NO: 11); XFE*GIYRLELLK-NH2 (SEQ ID NO: 12); XFE*AIYRLELLKAEEAN-NH2 (SEQ ID NO: 13); XFE*GIYRLELLKAibEEAibN-NH2 (SEQ ID NO: 14); and XAE*GIYRLELLKAEAAA-NH2 (SEQ ID NO: 15), respectively, where X represents a 4-pentenoic acid residue and the asterisk (*) indicates an N-allyl residue (*G, N-allylglycine). In one exemplary embodiment, the target binding moiety is a KRAS binding molecule HB3 according to the formula: XFE*GIYRLELLKAEEAN-NH2 (SEQ ID NO: 13). In one exemplary embodiment, the target binding moiety is a KRAS binding molecule HB7 according to the formula: XAE*GIYRLELLKAEAAA-NH2 (SEQ ID NO: 15). See Nickerson et al., An Orthosteric Inhibitor of the RAS-SOS Interaction, doi:10.1016 / B978-0-12-420146-0.00002-0 (incorporated herein by reference in its entirety), particularly Table 2.1.

[0473] In one exemplary embodiment, the target binding moiety has the formula: [ka] In one exemplary embodiment, the target binding moiety is a KRAS binding molecule according to 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 exemplary embodiment, the target binding moiety is a KRAS binding moiety and 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 (incorporated herein by reference in its entirety).

[0474] In one exemplary embodiment, the target binding moiety has the formula: [ka] JPEG2025533072000274.jpg179170JPEG2025533072000275.jpg169170JPEG2025533072000276.jpg195170JPEG2025533072000277.jpg191170JPEG2025533072000278.jpg255169JPEG2025533072000279.jpg100170. In one exemplary embodiment, the target binding moiety has the formula: Ac-FIGRLCTEILKLREGN-NH2 (SEQ ID NO: 16); Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 17); Ac-WIGRLCTEILRLRNGN-NH2 (SEQ ID NO: 18); Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 19); Ac-AIGRLCTEILRLRNGA-NH2 (SEQ ID NO: 20); Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 21); Ac-WIGRLCTEILRLRNGN-NH2 (SEQ ID NO: 22); Ac-LAWALRELERELARLC-NH2 (SEQ ID NO: 23); Ac-WIGRLCTEIR HAc-LAWRLRELERELARLC-NH2 (SEQ ID NO: 24); Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 25); Ac-WIGRLCTEIRRLRNGN-NH2 (SEQ ID NO: 26); Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 27); Ac-WIGRLCTEILRLRNGN-NH2 (SEQ ID NO: 28); Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 29); Ac-FIGRLCTEILKLREGN-NH2 (SEQ ID NO: 30); FITC-AβLAWRLRELERELAR LC-NH2 (SEQ ID NO: 31); Ac-WIGRLCTEILRLRNGN-NH2 (SEQ ID NO: 32); FITC-AβLAWRLRELERELARLC-NH2 (SEQ ID NO: 33); Ac-AIGRLCTEILRLRNGA-NH2 (SEQ ID NO: 34); FITC-AβLAWRLRELERELARLC-NH2 (SEQ ID NO: 35); Ac-WIGRLCTEILRLRNGN-NH2 (SEQ ID NO: 36); FITC-AβLAWALRELERELARLC-NH2 (SEQ ID NO: 37); Ac-WIGRLCTEIR H RLRNGN-NH2 (SEQ ID NO: 38); FITC-AβLAWRLRELERELARLC-NH2 (SEQ ID NO: 39); Ac-WIGRLCTEIRRLRNGN-NH2 (SEQ ID NO: 40); FITC-AβLAWRLRELERELARLC-NH2 (SEQ ID NO: 41); Ac-WIGRLCTEIR H RLRNGN-NH2 (SEQ ID NO: 42); DZ-GLAWRLRELERELARLC-NH2 (SEQ ID NO: 43); Ac-WIGRLCTEIK(DZ)RLRNGN-NH2 (SEQ ID NO: 44); or Ac-LAWRLRELERELARLC-NH2 (SEQ ID NO: 45) (wherein R H is L-homoarginine; Aβ is L-β-alanine; DZ is a diazirine photocrosslinker; and FITC is 5-isothiocyanate fluorescein linked to the N-terminal amine via a thiourea bond. See Hong et al., PNAS May 4, 2021 118(18)e2101027118; doi:10.1073 / pnas.2101027118 (incorporated herein by reference in its entirety), particularly Table S2.

[0475] In one exemplary embodiment, the target binding moiety has the formula: [ka] where the R group can be any substituent known in the art. In an exemplary embodiment, R4 is an electrophilic group. In an exemplary embodiment, R4 is [ka] (Wherein R is H, [ka] is) See Yoo et al., ACS Chem. Biol. 2020, 15, 6, 1604-1612 (incorporated herein by reference in its entirety).

[0476] FKBP12 F36V In another exemplary embodiment, the protein-binding moiety can be designed to bind to FK506-binding protein (FKBP). The FKBP can be FKBP12, which binds to intracellular calcium release channels and TGF-β type I receptors. In one exemplary embodiment, the FKBP protein-binding moiety binds to FKBP12. F36V In another exemplary embodiment, the binding molecule is [ka] or an analog thereof.

[0477] Tyrosine phosphorylation on FGFR1 can initiate a signaling cascade leading to PI3K / AKT / mTOR signaling and increased transcription of the circulating growth factor G-CSF (see, e.g., Turner et al., Nature Reviews Cancer 2010).

[0478] In one exemplary embodiment, ABL kinase is utilized to target FKBP12 F36V In some embodiments, the chimeric small molecule is [ka] Selected from JPEG2025533072000285.jpg241170JPEG2025533072000286.jpg146170.

[0479] In one exemplary embodiment, the chimeric small molecule comprises: [ka] In one exemplary embodiment, the molecule has the ability to activate FGFR1 / mTOR / G-CSF signaling in a dose-dependent manner.

[0480] EGFR In one exemplary embodiment, the protein-binding moiety is an EGFR-binding moiety. EGFR is a tyrosine kinase receptor belonging to the ErbB receptor family that mediates cell growth, differentiation, and repair in cells, particularly non-cancerous cells, whereas EGF is overexpressed in certain cells, such as many solid tumors, including colorectal cancer, non-small cell lung cancer, ovarian squamous cell carcinoma, renal cancer, head cancer, pancreatic cancer, neck cancer, and prostate cancer, and particularly breast cancer.

[0481] In one embodiment, the protein binding molecule has the formula: [ka] or an analog thereof.

[0482] HSP90 Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that, together with its co-chaperones, regulates proteins involved in cell cycle regulation and signal transduction. Like many ATP-dependent proteins, this protein undergoes a functional cycle that is linked to its ATPase cycle.

[0483] In one embodiment, the HSP90 binding molecule is [ka] or an analog thereof.

[0484] Additional HSP90 binding agents include geldanamycin and formula; [ka] Tanespimycin (IC50 of 5 nM in cell-free assay), according to the formula; [ka] Alvespimycin (IC50 of 62 nM in cell-free assay), according to the formula; [ka] EC141 relating to Eq. [ka] Novobiocin analogs can also be used, as described in Hall et al., J Med Chem. 2016 Feb 11; 59(3):925-933; doi:10.1021 / acs.jmedchem.5b01354 (incorporated by reference), which can be used as MAPK signaling disruptors.

[0485] BTK Bruton's tyrosine kinase (BTK) is a protein involved in multiple signaling cascades and is ubiquitously expressed in B cells. First, BTK is a cytoplasmic protein and is therefore available for interaction with cytoplasmic kinases such as AMPK.

[0486] In one embodiment, [ka] A BTK-binding molecule or analog thereof selected from the group consisting of:

[0487] MDM2 In certain embodiments, the target protein binding moiety is [ka] or a derivative or analogue thereof.

[0488] BRD4 In certain embodiments, the target protein binding moiety is [ka] or an analog thereof.

[0489] FGFR1 In one exemplary embodiment, the target protein binding moiety inhibits an FGFR1 fusion protein. In one exemplary embodiment, the FGFR1 fusion protein inhibitor has the formula [ka] The drug is dovitinib, also known as TKI258.

[0490] PtpA, PtpB In one exemplary embodiment, the target protein binding moiety is a PtpA binding moiety and has the formula [ka] or any derivative thereof.

[0491] In a preferred embodiment, the PtpB binding moiety has the formula [ka] or any derivative thereof.

[0492] SapM In one exemplary embodiment, the target protein binding moiety is a SapM binding moiety. In an exemplary embodiment, the SapM binding moiety contains a trihydroxybenzene group. In an exemplary embodiment, the SapM binding moiety comprises a benzylidenemalononitrile scaffold. In one exemplary embodiment, the SapM binding moiety has the formula: [ka] JPEG2025533072000301.jpg106170 or any derivative thereof. In one exemplary embodiment, the SapM binding moieties are L-ascorbic acid (L-AC) and 2-phospho-L-ascorbic acid (2P-AC).

[0493] UMPK In an exemplary embodiment, the targeted binding agent is an M.tb kinase inhibitor. In an exemplary embodiment, the M.tb kinase inhibitor is a UMPK inhibitor identified in U.S. Patent Application Publication No. 20090209022, which is incorporated herein by reference, and any derivatives thereof.

[0494] Colistin In a preferred embodiment, the PsA-associated target protein binding moiety is colistin, which has the formula: [ka] It has.

[0495] electrophilic reactive groups In one exemplary embodiment, a molecule or binding moiety as disclosed herein can be modified to include an electrophilic reactive group. In one embodiment, the electrophilic reactive group is located between the linker and the target-binding moiety. In some embodiments, the electrophilic reactive group is located between two linkers, between the first linker attached to the kinase-binding agent and the second linker attached to the target-binding moiety. An electrophilic reactive group, as used herein, 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 be directly attached to the target kinase. Once attached to the electrophilic reactive group, the kinase is now tagged with the target-binding moiety. In one exemplary embodiment, a molecule or binding moiety can be modified with an electrophilic reactive group to reduce or decrease the strength of the covalent binding capacity of the electrophilic reactive group or to increase the binding affinity or strength of the electrophilic reactive group, as needed for the application. In some embodiments, a binding molecule can be selected that can create an irreversible covalent bond to the target. When used in chimeric small molecules, such tight binding may be less desirable. Thus, modification of such electrophilic reactive groups is desirable and can be modified to reduce interactions; see, for example, sciencedirect.com / science / article / pii / S0968089618320807. Specifically, reactivity can be designed to allow for covalent binding with the target, with reversible or irreversible properties depending on the desired functional group. 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 a preferred embodiment, the electrophilic reactive group reacts with lysine.An exemplary database that can facilitate the identification of protein-ligand interactions around the binding site is described in Du et al., Nucleic Acids Research, Volume 49, Issue D1, 8 January 2021, Pages D1122-D1129 (incorporated herein by reference), along with the database CovalentInDB, accessible at cadd.zju.edu.cn / cidb / . This approach can be used with any design of electrophilic reactive groups for molecules as disclosed herein. The electrophilic reactive groups described herein can be attached to the chimeric small molecule via any terminal carbon group; a carbon on the cyclic moiety (e.g., a phenyl ring, a heterocycle) and / or a nitrogen on the cyclic moiety.

[0496] N-Acyl-N-Alkyl Sulfonimide (NASA) The design of electrophilic reactive groups can be achieved using NASA chemistry by forming reversible or irreversible bonds with nucleophilic functional groups located on kinases. NASA chemistry is outlined in Nat Commun 9, 1870 (2018), incorporated herein by reference. In a preferred embodiment, the kinase-binding moiety can be attached to a linker utilizing an N-acyl N-alkylsulfonamide (NASA) electrophilic reactive group further attached to the kinase-binding moiety. Upon non-covalent attachment to the target enzyme, the NASA-containing chimeric small molecule covalently binds to the enzyme because the NASA chemically reacts with a nearby lysine or other amino acid as described herein. The NASA-modified chimeric small molecule then dissociates from the kinase, leaving behind a protein target binder covalently attached to the kinase. This modified kinase then binds to the target protein through the newly attached binder and further modifies the protein. In an exemplary embodiment, NASA chemistry is used to label the kinase-binding moiety. Accordingly, embodiments include methods of making the compositions disclosed herein using NASA chemistry and as further described in the examples.

[0497] In an exemplary embodiment, the NASA analog has the formula: [ka] JPEG2025533072000304.jpg53170 or its derivatives.

[0498] Dibromophenyl benzoate In a preferred embodiment, the electrophilic reactive group is dibromophenylbenzoate (DB). DB can be used to functionalize the linker by reacting with a nucleophile located on the kinase. The dibromophenyl group acts as a leaving group to facilitate the reaction, while the benzoate stabilizes the currently attached moiety. In a preferred embodiment, the linker connecting the kinase-binding moiety and the protein-binding moiety is functionalized with DB, thereby labeling the target kinase with the protein-binding moiety. DB chemistry is outlined in Takaoka et al. Chem. Sci., (2015), 6, 3217-3224 (incorporated herein by reference).

[0499] N-Sulfonylpyridine In a preferred embodiment, the electrophilic reactive group is N-sulfonylpyridone (SP). SP can be used to functionalize the linker by sulfonylation with a nucleophile located on the kinase. In a preferred embodiment, the linker connecting the kinase-binding moiety and the protein-binding moiety is functionalized with SP, thereby labeling the target kinase with the protein-binding moiety. SP chemistry is outlined in K. Matsuo et al. Angew. Chem. Int. Ed. 2018, 57, 659 (incorporated herein by reference).

[0500] In an exemplary embodiment, the electrophilic reactive group is [ka] Includes one of the following.

[0501] In an exemplary embodiment, the electrophilic reactive group has the formula: [ka] or a derivative thereof.

[0502] Additional electrophilic reactive groups In an exemplary embodiment, the electrophilic reactive group is: [ka] It consists of any one or more of the following.

[0503] In an exemplary embodiment, the electrophilic reactive group is: [ka] wherein R1 is any one or more of: [ka] selected from the group consisting of: R2 is [ka] is selected from the group consisting of:

[0504] In an exemplary embodiment, the electrophilic reactive group is: [ka] It consists of any one or more of the following.

[0505] In an exemplary embodiment, the electrophilic reactive group is: [ka] It consists of any one or more of the following.

[0506] Photoreactive groups In one exemplary 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, which upon activation becomes a carbon-centered radical, a nitrene, or a carbene, respectively. In another embodiment, the photoreactive group is a thienyl-substituted α-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), 876-880.

[0507] Linker A linker or linking moiety is a bifunctional or multifunctional moiety that can be used to link one or more target-binding moieties, protein-binding moieties. In some embodiments, the linker has a functional group capable of reacting with these moieties to form a covalent bond. The linker moiety is preferably a chemical linker moiety and is represented as L in the formulas of the present invention. In certain embodiments, the linker moiety may preferably include one or more repeats, for example, 1, 2, 3, 4, 5, 6, 7, 8 or more repeats, and the use of repeats may facilitate or improve molecular spacing, conformation, and / or performance. A linker as described herein may refer to both L1 and L2, or L1 and L2 may be different linkers as described herein.

[0508] A linker or linking moiety can be used to link the kinase-binding moiety to the target-binding moiety and / or the electrophilic reactive group to either the kinase-binding agent, the target-binding moiety, or both. When more than one linker molecule is used in a molecule, the linkers can be the same or different from one another.

[0509] In an exemplary embodiment, the linker can be represented together with an exit vector. In an exemplary embodiment, the exit vector can be represented independently of the linker. Exit vector parameters can be identified based, in part, on the average orientation of the substituents attached to the variable points, which can be generated using cheminformatics software. The exit vector can include a bond exiting the chemical moiety. In some embodiments, the exit vector is provided as a bond on the linker or from the binding moiety to provide a conformational link between the linker and the activator moiety and / or the localization moiety.

[0510] Exit Vector One or more exit vectors may be utilized with the molecules described herein. In certain embodiments, a linker or kinase binding moiety may be represented with an exit vector included in the linker or kinase binding moiety. In some embodiments, an exit vector may be represented independently of the linker or kinase binding moiety. Exit vector parameters may be identified, in part, based on the average orientation of substituents attached to a variable point, which may be generated using cheminformatics software. An exit vector may comprise a bond exiting a chemical moiety. In some embodiments, an exit vector is provided as a bond on the linker or from the Abl binding moiety, providing a conformational link between the linker and the Abl binding moiety and / or second Abl binding moiety. An exit vector may also be represented independently of the linker in the formulas detailed herein. In some embodiments, an exit vector is included in W.

[0511] In some embodiments, the bond is selected to be energetically favorable, preferably increasing binding affinity. Exit vectors can be tailored depending on the linker utilized in the molecule. In embodiments, an exit vector is a chemical moiety or bond that promotes steric prominence, which may further promote subsequent coupling, binding, and / or accessibility.

[0512] In an exemplary embodiment, the kinase-binding moiety has an adapter or reactive handle, both of which are used interchangeably herein. The reactive handle comprises a group on the kinase-binding moiety that is attached to a linker. In an exemplary embodiment, the reactive handle is capable of performing click chemistry, amide coupling chemistry, crosslinking chemistry, alkylation, or sulfonation chemistry (see, e.g., I, K.; Brechbiel, M.W. Growing Applications of "Click Chemistry" for Bioconjugation in Contemporary Biomedical Research. Cancer Biotherapy and Radiopharmaceuticals, 2009, 24, 289-302).

[0513] In an exemplary embodiment, the bond is selected to be energetically favorable, preferably increasing binding affinity. The exit vector can be tailored depending on the linker utilized in the molecule. In an exemplary embodiment, the exit vector is a chemical moiety or bond that promotes steric protrusion, which may further promote subsequent coupling, binding, and / or accessibility.

[0514] In certain embodiments, L is [ka] or any combination thereof; any atom in the ring may be substituted with C, NO, S; the linker may be attached to one or more PEG molecules before being attached to A and optionally B; and m and n may be independently selected from 0 to 6.

[0515] In a preferred exemplary embodiment, the linker, L, has one point of covalent attachment to the kinase-binding molecule and two points of covalent attachment to the other kinase-binding molecule. The points of covalent attachment can be any single, double, triple, or quadruple bond between one component of the BFM / chimeric small molecule and another component. In a preferred exemplary embodiment, the linker has the formula [ka] The kinase-binding molecule is linked to one kinase-binding molecule, i.e., A, and the other, i.e., B.

[0516] In an exemplary embodiment, the PEG compound of the aforementioned linker can be replaced with any of the linkers mentioned herein, which are optimized for physiochemical properties such as solubility and / or permeability and / or pharmacokinetic properties such as microsomal stability or target binding.

[0517] A linker as described herein can be attached to a chimeric small molecule at any terminal carbon group; a carbon on a cyclic moiety (e.g., phenyl ring, heterocycle) and / or a nitrogen on a cyclic moiety.

[0518] In an exemplary embodiment, the kinase-binding agent has an adapter or reactive handle, both of which are used interchangeably herein. The reactive handle comprises a group on the kinase-binding agent that is attached to a linker. In an exemplary embodiment, the reactive handle can be implemented using click chemistry, amide coupling chemistry, crosslinking chemistry, alkylation, or sulfonation chemistry.

[0519] Bioorthogonal groups The chimeric small molecules disclosed herein may further comprise a bioorthogonal group. Chimeric small molecules can be configured to include a bioorthogonal group as a mechanism for removing the kinase-binding moiety from the target kinase. This occurs when a coupling molecule selected to react with the bioorthogonal molecule is introduced into a system containing the kinase-bound chimeric small molecule and binds to the bioorthogonal group. As a result, the kinase-binding molecule is no longer functional and cannot bind to the target kinase. In an exemplary embodiment, the kinase-binding agent comprises a bioorthogonal group. In an exemplary embodiment, the kinase-binding agent is modified to include a bioorthogonal group. Bioorthogonal chemistry involves chemical reactions that occur in a biological environment without reacting with endogenous systems, such as functional groups. Bioorthogonal groups include moieties capable of bioorthogonal chemistry. Non-limiting examples of bioorthogonal groups include tetrazine, triazine, cyclooctene, cyclopropene, and diazo groups.

[0520] In one exemplary embodiment, the bioorthogonal group is: [ka] Includes one of the following.

[0521] Electrophilic Reactive Linkers In another aspect, embodiments disclosed herein relate to electrophilic linkers with enhanced stability, reactivity, and tunability. In one exemplary embodiment, the linker molecule has the formula L1-El, EL-L1, or L1-El-L2, where L1 and L2 are independently selected from alkanes, alkenes, amines, ethers, thiols, sulfones, carbonyls, acyl groups, ketones, carboxylic acid esters, amides, enones, anhydrides, imides, and PEGs, and EL is an electrophilic reactive group. The electrophilic reactive linker can be used to attach all or a portion of L1 or L2 to a target polypeptide. The El group of the linker molecule can be selected based on its ability to covalently attach all or a portion of L1 or L2 to a cysteine, lysine, methionine, or tyrosine residue on the target polypeptide. In one exemplary embodiment, L1 and L2 can independently be any of the linkers described above.

[0522] Cysteine-reactive electrophilic linkers In one exemplary embodiment, the linker molecule comprises an El group suitable for attaching all or a portion of L1 or L2 to a cysteine, where El is: [ka] is selected from the group consisting of:

[0523] Lysine-reactive electrophilic linkers In one exemplary embodiment, the linker molecule comprises an El group suitable for attaching all or a portion of L1 or L2 to a lysine, where El is: [ka] (Wherein R1 is [ka] selected from the group consisting of: R2 is [ka] selected from the group consisting of is.

[0524] FIG. 32 demonstrates how the reactivity and specificity of the El group can be tuned to a desired level by modifying the substituents on the El group.

[0525] In another exemplary embodiment, the linker molecule comprises an El group suitable for attaching all or a portion of L1 or L2 to a lysine, the El group being: [ka] is selected from.

[0526] Methionine-reactive electrophilic linkers In another exemplary embodiment, the linker molecule comprises an El group suitable for attaching all or a portion of L1 or L2 to a methionine, the El group being: [ka] is selected from.

[0527] Use of electrophilic reactive linker groups The electrophilic reactive linkers disclosed in this section can be used with any bifunctional molecule, including the chimeric molecules disclosed herein. The electrophilic reactive linkers can also be used with other bifunctional molecules, such as PROTACs. Bekes et al., "PROTAC targeted protein degraders; the past is prologue," Nature Review Drug Discovery 21, 181-200 (2022). Additionally, electrophilic reactive groups can be used in conjunction with other bifunctional molecules. For example, a target polypeptide binding moiety can be attached to one end of the linker, and a modifying moiety can be attached to the other end of the linker. The target polypeptide binding moiety can be selected to selectively bind to a target polypeptide, such as an enzyme, at a catalytic or allosteric site, thereby enabling the electrophilic reactive linkers disclosed herein to covalently attach a modifying moiety to a target polypeptide at a cysteine, lysine, methionine, or tyrosine. In one exemplary embodiment, the target polypeptide binding moiety is a binding moiety capable of specifically binding to a kinase, phosphatase, ubiquitinase, deubiquitinase, acetyltransferase, deacetylase, methyltransferase, demethylase, or glycosyltransferase, and the modifying moiety binds to a neo-substrate of the kinase, phosphatase, ubiquitinase, deubiquitinase, acetyltransferase, deacetylase, methyltransferase, demethylase, or glycosyltransferase. In another exemplary embodiment, the modifying moiety is an immunogenic moiety, and the target polypeptide binding moiety is a polypeptide to which the immunogenic moiety is attached.

[0528] Exemplary Chimeric Small Molecules Any combination of the kinase-binding moieties, linkers, electrophilic activating groups, and target-binding moieties described above may be used to construct chimeric small molecules. The following description, for reference purposes only, provides certain chimeric small molecules that can be generated according to the design principles and exemplary moieties provided above.

[0529] In an exemplary embodiment, the chimeric small molecule has the formula: [ka] It has.

[0530] wherein X and Y are independently selected from CH2 or (CH2)2O, and n and m are independently selected from 1, 2, 3, 4, 5, or 6.

[0531] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] (Wherein JQ1 is a compound represented by the formula: [ka] or analogs thereof, and these two formulas include a single secondary amine (i.e., the right-most secondary amine) containing a linker. relates to.

[0532] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0533] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0534] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0535] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0536] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0537] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0538] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0539] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0540] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0541] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0542] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0543] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] where R1 is a target-binding moiety and R2 is a kinase-binding moiety. relates to.

[0544] In an exemplary embodiment, R1 is [ka] is.

[0545] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0546] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0547] In certain exemplary embodiments, the chimeric small molecule has the formula: [ka] relates to.

[0548] How to use In another aspect, the chimeric small molecules described above can be used in methods for conferring novel functions to cellular kinases or modulating the activity of cellular kinases. The chimeric small molecules find use in treating a variety of diseases and disorders. In exemplary embodiments, the target-binding moiety can bind to a target of interest and preferably localize to the region of the target of interest, thereby enabling the protein-binding moiety to modify the target. An exemplary application includes use in rewiring cell signaling. See Lim et al., Nat Rev Mol Cell Biol 2010, 11(6), 393-403. For example, cell signaling can be addressed by adding phosphoryl groups to specific signaling proteins of interest with controlled dosage and time to enable rewiring of kinase signaling pathways in disease or health. The chimeric small molecule systems herein may enable targeted degradation, where the phosphorylation site serves as a target for recruiting ubiquitin ligases to signal for 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, adding negatively charged phosphoryl groups to aggregation-prone proteins using the present chimeric small molecules 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, including methods for treating kinasopathies. Exemplary embodiments further include modulating nucleotide-binding proteins, which may include their use in conjunction with orthogonally tagged nucleases, such as Cas, and phosphorylating transcription factors to affect binding.In one exemplary embodiment, the invention described herein relates to a method of treatment in which cells are modified ex vivo with a chimeric small molecule to modify at least one target substrate, and the edited cells are then administered to a patient in need thereof.

[0549] Methods for modifying target substrates The chimeric small molecules disclosed herein can be utilized in methods for modifying target substrates. Methods for modifying target substrates can include generating repurposed / reprogrammed cellular proteins by delivering chimeric small molecules as described herein. In exemplary embodiments, chimeric small molecules can be used to inhibit nucleotide-binding proteins, inhibit oncogenic kinases, generate neoantigens as molecular prostheses for kinasopathies, treatment of pathogens, and induction of receptor tyrosine kinase signaling to elicit an immune response.

[0550] Methods for modifying a substrate are provided, which can be a substrate in a cell. In one exemplary embodiment, a chimeric small molecule as described herein is introduced. In one exemplary embodiment, the modification includes inducing a post-translational modification of a target protein. In one exemplary embodiment, the post-translational modification is phosphorylation. The method includes administering the chimeric small molecule to a cell or cell population. The method for modifying a target substrate can include contacting the target substrate with a chimeric small molecule of the invention, e.g., a bifunctional molecule. The contacting allows binding or association with the target substrate or a molecule in proximity to the target substrate. The modification can be by binding, changing structural stability, phosphorylation of the target, or by inducing a conformational change through another mechanism that affects the behavior of the target substrate. For example, activation or inactivation of the target substrate by binding of the chimeric small molecule results in modification of the target substrate at one or more novel modification sites that would otherwise remain unmodified when the chimeric small molecule is not bound to the target substrate. In one aspect, the method includes inducing phosphorylation of the target protein in a cell. The method can include contacting the target substrate with a chimeric small molecule.

[0551] In an exemplary embodiment, the chimeric small molecule can label cellular proteins with target binding moieties for target substrates via electrophilic reactive group moieties. The electrophilic reactive group reacts with and binds to nucleophilic side chains on cellular kinases. Labeling the cellular kinase can facilitate target substrate modification by allowing binding or association with the target substrate or molecules proximal to the target substrate. In an exemplary embodiment, this approach can enable targeted modification using protein inhibitor moieties, as well as activators and neutral binding molecules. Such kinase binding moieties tethered to 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. Generally, labeling proximal to the inhibitor binding site refers to a reactive group at, within, or at a distance from the binding moiety binding site that allows the electrophilic reactive group to react at or near the binding moiety binding site in time and / or space. The tethering of the electrophilic warhead may include a linker, bond, and / or exit vector or adapter, which may be present in some instances. In some instances, in one aspect, the target substrate is not a natural substrate of the protein, or activation of the protein by the binding moiety results in the protein modifying the target substrate at one or more novel modification sites that would otherwise remain unmodified when not activated by binding to the activator moiety. Modification may be due to a conformational change upon binding, a change in structural stability, phosphorylation of the target, or other mechanisms that affect the behavior of the target substrate, such as induced removal of groups by phosphatases, methyltransferases, etc. Modification may include post-translational modifications as 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 aspect, the method comprises inducing phosphorylation of a target protein in or on a cell. The method can include contacting the target substrate with a chimeric small molecule.In one exemplary embodiment, the target substrate is in proximity to a kinase specific for the protein-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-donating molecule can enhance phosphorylation.

[0552] In exemplary embodiments, inhibition of nucleotide-binding proteins can include inhibition of CRISPR-Cas protein binding to transcription factors that bind to nucleic acids or DNA. Thus, proteins modified to contain a binding domain that can be targeted by an orthogonal tag, such as Cas9 containing an FKBP-binding domain, can be inhibited by the use of small molecules containing an orthogonal tag, such as dTAG. Sequence-specific modular adapters consisting of a DNA-binding protein and a self-ligating protein tag can 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 can be modified by modification of transcription factors with chimeric small molecules. Because post-translational phosphorylation of transcription factors is required for direct binding interactions or conformational changes of transcription factors, which can lead to activation or inhibition of gene transcription, methods for modifying transcription factors are provided. In exemplary embodiments, methods of use may include eliciting an immune response, generating autoantigens, and target inactivation. In exemplary embodiments, hyperphosphorylation, or neophosphorylation, of a target protein can result in immune recruitment to the target, for example, by triggering presentation of a neoepitope and attacking cells presenting that epitope by T cells. In one application, the small molecules disclosed herein are utilized in human leukocyte antigen (HLA) presentation and immune responses. Eliciting an immune response through neophosphorylation can find use in cancer immunotherapy approaches. In an exemplary approach, kinases are selected for phosphorylation of p53, for example, at Ser33, Ser315, and / or Thr82. This phosphorylation leads to subsequent binding and conformational changes, which result in activation as a transcription factor. See, e.g., Ryan and Vousden, Nature, 419 (2002).Thus, by designing molecules containing binding moieties for phosphorylating or dephosphorylating kinases along with a target for p53, it may be possible to control nucleotide binding based on the desired conformation of the transcription factor. See, 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.

[0553] Quinasopathy Treatment of kinasopathies is also contemplated; for a review, see Lahiry et al., Nature Reviews Genetics, 2011 (Table 1 discloses hereditary kinasopathies), incorporated herein by reference. Thus, for kinasopathies with loss of function, the chimeric small molecules of the present invention can recruit effective kinases to provide the lost function. See, e.g., Lahiry et al., Nature Reviews Genetics volume 11, pages 60-74 (2010), incorporated herein by reference (discussing various germline disorders and cancers associated with kinase dysfunction), in particular Supplementary Table 1 for hereditary kinasopathies and Supplementary Table 2 for kinases associated with cancer. In an exemplary embodiment, Src-family protein tyrosine kinases (SFKs) are designed such that in their active conformation, they contain a conserved Y-terminal residue in an active A-loop conformation. ASFK is stabilized by phosphorylation. By modifying SFK, for example, targeting phosphorylation in the A-loop, SFK-related kinasopathies, such as ALL and CML, can be addressed by treating abnormal SFK. See, for example, Mechanism of Drug-Resistance in Kinases, Expert Opin Investig Drugs. 2011 Feb; 20(2): 153-208; doi: 10.1517 / 13543784.2011.546344.

[0554] Regulated Induced Proximity Targeting Chimera (RIPTAC) In an exemplary embodiment, the functional moieties of RIPTAC are used as the target-binding and kinase-binding moieties of the chimeric small molecule described herein. RIPTAC is a bifunctional molecule composed of a moiety that targets a protein of interest and a moiety that can bind to a ubiquitously expressed protein essential for cell survival. For example, RIPTAC may contain binding moieties directed against two disease-specific proteins (e.g., cancer-specific proteins). After binding, these two disease-specific proteins are anchored by RIPTAC and are in close proximity to each other, forcing them to interfere with each other's functional groups. See, for example, Raina, K. et al. Regulated Induced Proximity Targeting Chimeras (RIPTACs): A Novel Heterobifunctional Small Molecule Therapeutic Strategy for Killing Cancer Cells Selectively, 2023; Liu, J.O. Targeting Cancer with Molecular Glues. Science, 2023, 381, 729-730; Schulze, C.J. et al. Chemical Remodeling of a Cellular Chaperone to Target the Active State of Mutant KRAS. Science, 2023, 381, 794-799 (each of which is hereby incorporated by reference). Examples of chimeric small molecules that link these two parts of RIPTAC can be seen in Figure 2 of Raina et al. Figure 2 highlights the linker that links various RIPTACs. One of skill in the art would be able to substitute the linkers described in Raina et al. with the linkers and electrophilic reactive groups described herein using methods known in the art to achieve chimeric small molecules. Raina et al. demonstrate that the degree of selectivity can be tuned, advantageously by varying various linker compositions and lengths.

[0555] Thus, a RIPTAC could be modified to include a binding moiety, linker, and / or electrophilic reactive group as described herein without disrupting its functionality. In exemplary embodiments, a chimeric small molecule described herein can include a kinase-binding moiety directed to an overexpressed kinase and a target-binding moiety directed to a dysfunctional protein. In certain exemplary embodiments, a chimeric small molecule described herein is used as a RIPTAC.

[0556] Methods of Use with Kinase Inhibitor Binding Moieties In one exemplary embodiment, the method includes generating a reprogrammed cellular kinase by delivering a chimeric small molecule of the formula A-L1-EB or A-L1-E-L2-B, where A is a protein binding moiety specific for the cellular protein to be repurposed / reprogrammed; B is a target binding moiety specific for the target substrate to be modified; L1 and L2 are linkers; and E is an electrophilic reactive group through which the chimeric small molecule labels the cellular protein with the target binding moiety for the target substrate; and modifying the target substrate by binding the repurposed / reprogrammed protein to the target substrate via the target binding moiety, whereby the repurposed / reprogrammed cellular protein introduces one or more modifications to the target substrate. In one exemplary embodiment, the protein binding moiety has a half-life that is about 2, 3, 4, 5, 6, or 7 times shorter than the half-life of the protein to be repurposed / reprogrammed. In an exemplary embodiment, the protein to be reprogrammed is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In an exemplary embodiment, the inhibitor is a protein-binding moiety. In an exemplary embodiment, the protein to be repurposed / reprogrammed is a kinase, and the protein-binding moiety is a kinase inhibitor. In an exemplary embodiment, the kinase inhibitor is a "broad-spectrum" kinase inhibitor. In an exemplary embodiment, the method comprises administering a coupling molecule, thereby quenching the inhibitory activity of the protein inhibitor. In an exemplary 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, isocyanopropanoate, tetrazole, 2-acylboronic acid, or any derivative thereof. In an exemplary embodiment, the cyclooctyne derivative comprises a dibenzocyclooctyne, a biarylazacyclooctynone, or a dimethoxyazacyclooctyne. In an exemplary embodiment, the method comprises a strained alkyne comprising a bicyclononyne or a dioxabiaryldecyne.

[0557] In an exemplary embodiment, the method includes a chimeric small molecule that is a protein-binding moiety with a half-life that is about 2, 3, 4, 5, or 6 times shorter than the half-life of the protein to be reprogrammed. In an exemplary embodiment, the protein to be reprogrammed is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In an exemplary embodiment, the kinase-binding moiety is an inhibitor. In an exemplary embodiment, the protein to be reprogrammed is a kinase, and the kinase-binding moiety is a kinase inhibitor. For an exemplary kinase, the inhibitor is a broad-spectrum kinase inhibitor.

[0558] In an exemplary embodiment, the method includes a chimeric small molecule in which the kinase-binding moiety includes a bioorthogonal group capable of reacting with and binding to a coupling molecule. In an exemplary embodiment, the method further includes administering the coupling molecule. When administered, the coupling small molecule reacts with the bioorthogonal group on the chimeric small molecule, thereby quenching the kinase inhibitor from binding to the kinase. In an exemplary 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, isocyanopropanoate, tetrazole, 2-acylboronic acid, or any derivative thereof. In an exemplary embodiment, the cyclooctyne derivative includes dibenzocyclooctyne, biarylazacyclooctynone, or dimethoxyazacyclooctyne. In an exemplary embodiment, the strained alkyne includes bicyclononyne or dioxabiaryldecyne.

[0559] In one exemplary embodiment, the coupling molecule is co-administered with the chimeric small molecule. In an exemplary embodiment, the coupling molecule is administered after administration of the chimeric small molecule. In an exemplary embodiment, 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 chimeric small 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 protein targeting moiety, as described above. This reaction inhibits the binding moiety from binding to the protein. When utilized with a chimeric small molecule containing an electrophilic reactive group, the protein-binding moiety can be released from the chimeric small molecule and the coupling molecule can bind to the bioorthogonal group on the kinase-binding moiety, thereby rendering the kinase-binding moiety no longer able to bind to the kinase. In one exemplary embodiment, the coupling molecule is utilized with a kinase-binding moiety that is an inhibitor of the protein.

[0561] The coupling molecule can be administered in any pharmaceutical formulation, effective amount, and dosage form previously described. The coupling molecule can be delivered using any method previously described, or administered with any co-therapy or combination as described herein. The chimeric small molecule and the coupling molecule can be delivered or administered simultaneously or sequentially. Co-delivery of the coupling molecule and the chimeric small molecule can occur within the same delivery method or by separate delivery methods. The simultaneous but separate delivery of the coupling small molecules can be by the same or different types of delivery methods previously described. Sequential delivery of the coupling molecules can occur by the same or different types of delivery methods. The sequential delivery of the coupling molecules can occur 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 a preferred embodiment, a coupling molecule is introduced into a system containing the chimeric small molecule. As the coupling molecule contacts the chimeric small molecule bound to the target protein, it quenches the binding between the protein-binding moiety and the target protein. In an exemplary embodiment, the coupling molecule is a molecule capable of reacting with a bioorthogonal molecule that is a substituent of the protein-binding moiety. This reaction results in the coupling molecule being attached to the protein-binding moiety, such that the protein-binding moiety no longer binds to the protein. In an exemplary embodiment, the coupling molecule can react with the bioorthogonal moiety through an aldehyde / ketone nucleophile reaction, a dipolar cycloaddition, a phosphine ligation, a Diels-Alder cycloaddition, a [4+1] cycloaddition, a nitrile imine-alkene reaction, or a 2-acylboronic acid condensation, or any other bioorthogonal reaction.

[0563] In one exemplary embodiment, the coupling molecule and the bioorthogonal moiety are coupled via aldehyde / ketone nucleophile condensation. Classically, aldehydes are coupled with amine groups, such as alkoxyamines or hydrazines. Although intracellular metabolites also contain aldehydes and ketones, this approach is effective on the cell surface. In a preferred embodiment, the coupling molecule is an aldehyde.

[0564] In one exemplary embodiment, the coupling molecule and the bioorthogonal moiety are coupled via a dipolar cycloaddition. 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, thereby promoting the reaction. In most cases, the strained alkyne is a cyclooctyne or any derivative thereof. Non-limiting examples of cyclooctynes ​​include dibenzocyclooctynes, biarylazacyclooctynones, and dimethoxyazacyclooctynes. In one exemplary embodiment, the coupling molecule is an alkyne. In an exemplary embodiment, the coupling molecule is a strained alkyne. In a preferred 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 derivative thereof.

[0565] Dipolar cycloadditions can also involve the reaction between oxanorbornadiene and azide. In this case, the cycloaddition between the oxanorbornadiene and azide is followed by a spontaneous retro-Diels-Alder reaction to produce a triazole and a furan. In one exemplary embodiment, the coupling molecule is oxanorbornadiene or any of its derivatives.

[0566] Dipolar cycloaddition can also involve the reaction between norbornene and a nitrile oxide. In one exemplary embodiment, the coupling molecule is norbornene. The coupling molecule can also undergo dipolar cycloaddition with another dipolar molecule, such as a nitrone, (imino)sindone, or 1,3-dithiolium-4-olate, and can include the corresponding unsaturated hydrocarbon.

[0567] In an exemplary embodiment, the coupling molecule and the bioorthogonal moiety are coupled via phosphine ligation, or alternatively referred to as Staudinger ligation. Phosphine ligation typically occurs between an azide and a phosphine, typically forming a phosphine oxide and a stable amide bond, or when an electron-deficient aromatic azide is used, forming an iminophosphorane. In an exemplary embodiment, the coupling molecule is a phosphine or any derivative thereof. Phosphine ligation may also involve a cyclopropene instead of an azide. Non-limiting examples of cyclopropanes include cyclopropenone, cyclopropenethione, and cyclopropenium ion.

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

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

[0570] In one exemplary embodiment, the coupling molecule and the bioorthogonal moiety are coupled via nitrile imine-alkene cycloaddition. Classically, tetrazole is photolyzed to generate nitrile imines, which readily couple with unsaturated hydrocarbons. The wavelength required for photolysis depends on the substituents on the tetrazine. However, photolysis is not required in the presence of hydrazonoyl chloride, which spontaneously generates nitrile imines from tetrazole at neutral pH. In a preferred embodiment, the coupling molecule is an unsaturated hydrocarbon, optionally introduced with hydrazonoyl chloride. In one exemplary embodiment, the coupling molecule and the bioorthogonal moiety are coupled via 2-acylboronic acid condensation. In this reaction, a boronic acid couples with an amine to form a stable diazaborine. In a preferred 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 (incorporated herein by reference).

[0571] Carcinogenicity application In one exemplary embodiment, the disease is associated with cancer. Specifically, the disease is oncogenic. Many oncogenic targets are known and can be regulated by post-translational modifications. See, e.g., Chen, L., Liu, S. & Tao, Y. Regulating tumor suppressor genes: post-translational modifications. Sig Transduct Target Ther 5, 90 (2020); doi:10.1038 / s41392-020-0196-9. For exemplary types of post-translational modifications of proteins implicated in oncogenesis and their expression patterns, see Table 1 in Sharma, et al., (2019). Post-Translational Modifications (PTMs), from a Cancer Perspective: An Overview. Oncogen 2(3):12 (specifically incorporated herein by reference).

[0572] The chimeric small molecules disclosed herein can be used in methods for treating cancer. The method for treating cancer can include generating repurposed / reprogrammed cellular proteins by administering a chimeric small molecule as described herein. The chimeric small molecule labels a cellular protein with an oncogenic target kinase binding moiety via an electrophilic reactive group moiety. The electrophilic reactive group reacts with and binds to a nucleophilic side chain on the cellular protein. Labeling the cellular protein can enable binding or association with the oncogenic target protein or a molecule in proximity to the oncogenic protein, thereby facilitating modification of the target substrate. In one aspect, the method includes inducing phosphorylation of an oncogenic target protein in or on a cell. The method can include contacting the oncogenic target protein with the chimeric small molecule. In an exemplary embodiment, the oncogenic target protein is in proximity to a kinase specific for the protein-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 donating molecules may increase phosphorylation.

[0573] Methods of treating cancer are provided. The method of treating cancer includes administering to a subject in need thereof a chimeric small molecule of the formula: A-L1-EB or A-L1-E-L2-B, where A is a protein-binding moiety; E is an electrophilic reactive group; and B is an oncogenic target protein to be modified, thereby generating a reprogrammed cellular protein, whereby the chimeric small molecule labels the cellular protein with a target binding moiety for a target substrate; and modifying the oncogenic target protein by binding the repurposed / reprogrammed protein to the target substrate via the target binding moiety, whereby the repurposed / reprogrammed cellular protein introduces one or more modifications to the target substrate. In one exemplary embodiment, the target binding moiety is selected from KRAS, RAS, FKPB, and the like. 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 exemplary embodiment, the cellular protein to be reprogrammed is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or translocase. In one exemplary embodiment, the kinase-binding moiety is a kinase inhibitor. In one exemplary 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 exemplary embodiment, administering a coupling molecule, thereby quenching the inhibitory activity of the kinase inhibitor.

[0574] Methods for treating diseases associated with abnormal KRAS signaling are provided, comprising administering a composition comprising a chimeric small molecule comprising a KRAS-binding molecule and a kinase-binding molecule as described herein. In one exemplary embodiment, the kinase-binding molecule is a target of a kinase 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 certain embodiments, the kinase-binding molecule is an AMPK-binding moiety. In one exemplary embodiment, KRAS is KRAS G12C In one exemplary embodiment, the chimeric small 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.

[0575] In an exemplary embodiment, a method of treating cancer in a cell is provided, comprising administering a chimeric small molecule of the invention. In one example, the small molecule comprises a PI3K kinase-binding agent, a linker, an electrophilic reactive group, and a p53 target-binding moiety, e.g., based on idasanutlin. In one exemplary embodiment, the molecule comprises a PI3K-binding agent based on the inhibitor PIK108, optionally further comprising a bioorthogonal group, e.g., cyclopropenyl. The binding moiety PIK108 comprises a linker connected to an electrophilic reactive group, e.g., dibromophenylbenzoate. The electrophilic reactive group is then connected to a p53 protein target-binding moiety, optionally via a linker. Upon binding to PI3K kinase via PIK108, a nearby lysine in the binding pocket of PI3K reacts with the lysine reactive group (e.g., dibromophenylbenzoate), displacing the kinase inhibitor and leaving PI3K tagged with the p53-binding agent. Kinases covalently labeled with target-binding moieties can then hyperphosphorylate and / or neophosphorylate p53. Administration of the tetrazine-coupled molecule, when presented on the PI3K-binding molecule, can quench the cyclopropenyl bioorthogonal group and inactivate the displaced kinase-binding moiety.

[0576] Exemplary oncogenic fusion proteins that can be processed by the binding of multimeric kinases include fusions involving the ABL protein. The ABL protein is a non-receptor tyrosine kinase that is normally under well-regulated regulation. However, chromosomal translocations that link the ABL gene with genes encoding other proteins result in various oncogenic fusion proteins (BCR-ABL, TEL-ABL, NUP214-ABL, etc.), which are prone to dimerization (or oligomerization) and subsequent autophosphorylation. As a result, the ABL kinase becomes constitutively active, leading to diseases such as chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and other myeloproliferative disorders. An exemplary oncogenic fusion is BCR-ABL. This may be particularly true for kinases such as Abl, which must form a complex to become active.

[0577] In an exemplary embodiment, the cancer is characterized by an oncofusion of a kinase, e.g., ABL-kinase. Oncogenic ABL fusion proteins are known in the art and have been implicated in various proliferative disorders. Chromosomal translocations result in the splicing of the ABL gene with genes encoding other proteins, resulting in various proteins prone to dimerization (or oligomerization) and autophosphorylation, making ABL kinase constitutively active and leading to myeloproliferative disorders. In an exemplary embodiment, the oncofusion is TEL-ABL or NUP214-ABL.

[0578] 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 designed as molecular adhesives for fusion targeting using the small molecule design considerations described herein. Specifically, druggable kinases associated with fusions include, and have been identified as, 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.

[0579] Additional fusions that can be targeted with 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, ALK- SCEL, ALK--STK39, AP3B1-BRAF, ARHGEF25--NTRK1, ARID2--TMPRSS2, ATAD2--ERBB4, ATF7IP--TMPRSS2, ATG7-BRAF, ATP1A1--NOTCH2, ATP1B1--NRG1 , ATP2B4--ERBB4, B4GALT1--RAF1, BACE2--TMPRSS2, BAIAP2L1-MET, BCL2L11-BRAF, BCR--ABL1, BRAF--AP3B1, BRAF--ATG7, BRAF--CUL1, BRAF--DENND 2A, 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、N OTCH2--CWH43、NOTCH2-MNDA、NOTCH2--PSMA5、NOTCH2--REG4、NOTCH2--SEC22B、NOTCH2--SPAG17、NRG1--PMEPA1、NRG1--STMN2、NTRK1--DYNC2H1、NTR K3--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、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--TMPRSS, USP46-PDGFRA, VCL--FGFR2, VPS18--NTRK3, WRN--NRG1, ZBTB7B--NTRK1, ZC3HAV1-BRAF, ZEB2--AKT3 and ZNF430--BRD4 are included.,

[0580] It should be noted that there seems to be a small error in the original text where "USP28--TMPRSS2" is likely meant to be "USP28--TMPRSS2" (the second "2" might be a typo). The translation has been done as accurately as possible based on the provided text.Exemplary targetable fusions include ALK fusions, such as TFG-ALK. ALK fusions have been identified in several cancer types, including lung adenocarcinoma, bladder cancer, colorectal cancer, breast cancer, renal cell carcinoma, renal medullary carcinoma, and thyroid cancer. Specifically, EML4-ALK fusions have been found in lung adenocarcinoma, STRN-ALK fusions in thyroid cancer and papillary renal carcinoma, TPM1-ALK fusions in bladder cancer, SMEK2-ALK fusions in rectal adenocarcinoma, and GTF2IRD1-ALK fusions in thyroid cancer. Another targetable fusion is BRAF fusion, which is associated with prostate cancer, melanoma, radiation-induced thyroid cancer, and pediatric low-grade glioma. Specifically, TRIM-BRAF fusions have been found in rectal adenocarcinoma, ATG7-BRAF in melanoma, and ZC3HAV1-BRAF and FAM114A2-BRAF in thyroid cancer. Other exemplary fusions include AGK-BRAF, SND1-BRAF, MACF1-BRAF, TAX1BP1-BRAF, and CDC27-BRAF. It is known in the art that BRAF dimers are insensitive to RAF inhibitors and instead are subject to downstream inhibitory processes, for example, through MEK inhibition.

[0581] Other targetable fusions include FGFR fusions, which have been identified in glioblastoma multiforme, bladder urothelial carcinoma, lung squamous cell carcinoma, papillary renal cell carcinoma, brain low-grade glioma, prostate adenocarcinoma, head and neck squamous cell carcinoma, invasive breast cancer, and gastric adenocarcinoma tumor types. Specifically, FGFR3-TACC3 fusions have been found in papillary renal carcinoma, FGFR3-ELAVL3 in low-grade glioma, and FGFR3-BAIAP2L1 in bladder cancer. Another targetable fusion is the WASF2-FGR fusion, which has been found in lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, and skin melanoma. Another targetable fusion is the MET fusion, which has been found in low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, and thyroid cancer. Specifically, 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.

[0582] Other targetable fusions include NTRK fusions, which have been associated with congenital fibrosarcomas, human secretory breast cancer, and papillary thyroid cancer, including glioblastoma, cholangiocarcinoma, and pediatric high-grade gliomas. Specifically, PAN3-NTRK2 has been found in head and neck squamous cell carcinoma, AFAP1-NTRK2 in low-grade glioma, TRIM24-NTRK2 in lung adenocarcinoma, and TPM3-NTRK1 in sarcoma and thyroid cancer. Other targetable fusions include PIK3CA fusions, which have been found in endometrial cancer, invasive breast cancer, colorectal cancer, head and neck cancer. Specifically, TBL1XR1-PIK3CA fusions have been found in breast cancer and prostate adenocarcinoma, FNDC3B-PIK3CA fusions in uterine endometrial cancer, and TBL1XR1-PIK3CA fusions in invasive breast cancer and prostate cancer. Another targetable fusion is PKC fusion, which has been found in papillary glioneuronal tumors and benign fibrous histiocytomas. PRKCA fusion has been found in lung squamous cell carcinoma, and PRKCB fusion has been found in lung squamous cell carcinoma, lung adenocarcinoma, and low-grade glioma. Exemplary fusions include PRKCA, which has been fused with IGF2BP3. TANC2-PRKCA.

[0583] Another targetable fusion includes PKN1 fusions, which have been found in lung squamous cell carcinoma and hepatocellular carcinoma. Exemplary PKN1 fusions include ANXA4-PKN1 and TECR-PKN1. Another targetable fusion includes RAF1 fusions, also known as CRAF fusions, which have been found in melanoma and prostate adenocarcinoma. Specifically, AGGF1-RAF1 has been found in papillary thyroid carcinoma and prostate cancer. Another targetable fusion includes RET fusions, which have been found in lung adenocarcinoma and thyroid cancer. Specifically, CCDC6-RET fusions have been found in thyroid carcinoma and colon adenocarcinoma, while ERC1-RET fusions have been found in breast cancer. Other exemplary fusions include RET and AKAP13, FKBP15, SPECC1L, and TBL1XR1. Another targetable fusion is a ROS1 fusion, such as CEP85L-ROS1, which has been found in glioblastomas and single hemangiosarcomas. Another notable ROS1 fusion is CD74-ROS1, while other fusions have been found in 8 / 513 lung adenocarcinomas.

[0584] Tyrosine kinase fusion genes are a notable class of oncogenes. Tyrosine kinase fusions have been found in leukemias and solid tumors. Like other fusions, they are created by translocations and other chromosomal rearrangements of certain tyrosine kinase genes. These fusions include ABL, PDGFRA, PDGFRB, FGFR1, SYK, RET, JAK2, and ALK. The kinase domain is activated by forced oligomerization and inactivation of the inhibitory domain. Activated tyrosine kinase fusions then signal through a series of transduction cascades. The fusion partner recruits proteins, which contribute to signal transduction, protein stability, cellular localization, and oligomerization.

[0585] See, for example, Stransky, N., Cerami, E., Schalm, S. et al. The landscape of kinase fusions in cancer. Nat Commun 5, 4846 (2014). doi:10.1038 / ncomms5846 (including, in detail, Figure 1, which provides a landscape of recurrent kinase fusions in solid tumors, incorporated by reference); Medves et al., J Cell Mol Med. 2012 Feb;16(2):237-48; doi:10.1111 / j.1582-4934.2011.01415.x (including, in detail, TK fusions and their inhibitor molecules in Table 1, incorporated by reference); and Gao, Qingsong et al. "Driver Fusions and Their Implications in the Development and Treatment of Human Cancers." Cell Reports vol.23,1(2018):227-238.e3.doi:10.1016 / j.celrep.2018.03.050, each of which is incorporated herein by reference in its entirety.

[0586] Gao et al. provide a table of potentially druggable fusion events and their targets in Table S5, which is specifically incorporated herein by reference for its teachings on fusions, targets, and indications associated with fusion events.

[0587] Exemplary cancers associated with such fusions include adrenocortical carcinoma, urothelial carcinoma of the bladder, brain low-grade glioma, invasive breast cancer, cervical squamous cell carcinoma and adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, kidney clear cell carcinoma, kidney papillary renal cell carcinoma, acute myeloid leukemia, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid neoplasms diffuse large B-cell lymphoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, cutaneous melanoma of the skin, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid carcinoma, uterine carcinosarcoma, endometrial carcinoma of the uterine corpus, and uveal melanoma.

[0588] RAS isoforms share conserved amino acid sequences in the switch I and switch II regions of Ras. The switch region of Ras is the binding interface between effector proteins and Ras regulators, such as GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Several cancer mutations are located within switch II and the P-loop associated with switch I. Thus, phosphorylation of loop residues in switch I or switch II can mediate Ras activity, as post-translational modifications of loop residues are known to generally result in conformational changes.

[0589] KRAS is a key regulator of cell proliferation, differentiation, and survival and is the most frequently mutated oncogene in human cancers. Exemplary oncogenic driver mutations include KRAS G12C The active, GTP-bound state of KRAS is in a closed conformation, while the inactive, GDP-bound state is in an open conformation. KRAS G12C and other oncogenic RAS mutations result in dysregulated excess cellular GTP-bound RAS, and RAS function remains in an activated, open conformation, resulting in uncontrolled cell growth and proliferation, invasiveness, and evasion of immune surveillance. Thus, inhibition of GTPases (e.g., Ras) is within the scope of the chimeric small molecules disclosed herein.

[0590] Without being bound by any particular scientific theory, KRAS, especially KRAS G12C It has been proposed that phosphorylation of Mg may promote a conformational change, possibly by disrupting binding to GTPase-activating proteins, which may reduce Ras activity, which has been implicated in carcinogenesis. 2+ Phosphorylation of residues T35 or S17, which coordinate the ion and also the γ- and β-phosphates of GTP, could potentially disrupt four-way Mg2+ chelation. This tetrachelated Mg2+ state is characteristic of the active GTP-bound state and is in a "closed conformation," whereas in the inactive GDP-bound state, only S17 and the γ-phosphate of GDP are Mg2+ bound. 2+ Furthermore, without being bound by theory, it is possible that phosphorylation of any switch I or switch II or switch-adjacent residue can disrupt protein-protein interactions between the switch region and Ras regulators and activators, or that phosphorylation of loop residues in switch I or switch II can cause conformational changes, as it is known that post-translational modifications of loop residues often result in conformational changes. Thus, kinase modulation of KRAS signaling using the phosphorylation-inducing chimeric small molecules described herein may be useful as anti-cancer therapy by disrupting KRAS membrane localization or binding partners.

[0591] In one aspect, the method includes treating cancer as a result of KRAS. In one exemplary embodiment, the chimeric small molecule target binding moiety targets KRAS, 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.

[0592] In one exemplary embodiment, the chimeric small molecule target binding moiety targets MYC, K-RAS, N-RAS, TP53, KDM6A, NPM1, H-RAS, FGFR3, MSH6, TP53, EGFR, PIK3CA, ABLI, CTNNB1, KIT, INFlA, JAK2, BRAF, IDHI, RET, PDGFRA, MET, APC, CDC27, CDK4, prostate-specific antigen, alpha-fetoprotein, breast mucin, gp100, g250, p53, MART-I, MAGE, BAGE, GAGE, tyrosinase, tyrosinase-related protein 11, tyrosinase-related protein, or RAD50.

[0593] Additional cancer targets, disease indications, and small molecule target binding moieties are provided in Table 2 of Sharma BS (2019). Post-Translational Modifications (PTMs), from a Cancer Perspective: An Overview. Oncogen 2(3):12, specifically incorporated herein by reference.

[0594] Disease / Disorder In some embodiments, the disease is associated with aberrant protein expression or expression of a tumor antigen, such as, for example, a proliferative disorder, a precancerous condition, a cancer, or a non-cancer related indication associated with expression of a tumor antigen, which in some embodiments is B2M, CD247, CD3D, CD3E, CD3G, TRAC, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, DCK, CD52, FKBP1A, CIITA, NLRC5, RFXANK, RFX5, RFXAP, or NR3C1, HAVCR2, LAG3, PDCD1, PD-L2, CTLA4, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD113), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF-beta or PTPN11 DCK, CD52, NR3C1, LILRB1, CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21);Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); n-kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); elongation factor 2 mutated (ELF2M); Furin B2; fibroblast activation protein α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAIX); proteasome (prosome, macropein) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe ); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X-chromosome open CXORF61; CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globo-H glycoceramide (globo-H); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2);TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen 1 recognized by T cells (Melan-A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma a inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC (SEQ ID NO: 46) binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6);Human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); and targets selected from immunoglobulin lambda-like polypeptide 1 (IGLL1), CD19, BCMA, CD70, G6PC, dystrophin including modifications of exon 51 by deletion or truncation, DMPK, CFTR (cystic fibrosis transmembrane conductance regulator). In one exemplary embodiment, the targets include knock-in of CD70 or CD33 and knock-out of B2M. In one exemplary embodiment, the targets include knock-out of TRAC and B2M or TRAC, B2M, and PD1, with or without additional target genes. In one exemplary embodiment, the disease is cystic fibrosis, where the SCNN1A gene is targeted. In one exemplary embodiment, chimeric small molecule-mediated modification is used in multiple sclerosis, e.g., αB-crystallin, or SLE with multiple targets (see, e.g., Doyle and Mamula, Curr Opin Immunol. 2012). Treatment of intracellular pathogens.

[0595] Additional Diseases and Disorders In one exemplary embodiment, the treatment is treatment of a disease / disorder of a single organ, including liver disease, eye disease, muscle disease, heart disease, blood disease, brain disease, kidney disease, or may include treatment for autoimmune diseases, central nervous system diseases, cancer and other proliferative diseases, neurodegenerative disorders, inflammatory diseases, metabolic disorders, musculoskeletal disorders, etc.

[0596] Specific diseases / disorders include achondroplasia, color blindness, acid maltase deficiency, adrenoleukodystrophy, Aicardi syndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgen insensitivity syndrome, Apert syndrome, arrhythmogenic right ventricle, dysplasia, ataxia-telangiectasia, Barth syndrome, beta-thalassemia, blue rubber bleb nevus syndrome, Canavan disease, chronic granulomatous disease (CGD), cri-catch-all syndrome, cystic fibrosis, Dercum's disease, ectodermal dysplasia, Fanconi anemia, fibrodysplasia ossificans progressiva, fragile X syndrome, galactosemia, Gaucher disease, systemic gangliosidoses (e.g., GM1), hemochromatosis, hemoglobin C mutation at the sixth codon of beta-globin (HbC), hemophilia, Huntington's disease, Hurler syndrome, hypophosphatasia, Klinefelter syndrome, Krabbe disease, and Langer-Giedy syndrome. These include On syndrome, leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidoses (MPS), nail-patella syndrome, nephrogenic diabetes insipidus, neurofibromatosis, Niemann-Pick disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Schwabmann syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease, thrombocytopenic radial deficiency (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberous sclerosis complex, Turner syndrome, urea cycle disorders, von Hippel-Lindau disease, Waardenburg syndrome, Williams syndrome, Wilson disease, and Wiskott-Aldrich syndrome.

[0597] In an exemplary embodiment, the disease is associated with expression of a tumor antigen, such as, for example, a proliferative disease, a precancerous condition, a cancer, or a non-cancer related indication associated with expression of a tumor antigen, which in some embodiments is selected from the group consisting of B2M, CD247, CD3D, CD3E, CD3G, TRAC, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, DCK, CD52, FKBP1A, CIITA, NLRC5, RFXANK, RFX5, RFXAP, or NR3C1, HAVCR 2, LAG3, PDCD1, PD-L2, CTLA4, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD113), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF-beta or PTPN11 DCK, CD52, NR3C1, LILRB1, CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2);Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); n-kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FA P); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropein) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3) bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high-molecular-weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (AL...

Claims

1. formula A-L １ -E-B or A-L １ -E-L ２ -B wherein A is a kinase-binding moiety; B is a target binding moiety; L １ and L ２ are each a linker; and E is an electrophilic reactive group. Chimeric small molecules according to the present invention.

2. 2. The chimeric small molecule of claim 1, wherein the electrophilic reactive group is selected from N-acyl-N-alkylsulfonamide (NASA), dibromophenylbenzoate, or N-sulfonylpyridone.

3. The chimeric small molecule of claim 1 or 2, wherein the electrophilic reactive group reacts with a nucleophilic reactive group.

4. The electrophilic reactive group has the formula: 【Chemical 1】 (Wherein, R1 is C—O, SO ２ , Me-C-O or Me-SO ２ and 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, or one or more condensed rings thereof; -OCF ２ and the benzene ring is optionally substituted at any position. and the electrophilic reactive group is attached to the chimeric small molecule via a terminal carbon group; a carbon on the cyclic moiety and / or a nitrogen on the cyclic moiety.

5. The electrophilic reactive group is 【Chemistry 2】 【change】 and wherein the electrophilic reactive group is attached to the chimeric small molecule via a terminal carbon group; a carbon on the cyclic moiety and / or a nitrogen on the cyclic moiety.

6. The kinase binding moiety may be selected from the group consisting of FKBP, PKC, AMPK, ABL, PK, MAPK, e.g., MAPK1, MAPK11, MAPK12, MAPK13, MAPK14, p38α MAPK, EGFR, FGFR, NGFR, TrkA, ABL, CDK, e.g., CDK2, CDK4, CDK8, 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., 2. The chimeric small molecule of claim 1, which is an IRE-binding moiety, such as PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, such as PKC-ζ, NOP, the GPC family, such as the μ or δ opioid receptor, UMPK, SphK, GSK-3, IRTK, PDGFR, IDH, ITK, TAK, BMX, LIMK, or an IRE-binding moiety.

7. The chimeric small molecule of any one of claims 1 to 6, wherein the kinase binding moiety has a half-life that is shorter than the half-life of the kinase.

8. The chimeric small molecule of claim 7 , wherein the half-life of the kinase binding moiety is at least 2, 3, 4, or 5 times less than the half-life of the kinase.

9. The chimeric small molecule of any one of claims 1 to 8, wherein the kinase binding moiety is a kinase inhibitor or a kinase activator.

10. The chimeric small molecule of any one of claims 1 to 9, wherein the kinase inhibitor is a broad spectrum kinase inhibitor.

11. The electrophilic reactive group is 【Chemistry 3】 and the electrophilic reactive group is attached to the chimeric small molecule via a terminal carbon group; a carbon on the cyclic moiety and / or a nitrogen on the cyclic moiety.

12. 12. The chimeric small molecule of any one of claims 1 to 11, wherein 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; PEG, or any combination thereof.

13. 13. The chimeric small molecule of any one of claims 1 to 12, wherein L1 and L2 are the same or different molecules 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.

14. The chimeric small molecule of any one of claims 1 to 13, wherein the kinase binding moiety further comprises a bioorthogonal group.

15. The chimeric small molecule of any one of claims 1 to 14, wherein the bioorthogonal group is selected from tetrazine, triazine, cyclooctene, cyclopropene, and diazo.

16. The bioorthogonal group is 【Chemistry 4】 The chimeric small molecule of any one of claims 1 to 15, selected from the group consisting of:

17. The chimeric small molecule of any one of claims 1 to 16, wherein the target is a protein.

18. 18. The chimeric small molecule of claim 17, wherein the target protein is from a pathogen.

19. 19. The chimeric small molecule of claim 18, wherein the pathogen is a virus, bacterium, fungus, or protozoan.

20. 19. The chimeric small molecule of claim 18, wherein the microbial protein is an intracellular or extracellular pathogen protein.

21. 21. The chimeric small molecule of claim 20, wherein the intracellular pathogen is Mycobacterium tuberculosis or the extracellular pathogen is Pseudomonas aeruginosa.

22. 21. The chimeric small molecule of claim 20, wherein the kinase binding moiety is a phosphatase A (PtpA) binding moiety, a PtpB binding moiety.

23. 23. The chimeric small molecule of any one of claims 1 to 22, which is capable of covalently labeling a kinase with said kinase binding moiety.

24. 23. The molecule of claim 22, wherein the label is a label of a nucleophile disposed on the kinase.

25. The chimeric small molecule of claim 1 , wherein the target has the ability to bind to an oncogenic target.

26. 10. The chimeric small molecule of claim 9, wherein the kinase inhibitor is sorafenib, SB2035890, or skepinone B, or an analog or derivative thereof.

27. 10. The chimeric small molecule of claim 9, wherein the kinase inhibitor is gefitinib or an analog or derivative thereof.

28. 10. The chimeric small molecule of claim 9, wherein the kinase inhibitor is imatinib or an analog or derivative thereof.

29. 10. The chimeric small molecule of claim 9, wherein the kinase inhibitor is idelalisib or an analog or derivative thereof.

30. 10. The chimeric small molecule of claim 9, wherein the kinase inhibitor is enasidenib, erdafitinib, ivosidenib, pemigatinib, pralsetinib, infigratinib, dacomitinib, capmatinib, mobocertinib, gilteritinib, or an analog or derivative thereof.

31. The kinase inhibitor is 【Chemistry 5】 or an analog or derivative thereof.

32. The kinase inhibitor is 【Chemistry 6】 or an analog or derivative thereof.

33. The kinase inhibitor is 【Chemistry 7】 or an analog or derivative thereof.

34. The kinase inhibitor is 【Chemistry 8】 or an analog or derivative thereof.

35. The kinase inhibitor is 【Chemistry 9】 or an analog or derivative thereof.

36. The kinase inhibitor is 【Chemistry 10】 or an analog or derivative thereof.

37. The kinase inhibitor is 【Chemistry 11】 or an analog or derivative thereof.

38. The kinase inhibitor is 【Chemistry 12】 or an analog or derivative thereof.

39. The kinase inhibitor is 【Chemistry 13】 or an analog or derivative thereof.

40. The kinase inhibitor is 【Chemistry 14】 or an analog or derivative thereof.

41. The kinase inhibitor is 【Chemistry 15】 or an analog or derivative thereof.

42. The kinase inhibitor is 【Chemistry 16】 or an analog or derivative thereof.

43. The kinase inhibitor is 【Chemistry 17】 or an analog or derivative thereof.

44. The kinase inhibitor is 【Chemistry 18】 or an analog or derivative thereof.

45. The kinase inhibitor is 【Chemistry 19】 or an analog or derivative thereof.

46. The kinase inhibitor is 【Chemistry 20】 or an analog or derivative thereof.

47. The targeted binding agent comprises: 【Chemical Formula 21】 or an analog or derivative thereof.

48. A-L １ -E is a group of the formula: 【Chemical 22】 The chimeric small molecule of claim 1 , wherein

49. A-L １ -E is a group of the formula: 【Chemical 23】 The chimeric small molecule of claim 1 , wherein

50. A-L １ -E is a group of the formula: 【Chemistry 24】 The chimeric small molecule of claim 1 , wherein

51. A-L １ -E is a group of the formula: 【Chemistry 25】 The chimeric small molecule of claim 1 , wherein

52. A-L １ -E is a group of the formula: 【Chemical 26】 The chimeric small molecule of claim 1 , wherein

53. A-L １ -E is a group of the formula: 【Chemical 27】 The chimeric small molecule of claim 1 , wherein

54. A-L １ -E is a group of the formula: 【Chemical Formula 28】 The chimeric small molecule of claim 1 , wherein

55. A-L １ -E is a group of the formula: 【Chemical 29】 The chimeric small molecule of claim 1 , wherein

56. A-L １ -E is a group of the formula: 【Chemistry 30】 The chimeric small molecule of claim 1 , wherein

57. A-L １ -E is a group of the formula: 【Chemical 31】 The chimeric small molecule of claim 1 , wherein

58. A-L １ -E is a group of the formula: 【Chemical 32】 The chimeric small molecule of claim 1 , wherein

59. A-L １ -E is a group of the formula: 【Chemical 33】 The chimeric small molecule of claim 1 , wherein

60. A-L １ -E is a group of the formula: 【Chemical 34】 The chimeric small molecule of claim 1 , wherein

61. A-L １ -E is a group of the formula: 【Chemical 35】 The chimeric small molecule of claim 1 , wherein

62. A-L １ -E is a group of the formula: 【Chemical 36】 The chimeric small molecule of claim 1 , wherein

63. A-L １ -E is a group of the formula 【Chemical 37】 or an analog or derivative thereof.

64. formula: 【Chemical Formula 38】 (Wherein m=0 or 1; n=1, 2, 3, 4 or 5; and X=CH ２ or (CH ２ ) ２ O) 2. The chimeric small molecule of claim 1, having the formula:

65. formula: 【Chemical Formula 39】 (Wherein X and Y are CH ２ or (CH ２ ) ２ and n and m are independently selected from 1, 2, 3, 4, 5, or 6.

2. The chimeric small molecule of claim 1, having the formula:

66. formula: 【Chemistry 40】 (Wherein JQ1 is a compound represented by the formula: 【Chemistry 41】 or its analogues or derivatives) 2. The chimeric small molecule of claim 1, having the formula:

67. formula: 【Chemistry 42】 (In the formula, R １ is the target binding moiety, and R ２ is the kinase binding moiety 2. The chimeric small molecule of claim 1, having the formula:

68. formula: 【Chemistry 43】 (In the formula, R １ is the target binding moiety, and R ２ is the kinase binding moiety 2. The chimeric small molecule of claim 1, having the formula:

69. formula: 【Chemical 44】 (In the formula, R １ is the target binding moiety, and R ２ is the kinase binding moiety 2. The chimeric small molecule of claim 1, having the formula:

70. formula: 【Chemistry 45】 (In the formula, R １ is the target binding moiety, and R ２ is the kinase binding moiety 2. The chimeric small molecule of claim 1, having the formula:

71. R １ but, 【Chemistry 46】 or an analogue or derivative thereof, R ２ but, 【Chemistry 47】 71. The chimeric small molecule of any one of claims 67-70, having the formula:

72. formula: 【Chemistry 48】 2. The chimeric small molecule of claim 1, having the formula:

73. formula: 【Chemistry 49】 2. The chimeric small molecule of claim 1, having the formula:

74. formula: 【Chemistry 50】 2. The chimeric small molecule of claim 1, having the formula:

75. formula: 【Chemistry 51】 2. The chimeric small molecule of claim 1, having the formula:

76. formula: 【Chemistry 52】 2. The chimeric small molecule of claim 1, having the formula:

77. formula: 【Chemistry 53】 2. The chimeric small molecule of claim 1, having the formula:

78. formula: 【Chemical 54】 2. The chimeric small molecule of claim 1, having the formula:

79. formula: 【Chemistry 55】 2. The chimeric small molecule of claim 1, having the formula:

80. formula: 【Chemical Formula 56】 2. The chimeric small molecule of claim 1, having the formula:

81. formula: 【Chemical 57】 2. The chimeric small molecule of claim 1, having the formula:

82. formula: 【Chemistry 58】 2. The chimeric small molecule of claim 1, having the formula:

83. formula: 【Chemical 59】 2. The chimeric small molecule of claim 1, having the formula:

84. formula: 【Chemistry 60】 2. The chimeric small molecule of claim 1, having the formula:

85. 85. A method of inducing modification of a target substrate, comprising administering to a cell or population of cells a chimeric small molecule of any one of claims 1-84.

86. A method for modifying a substrate, the method comprising introducing into a cell a molecule according to any one of claims 1 to 84.

87. 1. A method for modifying a target substrate in a cell, comprising: formula A - L - E - B or A - L １ - E - L ２ - B where A is a kinase-binding moiety specific for the cellular kinase to be repurposed / reprogrammed; B is a target binding moiety specific for the target substrate to be modified; L is a linker; and El is an electrophilic reactive group. wherein the chimeric small molecule tags the cellular kinase with the target binding moiety for the target substrate; and modifying the target substrate by binding the repurposed / reprogrammed kinase to the target substrate via the target binding moiety, whereby the repurposed / reprogrammed cellular kinase introduces one or more modifications to the target substrate; A method comprising:

88. The kinase binding moiety may be selected from the group consisting of FKBP, PKC, AMPK, ABL, PK, MAPK, e.g., MAPK1, MAPK11, MAPK12, MAPK13, MAPK14, p38α MAPK, EGFR, FGFR, NGFR, TrkA, ABL, CDK, e.g., CDK2, CDK4, CDK8, 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., 88. The method of claim 87, wherein the binding moiety is, for example, mTORC-1, PAK, PDK, such as PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, such as PKC-ζ, NOP, GPC family, such as μ opioid receptor or δ opioid receptor, UMPK, SphK or GSK-3 binding moiety.

89. 89. The chimeric small molecule of claim 87 or 88, wherein the kinase binding moiety has a half-life that is shorter than the half-life of the target to which the target binding moiety is capable of binding.

90. 88. The method of claim 87, further comprising administering a coupling molecule, thereby quenching the inhibitory activity of the kinase inhibitor.

91. 88. The method of claim 87, wherein the modification comprises inducing a post-translational modification of the target protein.

92. 92. The method of claim 91, wherein the post-translational modification is phosphorylation.

93. 1. A method of treating cancer comprising administering to a subject in need thereof a compound of the formula: A-L-E-B, A-L １ -E-L ２ -B or A-(L) ｎ -B where A is a kinase-binding moiety; L is a linker; and n is 0-6; E is an electrophilic reactive group; and B is an oncogenic protein to be modified. generating a reprogrammed cellular kinase by administering a chimeric small molecule of the present invention, wherein the chimeric small molecule tags the cellular kinase with a target binding moiety for the target substrate; modifying the oncogenic protein by binding the repurposed / reprogrammed kinase to the target substrate via the target binding moiety, whereby the repurposed / reprogrammed cellular kinase introduces one or more modifications to the target substrate; and A method comprising:

94. 94. The method of claim 93, wherein the kinase binding moiety is an inhibitor.

95. The kinase binding moiety may be selected from the group consisting of FKBP, PKC, AMPK, ABL, PK, MAPK, e.g., MAPK1, MAPK11, MAPK12, MAPK13, MAPK14, p38α MAPK, EGFR, FGFR, NGFR, TrkA, ABL, CDK, e.g., CDK2, CDK4, CDK8, 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., 94. The method of claim 93, wherein the binding moiety is, for example, mTORC-1, PAK, PDK, such as PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, such as PKC-ζ, NOP, GPC family, such as μ opioid receptor or δ opioid receptor, UMPK, SphK or GSK-3 binding moiety.

96. 96. The chimeric small molecule of any one of claims 93 to 95, wherein the kinase binding moiety has a half-life that is shorter than the half-life of the kinase.

97. 97. The method of any one of claims 93 to 96, further comprising administering a quencher molecule, thereby quenching the inhibitory activity of said kinase inhibitor.

98. 1. A method of treating an infection by a pathogen, comprising: To those who need it, use the formula: A - L - E - B or A - L １ - E - L ２ - B where A is the kinase-binding moiety; L is a linker; E is an electrophilic reactive group, and B is the pathogen protein to be modified. generating a reprogrammed cellular kinase by administering a chimeric small molecule of the present invention, wherein the chimeric small molecule tags the cellular kinase with a target binding moiety for the target substrate; modifying the pathogen protein by binding the repurposed / reprogrammed kinase to the pathogen protein via the target binding moiety, whereby the repurposed / reprogrammed cellular kinase introduces one or more modifications to the target substrate; and A method comprising:

99. The kinase binding moiety may be selected from the group consisting of FKBP, PKC, AMPK, ABL, PK, MAPK, e.g., MAPK1, MAPK11, MAPK12, MAPK13, MAPK14, p38α MAPK, EGFR, FGFR, NGFR, TrkA, ABL, CDK, e.g., CDK2, CDK4, CDK8, 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., 99. The method of claim 98, wherein the binding moiety is, for example, mTORC-1, PAK, PDK, such as PDK1 or PDK2, PTK2 / FAK, pyruvate kinase, RAC-α, RIPK, TYK2, SHP, aPKC, such as PKC-ζ, NOP, GPC family, such as μ opioid receptor or δ opioid receptor, UMPK, SphK or GSK-3 binding moiety.

100. 100. The method of claim 98 or 99, wherein the kinase binding moiety has a half-life that is shorter than the half-life of the target to which the target binding moiety is capable of binding.

101. 101. The method of any one of claims 98 to 100, wherein the kinase binding moiety is an inhibitor.

102. 99. The method of claim 98, further comprising administering a quenching molecule, thereby quenching the inhibitory activity of the kinase inhibitor.

103. 99. The method of claim 98, wherein the pathogen is a virus, bacterium, fungus, or protozoan.

104. 104. The method of claim 103, wherein the bacterium is Mycobacterium tuberculosis (Mtb) or Pseudomonas aeruginosa (PsA).

105. 105. The method of claim 104, wherein the pathogen is Mtb and the pathogen protein is one or more of PtpA, PtpB, SapM, ESAT-6, and Rv2966c.

106. 106. The method of claim 105, wherein the pathogen is (PsA) and the target binding moiety is colistin.

107. 107. The method of any one of claims 86 to 106, wherein the electrophilic reactive group reacts with a nucleophilic group of one of cysteine, serine, threonine, tyrosine, glutamic acid, aspartic acid, lysine, arginine, and histidine.

108. Formula L １ -El, formula EL-L １ or formula L １ -El-L ２ (In the formula, L １ and L ２ are independently selected from alkanes, alkenes, amines, ethers, thiols, sulfones, carbonyls, acyl, ketones, carboxylic acid esters, amides, enones, anhydrides, imides, and PEGs, and EL is an electrophilic reactive group.

109. El is a cleavage of the linker and attachment of L to a cysteine, lysine, methionine or tyrosine amino acid on the target polypeptide. １ or L ２ 109. The linker of claim 108, configured to facilitate attachment of all or a portion of

110. L １ or L ２ and further comprising an attachment moiety configured to covalently attach to the target polypeptide via El.

111. 111. The linker of claim 110, further comprising a binding moiety located opposite the attachment moiety and capable of binding to the target polypeptide.

112. The El is an L to cysteine １ or L ２ and E1 is configured to facilitate attachment of all or a portion of 【Hua 61】 112. The linker of any one of claims 108 to 111, selected from the group consisting of:

113. The El is an L to lysine １ or L ２ and E1 is configured to facilitate attachment of all or a portion of 【Hua 62】 (In the formula, R １ teeth, 【Chemistry 63】 selected from the group consisting of: R ２ teeth, 【Hua 64】 selected from the group consisting of The linker according to any one of claims 108 to 111,

114. The El is an L to lysine １ or L ２ and E1 is configured to facilitate attachment of all or a portion of 【Chemistry 65】 112. The linker of any one of claims 108 to 111, selected from the group consisting of:

115. The El is an L to methionine １ or L ２ and E1 is configured to facilitate attachment of all or a portion of 【Hua 66】 112. The linker of any one of claims 108 to 111, selected from the group consisting of:

116. 116. A heterobifunctional molecule comprising an electrophilically reactive linker according to any one of claims 108 to 115, a target polypeptide binding moiety attached to the linker at one end, and a modifying moiety attached to the linker at an opposite end.

117. 117. The heterobifunctional molecule of claim 116, wherein the target polypeptide portion binds to a kinase, phosphatase, ubiquitinase, deubiquitinase, acetyltransferase, deacetylase, methyltransferase, demethylase, or glycosyltransferase, and the modifying portion binds to a neo-substrate of the kinase, phosphatase, ubiquitinase, deubiquitinase, acetyltransferase, deacetylase, methyltransferase, demethylase, or glycosyltransferase.

118. 117. The heterobifunctional molecule of claim 116, wherein the modifying moiety is an immunogenic moiety and the target polypeptide binding moiety is a polypeptide to which the immunogenic moiety is attached.

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