Methods and reagents for analyzing protein-protein interfaces

Abstract

This disclosure provides methods and reagents useful for analyzing protein-protein interfaces, such as the interface between a presenter protein (e.g., a member of the FKBP family, a member of the cyclophyllin family, or PIN1) and a target protein. [Solution] In some embodiments, the target and / or presenter protein is an intracellular protein. In some embodiments, the target and / or presenter protein is a mammalian protein.

Description

[Background technology]

[0001] The vast majority of small molecule drugs act by binding to functionally important pockets on target proteins, thereby modulating the activity of those proteins. For example, statins, cholesterol-lowering drugs, bind to the enzymatic active site of HMG-CoA reductase, thereby preventing the enzyme from engaging its substrate. The fact that many such drug / target interaction pairs are known can lead some to the misconception that, with the right amount of time, effort, and resources, it is possible to discover small molecule modulators for almost all, if not all, proteins. This is far from the truth. Current estimates suggest that only about 10% of all human proteins are potentially targets for small molecules. The remaining 90% are currently considered difficult or cumbersome to develop small molecule drugs for. Such targets are commonly referred to as "undruggable." These undruggable targets include a vast and largely undeveloped treasure trove of medically important human proteins. Therefore, there is great interest in discovering novel molecular modalities that can modulate the function of such undruggable targets.

[0002] The interaction of small molecules with targets is driven by adhesive forces, the strength of which is roughly proportional to the contact surface area; therefore, small molecules are limited in their targeting ability. Due to their small size, the only way for small molecules to increase the contact surface area sufficiently and effectively interact with target proteins is through literal incorporation by those proteins. Indeed, both numerous experimental and computational data support the idea that only those proteins that have hydrophobic "pockets" on their surfaces can bind to small molecules. In such cases, binding is made possible by incorporation.

[0003] Nature has developed strategies that allow small molecules to interact with target proteins outside of the hydrophobic pocket. This strategy is exemplified by the naturally occurring immunosuppressants cyclosporine A, rapamycin, and FK506. The bioactivity of these drugs involves the formation of high-affinity complexes of small molecules with small presenting proteins. The complex surface of the small molecule and the presenting protein associates with the target. Thus, for example, the binary complex formed between cyclosporine A and cyclophylline A targets calcineurin with high affinity and specificity, whereas neither cyclosporine A nor cyclophylline A alone binds to calcineurin with measurable affinity. [Overview of the project] [Problems that the invention aims to solve]

[0004] The inventors have developed compounds and conjugates useful for exploring the interfaces between presenter protein and target protein pairs, and for use in the development of small molecules that can identify these pairs and modulate their interactions. [Means for solving the problem]

[0005] Accordingly, this disclosure provides methods and reagents useful for analyzing protein-protein interfaces, such as the interface between a presenter protein (e.g., a member of the FKBP family, a member of the cyclophyllin family, or PIN1) and a target protein. Such analysis is useful in helping to design small molecules that can simultaneously bind to both the presenter protein and the target protein, and the resulting small molecule-presenter protein complex can bind to the target protein and modulate its activity. In some embodiments, the target and / or presenter protein is an intracellular protein. In some embodiments, the target and / or presenter protein is a mammalian protein.

[0006] In some embodiments, the disclosure provides compounds that can be used as crosslinking substrates. These compounds may comprise a protein-binding moiety that can covalently or noncovalently bind to a protein (e.g., a target protein or a presenter protein) and at least one crosslinking group that can chemoselectively react with an amino acid of the protein different from the one bound to the protein-binding moiety. In some embodiments, the compound comprises only one crosslinking group.

[0007] Accordingly, in one embodiment, the present disclosure provides a compound comprising a protein-binding moiety (e.g., a presenter protein-binding moiety or a target protein-binding moiety) and a crosslinking group (e.g., a moiety capable of chemoselectively reacting with amino acids of a different protein than the one bound to the protein-binding moiety). The protein-binding moiety can bind (covalently or non-covalently) to a protein (e.g., a presenter protein or a target protein, depending on whether it is a presenter protein-binding moiety or a target protein-binding moiety), while the crosslinking group can form a covalent bond with a protein (e.g., a presenter protein, a target protein, or another compound capable of binding to such other proteins). In some embodiments, when the compound comprises a presenter protein-binding moiety, the compound does not comprise a target protein-binding moiety.

[0008] In some embodiments, the crosslinking group is a sulfhydryl-reactive crosslinking group (for example, the crosslinking group includes a mixed disulfide, maleimide, vinyl sulfone, vinyl ketone, or alkyl halogen), an amino-reactive crosslinking group, a carboxyl-reactive crosslinking group, a carbonyl-reactive crosslinking group, or a triazole-forming crosslinking group.

[0009] In some embodiments, the crosslinking group comprises a mixed disulfide, for example, the crosslinking group has the structure of chemical formula I.

[0010]

Chem.

[0011] It contains, in the chemical formula, the wavy line exemplifies the attachment point of the crosslinking group to the rest of the compound, a is 0, 1, or 2, R A is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C6-C 10 aryl, or optionally substituted C2-C9 heteroaryl. In some embodiments, R A is optionally substituted C2-C9 heteroaryl (e.g., pyridyl). In some embodiments, the crosslinking group has the structure

[0012]

Chem.

[0013] It contains, in the chemical formula, the wavy line exemplifies the attachment point of the crosslinking group to the rest of the compound. In some embodiments, R A is optionally substituted C1-C6 alkyl (e.g., methyl). In some embodiments, the crosslinking group has the structure

[0014]

Chem.

[0015] It contains, in the chemical formula, the wavy line exemplifies the attachment point of the crosslinking group to the rest of the compound. In some embodiments, the crosslinking group contains maleimide, for example, the crosslinking group has the structure

[0016]

Chem.

[0017] The compound includes the hyphen, and in the chemical formula, the wavy line illustrates the attachment point of the bridging group to the remainder of the compound. In some embodiments, the crosslinking group includes vinyl sulfone, for example, the crosslinking group is structure

[0018] [ka]

[0019] The compound includes the hyphen, and in the chemical formula, the wavy line illustrates the attachment point of the bridging group to the remainder of the compound. In some embodiments, the crosslinking group includes a vinyl ketone, for example, the crosslinking group is structure

[0020] [ka]

[0021] The compound includes the hyphen, and in the chemical formula, the wavy line illustrates the attachment point of the bridging group to the remainder of the compound. In some embodiments, the crosslinking group includes an alkyl halide, for example, an alkyl chloride, and for example, the crosslinking group is structure

[0022] [ka]

[0023] The compound includes the hyphen, and in the chemical formula, the wavy line illustrates the attachment point of the bridging group to the remainder of the compound. In some embodiments of the above compounds, the protein-binding portion can interact with the protein in a non-covalent manner.

[0024] In some embodiments, the disclosure provides a compound comprising a presenter protein-binding moiety and a crosslinking group. In some embodiments, the protein-binding moiety and the crosslinking group are attached via a linker.

[0025] In some aspects, this disclosure is structure

[0026] [ka]

[0027] JPEG2026090316000010.jpg54170

[0028] The present invention provides compounds having the following properties. In some embodiments, the disclosure provides conjugates comprising a presenter protein binding moiety that can covalently or noncovalently bind to a presenter protein conjugating to a target protein via a linker, methods for synthesizing such conjugates, and uses thereof.

[0029] Accordingly, in another embodiment, the present disclosure provides a conjugate comprising a presenter protein-binding portion conjugated to a target protein. In some embodiments, the presenter protein-binding portion of the conjugate can engage in non-covalent interactions with the presenter protein. In some embodiments, the presenter protein-binding portion of the conjugate can engage in covalent interactions with the presenter protein.

[0030] In some embodiments, the disclosure provides a method for generating a conjugate comprising a presenter protein binding moiety conjugated to a target protein. The method comprises reacting (a) a compound comprising a presenter protein binding moiety and a crosslinking group with (b) a target protein under conditions that enable the generation of a conjugate.

[0031] In some embodiments, the Disclosure provides a method for generating a conjugate comprising a presenter protein-binding moiety conjugated to a target protein. The method comprises providing (a) a compound comprising a presenter protein-binding moiety and a crosslinking group; (b) a target protein; and (c) a presenter protein; and reacting the compound with the target protein under conditions that enable the generation of a conjugate.

[0032] In some embodiments, the disclosure provides complexes comprising a presenter protein and a conjugate comprising a presenter protein binding site and a target protein, methods for generating the same, and uses thereof.

[0033] Therefore, in another embodiment, the present disclosure provides a complex comprising (i) a conjugate including a presenter protein binding portion conjugated to a target protein, and (ii) a presenter protein.

[0034] In some embodiments, the disclosure provides a method for generating a complex comprising (i) a conjugate containing a presenter protein-binding portion conjugated to a target protein, and (ii) a presenter protein. The method comprises combining the conjugate containing a presenter protein-binding portion conjugated to a target protein with the presenter protein under conditions that enable the formation of the complex.

[0035] In some embodiments, the Disclosure provides a method for generating a complex comprising (i) a conjugate comprising a presenter protein binding moiety conjugated to a target protein, and (ii) a presenter protein. The method comprises providing (a) a compound comprising a presenter protein binding moiety and a crosslinking group; (b) a target protein; and (c) a presenter protein; and reacting the compound with the target protein under conditions that enable the formation of a complex.

[0036] In some embodiments of the above methods, the presenter protein binds to the compound in the absence of the target protein. In some embodiments of the above methods, the presenter protein does not substantially bind to the compound in the absence of the target protein. In some embodiments of the above methods, the compound and the target protein do not substantially react in the absence of the presenter protein. In some embodiments of the above methods, the compound and the target protein react in the absence of the presenter protein. In some embodiments of the above methods, the conditions do not include a reducing agent. In some embodiments of the above methods, the conditions include an excess of presenter protein.

[0037] In some embodiments, detectable binding between a compound and a presenter protein is observed in the absence of the target protein. However, in some embodiments, detectable binding between a compound and a presenter protein is not observed in the absence of the target protein (e.g., the presenter protein does not substantially bind to the compound). In some embodiments, a significant reaction between the crosslinking group and the target protein (e.g., significant conjugate formation) is not observed in the absence of the presenter protein. However, in some embodiments, a significant reaction between the crosslinking group and the target protein may be observed even in the absence of the presenter protein. In some embodiments, the rate and / or extent of such reactions (e.g., the rate and / or amount of conjugate formation) may differ in a given assay when the presenter protein is present compared to when it is not (e.g., the rate and / or amount of conjugate formation is 2, 3, 4, 5, 10, or 100 times greater in the presence of the presenter protein).

[0038] In some embodiments, the conjugate generation described herein is carried out under conditions that do not contain (or are substantially free of) reducing reagents. In some embodiments, the present invention provides a complex comprising (i) a presenter protein; (ii) a compound as described herein (e.g., a compound whose structure comprises a presenter protein binding site and a crosslinking site); and (iii) a target protein. In some embodiments, such a complex is exposed to conditions that allow a reaction between the crosslinking site and the target protein, and / or is maintained under these conditions so that a crosslink is formed between them. In some embodiments, the crosslink is with a heteroatom in an amino acid (e.g., an amino acid side chain) of the target protein. In some embodiments, the crosslink is with an -S- atom in cysteine ​​in the target protein. In some embodiments, the target protein is a variant of a native target protein. In some such embodiments, the mutant exhibits high (e.g., 80%, 81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) aspect of the native target protein, but has a different amino acid sequence due to the substitution or addition of at least one amino acid that is susceptible to involvement in crosslinking with a crosslinking group (e.g., its amino acid side chain contains a heteroatom that can participate in such crosslinking).

[0039] In some embodiments, the disclosure provides conjugates comprising a target protein binding moiety that can covalently or noncovalently bind to a target protein conjugated to a presenter protein via a linker, methods for synthesizing such conjugates, and uses thereof.

[0040] Therefore, in another embodiment, the present disclosure provides a conjugate comprising a target protein-binding portion conjugated to a presenter protein. In some embodiments, the target protein-binding portion of the conjugate can engage in non-covalent interactions with the target protein. In some embodiments, the target protein-binding portion of the conjugate can engage in non-covalent interactions with the target protein. In some embodiments, the target protein-binding portion and the presenter protein are conjugated via a linker.

[0041] In some embodiments, the disclosure provides a method for generating a conjugate comprising a target protein-binding moiety conjugated to a presenter protein. The method comprises reacting (a) a compound comprising a target protein-binding moiety and a crosslinking group with (b) a presenter protein under conditions that enable the generation of a conjugate.

[0042] In some embodiments, the Disclosure provides a method for generating a conjugate comprising a target protein-binding moiety conjugated to a presenter protein. The method comprises providing (a) a compound comprising a target protein-binding moiety and a crosslinking group; (b) a presenter protein; and (c) a target protein; and reacting the compound with the presenter protein under conditions that enable the generation of a conjugate.

[0043] In some embodiments, detectable binding between the compound and the target protein is observed in the absence of the presenter protein. However, in some embodiments, detectable binding between the compound and the target protein is not observed in the absence of the presenter protein (e.g., the presenter protein does not substantially bind to the compound). In some embodiments, a significant reaction between the crosslinking group and the presenter protein (e.g., significant conjugate formation) is not observed in the absence of the target protein. However, in some embodiments, a significant reaction between the crosslinking group and the presenter protein may be observed even in the absence of the target protein. In some embodiments, the rate and / or extent of such reactions (e.g., the rate and / or amount of conjugate formation) may differ in a given assay when the presenter protein is present compared to when it is not (e.g., the rate and / or amount of conjugate formation is 2, 3, 4, 5, 10, or 100 times greater in the presence of the presenter protein).

[0044] In some embodiments, the target protein binds to the compound in the absence of the presenter protein. In some embodiments, the target protein does not substantially bind to the compound in the absence of the presenter protein. In some embodiments, the presenter protein does not substantially bind to the compound in the absence of the target protein. In some embodiments, the reaction between the crosslinking group and the target protein (e.g., conjugate formation) is not observed in the absence of the presenter protein. However, in some embodiments, the reaction between the crosslinking group and the target protein is observed even in the absence of the presenter protein. In some embodiments, conjugate formation as described herein is carried out under conditions that do not contain a reducing agent (e.g., substantially without one).

[0045] In some embodiments, the present invention provides a complex comprising (i) a presenter protein; (ii) a compound as described herein (e.g., a compound whose structure comprises a presenter protein binding site and a crosslinking site); and (iii) a target protein. In some embodiments, such a complex is exposed to conditions that allow a reaction between the crosslinking site and the target protein, and / or is maintained under these conditions so that a crosslink is formed between them. In some embodiments, the crosslink is with a heteroatom in an amino acid (e.g., an amino acid side chain) of the target protein. In some embodiments, the crosslink is with an -S- atom in cysteine ​​in the target protein. In some embodiments, the target protein is a variant of a native target protein. In some such embodiments, the mutant exhibits high (e.g., 80%, 81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) aspect of the native target protein, but has a different amino acid sequence due to the substitution or addition of at least one amino acid that is susceptible to involvement in crosslinking with a crosslinking group (e.g., its amino acid side chain contains a heteroatom that can participate in such crosslinking).

[0046] In some embodiments, the disclosure provides complexes comprising a target protein and a conjugate comprising a target protein-binding portion conjugated to a presenter protein via a linker, methods for generating such complexes, and uses thereof.

[0047] In some embodiments, the disclosure provides a complex comprising (i) a conjugate including a target protein-binding moiety conjugated to a presenter protein; (ii) a target protein; and (iii) a presenter protein. In some embodiments, such a complex is exposed to and / or maintained under conditions that allow a crosslinking moiety with the presenter protein to react, and / or to form a crosslink between them. In some embodiments, the crosslinking is with heteroatoms in the amino acids (e.g., amino acid side chains) of the presenter protein.

[0048] In some embodiments, the crosslinking is with the -S- atom at cysteine ​​in the presenter protein. In some embodiments, the presenter protein is a variant of the native presenter protein. In some such embodiments, the variant exhibits a high degree of (e.g., 80%, 81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) than the native presenter protein but has a different amino acid sequence due to the substitution or addition of at least one amino acid that is susceptible to involvement in crosslinking with the crosslinking group (e.g., its amino acid side chain contains a heteroatom that can participate in such crosslinking).

[0049] In some embodiments, the Disclosure provides a method for generating a complex comprising (i) a conjugate comprising a target protein-binding moiety conjugated to a presenter protein, and (ii) a target protein. The method comprises combining the conjugate comprising a target protein-binding moiety conjugated to a presenter protein with the target protein under conditions that enable the formation of the complex. In some embodiments, the Invention is characterized by a method for generating a complex comprising (i) a conjugate as described herein (e.g., a conjugate comprising a target protein-binding moiety and a presenter protein), and (ii) a target protein. In some such embodiments, the method provided comprises combining the conjugate and the target protein under conditions that enable the formation of the complex. Alternatively or further, in some embodiments, such a method comprises, for example, (i) (a) a compound (e.g., a compound whose structure comprises a target protein-binding moiety and a crosslinking group); (b) a target protein; and (c) a presenter protein; and (ii) exposing the combination to conditions that enable the formation of the complex and / or maintaining the combination under conditions that enable the formation of the complex. In some such embodiments, the conditions allow for the reaction of the crosslinking group with the presenter protein so that a conjugate is formed.

[0050] In some embodiments, the Disclosure provides a method for generating a complex comprising (i) a conjugate comprising a target protein-binding moiety conjugated to a presenter protein, and (ii) a target protein. The method comprises providing (a) a compound comprising a target protein-binding moiety and a crosslinking group; (b) a presenter protein; and (c) a target protein; and reacting the compound with the presenter protein under conditions that enable the formation of a complex.

[0051] In some such embodiments, the conditions characterize the compound, presenter protein, and / or target protein in such a way that detectable binding between the compound and the target protein is observed in the absence of the presenter protein. However, in some embodiments, detectable binding between the compound and the target protein is not observed in the absence of the presenter protein under these conditions (e.g., the target protein does not substantially bind to the compound). In some embodiments, a significant reaction between the crosslinking group and the presenter protein is not observed in the absence of the target protein under these conditions. However, in some embodiments, a significant reaction between the crosslinking group and the presenter protein may be observed even in the absence of the target protein under these conditions. In some embodiments, the conditions do not include a reducing agent. In some embodiments, the conditions include an excess of presenter protein.

[0052] In some embodiments, the target protein binds to the compound in the absence of the presenter protein. In some embodiments, the target protein does not substantially bind to the compound in the absence of the presenter protein. In some embodiments, the compound and the presenter protein do not substantially react in the absence of the target protein. In some embodiments, the compound and the presenter protein react in the absence of the target protein. In some embodiments, the conditions do not include a reducing agent. In some embodiments, the conditions include an excess of the target protein.

[0053] In some embodiments, the disclosure provides a compound comprising a presenter protein-binding moiety capable of non-covalent interactions with a presenter protein and a target protein-binding moiety capable of covalent or non-covalent interactions with a target protein. In some embodiments, the presenter protein-binding moiety and the target protein-binding moiety are attached via a linker.

[0054] Therefore, in some embodiments, this disclosure relates to the structure of chemical formula VII. ALB Compound having Chemical Formula VII To provide a compound having the formula, in the formula, A is the structure of Chemical Formula VIII

[0055]

Chem.

[0056] which includes, in the formula, b and c are independently 0, 1, or 2, d is 0, 1, 2, 3, 4, 5, 6, or 7, X 1 and X 2 are each independently absent, CH2, O, S, SO, SO2, or NR 13 where, each R 1 and R 2 are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C 10 carbocyclic, optionally substituted C6-C 10 aryl, optionally substituted C6-C 10 aryl C1-C6 alkyl, optionally substituted C2-C9 heterocyclyl (e.g., optionally substituted C2-C9 heteroaryl), optionally substituted C2-C9 heterocyclyl C1-C6 alkyl (e.g., optionally substituted C2-C9 heteroaryl C1-C6 alkyl), or R 1 and R 2 together with the carbon atom to which they are attached form C=O, or R 1 and R 2 together form optionally substituted C3-C 10It forms carbocyclils or C2-C9 heterocyclils that are optionally substituted. Each R 3 These are independently: hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, and optionally substituted C3-C 10 Carbocyclyl, optionally substituted with C6~C 10 Aryl, C6~C which are replaced by any choice 10 It is an aryl C1-C6 alkyl, an optionally substituted C2-C9 heterocyclil (e.g., an optionally substituted C2-C9 heteroaryl), or an optionally substituted C2-C9 heterocyclil C1-C6 alkyl (e.g., an optionally substituted C2-C9 heteroaryl C1-C6 alkyl), or two R 8 These are combined and replaced by C3~C 10 Carbocyclyl, optionally substituted with C6~C 10 Aryls, optionally substituted C2-C9 heterocyclines, for example, forming optionally substituted C2-C9 heteroaryls, R 4 These are C1-C6 alkyl groups that are optionally substituted. L is an optional linker, B is the target protein binding site.

[0057] In some embodiments of the compound of chemical formula VII, the target protein binding site B can engage in non-covalent interactions with the target protein. In some embodiments of the compound of chemical formula VII, the target protein binding site B can engage in covalent interactions with the target protein. In some embodiments of the compound of chemical formula VII, the linker L is present. In some embodiments of the compound of chemical formula VII, the linker L is absent.

[0058] In some embodiments, the disclosure provides ternary complexes comprising a presenter protein, a target protein, and a compound comprising a presenter protein-binding portion and a target protein-binding portion, methods for producing the same, and uses thereof.

[0059] Therefore, in another embodiment, the present disclosure provides a complex comprising (i) a compound of chemical formula VII; (ii) a target protein; and (iii) a presenter protein. In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful for identifying conjugates comprising a presenter protein binding site and a target protein that can form a complex with a presenter protein.

[0060] In some embodiments, the present invention features a method for identifying and / or characterizing a conjugate as described herein that can form a complex with a presenter protein (e.g., a compound whose structure comprises a presenter protein binding site and a crosslinking group, which is conjugated to a target protein). In some embodiments, such a method comprises the steps of (a) (i) providing such a conjugate (e.g., a compound whose structure comprises a presenter protein binding site and a crosslinking group, which is conjugated to a target protein), and (ii) a presenter protein; (b) if the conjugate can form a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable for enabling complex formation; and (c) determining whether a complex comprising the conjugate and the presenter protein has been formed, the formation of which indicates that the conjugate can form a complex with the presenter protein.

[0061] Accordingly, in certain embodiments, the present disclosure provides a method for identifying and / or characterizing a conjugate that can form a complex with a presenter protein. The method comprises the steps of (a) (i) providing a conjugate comprising a presenter protein binding portion conjugating to a target protein, and (ii) a presenter protein; (b) if the conjugate can form a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable for enabling complex formation; and (c) determining whether a complex comprising the conjugate and the presenter protein has been formed, the formation of which indicates that the conjugate is capable of forming a complex with the presenter protein.

[0062] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful for identifying target proteins that can form covalent bonds with the compounds. Accordingly, in another aspect, the present disclosure provides a method for identifying and / or characterizing a target protein that can react with a compound in the presence of a presenter protein, wherein the compound comprises a presenter protein binding portion and a crosslinking portion. The method comprises the steps of (a) providing (i) a compound comprising a presenter protein binding portion and a crosslinking portion; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, the target protein, and the presenter protein under conditions suitable for enabling complex formation, if the conjugate can form a complex with the presenter protein; and (c) determining whether the target protein and the compound react during complex formation to form a conjugate, wherein if the target protein and the compound form a conjugate, the target protein is identified as being able to react with the compound in the presence of the presenter protein.

[0063] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful for identifying target proteins that can form complexes with presenter proteins.

[0064] Accordingly, in another aspect, the present disclosure provides a method for identifying and / or characterizing a target protein that binds to a presenter protein. The method comprises the steps of (a) (i) providing a conjugate comprising a presenter protein-binding portion conjugated to a target protein, and (ii) the presenter protein; (b) if the conjugate can form a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable for enabling complex formation; and (c) determining whether the target protein is bound to the presenter protein in the complex, wherein if the target protein is bound to the presenter protein, the target protein is identified as binding to the presenter protein.

[0065] In certain embodiments, the Disclosure provides a method for identifying and / or characterizing a target protein that binds to a presenter protein. The method comprises the steps of (a) providing (i) a compound comprising a presenter protein binding portion and a crosslinking portion; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, the target protein, and the presenter protein under conditions suitable for enabling complex formation, if the conjugate can form a complex with the presenter protein; and (c) determining whether the target protein is bound to the presenter protein in the complex, wherein if the target protein is bound to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein.

[0066] In some embodiments, the Disclosure provides a method for identifying and / or characterizing a target protein that can form a complex with a presenter protein. The method comprises the steps of (a) providing (i) a compound of chemical formula VII; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, target protein, and presenter protein under conditions suitable for enabling complex formation, if the conjugate can form a complex with the presenter protein; and (c) determining whether the compound, target protein, and presenter protein form a complex, wherein if the compound, target protein, and presenter protein form a complex, the target protein is identified as a target protein that can form a complex with the presenter protein.

[0067] In certain embodiments, the Disclosure provides a method for identifying and / or characterizing a target protein that binds to a presenter protein. The method comprises the steps of (a) providing (i) a compound of chemical formula VII; (ii) a target protein; and (iii) a presenter protein; (b) if the compound can form a complex with the presenter protein, combining the compound, the target protein, and the presenter protein under conditions suitable for enabling complex formation; and (c) determining whether the target protein binds to the presenter protein in the complex, wherein if the target protein binds to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein.

[0068] In some embodiments, the Disclosure provides a method for identifying target proteins that can complex with a presenter protein by (a) providing a presenter protein comprising (i) one or more target proteins, (ii) any of the above compounds, and (iii) a tag (e.g., an affinity tag); (b) combining one or more target proteins, a compound, and a presenter protein under conditions suitable for enabling complex formation, if one or more target proteins can complex with the presenter protein; and (c) determining whether one or more target proteins complex with the compound and the presenter protein, wherein target proteins that complex with the presenter protein are identified as target proteins that can complex with the presenter protein.

[0069] In some embodiments, the determination step includes utilizing a tag on the presenter protein to selectively isolate the target protein that is complexed with the presenter protein (e.g., by use in a pull-down experiment). In some embodiments, the complex comprises the target protein, the presenter protein, and the compound of the present invention. In some embodiments, the complex comprises a conjugate containing the target protein and presenter protein binding portions (e.g., a conjugate formed by a reaction between a crosslinking group of the compound of the present invention and a reactive amino acid of the target protein), as well as the presenter protein. In some embodiments, the method further includes (d) identifying the target protein in the complex formed between one or more target proteins, the compound, and the presenter protein (e.g., determining the structure of the target protein). In some embodiments, identifying the structure of the target protein includes performing mass spectrometry on the complex. In some embodiments, the determination of whether the target protein and the presenter protein form a complex and / or whether the target protein binds to the presenter protein in the complex may be made using a pull-down experiment in which the target protein or presenter protein is labeled (e.g., the complex may be selectively pulled down in the presence of the target protein and / or presenter protein that are not in the complex).

[0070] In some embodiments, the Disclosure provides a method for identifying target proteins that can complex with presenter proteins by (a) providing a presenter protein comprising (i) two or more target proteins; (ii) any of the above compounds; and (iii) an affinity tag; (b) combining two or more target proteins, compounds, and presenter proteins under conditions suitable for enabling complex formation, if the target proteins can complex with the presenter proteins; (c) selectively isolating one or more complexes of target proteins, compounds, and presenter proteins formed in (b); and (d) identifying the target proteins in one or more complexes isolated in step (c) by mass spectrometry (e.g., determining the structure of the target proteins), thereby identifying the target proteins that can complex with presenter proteins.

[0071] In some embodiments, the determination step includes utilizing a tag on the presenter protein to selectively isolate the target protein that is complexed with the presenter protein (e.g., by use in a pull-down experiment). In some embodiments, the complex comprises the target protein, the presenter protein, and the compound of the present invention. In some embodiments, the complex comprises a conjugate containing target protein and presenter protein binding sites (e.g., a conjugate formed by a reaction between a crosslinking group of the compound of the present invention and a reactive amino acid of the target protein), as well as the presenter protein. In some embodiments, the determination of whether the target protein and presenter protein form a complex and / or whether the target protein binds to the presenter protein in the complex may be made using a pull-down experiment in which the target protein or presenter protein is labeled (e.g., the complex may be selectively pulled down in the presence of the target protein and / or presenter protein that are not in the complex).

[0072] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful in identifying a site on a target protein to which a presenter protein binding portion can be attached, resulting in a conjugate that can form a complex with a presenter protein.

[0073] Accordingly, in another embodiment, the present disclosure provides a method for identifying and / or characterizing a location on a target protein that forms a conjugate with a presenter protein binding site, where the conjugate can form a complex with the presenter protein. The method comprises the steps of (a)(i) providing a conjugate containing a presenter protein binding site conjugating to a target protein at a certain site, and (ii) a presenter protein; (b) combining the conjugate and the presenter protein; (c) determining whether the conjugate and the presenter protein form a complex; and (d) optionally repeating steps (a) to (c) with presenter protein binding sites conjugating at different locations on the target protein until the conjugate and the presenter protein form a complex, wherein if the conjugate and the presenter protein form a complex, the location on the target protein that forms a conjugate with the presenter protein binding site (where the conjugate can form a complex with the presenter protein) is identified. In some embodiments, the presenter protein is a variant of a naturally occurring target protein.

[0074] In some embodiments, the disclosure provides a method for identifying and / or characterizing a location on a target protein where a presenter protein binding site forms a conjugate, the conjugate which can form a complex with the presenter protein. The method includes the steps of (a) providing (i) a compound comprising a presenter protein binding moiety and a crosslinking group; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound and the target protein under conditions that allow for the formation of a conjugate comprising a presenter protein binding moiety conjugating to the target protein at a site in the presence of the presenter protein; (c) determining whether the conjugate and the presenter protein form a complex; and (d) optionally repeating steps (a) to (c) until the conjugate and the presenter protein form a complex (the presenter protein binding moiety conjugates at different sites on the target protein) (if the conjugate and the presenter protein form a complex, the site on the target protein that forms the presenter protein binding moiety and conjugate (the conjugate can form a complex with the presenter protein) is identified), thereby identifying the site on the target protein that forms a conjugate that can form a complex with the presenter protein. In some embodiments, the target protein is a variant of a naturally occurring target protein.

[0075] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful in identifying compounds that can form covalent bonds with target proteins in the presence of a presenter protein. In some embodiments, compounds are identified as selectively forming covalent bonds with target proteins in the presence of a presenter protein.

[0076] Accordingly, in another aspect, the present disclosure provides a method for identifying and / or characterizing a compound that can covalently bind to a target protein in the presence of a presenter protein. The method comprises the steps of (a) providing a sample comprising (i) a compound comprising a presenter protein binding moiety and a crosslinking group; (ii) a target protein; and (iii) a presenter protein; and (b) determining whether the compound and the target protein form a covalent bond via the crosslinking group in the compound in the sample, wherein if the compound and the target protein react in the sample, the compound is identified as covalently binding to the target protein in the presence of the presenter protein.

[0077] In certain embodiments, the Disclosure provides a method for identifying and / or characterizing compounds that can selectively and covalently bind to a target protein in the presence of a presenter protein. The method comprises the steps of: (a) providing a first sample comprising (i) a compound comprising a presenter protein binding moiety and a crosslinking group; (ii) a target protein; and (iii) a presenter protein; and a second sample comprising (i) the same compound as in the first sample, comprising a presenter protein binding moiety and a crosslinking group, and (ii) the same target protein as in the first sample; and (b) determining the extent to which the compound and the target protein react in the first sample compared to the second sample, wherein if the compound and the target protein react more in the first sample than in the second sample, the compound is identified as selectively covalently binding to the target protein in the presence of a presenter protein.

[0078] In some embodiments, if the compound and target protein react at least five times more in the first sample than in the second sample, the compound is identified as selectively covalently binding to the target protein in the presence of the presenter protein. In some embodiments, if the compound and target protein react in the first sample but substantially do not react in the second sample, the compound is identified as selectively covalently binding to the target protein in the presence of the presenter protein.

[0079] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful in identifying conjugates that include a presenter protein binding moiety capable of forming a complex with a target protein and a presenter protein.

[0080] Accordingly, in another aspect, the present disclosure provides a method for identifying and / or characterizing conjugates that can form complexes with presenter proteins. The method includes (a) providing a conjugate comprising a presenter protein binding portion conjugating to a target protein, and (ii) a presenter protein; (b) combining the conjugate and the presenter protein under conditions suitable for complex formation; and (c) determining whether the conjugate and the presenter protein form a complex (if the conjugate and the presenter protein do, the conjugate is identified as being able to form a complex with the presenter protein), thereby identifying a conjugate that can form a complex with the presenter protein.

[0081] In some embodiments, the binding between the conjugate and the protein may be determined by methods including ternary time-resolved fluorescence energy transfer assays, ternary amplified luminescence proximity homogeneous assays, isothermal titration calorimetry, surface plasmon resonance, or nuclear magnetic resonance.

[0082] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful in determining the structure of the protein-protein interface between a presenter protein and a target protein.

[0083] Accordingly, in another aspect, the present disclosure provides a method for determining the structure of an interface in a complex comprising a presenter protein and a target protein, and / or assessing one or more of its structural features. The method comprises the steps of (a) (i) providing a conjugate comprising a presenter protein binding portion conjugated to a target protein, and (ii) the presenter protein; (b) contacting the conjugate and the presenter protein to form a complex (e.g., in a vial); and (c) determining the crystal structure of the complex (the interface structure comprises at least a portion of the crystal structure between the presenter protein and the target protein), thereby determining the structure of the interface in a complex comprising a presenter protein and a target protein.

[0084] In some embodiments, the Disclosure provides a method for determining the structure of an interface in a complex comprising a presenter protein and a target protein, and / or assessing one or more of its structural features. The method includes the steps of: (a) providing (i) a compound comprising a presenter protein binding portion and a crosslinking portion; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, the target protein, and the presenter protein under conditions suitable for the formation of a conjugate between the compound and the target protein, and the formation of a complex between the conjugate and the presenter protein (e.g., in a vial); and (c) determining the crystal structure of the complex (the interface structure includes at least a portion of the crystal structure between the presenter protein and the target protein), thereby determining the structure of the interface in a complex comprising a presenter protein and a target protein.

[0085] In some embodiments, the Disclosure provides a method for determining the structure of an interface in a complex comprising a presenter protein and a target protein, and / or assessing one or more of its structural features. The method includes (a) providing (i) a compound of chemical formula VII; (ii) a target protein; and (iii) a presenter protein; (b) forming a complex comprising the compound, the target protein, and the presenter protein (e.g., in a vial); and (c) determining the crystal structure of the complex (the interface structure comprises at least a portion of the crystal structure between the presenter protein and the target protein), thereby determining the structure of the interface in the complex comprising the presenter protein and the target protein.

[0086] In some embodiments, the Disclosure provides a method for determining the structure of a protein-protein interface in a complex comprising a presenter protein and a target protein, and / or assessing one or more of its structural features. The method includes the steps of (a) providing a crystal of any of the above complexes; and (b) determining the structure of the crystal (the interface structure includes at least a portion of the crystal structure between the presenter protein and the target protein), thereby determining the structure of the protein-protein interface in the complex comprising the presenter protein and the target protein. In some embodiments, the Disclosure provides a method for identifying and / or characterizing compounds that can modulate the biological activity of a target protein. The method includes the steps of (a) providing the structure of a protein-protein interface in a complex comprising a presenter protein and a target protein (e.g., the structure determined by any of the above methods); and (b) determining the structure of a compound that can bind at the interface, thereby identifying a compound that can modulate the biological activity of a target protein. In some embodiments, the structure of a compound that can bind at the interface is determined using a computational method. In some embodiments, the structure of a compound that can bind at the interface is determined by screening compounds containing the presenter protein binding moiety described herein for complex formation, in the presence of the target protein and the presenter protein.

[0087] In certain embodiments, the disclosure provides a method for obtaining X-ray crystal coordinates for a complex. The method includes (a) providing a conjugate comprising (i) a presenter protein binding portion conjugated to a target protein, and (ii) a presenter protein; (b) if the conjugate can form a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable for enabling complex formation; and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0088] In certain embodiments, the present disclosure provides a method for obtaining X-ray crystal coordinates for a complex. The method includes the steps of: (a) providing (i) a compound comprising a presenter protein binding portion and a crosslinking portion; (ii) a target protein; and (iii) a presenter protein; (b) if the compound can form a complex with the presenter protein, combining the compound, the target protein, and the presenter protein under conditions suitable for enabling complex formation; and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0089] In certain embodiments, the Disclosure provides a method for obtaining X-ray crystal coordinates for a complex. This method includes the steps of (a) providing (i) a compound of the present invention; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, the target protein, and the presenter protein under conditions suitable for enabling complex formation, if the compound can form a complex with the presenter protein; and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0090] In certain embodiments, the Disclosure provides a method for determining residues on a target protein involved in binding to a presenter protein. This method comprises the steps of (a) providing the X-ray crystal coordinates of the complex obtained by the method of the Invention; and (b) identifying residues of the target protein that include atoms within 4 Å of atoms on the presenter protein, thereby determining residues on the target protein involved in binding to the presenter protein.

[0091] In certain embodiments, the Disclosure provides a method for determining the biochemical and / or biophysical properties of any of the presenter protein / target protein complexes described herein. The method comprises the steps of (a) providing the X-ray crystal coordinates of the complex described herein obtained by the method described herein; and (b) calculating the biochemical and / or biophysical properties of the complex, thereby determining the biochemical and / or biophysical properties of the presenter protein / target protein complex.

[0092] In some embodiments, the biochemical and / or biophysical properties are the free energy of the complex bond, the K of the complex. d , the K of the complex i , the K of the complex inact , and / or the K of the complex i / K inact This includes. In some embodiments, biochemical and / or biophysical properties are determined by isothermal titration calorimetry, surface plasmon resonance, and / or mass spectrometry.

[0093] In some embodiments, the interface in a complex comprising a presenter protein and a target protein is a binding pocket, or includes a binding pocket. In some embodiments, the disclosure provides a composition comprising any of the above-mentioned compounds, target proteins, and presenter proteins in solution.

[0094] In some embodiments, the present disclosure features a pharmaceutical composition comprising any of the compounds, conjugates, or complexes of the present invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a unit formulation.

[0095] In some embodiments, the present invention features a method for modulating a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein such as a bacterial target protein). In some embodiments, such a method includes contacting the target protein with a modulation amount (e.g., positive or negative modulation) of the compound of the present invention (e.g., in the presence of a presenter protein), a conjugate containing a target protein binding moiety, or a composition.

[0096] In some embodiments, the Disclosure provides methods for modulating (e.g., positively or negatively) a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein such as a bacterial target protein). In some embodiments, such methods include the step of modulating (e.g., positively or negatively) a target protein by contacting a cell expressing a target protein and a presenter protein with an effective amount of the compound or composition of the Invention, under conditions that the compound is capable of forming a complex with a presenter protein and the resulting complex is capable of binding to the target protein.

[0097] In some embodiments, the Disclosure provides methods for modulating (e.g., positively or negatively) a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein such as a bacterial target protein). In some embodiments, such methods include the step of modulating the target protein by contacting the target protein with a conjugate of the Invention comprising a target protein binding moiety.

[0098] In some embodiments, the present disclosure provides methods for inhibiting prolyl isomerase activity. In some embodiments, such methods include the step of inhibiting prolyl isomerase activity by contacting a compound or composition of the present invention with cells expressing prolyl isomerase under conditions that allow for the formation of a complex between the compound and prolyl isomerase.

[0099] In some embodiments, the present disclosure provides a method for forming a presenter protein / compound complex within a cell. In some embodiments, such a method includes contacting a cell expressing a presenter protein with the compound or composition of the present invention under conditions that allow for the formation of a complex between the compound and the presenter protein.

[0100] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding portion can bind a protein encoded by any one of the genes in Table 1. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding portion is a prolyl isomerase binding portion. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding portion is an FKBP binding portion (for example, the presenter protein binding portion can bind FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52), a cyclophylline binding portion (for example, the presenter protein binding portion can bind PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1), or a PIN1 binding portion. In some embodiments of the above methods, it is known that the presenter protein binds to the presenter protein binding site.

[0101] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding portion is an FKBP binding portion (e.g., a selective FKBP binding portion or a non-selective FKBP binding portion). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the FKBP binding portion has a structure of chemical formula IIa or IIb.

[0102] [ka]

[0103] It includes, and in the chemical formula, Z 1 and Z 2 These are, independently, either a C1-C6 alkyl group that is optionally substituted, a C1-C6 heteroalkyl group that is optionally substituted, or Z 1 and Z 2 These combine to form a macrocyclic molecule of 10 to 40 members, which are optionally substituted, along with the atoms to which they are attached, Z 1 or Z 2 At least one of them includes an attachment point to a crosslinking group, b and c are independently 0, 1, or 2. d is 0, 1, 2, 3, 4, 5, 6, or 7. X 1 and X 2 These are, independently, non-existent, CH2, O, S, SO, SO2, or NR. 4 And, Each R 1 and R 2 These are independently: hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, and optionally substituted C3-C 10Carbocyclyl, optionally substituted with C6~C 10 Aryl, C6~C which are replaced by any choice 10 It is an aryl C1-C6 alkyl, an optionally substituted C2-C9 heterocyclil (e.g., an optionally substituted C2-C9 heteroaryl), an optionally substituted C2-C9 heterocyclil C1-C6 alkyl (e.g., an optionally substituted C2-C9 heteroaryl C1-C6 alkyl), or R 1 and R 2 These combine with the carbon atoms to which they are bonded to form C=O, or R 1 and R 2 These are combined and replaced by C3~C 10 It forms carbocyclils or C2-C9 heterocyclils that are optionally substituted. Each R 3 These are independently: hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, and optionally substituted C3-C 10 Carbocyclyl, optionally substituted with C6~C 10 Aryl, C6~C which are replaced by any choice 10 It is an aryl C1-C6 alkyl, an optionally substituted C2-C9 heterocyclil (e.g., an optionally substituted C2-C9 heteroaryl), or an optionally substituted C2-C9 heterocyclil C1-C6 alkyl (e.g., an optionally substituted C2-C9 heteroaryl C1-C6 alkyl), or two R 8 These are combined and replaced by C3~C 10 Carbocyclyl, optionally substituted with C6~C 10 Forming aryls, for example, C2-C9 heteroaryls that are optionally substituted, Each R 4 These are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocykyl, and optionally substituted C6-C 10 These are aryl C1-C6 alkyl groups and optionally substituted C3-C7 carbocyacrylic C1-C6 alkyl groups.

[0104] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety is structure

[0105] [ka]

[0106] Includes. In some embodiments of the compounds, conjugates, complexes, compositions, or methods described above, the presenter protein binding portion is a cyclophylline binding portion (e.g., a selective cyclophylline binding portion or a non-selective cyclophylline binding portion). In some embodiments of the compounds, conjugates, complexes, compositions, or methods described above, the cyclophylline binding portion has a structure of chemical formula III or IV.

[0107] [ka]

[0108] It includes, and in the chemical formula, Z 3 , Z 4 , Z 5 , and Z 6 Each of these is independently either a hydroxyl group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 heteroalkyl group, or Z 3 and Z 4 or Z 5 and Z 6These combine to form a macrocyclic molecule of 10 to 40 members, which are optionally substituted along with the atoms to which they are attached. Z 3 , Z 4 , Z 5 , Z 6 , or R 5 At least one of them includes an attachment point to a crosslinking group, e is 0, 1, 2, 3, or 4. R 5 This includes C1-C6 alkyl groups that are optionally substituted, C2-C6 alkenyl groups that are optionally substituted, C2-C6 alkynyl groups that are optionally substituted, C1-C6 heteroalkyl groups that are optionally substituted, C2-C6 heteroalkenyl groups that are optionally substituted, C2-C6 heteroalkynyl groups that are optionally substituted, and C3-C6 groups that are optionally substituted. 10 Carbocyclyl, optionally substituted with C6~C 10 Aryl, C6~C which are replaced by any choice 10 These are aryl C1-C6 alkyl groups, optionally substituted C2-C9 heteroaryl groups, optionally substituted C2-C9 heteroaryl C1-C6 alkyl groups, optionally substituted C2-C9 heterocyclil groups, or optionally substituted C2-C9 heterocyclil C1-C6 alkyl groups. R 6 These are C1-C6 alkyl groups that are optionally substituted. Each R 7 These are independently hydroxyl, cyano, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, and optionally substituted C3-C 10 Carbocyclyl, optionally substituted with C6~C 10 Aryl, C6~C which are replaced by any choice 10The aryl C1-C6 alkyl, optionally substituted C2-C9 heterocyclil (e.g., optionally substituted C2-C9 heteroaryl), or optionally substituted C2-C9 heterocyclil C1-C6 alkyl (e.g., optionally substituted C2-C9 heteroaryl C1-C6 alkyl), R 8 This includes hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocykyl, and optionally substituted C6-C 10 These are aryl C1-C6 alkyl groups and optionally substituted C3-C7 carbocyacrylic C1-C6 alkyl groups.

[0109] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety is structure

[0110] [ka]

[0111] Includes. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the target protein includes an undruggable surface. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the target protein does not have a traditional binding pocket.

[0112] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the target protein is modified, with at least one native amino acid being replaced by a reactive amino acid (e.g., a native amino acid, e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine, or a non-native amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the target protein is modified, with at least one native reactive amino acid (e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine) being replaced by a non-reactive amino acid (e.g., a native amino acid, e.g., serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine, or a non-native amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the at least one native reactive amino acid is an amino acid exposed to a solvent. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the target protein is modified so that all reactive amino acids are replaced with non-reactive amino acids. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the substitutions are conservative substitutions. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the target protein contains only one solvent-exposed reactive amino acid.

[0113] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein is a protein encoded by any one of the genes in Table 1. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein is prolyl isomerase. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the prolyl isomerase is a member of the FKBP family (e.g., FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52), a member of the cyclophylline family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1), or PIN1.

[0114] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein is modified, and at least one native amino acid is replaced with a reactive amino acid (e.g., a native amino acid, e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine, or a non-native amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein is modified, and at least one native reactive amino acid (e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine) is replaced with a non-reactive amino acid (e.g., a native amino acid, e.g., serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine, or a non-native amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, at least one native reactive amino acid is an amino acid exposed to a solvent. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein is modified so that all reactive amino acids are replaced with non-reactive amino acids. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the substitutions are conservative substitutions.

[0115] In some embodiments of the above compounds, conjugates, complexes, compositions, or methods, the linker is 1 to 20 atoms in length. In some embodiments of the above compounds, conjugates, complexes, compositions, or methods, the linker is 1.5 to 30 angstroms in length.

[0116] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the linker has a structure of chemical formula V. A 1 -( B 1 ) f -(C 1 ) g -( B2 ) h -(D)-(B 3 ) i -(C 2 ) j -(B 4 ) k -A 2 Chemical formula V having, In the chemical formula, A 1 is a bond between a linker and a protein-binding moiety, and A 2 is a bond between a cross-linking group and a linker, and B 1 , B 2 , B 3 , and B 4 are each independently optionally substituted C1-C2 alkyl, optionally substituted C1-C3 heteroalkyl, O, S, and NR N selected from, and R N is hydrogen, optionally substituted C 1~4 alkyl, optionally substituted C 2~4 alkenyl, optionally substituted C 2~4 alkynyl, optionally substituted C 2~6 heterocyclyl, optionally substituted C 6~12 aryl, or optionally substituted C 1~7 heteroalkyl, and C 1 and C 2 are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, f, g, h, I, j, and k are each independently 0 or 1, and D is optionally substituted C 1~10 alkyl, optionally substituted C 2~10 alkenyl, optionally substituted C 2~10 alkynyl, optionally substituted C 2~6 heterocyclyl, optionally substituted C 6~12 aryl, optionally substituted C2-C 10 polyethylene glycol, or optionally substituted C 1~10 heteroalkyl, or A 1 -(B1 ) f -(C 1 ) g -( B 2 ) h -to-(B 3 ) i -(C 2 ) j -( B 4 ) k -A 2 It is a chemical bond that connects them.

[0117] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the linker is a structure of chemical formula VI.

[0118] [ka]

[0119] It includes, in the chemical formula, A 1 This is the bond between the linker and the protein binding site. A 2 This is the bond between the crosslinking group and the linker. l is 0, 1, 2, or 3. m is either 0 or 1. n is 0, 1, or 2. X 3 , X 4 , and X 5 These are, independently, non-existence, O, S, -C≡C-, and CR. 9 R 10 or NR 11 And, Each R 9 , R 10 , and R 11 These are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocykyl, and optionally substituted C6-C 10These are aryl C1-C6 alkyl groups and optionally substituted C3-C7 carbocykyl C1-C6 alkyl groups. In some embodiments, each R 9 , R 10 , and R 11 These are independently hydrogen, unsubstituted C1-C6 alkyl, unsubstituted C2-C6 alkenyl, unsubstituted C2-C6 alkynyl, unsubstituted aryl, C3-C7 carbocykyl, and unsubstituted C6-C6. 10 These are aryl C1-C6 alkyl groups and unsubstituted C3-C7 carbocyclyl C1-C6 alkyl groups.

[0120] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the linker is structure

[0121] [ka]

[0122] Includes. chemical terms Those skilled in the art will see that certain compounds described herein can exist in the form of one or more different isomers (e.g., stereoisomers, geometric isomers, tautomers) and / or isotopes (in which one or more atoms are replaced with different isotopes of that atom, e.g., hydrogen replaced with deuterium). Unless otherwise specified or made clear from the context, the structures represented can be understood to represent any such isomer or isotope form, either alone or in combination.

[0123] The compounds described herein may be asymmetric (for example, having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of this disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, for example, by the resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers, such as olefins and C=N double bonds, can also be present in the compounds described herein, and all such stable isomers are intended in this disclosure. Cis and trans geometric isomers of the compounds of this disclosure are described and can be isolated as mixtures of isomers or as separated isomers.

[0124] In some embodiments, one or more compounds represented herein may exist in various tautomerized forms. As will be evident from the context, unless explicitly excluded, references to such compounds encompass all such tautomerized forms. In some embodiments, the tautomerized form arises from the exchange of a single bond with an adjacent double bond and the accompanying transfer of protons. In certain embodiments, the tautomerized form may be a prototropic tautomer of an isomer-protonated state having the same empirical formula and total charge as the reference form. Examples of prototropic tautomerized moieties include ketone-enol pairs, amide-imidolic acid pairs, lactam-lactim pairs, amide-imidolic acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in a heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H-, and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, and 1H- and 2H-pyrazoles. In some embodiments, the tautomers may be in equilibrium or sterically locked into one form by appropriate substitution. In certain embodiments, the tautomers are acetal interconverted, for example, in the following scheme:

[0125] [ka]

[0126] It arises from the mutual conversions exemplified therein. Those skilled in the art will see that in some embodiments, isotopes of the compounds described herein can be prepared and / or utilized according to the present invention. “Isotopes” means atoms having the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, isotopic substitution (e.g., substitution of hydrogen with deuterium) can alter the physicochemical properties of molecules, such as metabolic and / or chiral center racemization rates.

[0127] As is known in the art, many chemical entities (in particular many organic molecules and / or many small molecules) can exist in a wide variety of solid forms, such as amorphous and / or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.). In some embodiments, such entities can be used in any form, including any solid form. In some embodiments, such entities are used in a specific form, for example, a specific solid form.

[0128] In some embodiments, the compounds described and / or represented herein may be provided and / or utilized in salt form. In certain embodiments, the compounds described and / or represented herein may be provided and / or utilized in hydrate or solvate form.

[0129] Substituents of the compounds of this disclosure are disclosed in groups or as a range in various parts of this specification. This disclosure is particularly intended to include any individual partial combination of members of such groups and ranges. For example, "C 1~6The term “alkyl” is particularly intended to disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl individually. Furthermore, where a compound includes multiple positions for which substitution is disclosed as a group or range, unless otherwise specified, this disclosure is intended to cover individual compounds and groups of compounds (e.g., groups and subgroups) containing any individual partial combination of members at each position.

[0130] In this specification, phrases of the form “arbitrarily substituted X” (e.g., arbitrarily substituted alkyl) are intended to be equivalent to “X when X is arbitrarily substituted” (e.g., “alkyl when alkyl is arbitrarily substituted”). It is not intended to mean that the feature “X” (e.g., alkyl) itself is arbitrarily substituted.

[0131] As used herein, the term “alkyl” means a saturated hydrocarbon group containing 1 to 20 (e.g., 1 to 10 or 1 to 6) carbon atoms. In some embodiments, the alkyl group is unbranched (i.e., linear). In some embodiments, the alkyl group is branched. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and (1) alkoxy, (2) alkylsulfinyl, (3) amino as defined herein (e.g., unsubstituted amino (i.e., -NH2) or substituted amino (i.e., -N(R) N1 )2(in the chemical formula, R N1 (4)C 6~10 Aryl C 1~6 Alkoxy, (5) Azide, (6) Halo, (7) (C 2~9 (8) Hydroxyl (heterocyclyl), (9) Hydroxyl (optionally substituted with an O-protecting group), (10) Nitro, (11) Oxo (e.g., carboxyaldehyde or acyl), (12) C 1~7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) -CO2R optionally substituted with an O-protecting groupA’ (In the chemical formula, R A’ (a)C 1~20 Alkyl (for example, C 1~6 (b)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c)C 6~10 (d) Aryl, (d) Hydrogen, (e) C 1~6 Alk-C 6~10 Aryl, (f)amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (In the chemical formula, s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (Selected from the group consisting of amino-polyethylene glycol represented by alkyl), (15)-C(O)NR B’ R C’ (In the chemical formula, R B’ and R C’ Each of them independently consists of (a) hydrogen and (b) C 1~6 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (16)-SO2R D’ (In the chemical formula, R D’ (a)C 1~6 Alkyl, (b)C 6~10 Ariel, (c)C1~6 Alk-C 6~10 (Selected from the group consisting of aryl and (d)hydroxyl), (17)-SO2NR E’ R F’ (In the chemical formula, R E’ and R F’ Each of them independently consists of (a) hydrogen and (b) C 1~6 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (18)-C(O)R G’ (In the chemical formula, R G’ (a)C 1~20 Alkyl (for example, C 1~6 (b)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c)C 6~10 (d) Aryl, (d) Hydrogen, (e) C 1~6 Alk-C 6~10 Aryl, (f)amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (Selected from the group consisting of amino-polyethylene glycol represented by alkyl), (19)-NR H’C(O)R I’ (In the formula, R H’ (a1) hydrogen and (b1) C 1~6 Selected from the group consisting of alkyl, and R I’ (a2)C 1~20 Alkyl (for example, C 1~6 (Alkyl), (b2)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c2)C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 Alk-C 6~10 Aryl, (f2)amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (Selected from the group consisting of amino-polyethylene glycol represented by alkyl), (20)-NR J’ C(O)OR K’ (In the formula, R J’ (a1) hydrogen and (b1) C 1~6 Selected from the group consisting of alkyl, and R K’ (a2)C 1~20 Alkyl (for example, C 1~6 (Alkyl), (b2)C 2~20 Alkenyl (for example, C 2~6Alkenil), (c2)C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 Alk-C 6~10 Aryl, (f2)amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (1) C1-alkalyl alkyl group (selected from the group consisting of amino-polyethylene glycol represented by (21) amidine, and (22) silyl groups such as trimethylsilyl, t-butyldimethylsilyl, and triisopropylsilyl) can be optionally substituted with one, two, or three substituents independently selected from the group consisting of (21) amidine, and (22) silyl groups such as trimethylsilyl, t-butyldimethylsilyl, and triisopropylsilyl, or four substituents in the case of alkyl groups of two or more carbons. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of C1-alkalyl can be further substituted with an oxo group to give the respective allyloyl substituents.

[0132] As used herein, the terms "alkylene" and the prefix "alk-" refer to saturated divalent hydrocarbon groups derived from linear or branched saturated hydrocarbons by removing two hydrogen atoms, exemplified by methylene, ethylene, isopropylene, and the like.x~y The term "alkylene" and "C x~y The prefix "alk-" represents an alkylene group having x to y carbon atoms. Exemplary values ​​for x are 1, 2, 3, 4, 5, and 6, and exemplary values ​​for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (for example, C 1~6 , C 1~10 , C 2~20 , C 2~6 , C 2~10 , or C 2~20 Alkylene). In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituents defined herein for the alkyl group.

[0133] As used herein, the term “alkenyl” refers to a monovalent linear or branched group of 2 to 20 carbon atoms (e.g., 2 to 6 or 2 to 10 carbon atoms) containing one or more carbon-carbon double bonds, unless otherwise specified, and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyls include both cis and trans isomers. The alkenyl group may be optionally substituted with 1, 2, 3, or 4 substituents independently selected from any of the amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl) substituents defined herein or the exemplary alkyl substituents described herein.

[0134] As used herein, the term “alkynyl” refers to a monovalent linear or branched group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond, exemplified by ethynyl, 1-propynyl, and the like. The alkynyl group may be optionally substituted with 1, 2, 3, or 4 substituents independently selected from any of the aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl) substituents defined herein or the exemplary alkyl substituents described herein.

[0135] As used herein, the term "amino" means -N(R N1 )2(in the formula, each R N1 These are independently H, OH, NO2, N(R) N2 )2, SO2OR N2 SO2R N2 SOR N2 , N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkalyl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted with an optionally substituted arylalkoxycarbonyl group or any of the O-protecting groups described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted with an optionally substituted arylalkoxycarbonyl group or any of the O-protecting groups described herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), and these listed R N1 Each of the groups is optionally substituted for each group as defined herein, or two R groups N1 These combine to form a heterocycline or an N-protecting group, and each R N2 The amino group of this invention is either an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(R)). N1 )2) may be. In preferred embodiments, the amino is -NH2 or -NHR N1 (In the formula, R N1 These are independently OH, NO2, NH2, NR N2 2. SO2OR N2 SO2R N2 SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others as described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, and each R N2 H, C1~20 Alkyl (for example, C 1~6 Alkyl), or C 6~10 (It could be an allele.)

[0136] As used herein, the term “amino acid” means a molecule having a side chain, an amino group, and an acid group (e.g., a carboxyl group of -CO2H or a sulfo group of -SO3H), where an amino acid is bonded to a parent molecule by a side chain, an amino group, or an acid group (e.g., a side chain). As used herein, the broadest definition of “amino acid” means any compound and / or substance that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. “Standard amino acid” means any of the 20 standard L-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” means any amino acid other than a standard amino acid, whether prepared synthetically or obtained from natural sources. In some embodiments, amino acids may include structural modifications to the general structure described above, including amino acids at the carboxyl terminus and / or amino terminus of the polypeptide. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, and / or substitution of the general structure. In some embodiments, such modifications may alter the cyclic half-life of the polypeptide containing the modified amino acids compared to one containing otherwise identical unmodified amino acids. In some embodiments, such modifications do not significantly alter the related activity of the polypeptide containing the modified amino acids compared to one containing otherwise identical unmodified amino acids. As will be apparent from the context, in some embodiments, the term “amino acid” is used to mean a free amino acid. In some embodiments, it is used to mean an amino acid residue of a polypeptide. In some embodiments, the amino acid is bonded to the parent molecule by a carbonyl group with the side chain or amino group bonded to the carbonyl group. In some embodiments, the amino acid is an α-amino acid.In certain embodiments, the amino acid is a β-amino acid. In some embodiments, the amino acid is a γ-amino acid. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl groups. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolicine, selenocystine, serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid group is (1) C. 1~6 Alkoxy, (2)C 1~6 (3) Alkyl sulfinyl, amino as defined herein (e.g., unsubstituted amino (i.e., -NH2) or substituted amino (i.e., -N(R) N1 )2(wherein, R N1 (4)C 6~10 Aryl C 1~6 Alkoxy, (5) Azide, (6) Halo, (7) (C 2~9 (Heterocyclyl) oxy, (8) hydroxyl, (9) nitro, (10) oxo (e.g., carboxyaldehyde or acyl), (11) C 1~7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14)-CO2R A’ (In the formula, R A’ (a)C 1~20 Alkyl (for example, C 1~6 (b)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c)C 6~10 (d) Aryl, (d) Hydrogen, (e) C 1~6 Alk-C 6~10 Aryl, (f)amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3OR'(wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (Selected from the group consisting of amino-polyethylene glycol represented by alkyl), (15)-C(O)NR B’ R C’ (In the formula, R B’ and R C’ Each of them independently consists of (a) hydrogen and (b) C 1~6 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (16)-SO2R D’ (In the formula, R D’ (a)C 1~6 Alkyl, (b)C 6~10 Ariel, (c)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryl and (d)hydroxyl), (17)-SO2NR E’ R F’ (In the formula, R E’ and R F’ Each of them independently consists of (a) hydrogen and (b) C 1~6 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (18)-C(O)R G’ (In the formula, R G’(a)C 1~20 Alkyl (for example, C 1~6 (b)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c)C 6~10 (d) Aryl, (d) Hydrogen, (e) C 1~6 Alk-C 6~10 Aryl, (f)amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (Selected from the group consisting of amino-polyethylene glycol represented by alkyl), (19)-NR H’ C(O)R I’ (In the formula, R H’ (a1) hydrogen and (b1) C 1~6 Selected from the group consisting of alkyl, and R I’ (a2)C 1~20 Alkyl (for example, C 1~6 (Alkyl), (b2)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c2)C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 Alk-C 6~10 Aryl, (f2)amino-C 1~20Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 Polyethylene glycol represented by (alkyl), and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (Selected from the group consisting of amino-polyethylene glycol represented by alkyl), (20)-NR J’ C(O)OR K’ (In the formula, R J’ (a1) hydrogen and (b1) C 1~6 Selected from the group consisting of alkyl, and R K’ (a2)C 1~20 Alkyl (for example, C 1~6 (Alkyl), (b2)C 2~20 Alkenyl (for example, C 2~6 Alkenil), (c2)C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 Alk-C 6~10 Aryl, (f2)amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20Polyethylene glycol represented by (alkyl), and (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 This is independently of hydrogen or optionally substituted C 1~6 (21) The amino acids can be optionally substituted with one, two, or three substituents, or in the case of an amino acid group of two or more carbon atoms, four substituents, which are independently selected from the group consisting of amino-polyethylene glycols represented by (alkyl) and the group consisting of amidine. In some embodiments, these groups Each of these can be further substituted as described herein.

[0137] As used herein, the term "N-alkylated amino acid" refers to an amino acid containing a C1-C6 alkyl group optionally substituted on the nitrogen atom of the amino acid forming the peptide bond. Examples of N-alkylated amino acids, but not limited to, include N-methylalanine, N-methylthreonine, N-methylphenylalanine, N-methylaspartic acid, N-methylvaline, N-methylleucine, N-methylglycine, N-methylisoleucine, N(α)-methyllysine, N(α)-methylasparagine, and N(α)-methylglutamine.

[0138] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or polycyclic carbocyclic system having one or two aromatic rings, exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenantrenyl, fluorenyl, indanyl, indenyl, and (1)C 1~7 Acyl (e.g., carboxyaldehyde), (2)C1~20 Alkyl (for example, C 1~6 Alkyl, alkoxy-C 1~6 Alkyl, C 1~6 Alkylsulfinyl-C 1~6 Alkyl, amino-C 1~6 Alkyl, Azide-C 1~6 Alkyl, (carboxyaldehyde)-C 1~6 Alkyl, Halo-C 1~6 Alkyl (e.g., perfluoroalkyl), hydroxy-C 1~6 Alkyl, Nitro-C 1~6 Alkyl, or C 1~6 Thioalkoxy-C 1~6 (Alkyl), (3)C 1~20 Alkoxy (for example, C 1~6 Alkoxy, for example, perfluoroalkoxy), (4)C 1~6 Alkyl sulfinyl, (5)C 6~10 (6) Aryl, (7) Amino, C 1~6 Alk-C 6~10 (8) Azid, (9) C 3~8 Cycloalkyl, (10)C 1~6 Alk-C 3~8 Cycloalkyl, (11) Halo, (12) C 1~12 Heterocyclines (for example, C 1~12 (heteroaryl), (13)(C 1~12 (14)Hydroxy, (15)Nitro, (16)C 1~20 Thioalkoxy (e.g., thioalkoxy), (17)-(CH2) q CO2R A’ (In the formula, q is an integer from 0 to 4, and R A’ (a) alkyl, (b) C 6~10 (c) aryl, and (d) C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (18)-(CH2) q CONR B’ R C’ (In the formula, q is an integer from 0 to 4, and R B’ and R C’ (a) hydrogen, (b) C 1~6Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Independently selected from the group consisting of aryls), (19)-(CH2) q SO2R D’ (In the formula, q is an integer from 0 to 4, and R D’ (a) alkyl, (b) C 6~10 Aryl, and (c) ALC-C 6~10 (Selected from the group consisting of aryls), (20)-(CH2) q SO2NR E’ R F’ (In the formula, q is an integer from 0 to 4, and R E’ and R F’ Each of them is (a) hydrogen, (b) C 1~6 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Independently selected from the group consisting of aryls), (21) thiol, (22) C 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24)C 6~10 Aryl C 1~6 Alkoxy, (25)C 1~6 Alk-C 1~12 Heterocyclines (for example, C 1~6 Alk-C 1~12 (Heteroaryl), (26)C 2~20 Alkenyl, and (27)C 2~20 The substituents can be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkynyls. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of C1-alkaryl or C1-alkheterocyclyl can be further substituted with an oxo group to give the respective allyroyl substituent and (heterocyclyl)oil substituent.

[0139] As used herein, the “arylalkyl” group represents an aryl group as defined herein, bonded to a parent molecule by an alkylene group as defined herein. An example of an unsubstituted arylalkyl group is one with 7 to 30 carbon atoms (for example, 7 to 16 or 7 to 20 carbon atoms, e.g., C 1~6 Alk-C 6~10 Ariel, C 1~10 Alk-C 6~10 Aryl, or C 1~20 Alk-C 6~10 (aryl). In some embodiments, alkylenes and aryls can each be further substituted with one, two, three, or four substituents as defined herein for each group. Other groups preceded by the prefix "alk-" are similarly defined, in which case "alk-" is C unless otherwise specified. 1~6 This refers to alkylene, and the chemical structure to which it is bonded is as defined herein.

[0140] The term "azide" represents a -N3 group, and can also be expressed as -N=N=N. As used herein, the terms "carbocyclic" and "carbocykryl" refer to a ring formed by optionally substituted carbon atoms. 3~12 This refers to monocyclic, bicyclic, or tricyclic non-aromatic ring structures. Examples of carbocyclic structures include cycloalkyl groups, cycloalkenyl groups, and cycloalkynyl groups.

[0141] As used herein, the “carbocykylalkyl” group represents a carbocyclic group as defined herein, bonded to a parent molecule by an alkylene group as defined herein. An example of an unsubstituted carbocyclylalkyl group is one with 7 to 30 carbon atoms (e.g., 7 to 16 or 7 to 20 carbon atoms, e.g., C 1~6 Alk-C 6~10 Carbocyclyl, C 1~10 Alk-C 6~10 Carbocyclyl, or C 1~20 Alk-C 6~10(Carbocyclyl). In some embodiments, alkylenes and carbocyclyls can each be further substituted with one, two, three, or four substituents as defined herein for each group. Other groups preceded by the prefix "alk-" are similarly defined, in which case "alk-" is C unless otherwise specified. 1~6 This refers to alkylene, and the chemical structure to which it is bonded is as defined herein.

[0142] As used herein, the term "carbonyl" refers to a C(O) group, and can also be expressed as C=O. As used herein, the term "carboxyl" means -CO2H.

[0143] As used herein, the term "cyano" refers to the -CN group. As used herein, the term "cycloalkyl" refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of 3 to 8 carbon atoms, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, dicyclicheptyl, and the like. When a cycloalkyl group contains one carbon-carbon double bond, it can be referred to as a "cycloalkenyl" group. Exemplary cycloalkenyl groups include cyclopentenyl and cyclohexenyl. The cycloalkyl groups of the present invention are (1)C 1~7 (2) Acyl (e.g., carboxyaldehyde), (2) C1-20 alkyl (e.g., alkyl, alkoxy-C1-6 alkyl, alkylsulfinyl-C1-6 alkyl, amino-C1-6 alkyl, azide-C1-6 alkyl, (carboxyaldehyde)-C1-6 alkyl, halo-C1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C1-6 alkyl, nitro-C1-6 alkyl, or C1-6 thioalkoxy-C1-6 alkyl), (3) C 1~20 Alkoxy (for example, C 1~6 Alkoxy, for example, perfluoroalkoxy), (4)C 1~6 Alkyl sulfinyl, (5)C 6~10(6) Aryl, (7) Amino, C 1~6 Alk-C 6~10 (8) Azid, (9) C 3~8 Cycloalkyl, (10)C 1~6 Alk-C 3~8 Cycloalkyl, (11) Halo, (12) C 1~12 Heterocyclines (for example, C 1~12 (heteroaryl), (13)(C 1~12 (Heterocyclyl)oxy, (14)hydroxyl, (15)nitro, (16)C1-20 thioalkoxy (e.g., C1-6 thioalkoxy), (17)-(CH2) q CO2R A’ (In the formula, q is an integer from 0 to 4, and R A’ (a)C 1~6 Alkyl, (b)C 6~10 (c) aryl, and (d) C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (18)-(CH2) q CONR B’ R C’ (In the formula, q is an integer from 0 to 4, and R B’ and R C’ (a) hydrogen, (b) C 6~10 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Independently selected from the group consisting of aryls), (19)-(CH2) q SO2R D’ (In the formula, q is an integer from 0 to 4, and R D’ (a)C 6~10 Alkyl, (b)C 6~10 Aryl, and (c)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (20)-(CH2) q SO2NR E’ R F’ (In the formula, q is an integer from 0 to 4, and R E’ and R F’ Each of them is (a) hydrogen, (b) C 6~10 Alkyl, (c)C 6~10Aryl, and (d)C 1~6 Alk-C 6~10 (21) Thiol, (22) C (independently selected from the group consisting of aryls) 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24)C 6~10 Aryl C 1~6 Alkoxy, (25)C 1~6 Alk-C 1~12 Heterocyclines (for example, C 1~6 Alk-C 1~12 (heteroaryl), (26) oxo, (27) C 2~20 Alkenyl, and (28)C 2~20 They can be optionally substituted with alkynyl groups. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of C1-alkalyl or C1-alk heterocyclyl can be further substituted with an oxo group to give the respective allyroyl substituent and (heterocyclyl)oil substituent.

[0144] As used herein, the “cycloalkylalkyl” group represents a cycloalkyl group as defined herein, bonded to a parent molecule by an alkylene group as defined herein (for example, an alkylene group with 1 to 4, 1 to 6, 1 to 10, or 1 to 20 carbon atoms). In some embodiments, the alkylene and cycloalkyl groups can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for their respective groups.

[0145] As used herein, the term "diastereomer" refers to stereoisomers that are not mirror images of each other and cannot be superimposed on each other. As used herein, the term "enantiomer" means each individual optically active form of the compound of the present invention having an optical purity or enantiomer excess (determined by standard methods of the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90%, and more preferably at least 98%.

[0146] As used herein, the term "halo" refers to a halogen selected from bromine, chlorine, iodine, or fluorine. As used herein, the term “heteroalkyl” means an alkyl group as defined herein in which one or two of the component carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, the heteroalkyl group may be further substituted with one, two, three, or four substituents as described herein for the alkyl group. As used herein, the terms “heteroalkenyl” and “heteroalkynyl” mean an alkenyl group and an alkynyl group as defined herein in which one or two of the component carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, the heteroalkenyl group and the heteroalkynyl group may be further substituted with one, two, three, or four substituents as described herein for the alkyl group.

[0147] As used herein, the term “heteroaryl” refers to a subset of heterocyclyls as defined herein that are aromatic; that is, they contain 4n+2 π electrons within a monocyclic or polycyclic system. Exemplary unsubstituted heteroaryl groups have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbon atoms. In some embodiments, the heteroaryl is substituted with 1, 2, 3, or 4 substituents defined for the heterocyclyl group.

[0148] The term "heteroarylalkyl" means a heteroaryl group as defined herein, bonded to a parent molecule by an alkylene group as defined herein. Exemplary unsubstituted heteroarylalkyls include 2 to 32 carbon atoms (e.g., 2 to 22, 2 to 18, 2 to 17, 2 to 16, 3 to 15, 2 to 14, 2 to 13, or 2 to 12 carbon atoms, e.g., C 1~6 Alk-C 1~12 Heteroaryl, C 1~10 Alk-C 1~12 Heteroaryl, or C 1~20 Alk-C 1~12 Heteroarylalkyl groups are heteroaryl groups. In some embodiments, alkylenes and heteroaryl groups can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for their respective groups. Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

[0149] As used herein, the term “heterocyclyl” refers to a five-membered, six-membered, or seven-membered ring containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, unless otherwise specified. A five-membered ring has zeros to two double bonds. Six- and seven-membered rings have zeros to three double bonds. An example of an unsubstituted heterocyclyl group is one to twelve (e.g., one to eleven, one to ten, one to nine, two to twelve, two to one, two to ten, or two to nine) carbon atoms. The term “heterocyclyl” also refers to a heterocyclic compound having a bridging polycyclic structure in which one or more carbons and / or heteroatoms bridge two non-adjacent members of a monoring, such as a quinuclidinyl group. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocycles are condensed with one, two, or three carbon rings, such as an aryl ring, cyclohexane ring, cyclohexene ring, cyclopentane ring, cyclopentene ring, or other monocyclic heterocycles, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, etc. Examples of condensed heterocyclyls include tropane and 1,2,3,5,8,8a-hexahydroindidine, including dihydro and tetrahydro forms in which one or more double bonds are reduced and replaced by hydrogen. Heterocyclic compounds include pyrrolyl, pyrrolinil, pyrrolidinyl, pyrazolyl, pyrazolinil, pyrazolidinyl, imidazolyl, imidazolinil, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridadinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinil, thiomorpholinil, thiazolyl, thiazolidinyl, isothiazolyl, isothi Azolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), prinyl, thiadiazolyl (e.g., 1,Examples of heterocyclyls include 2,3-thiadiazolyl, tetrahydrofuranil, dihydrofuranil, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranil, isobenzofuranil, and benzothienyl. Further exemplary heterocyclyls include 2,3,4,5-tetrahydro-2-oxoxoxazolyl, 2,3-dihydro-2-oxo-1H-imidazolyl, 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl), and 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g., 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl). Xo-5-methyl-5-phenyl-1H-imidazolyl), 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl), 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl4-amino-5-oxo-1H-triazolyl), 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g., 1,2 ,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl), 2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl), 1,6-dihydro-6-oxopyrimidinyl, 1,6-dihydro-4-oxopyrimidinyl (e.g., 2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidine-1-yl), 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl ( For example, 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl), 1,6-dihydro-6-oxo-pyridazinyl (for example, 1,6-dihydro-6-oxo-3-ethylpyridazinyl), 1,6-dihydro-6-oxo-1,2,4-triazinyl (for example, 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl), 2,3-dihydro-2-oxo-1H-indolyl (for example, 3,3-dimethyl-2,3-Dihydro-2-oxo-1H-indolyl and 2,3-Dihydro-2-oxo-3,3'-Spiropropan-1H-indole-1-yl), 1,3-Dihydro-1-oxo-2H-iso-indolyl, 1,3-Dihydro-1,3-Dioxo-2H-iso-indolyl, 1H-Benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-Benzopyrazolyl), 2,3-Dihydro-2-oxo-1H-Benzimidazo Lyl (e.g., 3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl), 2,3-dihydro-2-oxo-benzoxazolyl (e.g., 5-chloro-2,3-dihydro-2-oxobenzoxazolyl), 2,3-dihydro-2-oxo-benzoxazolyl, 2-oxo-2H-benzopyranyl, 1,4-benzodioxanyl, 1,3-benzodioxanyl, 2,3-dihydro-3-oxo,4H-1, 3-Benzothiadinyl, 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g., 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl), 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g., 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl), 1,2,3,6-tetrahydro-2,6-dioxo-7H-prinyl (e.g., 1,2,3 Examples include ,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-prinyl), 1,2,3,6-tetrahydro-2,6-dioxo-1H-prinyl (for example, 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-prinyl), 2-oxobenz[c,d]indolyl, 1,1-dioxo-2H-naphtho[1,8-c,d]isothiazolyl, and 1,8-naphthylenedicarboxamide. Additional heterocyclic compounds include 3,3a,4,5,6,6a-hexahydropyrrolo[3,4-b]pyrrole-(2H)-yl, and 2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanil), tetrahydropyranil, dithiazolyl, benzofuranil, benzothienyl, oxepanil, thiepanil, azocanil, oxecanil, and thiocanil. Heterocyclic groups also include those with the chemical formula,

[0150] [ka]

[0151] (In the formula, Other examples include groups represented by (1)C 1~7 Acyl (e.g., carboxyaldehyde), (2)C 1~20 Alkyl (for example, alkyl, alkoxy-C) 1~6 Alkyl, alkylsulfinyl-C 1~6 Alkyl, amino-C 1~6 Alkyl, Azide-C 1~6 Alkyl, (carboxyaldehyde)-C 1~6 Alkyl, Halo-C 1~6 Alkyl (e.g., perfluoroalkyl), hydroxy-C 1~6 Alkyl, Nitro-C 1~6 Alkyl, or C 1~6 Thioalkoxy-C 1~6 (Alkyl), (3)C 1~20 Alkoxy (for example, C 1~6 Alkoxy, for example, perfluoroalkoxy), (4)C 1~6 Alkyl sulfinyl, (5)C 6~10 (6) Aryl, (7) Amino, C 1~6 Alk-C 6~10 (8) Azid, (9) C 3~8 Cycloalkyl, (10)C 1~6 Alk-C 3~8 Cycloalkyl, (11) Halo, (12) C 1~12 Heterocyclyls (e.g., C2-12 heteroaryls), (13)(C 1~12(14)Hydroxy, (15)Nitro, (16)C 1~20 Thioalkoxy (receive C 1~6 Thiokoxy), (17)-(CH2) q CO2R A’ (In the formula, q is an integer from 0 to 4, and R A’ (a) alkyl, (b) C 6~10 (c) aryl, and (d) C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (18)-(CH2) q CONR B’ R C’ (In the formula, q is an integer from 0 to 4, and R B’ and R C’ (a) hydrogen, (b) alkyl, (c) C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Independently selected from the group consisting of aryls), (19)-(CH2) q SO2R D’ (In the formula, q is an integer from 0 to 4, and R D’ (a)C 1~6 Alkyl, (b)C 6~10 Aryl, and (c)C 1~6 Alk-C 6~10 (Selected from the group consisting of aryls), (20)-(CH2) q SO2NR E’ R F’ (In the formula, q is an integer from 0 to 4, and R E’ and R F’ Each of them is (a) hydrogen, (b) C 1~6 Alkyl, (c)C 6~10 Aryl, and (d)C 1~6 Alk-C 6~10 (Independently selected from the group consisting of aryls), (21) thiol, (22) C 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24) Arylalkoxy, (25) C 1~6 Alk-C 1~12 Heterocyclines (for example, C 1~6 Alk-C 1~12(heteroaryl), (26) oxo, (27) (C 1~12 Heterocyclyl(imino), (28)C 2~20 Alkenil, and (29)C 2~20 The substituents can be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkynyls. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of C1-alkaryl or C1-alkheterocyclyl can be further substituted with an oxo group to give the respective allyroyl substituent and (heterocyclyl)oil substituent.

[0152] As used herein, the “heterocyclylalkyl” group represents a heterocyclyl group as defined herein, bonded to a parent molecule by an alkylene group as defined herein. An example of an unsubstituted heterocyclylalkyl group is one with 2 to 32 carbon atoms (e.g., 2 to 22, 2 to 18, 2 to 17, 2 to 16, 3 to 15, 2 to 14, 2 to 13, or 2 to 12 carbon atoms, e.g., C 1~6 Alk-C 1~12 Heterocyclyl, C 1~10 Alk-C 1~12 Heterocyclyl, or C 1~20 Alk-C 1~12 (Heterocyclyl). In some embodiments, the alkylene and heterocyclyl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for each group.

[0153] As used herein, the term "hydrocarbon" refers to a group consisting only of carbon atoms and hydrogen atoms. The term "hydroxyl as used herein" refers to an -OH group. In some embodiments, the hydroxyl group can be substituted for the alkyl group with one, two, three, or four substituents (e.g., O-protecting groups) as defined herein.

[0154] As used herein, the term “isomer” means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the present invention. Compounds of the present invention may have one or more chiral centers and / or double bonds, and are therefore recognized as being able to exist as stereoisomers such as double bond isomers (i.e., E / Z geometric isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the present invention, the chemical structures represented herein, i.e., compounds of the present invention, encompass all corresponding stereoisomers, i.e., stereomeric pure forms (e.g., geometric pure forms, enantimeric pure forms, or diasteremeric pure forms) as well as enantiomer mixtures and stereoisomer mixtures (e.g., racemates). Enantiomer and stereoisomer mixtures of the compounds of the present invention are typically separable into enantiomers or stereoisomers of their components by known methods such as chiral phase gas chromatography, chiral phase high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, and crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantimerically pure intermediates, reagents, and catalysts by known asymmetric synthesis methods.

[0155] As used herein, the term “N-protected amino” means an amino group as defined herein that is bonded to one or two N-protecting groups as defined herein. As used herein, the term “N-protecting group” refers to a group intended to protect an amino group from undesirable reactions during a synthetic procedure. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd edition (John Wiley & Sons, New York, 1999) (incorporated herein by reference).N-protecting groups include acyl groups, allyl groups, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries, such as protected or deprotected D,L, or D,L-amino acids, even For example, sulfonyl-containing groups such as alanine, leucine, and phenylalanine, carbamate-forming groups such as benzenesulfonyl and p-toluenesulfonyl, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-Dimethoxybenzyloxycarbonyl, 4-Methoxybenzyloxycarbonyl, 2-Nitro-4,5-Dimethoxybenzyloxycarbonyl, 3,4,5-Trimethoxybenzyloxycarbonyl, 1-(p-Biphenylyl)-1-Methylethoxycarbonyl, α,α-Dimethyl-3,5-Dimethoxybenzyloxycarbonyl, Benzhydryloxycarbonyl, t-Butyloxycarbonyl, Diisopropylmethoxycarbonyl, Isopropyloxycarbonyl, Ethoxycarbonyl Examples of N-protecting groups include methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc., as well as alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, etc., and silyl groups such as trimethylsilyl. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0156] As used herein, the term "nitro" refers to the -NO2 group. As used herein, the term “O-protecting group” refers to a group intended to protect an oxygen-containing group (e.g., phenol, hydroxyl, or carbonyl) from undesirable reactions during a synthetic procedure. Commonly used O-protecting groups are described in "Protective Groups in Organic Synthesis," 3rd edition, by John Wiley & Sons, by Greene. Disclosed in Wiley & Sons, New York, 1999 (incorporated herein by reference). Exemplary O-protecting groups include acyl groups, allyloyl groups, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, and fu. Phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl, alkylcarbonyl groups such as acyl, acetyl, propionyl, pivaloyl, etc., optionally substituted arylcarbonyl groups such as benzoyl, silyl groups such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), etc., with hydroxyl Tel-forming groups, such as methyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, trityl, etc., alkoxycarbonyls, such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbonyl, etc. Coxyalkoxycarbonyl groups, for example, methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl, etc., haloalkoxycarbonyl groups, for example, 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,Optionally substituted aryl alkoxycarbonyl groups such as 2-trichloroethoxycarbonyl, for example benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, fluorenylmethyloxycarbonyl, and optionally substituted aryl oxycarbonyl groups, For example, phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p-methylphenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxycarbonyl, etc.), substituted alkyl, aryl, and alkaryl ethers (for example, trityl, methylthiomethyl, methoxymethyl, benzyloxymethyl, siloxymethyl) , 2,2,2,-trichloroethoxymethyl, tetrahydropyranyl, tetrahydrofuranyl, ethoxyethyl, 1-[2-(trimethylsilyl)ethoxy]ethyl, 2-trimethylsilylethyl, t-butyl ether, p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl), silyl ethers (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzyme) Lucilyl, triphenylsilyl, and diphenylmethylsilyl), carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, vinyl, allyl, nitrophenyl, benzyl, methoxybenzyl, 3,4-dimethoxybenzyl, and nitrobenzyl), carbonyl protecting groups (e.g., acetal groups and ketal groups, e.g., dimethylacetal, 1,3-dioxolane, etc., acylar groups, and dithiane groups, e.g., 1,3-dithiane, 1,Examples include 3-dithiolane, carboxylic acid protecting groups (e.g., ester groups such as methyl esters, benzyl esters, t-butyl esters, ortho esters, etc.), and oxazoline groups.

[0157] As used herein, the term "oxo" represents =O. As used herein, the prefix "perfluoro" refers to any group as defined herein in which each hydrogen group bonded to an alkyl group is replaced by a fluoride group. Examples of perfluoroalkyl groups include trifluoromethyl and pentafluoroethyl.

[0158] As used herein, the term "protected hydroxyl" refers to an oxygen atom bonded to an O-protecting group. As used herein, the term “spirocyclyl” refers to a C group in which both ends are bonded to the same carbon atom of the parent group to form a spirocyclic group. 2~7 The term represents both a divalent alkylene group and a divalent heteroalkylene group in which both ends are bonded to the same atom. The heteroalkylene group forming the spirocyclyl group can contain 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl group contains 1 to 7 carbon atoms, excluding the carbon atom to which the divalent group is bonded. The spirocyclyl group of the present invention may be optionally substituted with 1, 2, 3, or 4 substituents provided herein as optional substituents for cycloalkyl groups and / or heterocyclyl groups.

[0159] As used herein, the term “stereoisomer” means all possible different isomers and conformational forms that a compound (for example, a compound of any chemical formula described herein) can take, in particular all possible stereochemical and conformational isomers of the basic molecular structure, all diastereomers, enantiomers, and / or conformers. Some of the compounds of the present invention may exist in different tautomers, and all of these tautomers are included within the scope of the present invention.

[0160] As used herein, the term "sulfonyl" refers to a -S(O)2- group. As used herein, the term "thiol" refers to the -SH group. definition In this application, unless otherwise clearly indicated by the context, (i) the term "a" can be understood to mean "at least one"; (ii) the term "or" can be understood to mean "and / or"; (iii) the terms "comprising" and "including" can be understood to encompass itemized components or processes, whether presented by themselves or together with one or more additional components or processes; (iv) the terms "about" and "approximately" can be understood to allow a standard deviation as understood by those skilled in the art; and (v) where a range is provided, it includes endpoints.

[0161] As is well known in the art, "affinity" is a measure of the tightness with which a particular ligand binds to its partner. Affinity can be measured in various ways. In some embodiments, affinity is measured by quantitative assays. In some such embodiments, the binding partner concentration may be fixed in excess of the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the binding partner concentration and / or ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under equivalent conditions (e.g., concentration).

[0162] As used herein, the terms “approximately” and “about” are intended to encompass the normal statistical variation understood by those skilled in the art, where appropriate in the relevant context. In certain embodiments, the terms “approximately” or “about” mean, respectively, a range of values ​​that fall within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, in either direction (greater or less) of the stated value, unless otherwise specified or is particularly obvious from the context (for example, if such number exceeds 100% of the possible value).

[0163] It will be understood that the term “bonding” as used herein typically means an association between two or more entities (e.g., non-covalent or covalent). “Direct” bonding includes physical contact between entities or parts, while indirect bonding includes physical interaction through physical contact with one or more intermediate entities. Bonding between two or more entities can typically be evaluated in any of a variety of situations, including when studying the interacting entities or parts in isolation or in more complex systems (e.g., covalently or otherwise associated with a carrier entity and / or a biological system or cell).

[0164] The affinity between molecule X and its partner Y is generally expressed by the dissociation constant (K D ) can be expressed by. Affinity can be measured by conventional methods known in the art, including those described herein. Specific exemplary and typical embodiments for measuring binding affinity are described below. The term "K" used herein refers to D The term "compound-protein" is intended to mean the dissociation equilibrium constant of a particular compound-protein or complex-protein interaction. Typically, the compounds of the present invention, when determined, for example, by surface plasmon resonance (SPR) technique using a presenter protein as an analyte and the compound as a ligand, have a dissociation equilibrium constant of about 10. -6 Less than M, for example, about 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M, or even smaller dissociation equilibrium constants (K D ) binds to the presenter protein. The presenter protein / compound complex of the present invention, when determined by surface plasmon resonance (SPR) technique using, for example, the target protein as an analyte and the complex as a ligand, has a coefficient of approximately 10. -6 Less than M, for example, about 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M, or even smaller dissociation equilibrium constants (K D It binds to target proteins (for example, eukaryotic target proteins such as mammalian target proteins or fungal target proteins, or prokaryotic target proteins such as bacterial target proteins).

[0165] As used herein, the term “crosslinking group” refers to a group comprising a reactive functional group that can chemically attach to certain functional groups on proteins or other molecules (e.g., primary amines, sulfhydryls). “Molotion capable of chemoselective reaction with amino acids” refers, as used herein, to a molotion comprising a reactive functional group that can chemically attach to the functional groups of natural or unnatural amino acids (e.g., primary and secondary amines, sulfhydryls, alcohols, carboxyl groups, carbonyls, or triazole-forming functional groups, e.g., azides or alkynes). Examples of crosslinking groups include sulfhydryl-reactive crosslinking groups (e.g., groups containing maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, or vinyl sulfone), amine-reactive crosslinking groups (e.g., groups containing esters, e.g., NHS esters, imide esters, and pentafluorophenyl esters, or hydroxymethylphosphine), carboxyl-reactive crosslinking groups (e.g., groups containing primary or secondary amines, alcohols, or thiols), carbonyl-reactive crosslinking groups (e.g., groups containing hydrazides or alkoxyamines), and triazole-forming crosslinking groups (e.g., groups containing azides or alkynes).

[0166] As used herein, the term “complex” refers to a group of two or more compounds and / or proteins that are bound together by binding interactions (e.g., non-covalent interactions, e.g., hydrophobic interaction, electrostatic interaction, van der Waals interaction, or π-effect interaction). Examples of complexes include “presenter protein / conjugate complexes” and “target protein / conjugate complexes,” which include the conjugate of the present invention bound to a presenter protein or a target protein. As used herein, the term “conjugate” refers to a compound formed by the joining (e.g., by a covalent bond formation reaction) of two or more compounds (e.g., a crosslinking group and a protein, e.g., a compound comprising a target protein or presenter protein).

[0167] As used herein, an atom “involved in bonding” is defined as an atom located within 4 Å of the entity to which it is bonded, or an atom attached to an atom located within 4 Å of the entity to which it is bonded.

[0168] The term “presenter protein” refers to a protein that binds to a small molecule to form a complex that binds to a target protein (e.g., a eukaryotic target protein such as a mammalian or fungal target protein, or a prokaryotic target protein such as a bacterial target protein) and modulates its activity. In some embodiments, the presenter protein is a relatively abundant protein (e.g., the presenter protein is abundant enough that its involvement in the tripartite complex does not substantially affect the presenter protein’s biological role in the cell and / or the cell’s viability or other attributes). In certain embodiments, the presenter protein is a protein that has chaperone activity in the cell. In some embodiments, the presenter protein is a protein that has multiple innate interaction partners in the cell. In certain embodiments, the presenter protein is one that is known to bind to a small molecule to form a binary complex that is known or presumed to bind to a target protein and modulate its biological activity.

[0169] The term "presenter protein binding site" means that the compound specifically binds to the presenter protein with a KD of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM), or with an IC of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM). 50Therefore, this refers to groups of atoms involved in binding to the presenter protein and the regions bound to them (e.g., atoms in 20 atoms, atoms in 15 atoms, atoms in 10 atoms, atoms in 5 atoms) in order to inhibit the peptidyl-prolyl isomerase activity of the presenter protein. It should be understood that the presenter protein binding region does not necessarily encompass all the atoms in the compound that interacts with the presenter protein. It should also be understood that one or more atoms of the presenter protein binding region may be located within the target protein binding region (e.g., eukaryotic target protein binding regions such as mammalian target protein binding regions or fungal target protein binding regions, or prokaryotic target protein binding regions such as bacterial target protein binding regions).

[0170] As used herein, “FKBP binding site” refers to a presenter protein binding site that is selective for a presenter protein in the FKBP family of proteins (e.g., FKBP12, FKBP12.6, FKBPP13, FKBP25, FKBP51, or FKBP52). “Selective FKBP binding site” refers to a binding site that is specific to one or more members (e.g., two, three, four, or five) of the FKBP family, rather than all other members of the FKBP family. “Non-selective FKBP binding site” refers to a binding site that has comparable affinity (up to 2x, up to 3x, up to 4x, up to 5x, or up to 10x) for all members of the FKBP family.

[0171] The term "protein binding site" refers to the group of atoms involved in binding to a protein (e.g., presenter protein or target protein), and the attached portion (e.g., up to 20 atoms, up to 15 atoms, up to 10 atoms, up to 5 atoms), and the compound has a K content of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM). D In conjunction with this, ICs specifically bind to the protein, or, for example, ICs in amounts less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM) 50 This inhibits the peptidyl-prolyl isomerase activity of the presenter protein. It is understood that the protein-binding region does not necessarily contain all atoms in the compound that interact with the protein.

[0172] As used herein, the term “react” refers to the process by which atoms of the same or different elements reconfigure themselves to form a novel substance. For example, the formation of a covalent bond between two atoms, or, for example, the reaction between a reactive amino acid on a protein and a crosslinking group that causes the covalent bond to form. Reactions can be measured by any method known in the art; for example, the formation of a reaction product can be determined by LC-MS or NMR.

[0173] As used herein, the term “reactive amino acid” refers to a natural or unnatural amino acid that contains a functional group (e.g., a nucleophilic functional group) that can be chemically attached to a specific functional group (e.g., a crosslinking group). Examples of reactive amino acids include cysteine, lysine, serine, and amino acids having an azide on their side chain. “Unreactive amino acid” refers to a natural or unnatural amino acid that does not contain a functional group that can be chemically attached to a specific functional group. Examples of unreactive amino acids include valine, alanine, isoleucine, telonine, and leucine.

[0174] The term “reference” is often used herein to describe a standard or control compound, individual, group, sample, sequence, or value that is compared to the compound, individual, group, sample, sequence, or value of interest. In some embodiments, the reference compound, individual, group, sample, sequence, or value is examined and / or determined substantially simultaneously with the examination or determination of the compound, individual, group, sample, sequence, or value of interest. In some embodiments, the reference compound, individual, group, sample, sequence, or value is a historical reference optionally embodied in tangible media. Typically, as understood by those skilled in the art, the reference compound, individual, group, sample, sequence, or value is determined or characterized under conditions equivalent to those used to determine or characterize the compound, individual, group, sample, sequence, or value of interest.

[0175] As used herein, the term “solvent-exposed amino acid” refers to an amino acid that is accessible to the solvent surrounding a protein. In some embodiments, the solvent-exposed amino acid is one that, when substituted, does not substantially alter the three-dimensional structure of the protein.

[0176] As used herein, the terms “specific binding” or “specific” mean the interaction between the binder and the target entity. As will be understood by those skilled in the art, an interaction is considered “specific” if, in the presence of alternative interactions, binding by KD at concentrations of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM) is favorable. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitopes, clefts, binding sites). It should be understood that specificity does not have to be absolute. In some embodiments, specificity may be evaluated in comparison to the specificity of the binder to one or more other possible target entities (e.g., competitors). In some embodiments, specificity is evaluated in comparison to the specificity of a reference specific binder. In some embodiments, specificity is evaluated in comparison to the specificity of a reference nonspecific binding agent.

[0177] The term “specific,” when used in relation to an active compound, is understood by those skilled in the art to mean that the compound identifies a possible target entity or state. For example, in some embodiments, a compound is said to bind “specifically” to a target if it preferentially binds to that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction depends on the presence of specific structural features of the target entity (e.g., epitopes, clefts, binding sites). It should be understood that specificity does not have to be absolute. In some embodiments, specificity may be evaluated in comparison to the specificity of the binder to one or more other possible target entities (e.g., competitors). In some embodiments, specificity is evaluated in comparison to the specificity of a reference-specific binder. In some embodiments, specificity is evaluated in comparison to the specificity of a reference-nonspecific binding activator. In some embodiments, the activator or entity does not detectably bind to competing alternative targets under the conditions of binding to its target entity. In some embodiments, the binder binds to its target entity with a higher on-rate, lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to competing alternative targets.

[0178] The term "substantial" means a qualitative condition that exhibits the whole or nearly whole range or degree of the characteristics or properties of an object. Those skilled in the field of biological technology will understand that it is rare for biological and chemical phenomena to reach a final state and / or proceed completely, or to achieve or avoid absolute results. Therefore, the term "substantial" is used herein to capture the possibility of a lack of completeness inherent in many biological and chemical phenomena.

[0179] As used herein, the term “substantially unbinding” to a particular protein means, for example, 10 times against the target. -4 M or higher, alternatively 10 -5 M or higher, alternatively 10 -6 M or higher, alternatively 10-7 M or higher, alternatively 10 -8 M or higher, alternatively 10 -9 M or higher, alternatively 10 -10 M or higher, alternatively 10 -11 M or higher, alternatively 10 -12 M or higher K D or 10 -4 M~10 -12 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 K within the range of M D It can be represented by a molecule or a part of a molecule that possesses [this characteristic].

[0180] The term “target protein” refers to any protein involved in a biological pathway associated with a disease, disorder, or condition. In some embodiments, the target protein is not mTOR or calcineurin. In some embodiments, the target protein can form a tripartite complex with a presenter protein and small molecules. In some embodiments, the target protein is a naturally occurring protein. In some such embodiments, the target protein is found naturally in certain mammalian cells (e.g., mammalian target protein), fungal cells (e.g., fungal target protein), bacterial cells (e.g., bacterial target protein), or plant cells (e.g., plant target protein). In some embodiments, the target protein is characterized by innate interactions with one or more naturally occurring presenter protein / natural small molecule complexes. In some embodiments, the target protein is characterized by innate interactions with multiple different naturally occurring presenter protein / natural small molecule complexes, and in some such embodiments, some or all of the complexes utilize the same presenter protein (and different small molecules). In some embodiments, the target protein does not substantially bind to a complex with cyclosporine, rapamycin, or FK506 and a presenter protein (e.g., FKBP). Target proteins may be naturally occurring, for example, wild-type. Alternatively, target proteins may differ from wild-type proteins but still maintain biological function, such as allele variants, splice mutants, or biologically active fragments. Exemplary mammalian target proteins include GTPases, GTPase-activating proteins, guanine nucleotide exchange factors, heat shock proteins, ion channels, coiled-coil proteins, kinases, phosphatases, ubiquitin ligases, transcription factors, chromatin modifiers / remodelers, proteins with classical protein-protein interaction domains and motifs, or any other proteins involved in biological pathways associated with disease, disorder, or pathology.

[0181] In some embodiments, the target protein is a modified target protein. The modified target protein may include conserved or non-conserved insertions, deletions, or substitutions of amino acids in the protein sequence (e.g., D-amino acids, des-amino acids) (for example, here such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine ​​residues to the amino or carboxyl terminus of any of the polypeptides of the present invention can, for example, promote the conjugation of these proteins by disulfide bonds. In some embodiments, one or more reactive amino acid residues (e.g., cysteine) are removed to reduce the number of possible conjugation sites on the protein. Amino acid substitutions may be conserved (i.e., here the residue is replaced by another of the same general type or group) or non-conserved (i.e., here the residue is replaced by a different type of amino acid). Furthermore, non-natural amino acids (i.e., conserved amino acid substitutions or non-conserved amino acid substitutions that are not of natural origin) may be substituted with natural amino acids. The term "target protein binding region" refers to the group of ring atoms and their associated regions (e.g., atoms in 20 atoms, atoms in 15 atoms, atoms in 10 atoms, atoms in 5 atoms) that are involved in binding a compound to a target protein (e.g., eukaryotic target proteins such as mammalian or fungal target proteins, or prokaryotic target proteins such as bacterial target proteins) when the compound is in complex with a presenter protein. It should be understood that the target protein binding region does not necessarily encompass all atoms in the compound that interact with the target protein. It should also be understood that one or more atoms in the presenter protein binding region may also be present in the target protein binding region.

[0182] The term "conventional binding pocket" refers to a cavity or pocket in a protein structure that has physiological and / or geometric properties comparable to those of a protein whose activity is modulated by one or more small molecules. In some embodiments, the conventional binding pocket is 1000A 3 It is a clearly defined pocket having a volume greater than or equal to. Those skilled in the art are familiar with the concept of a conventional binding pocket and are aware of its relationship to "druggability." In certain embodiments, a protein is considered to lack a conventional binding pocket if it is undruggable as defined herein.

[0183] The term "undruggable target" refers to a protein that is not a member of a protein family known to be targeted by a drug and / or does not have a binding site suitable for high-affinity binding to a small molecule. Methods for determining whether a target protein is undruggable are known in the art. For example, whether a target protein is undruggable can be determined using a structure-based algorithm, such as the DOGSITESCORER® program (Universitat Hamburg, Hamburg, Germany), which evaluates draggability based on parameters calculated for the binding pocket on the protein, including volume, surface area, lipophilic surface area, depth, and / or hydrophobicity ratio. [Brief explanation of the drawing]

[0184] [Figure 1] This image illustrates the SDS-PAGE analysis of the KRASGTP / S39Clite / C2-FK506 conjugate. Lane 1: KRASGTP / S39Clite; Lane 2: KRASGTP / S39Clite / C2-FK506 reaction mixture; Lane 3: KRASGTP / S39Clite / C2-FK506 reaction mixture + 100 mM DTT. [Figure 2]This image illustrates the SDS-PAGE analysis of the KRASGTP / G12Clite / SFAX9DS conjugate. [Figure 3A] This image illustrates SEC and SDS-PAGE analysis of KRASGTP / S39Clite / C2-Holt / FKBP12 complex formation. SEC purification profile. The blue dotted line indicates the peak corresponding to the elution of the KRASGTP / S39Clite / C2-Holt / FKBP12 ternary complex. [Figure 3B] This image illustrates SEC and SDS-PAGE analysis of KRASGTP / S39Clite / C2-Holt / FKBP12 complex formation. The image shows SDS-PAGE analysis of the SEC elution peak. The blue dotted line corresponds to the fraction collected for the KRASGTP / S39Clite / C2-Holt / FKBP12 elution peak. [Figure 4] This image illustrates the SEC profile and SDS-PAGE analysis of the elution peak, confirming the formation of the KRASGDP / S39Clite / SFAC4DS / CypAC52S complex. [Figure 5A] These images illustrate the SEC profiles and SDS-PAGE analyses of free PTP1BS187Clite and FKBP12 proteins, and the PTP1BS187Clite / C3-SLF / FKBP12 complex. [Figure 5B] These images illustrate the SEC profiles and SDS-PAGE analyses of free PTP1BS187Clite and FKBP12 proteins, and the PTP1BS187Clite / C3-SLF / FKBP12 complex. [Figure 6] This image illustrates the crosslinking efficiency of C3- and C4-SLF by SDS-PAGE. [Figure 7A] This image illustrates the crystal structure of the FKBP12-compound 1-KRASGTP / S39C complex. The ribbon-like representation shows FKBP12, KRASGTP / S39C, and the ligand. The Fo-Fc electron density at 3σ is shown for the ligand in the close-up image. [Figure 7B]This image illustrates the crystal structure of the FKBP12-compound 1-KRASGTP / S39C complex. It represents the surface of the complex, with atoms within 4 Å of the ligand or partner protein colored red. [Figure 8] This image illustrates the crystal structure of CypAC52S-SFAC4DS-KRASGDP / S39C. [Figure 9A] This image illustrates the crystal structure of FKBP12-C3SLF-PTP1BS187C. It illustrates that the crystal contains a complex molecule of two asymmetric units of FKBP12-C3SLF-PTP1BS187C. [Figure 9B] This image illustrates the crystal structure of FKBP12-C3SLF-PTP1BS187C. It illustrates that the embedded area of ​​PTP1BS187C is 427 Å2 and the embedded area of ​​C3-SLF is 615 Å2. [Figure 10] This image illustrates the crystal structure of MCL1S245C / C3SLF / FKBP52. [Figure 11] This image illustrates the binding curve for the W21487-dependent complex formation of the CYPA-W21487-KRASG12C-GTP ternary complex. [Figure 12] This image illustrates the binding curve for the W21487-dependent complex formation of the CYPA-W21487-KRASG12C-GTP ternary complex. [Figure 13] This image illustrates ITC measurements for the binding of the FKBP12-compound 1 and FKBP12-compound 2 binary complexes to CEP250. [Figure 14-1] This image illustrates an SPR sensorgram for the binding of FKBP12 / compound 1 to CEP25011.4 and CEP25029.2. [Figure 14-2] This image illustrates an SPR sensorgram for the binding of FKBP12 / compound 1 to CEP25011.4 and CEP25029.2. [Figure 15]This image illustrates the sensorogram and steady-state fitting curve for the binding of CYPA / compound 3 to KRASG12C-GTP. [Figure 16] This image illustrates the fluorescence polarization curve for CypA:C3DS:KRAS complex formation. [Figure 17A] This image (Figure 17A) illustrates the 2D1H-15N TROSY-HSQC spectrum of KRASG12C-GTP. [Figure 17B] This image illustrates the addition of stoichiometric amounts of CYPA. [Figure 17C] These images illustrate KRAS and CYPA individually. [Modes for carrying out the invention]

[0185] Small molecules have limited targeting capabilities because their interactions with targets are driven by adhesive forces, the strength of which is roughly proportional to the contact surface area. Due to their small size, the only way for small molecules to build a sufficient intermolecular contact surface area to effectively interact with target proteins is to encapsulate them. In fact, a large number of experimental and computational data support the view that only proteins with hydrophobic "pockets" on their surface can bind to small molecules. In this case, binding is made possible by encapsulation.

[0186] Nature has evolved strategies to interact small molecules with target proteins at sites other than the hydrophobic pocket. This strategy is exemplified by the naturally occurring immunosuppressants cyclosporine A, rapamycin, and FK506. The biological activity of these drugs requires the formation of high-affinity complexes between the small molecule and a small presenting protein. The complex surface of the small molecule and the presenting protein engages with the target. For example, the binary complex formed between cyclosporine A and cyclophylline A targets calcineurin with high affinity and specificity, whereas neither cyclosporine A nor cyclophylline A binds to calcineurin with measurable affinity on their own.

[0187] Many important therapeutic targets exert their functions through complexation with other proteins. Protein / protein interaction surfaces, in many of these systems, contain an inner core of hydrophobic side chains surrounded by a broad ring of polar residues. Since hydrophobic residues contribute to almost all energetically favorable contacts, this cluster has been represented as a "hot spot" for protein-protein interaction engagement. Importantly, in the aforementioned complexes of naturally occurring small molecules and small presenting proteins, the small molecule provides a cluster of hydrophobic functions similar to the hot spot, while the protein provides the ring of primarily polar residues. In other words, the presented small molecule system mimics a surface architecture widely utilized in natural protein / protein interaction systems.

[0188] Nature has demonstrated the ability to reprogram the target specificity of presented small molecule-portable hotspots through evolutionary diversification. In the best-characterized example, the complex formed between FK506-binding protein (FKBP) and FK506 targets calcineurin. However, FKBP can also form a complex with the related molecule rapamycin, and this complex interacts with a completely different target, TorC1. To date, no methods have been developed to reprogram the binding and modulating capabilities of presenter protein / ligand interfaces so that they can interact with and modulate other target proteins that have previously been considered untruggable.

[0189] Furthermore, it is widely acknowledged that several drug candidates fail to function effectively because they modulate the activity of both the intended target and other unintended proteins in the same way. This problem is particularly difficult when the drug binding site on the target protein is similar to the binding site on the non-target protein. Insulin-like growth factor receptor (IGF-1R), whose ATP binding pocket is structurally similar to that of the non-target insulin receptor (IR), is one such example. Small molecule development candidates designed to target IGF-1R also typically have the unacceptable side effect of similarly modulating the insulin receptor. However, structural dissimilarities exist between these two proteins in the region surrounding the ATP binding pocket. Despite these findings, there is currently no known method to leverage these differences to develop drugs that are more specific to IGF-1R than to IR.

[0190] This disclosure provides methods and reagents useful for analyzing protein-protein interfaces, such as the interface between a presenter protein (e.g., a member of the FKBP family, a member of the cyclophyllin family, or PIN1) and a target protein. In some embodiments, the target and / or presenter protein is an intracellular protein. In some embodiments, the target and / or presenter protein is a mammalian protein. In some embodiments, these methods and reagents may be useful for identifying target proteins that are susceptible to inhibition or activation by forming complexes with the presenter protein and small molecules. In some embodiments, these methods and reagents may be useful in identifying compounds that can inhibit or activate target proteins by forming complexes with the presenter protein and target protein. Compounds and Conjugates This disclosure provides compounds comprising a protein-binding moiety (e.g., a presenter protein-binding moiety or a target protein-binding moiety) and a crosslinking group. The present invention also features a conjugate comprising a protein-binding moiety conjugated to a protein, e.g., a presenter protein-binding moiety conjugated to a target protein, or a target protein-binding moiety conjugated to a presenter protein.

[0191] This invention also relates to the compound ALB of chemical formula VII. (Formula VII) It is characterized by the fact that in the chemical formula, A is the structure of chemical formula VIII.

[0192] [ka]

[0193] Includes. In some embodiments, the compounds of the present invention are

[0194] [ka]

[0195] JPEG2026090316000022.jpg57170

[0196] That is the case. crosslinking group In some embodiments, the compounds of the present invention include a crosslinking group. A crosslinking group refers to a group containing a reactive functional group that can chemically attach to specific functional groups (e.g., primary amines, sulfhydryls) on proteins or other molecules. Examples of crosslinking groups include sulfhydryl reactive crosslinking groups (e.g., groups containing maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, or vinyl sulfone), amine reactive crosslinking groups (e.g., groups containing esters, e.g., NHS esters, imide esters, and pentafluorophenyl esters, or hydroxymethylphosphine), carboxyl reactive crosslinking groups (e.g., groups containing primary or secondary amines, alcohols, or thiols), carbonyl reactive crosslinking groups (e.g., groups containing hydrazides or alkoxyamines), and triazole-forming crosslinking groups (e.g., groups containing azides or alkynes).

[0197] Exemplary crosslinking groups include 2'-pyridyl disulfide, 4'-pyridyl disulfideiodoacetyl, maleimide, thioesters, alkyl disulfides, alkylamine disulfides, nitrobenzoic acid disulfide, anhydrides, NHS esters, aldehydes, alkyl chlorides, alkynes, and azides.

[0198] Presenter protein binding site In some embodiments, the compounds of the present invention include a presenter protein binding moiety. In some embodiments, the presenter protein binding moiety is configured such that the provided compound specifically binds to the presenter protein with a KD of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM) or with an IC of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM). 50Therefore, to inhibit the peptidyl-prolyl isomerase activity of the presenter protein, the presenter protein may include groups of atoms involved in binding to the presenter protein (e.g., 5-20 atoms, 5-10 atoms, 10-20 atoms) and the parts bound to them (e.g., atoms contained in 20 atoms, e.g., atoms contained in 15 atoms, atoms contained in 10 atoms, atoms contained in 5 atoms). In some embodiments, the presenter protein binding part does not include all of the atoms in the provided compound that interact with the presenter protein. In certain embodiments, one or more atoms of the presenter protein binding part do not interact with the presenter protein.

[0199] In some embodiments, the presenter protein binding moiety comprises an N-acylproline moiety, an N-acylpipecolic acid moiety, an N-acyl-3-morpholinocarboxylic acid moiety, and / or an N-acylpiperazine acid moiety (for example, any of the nitrogen atoms are acylated). In certain embodiments, the presenter protein binding moiety comprises an N-acylpipecolic acid moiety. In some embodiments, the presenter protein binding moiety comprises an N-acylproline moiety. In certain embodiments, the presenter protein binding moiety comprises an N-acyl-3-morpholinocarboxylic acid moiety. In some embodiments, the presenter protein binding moiety comprises an N-acylpiperazine acid moiety.

[0200] In some embodiments, at least one atom of the presenter protein binding moiety is involved in binding to one or more of the following FKBP12 atoms: Tyr27, Phe37, Asp38, Arg41, Phe47, Gln54, Glu55, Val56, Ile57, Trp60, Ala82, Try83, His88, Ile92, and / or Phe100 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In some embodiments, at least one of the presenter protein binding moiety is involved in binding to at least one of the following FKBP12 atoms: Arg41, Gln54, Glu55, and / or Ala82 (e.g., 2, 3, or 4).

[0201] In some embodiments, the presenter protein binding moiety is chemical formula II-IV:

[0202] [ka]

[0203] It has the structure shown in [image / diagram]. In some embodiments, the presenter protein binding portion is structure

[0204] [ka]

[0205] JPEG2026090316000025.jpg203170

[0206] JPEG2026090316000026.jpg223170

[0207] JPEG2026090316000027.jpg133170

[0208] It includes or consists of stereoisomers thereof. The presenter protein can bind to an atom at the presenter protein binding site. Alternatively, or further, the presenter protein can bind to two or more atoms at the presenter protein binding site. Another alternative is that the presenter protein can bind to a substituent attached to one or more atoms at the presenter protein binding site. Furthermore, in some embodiments, the presenter protein can bind to an atom at the presenter protein binding site and to substituents attached to one or more atoms at the presenter protein binding site. In some embodiments, the presenter protein binds to a group that mimics the presenter protein's native ligand, where the group mimicking the presenter protein's native ligand is attached to the presenter protein binding site. In some embodiments, the presenter protein binds to the presenter protein, and the affinity of the presenter protein for the presenter protein in the binary complex increases compared to the affinity of the presenter protein for the presenter protein in the absence of the complex. Binding in such examples is typically, but not limited to, due to non-covalent interactions of the presenter protein to the presenter protein binding site.

[0209] Target protein binding site In some embodiments, the compounds of the present invention include a target protein binding moiety (e.g., a eukaryotic target protein binding moiety, e.g., a mammalian target protein binding moiety or a fungal target protein binding moiety or a prokaryotic target protein binding moiety, e.g., a bacterial target protein binding moiety). In some embodiments, the target protein binding moiety includes a group of atoms (e.g., 5 to 20 atoms, 5 to 10 atoms, 10 to 20 atoms) and may include any portion attached thereto that specifically binds to the target protein (e.g., up to 20 atoms, up to 15 atoms, up to 10 atoms, up to 5 atoms). In some embodiments, the target protein binding moiety includes multiple atoms in the compound that interact with the target protein. In certain embodiments, one or more atoms of the target protein binding moiety do not interact with the target protein.

[0210] The target protein can bind to an atom in the target protein binding site. Alternatively, or further, the target protein can bind to two or more atoms in the target protein binding site. Another option is that the target protein can bind to substituents attached to one or more atoms in the target protein binding site. Yet another option is that the target protein can bind to an atom in the target protein binding site and substituents attached to one or more atoms in the target protein binding site. Yet another option is that the target protein binds to a group that mimics the native ligand of the target protein, and the group that mimics the native ligand of the target protein binds to the target protein binding site. Yet another option is that the target protein binds to a presenter protein, and the affinity of the target protein to the presenter protein in the binary complex increases in contrast to the affinity of the target protein to the presenter protein in the absence of the complex. Binding in these examples typically, but are not limited to, mediated by non-covalent interactions of the target protein to the target protein binding site. Linker The compounds of the present invention include a linker (e.g., a partial linker) that connects a protein-binding portion (e.g., a presenter protein-binding portion or a target protein-binding portion) to a crosslinking group, or a linker that connects a protein-binding portion to a protein (e.g., a presenter protein or a target protein). The linker components of the present invention, in their simplest form, are bonds, but can also provide a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking two portions.

[0211] In some embodiments, at least one atom of the linker is involved in binding to the presenter protein and / or target protein. In certain embodiments, at least one atom of the linker is not involved in binding to the presenter protein and / or target protein.

[0212] Thus, when a linker is included in a compound and / or conjugate as described herein, it achieves linkage of two (or more) parts by covalent means involving bond formation with one or more functional groups located on either part. Examples of chemically reactive functional groups that can be used for this purpose include, but are not limited to, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, adjacent diols, thioethers, 2-amino alcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenol groups.

[0213] In some embodiments, covalent bonding of two or more parts can be achieved using a linker containing a reactive moiety capable of reacting with such functional groups present in both parts. For example, the amine group of one part can react with the carboxyl group or an activated derivative of the linker to form an amide that bonds the two.

[0214] Examples of moieties that can react with sulfhydryl groups include α-haloacetyl compounds of the XCH2CO- (where X = Br, Cl, or I) type. As described in Gurd's *Methods Enzymol.*, Vol. 11, p. 532, 1967, this type not only exhibits specific reactivity with sulfhydryl groups but can also be used to modify imidazolyl, thioether, phenol, and amino groups. N-maleimide derivatives are also considered to be selective for sulfhydryl groups, and may also be useful for coupling with amino groups under certain conditions. Reagents such as 2-iminothiolanes, which introduce thiol groups by converting amino groups (Traut et al., *Biochemistry*, Vol. 12, p. 3266, 1973), can be considered sulfhydryl reagents if the coupling occurs through the formation of disulfide bridges.

[0215] Examples of reactive moieties that can react with amino groups include alkylating agents and acylating agents. Typical alkylating agents include: (i) α-haloacetyl compounds, which, as described for example in Wong, Biochemistry, Vol. 24, p. 5337, 1979, exhibit specificity for amino groups in the absence of reactive thiol groups and are of the XCH2CO- type (where X = Br, Cl, or I), (ii) N-maleimide derivatives, which can react with an amino group via a Michael-type reaction or via acylation by addition to a ring carbonyl group, as described, for example, in Smyth et al., Journal of the American Chemical Society (J.Am.Chem.Soc.), Vol. 82, p. 4600, 1960, and Biochemical Journal (Biochem.J.), Vol. 91, p. 589, 1964. (iii) Aryl halides, for example, reactive nitro-aromatic compounds, (iv) Alkyl halides, e.g., McKenzie et al., Journal of Protein Chemistry (J. Protein Chem.), Vol. 7, p. 581, 1988. (v) Aldehydes and ketones capable of forming an amino group and a Schiff base, the adducts formed usually yield amines that are stable upon reduction. (vi) Epoxide derivatives such as epichlorohydrin and bisoxiran, which can react with amino groups, sulfhydryl groups, or phenolic hydroxyl groups. (vii) chlorine-containing derivatives of (vii)s-triazines, which are highly reactive with nucleophiles such as amino groups, sulfhydryl groups, and hydroxyl groups. (viii) Aziridines based on s-triazine compounds, as detailed above, for example, described by Ross in the Journal of Advanced Cancer Research (J.Adv.Cancer Res.), Vol. 2, p. 1, 1954, which react with nucleophiles such as amino groups upon ring opening, (ix) Diethyl squalate, described in Tietze, Hemische Berchte, Vol. 124, p. 1215, 1991, and (x)α-haloalkyl ethers, as described by Benneche et al. in the European Journal of Medicinal Chemistry (Eur.J.Med.Chem.), Vol. 28, p. 463, 1993, are alkylating agents that are more reactive than ordinary alkyl halides due to activation caused by the ether oxygen atom. These are some examples.

[0216] Typical amino-reactive acylating agents include: (i) Isocyanates and isothiocyanates, in particular aromatic derivatives, which form stable urea derivatives and thiourea derivatives, respectively. (ii) Sulfonyl chloride, which is described in Herzig et al., Biopolymers, Vol. 2, p. 349, 1964. (iii) Acid halides, (iv) Active esters, for example, ester nitrophenyl esters or N-hydroxysuccinimidyl esters, (v) Acid anhydrides, e.g., mixed type, symmetric type, or N-carboxyanhydride, (vi) Other useful reagents for amide bond formation, e.g., Principles of Peptide Synthesis by M. Bodansky. (Synthesis), described in Springer-Verlag, 1984. (vii) Acyl azide, this azide group is generated from hydrazide derivatives pre-formed using sodium nitrite, as described in Wetz et al., Analytical Biochem., Vol. 58, p. 347, 1974. (viii) Imide esters, which, as described for example, in Hunter and Ludwig, Journal of the American Chemical Society (J.Am.Chem.Soc.), Vol. 84, p. 3491, 1962, react with an amino group to form a stable amidine, and (ix) Haloheteroaryl group, for example, halopyridine or halopyrimidine These are some examples.

[0217] Aldehydes and ketones can react with amines to form Schiff bases, which can be favorably stabilized by reductive amination. The alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, as described, for example, in Webb et al., Bioconjugate Chem., Vol. 1, p. 96, 1990.

[0218] Examples of reactive moieties that can react with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides. These react with high specificity to produce ester groups, as described, for example, in Herriot's *Adv. Protein Chem.*, Vol. 3, p. 169, 1947. Carbodiimides, which react via O-acylurea formation and subsequent amide bond formation, are also available as carboxyl modification reagents.

[0219] For example, it may be seen that, if desired, the functional groups of any part may be converted to other functional groups before the reaction in order to impart additional reactivity or selectivity. Examples of methods useful for this purpose include the conversion of amines to carboxyls using reagents such as dicarboxylic acid anhydrides, the conversion of amines to thiols using reagents such as N-acetylhomocysteine ​​thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, and thiol-containing succinimidyl derivatives, the conversion of thiols to carboxyls using reagents such as α-haloacetates, the conversion of thiols to amines using reagents such as ethyleneimine and 2-bromoethylamine, the conversion of carboxyls to amines using reagents such as carbodiimide and subsequently diamines, and the conversion of alcohols to thiols using reagents such as tosyl chloride, followed by transesterification with thioacetate, and hydrolysis to thiols with sodium acetate.

[0220] If desired, a so-called zero-length linker may be used, which involves directly covalently bonding the reactive chemical groups of one portion to the reactive chemical groups of the other portion without introducing additional binding material, in accordance with the present invention.

[0221] However, more generally, a linker comprises two or more reactive moieties connected by a spacer element as described above. The presence of such spacers allows a bifunctional linker to react with a specific functional group in either moiety, generating a covalent bond between the two. The reactive moieties in the linker may be identical (homobifunctional linker) or different (heterobifunctional linker or polyfunctional linker if several dissimilar reactive moieties are present), providing a variety of possible reagents that can generate a covalent bond between the two moieties.

[0222] The spacer elements in the linker typically consist of a straight chain or a branched chain, C 1~10 Alkyl, C 2~10 Alkenil, C 2~10 Alkinyl, C 2~ 6 heterocyclyl, C 6~12 Ariel, C 7~14 Alkaline, C 3~10 Alkheterocykrill, C2~C 100 Polyethylene glycol, or C 1~10 It may contain heteroalkyl groups.

[0223] In some cases, the linker is described by the chemical formula V. Examples of homobifunctional linkers useful for preparing the conjugates of the present invention include, but are not limited to, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactonediol.

[0224] In some embodiments, the linker is a bond or a linear chain of up to 10 atoms independently selected from carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, or phosphorus atoms, each atom in the chain being optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamide, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and any two atoms in the chain may form a ring together with substituents attached to them, and the ring may undergo further substitution and / or fusion to one or more optionally substituted carbocyclic, heterocyclic, arylcyclic, or heteroarylcyclic rings.

[0225] In some embodiments, the linker is of chemical formula XIX: A 1 -( B 1 ) a -(C 1 ) b -( B 2 ) c -(D)-(B 3 ) d -(C 2 )e-(B 4 ) f -A 2 Chemical formula XIX (In the formula, A 1 This is the bond between the linker and the presenter protein binding site, A 2 This is the bond between the mammalian target interaction portion and the linker, B 1 B 2 B 3 , and B 4 These are, independently, optionally substituted C1-C2 alkyl groups, optionally substituted C1-C3 heteroalkyl groups, O, S, and NR. N Selected from, R N C is a hydrogen atom that is optionally substituted with other carbon atoms.1~4 Alkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkinyl, optionally substituted C 2~6 Heterocyclines, optionally substituted C 6~12 Aryl, or optionally substituted C 1~7 It is a heteroalkyl group, C 1 and C 2 Each is independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, and a, b, c, d, e, and f are independently 0 or 1, and D is optionally substituted with C 1~10 Alkyl, optionally substituted C 2~10 Alkenyl, optionally substituted C 2~10 Alkinyl, optionally substituted C2-6 heterocyclyl, optionally substituted C 6~12 Aryl, optionally substituted C2-C 10 Polyethylene glycol, or optionally substituted C 1~10 Heteroalkyl, or A 1 -( B 1 )a-(C 1 )b-(B 2 )c- and -(B 3 )d-(C 2 )e-(B 4 )fA 2 It has a structure that is a chemical bond connecting two things.

[0226] protein Presenter protein Presenter proteins can bind to small molecules to form complexes, and these complexes can bind to target proteins (e.g., eukaryotic target proteins such as mammalian or fungal target proteins, or prokaryotic target proteins such as bacterial target proteins) to modulate their activity. In some embodiments, the presenter protein is a mammalian presenter protein (e.g., a human presenter protein). In some embodiments, the presenter protein is a fungal presenter protein. In certain embodiments, the presenter protein is a bacterial presenter protein. In some embodiments, the presenter protein is a plant presenter protein. In some embodiments, the presenter protein is a relatively abundant protein (e.g., the presenter protein is sufficiently abundant that its involvement in the tripartite complex does not substantially adversely affect the presenter protein's biological role and / or the cell's viability or other attributes). In some embodiments, the presenter protein is a more abundant target protein. In certain embodiments, the presenter protein is a protein with chaperone activity in the cell. In some embodiments, the presenter protein has multiple intrinsic interaction partners in the cell. In certain embodiments, the presenter protein is known to bind to a target protein and form a binary complex that modulates its biological activity, or is presumed to do so. Immunophilins are a class of presenter proteins known to have such functions, and include FKBP and cyclophilin. In some embodiments, the reference presenter protein exhibits peptidyl prolyl isomerase activity. In some embodiments, the presenter protein exhibits equivalent activity to the reference presenter protein.In certain embodiments, the presenter protein is a member of the FKBP family (e.g., FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36, FKBP38, FKBP51, FKBP52, FKBP60, FKBP65, and FKBP133), a member of the cyclophyllin family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, or PPIAL4G), or PIN1. The "FKBP family" is a family of proteins that possess prolyl isomerase activity and function as protein folding chaperones for proteins containing proline residues. Genes encoding proteins in this family include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and LOC541473.

[0227] The "cyclophyllin family" is a family of proteins that bind to cyclosporine. Genes encoding proteins in this family include PPIA, PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1, PPIL2, PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary cyclophyllins include PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, and PPIAL4G.

[0228] In some embodiments, the presenter protein is a chaperone protein, such as GRP78 / BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase (PDI), and ERp57.

[0229] In some embodiments, the presenter protein is an allele variant or splice variant of FKBP or cyclophyllin as disclosed herein. In some embodiments, the presenter protein is a polypeptide whose amino acid sequence includes i) a significant identity with that of a reference presenter protein, ii) a portion that shows significant identity with a corresponding portion of the reference presenter protein, and / or iii) a portion that includes at least one characteristic sequence found in the presenter protein. In many embodiments, identity is considered "significant" for the purpose of defining a presenter protein if it is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. In some embodiments, portions showing significant identity are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4 It has a length of 6, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 450, 500, 550, 600 or more amino acids.

[0230] Representative presenter proteins are encoded by the genes or homologs listed in Table 1. In some embodiments, the reference presenter protein is encoded by the set of genes shown in Table 1. Furthermore, those skilled in the art can easily identify sequences characteristic of presenter proteins in general and / or specific subsets of presenter proteins by referring to Table 1.

[0231] [Table 1]

[0232] target protein Target proteins (e.g., eukaryotic target proteins such as mammalian target proteins or fungal target proteins, or prokaryotic target proteins such as bacterial target proteins) are proteins that mediate disease pathology or the symptoms of disease pathology. Therefore, desirable therapeutic effects can be achieved by modulating (inhibiting or increasing) their activity. Useful target proteins for the complex and method of the present invention include those that are not associated with presenter proteins in nature, for example, those that have an affinity of more than 1 μM, preferably more than 5 μM, and more preferably more than 10 μM, for presenter proteins in the absence of a binary complex with the compound of the present invention. Alternatively, target proteins that are not associated with presenter proteins in nature are those that have an affinity of more than 1 μM, preferably more than 5 μM, and more preferably more than 10 μM, for the compound of the present invention in the absence of a binary complex. Other options include target proteins that are not naturally associated with the presenter protein and have an affinity greater than 1 μM, preferably greater than 5 μM, more preferably greater than 10 μM, for a binary complex of cyclosporine, rapamycin, or FK506 with the presenter protein (e.g., FKBP). Still other options include target proteins other than calcineurin and mTOR that are not naturally associated with the presenter protein. The selection of a suitable target protein for the complex and method of the present invention may depend on the presenter protein. For example, a target protein with low affinity for cyclophylline may have high affinity for FKBP, in which case it would not be used together with the latter.

[0233] The target protein may be naturally occurring, for example, in its wild-type form. Alternatively, the target protein may differ from the wild-type protein but still maintain its biological function, such as an allele variant, splice mutant, or biologically active fragment.

[0234] In some embodiments, the target protein is a transmembrane protein. In some embodiments, the target protein has a coiled-coil structure. In certain embodiments, the target protein is one protein in a dimer complex.

[0235] In some embodiments, the target protein of the present invention comprises one or more surface sites (e.g., flat surface sites) characterized by low or undetectable binding of small molecules to the site in the absence of presenter protein / compound complex formation. In some embodiments, the target protein comprises one or more surface sites (e.g., flat surface sites) that exhibit low or undetectable binding of a particular small molecule (e.g., a compound) in the absence of presenter protein / compound complex formation (e.g., binding of 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, or less, compared to the binding observed in a presenter protein / compound complex containing the same compound). In some embodiments, the target protein has a surface (in some embodiments, the entire surface) characterized by one or more sites lacking cavities or pockets on the protein structure that have physiological and / or geometric properties comparable to, for example, a protein whose activity is modulated by one or more small molecules, lacking any conventional binding pockets. In certain embodiments, the target protein has conventional binding pockets and sites suitable for protein-protein interactions. In some embodiments, the target protein is an untruggable target, for example, the target protein is not a member of a protein family known to be targeted by a drug and / or does not have a binding site that is expected to be suitable for binding to a small molecule (for example, according to the established understanding in the art as discussed herein). In some embodiments, the protein contains at least one reactive cysteine.

[0236] The most common type is the GTP interfaceば、DIRAS1、DIRAS2、DIRAS3、ERAS、GEM HRAS, KRAS, MRAS, NKIRAS1, NKIRAS2, NRAS, RALA, RALB, RAP1A, RAP1B, RAP 2A, RAP2B, RAP2C, RASD1, RASD2, RASL10A, RASL10B, RASL11A, RASL11B, RAS L12, REM1, REM2, RERG, RERGL, RRAD, RRAS, RRAS2, RHOA, RHOB, RHOBTB1, RHO BTB2、RHOBTB3、RHOC、RHOD、RHOF、RHOG、RHOH、RHOJ、RHOQ、RHOU、RHOV、RND1 RND2, RND3, RAC1, RAC2, RAC3, CDC42, RAB1A, RAB1B, RAB2, RAB3A, RAB3B AB3C, RAB3D, RAB4A, RAB4B, RAB5A, RAB5B, RAB5C, RAB6A, RAB6B, RAB6C, RAB 7A、RAB7B、RAB7L1、RAB8A、RAB8B、RAB9、RAB9B、RABL2A、RABL2B、RABL4、RAB1 0、RAB11A、RAB11B、RAB12、RAB13、RAB14、RAB15、RAB17、RAB18、RAB19、RAB2 0、RAB21、RAB22A、RAB23、RAB24、RAB25、RAB26、RAB27A、RAB27B、RAB28、RAB 2B, RAB30, RAB31, RAB32, RAB33A, RAB33B, RAB34, RAB35, RAB36, RAB37, RAB 38. RAB39, RAB39B, RAB40A, RAB40AL, RAB40B, RAB40C, RAB41, RAB42, RAB43 RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, ARF1, ARF3, ARF4, ARF5, ARF6, ARL1, A RL2, ARL3, ARL4, ARL5, ARL5C, ARL6, ARL7, ARL8, ARL9, ARL10A, ARL10B, ARL 10C、ARL11、ARL13A、ARL13B、ARL14、ARL15、ARL16、ARL17、TRIM23、ARL4D、A RFRP1, ARL13B, RAN, RHEB, RHEBL1, RRAD, GEM, REM, REM2, RIT1, RIT2, RHOT1or RHOT2. In some embodiments, the target protein is a GTPase-activating protein, such as NF1, IQGAP1, PLEXIN-B1, RASAL1, RASAL2, ARHGAP5, ARHGAP8, ARHGAP12, ARHGAP22, ARHGAP25, BCR, DLC1, DLC2, DLC3, GRAF, RALBP1, RAP1GAP, SIPA1, TSC2, AGAP2, ASAP1, or ASAP3. In some embodiments, the target protein is a guanine nucleotide exchange factor, such as CNRASGEF, RASGEF1A, RASGRF2, RASGRP1, RASGRP4, SOS1, RALGDS, RGL1, RGL2, RGR, ARHGEF10, ASEF / ARHGEF4, ASEF2, DBS, ECT2, GEF-H1, LARG, NET1, OBSCURIN, P-REX1, P-REX2, PDZ-RHOGEF, TEM4, TIAM1, TRIO, VAV1, VAV2, VAV3, DOCK1, DOCK2, DOCK3, DOCK4, DOCK8, DOCK10, C3G, BIG2 / ARFGEF2, EFA6, FBX8, or GEP100. In certain embodiments, the target protein is a protein having a protein-protein interaction domain, such as ARM, BAR, BEACH, BH, BIR, BRCT, BROMO, BTB, C1, C2, CARD, CC, CALM, CH, CHROMO, CUE, DEATH, DED, DEP, DH, EF-hand, EH, ENTH, EVH1, F-box, FERM, FF, FH2, FHA, FYVE, GAT, GEL, GLUE, GRAM, GRIP, GY F, HEAT, HECT, IQ, LRR, MBT, MH1, MH2, MIU, NZF, PAS, PB1, PDZ, PH, POLO-Box, PTB, PUF, PWWP, PX, RGS, RING, SAM, SC, SH2, SH3, SOCS, SPRY, START, SWIRM, TIR, TPR, TRAF, SNARE, TUBBY, TUDOR, UBA, UEV, UIM, VHL, VHS, WD40, WW, SH2, SH3, TRAF, bromodomain, or TPR. In some embodiments, the target protein is a heat shock protein, e.g., Hsp20, Hsp27, Hsp70, Hsp84,The target protein is αB crystallin, TRAP-1, hsfl, or Hsp90. In certain embodiments, the target protein is an ion channel, e.g., Cav2.2, Cav3.2, IKACh, Kv1.5, TRPA1, NAv1.7, Nav1.8, Nav1.9, P2X3, or P2X4. In some embodiments, the target protein is a coiled-coil protein, e.g., geminin, SPAG4, VAV1, MAD1, ROCK1, RNF31, NEDP1, HCCM, EEA1, vimentin, ATF4, Nemo, SNAP25, syntaxin 1a, FYCO1, or CEP250. In certain embodiments, the target protein is a kinase, for example, CyclinD1, ABL, ALK, AXL, BTK, EGFR, FMS, FAK, FGFR1, 2, 3, 4, FLT3, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, IGF1R, INSR, JAK1, JAK2, JAK3, KIT, MET, PDGFRA, PDGFRB, RETRON, ROR1, ROR2, ROS, SRC, SYK, TIE1, TIE2, TRKA, TRKB, KDR, AKT1 These include AKT2, AKT3, PDK1, PKC, RHO, ROCK1, RSK1, RKS2, RKS3, ATM, ATR, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, ERK1, ERK2, ERK3, ERK4, GSK3A, GSK3B, JNK1, JNK2, JNK3, AurA, AurB, PLK1, PLK2, PLK3, PLK4, IKK, KIN1, cRaf, PKN3, c-Src, Fak, PyK2, or AMPK. In some embodiments, the target protein is a phosphatase, such as WIP1, SHP2, SHP1, PRL-3, PTP1B, or STEP. In certain embodiments, the target protein is ubiquitin or ubiquitin-like protein (e.g., NEDD8, ATG8 protein, SUMO protein, ISG15), activating enzyme (E1, e.g., UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, SAE1), conjugation enzyme (E2, e.g., UBE protein, ATG3, BIRC6), ligation enzyme (E3, e.g., BMI-1,The target proteins are MDM2, NEDD4-1, beta-TRCP, SKP2, E6AP, CBL-B, or APC / C), and ubiquitin or ubiquitin-like protein proteases. In some embodiments, the target protein is a chromatin modifier / remodeler, such as a chromatin modifier / remodeler encoded by the gene BRG1, BRM, ATRX, PRDM3, ASH1L, CBP, KAT6A, KAT6B, MLL, NSD1, SETD2, EP300, KAT2A, or CREBBP. In some embodiments, the target protein is a transcription factor, for example, the genes EHF, ELF1, ELF3, ELF4, ELF5, ELK1, ELK3, ELK4, ERF, ERG, ETS1, ETV1, ETV2, ETV3, ETV4, ETV5, ETV6, FEV, FLI1, GAVPA, SPDEF, SPI1, SPIC, SPIB, E2F1, E2F2, E2F3, E2F4, E2F7, E2F8, ARNTL, B HLHA15, BHLHB2, BHLBHB3, BHLHE22, BHLHE23, BHLHE41, CLOCK, FIGLA, HAS5, HES7, HEY1, HEY2, ID4, MAX, MESP1, ML X, MLXIPL, MNT, MSC, MYF6, NEUROD2, NEUROG2, NHLH1, OLIG1, OLIG2, OLIG3, SREBF2, TCF3, TCF4, TFAP4, TFE3, TFEB, TFEC, USF1, ARF4, ATF7, BATF3, CEBPB, CEBPD, CEBPG, CREB3L1, DBP, HLF, JDP2, MAFF, MAFG, MAFK, NRL, NFE2, NFIL3 , TEF, XBP1, PROX1, TEAD1, TEAD3, TEAD4, ONECUT3, ALX3, ALX4, ARX, BARHL2, BARX, BSX, CART1, CDX1, CDX2, DLX1, DL X2, DLX3, DLX4, DLX5, DLX6, DMBX1, DPRX, DRGX, DUXA, EMX1, EMX2, EN1, EN2, ESX1, EVX1, EVX2, GBX1, GBX2, GSC, GSC2 , GSX1, GSX2, HESX1, HMX1, HMX2, HMX3, HNF1A, HNF1B, HOMEZ, HOXA1, HOXA1, HOXA13, HOXA2, HOXAB13, HOXB2, HOXB3,HOXB5、HOXC10、HOXC11、HOXC12、HOXC13、HOXD11、HOXD12、HOXD13、HOXD8、IRX2、IRX5、ISL2、ISX、LBX2、LHX2、LHX6、LHX9、LMX1A、LMX1B、MEIS1、MEIS2、MEIS3、MEOX1、MEOX2、MIXL1、MNX1、MSX1、MSX2、NKX2-3、NKX2-8、NKX3-1、NKX3-2、NKX6-1、NKX6-2、NOTO、ONECUT1、ONECUT2、OTX1、OTX2、PDX1、PHOX2A、PHOX2B、PITX1、PITX3、PKNOX1、PROP1、PRRX1、PRRX2、RAX、RAXL1、RHOXF1、SHOX、SHOX2、TGIF1、TGIF2、TGIF2LX、UNCX、VAX1、VAX2、VENTX、VSX1、VSX2、CUX1、CUX2、POU1F1、POU2F1、POU2F2、POU2F3、POU3F1、POU3F2、POU3F3、POU3F4、POU4F1、POU4F2、POU4F3、POU5F1P1、POU6F2、RFX2、RFX3、RFX4、RFX5、TFAP2A、TFAP2B、TFAP2C、GRHL1、TFCP2、NFIA、NFIB、NFIX、GCM1、GCM2、HSF1、HSF2、HSF4、HSFY2、EBF1、IRF3、IRF4、IRF5、IRF7、IRF8、IRF9、MEF2A、MEF2B、MEF2D、SRF、NRF1、CPEB1、GMEB2、MYBL1、MYBL2、SMAD3、CENPB、PAX1、PAX2、PAX9、PAX3、PAX4、PAX5、PAX6、PAX7、BCL6B、EGR1、EGR2、EGR3、EGR4、GLIS1、GLIS2、GLI2、GLIS3、HIC2、HINFP1、KLF13、KLF14、KLF16、MTF1、PRDM1、PRDM4、SCRT1、SCRT2、SNAI2、SP1、SP3、SP4、SP8、YY1、YY2、ZBED1、ZBTB7A、ZBTB7B、ZBTB7C、ZIC1、ZIC3、ZIC4、ZNF143、ZNF232、ZNF238、ZNF282、ZNF306、ZNF410、ZNF435、ZBTB49、ZNF524、ZNF713、ZNF740、ZNF75A、ZNF784、ZSCAN4、CTCF、LEF1、SOX10, SOX14, SOX15, SOX18, SOX2, SOX21, SOX4, SOX7, SOX8, SOX9, SRY, TCF7L1, FOX03, FOXB1, FOXC1, FOXC2, FOXD2, FOXD3, FOXG1, FOXI1, FOXJ2, FOXJ3, FOXK1, F, OXL1, FOXO1, FOXO4, FOXO6, FOXP3, EOMES, MGA, NFAT5, NFATC1, NFKB1, NFKB2, TΡ63, RUNX2, RUNX3, T, TBR1, TΒΧ1, TΒΧ15, TΒ Χ19, TΒΧ2, TΒΧ20, TΒΧ21, TΒΧ4, TΒΧ5, AR, ESR1, ESRRA, ESRRB, HNF4A, NR2C2, NR2E1, NR2F1, NR2F6, NR3C1, NR3C2, NR4A2, RAR A is a transcription factor encoded by RARB, RARG, RORA, RXRA, RXRB, RXRG, THRA, THRB, VDR, GATA3, GATA4 or GATA5, or C-myc, Max, Stat3, Stat4, Stat6, androgen receptor, C-Jun, C-Fox, N-Myc, L-Myc, MITF, Hif-1α, Hif-2α, Bcl6, E2F1, NF-κB, Stat5, or ER(coact). In certain embodiments, the target proteins are TrkA, P2Y14 and mPEGS, ASK1, ALK, Bcl-2, BCL-XL, mSIN1, RORγt, IL17RA, eIF4E, TLR7R, PCSK9, IgER, CD40, CD40L, Shn-3, TNFR1, TNFR2, IL31RA, OSMR, IL12beta1, 2, Tau, FASN, KCTD6, KCTD9, Raptor, Rictor, RALGAPA, and RALGAPB. These include annexin family members, BCOR, NCOR, β-catenin, AAC, PLD1, PLD2, Frizzled7, RaLP11, MLL-1, Myb, Ezh2, RhoGD12, EGFR, CTLA4R, GCGC(coact), adiponectin R2, GPR81, IMPDH2, IL-4R, IL-13R, IL-1R, IL2-R, IL-6R, IL-22R, TNF-R, TLR4, MyD88, Keap1, or Nrlp3.

[0237] Protein mutants Protein or polypeptide variants as described herein generally exhibit a significant identity (e.g., 80% or more, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) with the amino acid sequence of a reference polypeptide (e.g., a presenter protein or target protein as described herein, e.g., a mammalian presenter protein or target protein), but have an amino acid sequence containing a limited number of specific amino acid changes (e.g., conserved or non-conserved insertions, deletions, or substitutions) and / or contain one or more amino acid variants or analogs (e.g., D-amino acids, des-amino acids) with respect to the reference polypeptide. In certain embodiments, the variant shares relevant biological activity (e.g., binding to a particular compound or part thereof) with the reference polypeptide. In some such embodiments, the mutant exhibits such activity at a level of more than 50% of the activity of the reference polypeptide and / or less than 1 / 0.5 of the activity of the reference polypeptide.

[0238] In some embodiments, the mutant polypeptide has an amino acid sequence that differs from the amino acid sequence of the reference polypeptide in that the mutant has a number of cysteine ​​residues and / or one or more cysteine ​​residues at positions corresponding to non-cysteine ​​residues in the reference polypeptide. For example, in some embodiments, the addition of one or more cysteine ​​residues to the amino or carboxyl terminus of any polypeptide described herein (e.g., presenter protein and / or target protein) can promote the conjugation of such polypeptides, for example, by disulfide bonds.

[0239] In some embodiments, amino acid substitutions may be conserved (i.e., the residue is replaced with another of the same general type or group) or non-conservative (i.e., the residue is replaced with a different type of amino acid). In some embodiments, natural amino acids may be substituted for non-natural amino acids (i.e., conserved amino acid substitutions or non-conservative amino acid substitutions that are not naturally derived), and vice versa.

[0240] Synthetically produced polypeptides may contain substitutions of amino acids not naturally encoded by DNA (e.g., non-natural amino acids or unnatural amino acids). Examples of unnatural amino acids include D-amino acids, amino acids with azide-containing side chains, amino acids with an acetylaminomethyl group attached to the sulfur atom of cysteine, pegylated amino acids, and those with the chemical formula NH2(CH2). n The formula includes omega amino acids of COOH (where n is 2-6), neutral nonpolar amino acids, such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe. Citrulline and methionine sulfoxides are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline to retain conformation-granting properties.

[0241] Analogues are produced by substitutional mutagenesis and may retain the structure of the original protein (e.g., local or global structure). Examples of substitutions identified as “conservative substitutions” are shown in Table 2. If such substitutions result in undesirable changes, other types of substitutions, referred to as “exemplary substitutions” in Table 2 or further described herein in relation to amino acid classes, are introduced and the product is screened.

[0242] Substantial modifications in functional or immunological identity are achieved by selecting substitutions that are significantly different in (a) the structure of the protein backbone in the region of substitution, for example, as a sheet or helical conformation, (b) molecular changes or hydrophobicity at the target site, or (c) their effect on maintaining the bulk of the side chain. Naturally occurring residues are grouped based on their general side-chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), histidine (His), tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), (2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), (3) Acidic / negatively charged: aspartic acid (Asp), glutamic acid (Glu), (4) Basic: asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg), (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His), (7) Polar: Ser, Thr, Asn, Gln, (8) Basic and positively charged: Arg, Lys, His, and; (9) Charged: Asp, Glu, Arg, Lys, His Other amino acid substitutions are listed in Table 2.

[0243] [Table 2]

[0244] Protein variants with altered reactive amino acid profiles In some embodiments, a protein or polypeptide variant may involve the addition of one or more reactive amino acid residues (e.g., cysteine) to the protein (e.g., at the amino or carboxyl terminus of any of the proteins described herein), which can promote conjugation of these proteins, for example, by disulfide bonds. In some embodiments, one or more reactive amino acids (e.g., cysteine) may be removed to reduce the number of possible conjugation sites on the protein. Amino acid substitutions may be conserved (i.e., the residue is replaced with another of the same general type or group) or non-conserved (i.e., the residue is replaced with a different type of amino acid). Furthermore, native amino acids may be substituted for non-native amino acids (i.e., conserved or non-native amino acid substitutions).

[0245] As is well known in the art, for example, JW Chin, "Expanding and Reprogramming the Genetic Code of Cells and Animals" Reprogramming the Genetic Code of Cells As described in the Annual Review of Biochemistry, Vol. 83, pp. 379-408 (and Animals), non-natural amino acids can be incorporated into proteins produced in vitro. For example, in one system, the UAG amber (stop) codon has been used to incorporate pyrrolidine via archaeal tRNA synthetase and tRNA, and the UAG amber (stop) codon can also be used to incorporate azides and alkynes via feeding. Other side chains on non-natural amino acids shown in the art include cyclopropene, trans-cyclooctene, bicyclo[6.1.0]nonine-lysine, coumarin, p-azidophenylalanine, N6-[(2-propynloxy))carbonyl]-L-lysine, bicyclo[6.1.0]nona-4-in-9-ylmethanol (BCN), N-5-norbornene-2-yloxycarbonyl-l-lysine, and N-tert-butyl It contains oxycarbonyl-l-lysine, N-2-azidoethyloxycarbonyl-l-lysine, NL-thiaprolyl-L-lysine, ND-cysteinyl-L-lysine, NL-cysteinyl-L-lysine, N6-[(2-propynyloxy)carbonyl]-L-lysine, N6-[(2-azidoethoxy)carbonyl]-L-lysine, benzophenone, 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine, and cyclooctin.

[0246] complex In contrast to many small molecule-protein interactions, which are driven by interactions between small molecules in cavities or pockets on proteins, in naturally occurring protein-protein interactions, binding events are typically largely driven by hydrophobic residues on the flat surface sites of the interacting proteins. Generally, hydrophobic residues on the flat surface sites of proteins form hydrophobic hotspots, where the majority of binding interactions between or within interacting proteins are van der Waals interactions. In some situations, small molecules engage with or create hydrophobic interaction sites on proteins (e.g., presenter proteins), where the small molecule can provide a “mobile hotspot” (or a portion thereof) in that it does not exist in the absence of the small molecule. Aspects of this disclosure are particularly applicable to such situations. For example, in some embodiments, compounds (and / or tagged forms thereof) as described herein form complexes with proteins (e.g., presenter protein / compound complexes) and engage in pseudo-protein-protein interactions (e.g., the formation of tripartite complexes with target proteins).

[0247] Many mammalian proteins can bind to one of several different partners, and in some cases, these alternative binding interactions contribute to the protein's biological activity. Many of these proteins present the same residues in various structural configurations, adapting to the intrinsic variability of the hotspot protein region. More specifically, protein-protein interactions can be mediated by a class of natural products produced by selected groups of fungal and bacterial species. These molecules exhibit a common structural organization and consequently possess the function of providing the ability to modulate protein-protein interactions. These molecules contain a highly conserved presenter protein-binding moiety and a target protein-interacting moiety that exhibits a high degree of variability between different natural products. The presenter protein-binding moiety confers specificity to the presenter protein, causing the molecule to bind to the presenter protein and form a complex. The mammalian target protein-binding moiety confers specificity to the target protein, causing the binary complex to bind to the target protein, typically modulating its activity (e.g., positively or negatively). In the present invention, binary complexes (e.g., between a compound and a presenter protein, or between a compound and a target protein) are mimicked by conjugating the presenter protein-binding portion to the target protein, or by conjugating the target protein-binding portion to the presenter protein. The conjugate thus obtained in the present invention can then bind to the presenter protein or target protein to form a complex that mimics a tripartite complex. These complexes can be used, for example, to determine the structure of the interface between the presenter protein and the target protein. Furthermore, by simplifying the formation of the complex, for example by conjugating the presenter protein-binding portion to the target protein, the compounds of the present invention can be used, for example, to identify target proteins that can bind to the presenter protein.

[0248] use Identification of target proteins In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful for identifying target proteins that can form complexes with presenter proteins (e.g., in the presence of small molecules). Target proteins can be identified by determining the formation of a conjugate containing a presenter protein-binding moiety conjugated to a target portion, and whether the conjugate forms a complex with the presenter protein.

[0249] Most target proteins known in the art that form ternary complexes with presenter proteins and small molecules were fortunately identified during the determination of the small molecule's mechanism of action. This method enables the rational identification of target proteins that can complex with presenter proteins in the presence of small molecules by covalently conjugating the presenter protein binding portion to the target molecule and allowing complex formation before the identification of a compound that can simultaneously bind both the presenter protein and the target protein.

[0250] Screening of small molecules for their ability to promote complex formation between presenter proteins and identified target proteins can then be used to identify promising therapies that can modulate the biological activity of target proteins.

[0251] In some embodiments, the compounds of the present invention can be used to identify target proteins that can form a complex with a presenter protein. For example, target proteins can be identified by combining one or more target proteins with a labeled presenter protein (e.g., labeled with biotin) under conditions that allow the formation of a presenter protein / target protein complex in the presence of the compounds of the present invention. Target proteins that do not form a complex with the presenter protein may then be removed (e.g., washed away), and the target proteins that do form a complex can then be pulled down using a label on the presenter protein and analyzed. In some embodiments, the pulled-down target proteins can be analyzed by mass spectrometry to determine their identity.

[0252] Compound design In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful for designing compounds that can modulate the biological activity of target proteins for use in the treatment of diseases.

[0253] For example, the formation of a complex of the presenter protein and conjugate of the present invention can facilitate the determination of the structure of the protein-protein interface between the presenter protein and the target protein by crystallization and crystal structure determination of the complex. Once the crystal structure of the complex of the present invention is determined, small molecules that can facilitate complex formation between the presenter protein and the target protein can be developed using methods known in the art for rational drug design, such as computational chemistry methods and / or methods for constructing novel structures, such as fragment immersion of crystals of the complex of the present invention, and fragment-based drug design using the structure determination thus obtained.

[0254] The compounds designed as described above may then be screened to determine their ability to modulate the biological activity of target proteins, and may be modified using medicinal chemistry techniques as needed to produce therapeutically useful compounds.

[0255] Identification of covalent small molecule therapies In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful in identifying compounds that can modulate the biological activity of target proteins through covalent interactions.

[0256] For example, the compounds of the present invention can be screened for their ability to covalently bind to a target protein in the presence and absence of a presenter protein to identify compounds that can selectively bind to a target protein only in the presence of a presenter protein. These compounds may then be tested for their ability to modulate the biological activity of the target protein and may be modified using medicinal chemistry techniques as needed to produce therapeutically useful compounds.

[0257] Determination of biochemical and / or biophysical properties In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful for determining the biochemical and / or biophysical properties of proteins or complexes.

[0258] For example, the free energy of binding between a conjugate containing a presenter protein binding site and a target protein and the presenter protein can be determined, for example, by isothermal titration calorimetry. d This can be determined, for example, by surface plasmon resonance. The K of the compound and the presenter protein for the target protein. i , K inact , and / or K i / Kinact This can be determined, for example, by mass spectrometry.

[0259] Treatment of disease or disability The compounds, conjugates, and complexes described herein may be useful in methods for treating diseases or disorders associated with the target proteins described herein, and are not limited to theory, but are thought to exert these desirable effects through interactions with presenter proteins and target proteins by their ability to modulate (e.g., positively or negatively) the activity of target proteins (e.g., eukaryotic target proteins, e.g., mammalian target proteins or fungal target proteins or prokaryotic target proteins, e.g., bacterial target proteins).

[0260] kit In some embodiments, the present invention relates to a kit for conveniently and effectively carrying out a method according to the present invention. Generally, a pharmaceutical pack or kit comprises one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Such a kit is particularly suitable for the delivery of solid oral formulations such as tablets or capsules. Such a kit may preferably include several unit doses and may include a card indicating the doses in accordance with the intended order of use. If desired, memory aids can be provided, for example, if the subject has Alzheimer's disease, for example, by writing the doses on a calendar in the form of numbers, letters, or other indications, or by specifying the dates on which the doses can be administered in the treatment schedule. Alternatively, a kit can be provided in which the doses are taken daily, including a placebo dose or calcium dietary supplement in a form similar to or different from the doses of the pharmaceutical composition. Optionally associated with such containers may be a warning in the form specified by the government agency regulating the manufacture, use, or sale of the pharmaceutical. This warning reflects that the manufacture, use, or sale for administration to humans has been authorized by the supervisory authority. Pharmaceutical composition For use in the treatment of human and animal subjects, the compounds and conjugates of the present invention can be formulated as pharmaceutical or veterinary drug compositions. Depending on the subject to be treated, the mode of administration, and the desired type of treatment (e.g., prevention, prophylaxis, or treatment), the compounds are formulated in a manner that conforms to these parameters. An overview of such techniques can be found in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005; and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J.C. Boylan, 1988–1999, Marcel Dekker, New York (both incorporated herein by reference).

[0261] The compounds described herein may be present in a total amount of 1 to 95% by weight of the total weight of the composition. The composition may be provided in dosage forms suitable for intra-articular, oral, parenteral (e.g., intravenous, intramuscular), transrectal, cutaneous, subcutaneous, topical, transdermal, sublingual, transnasal, transvaginal, intravesicular, intraurethral, ​​intrathecal, epidural, transaural, or transocular administration, or for injection, inhalation, or direct contact with the mucous membranes of the nose, genitourinary tract, genitals, or oral cavity. Therefore, the pharmaceutical composition may take the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, preparations suitable for ionophoretic delivery, or aerosols. The composition may be formulated according to conventional pharmaceutical regulations.

[0262] Generally, for therapeutic use, the compounds described herein may be used alone or in combination with one or more other activators. Examples of other pharmaceuticals combined with the compounds described herein would include pharmaceuticals for the treatment of the same indication. Other examples of pharmaceuticals that may be combined with the compounds described herein would include pharmaceuticals for the treatment of different but related symptoms or indications. Depending on the mode of administration, the compounds are formulated as compositions suitable for easy delivery. Each compound in a combination therapy may be formulated in various ways known in the art. For example, the first and second activators of a combination therapy may be formulated together or individually. Preferably, the first and second activators are formulated together for simultaneous or near-simultaneous administration of the activators.

[0263] The compounds of the present invention can be prepared and used as pharmaceutical compositions comprising an effective amount of the compound described herein and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, the composition comprises at least two different pharmaceutically acceptable excipients or carriers.

[0264] Formulations can be prepared in a manner suitable for systemic or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, or subcutaneous injection) or can be prepared for transdermal, transmucosal, or oral administration. Formulations generally include diluents and, in some cases, adjuvants, buffers, and preservatives. Compounds can also be administered as liposome compositions or microemulsions.

[0265] When intended for injection, the formulation can be conventionally prepared as a liquid solution, a suspension, a solid formulation suitable for dissolving or suspending in liquid before injection, or an emulsion. Suitable excipients include, for example, water, saline, dextrose, and glycerol. Such compositions may also contain various amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., such as sodium acetate and sorbitan monolaurate.

[0266] Various sustained-release systems for drugs have also been devised. For example, see U.S. Patent No. 5,624,677 (incorporated herein by reference). Systemic administration methods may include relatively non-invasive methods such as suppositories, transdermal patches, transmucosal delivery, and intranasal administration. Oral administration is also suitable for the compounds of the present invention. Suitable formulations include syrups, capsules, and tablets, as is understood in the art.

[0267] Each compound in the combination therapies described herein can be formulated in various ways known in the art. For example, the first and second agents of a combination therapy can be formulated together or separately.

[0268] Individually or in divided formulations of agonists can be packaged together as a kit. Examples, though not limited to, include kits containing two pills, pills + powder, suppositories + liquid in a vial, or two topical creams. The kit may include optional components to assist in administering the unit dose to the subject, such as vials for reconstituting the powder form, syringes for injection, customized IV delivery systems, or inhalers. Additionally, the unit dose kit may include instructions for the preparation and administration of the composition. The kit may be manufactured as a single-use unit dose for a single subject, as a multiple-use unit dose for a specific subject (with varying potency of individual compounds at a constant dose or as treatment progresses), or the kit may contain multiple doses suitable for administration to multiple subjects ("bulk packaging"). The kit components can be assembled as cartons, blister packs, bottles, tubes, etc.

[0269] Formulations intended for oral use include tablets containing the active ingredient as a mixture with non-toxic, pharmaceutically acceptable excipients. Such excipients may include, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches such as potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives such as microcrystalline cellulose, starches such as potato starch, croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), as well as lubricants, adhesives, and anti-adhesion agents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients may include colorants, flavorings, plasticizers, humectants, and buffering agents.

[0270] Two or more compounds may be mixed together or divided in a tablet, capsule, or other medium. For example, the first compound may be contained on the inside of a tablet and the second compound on the outside, such that a substantial portion of the second compound is released before the first compound is released.

[0271] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets may be prepared conventionally using the above-mentioned ingredients in tablet and capsule form, for example, by a mixer, fluidized bed apparatus, or spray dryer.

[0272] Dissolution or diffusion-controlled release can be achieved by applying an appropriate coating to the compound in tablet, capsule, pellet, or granular form, or by incorporating the compound into an appropriate matrix. Examples of controlled-release coatings include one or more of the coating materials listed above, and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, two hydroxymethacrylates, methacrylate hydrogels, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In addition, in controlled-release matrix formulations, the matrix material may include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, Carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0273] Liquid formulations that can incorporate the compounds and compositions of the present invention for oral administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions, which contain edible oils such as barnyard grass oil, sesame oil, coconut oil, and peanut oil, as well as elixirs and similar pharmaceutical media.

[0274] Generally, when administered to humans, the oral dose of any of the compounds in the combination of the present invention depends on the properties of the compound, but can be easily determined by those skilled in the art. Typically, such doses are usually about 0.001 mg to 2000 mg / day, preferably about 1 mg to 1000 mg / day, and more preferably about 5 mg to 500 mg / day. Doses up to 200 mg / day may be required.

[0275] The administration of each drug in the combination therapies described herein may be independently administered four times daily for a period of 1 day to 1 year, or even for the lifespan of the subject. Chronic long-term administration may be required. [Examples]

[0276] Example 1: Synthesis of a specific crosslinking reagent Synthesis of (R)-3-(3,4-dimethoxyphenyl)-1-(3-(3-(pyridine-2-yldisulfanyl)propanamide)phenyl)propyl(S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate (C3-SLF):

[0277] [ka]

[0278] To a 3 mL solution of DNF containing aniline 1 (90 mg, 172 μmol, 1 equivalent), disulfide 2 (74 mg, 343 μmol, 2 equivalents), and diisopropylethylamine (149 μL, 111 mg, 858 μmol, 5 equivalents), HATU (130 mg, 343 μmol, 2 equivalents) was added, and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with water and extracted with ethyl acetate (3×). The organic extract was washed with water and saturated sodium chloride, dried on magnesium sulfate, and evaporated. The residue was purified on silica gel gradient elution (20% ethyl acetate:80% heptane → 100% ethyl acetate) to obtain the title compound C3-SLF (50 mg, 40%). MS(ESI) calculation = 722.3 (M+H), observed = 722.3.

[0279] Synthesis of (R)-3-(3,4-dimethoxyphenyl)-1-(3-(4-(pyridine-2-yldisulfanyl)butanamide)phenyl)propyl(S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate(C4-SLF)):

[0280] [ka]

[0281] To a solution of aniline 1 (90 mg, 172 μmol, 1 equivalent), disulfide 2 (79 mg, 343 μmol, 2 equivalents), and diisopropylethylamine (149 μL, 111 mg, 858 μmol, 5 equivalents) in DMF (3 mL), HATU (130 mg, 343 μmol, 2 equivalents) was added, and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with water and extracted with ethyl acetate (3×). The organic extract was washed with water and saturated sodium chloride, dried on magnesium sulfate, and evaporated. The residue was purified on silica gel gradient elution (20% ethyl acetate:80% heptane → 100% ethyl acetate) to obtain the title compound C4-SLF (98 mg, 77%). MS(ESI) calculation = 736.3 (M+H), observed value = 736.3.

[0282] Synthesis of methyl(S)-1-((S)-3-(3-hydroxyphenyl)-2-((S)-3-methyl-2-(4-(pyridine-2-yldisulfanyl)butanamide)butanamide)propanoyl)hexahydropyridazine-3-carboxylate (SFAC4DS)):

[0283] [ka]

[0284] Amine 1 was prepared according to Paquette et al., JACS, 2002 (Vol. 124), pp. 4257-4270. To a solution of amine 1 (20 mg, 49.2 μmol) in acetonitrile (1 mL), triethylamine (16.5 μL, 118 μmol, 2.4 equivalents) was added, followed by acid chloride 2 (13.8 mg, 59.2 μmol, 1.2 equivalents). The reaction mixture was stirred at room temperature for 14 hours, then concentrated, and the residue was purified by preparative TLC (dichloromethane: MeOH:NH4OH, 20:1:0.1) to obtain 14.0 mg (47%) of the product as a colorless foam. f =0.59 (Dichloromethane:MeOH:NH4OH, 10:1:0.1). MS(ESI) calculated value = 618.2 (M+H), observed value = 618.2.

[0285] Synthesis of methyl(S)-1-((S)-3-(3-hydroxyphenyl)-2-((S)-3-methyl-2-(3-(2-(pyridine-2-yldisulfanyl)ethoxy)propanamide)butanamide)propanoyl)hexahydropyridazine-3-carboxylate (SFAX6)):

[0286] [ka]

[0287] Carboxylic acid 2 (70 mg, 0.270 mmol) and HBTU (204 mg, 0.540 mmol, 2.00 equivalents) were mixed in 3 mL of acetonitrile, and the resulting suspension was stirred at room temperature for 15 minutes. Subsequently, amine 1 (110 mg, 0.270 mmol, 1.00 equivalent), followed by triethylamine (113 μL, 0.810 mmol, 3.00 equivalents), was added, and the mixture was stirred at room temperature for 18 hours. The mixture was then treated with 20 mL of saturated sodium bicarbonate and extracted with ethyl acetate in 2 × 30 mL portions. The pooled organic extract was washed with brine in 2 × 20 mL portions, dried over saturated sodium sulfate, filtered, and concentrated under vacuum. The residues were purified using silica gel chromatography eluting with dichloromethane:MeOH, 100:1 to 50:1, and 70 mg (40%) of the product was obtained as a colorless oil. f =0.31 (Dichloromethane:MeOH, 20:1). MS(ESI) calculated value = 648.2 (M+H), observed value = 648.2.

[0288] Synthesis of N-(4-((2S,11R,14S,17S,20S,23S,26S)-26-ethyl-23-((1R,2R,E)-1-hydroxy-2-methylhexa-4-en-1-yl)-14,17-diisobutyl-20-isopropyl-4,11,13,16,19,22,28,31-octamethyl-3,6,9,12,15,18,21,24,27,30,33-undekaoxo-1,4,7,10,13,16,19,22,25,28,31-undekaazacyclotriacontan-2-yl)butyl)-4-(pyridine-2-yldisulfanyl)butanamide (CsA3))

[0289] [ka]

[0290] To a 6 mL NMP (Natural Microwave Propagation) solution of amine 1 (100 mg, 90.5 μmol) and carboxylic acid 2 (31 mg, 135.2 μmol, 1.5 equivalents), HATU (51 mg, 134.1 μmol, 1.48 equivalents) and DIPEA (70 μL, 401.9 μmol, 4.4 equivalents) were added. The reaction mixture was stirred at room temperature for 1 hour, then diluted with water and extracted with 3 × 30 mL portions of ethyl acetate. The extract was washed with saturated sodium chloride solution and concentrated under vacuum. The crude material was purified by reverse-phase chromatography on C18 medium, eluting with a gradient from 15% acetonitrile:85% water (both containing 0.1% formic acid) to 100% acetonitrile (containing 0.1% formic acid). MS(ESI) calculated value = 658.9 (M+2H), observed value = 659.0.

[0291] Example 2: Synthesis of a specific conjugate General Protocol: This protocol describes a method for forming target protein-compound conjugates. Reagents: Compounds in 100% DMSO (proprietary) and mammalian target proteins (proprietary) Device: Mini-PROTEAN TGX Gel (Bio-Rad) Experimental protocol: The target protein and compound are mixed together in a 1:2 molar ratio in 75 mM NaCl buffer containing 12.5 mM HEPES, pH 7.4, and 2% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation overnight at room temperature. Crosslinking efficiency is assessed by SDS-PAGE gel. The conjugate migrates more slowly than the uncrosslinked target protein. For thiol-reactive compounds, Cys-specific attachment of the compound to the target protein can be further confirmed by SDS-PAGE after adding 100 mM DTT to the reaction mixture to return the conjugate to its components.

[0292] A.KRAS GTP / S39C Formation of lite / C2-FK506 conjugate Reagents: C2-FK506 (proprietary product) in 100% DMSO, KRASGTP / S39C lite (proprietary product; containing residues 1-169 with G12V / S39C / C51S / C80L / C118S). Equipment: Mini-PROTEAN TGX gel (Bio-Rad) Experimental protocol: KRAS in a 1:2 molar ratio GTP / S39C lite and C2-FK506 are mixed together in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation at room temperature overnight. Crosslinking efficiency is assessed by SDS-PAGE gel. KRAS GTP / S39C The adhesion of C2-FK506 to cysteine ​​39 on lite is also assessed by incubation of a reaction mixture with 100 mM DTT. Result: C2-FK506 is KRAS GTP / S39C It efficiently crosslinks with lite and is specific to cysteine ​​39 (Figure 1).

[0293] B.KRAS GTP / G12C Formation of the lite / SFAX9DS conjugate Reagents: SFAX9DS (proprietary product) in 100% DMSO, KRAS GTP / G12C lite (proprietary product; residues 1-169 containing G12C / C51S / C80L / C118S). Device: Mini-PROTEAN TGX Gel (Bio-Rad) Experimental protocol: KRAS in a 1:2 molar ratio GTP / G12C lite and SFAX9DS were mixed together in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction mixture was incubated at 37°C for 30 minutes, followed by incubation overnight at room temperature. Crosslinking efficiency was assessed by SDS-PAGE gel. Wild-type CypA also crosslinked with the compound. Cysteine ​​52, as a reactive cysteine ​​on CypA, was mutated to serine, inhibiting presenter crosslinking. Results: SFAX9DS is KRAS GTP / G12CEfficiently crosslinks with lite proteins, CypA C52S It does not crosslink to SFAX9DS (Figure 2).

[0294] Example 3: Formation of a specific complex General Protocol: This protocol describes two methods for the formation and isolation of a complex consisting of a presenter protein, a compound, and a mammalian target protein. Reagents: Compounds in 100% DMSO (proprietary), presenter proteins (proprietary), and mammalian target proteins (proprietary) Devices: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare, CV120mL).

[0295] Experimental protocol A: Pre-conjugated compounds and proteins The conjugate and presenter proteins in a 1:2 molar ratio are mixed together in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation overnight at room temperature. The pure complex is isolated by size exclusion chromatography (SEC) purification. The reaction mixture is injected directly into a Superdex 75 column (CV 120 mL) pre-equilibriumated with a buffer containing 12.5 mM HEPES, pH 7.4, 75 mM NaCl. The complex elutes at a higher molecular weight than the unreacted target and presenter proteins. The sample is assessed by SDS-PAGE to confirm the presence of the complex in the elution peak.

[0296] Experimental protocol B: Crosslinking reagent, presenter protein, and target protein The compound, presenter protein, and target protein in a 1:2:2 molar ratio are mixed together in 12.5 mM HEPES, pH 7.4, and 75 mM NaCl buffer containing 2% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation overnight at room temperature. The pure complex is isolated by size exclusion chromatography (SEC) purification. The reaction mixture is injected directly into a Superdex 75 column (CV 120 mL) pre-equilibrium with a buffer containing 12.5 mM HEPES, pH 7.4, and 75 mM NaCl. The complex elutes at a higher molecular weight than the unreacted target and presenter proteins. The sample is assessed by SDS-PAGE to confirm the presence of the complex in the elution peak.

[0297] A.KRAS GTP / S39C Formation of the lite / C2-Holt / FKBP12 ternary complex Reagents: C2-Holt (proprietary product) in 100% DMSO, KRAS GTP / S39C lite (proprietary product; residues 1-169 containing G12V / S39C / C51S / C80L / C118S), and FKBP12 (proprietary product). Devices: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare, CV120mL) Experimental protocol: C2-Holt, FKBP12, and KRAS in a 1:2:2 molar ratio. GTP / S39CThe lite is mixed with 12.5 mM HEPES, pH 7.4, and 75 mM NaCl buffer containing 2% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation at room temperature overnight. The pure complex is isolated by size exclusion chromatography (SEC). The reaction mixture is injected directly into a Superdex 75 column (CV 120 mL) pre-equilibrium with a buffer containing 12.5 mM HEPES, pH 7.4, and 75 mM NaCl. The complex elutes approximately 69 mL after injection, and unreacted KRAS is eluted. GTP / S39C Lite and FKBP12 elute at approximately 75 mL and 87 mL after injection, respectively. KRAS at the elution peak. GTP / S39C To confirm the presence of lite and FKBP12, the sample is again assessed by SDS-PAGE. Results: SEC profile of elution peak and SDS-PAGE analysis were performed on KRAS GTP / S39C Confirm the formation of the lite / C2-Holt / FKBP12 complex (Figures 3A and 3B).

[0298] B.KRAS GDP / S39C lite / SFAC4DS / CypA C52S Formation of a three-component complex Reagents: SFAC4DS (proprietary product) in 100% DMSO, KRAS GDP / S39C lite (proprietary product; residues 1-169 containing G12V / S39C / C51S / C80L / C118S), and CypA C52S (Manufactured in-house). Devices: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare, CV120mL) Experimental protocol: SFAC4DS in a 1:2:2 molar ratio, CypA C52S , and KRAS GDP / S39CThe lite is mixed with 12.5 mM HEPES, pH 7.4, and 75 mM NaCl buffer containing 2% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation at room temperature overnight. The pure complex is isolated by size exclusion chromatography (SEC). The reaction mixture is injected directly into a Superdex 75 column (CV 120 mL) pre-equilibrium with a buffer containing 12.5 mM HEPES, pH 7.4, and 75 mM NaCl. The complex elutes approximately 69 mL after injection, and unreacted KRAS is eluted. GDP / S39C lite and CypA C52S After injection, the substances eluted at approximately 75 mL and 80 mL, respectively. KRAS at the elution peak. GDP / S39C lite and CypA C52S To confirm the presence of [the substance], the sample is assessed again by SDS-PAGE. Results: KRAS was determined by SEC profile of the elution peak and SDS-PAGE analysis. GDP / S39C lite / SFAC4DS / CypA C52S Confirm the formation of the complex (Figure 4).

[0299] C.PTP1B S187C Formation of the lite / C3-SLF / FKBP12 ternary complex Reagents: C3-SLF (proprietary product) in 100% DMSO, PTP1B E186C lite (proprietary product; residues 1-293 containing C32S / C92V / C121S / S187C) and FKBP12 (proprietary product). Devices: Mini-PROTEAN TGX Gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare, CV120mL) Experimental protocol: C3-SLF, FKBP12, and PTP1B in a 1:3:3 molar ratio. S187CThe lite is mixed with 12.5 mM HEPES, pH 7.4, and 75 mM NaCl buffer containing 4% DMSO. The reaction mixture is incubated at 37°C for 30 minutes, followed by incubation at room temperature overnight. The pure complex is isolated by size exclusion chromatography (SEC). The reaction mixture is injected directly into a Superdex 75 column (CV 120 mL) pre-equilibrium with a buffer containing 12.5 mM HEPES, pH 7.4, and 75 mM NaCl. The complex elutes approximately 62 mL after injection, while unreacted FKBP12 elutes approximately 75 mL (dimer) and 90 mL (monomer), respectively, after injection. PTP1B at the elution peak. S187C To confirm the presence of lite and FKBP12, the sample is again assessed by SDS-PAGE. Free PTP1B S187C The lite and FKBP12 mixtures were subjected to a Superdex 75 column under the same conditions to determine their elution times. Results: Free PTP1B elutes in approximately 64-65 ml. S187C Check the lite, free PTP1B S187C SEC profiles of lite and FKBP12 proteins (Figure 5A), as well as SDS-PAGE analysis. SEC profiles of elution peaks and SDS-PAGE analysis revealed PTP1B S187C The formation of the lite / C3-SLF / FKBP12 complex was confirmed, and it eluted at approximately 61 ml (Figure 5B).

[0300] Example 4: When the presenter protein is present, conjugate formation occurs; when it is absent, conjugate formation does not occur. This protocol describes a method for analyzing crosslinking efficiency using mass spectrometry and gel shift assays in an attempt to assess the presenter dependence of conjugate formation. Reagents: Compound in 100% DMSO (proprietary), FKBP12 (proprietary), KRAS GTP / G12C (Manufactured in-house, residues 1-169). Experimental protocol: To track the kinetics of the disulfide crosslinking reaction, an Agilent 6230 TOF-LC / MS and an Agilent 1260 HPLC instrument equipped with an AdvanceBio RP-mAb C4 column (2.1 × 100 mm, 3.5 μm) and an autosampler were used. HPLC-grade acetonitrile and water (containing 1.0 mM ammonium formate and 1 vol% formic acid, respectively) were used as mobile phases with the following ramps: a flow rate of 0.6 ml / min, water:acetonitrile = 95:5, ramping from 0.0 to 13.0 minutes, then water:acetonitrile = 5:95, from 13.0 to 17.0 minutes. Total time = 17.0 minutes.

[0301] All crosslinking reactions were performed in 1.5 mL amber glass vials with 0.5 mL glass inserts. A water-soluble peptide (SEQ ID NO: 1: YQNLLVGRNRGEEILD) was used as an internal standard. While the actual sequence of the internal standard was not critical, the selection of amino acid residues was important to avoid interference in the crosslinking assay. Therefore, proline (which interferes with FKBP12) and cysteine ​​(which interferes with disulfide bond formation) residues were excluded. All reactants and standard solutions were prepared in HEPES (pH 7.4, 1.0 mM MgCl2) buffer.

[0302] Prior to all reactions, standard curves were obtained for each component using a series of standard solutions (an example of standard curve analysis for FKBP12 is shown in Table 3 below). Using data from the standard curves, μmol of protein samples were plotted against area ratio (sample:std), and slope and intercept were obtained using linear fit (y=mx+c). The slope and intercept values ​​related to these standard curves were explained before and during the reaction, and during the evaluation of substrate and product concentrations. For all substrates / products, a blank injection was performed following the initial injection to verify the presence of residual protein / reagent. Based on this analysis, the autosampler sequence can be adjusted to include a suitable number of blank injections to remove any residual components, if any. MS spectra were analyzed using Agilent MassHunter vB.07.0 software.

[0303] [Table 3]

[0304] In a typical experiment to assess the presenter dependence of ligand crosslinking to target proteins, KRAS GTP / G12C The C3- or C4-SLF ligands were incubated for 4 hours at room temperature in 12.5 mM HEPES, pH 7.4, 75 mM NaCl, 1 mM MgCl2, and 3% DMSO at concentrations of 2 μM KRAS, 10 μM FKBP12, and 10 μM C3- or C4-SLF, with or without FKBP12. The amount of KRAS disulfide crosslinked with the ligand using the method described above was analyzed. As shown in Table 4, a 5- to 10-fold increase in crosslinking efficiency was observed in the presence of the presenter.

[0305] [Table 4]

[0306] In parallel with mass spectrometry, crosslinking reactions with C3- or C4-SLF were subjected to gel shift assays using 12% SDS-PAGE under the same experimental conditions as above, except that the crosslinking reaction was set at higher concentrations (60 μM KRAS, 180 μM FKBP12, and 180 μM C3- or C4-SLF), with or without FKBP12. These were quenched by MMTS to terminate the reaction. Similar to the MS data, ligand crosslinking efficiency was significantly boosted in the presence of FKBP12, which was more evident for C4-SLF (Figure 6).

[0307] Example 5: Determination of presenter protein / target protein interface structure by X-ray analysis This protocol is FKBP12-C2Holt-KRAS GTP / S39C This paper describes the crystallization of the ternary complex and the method for determining its crystalline structure.

[0308] A.FKBP12-C2Holt-KRAS GTP / S39C Determination of the crystal structure of the ternary complex Reagents: Ligand (C2Holt) in 100% DMSO (proprietary product), FKBP12 (proprietary product), KRAS GTP / S39C lite (proprietary product, containing residues 1-169 with G12V / S39C / C51S / C80L / C118S). Device: Superdex 75 (GE Healthcare) Experimental protocol: C Holt and FKBP12 were subjected to KRAS in 3:1 and 1.5:1 excess molar concentrations in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 1 mM MgCl2, and 2% DMSO. GTP / S39CIn addition to lite, the complex was incubated overnight at 20°C or at 4°C for 36–72 hours. The pure complex was isolated by size exclusion chromatography on a Superdex 75 column in 12.5 mM HEPES, pH 7.4, 75 mM NaCl, and 1 mM MgCl2. The purified complex (at 15–20 mg / ml) was subjected to crystallization screening at 20°C using sitting-drop vapor diffusion. Crystals were grown in well solutions containing 0.1 M MES, pH 6.5, and 20–22% PEG20,000. For data collection, the crystals were transferred to a solution containing mother liquor supplemented with 15% glycerol and then frozen in liquid nitrogen. Diffraction data sets were obtained at the Advanced Synchrotron Radiation Facility (APT). Samples were collected at a Photon Source (APS) and processed with the HKL program. Molecular substitution solutions were obtained using the PHASER program in the CCP4 suite, with the published structures of FKBP12 (PDB-ID1FKD) and KRAS (PDB-ID3GFT) used as search models. Subsequent model construction and refinement were performed using standard protocols with the CCP4 and COOT software packages.

[0309] Result:FKBP12-C2Holt-KRAS GTP / S39C Overall structure: The crystal is composed of asymmetric units FKBP12 and KRAS GTP / S39C It contains one heterodimer (Figure 7). The model consists of residues Met1 to Glu108 of FKBP12 and KRAS GTP / S39C The electron density was calculated to include Met1 through Lys169. The resulting electron density exhibits a clear binding mode, including ligand orientation and conformation. Continuous electron densities were observed for the cysteine ​​of the protein and the disulfide derived from the sulfur of the ligand.

[0310] KRAS involved in C2Holt binding GTP / S39C The residues (4 Å distance cutoff) are Glu37, Cys39, Leu56, and Met67. KRAS is involved in binding to FKBP12. GTP / S39CThe residues are Glu3, Lys5, Ile36, Cys39, Tyr40, Arg41, Asp54, Glu63, Tyr64, Met67, and Arg73. KRAS GTP / S39C The FKBP12 residues involved in binding are Arg43, Lys53, Gln54, Glu55, Thr86, Pro89, Gly90, and Ile92. The FKBP12 residues involved in C2Holt binding are Tyr27, Phe37, Asp38, Phe47, Glu55, Val56, Ile57, Trp60, Tyr83, His88, Ile91, Ile92, and Phe100.

[0311] The total embedded area of ​​the complex is 1,947 Å. 2 KRAS GTP / S39C The burial area is 600 Å. 2 Of these, 501 Å 2 This is due to FKBP12 (83%) contributing to 99 Å 2 This is due to C2Holt (17%). The embedded area of ​​FKBP12 is 762 Å. 2 Of these, 500 Å 2 KRAS GTP / S39C (66%) contributed, 262 Å 2 This is due to C2Holt contributing (34%). The embedded area of ​​C2Holt is 584 Å. 2 Of these, 132 Å 2 KRAS GTP / S39C This contributed (23%), 452 Å 2 FKBP12 contributes to this (77%). KRAS GTP / S39C The protein-protein interface between C2Holt and FKBP12 is formed by both hydrophobic and polar interactions, including three intermolecular hydrogen bonds. The binding interface between C2Holt and FKBP12 is largely contributed to by hydrophobic interactions, but also by the three carbonyl groups of the ligand, as well as the three hydrogen bonds between Tyr27, Ile57, and Tyr83 of FKBP12. C2Holt is designed (99Å). 2 ) by KRAS GTP / S39C It forms minimal contact with KRAS GTP / S39CIt forms one H bond with Glu37. The data collection and refinement statistics for the final structure are listed in Table 5 below.

[0312] B.KRAS GDP / S39C / SFAC4DS / CypA C52S Determination of the crystal structure of the ternary complex Reagents: Ligand (SFAC4DS) in 100% DMSO (proprietary product), CypA C52S (Manufactured in-house), KRAS GDP / S39C lite (proprietary product, containing residues 1-169 with G12V / S39C / C51S / C80L / C118S). Device: Superdex 75 (GE Healthcare) Experimental protocol: SFAC4DS and CypA C52S KRAS is used in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 1 mM MgCl2, and 2% DMSO in a 2:1 and 2:1 excess molar ratio. GDP / S39CIn addition to lite, the complex was incubated overnight at 20°C. The pure complex was isolated by size exclusion chromatography on a Superdex 75 column in 12.5 mM HEPES, pH 7.4, 75 mM NaCl, and 1 mM MgCl2. The purified complex (at 15 mg / ml) was subjected to crystallization screening at 20°C using sitting-drop vapor diffusion. Crystals were grown in well solutions containing 0.1 M bis-Tris, pH 6.5, and 25% PEG3350. For data acquisition, the crystals were transferred to a solution containing the mother liquor, supplemented with additional PEG3350 to make it 40% PEG, and then frozen in liquid nitrogen. Diffraction datasets were collected with an Advanced Light Source (ALS) and processed with the HKL program. Molecular substitution solutions were obtained using the PHASER program in the CCP4 suite, with the published structures of CypA (PDB-ID1CWA) and KRAS (PDB-ID3GFT) used as search models. Subsequent model construction and refinement were performed using standard protocols with the CCP4 and COOT software packages.

[0313] Result: CypA C52S -SFAC4DS-KRAS GDP / S39C Overall structure: The crystal is CypA in asymmetric units. C52S and KRAS GDP / S39C It contains one heterodimer (Figure 8). The model consists of residues Met1 to Glu165 of CypA and KRAS GDP / S39C The electron density was calculated to include Met1 through Lys169. The resulting electron density exhibits a clear binding mode, including ligand orientation and conformation. Continuous electron densities were observed for the cysteine ​​of the protein and the disulfide derived from the sulfur of the ligand.

[0314] KRAS involved in SFAC4DS coupling GDP / S39CThe residues (4 Å distance cutoff) are Glu3, Lys5, Cys39, Arg41, Leu52, Asp54, Ile55, and Leu56. CypA C52S KRAS involved in the binding to GDP / S39C The residues are Glu37, Asp38, Cys39, Arg41, Gln43, Leu56, Ala66, Met67, Gln70, and Thr74. KRAS GDP / S39C CypA involved in the binding C52S The residues are Arg55, Ile57, Arg69, Asn71, Thr73, Ala81, Ala103, Arg148, and Asn149. CypA is involved in the binding of SFAC4DS. C52S The residues are Arg55, Phe60, Met61, Gln63, Gly72, Ala101, Asn102, Gln111, Phe113, and His126.

[0315] The total embedding area of ​​this complex cannot be calculated due to partial structural defects at the protein-protein interface. Excluding the disordered region for calculation, the embedding area at the protein-protein interface is 1,350 Å. 2 It is more than 30% of which is SFAC4DS(443Å). 2 ) contributes to this. KRAS GDP / S39C and CypA C52S The protein-protein interface between SFAC4DS and CypA is formed by both hydrophobic and polar interactions, including two intermolecular H bonds. The binding interface between SFAC4DS and CypA is also contributed to by both hydrophobic and polar interactions. The carbonyl and NH groups of the ligand and CypA C52S Six H bonds exist between the residues Arg55, Gln63, Asn102, and His126. SFAC4DS is KRAS GDP / S39C Together, they form minimal direct contact, but KRAS GDP / S39C It forms one H bond with Arg41. The data collection and refinement statistics for the final structure are listed in Table 5 below.

[0316] C.PTP1BS187C Crystal structure determination of the / C3SLF / FKBP12 ternary complex Reagents: Ligand (C3-SLF) in 100% DMSO (proprietary product), FKBP12 (proprietary product), PTP1B S187C lite (proprietary product, containing residues 1-169 with C32S / C92V / C121S / S187C). Equipment: Superdex 75 (GE Healthcare), Gryphon (Art Robbins Instruments) Experimental protocol: C3SLF and FKBP12 were mixed in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, and 4% DMSO in a 3:1 excess molar ratio in PTP1B. S187C In addition to lite, the complex was incubated at 4°C for 36–72 hours. The pure complex was isolated by size exclusion chromatography on a Superdex 75 column in 12.5 mM HEPES, pH 7.4, and 75 mM NaCl. The purified complex (at 15 mg / ml) was subjected to crystallization screening at 20°C using sitting-drop vapor diffusion. Crystals were grown in well solutions containing 0.2 M magnesium acetate and 20% w / v PEG3350. For data acquisition, the crystals were transferred to a solution containing mother liquor supplemented with 25% PEG400 and then frozen in liquid nitrogen. Diffraction datasets were collected at the Advanced Photon Source (APS) and processed with the XDS program. Molecular substitution solutions were obtained using the PHASER program in the CCP4 suite, with the published structures of FKBP12 (PDB-ID2PPN) and PTP1B (PDB-ID2NT7) used as search models. The subsequent model configuration and refinement were carried out using a standard protocol with the software packages CCP4 and COOT.

[0317] Result:FKBP12-C3SLF-PTP1B S187C Overall structure: The crystal is asymmetric unit FKBP12-C3SLF-PTP1BS187C It contains two complex molecules (Figure 9A). The model is derived from residues Gly2 to Glu108 of FKBP12, and PTP1B S187C The electron density obtained contained Phe280 from Glu6. The electron density thus obtained shows a clear binding mode, including ligand orientation and conformation. Continuous electron densities were observed for the cysteine ​​of the protein and the disulfide derived from the sulfur of the ligand.

[0318] The total embedded area of ​​the complex is 1,042 Å. 2 This is PTP1B. S187C The burial area is 427 Å. 2 The embedding area of ​​C3-SLF is 615 Å. 2 This is the case (Figure 9B). PTP1B S187C The protein-protein interface between FKBP12 and FKBP12 is formed by both hydrophobic and polar interactions.

[0319] D.MCL1 S245C Crystal structure determination of the / C3SLF / FKBP52 ternary complex Reagents: Ligand (C3-SLF) in 100% DMSO (proprietary product), FKBP52 (proprietary product, residues 1-140), MCL1 S245C lite (proprietary product, containing residues 172-327 with S245C / C286S). Equipment: Superdex 75 (GE Healthcare), Gryphon (Art Robbins Instruments) Experimental protocol: C3SLF and FKBP52 were mixed in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 2% DMSO, with MCL1 in a 3:1 excess molar ratio. S245CThe complex was mixed with lite and incubated at 4°C for 24–48 hours. The pure complex was isolated by size exclusion chromatography on a Superdex 75 column in 12.5 mM HEPES, pH 7.4, and 75 mM NaCl. The purified complex (at 15 mg / ml) was subjected to crystallization screening at 20°C using sitting-drop vapor diffusion. Crystals were grown in well solutions containing 2.1 M malic acid. For data acquisition, the crystals were transferred to a solution containing mother liquor supplemented with 20% glycerol and then rapidly frozen in liquid nitrogen. 3.0 Å resolution diffraction datasets were measured at the Advanced Photon Source (APS) and processed with the XDS program. Molecular substitution solutions were obtained using the PHASER program in the CCP4 suite, with the published structures of FKBP52 (PDB-ID1N1A) and PTP1B (PDB-ID3MK8) used as search models. The subsequent model configuration and refinement were carried out using a standard protocol with the software packages CCP4 and COOT.

[0320] Results: The crystal is MCL1 in asymmetric units. S245C The complex contains one molecule of / C3SLF / FKBP52 (Figure 10). The electron density thus obtained revealed a clear binding between the two proteins, including ligand orientation and conformation. Continuous electron density was observed for the cysteine ​​of the protein and the disulfide derived from the sulfur of the ligand. The embedding area of ​​the complex was 1,410 Å. 2 Of these, approximately 60% are FKBP52(804Å). 2 ) contributes to this, and approximately 40% is C3-SLF (606Å 2 ) contributes to this. Due to limited resolution, detailed analysis of protein-protein and protein-ligand interactions was not feasible.

[0321] [Table 5]

[0322] Example 6: Determination of composite formation by TR-FRET TR-FRET technology (LANCE, Perkin Elmer) is a standard method for detecting the binary association of two fusion-tagged proteins, e.g., protein 1 / tag A and protein 2 / tag B (where A and B may be any of glutathione-S-transferase (GST), hexahistidine (His6), FLAG, biotin-avi, Myc, and hemagglutinin (HA)). In this example, the technology is used to measure the compound-promoted association between a presenter protein and a target protein. A mixture of presenter protein / tag A and target protein / tag B is added to a 384-well assay plate containing the compound of the present invention and incubated for 15 minutes. A mixture of anti-fusion-tag A or B europium-chelate donor and anti-fusion-tag A or B allophycocyanin acceptor or Ulight acceptor reagent is added, and the reaction is incubated for 240 minutes. The TR-FRET signal is read on an EnVision microplate reader (Perkin Elmer) using excitation = 320 nm and emission = 665 / 615 nm. Compounds that promote ternary complex formation are identified as those that elicit an increase in the TR-FRET ratio compared to the DMSO control well.

[0323] TR-FRET-mediated CYPA compound 3-KRAS G12C-GTP Determination of complex formation Avi-tagged cyclophyllin A and His-tagged KRAS G12C-GTPThe compound was mixed with increasing concentrations of ligand (compound 3) and incubated at room temperature for 15 minutes to allow for the formation of a ternary complex. A pre-mixture of anti-His Eu-W1024 and streptavidin APC was then added and incubated for 60 minutes. The TR-FRET signal was read on an EnVision microplate reader (Perkin Elmer, Ex320nm, Em665 / 615nm). A counterscreen without presenter and target proteins was also performed to exclude contributions from the compound alone.

[0324] Reagents and Instruments His6-KRAS G12C-GTP (Proprietary product; residues 1-169); 1.2 mM in PBS buffer, pH 7.4 • Avi-CYPA (proprietary product; residues 1-165); 556 μM in PBS buffer, pH 7.4 • Anti-His Eu-W1024 (Perkin Elmer) • Streptoavidin APC (PerkinElmer) • Ligand (W21487), 10 mM in 100% DMSO • EnVision (PerkinElmer) • Combi Multidrop liquid dispenser with 8-channel low-capacity cassettes • 384-well proxy plate (ProxiPlate) (Black) Experimental protocol 1. Using Mosquito, dispense 100 nL / well of the compound (concentration that changes in DMSO) into a 384-well black proxy plate (ProxiPlate) to prepare an assay-ready plate (ARP).

[0325] Prepare a 2× assay buffer containing 2.40 mM Hepes, pH 8.0, 200 mM NaCl, 2 mM MgCl2, 0.1% BSA, and 0.004% Tween-20.

[0326] 3. Prepare 2×PRE-MIX A: 100nM His6-KRas G12C-GTP (1-169) and 1000nM Avi-CypA (1-165) in 1× assay buffer.

[0327] 4. Using a MultiDrop Combi dispenser, dispense 2×PRE-MIX A into ARP, 5 μl / well. Incubate at room temperature for 15 minutes.

[0328] 5.2×PRE-MIX B: 10nM anti-His Eu-W1024 and 40nM anti-SA APCs are fabricated. 6. Dispense 2×PRE-MIX B into ARP, 5 μl / well using a MutiDrop Combi dispenser. Shake briefly on the Combi dispenser and incubate at room temperature for 60 minutes.

[0329] 7. Read the data on EnVision (Ex: 320nm; Em1: 615nm; Em2: 665nm). 8. Process the data using Dotmatics. Fit the curve using a 4-parameter nonlinear fit to determine the EC50 value for the formation of the ternary complex.

[0330] Results: The binding curve (Figure 11) shows the CYPA-compound 3-KRAS with a calculated EC50 value of 2.1 μM. G12C-GTP This complex formation is dependent on compound 3 of the ternary complex. Example 7: Determination of complex formation by amplified luminescence proximity homogeneous assay AlphaScreen technology (Perkin Elmer) is a standard method for detecting the binary association of two fusion-tagged proteins, e.g., protein 1 / tag A and protein 2 / tag B (where A and B may be any of glutathione-S-transferase (GST), hexahistidine (His6), FLAG, biotin-avi, Myc, and hemagglutinin (HA)). In this example, the technology is used to measure the compound-promoted association between a presenter protein and a target protein. A mixture of presenter protein / tag A and target protein / tag B is added to a 384-well assay plate containing the compound of the present invention and incubated for 15 minutes. A mixture of anti-fusion-tagged A or B AlphaScreen donor beads and anti-fusion-tagged A or B AlphaScreen acceptor beads is added, and the reaction is incubated for 240 minutes. The AlphaScreen signal is read on an EnVision microplate reader (Perkin Elmer) using excitation = 680 nm and emission = 585 nm. Compounds that promote ternary complex formation are identified as those that elicit an increase in the AlphaScreen signal compared to the DMSO control well.

[0331] Alpha-LISA-mediated CYPA-compound 3-KRAS G12C-GTP Determination of complex formation Avi-tagged cyclophyllin A and His-tagged KRAS G12C-GTPThe compound was mixed with increasing concentrations of ligand (compound 3) and incubated at room temperature for 60 minutes to allow for the formation of a ternary complex. A pre-mixture of nickel chelate donor beads and streptavidin acceptor beads was then added and incubated for another 60 minutes. The AlphaLISA signal was read on an EnVision microplate reader (Perkin Elmer, Ex680nm, Em615nm). A counterscreen without presenter and target proteins was also performed to exclude contributions from the compound alone.

[0332] Reagents and equipment: His6-KRAS G12C-GTP (Proprietary product; residues 1-169); 1.2 mM in PBS buffer, pH 7.4 • Avi-CYPA (proprietary product; residues 1-165); 556 μM in PBS buffer, pH 7.4 • Nickel chelate donor beads (Perkin Elmer) • Streptoavidin acceptor beads (Perkin Elmer) • Ligand (W21487), 10 mM in 100% DMSO • EnVision (PerkinElmer) • Combi Multidrop liquid dispenser with 8-channel low-capacity cassettes • AlphaPlate - 384 Plate (White) Experimental protocol: 1. Using Mosquito, dispense 100 nL / well of the compound (concentration that changes in DMSO) into a 384-well black proxy plate (ProxiPlate) to prepare an assay-ready plate (ARP).

[0333] Prepare a 2× assay buffer containing 2.40 mM Hepes, pH 8.0, 200 mM NaCl, 2 mM MgCl2, and 0.004% Tween-20. 3. Prepare 2×PRE-MIX A:300nM His6-KRas G12C-GTP (1-169) and 300nM Avi-CypA (1-165) in 1× assay buffer.

[0334] 4. Using a MultiDrop Combi dispenser, dispense 2×PRE-MIX A into ARP, 5 μl / well. Incubate at room temperature for 60 minutes.

[0335] Prepare streptavidin acceptor beads and nickel chelate donor beads containing 30 μg / ml of 5.2×PRE-MIX B. 6. Dispense 2×PRE-MIX B into ARP, 5 μl / well using a MutiDrop Combi dispenser. Shake briefly on the Combi dispenser and incubate at room temperature for 60 minutes.

[0336] 7. Read on EnVision (Ex: 680nm; Em1: 615nm). 8. Process the data using Dotmatics. Fit the curve using a 4-parameter nonlinear fit to determine the EC50 value for the formation of the ternary complex.

[0337] Results: The binding curve (Figure 12) shows the CYPA-compound 3-KRAS with a calculated EC50 value of 0.99 μM. G12C-GTP This complex formation is dependent on compound 3 of the ternary complex.

[0338] Example 8: Determination of composite formation by isothermal titration calorimetry Isothermal titration calorimetry (ITC) is an established biophysical technique used to directly measure the thermal changes associated with the binary interaction of two proteins, or proteins to a ligand. The measurement of thermal changes involves the association constant (K). a This allows for the precise determination of the reaction stoichiometry (N) and the change in bond enthalpy (ΔH). The Gibbs free change (ΔG) and the entropy change (ΔS) are also related by: ΔG = -RTlnK a This can be determined using the formula =ΔH-TΔS (where R is the gas constant and T is the absolute temperature). In this example, this method is used to measure the binding (e.g., non-covalent or covalent) of the compound or conjugate of the present invention to a presenter protein.

[0339] Kinetic and thermodynamic determination of the bond between FKBP12-compound 1 and CEP250 by ITC Reagents: Compound 1 and Compound 2 in 100% DMSO (proprietary), protein buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer + 1% DMSO), FKBP12 (proprietary), CEP250 29.4 (In-house manufactured, residues 1982-2231) and CEP250 11.4 (Manufactured in-house, residues 2134-2231).

[0340] Equipment: MicroCal(TM) ITC 200 (GE Healthcare). The device parameters are shown in Table 6.

[0341] [Table 6]

[0342] Experimental protocol: Dilute the FKBP12 stock solution to 10 μM with assay buffer (final 1% DMSO). Add the compound to FKBP12 to a total concentration of 20 μM (final 1% DMSO), and after a pre-incubation time of 5-10 minutes, fill the reaction cell of the ITC instrument with the binary complex. Dilute the CEP250 protein stock to 50 μM in assay buffer, add 20 μM of the compound (final 1% DMSO), and then fill the injection syringe. When injecting from the syringe into the reaction cell, perform a control experiment in the absence of the compound to determine operational artifacts and the heat associated with the dilution of the titrant. More detailed experimental parameters are shown in Table 7 below.

[0343] [Table 7]

[0344] Data fitting: Data fitting was performed using Origin ITC200 software following the procedure below. 1) Read the raw data.

[0345] 2) "mRawlTC": Integrates all peaks while adjusting the integral peak and baseline. 3) "ΔH" - Data Management: Remove bad data (injection #1 and other artifacts) and subtract straight lines (background removal).

[0346] 4) "ΔH" - Model Fitting: Select a set of site models and fit them using the Levenberg-Marquardt algorithm until χ² no longer decreases further, ending with "done" (parameters N, Ka, and ΔH are calculated based on the fitting).

[0347] Results: ITC measurements of the binding of the FKBP12-compound 1 and FKBP12-compound 2 binary complexes to CEP250 are summarized in Table 8 and Figure 13 below. Overall, the data for the FKBP12-compound 1 and FKBP12-compound 2 binary complexes binding to CEP25011.4 and CEP25029.4 show similar interaction parameters. The Kd values ​​were similar for all combinations. All interactions showed almost identical thermodynamic profiles, and the binding was characterized by a pure enthalpy binding mode (-T). * The AS term is positive and does not contribute to the Gibbs free energy. The bond stoichiometric ratio for all interactions is N=0.5~0.6, and CEP250 11.4 The crystalline structure of compound 1 / FKBP12 supports a 1:2 binding ratio for one CEP250 homodimer to two FKBP12 molecules.

[0348] [Table 8]

[0349] Example 9: Determination of the kinetics of binding between conjugates and proteins by surface plasmon resonance Surface plasmon resonance (SPR) is a biophysical technique used to measure the binary interaction and associated kinetics of two proteins, or proteins to a ligand. Typically, one component of a binary interaction pair is immobilized on a flow cell of an activated sensor chip via a fusion tag. Then, a second component (analyte) is injected onto the activated surface at increasing concentrations for a set period of time. The increase in the SPR signal (represented by resonance units, RU) during the association phase and the decrease in the SPR signal during the dissociation phase indicate the interaction and fit the binding model, as well as the associated K D , K a , K dThe value can be determined. In this embodiment, this method is used to measure the kinetics of the binding of the conjugate of the present invention to a presenter protein, where (i) the conjugate is immobilized on a chip via a fusion tag and the presenter protein is injected onto the surface, or (ii) the presenter protein is immobilized on a chip via a fusion tag and the conjugate is injected onto the surface.

[0350] Kinetic determination of the bond between FKBP12-compound 1 and CEP250 using SPR. This protocol describes the kinetics (K) of the binding of CEP250 (analyte) to the immobilized FKBP12-compound 1 binary complex (ligand). D , K a , K d Surface plasmon resonance (SPR) is used as a method to determine the )

[0351] Reagents: Compound 1 in 100% DMSO (proprietary), 10× HBS-P+ buffer (GE Healthcare BR-1006-71), assay buffer (1× HBS-P+ buffer, 1% DMSO, 1 μM Compound 1), 12× HIS-tagged FKBP12 (proprietary), CEP250 29.2 (residues 1982-2231) and CEP250 11.4 (Residues 2134-2231) (Manufactured in-house).

[0352] Device: BIACORE(trademark)X100 (GE Healthcare) Device: NTA sensor chip (GE Healthcare BR-1000-34) Experimental Protocol: The experiment is performed at 25°C. A stock solution of 12XHIS-tagged FKBP12 is diluted to 100 nM in assay buffer containing 1 μM compound 1 (final 1% DMSO). Approximately 200–400 RU of FKBP12 is immobilized on one of the two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for nonspecific interactions of the analyte to the sensor chip. Various concentrations of CEP250 (1 nM–1 μM range), serially diluted in the same assay buffer containing 1 μM compound 1 (final 1% DMSO), are injected onto the FKBP12 surface and the reference surface at a flow rate of 10 μl / min. The surface is regenerated between injections of the analyte with 350 mM EDTA.

[0353] Data fitting: The BiaEvaluation software program is used for data fitting. All data are subtracted for both the reference flow cell and buffer injection. For kinetic analysis, the data are locally fitted to a 1:1 interaction model.

[0354] Results: SPR sensorgrams are shown in Figure 14. Dissociation constants (K) for 5.4 nM and 0.29 nM. D ) are, respectively, CEP250 11.4 and CEP250 29.2 The binding of FKBP12 / compound 1 to was determined.

[0355] Example 10: Determination of the kinetics of binding between conjugates and proteins by biolayer interferometry. Biolayer interferometry (BLI) is a biophysical technique used to measure the binary interaction and associated kinetics of two proteins to a ligand. Typically, one component of a binary interaction pair is immobilized on a biosensor chip via a fusion tag. Then, a second component (analyte) is injected onto the biosensor chip at increasing concentrations for a set period of time. The increase in the BLI signal (expressed in optical thickness, nm) during the association phase and the decrease in the BLI signal during the dissociation phase indicate the interaction and fit the binding model to the associated K D , K a , K d The value can be determined. In this embodiment, this method is used to measure the kinetics of the binding of the conjugate of the present invention to a presenter protein, where (i) the conjugate is immobilized on a chip via a fusion tag and the presenter protein is injected onto the surface, or (ii) the presenter protein is immobilized on a chip via a fusion tag and the conjugate is injected onto the surface.

[0356] CYPA compound 3 and KRAS by BLI G12C-GTP Determination of the kinetics of the bond between This protocol involves KRAS to immobilized CYPA-compound 3 binary complex (ligand). G12C-GTP Dissociation constant (K) for the binding of (analyte) D Biolayer interferometry (BLI) is used as a method to determine the ).

[0357] Reagents: Compound 3 in 100% DMSO (proprietary), ForteBio kinetic buffer (ForteBio Corporation, Menlo Park, CA), assay buffer (kinetic buffer, 1% DMSO, 2 μM of Compound 3), Avi-tagged CYPA (proprietary), KRAS G12C-GTP (Residues 1-169) (Manufactured in-house). Equipment: Octet Red 96 units (ForteBio Co., Ltd., Menlo Park, CA) Device: Streptoavidin (SA) biosensor (ForteBio) Experimental protocol: Streptoavidin (SA) biosensors were coated at 25°C in a solution containing 10 μM Avi-CYPA protein until a packing signal of 0.6 nm was reached. Protein packing demonstrated long-term stability and the absence of baseline drift. Ternary complex formation was initiated in a 1:2 dilution series starting from 200 μM in KRAS. G12C-GTP The response was evaluated in dose-response experiments involving protein concentration. For the negative control, sensors coated with Avi-CYPA protein were immersed in wells containing only screening buffer (supplemented with 2 μM compound 3). Corrected binding response sensorograms were recorded and analyzed.

[0358] Data fitting: Analysis on the ForteBio Octet RED instrument was performed using ForteBio software. The analysis accounted for nonspecific binding, background, and signal drift, minimizing well-base and sensor variability. Dose-dependent formation of the ternary complex was observed, along with the corresponding equilibrium dissociation constant (K). D ) was decided.

[0359] Results: The sensorogram and steady-state fitting curve are shown in Figure 15. Dissociation constant (K) of 44 μM. D ) is KRAS G12C-GTP The binding of CYPA / compound 3 to the target compound was determined.

[0360] Example 11: Proteomics of FKBP12-binding target proteins for crosslinking reagents Identification reagents: Compound in 100% DMSO (proprietary product), N-terminal biotin-FKBP12 (proprietary product), HEK293T cell lysate (proprietary product).

[0361] Experimental protocol: HEK293 T cell lysates were prepared using sonication on ice (20% power, 4-10 second pulses) with a lysis buffer consisting of 40 mM HEPES, pH 7.3, 120 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.5% octyl-β-glucoside, and an EDTA-free protease inhibitor cocktail (Roche). The lysates were first purified by centrifugation, and the resulting supernatant was passed through a 0.2 mm syringe filter on ice. N-terminal biotin-labeled FKBP12 was added to 500 ml of lysate to a final concentration of 4 mM and mixed by pipetting, and then the compound was added to a final concentration of 10 mM, with the reactants mixed by pipetting. 60 mL of 50% slurry agarose-streptavidin resin (pre-equilibrium in lysis buffer) was added, and the reaction was allowed to proceed at 4°C for 1 hour with gentle shaking. After incubation, the resin was gently pelletized and washed four times with 1 mL of lysis buffer on ice by addition, centrifugation, and aspiration, and then washed four more times in the same physical manner with 1 mL of lysis buffer without detergent. The retained protein was eluted from the resin using 8 M urea in HEPES buffer, pH 8.0, diluted to 7 M urea with 100 mM HEPES, pH 8.0, and endoprotease Lys-C was added at 37°C for 2 hours for protein digestion. Next, the sample was diluted to 0.8 M urea with 100 mM HEPES, pH 8.0, trypsin was added, and the sample was digested further at 37°C for 16 hours. After digestion was complete, the sample was prepared for LC-MS / MS analysis using a C18SPE filter. The sample was packed onto this filter, washed, eluted, dried in a speed-vac, and finally suspended in 10 ml of 5% acetonitrile, 5% formate buffer for LC-MS / MS analysis. LC-MS / MS analysis was performed on a Thermo-Fisher LTQ-Velos-Pro OrbiTrap mass spectrometer using a top 20 data-dependent acquisition method and an 8-35% acetonitrile gradient for HPLC.The peptide sequence was assigned using the Sequest algorithm, and the identified protein was compared to a control sample (DMSO only) to identify candidate target proteins.

[0362] Results: Using the protocol described above, >100 target proteins have been identified that can bind to presenter proteins in the presence of crosslinking compounds. Identified target proteins include kinases, phosphatases, ubiquitin ligases, DNA-binding proteins, heat shock proteins, DNA helicases, GTPase-activating proteins, nucleotide-binding proteins, and various protein-binding proteins.

[0363] Example 12: Determination of binding between conjugate and protein by fluorescence polarization The fluorescence polarization (FP) technique is based on the observation that when a fluorescently labeled molecule is excited by polarization, it emits light with a degree of polarization inversely proportional to its molecular rotation. Small molecules rotate rapidly during the excited state and, due to emission, have a low polarization value. Larger complexes formed by the binding of a labeled molecule to a second molecule rotate only slightly during the excited state and therefore have a high polarization value. This property of fluorescence can be used to measure the interaction of a labeled ligand with larger proteins and provides a basis for direct and competitive binding assays. In this example, the method is used to measure the binding of the compound or conjugate of the present invention to a presenter protein and to establish ternary complex formation with a target protein.

[0364] Determination of CypA:C3DS:KRAS complex formation by FP Reagents: C3DS (proprietary) in 100% DMSO, protein buffer (12.5 mM HEPES, pH=7.4, 1 mM MgCl2), assay buffer (25 mM HEPES, pH7.3, 0.002% Tween20, 0.1% BSA, 10 mM NaCl, 1 mM MgCl2), CYPA (proprietary), Mant-GMP-PNP-filled KRAS (residues 1-169).

[0365] Device: SpectraMax Experimental protocol: The KRAS stock solution is packed to a final concentration of 0.8 μM in assay buffer (final 1% DMSO). The compound (C3DS) is added to a final concentration of 10 μM, and the reaction mixture is dispensed into 384-well Costal Black plates. CYPA is serially diluted in the wells of the plate and incubated at room temperature for 15 minutes. A control experiment in the absence of the compound is also performed to determine the association of CYPA to KRAS in the absence of the compound. The reaction mixture is excited at 355 nm, and the emission signal is recorded at 455 nm. The signal is measured in the vertical and parallel planes, and the polarization is recorded using the following equation.

[0366] FP (polarization unit × 10^-3) = signal(parallel) - signal(perpendicular) / [signal(parallel) + signal(perpendicular)] Results: Representative curves are shown in Figure 16, and a table listing EC50 (the concentration required to enhance the FP signal of KRAS by 50%) is listed below. The curves were fitted to a four-parameter equation, and the resulting EC50 values ​​show the effect of ligand C3DS on enhancing the binding between CYPA and KRAS.

[0367] [Table 9]

[0368] Example 13: Determination of binding between conjugates and proteins by nuclear magnetic resonance. Nuclear magnetic resonance (NMR) spectroscopy is a technique used to elucidate three-dimensional structures and study the dynamics of proteins and protein-ligand complexes. Furthermore, it can be used to identify ligand binding sites in protein-ligand interactions. Among several available NMR approaches, protein structure-based ligand screening (highly sensitive 2D) is one of them. 1 H- 15Identifying critical residues involved in ligand (drug) binding (N TROSY-HSQC spectroscopy) is the most sensitive method for such studies. This involves sequentially increasing concentrations of ligand added to the NMR sample of the protein, and 2D 1 H- 15 The collection of N TROSY-HSQCs provides highly degraded residue perturbation information at the atomic level, referred to as chemical shift perturbations (CSPs), which directly provides more accurate information about ligand binding sites that is not possible with any other available biophysical techniques. This approach allows for the study of weak, intermediate, and strong affinities of ligand binding to proteins or binary protein complexes, and this information can be directly linked to existing structural, dynamic, and kinetic information. In this embodiment, this method is used to demonstrate the binding (e.g., non-covalent or covalent) of the compound (drug) or conjugate of the present invention to a presenter protein.

[0369] Determination of the KRAS(G12C)-cyclophylline-compound 3-bond in the ternary complex by solution MR spectroscopy. Reagents: Compound 3 (proprietary product) in 100% DMSO, protein buffer (50 mM TRIS-d 11 50 mM NaCl, pH 7.0, 1 mM TCEP-d 16 (1 mM MgCl2), additives in the KRAS NMR sample (100 μM DSS in 93% H2O and 7% D2O), assay buffer (protein buffer in DMSO (≤5%) + increasing equivalent amount of drug), GMP- 15 N-KRAS(G12C)-16 (N-His, residues 1-169, proprietary), unlabeled (UL) cyclophylline (CYPA; residues 1-165) (proprietary).

[0370] Device: 5mm CPTCI 1 H- 13 C / 15A Bruker Avance 800MHz spectrometer equipped with an N / D Z-GRD Z44909 / 0026 frozen probe (Bruker). High-precision 5mm NMR tubes are used in these experiments.

[0371] NMR data processing and analysis: A Linux computer running Topspin v3.1 and NMRPipe / NMRDraw for processing, and the CCPNMR "Analysis" program for data analysis.

[0372] Experimental protocol: 0.72 mM GMP- in protein buffer 15 N-KRAS(G12C)-16 stock solution was used to prepare a 0.18 mM NMR sample in 600 μl (including NMR additives). DSS was used as an internal standard for chemical shift reference (at 0.0 ppm). 1 (H peak) 15 N KRAS's 2D 1 H- 15 N TROSY-HSQC spectra were collected (data size, 2048 × 128). One equivalent (0.18 mM) of CYPA in protein buffer was added to the NMR sample (from a 0.4 mM stock solution) and stirred for 10 minutes. The final NMR sample volume was maintained at 600 μl. Binary complex ( 15 2D (N-KRAS + UL-CYPA) 1 H- 15 The N TROSY-HSQC spectra were collected while maintaining the same other acquisition parameters (data size 2048×128) (KRAS) 1 H- 15 Only the N-correlated cross peak is visible in the spectrum. A stock solution of compound 3 in 100% DMSO at 20 mM was used for NMR titration. Compound 3 was added sequentially to the NMR sample to form a binary complex. 15In the NMR sample of (15N-KRAS + UL-CYPA), 0.5 equivalents, 1.0 equivalents, 2.5 equivalents, and 5.0 equivalents thereof (relative to the 15N-KRAS concentration) were obtained. At each stage, while maintaining the acquisition parameters the same, a sample volume of 600 μl was maintained. At each stage of the addition of Compound 3, 2D 1 H- 15 N TROSY-HSQC spectra were acquired to investigate the chemical shift perturbation (CSP) of the KRAS residues. All spectra were overlaid with each other. The effective CSP at each Compound 3 titration point was determined using the difference in the chemical shifts of each residue of KRAS in the ternary complex (KRAS + CYPA + Compound 3) relative to the binary complex (KRAS + CYPA). Subsequently, the weighted average chemical shift (Δδ 加重 ) of each KRAS residue was determined using the following equation: Δδ 加重 =[(Δ 1 H) 2 +(Δ 15 N / 5) 2 1 / 2 Residues with a Δδ 加重 exceeding one standard deviation from the total average are considered to be significantly perturbed and are used in binding site mapping. In separate titration experiments, the inventors added DMSO equivalents sequentially (to achieve the same solvent concentration as in the above experiments) and subtracted the contribution from the DMSO addition to collect 2D 1 H- 15 N TROSY-HSQC spectra for the binary complex (KRAS + CYPA). In a second control experiment, the inventors collected a series of 2D 1 H- 15 N TROSY-HSQC spectra of 15N-KRAS titrated with Compound 3 at different equivalents (in the absence of CYPA).

[0373] The effective CSPs are tabulated and analyzed. The drug-binding residues of KRAS (in the presence of CYPA) are mapped on the protein surface. The dissociation constant, K D is determined. Experimental and processing parameters:​ Spectral data size: 2048 (1H dimension) × 128 (15N dimensions) Number of scans: 4 Temperature: 298K Quadrature detection modes: DQD (1H) and Echo-AntiEcho (15N) The data size was expanded by applying forward-backward linear prediction within the indirect dimension. The dataset was extrapolated by zeroing each dimension once before the Fourier transform.

[0374] Result: KRAS G12C-GTP The 2D1H-15N TROSY-HSQC spectrum is shown in Figure 17A. Adding a stoichiometric amount of CYPA has no effect on the KRAS amide skeleton cross-peak (Figure 17B), indicating that KRAS and CYPA do not directly interact. Titration of W21487 to a 1:1 CYPA:KRAS sample yields a clear chemical shift indicating a direct interaction with KRAS (Figure 17C).

[0375] Example 14. Determination of binding between conjugates and proteins by microscale thermophoresis. Microscale thermophoresis (MST) is a technique for characterizing biomolecular interactions by correlating molecular properties of molecules, such as size, and changes in conformation with respect to their mobility in a constant temperature gradient. The gradient is induced by an infrared laser. The movement of biomolecules is often characterized by labeling the molecules using covalently attached fluorophores or, conversely, chromatic fluorescence. In this embodiment, this method is used to measure the binding of the compound or conjugate of the present invention to a presenter protein and to establish ternary complex formation with a target protein, where (i) the conjugate is labeled with a fluorophore and the presenter protein is titrated, or (ii) the presenter protein is labeled with a fluorophore and the conjugate is titrated.

[0376] Example 15. Determination of the binding between a conjugate and a protein by second harmonic generation technology Second harmonic generation (SHG) is an optical phenomenon that can be used to measure conformational changes in aqueous solutions in real time. The SHG signal intensity is sensitive to the average angular orientation of the dye labeled protein linked to the surface, and the magnitude of the signal change is directly correlated with the amount of angular change. Different conformations can be classified by the magnitude of the signal change due to binding, the signal relative to the baseline (more perpendicular orientation to the surface generated positive signal change, same for the reverse), and kinetics. In this example, this method is used to measure the binding of the compounds or conjugates of the invention to the presenter protein and to establish the formation of a ternary complex with the target protein, where (i) the dye labeled conjugate is immobilized on the surface via a fusion tag and the presenter protein is injected onto the surface or (ii) the dye labeled presenter protein is immobilized on the surface via a fusion tag and the conjugate is injected onto the surface.

[0377] Example 16. Determination of the binding between a conjugate and a protein by differential scanning fluorimetry Differential scanning fluorimetry (DSF) is a solution-based biophysical technique used to measure the melting temperature (T m ) of a protein. In a typical experiment, the protein of interest is subjected to increasing heat (typically 4 °C to 95 °C) in the presence of a fluorescent dye (e.g., SYPRO Orange). The fluorescence intensity is plotted as a function of temperature, and T m is calculated from the minimum of the negative derivative of the fluorescence signal. For the target protein, the thermal shift (ΔT m) can be measured to assess whether a small molecule binds to and stabilizes a protein. In this example, the method is used to measure the thermal shift (e.g., non-covalent or covalent) of the compound or conjugate of the present invention to a presenter protein, where (i) the conjugate is labeled with a fluorescent dye and the presenter protein is titrated, or (ii) the presenter protein is labeled with a fluorescent dye and the conjugate is titrated.

[0378] Example 17. Determination of binding between conjugates and proteins using nanoDSF. NanoDSF uses altered tryptophan or tyrosine fluorescence to fluoresce in proteins. m This is an advanced DSF method for measuring [the fluorescence intensity of endogenous tryptophan or tyrosine residues]. In a typical experiment, the protein of interest is subjected to increasing heat (typically 4°C to 95°C), and the fluorescence intensity of endogenous tryptophan or tyrosine residues is monitored as a function of temperature. m This can be calculated from the change in tryptophan fluorescence intensity or from the rate of tryptophan emission at 330 nm and 350 nm, which explains the shift in tryptophan emission due to unfolding. For target proteins, ΔT in the presence of small molecules m This can be measured to assess whether a small molecule binds to and stabilizes a protein. In this example, the method is used to measure the thermal shift (e.g., non-covalent or covalent) of the compound or conjugate of the present invention to a presenter protein, where (i) the fluorescence of the conjugate is measured and the presenter protein is titrated, or (ii) the fluorescence of the presenter protein is monitored and the conjugate is titrated.

[0379] Example 18. Determination of composite formation by differential light scattering. Dynamic light scattering (DLS) is an established biophysical method used to measure time-dependent fluctuations in scattering intensity that exhibit random Brownian motion. Diffusion coefficient and particle size information can be obtained from the analysis of these fluctuations. More specifically, this method provides the ability to measure size characteristics, including radius and molecular weight, of proteins in aqueous solutions. In this embodiment, this method is used to measure changes in radius or molecular weight in (i) a presenter protein upon binding of the conjugate of the present invention, or (ii) a conjugate of the present invention upon binding to a presenter protein.

[0380] Example 19. Determination of binding between conjugates and proteins using sonic acoustic technology. Surface acoustic wave (SAW) technology is a biophysical method used for the real-time detection of binding-induced conformational changes by monitoring the phase shift of surface acoustic waves traveling along a biosensor. It can be used to measure the binary interaction of proteins to two proteins or ligands and the associated kinetics. Typically, one component of a binary interaction pair is immobilized on the biosensor via a fusion tag. Then, an increasing concentration of the second component (analyte) is injected onto the biosensor for a set period of time. The increase in signal during the association phase (measured by changes in wave phase or amplitude) and the decrease in signal during the dissociation phase indicate the interaction and, fitting it to a binding model, the associated K D , K a , K d The value can be determined. In this embodiment, this method is used to measure the kinetics of the binding of the conjugate of the present invention to a presenter protein, where (i) the conjugate is immobilized on a biosensor chip via a fusion tag and the presenter protein is injected onto the surface or (ii) the presenter protein.

[0381] Example 20: Determination of composite formation by small-angle X-ray scattering Small-angle X-ray scattering (SAXS) is a solution-based method used to determine the structure of proteins in terms of average particle size and shape. It can deliver structural information on repeating distances in partially ordered systems up to 150 nm in size, with a resolution range of 1–25 nm. Ultra-small-angle scattering (USAS) can resolve to even larger dimensions. In a typical scattering experiment, a solution of a protein or protein complex is exposed to X-rays (typically with a wavelength λ of approximately 0.15 nm). The scattering intensity I(s) is recorded as a function of momentum transport (s = 4πsinθ / λ, where 2θ is the angle between the incident and scattered radiation). Scattering from the solvent alone is subtracted from the intensity of the solution. The X-ray scattering curve (intensity against scattering angle) is then used to produce a low-resolution model of the protein or protein complex. In this example, this method is used to identify the presence of a ternary complex (e.g., non-covalent or covalent) of the compound or conjugate of the present invention to a presenter protein.

[0382] Other Embodiments While this disclosure has been described in conjunction with a detailed explanation, it should be understood that the above explanation exemplifies the scope of this disclosure but is not intended to limit the scope of this disclosure as defined by the appended claims. Other aspects, advantages, and modifications are within the claims below. Those skilled in the art can recognize or confirm many equivalents of the particular embodiments of the invention described herein by simply using conventional experimental methods. The scope of the invention is not intended to be limited to the above explanation, but rather as described in the appended claims. In the claims, articles, e.g., "a," "an," and "the," may mean one or more unless otherwise indicated or otherwise evident from the context. Any claim or description containing "or" between one or more members of a group is considered satisfied unless otherwise indicated or otherwise evident from the context, if one, more, or all of the group members are present, used, or otherwise related in a given product or process. The present invention includes embodiments in which exactly one member of the group is present, used, or otherwise related in a given product or process. The present invention also includes embodiments in which multiple or all of the group members are present, used, or otherwise related in a given product or process.

[0383] The term “including” is intended to be open and may include further elements or steps, but it should also be noted that it is not required to include them. When the term “including” is used herein, the term “consisting of” is thus also included and disclosed.

[0384] Where a range is given, it includes the endpoint. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of those skilled in the art, the values ​​expressed as a range may be assumed to be any specific value in different embodiments of the Invention, or a subrange within the described range, up to one-tenth of the lower limit unit of the range, unless otherwise clearly indicated by the context. Furthermore, it should be understood that any specific embodiment of the Invention that lies within the scope of the prior art may be expressly excluded from any one or more of the claims. Since such embodiments are considered to be known to those skilled in the art, such embodiments may be excluded even if the exclusion is not expressly stated herein. Any specific embodiment of the composition of the Invention (e.g., any polynucleotide or protein encoded thereby; any method of production; any method of use) may be excluded from any one or more of the claims for any reason, whether or not it relates to the existence of the prior art.

Claims

1. A compound containing a protein-binding site and a crosslinking group.

2. The compound according to claim 1, wherein the crosslinking group is a portion capable of chemoselectively reacting with an amino acid.

3. The compound according to claim 1 or 2, wherein the crosslinking group is a sulfhydryl-reactive crosslinking group, an amino-reactive crosslinking group, a carboxyl-reactive crosslinking group, a carbonyl-reactive crosslinking group, or a triazole-forming crosslinking group.

4. The compound according to claim 3, wherein the crosslinking group is a sulfhydryl-reactive crosslinking group.

5. The compound according to any one of claims 1 to 4, wherein the crosslinking group comprises a mixed disulfide.

6. The aforementioned crosslinking group has the structure of chemical formula I. 【Chemistry 1】 The formula includes, where the dashed line illustrates the attachment point of the crosslinking group to the remainder of the compound, a is 0, 1, or 2, R Ａ is optionally substituted C １ -C ６ alkyl, optionally substituted C １ -C ６ heteroalkyl, optionally substituted C ６ -C １０ aryl, or optionally substituted C ２ -C ９ heteroaryl, the compound according to claim 5.

7. R Ａ However, C is replaced by arbitrary selection. ２ ~C ９ The compound according to claim 6, which is a heteroaryl compound.

8. C which is replaced by the aforementioned optional selection ２ ~C ９ The compound according to claim 7, wherein the heteroaryl is pyridyl.

9. The aforementioned crosslinking base has structure 【Chemistry 2】 The compound according to claim 6, wherein the wavy line in the formula illustrates the attachment point of the crosslinking group to the remainder of the compound.

10. R Ａ However, C is replaced by arbitrary selection. １ ~C ６ The compound according to claim 6, wherein it is alkyl.

11. C which is replaced by the aforementioned optional selection １ ~C ６ The compound according to claim 10, wherein the alkyl group is methyl.

12. The aforementioned crosslinking base has structure 【Transformation 3】 The compound according to claim 6, wherein the wavy line in the formula illustrates the attachment point of the crosslinking group to the remainder of the compound.

13. The compound according to any one of claims 1 to 4, wherein the crosslinking group comprises maleimide.

14. The aforementioned crosslinking base has structure 【Chemistry 4】 The compound according to claim 13, comprising, wherein the dashed line in the formula illustrates the attachment point of the crosslinking group to the remainder of the compound.

15. The compound according to claims 1 to 4, wherein the crosslinking group comprises a vinyl sulfone.

16. The aforementioned crosslinking base has structure 【Transformation 5】 The compound according to claim 15, comprising, wherein the dashed line illustrates the attachment point of the crosslinking group to the remainder of the compound.

17. The compound according to claims 1 to 4, wherein the crosslinking group comprises a vinyl ketone.

18. The aforementioned crosslinking base has structure 【Transformation 6】 The compound according to claim 17, comprising, wherein the dashed line in the formula illustrates the attachment point of the crosslinking group to the remainder of the compound.

19. The compound according to any one of claims 1 to 4, wherein the crosslinking group comprises an alkyl halogen.

20. The compound according to claim 19, wherein the alkyl halogen is an alkyl chloride.

21. The aforementioned crosslinking base has structure 【Transformation 7】 The compound according to claim 20, comprising, wherein the dashed line illustrates the attachment point of the crosslinking group to the remainder of the compound.

22. The compound according to any one of claims 1 to 21, wherein the interaction between the protein-binding portion and the protein is non-covalent.

23. The compound according to any one of claims 1 to 21, wherein the interaction between the protein-binding portion and the protein is covalent.

24. A compound containing a presenter protein binding site and a crosslinking group.

25. The compound according to claim 24, wherein the presenter protein binding portion can bind a protein encoded by any one of the genes in Table 1.

26. The compound according to claim 24, wherein the presenter protein binding portion is a prolyl isomerase binding portion.

27. The compound according to any one of claims 24 to 26, wherein the presenter protein binding portion is an FKBP binding portion, a cyclophylline binding portion, or a PIN1 binding portion.

28. The compound according to claim 27, wherein the presenter protein binding portion is an FKBP binding portion.

29. The compound according to claim 28, wherein the presenter protein binding portion can bind FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

30. The compound according to claim 28 or 29, wherein the FKBP binding site is a selective FKBP binding site.

31. The compound according to claim 28 or 29, wherein the FKBP binding site is a non-selective FKBP binding site.

32. The FKBP bond portion has the structure of chemical formula IIa or IIb. 【Transformation 8】 Including Z １ and Z ２ Each of these is independently and arbitrarily replaced C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ It is heteroalkyl, or Z １ and Z ２ These combine to form a macrocyclic molecule of 10 to 40 members, which are optionally substituted, along with the atoms to which they are attached, Z １ or Z ２ At least one of these includes an attachment point to the crosslinking group, b and c are independently 0, 1, or 2. d is 0, 1, 2, 3, 4, 5, 6, or 7. X １ and X ２ These are, independently, non-existent, CH ２ ,O,S,SO,SO ２ , or NR ４ And, Each R １ and R ２ These are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, C replaced by optional substitution ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, or R １ and R ２ These combine with the carbon atoms to which they are bonded to form C=O, or R １ and R ２ These are combined and replaced by C by choice. ３ ~C １０ Carbocyclyl or optionally replaced C ２ ~C ９ Forming heterocyclines, Each R ３ is, independently, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C １ to C ６ alkyl, optionally substituted C ２ to C ６ alkenyl, optionally substituted C ２ to C ６ alkynyl, optionally substituted C １ to C ６ heteroalkyl, optionally substituted C ２ to C ６ heteroalkenyl, optionally substituted C ２ to C ６ heteroalkynyl, optionally substituted C ３ to C １０ carbocyclic, optionally substituted C ６ to C １０ aryl, optionally substituted C ６ to C １０ aryl C １ to C ６ alkyl, optionally substituted C ２ to C ９ heterocyclic, or optionally substituted C ２ to C ９ heterocyclic C １ to C ６ alkyl, or two Rs ８ combine to form optionally substituted C ３ to C １０ carbocyclic, optionally substituted C ６ to C １０ aryl, or optionally substituted C ２ to C ９ heteroaryl, and Each R ４ These are C, which is independently substituted with hydrogen of any choice. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ The compound according to any one of claims 28 to 31, wherein it is alkyl.

33. The presenter protein binding portion is structure 【Chemistry 9】 The compound according to claim 32, having the following characteristics.

34. The compound according to claim 27, wherein the presenter protein binding portion is a cyclophylline binding portion.

35. The compound according to claim 34, wherein the presenter protein binding portion can bind PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

36. The compound according to claim 34 or 35, wherein the cyclophylline binding portion is a selective cyclophylline binding portion.

37. The compound according to claim 34 or 35, wherein the cyclophylline binding portion is a non-selective cyclophylline binding portion.

38. The cyclophylline bond portion has a structure of chemical formula III or IV. 【Chemistry 10】 It has, in the formula, Z ３ Z ４ Z ５ , and Z ６ Each of these is independently a hydroxyl, or optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ It is heteroalkyl, or Z ３ and Z ４ or Z ５ and Z ６ These combine to form a macrocyclic molecule of 10 to 40 members, which are optionally substituted with the atoms to which they are attached. Z ３ Z ４ Z ５ Z ６ , or R ５ At least one of these includes an attachment point to the crosslinking group, e is 0, 1, 2, 3, or 4. R ５ C is replaced by an optional substitution. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, R ６ C is replaced by an optional substitution. １ ~C ６ It is alkyl, Each R ７ These are independently hydroxyl, cyano, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, R ８ C is a hydrogen atom that is optionally substituted. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ The compound according to any one of claims 34 to 37, wherein it is alkyl.

39. The presenter protein binding portion is structure 【Chemistry 11】 The compound according to claim 38, having the following characteristics.

40. A compound containing a target protein binding site and a crosslinking group.

41. The compound according to claim 40, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs.

42. The compound according to claim 40 or 41, wherein the target protein includes an undruggable surface.

43. The compound according to any one of claims 40 to 42, wherein the target protein does not have a traditional binding pocket.

44. The compound according to any one of claims 1 to 43, wherein the protein binding portion and the crosslinking group are joined via a linker.

45. The compound according to claim 44, wherein the linker has 1 to 20 atoms in length.

46. The linker has the structure of chemical formula V. A １ -(B １ ) ｆ -(C １ ) ｇ -(B ２ ) ｈ -(D)-(B ３ ) ｉ -(C ２ ) ｊ -(B ４ ) ｋ -A ２ Chemical formula V It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the crosslinking group and the linker, B １ , B ２ , B ３ , and B ４ Each of these is independently and arbitrarily replaced C １ ~C ２ Alkyl, optionally substituted C １ ~C ３ Heteroalkyl, O, S, and NR Ｎ Selected from, R Ｎ C is a hydrogen atom that is optionally substituted. １～４ Alkyl, optionally substituted C ２～４ Alkenyl, C which is optionally replaced. ２～４ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ C is replaced by an aryl or any other character. １～７ It is heteroalkyl, C １ and C ２ Each is independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, f, g, h, I, j, and k are independently 0 or 1, and D is optionally substituted with C １～１０ Alkyl, optionally substituted C ２～１０ Alkenyl, C which is optionally replaced. ２～１０ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ Aaryl is replaced by C of any choice. ２ ~C １０ Polyethylene glycol, or optionally substituted C １～１０ Heteroalkyl, or A １ - (B １ ) ｆ - (C １ ) ｇ - (B ２ ) ｈ - to - (B ３ ) ｉ - (C ２ ) ｊ - (B ４ ) ｋ -A ２ The compound according to claim 44 or 45, wherein the chemical bond connecting the two is a chemical bond.

47. The linker has the structure of chemical formula VI. 【Chemistry 12】 It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the bridging group and the linker, l is 0, 1, 2, or 3. m is either 0 or 1. n is 0, 1, or 2. X ３ , X ４ , and X ５ These are, independently, non-existence, O, S, -C≡C-, and CR. ９ R １０ Or NR １１ And, Each R ９ , R １０ , and R １１ These are C, which is independently substituted with hydrogen of any choice. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ The compound according to any one of claims 44 to 46, wherein it is alkyl.

48. The linker has a structure 【Chemistry 13】 The compound according to claim 47, comprising:

49. structure 【Chemistry 14】 【change】 A compound having the following properties.

50. A conjugate containing a presenter protein binding site that is conjugated to the target protein.

51. The conjugate according to claim 50, wherein the presenter protein-binding portion of the conjugate is capable of non-covalent interaction with the presenter protein.

52. The conjugate according to claim 50, wherein the presenter protein-binding portion of the conjugate is capable of covalent interaction with the presenter protein.

53. The conjugate according to any one of claims 50 to 52, wherein the presenter protein binding portion can bind a protein encoded by any one of the genes in Table 1.

54. The conjugate according to any one of claims 50 to 52, wherein the presenter protein binding portion is a prolyl isomerase binding portion.

55. The conjugate according to any one of claims 50 to 54, wherein the presenter protein binding portion is an FKBP binding portion, a cyclophylline binding portion, or a PIN1 binding portion.

56. The conjugate according to claim 55, wherein the presenter protein binding portion is an FKBP binding portion.

57. The conjugate according to claim 56, wherein the presenter protein binding portion can bind FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

58. The conjugate according to claim 56 or 57, wherein the FKBP binding portion is a selective FKBP binding portion.

59. The conjugate according to claim 56 or 57, wherein the FKBP binding portion is a non-selective FKBP binding portion.

60. The FKBP bond portion has the structure of chemical formula IIa or IIb. 【Chemistry 15】 It has, in the formula, Z １ and Z ２ Each of these is independently and arbitrarily replaced C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ It is heteroalkyl, or Z １ and Z ２ These combine to form a macrocyclic molecule of 10 to 30 members, which are optionally substituted with the atoms to which they are attached, and Z １ or Z ２ At least one of these includes an attachment site to the target protein, b and c are independently 0, 1, or 2. d is 0, 1, 2, 3, 4, 5, 6, or 7. X １ and X ２ These are, independently, non-existent, CH ２ ,O,S,SO,SO ２ , or NR １３ And, Each R １ and R ２ These are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, C replaced by optional substitution ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, or R １ and R ２ These combine with the carbon atoms to which they are bonded to form C=O, or R １ and R ２ These are combined and replaced by C by choice. ３ ~C １０ Carbocyclyl or optionally replaced C ２ ~C ９ Forming heterocyclines, Each R ３ These are independently hydroxyl, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Heterocyclyl C １ ~C ６ Alkyl or two R ８ These are combined and replaced by C by choice. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ C is replaced by an aryl or any other character. ２ ~C ９ A conjugate according to any one of claims 56 to 59, which forms a heteroaryl.

61. The presenter protein binding portion is structure 【Chemistry 16】 The conjugate according to claim 60, including the following:

62. The compound according to claim 55, wherein the presenter protein binding portion is a cyclophylline binding portion.

63. The compound according to claim 62, wherein the presenter protein binding portion can bind PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

64. The compound according to claim 62 or 63, wherein the cyclophylline binding portion is a selective cyclophylline binding portion.

65. The compound according to claim 62 or 63, wherein the cyclophylline binding portion is a non-selective cyclophylline binding portion.

66. The cyclophylline bond portion has a structure of chemical formula III or IV. 【Chemistry 17】 Including Z ３ Z ４ Z ５ , and Z ６ Each of these is independently a hydroxyl, or optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ It is heteroalkyl, or Z ３ and Z ４ or Z ５ and Z ６ These combine to form a macrocyclic molecule of 10 to 40 members, which are optionally substituted with the atoms to which they are attached. Z ３ Z ４ Z ５ Z ６ , or R ５ At least one of these includes an attachment point to the crosslinking group, d is 0, 1, 2, 3, or 4. R ５ C is replaced by an optional substitution. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, R ６ C is replaced by an optional substitution. １ ~C ６ It is alkyl, Each R ７ These are independently hydroxyl, cyano, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, R ８ C is a hydrogen atom that is optionally substituted. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ The compound according to any one of claims 62 to 65, wherein it is alkyl.

67. The presenter protein binding portion is structure [Chemistry 18] The compound according to claim 66, having the following characteristics.

68. The conjugate according to any one of claims 50 to 67, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs.

69. The conjugate according to any one of claims 50 to 68, wherein the target protein includes an undruggable surface.

70. The conjugate according to any one of claims 50 to 69, wherein the target protein does not have a traditional binding pocket.

71. The conjugate according to any one of claims 50 to 70, wherein the amino acid sequence of the target protein is modified, and at least one native amino acid is substituted with a reactive amino acid.

72. The conjugate according to claim 71, wherein the reactive amino acid is a natural amino acid.

73. The conjugate according to claim 72, wherein the reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

74. The conjugate according to claim 71, wherein the reactive amino acid is a non-natural amino acid.

75. The conjugate according to any one of claims 50 to 74, wherein the amino acid sequence of the target protein is modified, and at least one native reactive amino acid is substituted with a non-reactive amino acid.

76. The conjugate according to claim 75, wherein the at least one natural reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

77. The conjugate according to claim 75 or 76, wherein the at least one native reactive amino acid is an amino acid exposed to a solvent.

78. The conjugate according to any one of claims 75 to 77, wherein the amino acid sequence of the target protein is modified so that all reactive amino acids are replaced with non-reactive amino acids.

79. The conjugate according to any one of claims 75 to 78, wherein the nonreactive amino acid is a natural amino acid.

80. The conjugate according to any one of claims 71 to 79, wherein the substitution is a conservative substitution.

81. The conjugate according to any one of claims 74 to 80, wherein the reactive amino acid is substituted with serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine.

82. The conjugate according to any one of claims 74 to 77, wherein the nonreactive amino acid is a non-natural amino acid.

83. The conjugate according to any one of claims 50 to 82, wherein the presenter protein binding portion and the target protein are conjugated via a linker.

84. The conjugate according to claim 83, wherein the linker has 1 to 20 atoms in length.

85. The linker has the structure of chemical formula III. A １ -(B １ ) ｆ -(C １ ) ｇ -(B ２ ) ｈ -(D)-(B ３ ) ｉ -(C ２ ) ｊ -(B ４ ) ｋ -A ２ Chemical formula V It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the crosslinking group and the linker, B １ , B ２ , B ３ , and B ４ Each of these is independently and arbitrarily replaced C １ ~C ２ Alkyl, optionally substituted C １ ~C ３ Heteroalkyl, O, S, and NR Ｎ Selected from, R Ｎ C is a hydrogen atom that is optionally substituted. １～４ Alkyl, optionally substituted C ２～４ Alkenyl, C which is optionally replaced. ２～４ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ C is replaced by an aryl or any other character. １～７ It is heteroalkyl, C １ and C ２ Each is independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, f, g, h, I, j, and k are independently 0 or 1, and D is optionally substituted with C １～１０ Alkyl, optionally substituted C ２～１０ Alkenyl, C which is optionally replaced. ２～１０ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ Aaryl is replaced by C of any choice. ２ ~C １０ Polyethylene glycol, or optionally substituted C １～１０ Heteroalkyl, or A １ - (B １ ) ｆ - (C １ ) ｇ - (B ２ ) ｈ - to - (B ３ ) ｉ - (C ２ ) ｊ - (B ４ ) ｋ -A ２ The conjugate according to claim 83 or 84, wherein the chemical bond connecting the two is a chemical bond.

86. The linker has the structure of chemical formula IV. 【Chemistry 19】 It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the bridging group and the linker, l is 0, 1, 2, or 3. m is either 0 or 1. n is 0, 1, or 2. X ３ , X ４ , and X ５ These are, independently, non-existence, O, S, -C≡C-, and CR. ９ R １０ Or NR １１ And, Each R ９ , R １０ , and R １１ These are C, which is independently substituted with hydrogen of any choice. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ The conjugate according to any one of claims 83 to 85, wherein it is alkyl.

87. The linker has a structure 【Chemistry 20】 The conjugate according to claim 86, having the following characteristics.

88. A method for generating a conjugate that includes a presenter protein binding portion conjugated to a target protein, comprising reacting (a) a compound containing a presenter protein binding portion and a crosslinking group with (b) a target protein under conditions that enable the generation of the conjugate.

89. A method for generating a conjugate that includes a presenter protein binding site conjugated to a target protein, (a) A compound comprising a presenter protein binding site and a crosslinking group; (b) A target protein; and (c) A presenter protein; The compound and the target protein are reacted under conditions that enable the formation of the conjugate. Methods that include...

90. The method according to claim 89, wherein the presenter protein binds to the compound in the absence of the target protein.

91. The method according to claim 89, wherein the presenter protein does not substantially bind to the compound in the absence of the target protein.

92. The method according to any one of claims 89 to 91, wherein the compound and the target protein do not substantially react in the absence of the presenter protein.

93. The method according to any one of claims 89 to 91, wherein the compound and the target protein react in the absence of the presenter protein.

94. The method according to any one of claims 88 to 93, wherein the above conditions do not include a reducing reagent.

95. A complex comprising (i) a conjugate containing a presenter protein binding portion conjugated to a target protein, and (ii) the presenter protein.

96. The complex according to claim 95, wherein the presenter protein is a protein encoded by any one of the genes in Table 1.

97. The complex according to claim 95, wherein the presenter protein is prolyl isomerase.

98. The complex according to any one of claims 95 to 97, wherein the prolyl isomerase is a member of the FKBP family, a member of the cyclophylline family, or PIN1.

99. The composite according to claim 98, wherein the member of the FKBP family is FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

100. The composite according to claim 98, wherein the member of the cyclophylline family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCYp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

101. A method for generating a complex comprising (i) a conjugate containing a presenter protein binding portion conjugated to a target protein, and (ii) a presenter protein, the method comprising combining the conjugate containing a presenter protein binding portion conjugated to a target protein and the presenter protein under conditions that enable the generation of the complex.

102. A method for generating a complex comprising (i) a conjugate containing a presenter protein binding portion conjugated to a target protein, and (ii) a presenter protein, (a) A compound comprising a presenter protein binding site and a crosslinking group; (b) A target protein; and (c) A presenter protein; The compound and the target protein are reacted under conditions that enable the formation of the complex. Methods that include...

103. The method according to claim 102, wherein the presenter protein binds to the compound in the absence of the target protein.

104. The method according to claim 102, wherein the presenter protein does not substantially bind to the compound in the absence of the target protein.

105. The method according to any one of claims 102 to 104, wherein the compound and the target protein do not substantially react in the absence of the presenter protein.

106. The method according to any one of claims 102 to 104, wherein the compound and the target protein react in the absence of the presenter protein.

107. The method according to any one of claims 101 to 106, wherein the above conditions do not include a reducing reagent.

108. The method according to any one of claims 101 to 107, wherein the conditions include an excess of presenter protein.

109. A conjugate containing a target protein binding site that is conjugated to the presenter protein.

110. The conjugate according to claim 109, wherein the target protein-binding portion of the conjugate is capable of non-covalent interaction with the target protein.

111. The conjugate according to claim 109, wherein the target protein-binding portion of the conjugate is capable of covalent interaction with the target protein.

112. The conjugate according to any one of claims 109 to 111, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs.

113. The conjugate according to any one of claims 109 to 112, wherein the target protein includes an undruggable surface.

114. The conjugate according to any one of claims 109 to 113, wherein the target protein does not have a traditional binding pocket.

115. The conjugate according to any one of claims 109 to 114, wherein the presenter protein is a protein encoded by any one of the genes in Table 1.

116. The conjugate according to any one of claims 109 to 114, wherein the presenter protein is prolyl isomerase.

117. The conjugate according to any one of claims 109 to 116, wherein the presenter protein is a member of the FKBP family, a member of the cyclophyllin family, or PIN1.

118. The conjugate according to claim 117, wherein the member of the FKBP family is FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

119. The conjugate according to claim 117, wherein the member of the cyclophylline family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

120. The conjugate according to any one of claims 109 to 119, wherein the amino acid sequence of the presenter protein is modified, and at least one amino acid is substituted with a reactive amino acid.

121. The conjugate according to claim 120, wherein the reactive amino acid is a natural amino acid.

122. The conjugate according to claim 121, wherein the reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

123. The conjugate according to claim 120, wherein the reactive amino acid is a non-natural amino acid.

124. The conjugate according to any one of claims 109 to 123, wherein the amino acid sequence of the presenter protein is modified, and at least one native reactive amino acid is replaced with a non-reactive amino acid.

125. The conjugate according to claim 124, wherein the at least one naturally occurring reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

126. The conjugate according to claim 124 or 125, wherein the at least one innate reactive amino acid is an amino acid exposed to a solvent.

127. The conjugate according to any one of claims 124 to 126, wherein the amino acid sequence of the presenter protein is modified so that all reactive amino acids are replaced with non-reactive amino acids.

128. The conjugate according to any one of claims 124 to 127, wherein the nonreactive amino acid is a natural amino acid.

129. The conjugate according to any one of claims 120 to 128, wherein the substitution is a conservative substitution.

130. The conjugate according to any one of claims 124 to 129, wherein the reactive amino acid is substituted with serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine.

131. The conjugate according to any one of claims 124 to 127, wherein the nonreactive amino acid is a non-natural amino acid.

132. The conjugate according to any one of claims 109 to 131, wherein the target protein binding portion and the presenter protein are joined via a linker.

133. The conjugate according to claim 132, wherein the linker has 1 to 20 atoms in length.

134. The linker has the structure of chemical formula V. A １ -(B １ ) ｆ -(C １ ) ｇ -(B ２ ) ｈ -(D)-(B ３ ) ｉ -(C ２ ) ｊ -(B ４ ) ｋ -A ２ Chemical formula V It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the crosslinking group and the linker, B １ , B ２ , B ３ , and B ４ Each of these is independently and arbitrarily replaced C １ ~C ２ Alkyl, optionally substituted C １ ~C ３ Heteroalkyl, O, S, and NR Ｎ Selected from, R Ｎ C is a hydrogen atom that is optionally substituted. １～４ Alkyl, optionally substituted C ２～４ Alkenyl, C which is optionally replaced. ２～４ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ C is replaced by an aryl or any other character. １～７ It is heteroalkyl, C １ and C ２ Each is independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, f, g, h, I, j, and k are independently 0 or 1, and D is optionally substituted with C １～１０ Alkyl, optionally substituted C ２～１０ Alkenyl, C which is optionally replaced. ２～１０ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ Aaryl is replaced by C of any choice. ２ ~C １０ Polyethylene glycol, or optionally substituted C １～１０ Heteroalkyl, or A １ - (B １ ) ｆ - (C １ ) ｇ - (B ２ ) ｈ - to - (B ３ ) ｉ - (C ２ ) ｊ - (B ４ ) ｋ -A ２ The conjugate according to claim 132 or 133, wherein the chemical bond connecting the two is a chemical bond.

135. The linker has the structure of chemical formula IV. 【Chemistry 21】 It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the bridging group and the linker, l is 0, 1, 2, or 3. m is either 0 or 1. n is 0, 1, or 2. X ３ , X ４ , and X ５ These are, independently, non-existence, O, S, -C≡C-, and CR. ９ R １０ Or NR １１ And, Each R ９ , R １０ , and R １１ These are C, which is independently substituted with hydrogen of any choice. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ The conjugate according to any one of claims 132 to 134, wherein it is alkyl.

136. The linker has a structure 【Chemistry 22】 The conjugate according to claim 135, having the following characteristics.

137. A method for generating a conjugate containing a target protein binding portion conjugated to a presenter protein, comprising reacting (a) a compound containing a target protein binding portion and a crosslinking group with (b) a presenter protein under conditions that enable the generation of the conjugate.

138. A method for generating a conjugate that includes a target protein binding site conjugated to a presenter protein, (a) A compound comprising a target protein binding site and a crosslinking group; (b) A presenter protein; and (c) A target protein; The compound and the presenter protein are reacted under conditions that enable the formation of the conjugate. Methods that include...

139. The method according to claim 138, wherein the target protein binds to the compound in the absence of the presenter protein.

140. The method according to claim 138, wherein the target protein does not substantially bind to the compound in the absence of the presenter protein.

141. The method according to any one of claims 138 to 140, wherein the compound and the presenter protein do not substantially react in the absence of the target protein.

142. The method according to any one of claims 138 to 140, wherein the compound and the presenter protein react in the absence of the target protein.

143. The method according to any one of claims 137 to 142, wherein the above conditions do not include a reducing reagent.

144. (i) A complex comprising a conjugate containing a target protein-binding portion conjugated to a presenter protein, and (ii) the target protein.

145. The complex according to claim 144, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs.

146. The complex according to claim 144 or 145, wherein the target protein includes an undruggable surface.

147. The complex according to any one of claims 144 to 146, wherein the target protein does not have a traditional binding pocket.

148. A method for generating a complex comprising (i) a conjugate containing a target protein binding portion conjugated to a presenter protein, and (ii) a target protein, the method comprising combining the conjugate containing a target protein binding portion conjugated to a presenter protein with the target protein under conditions that enable the generation of the complex.

149. A method for generating a complex comprising (i) a conjugate containing a target protein binding portion conjugated to a presenter protein, and (ii) a target protein, (a) A compound comprising a target protein binding site and a crosslinking group; (b) A presenter protein; and (c) A target protein; The compound and the presenter protein are reacted under conditions that enable the formation of the complex. Methods that include...

150. The method according to claim 149, wherein the target protein binds to the compound in the absence of the presenter protein.

151. The method according to claim 149, wherein the target protein does not substantially bind to the compound in the absence of the presenter protein.

152. The method according to any one of claims 149 to 151, wherein the compound and the presenter protein do not substantially react in the absence of the target protein.

153. The method according to any one of claims 149 to 151, wherein the compound and the presenter protein react in the absence of the target protein.

154. The method according to any one of claims 148 to 153, wherein the above conditions do not include a reducing reagent.

155. The method according to any one of claims 149 to 154, wherein the conditions include an excess of target protein.

156. Structure of chemical formula VII A-L-B Chemical formula VII Compounds containing (In the formula, A is the structure of chemical formula VIII) 【Chemistry 23】 The formula has such that b and c are independently 0, 1, or 2. d is 0, 1, 2, 3, 4, 5, 6, or 7. X １ and X ２ These are, independently, non-existent, CH ２ ,O,S,SO,SO ２ , or NR １３ And, Each R １ and R ２ These are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, C replaced by optional substitution ２ ~C ９ Heterocyclyl C １ ~C ６ It is alkyl, or R １ and R ２ These combine with the carbon atoms to which they are bonded to form C=O, or R １ and R ２ These are combined and replaced by C by choice. ３ ~C １０ Carbocyclyl or optionally replaced C ２ ~C ９ Forming heterocyclines, Each R ３ These are independently hydroxyl, optionally substituted amino, halogen, thiol, and optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl is optionally replaced with C. １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, C which is optionally substituted. ２ ~C ６ Heteroalkynyl, C which is optionally substituted. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Heterocyclyl C １ ~C ６ Alkyl or two R ８ These are combined and replaced by C by choice. ３ ~C １０ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aaryl is replaced by C of any choice. ２ ~C ９ Heterocyclines, or C as optionally replaced. ２ ~C ９ Forms a heteroaryl, R ４ C is replaced by an optional substitution. １ ~C ６ It is alkyl, L is an optional linker, B is the target protein binding site.

157. The compound according to claim 156, wherein the interaction between the target protein binding site and the target protein is non-covalent.

158. The compound according to claim 156, wherein the interaction between the target protein binding site and the target protein is covalent.

159. The compound according to any one of claims 156 to 158, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs.

160. The compound according to any one of claims 156 to 159, wherein the target protein includes an undruggable surface.

161. The compound according to any one of claims 156 to 160, wherein the target protein does not have a traditional binding pocket.

162. A is structure 【Chemistry 24】 A compound according to any one of claims 156 to 161, having the following characteristics.

163. The compound according to any one of claims 156 to 162, wherein the target protein binding portion and the presenter protein are conjugated via a linker.

164. The compound according to claim 163, wherein the linker has 1 to 20 atoms in length.

165. The linker has the structure of chemical formula V. A １ -(B １ ) ｆ -(C １ ) ｇ -(B ２ ) ｈ -(D)-(B ３ ) ｉ -(C ２ ) ｊ -(B ４ ) ｋ -A ２ Chemical formula V It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the crosslinking group and the linker, B １ , B ２ , B ３ , and B ４ Each of these is independently and arbitrarily replaced C １ ~C ２ Alkyl, optionally substituted C １ ~C ３ Heteroalkyl, O, S, and NR Ｎ Selected from, R Ｎ C is a hydrogen atom that is optionally substituted. １～４ Alkyl, optionally substituted C ２～４ Alkenyl, C which is optionally replaced. ２～４ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ C is replaced by an aryl or any other character. １～７ It is heteroalkyl, C １ and C ２ Each is independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl, f, g, h, I, j, and k are independently 0 or 1, and D is optionally substituted with C １～１０ Alkyl, optionally substituted C ２～１０ Alkenyl, C which is optionally replaced. ２～１０ Alkinyl is optionally replaced with C. ２～６ Heterocyclines, C replaced by optional substitution ６～１２ Aaryl is replaced by C of any choice. ２ ~C １０ Polyethylene glycol, or optionally substituted C １～１０ Heteroalkyl, or A １ - (B １ ) ｆ - (C １ ) ｇ - (B ２ ) ｈ - to - (B ３ ) ｉ - (C ２ ) ｊ - (B ４ ) ｋ -A ２ The compound according to claim 163 or 164, wherein the chemical bond is connecting the two.

166. The linker has the structure of chemical formula IV. 【Chemistry 25】 It has, in the formula, A １ This is the bond between the linker and the protein binding portion, A ２ This is the bond between the bridging group and the linker, l is 0, 1, 2, or 3. m is either 0 or 1. n is 0, 1, or 2. X ３ , X ４ , and X ５ These are, independently, non-existence, O, S, -C≡C-, and CR. ９ R １０ Or NR １１ And, Each R ９ , R １０ , and R １１ These are C, which is independently substituted with hydrogen of any choice. １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, C which is optionally replaced. ２ ~C ６ Alkinyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, C which is optionally substituted. ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ A compound according to any one of claims 163 to 165, wherein it is alkyl.

167. The linker has a structure 【Chemistry 26】 The compound according to claim 166, having the following characteristics.

168. (i) the compound according to claim 156; (ii) a target protein; and (iii) a complex comprising a presenter protein.

169. The complex according to claim 168, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having classical protein-protein interaction domains and motifs.

170. The complex according to claim 168 or 169, wherein the target protein includes an undruggable surface.

171. The complex according to any one of claims 168 to 170, wherein the target protein does not have a traditional binding pocket.

172. The complex according to any one of claims 168 to 171, wherein the presenter protein is a protein encoded by any one of the genes in Table 1.

173. The complex according to any one of claims 168 to 171, wherein the presenter protein is prolyl isomerase.

174. The compound according to any one of claims 168 to 173, wherein the presenter protein is a member of the FKBP family, a member of the cyclophylline family, or PIN1.

175. The compound according to claim 174, wherein the member of the FKBP family is FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

176. The composite according to claim 174, wherein the member of the cyclophylline family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

177. A method for identifying a conjugate that includes a presenter protein binding site that conjugates to a target protein capable of forming a complex with a presenter protein, (a) (i) a conjugate including a presenter protein binding site conjugated to a target protein, and (ii) a presenter protein; (b) If the conjugate can form a complex with the presenter protein, the conjugate and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the conjugate and the presenter protein form a complex (if the conjugate and the presenter protein form a complex, the conjugate is identified as a conjugate that can form a complex with the presenter protein), thereby identifying a conjugate that includes a presenter protein binding portion that conjugates to a target protein that can form a complex with the presenter protein. Methods that include...

178. A method for identifying a target protein that binds to a presenter protein, (a) (i) a conjugate including a presenter protein binding site conjugated to a target protein, and (ii) a presenter protein; (b) If the conjugate can form a complex with the presenter protein, the conjugate and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the target protein is bound to the presenter protein in the complex (if the target protein is bound to the presenter protein, the target protein is identified as being bound to the presenter protein), thereby identifying the target protein that is bound to the presenter protein. Methods that include...

179. A method for identifying a target protein that can react with a compound in the presence of a presenter protein, wherein the compound comprises a presenter protein binding portion and a crosslinking portion. (a)(i) a compound comprising a presenter protein binding site and a crosslinking site; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the target protein and the compound react during the formation of the complex to form a conjugate (if the target protein and the compound react during the formation of the complex to form a conjugate, the target protein is identified as a target protein that can react with the compound in the presence of the presenter protein), thereby identifying a target protein that can react with a compound containing a presenter protein binding portion and a crosslinking portion in the presence of the presenter protein. Methods that include...

180. A method for identifying a target protein that binds to a presenter protein, (a)(i) a compound comprising a presenter protein binding site and a crosslinking site; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the target protein is bound to the presenter protein in the complex (if the target protein is bound to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein), thereby identifying the target protein that binds to the presenter protein. Methods that include...

181. A method for identifying target proteins that can form a complex with a presenter protein, (a)(i) the compound according to claim 134; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the compound, the target protein, and the presenter protein form a complex (if the compound, the target protein, and the presenter protein form a complex, the target protein is identified as a target protein that can form a complex with the presenter protein), thereby identifying a target protein that can form a complex with the presenter protein. Methods that include...

182. A method for identifying a target protein that binds to a presenter protein, (a)(i) the compound according to claim 134; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the target protein is bound to the presenter protein in the complex (if the target protein is bound to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein), This allows us to identify the target protein that binds to the presenter protein. Methods that include...

183. A method for identifying the location on a target protein where a presenter protein binding site forms a conjugate, wherein the conjugate can form a complex with the presenter protein. (a) (i) a conjugate comprising a presenter protein binding site conjugated to a target protein at a certain site, and (ii) a presenter protein; (b) If the conjugate can form a complex with the presenter protein, the conjugate and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determining whether the conjugate and the presenter protein form a complex; (d) Repeat steps (a) to (c) at different locations on the target protein where the conjugate and the presenter protein are conjugated until the conjugate and the presenter protein form a complex (when the conjugate and the presenter protein form a complex, the location on the target protein where the presenter protein binding site and the conjugate form (the conjugate can form a complex with the presenter protein) are identified), thereby identifying the location on the target protein where the conjugate that can form a complex with the presenter protein is formed. Methods that include...

184. A method for identifying the location on a target protein where a presenter protein binding site forms a conjugate, wherein the conjugate can form a complex with the presenter protein. (a)(i) a compound comprising a presenter protein binding site and a crosslinking group; (ii) a target protein; and (iii) a presenter protein; (b) Combining the compound with the target protein in the presence of the presenter protein under conditions that enable the formation of a conjugate including a presenter protein binding portion that is conjugated to the target protein at a certain site; (c) Determining whether the conjugate and the presenter protein form a complex; (d) Repeat steps (a) to (c) until the conjugate and the presenter protein form a complex (the presenter protein binding portion is conjugated at different locations on the target protein); (The location on the target protein that forms a conjugate that can form a complex with the presenter protein is identified when the conjugate and the presenter protein form a complex), thereby identifying the location on the target protein that forms a conjugate with the presenter protein (the conjugate can form a complex with the presenter protein). Methods that include...

185. The method according to claim 183 or 184, wherein the amino acid sequence of the target protein is modified, and at least one amino acid is substituted with a reactive amino acid.

186. The method according to claim 185, wherein the reactive amino acid is a natural amino acid.

187. The method according to claim 186, wherein the reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

188. The method according to claim 185, wherein the reactive amino acid is a non-natural amino acid.

189. The method according to any one of claims 183 to 188, wherein the amino acid sequence of the target protein is modified, and at least one naturally reactive amino acid is replaced with a non-reactive amino acid.

190. The method according to claim 189, wherein the at least one natural reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

191. The method according to claim 189 or 190, wherein the at least one innate reactive amino acid is an amino acid exposed to a solvent.

192. The method according to any one of claims 189 to 191, wherein the amino acid sequence of the target protein is modified so that all reactive amino acids are replaced with non-reactive amino acids.

193. The method according to any one of claims 189 to 192, wherein the nonreactive amino acid is a natural amino acid.

194. The method according to any one of claims 183 to 193, wherein the substitution is a conservative substitution.

195. The method according to any one of claims 185 to 194, wherein the reactive amino acid is substituted with serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine.

196. The method according to any one of claims 185 to 195, wherein the nonreactive amino acid is a non-natural amino acid.

197. A method for identifying compounds that can covalently bind to a target protein in the presence of a presenter protein, (a)(i) a compound comprising a presenter protein binding site and a crosslinking group; (ii) a target protein; and (iii) a sample comprising a presenter protein; (b) A method comprising determining whether the compound and the target protein form a covalent bond via the crosslinking group of the compound in the sample, wherein if the compound and the target protein react in the sample, the compound is identified as covalently bonded to the target protein in the presence of a presenter protein.

198. A method for identifying compounds that can selectively and covalently bond to a target protein in the presence of a presenter protein, (a) To provide a first sample comprising (i) a compound comprising a presenter protein binding moiety and a crosslinking group; (ii) a target protein; and (iii) a presenter protein; and a second sample comprising the same compound as in the first sample, comprising (i) a presenter protein binding moiety and a crosslinking group, and (ii) the same target protein as in the first sample; and (b) To determine the extent to which the compound and the target protein react in the first sample compared to the second sample. A method comprising a compound and a target protein, wherein if the compound and the target protein react more in the first sample than in the second sample, the compound is identified as selectively covalently binding to the target protein in the presence of a presenter protein.

199. The method according to claim 198, wherein the compound and the target protein react in the first sample at least five times more than in the second sample, the compound is identified as selectively covalently binding to the target protein in the presence of a presenter protein.

200. The method according to claim 198 or 199, wherein if the compound and the target protein react in the first sample but substantially do not react in the second sample, the compound is identified as selectively covalently binding to the target protein in the presence of a presenter protein.

201. A method for identifying conjugates that can form a complex with a presenter protein, (a) (i) a conjugate including a presenter protein binding site conjugated to a target protein, and (ii) a presenter protein; (b) If the conjugate can form a complex with the presenter protein, the conjugate and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine whether the conjugate and the presenter protein form a complex (if the conjugate and the presenter protein form a complex, the conjugate is identified as being able to form a complex with the presenter protein), thereby identifying the conjugate that is capable of forming a complex with the presenter protein. Methods that include...

202. A method for determining the structure of an interface in a complex comprising a presenter protein and a target protein, (a) (i) a conjugate including a presenter protein binding site conjugated to a target protein, and (ii) a presenter protein; (b) If the conjugate can form a complex with the presenter protein, the conjugate and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine the crystal structure of the complex (the structure of the interface includes at least a portion of the crystal structure between the presenter protein and the target protein), thereby determining the structure of the interface in the complex comprising the presenter protein and the target protein. Methods that include...

203. A method for determining the structure of an interface in a complex comprising a presenter protein and a target protein, (a)(i) a compound comprising a presenter protein binding site and a crosslinking site; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine the crystal structure of the complex (the structure of the interface includes at least a portion of the crystal structure between the presenter protein and the target protein), thereby determining the structure of the interface in the complex comprising the presenter protein and the target protein. Methods that include...

204. A method for determining the structure of an interface in a complex comprising a presenter protein and a target protein, (a)(i) the compound according to claim 156; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine the crystal structure of the complex (the structure of the interface includes at least a portion of the crystal structure between the presenter protein and the target protein); This allows us to determine the structure of the interface in the complex containing the presenter protein and the target protein. Methods that include...

205. The method according to any one of claims 202 to 204, wherein the interface in a complex comprising a presenter protein and a target protein is a binding pocket.

206. A method for obtaining X-ray crystal coordinates for a composite, (a) (i) a conjugate including a presenter protein binding site conjugated to a target protein, and (ii) a presenter protein; (b) If the conjugate can form a complex with the presenter protein, the conjugate and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine the crystal structure of the composite and thereby obtain the X-ray crystal coordinates for the composite. Methods that include...

207. A method for obtaining X-ray crystal coordinates for a composite, (a)(i) a compound comprising a presenter protein binding site and a crosslinking site; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine the crystal structure of the composite and thereby obtain the X-ray crystal coordinates for the composite. Methods that include...

208. A method for obtaining X-ray crystal coordinates for a composite, (a)(i) the compound according to claim 156; (ii) a target protein; and (iii) a presenter protein; (b) If the compound can form a complex with the presenter protein, the compound, the target protein, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Determine the crystal structure of the composite and thereby obtain the X-ray crystal coordinates for the composite. Methods that include...

209. A method for determining residues on a target protein involved in binding to a presenter protein, (a) to provide the X-ray crystal coordinates of a composite obtained by the method described in any one of claims 206 to 208; (b) Identify the residue of the target protein that includes an atom within 4 Å of the atom on the presenter protein, This allows us to determine the residues on the target protein that are involved in binding to the presenter protein. Methods that include...

210. A method for determining the biochemical and / or biophysical properties of a complex according to any one of claims 95 to 100, 144 to 147, or 168 to 176, (a) To provide the X-ray crystal coordinates of the composite according to any one of claims 95-100, 144-147, or 168-176, obtained by the method according to any one of claims 206-208; (b) Calculate the biochemical and / or biophysical properties of the complex, This allows for the determination of the biochemical and / or biophysical properties of the presenter protein / target protein complex. Methods that include...

211. The biochemical and / or biophysical properties are the free energy of the complex and the K of the complex. ｄ , K of the composite ｉ , K of the composite ｉｎａｃｔ , and / or K of the composite ｉ / K ｉｎａｃｔ The method according to claim 210, including the method described in claim 210.

212. The method according to claim 211, wherein the biochemical and / or biophysical properties include the free energy of the complex.

213. The method according to claim 212, wherein the free energy of the bond of the composite is determined by isothermal titration calorimetry.

214. The biochemical and / or biophysical properties of the complex ｄ The method according to claim 211, including the method described in claim 211.

215. The K of the composite ｄ The method according to claim 214, wherein the determination is made by surface plasmon resonance.

216. The biochemical and / or biophysical properties of the complex are ｉ , the K of the composite ｉｎａｃｔ , and / or the K of the composite ｉ / K ｉｎａｃｔ The method according to claim 211, including the method described in claim 211.

217. The K of the composite ｉ , the K of the composite ｉｎａｃｔ , and / or the K of the composite ｉ / K ｉｎａｃｔ The method according to claim 216, wherein the result is determined by mass spectrometry.

218. A composition comprising a compound according to any one of claims 1 to 49 or 156 to 167 and a suitable carrier.

219. A pharmaceutical composition comprising a compound according to any one of claims 1 to 49 or 156 to 167 and a pharmaceutically acceptable carrier.

220. A method for modulating a target protein, comprising contacting the target protein with a modulation amount of a compound according to any one of claims 1 to 49 or 156 to 167, or a composition according to claim 218 or 219.

221. A method for modulating a target protein, comprising contacting a cell with an effective amount of a composition according to any one of claims 1 to 49 or 156 to 167, or claim 218 or 219, thereby causing the cell to form a complex according to any one of claims 95 to 100, 144 to 147, or 168 to 176.

222. A method for modulating a target protein, comprising contacting the target protein with a conjugate according to any one of claims 108 to 136.

223. A method for inhibiting prolyl isomerase activity, comprising contacting cells expressing the prolyl isomerase with the compound according to any one of claims 1 to 49 or 156 to 167, or the composition according to claim 218 or 219, under conditions that allow for the formation of a complex between the compound and the prolyl isomerase, thereby inhibiting prolyl isomerase activity.

224. A method for forming a complex according to any one of claims 95-100, 144-147, or 168-176 in a cell, comprising contacting a cell expressing a presenter protein with a compound according to any one of claims 1-49 or 156-167, or a composition according to claim 218 or 219, under conditions that enable the formation of a complex between the compound and the presenter protein.

225. A method for identifying target proteins that can form a complex with a presenter protein, (a) (i) one or more target proteins, (ii) a compound according to any one of claims 1 to 49; and (iii) a presenter protein comprising a tag; (b) If the target protein can form a complex with the presenter protein, combining the one or more target proteins, the compound, and the presenter protein under conditions suitable for enabling complex formation; (c) Determining whether one or more target proteins form a complex with the compound and the presenter protein. Includes, A method wherein a target protein that forms a complex with the compound and the presenter protein is identified as a target protein capable of forming a complex with the presenter protein.

226. The method according to claim 225, wherein the presenter protein includes an affinity tag.

227. The method according to claim 225 or 226, wherein the determination step includes utilizing the tag of the presenter protein to selectively isolate a target protein that forms a complex with the presenter protein.

228. (d) The method according to any one of claims 225 to 227, further comprising identifying the target protein in a complex formed between one or more target proteins and the presenter protein.

229. The method according to claim 228, wherein identifying the structure of the target protein includes performing mass spectrometry on the complex.

230. A method for identifying target proteins that can form a complex with a presenter protein, (a) (i) two or more target proteins; (ii) a compound according to any one of claims 1 to 49; and (iii) a presenter protein comprising an affinity tag; (b) If the target protein can form a complex with the presenter protein, the two or more target proteins, the compound, and the presenter protein are brought together under conditions suitable for enabling complex formation; (c) Selectively isolating one or more complexes of the target protein, the compound, and the presenter protein formed in step (b); (d) Determine the structure of the target protein in one or more complexes isolated in step (c) by mass spectrometry, This allows us to identify target proteins that can form complexes with presenter proteins. Methods that include...

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