Methods and reagents for analyzing protein-protein interfaces

Abstract

The present disclosure provides methods and reagents useful for analyzing protein-protein interfaces, for example, the interface between a presenter protein (eg, a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein. In some embodiments, the target and / or presenter proteins are intracellular proteins, hi some embodiments, the target and / or presenter proteins are mammalian proteins.

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, cholesterol-lowering drugs such as statins bind to the enzyme 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 may lead some to mistakenly believe that, with the appropriate amount of time, effort, and resources, it is possible to discover small molecule modulators for most, if not all, proteins. This is far from the truth. Current estimates suggest that only about 10% of all human proteins are potential small molecule targets. The remaining 90% are currently considered too cumbersome or difficult for small molecule drug discovery. Such targets are commonly referred to as "undruggable." These undruggable targets represent a vast and largely untapped reservoir of medically important human proteins. Therefore, there is significant interest in discovering new molecular modalities that can modulate the function of such undruggable targets.

[0002] The interaction of small molecules with targets is driven by adhesive forces, and the strength of this is roughly proportional to the contact surface area, so small molecules are limited in their targeting ability.Due to their small size, the only way for small molecules to increase sufficient intermolecular contact surface area and effectively interact with target proteins is to be literally incorporated by the protein.In fact, both a large amount of experimental and computational data supports the idea that only those proteins that have hydrophobic "pockets" on their surface can bind small molecules.In such cases, binding is possible through incorporation.

[0003] Nature has developed strategies that allow small molecules to interact with target proteins at sites other than hydrophobic pockets. This strategy is exemplified by the naturally occurring immunosuppressants cyclosporin A, rapamycin, and FK506. The biological activity of these drugs involves the formation of high-affinity complexes of small molecules with small display proteins. The combined surface of the small molecule and the display protein associates with the target. Thus, for example, the binary complex formed between cyclosporin A and cyclophilin A targets calcineurin with high affinity and specificity, but neither cyclosporin A nor cyclophilin A alone binds calcineurin with measurable affinity. Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have developed compounds and conjugates useful for identifying presenter and target protein pairs and exploring the interface between them for use in developing small molecules that can modulate these interactions. [Means for solving the problem]

[0005] Thus, the present 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 cyclophilin family, or PIN1) and a target protein. Such analyses are useful in aiding in the design of 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 and modulate the activity of the 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.

[0006] In some aspects, the present disclosure provides compounds that can be used as cross-linking substrates. These compounds can include a protein-binding moiety that can be covalently or non-covalently bound to a protein (e.g., a target protein or a presenter protein) and at least one cross-linking group that can chemoselectively react with an amino acid of the protein that is different from the one that is bound to the protein-binding moiety. In some embodiments, the compound includes only one cross-linking group.

[0007] Thus, in one aspect, the present disclosure provides compounds comprising a protein-binding moiety (e.g., a presenter protein-binding moiety or a target protein-binding moiety) and a cross-linking group (e.g., a moiety capable of chemoselectively reacting with an amino acid of a protein different from that 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 cross-linking group can form a covalent bond with a protein (e.g., a presenter protein, a target protein, or another compound capable of binding such other proteins). In some embodiments, when a compound comprises a presenter protein-binding moiety, the compound does not comprise a target protein-binding moiety. In some embodiments, when a compound comprises a target protein-binding moiety, the compound does not comprise a presenter protein-binding moiety.

[0008] In some embodiments, the crosslinking group is a sulfhydryl-reactive crosslinking group (e.g., the crosslinking group comprises a mixed disulfide, maleimide, vinyl sulfone, vinyl ketone, or alkyl halide), 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 bridging group comprises a mixed disulfide, e.g., the bridging group has the structure of Formula I

[0010] [ka]

[0011] wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; a is 0, 1, or 2; R A is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C6-C 10 aryl, or optionally substituted C2-C9 heteroaryl. In some embodiments, R A is an optionally substituted C2-C9 heteroaryl (e.g., pyridyl). In some embodiments, the bridging group has the structure

[0012] [ka]

[0013] where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments, R A is an optionally substituted C1-C6 alkyl (e.g., methyl). In some embodiments, the bridging group has the structure

[0014] [ka]

[0015] where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments, the bridging group comprises a maleimide, e.g., the bridging group has the structure

[0016] [ka]

[0017] where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments, the bridging group comprises a vinyl sulfone, e.g., the bridging group has the structure

[0018] [ka]

[0019] where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments, the crosslinking group comprises a vinyl ketone, e.g., the crosslinking group has the structure

[0020] [ka]

[0021] where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments, the bridging group comprises an alkyl halide, e.g., an alkyl chloride, e.g., the bridging group has the structure

[0022] [ka]

[0023] where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments of any of the above compounds, the protein-binding moiety portion is capable of non-covalent interactions with proteins. In some embodiments of any of the above compounds, the protein-binding moiety portion is capable of covalent interactions with proteins.

[0024] In some aspects, the present disclosure provides compounds comprising a presenter protein-binding moiety and a cross-linking group. In some embodiments, the protein-binding moiety and the cross-linking group are attached via a linker.

[0025] In some aspects, the present disclosure provides a method for producing a compound comprising:

[0026] [ka]

[0027] JPEG0007812813000009.jpg55170

[0028] The present invention provides a compound having the formula: In some aspects, the present disclosure provides conjugates comprising a presenter protein binding moiety that can be covalently or non-covalently bound to a presenter protein that is conjugated to a target protein via a linker, methods for their synthesis, and uses thereof.

[0029] Thus, in another aspect, the present disclosure provides a conjugate comprising a presenter protein-binding moiety conjugated to a target protein. In some embodiments, the presenter protein-binding moiety portion of the conjugate is capable of non-covalent interactions with the presenter protein. In some embodiments, the presenter protein-binding moiety portion of the conjugate is capable of covalent interactions with the presenter protein.

[0030] In some aspects, the present disclosure provides methods for producing a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, the method comprising reacting (a) a compound comprising a presenter protein-binding moiety and a cross-linking group with (b) the target protein under conditions that allow for the production of the conjugate.

[0031] In some aspects, the present disclosure provides a method for producing a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, the method comprising providing (a) a compound comprising a presenter protein-binding moiety and a cross-linking group, (b) a target protein, and (c) a presenter protein, and reacting the compound with the target protein under conditions that allow the production of the conjugate.

[0032] In some aspects, the present disclosure provides complexes comprising a presenter protein and a conjugate comprising a presenter protein-binding moiety and a target protein, methods for their production, and uses thereof.

[0033] Thus, in another aspect, the present disclosure provides a complex comprising: (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein; and (ii) a presenter protein.

[0034] In some aspects, the disclosure provides methods 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 comprising combining the conjugate comprising a presenter protein-binding moiety conjugated to a target protein with the presenter protein under conditions that allow the formation of the complex.

[0035] In some aspects, the present disclosure provides methods for producing a complex comprising (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, and (ii) a presenter protein, the method comprising providing (a) a compound comprising a presenter protein-binding moiety and a cross-linking group, (b) a target protein, and (c) the presenter protein, and reacting the compound with the target protein under conditions that allow the formation of the 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 reagent. In some embodiments of the above methods, the conditions include an excess of presenter protein.

[0037] In some embodiments, detectable binding between the compound and the presenter protein is observed in the absence of the target protein. However, in some embodiments, detectable binding between the compound and the 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, no significant reaction (e.g., significant conjugate formation) between the crosslinking group and the target protein is observed in the absence of the presenter protein. However, in some embodiments, 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 reaction (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 absent (e.g., the rate and / or amount of conjugate formation is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 100-fold greater in the presence of the presenter protein).

[0038] In some embodiments, conjugation as described herein is carried out under conditions that are free of (eg, 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 moiety and a cross-linking group); and (iii) a target protein. In some embodiments, such a complex is exposed to and / or maintained under conditions that allow the cross-linking moiety to react with the target protein, such that a cross-link between them is formed. In some embodiments, the cross-link is with a heteroatom in an amino acid (e.g., an amino acid side chain) of the target protein. In some embodiments, the cross-link is with the -S- atom of a cysteine ​​in the target protein. In some embodiments, the target protein is a mutant of a naturally occurring target protein. In some such embodiments, the variant has an amino acid sequence that is highly identical (e.g., 80%, 81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the native target protein, but differs by the substitution or addition of at least one amino acid that is susceptible to participating in cross-linking with a cross-linking group (e.g., the amino acid side chain contains a heteroatom that can participate in such cross-linking).

[0039] In some aspects, the present disclosure provides conjugates comprising a target protein binding moiety that can be covalently or non-covalently bound to a target protein that is conjugated to a presenter protein via a linker, methods for their synthesis, and uses thereof.

[0040] Thus, in another aspect, the present disclosure provides a conjugate comprising a target protein binding moiety conjugated to a presenter protein. In some embodiments, the target protein binding moiety portion of the conjugate is capable of non-covalent interaction with the target protein. In some embodiments, the target protein binding moiety portion of the conjugate is capable of non-covalent interaction with the target protein. In some embodiments, the target protein binding moiety and the presenter protein are conjugated via a linker.

[0041] In some aspects, the present disclosure provides methods for producing a conjugate comprising a target protein binding moiety conjugated to a presenter protein, the method comprising reacting (a) a compound comprising a target protein binding moiety and a cross-linking group with (b) the presenter protein under conditions that allow for the production of the conjugate.

[0042] In some aspects, the present disclosure provides a method for producing a conjugate comprising a target protein-binding moiety conjugated to a presenter protein, the method comprising providing (a) a compound comprising a target protein-binding moiety and a cross-linking group, (b) a presenter protein, and (c) a target protein, and reacting the compound with the presenter protein under conditions that allow the production of the 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, no significant reaction (e.g., significant conjugate formation) between the crosslinking group and the presenter protein is observed in the absence of the target protein. However, in some embodiments, 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 reaction (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 absent (e.g., the rate and / or amount of conjugate formation is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold 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 (e.g., conjugate formation) between the crosslinking group and the target protein 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 performed under conditions that do not include (e.g., are substantially free of) reducing agents.

[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 moiety and a cross-linking group); and (iii) a target protein. In some embodiments, such a complex is exposed to and / or maintained under conditions that allow the cross-linking moiety to react with the target protein, such that a cross-link between them is formed. In some embodiments, the cross-link is with a heteroatom in an amino acid (e.g., an amino acid side chain) of the target protein. In some embodiments, the cross-link is with the -S- atom of a cysteine ​​in the target protein. In some embodiments, the target protein is a mutant of a naturally occurring target protein. In some such embodiments, the variant has an amino acid sequence that is highly identical (e.g., 80%, 81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the native target protein, but differs by the substitution or addition of at least one amino acid that is susceptible to participating in cross-linking with a cross-linking group (e.g., the amino acid side chain contains a heteroatom that can participate in such cross-linking).

[0046] In some aspects, the present disclosure provides complexes comprising a target protein and a conjugate comprising a target protein binding moiety conjugated to a presenter protein via a linker, methods for their production, and uses thereof.

[0047] In some aspects, the present disclosure provides a complex comprising: (i) a conjugate comprising 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 reaction of the cross-linking moiety with the presenter protein, such that a cross-link is formed therebetween. In some embodiments, the cross-link is with a heteroatom in an amino acid (e.g., an amino acid side chain) of the presenter protein.

[0048] In some embodiments, the crosslink is with the -S- atom of a cysteine ​​in the presenter protein. In some embodiments, the presenter protein is a variant of a naturally occurring presenter protein. In some such embodiments, the variant has an amino acid sequence that is highly identical (e.g., 80%, 81%, 82%; 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the naturally occurring presenter protein, but differs by the substitution or addition of at least one amino acid that is susceptible to participating in crosslinking with a crosslinking group (e.g., the amino acid side chain contains a heteroatom that can participate in such crosslinking).

[0049] In some aspects, the disclosure provides methods 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 the target protein-binding moiety conjugated to a presenter protein with the target protein under conditions that allow for the formation of the complex. In some aspects, the invention features methods 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 provided methods comprise combining the conjugate and the target protein under conditions that allow for the formation of the complex. Alternatively or additionally, in some embodiments, such methods comprise, for example, (i) combining (a) a compound (e.g., a compound whose structure comprises a target protein-binding moiety and a cross-linking group); (b) the target protein; and (c) the presenter protein; and (ii) exposing the combination to and / or maintaining the combination under conditions that allow for the formation of the complex. In some such embodiments, the conditions allow reaction of the cross-linking group with the presenter protein so as to produce a conjugate.

[0050] In some aspects, the present disclosure provides methods for producing a complex comprising (i) a conjugate comprising a target protein-binding moiety conjugated to a presenter protein, and (ii) a target protein, the method comprising providing (a) a compound comprising a target protein-binding moiety and a cross-linking group, (b) a presenter protein, and (c) the target protein, and reacting the compound with the presenter protein under conditions that allow the formation of the complex.

[0051] In some such embodiments, the conditions are such that the compound, presenter protein, and / or target protein are characterized in 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 under these conditions in the absence of the presenter protein (e.g., the target protein does not substantially bind to the compound). In some embodiments, no significant reaction between the crosslinking group and the presenter protein is observed under these conditions in the absence of the target protein. However, in some embodiments, significant reaction between the crosslinking group and the presenter protein may be observed under these conditions even in the absence of the target protein. In some embodiments, the conditions do not include a reducing reagent. 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 reagent. In some embodiments, the conditions include an excess of target protein.

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

[0054] Thus, in some aspects, the present disclosure provides a compound of formula VII ALB Chemical formula VII wherein A is a compound having the structure of formula VIII

[0055] [ka]

[0056] wherein 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 and 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-C6 10 Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl (e.g., optionally substituted C2-C9 heteroaryl), optionally substituted C2-C9 heterocyclylC1-C6 alkyl (e.g., optionally substituted C2-C9 heteroarylC1-C6 alkyl), or R 1 and R 2 combine with the carbon atom to which they are attached to form C=O, or R 1 and R 2 are combined to form optionally substituted C3 to C 10forming a carbocyclyl or an optionally substituted C2-C9 heterocyclyl; Each R 3 are independently selected from 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-C6 10 Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl (e.g., optionally substituted C2-C9 heteroaryl), or optionally substituted C2-C9 heterocyclylC1-C6 alkyl (e.g., optionally substituted C2-C9 heteroarylC1-C6 alkyl), or two R 8 are combined to form optionally substituted C3 to C 10 Carbocyclyl, optionally substituted C6-C 10 aryl, optionally substituted C2-C9 heterocyclyl, for example, optionally substituted C2-C9 heteroaryl; R 4 is an optionally substituted C1-C6 alkyl; L is an optional linker; B is a target protein binding moiety.

[0057] In some embodiments of compounds of Formula VII, the target protein binding moiety, B, is capable of non-covalent interactions with the target protein. In some embodiments of compounds of Formula VII, the target protein binding moiety, B, is capable of covalent interactions with the target protein. In some embodiments of compounds of Formula VII, the linker, L, is present. In some embodiments of compounds of Formula VII, the linker, L, is absent.

[0058] In some aspects, the present disclosure provides ternary complexes comprising a presenter protein, a target protein, and a compound comprising a presenter protein-binding moiety and a target protein-binding moiety, methods for their production, and uses thereof.

[0059] Thus, in another aspect, the present disclosure provides a complex comprising: (i) a compound of 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 moiety and a target protein that can form a complex with the presenter protein.

[0060] In some aspects, the invention features methods for identifying and / or characterizing a conjugate as described herein that is capable of forming a complex with a presenter protein (e.g., where a compound whose structure comprises a presenter protein-binding moiety and a cross-linking group is conjugated to a target protein). In some embodiments, such methods include: (a) providing (i) such a conjugate (e.g., where a compound whose structure comprises a presenter protein-binding moiety and a cross-linking group is conjugated to a target protein) and (ii) a presenter protein; (b) if the conjugate is capable of forming a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable to allow complex formation; and (c) determining whether a complex comprising the conjugate and the presenter protein is formed, where the formation of a complex indicates that the conjugate is capable of forming a complex with the presenter protein.

[0061] Thus, in some aspects, the present disclosure provides a method for identifying and / or characterizing a conjugate capable of forming a complex with a presenter protein, the method comprising the steps of: (a) providing (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, and (ii) the presenter protein; (b) if the conjugate is capable of forming a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable to allow complex formation; and (c) determining whether a complex comprising the conjugate and the presenter protein is formed, wherein the formation of a complex 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 are capable of forming covalent bonds with the compounds. Thus, in another aspect, the present disclosure provides a method for identifying and / or characterizing a target protein capable of reacting with a compound in the presence of a presenter protein, the compound comprising a presenter protein-binding moiety and a bridging moiety. The method includes the steps of: (a) providing (i) a compound comprising a presenter protein-binding moiety and a bridging moiety; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, target protein, and presenter protein under conditions suitable to allow complex formation if the conjugate is capable of forming a complex with the presenter protein; and (c) determining whether the target protein and compound react to form a conjugate during complex formation; if the target protein and compound form a conjugate, the target protein is identified as being capable of reacting 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 are capable of forming complexes with presenter proteins.

[0064] Thus, 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 comprising the steps of: (a) providing a conjugate comprising (i) a presenter protein-binding moiety conjugated to a target protein, and (ii) the presenter protein; (b) if the conjugate is capable of forming a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable to allow complex formation; and (c) determining whether the target protein is bound to the presenter protein in the complex, wherein if the target protein binds to the presenter protein, the target protein is identified as binding to the presenter protein.

[0065] In some aspects, the present disclosure provides a method for identifying and / or characterizing a target protein that binds to a presenter protein, the method comprising the steps of: (a) providing (i) a compound comprising a presenter protein-binding moiety and a cross-linking moiety; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, the target protein, and the presenter protein under conditions suitable to allow 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 binds to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein.

[0066] In some aspects, the present disclosure provides a method for identifying and / or characterizing a target protein capable of forming a complex with a presenter protein, the method comprising the steps of: (a) providing (i) a compound of Formula VII; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, target protein, and presenter protein under conditions suitable to allow complex formation if the conjugate is capable of forming a complex with the presenter protein; and (c) determining whether the compound, target protein, and presenter protein form a complex, whereby the target protein is identified as a target protein capable of forming a complex with the presenter protein.

[0067] In some aspects, the present disclosure provides a method for identifying and / or characterizing a target protein that binds to a presenter protein, the method comprising the steps of: (a) providing (i) a compound of 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 to allow complex formation; and (c) determining whether the target protein binds to the presenter protein in the complex, where 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 aspects, the present disclosure provides methods for identifying target proteins that can form a complex with a presenter protein by: (a) providing a presenter protein comprising: (i) one or more target proteins; (ii) any of the compounds described above; and (iii) a tag (e.g., an affinity tag); (b) combining the one or more target proteins, the compounds, and the presenter protein under conditions suitable to allow complex formation if one or more of the target proteins can form a complex with the presenter protein; and (c) determining whether the one or more target proteins form a complex with the compounds and the presenter protein, wherein target proteins that form a complex with the presenter protein are identified as target proteins that can form a complex with the presenter protein.

[0069] In some embodiments, the determining step comprises utilizing a tag on the presenter protein to selectively isolate a target protein complexed with the presenter protein (e.g., by use in a pull-down experiment). In some embodiments, the complex comprises a target protein, a presenter protein, and a compound of the present invention. In some embodiments, the complex comprises a conjugate comprising a target protein and a presenter protein-binding moiety (e.g., a conjugate formed by reaction between a crosslinking group of a compound of the present invention and a reactive amino acid of the target protein), and the presenter protein. In some embodiments, the method further comprises (d) identifying the target protein in a 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 comprises performing mass spectrometry on the complex. In some embodiments, determining 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 can be performed using a pull-down experiment in which the target protein or presenter protein is labeled (e.g., where the complex can be selectively pulled down in the presence of the target protein and / or presenter protein that are not in the complex).

[0070] In some aspects, the present disclosure provides methods for identifying target proteins that can form complexes with presenter proteins by: (a) providing a presenter protein comprising: (i) two or more target proteins; (ii) any of the compounds described above; and (iii) an affinity tag; (b) if the target proteins can form complexes with the presenter proteins, combining the two or more target proteins, the compounds, and the presenter proteins under conditions suitable to allow complex formation; (c) selectively isolating one or more complexes of the target proteins, the compounds, and the presenter proteins formed in (b); and (d) identifying the target proteins in the 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 form complexes with the presenter proteins.

[0071] In some embodiments, the determining step involves utilizing a tag on the presenter protein to selectively isolate a 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 a compound of the present invention. In some embodiments, the complex comprises a conjugate comprising a target protein and a presenter protein-binding moiety (e.g., a conjugate formed by reaction between a cross-linking group of a compound of the present invention and a reactive amino acid of the target protein), and the presenter protein. In some embodiments, determining whether the target protein and the presenter protein form a complex and / or whether the target protein binds to the presenter protein in a complex can be performed using a pull-down experiment in which the target protein or the presenter protein is labeled (e.g., where the complex can 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 for identifying locations on a target protein to attach a presenter protein binding moiety, resulting in a conjugate that is capable of forming a complex with the presenter protein.

[0073] Thus, in another aspect, 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 moiety, where the conjugate can form a complex with the presenter protein. The method includes the steps of: (a) providing (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein at a location, 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)-(c) with the presenter protein-binding moiety conjugated to a different location on the target protein until the conjugate and the presenter protein form a complex; whereby, 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 moiety (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 aspects, the present disclosure provides methods for identifying and / or characterizing locations on a target protein that form conjugates with presenter protein binding moieties, where the conjugates are capable of forming complexes with the presenter protein. The method includes the steps of: (a) providing (i) a compound comprising a presenter protein-binding moiety and a cross-linking group; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound with the target protein under conditions that allow the formation of a conjugate comprising the presenter protein-binding moiety conjugated to the target protein at a location in the presence of the presenter protein; (c) determining whether the conjugate and presenter protein form a complex; and (d) optionally repeating steps (a)-(c) (the presenter protein-binding moiety is conjugated to a different location on the target protein) until the conjugate and presenter protein form a complex (if the conjugate and presenter protein form a complex, a location on the target protein that forms a conjugate with the presenter protein-binding moiety (the conjugate is capable of forming a complex with the presenter protein) is identified, thereby identifying a location on the target protein that forms a conjugate that is capable of forming 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 for identifying compounds that can form covalent bonds with target proteins in the presence of a presenter protein, hi some embodiments, compounds are identified that selectively form covalent bonds with target proteins in the presence of a presenter protein.

[0076] Thus, in another aspect, the present disclosure provides a method for identifying and / or characterizing a compound capable of covalently binding to a target protein in the presence of a presenter protein, the method comprising the steps of: (a) providing a sample comprising: (i) a compound comprising a presenter protein-binding moiety and a cross-linking 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 cross-linking 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 some aspects, the present disclosure provides a method for identifying and / or characterizing a compound that can selectively and covalently bind to a target protein in the presence of a presenter protein. The method includes the steps of: (a) providing a first sample containing (i) a compound comprising a presenter protein-binding moiety and a cross-linking group; (ii) a target protein; and (iii) a presenter protein; and a second sample containing the same compound as in the first sample, the compound comprising the presenter protein-binding moiety and a cross-linking group, and the same target protein as in the first sample; and (b) determining the degree 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 the presenter protein.

[0078] In some embodiments, a compound is identified as selectively covalently binding to a target protein in the presence of a presenter protein if the compound and the target protein react at least 5 times more in the first sample than in the second sample. In some embodiments, a compound is identified as selectively covalently binding to a target protein in the presence of a presenter protein if the compound and the target protein react in the first sample but do not substantially react in the second sample.

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

[0080] Thus, in another aspect, the present disclosure provides a method for identifying and / or characterizing a conjugate capable of forming a complex with a presenter protein, the method comprising the steps of: (a) providing (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, and (ii) the 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 form a complex, the conjugate is identified as being capable of forming a complex with the presenter protein), thereby identifying the conjugate capable of forming 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 assay, ternary amplified luminescence proximity homogeneous assay, 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 for determining the structure of the protein-protein interface between a presenter protein and a target protein.

[0083] Therefore, 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 structural features thereof, the method comprising the steps of: (a) providing a conjugate comprising (i) a presenter protein-binding moiety conjugated to a target protein, and (ii) the presenter protein; (b) contacting the conjugate with the presenter protein to form a complex (e.g., in a vial); and (c) determining a crystal structure of the complex (the structure of the interface 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.

[0084] In some aspects, 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 structural features thereof, the method comprising: (a) providing (i) a compound comprising a presenter protein-binding moiety and a cross-linking moiety; (ii) a target protein; and (iii) a presenter protein; (b) combining the compound, the target protein, and the presenter protein under conditions suitable for forming a conjugate between the compound and the target protein, and for forming a complex between the conjugate and the presenter protein (e.g., in a vial); and (c) determining a crystal structure of the complex (the structure of the interface 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.

[0085] In some aspects, 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 structural features thereof, the method comprising: (a) providing (i) a compound of Formula VII; (ii) a target protein; and (iii) a presenter protein; (b) forming a complex (e.g., in a vial) comprising the compound, the target protein, and the presenter protein; and (c) determining a crystal structure of the complex (wherein the structure of the interface 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 aspects, the present disclosure provides methods 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 structural features thereof. The methods include: (a) providing a crystal of any of the complexes; and (b) determining the structure of the crystal (the structure of the interface comprising 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 aspects, the present disclosure provides methods for identifying and / or characterizing compounds capable of modulating the biological activity of the target protein. The methods include: (a) providing a structure of the protein-protein interface in a complex comprising the presenter protein and the target protein (e.g., a structure determined by any of the methods described above); 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 the target protein. In some embodiments, the structure of a compound that can bind at the interface is determined using computational methods. In some embodiments, the structures of compounds that can bind at the interface are determined by screening compounds comprising a presenter protein binding moiety described herein for complex formation in the presence of a target protein and a presenter protein.

[0087] In some aspects, the present disclosure provides a method for obtaining X-ray crystal coordinates for a complex, the method comprising the steps of: (a) providing (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, and (ii) the presenter protein; (b) if the conjugate is capable of forming a complex with the presenter protein, combining the conjugate and the presenter protein under conditions suitable to allow complex formation; and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0088] In some aspects, the present disclosure provides a method for obtaining X-ray crystal coordinates for a complex, the method comprising the steps of: (a) providing (i) a compound comprising a presenter protein binding moiety and a cross-linking moiety; (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 to allow complex formation; and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0089] In some aspects, the present disclosure provides a method for obtaining X-ray crystal coordinates for a complex, the method comprising the steps of: (a) providing (i) a compound of the present invention; (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 to allow complex formation; and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0090] In some aspects, the present disclosure provides a method for determining residues on a target protein that are involved in binding with a presenter protein, the method comprising the steps of: (a) providing X-ray crystal coordinates of a complex obtained by the method of the present invention; and (b) identifying residues on the target protein that include atoms within 4 Å of an atom on the presenter protein, thereby determining the residues on the target protein that are involved in binding with the presenter protein.

[0091] In some aspects, the present disclosure provides methods for determining the biochemical and / or biophysical properties of any of the presenter protein / target protein complexes described herein, comprising the steps of: (a) providing X-ray crystal coordinates of a complex described herein obtained by a 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 include the free energy of binding of the complex, the K d , complex K i , complex K inact , and / or complex K i / K inact In some embodiments, the 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 or comprises a binding pocket. In some aspects, the present disclosure provides a composition comprising any of the above-described compounds, a target protein, and a presenter protein in solution.

[0094] In some aspects, the disclosure features a pharmaceutical composition including any of the compounds, conjugates, or complexes of the invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a unit dosage form.

[0095] In some aspects, the invention features methods of 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 methods include contacting the target protein with a modulating (e.g., positive or negative modulating) amount of a compound of the invention (e.g., in the presence of a presenter protein), a conjugate comprising a target protein-binding moiety, or a composition.

[0096] In some aspects, the present disclosure provides methods for modulating (e.g., positively or negatively 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 methods comprise the step of modulating (e.g., positively or negatively modulating) the target protein by contacting a cell expressing the target protein and a presenter protein with an effective amount of a compound or composition of the invention under conditions in which the compound is capable of forming a complex with the presenter protein and the resulting complex is capable of binding to the target protein.

[0097] In some aspects, the present disclosure provides methods of modulating (e.g., positively or negatively 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 methods comprise modulating the target protein by contacting the target protein with a conjugate of the invention comprising a target protein-binding moiety.

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

[0099] In some aspects, the present disclosure provides methods for forming a presenter protein / compound complex in a cell, hi some embodiments, such methods comprise contacting a cell expressing a presenter protein with a compound or composition of the 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 moiety is capable of binding 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 moiety is a prolyl isomerase binding moiety. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety is an FKBP binding moiety (e.g., the presenter protein binding moiety can bind FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52), a cyclophilin binding moiety (e.g., the presenter protein binding moiety can bind PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1), or a PIN1 binding moiety. In some embodiments of any of the above methods, the presenter protein is known to bind to the presenter protein binding moiety.

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

[0102] [ka]

[0103] In the chemical formula, Z 1 and Z 2 are each independently an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, or Z 1 and Z 2 combine with the atoms to which they are attached to form an optionally substituted 10-40 member macrocycle, and Z 1 or Z 2 at least one of which contains a point of attachment 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 are each independently absent, CH2, O, S, SO, SO2, or NR 4 and 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-C6 10Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl (e.g., optionally substituted C2-C9 heteroaryl), optionally substituted C2-C9 heterocyclylC1-C6 alkyl (e.g., optionally substituted C2-C9 heteroarylC1-C6 alkyl), or R 1 and R 2 combine with the carbon atom to which they are attached to form C=O, or R 1 and R 2 are combined to form optionally substituted C3 to C 10 forming a carbocyclyl or an optionally substituted C2-C9 heterocyclyl; Each R 3 are independently selected from 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-C6 10 Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl (e.g., optionally substituted C2-C9 heteroaryl), or optionally substituted C2-C9 heterocyclylC1-C6 alkyl (e.g., optionally substituted C2-C9 heteroarylC1-C6 alkyl), or two R 8 are combined to form optionally substituted C3 to C 10 Carbocyclyl, optionally substituted C6-C 10 forming an aryl, for example an optionally substituted C2-C9 heteroaryl; Each R 4 are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl.

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

[0105] [ka]

[0106] Includes: In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein-binding moiety is a cyclophilin-binding moiety (e.g., a selective cyclophilin-binding moiety or a non-selective cyclophilin-binding moiety). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the cyclophilin-binding moiety is represented by the structure of Formula III or IV.

[0107] [ka]

[0108] In the chemical formula, Z 3 , Z 4 , Z 5 , and Z 6 are each independently hydroxyl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, or Z 3 and Z 4 or Z 5 and Z 6combine with the atoms to which they are attached to form an optionally substituted 10-40 member macrocycle; Z 3 , Z 4 , Z 5 , Z 6 , or R 5 at least one of which contains a point of attachment to a crosslinking group; e is 0, 1, 2, 3, or 4; R 5 is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 10 Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; R 6 is an optionally substituted C1-C6 alkyl; Each R 7 are independently selected from 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, optionally substituted C3-C6 10 Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10arylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl (e.g., optionally substituted C2-C9 heteroaryl), or optionally substituted C2-C9 heterocyclylC1-C6 alkyl (e.g., optionally substituted C2-C9 heteroarylC1-C6 alkyl); R 8 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl.

[0109] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety has the 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 with a classical protein-protein interaction domain and motif. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the target protein comprises 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 has been modified to replace at least one naturally occurring amino acid with a reactive amino acid (e.g., a naturally occurring amino acid, such as cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine, or an unnatural amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the target protein has been modified to replace at least one naturally occurring reactive amino acid (e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine) with a non-reactive amino acid (e.g., a naturally occurring amino acid, such as serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine, or an unnatural amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the at least one naturally occurring reactive amino acid is an amino acid exposed to 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 to replace all reactive amino acids with non-reactive amino acids.In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the substitution is a conservative substitution.In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the target protein contains only one reactive amino acid that is exposed to solvent.

[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 a 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 cyclophilin 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 has been modified, whereby at least one naturally occurring amino acid has been replaced with a reactive amino acid (e.g., a naturally occurring amino acid, such as cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine, or an unnatural amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein has been modified, whereby at least one naturally occurring reactive amino acid (e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine) has been replaced with a non-reactive amino acid (e.g., a naturally occurring amino acid, such as serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine, or an unnatural amino acid). In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the at least one naturally occurring reactive amino acid is an amino acid exposed to solvent. In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein has been modified to replace all reactive amino acids 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 any of the above compounds, conjugates, complexes, compositions, or methods, the linker is 1 to 20 atoms in length. In some embodiments of any 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 the structure of 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 and In the chemical formula, A 1 is the bond between the linker and the protein-binding moiety, and A 2 is the bond between the bridging group and the linker, and B 1 , B 2 , B 3 , and B 4 are each independently an optionally substituted C1-C2 alkyl, an optionally substituted C1-C3 heteroalkyl, O, S, and NR N Selected from R N is hydrogen, optionally substituted with 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, 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 an 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 C-C 10 Polyethylene glycol or optionally substituted C 1~10 Heteroalkyl, or A 1 -(B1 ) f -(C 1 ) g -(B 2 ) h -(B 3 ) i -(C 2 ) j -(B 4 ) k -A 2 is a chemical bond that connects

[0117] In some embodiments of any of the above compounds, conjugates, complexes, compositions, or methods, the linker has the structure of Formula VI

[0118] [ka]

[0119] In the chemical formula, A 1 is the bond between the linker and the protein-binding moiety, A 2 is the bond between the bridging group and the linker, l is 0, 1, 2, or 3; m is 0 or 1, n is 0, 1, or 2; X 3 , X 4 , and X 5 are each independently absent, O, S, -C≡C-, CR 9 R 10 or NR 11 and Each R 9 , R 10 , and R 11 are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C 10arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl. In some embodiments, each R 9 , R 10 , and R 11 are independently hydrogen, unsubstituted C1-C6 alkyl, unsubstituted C2-C6 alkenyl, unsubstituted C2-C6 alkynyl, unsubstituted aryl, C3-C7 carbocyclyl, unsubstituted C6-C 10 aryl C1-C6 alkyl, and unsubstituted C3-C7 carbocyclyl C1-C6 alkyl.

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

[0121] [ka]

[0122] Includes: chemical terms It will be appreciated by those of skill in the art that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, tautomers) and / or isotopic (replacement of one or more atoms with a different isotope of that atom, e.g., replacement of hydrogen with deuterium) forms. Unless otherwise specified or apparent from context, depicted structures can be understood to represent any such isomeric or isotopic forms, either alone or in combination.

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

[0124] In some embodiments, one or more compounds depicted herein may exist in various tautomeric forms. Unless explicitly excluded, as will be clear from the context, reference to such a compound encompasses all such tautomeric forms. In some embodiments, tautomeric forms result from the swapping of a single bond with an adjacent double bond and the accompanying migration of a proton. In certain embodiments, a tautomeric form may be a prototropic tautomer of an isomeric protonation state having the same empirical formula and total charge as the referenced form. Examples of prototropic tautomeric moieties are ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position on a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. In some embodiments, tautomeric forms may be in equilibrium or sterically locked to one form by appropriate substitution. In certain embodiments, tautomeric forms may undergo acetal interconversion, for example, as shown in the following scheme:

[0125] [ka]

[0126] This arises from interconversions exemplified by: Those skilled in the art will appreciate that in some embodiments, isotopes of the compounds described herein may be prepared and / or utilized in accordance with the present invention. "Isotopes" refer to atoms having the same atomic number but different mass numbers resulting from different numbers of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, isotopic substitution (e.g., replacement of hydrogen with deuterium) may alter the physicochemical properties of a molecule, such as metabolism and / or the racemization rate of a chiral center.

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

[0129] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. The present disclosure is specifically intended to include all individual subcombinations of the members of such groups and ranges. For example, "C 1~6The term "alkyl" is specifically intended to individually disclose methyl, ethyl, C alkyl, C alkyl, C alkyl, and C alkyl. Furthermore, where a compound contains multiple positions where substitution is disclosed in a group or range, unless otherwise specified, the disclosure is intended to cover individual compounds and groups of compounds (e.g., families and subfamily) containing all individual subcombinations of members at each position.

[0130] As used herein, phrases of the form "optionally substituted X" (e.g., optionally substituted alkyl) are intended to be equivalent to "X when X is optionally substituted" (e.g., "alkyl when alkyl is optionally substituted"). It is not intended to imply that the feature "X" (e.g., alkyl) itself is optional.

[0131] As used herein, the term "alkyl" refers to a saturated hydrocarbon group containing 1 to 20 (e.g., 1 to 10 or 1 to 6) carbons. 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 the like, and include (1) alkoxy, (2) alkylsulfinyl, (3) amino, as defined herein (e.g., unsubstituted amino (i.e., -NH) or substituted amino (i.e., -N(R N1 )2(in the chemical formula, R N1 is as defined for amino), (4) C 6~10 Aryl C 1~6 Alkoxy, (5) Azido, (6) Halo, (7) (C 2~9 heterocyclyl)oxy, (8) hydroxyl optionally substituted with an O-protecting group, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1~7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) —COR optionally substituted with an O-protecting groupA’ (In the chemical formula, R A’ is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (15) —C(O)NR B’ R C’ (In the chemical formula, R B’ and R C’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (16)-SO2R D’ (In the chemical formula, R D’ is (a)C 1~6 Alkyl, (b) C 6~10 Aryl, (c) C1~6 ALC-C 6~10 (17) -SO2NR E’ R F’ (In the chemical formula, R E’ and R F’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (18)—C(O)R G’ (In the chemical formula, R G’ is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (19)-NR H’C(O)R I’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R I’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (20)-NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R K’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (21) amidine; and (22) silyl groups, such as trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyl, and the like. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl can be further substituted with an oxo group to provide the respective aryloyl substituent.

[0132] The terms "alkylene" and the prefix "alk-" as used herein refer to a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and are exemplified by methylene, ethylene, isopropylene, and the like.x~y The terms "alkylene" and "C x~y The prefix "alk-" denotes an alkylene group having x to y carbons. Exemplary values ​​of x are 1, 2, 3, 4, 5, and 6, and exemplary values ​​of y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C 1~6 , C 1~10 , C 2~20 , C 2~6 , C 2~10 , or C 2~20 In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituents, as defined herein for an alkyl group.

[0133] The term "alkenyl," as used herein, unless otherwise specified, represents a monovalent straight- or branched-chain group of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyl includes both cis and trans isomers. Alkenyl groups can be optionally substituted with 1, 2, 3, or 4 substituents independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0134] The term "alkynyl," as used herein, refers to a monovalent straight- or branched-chain 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, and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups can be optionally substituted with 1, 2, 3, or 4 substituents independently selected from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0135] As used herein, the term "amino" refers to -N(R N1 )2(in the formula, each R N1 are independently H, OH, NO2, N(R N2 )2, SO2OR N2 , SO2R N2 , SOR N2 , an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group such as an optionally substituted arylalkoxycarbonyl group or any described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group such as an optionally substituted arylalkoxycarbonyl group or any described herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), and these enumerated R N1 Each of the groups can be optionally substituted as defined herein for each group, or two R N1 are combined to form a heterocyclyl or N-protecting group, and each R N2 are independently H, alkyl, or aryl). The amino groups of the present invention can be unsubstituted amino (i.e., —NH) or substituted amino (i.e., —N(R N1 )2). In a preferred embodiment, amino can be -NH2 or -NHR N1 (In the formula, R N1 are independently OH, NO2, NH2, and NR N2 2. SO2OR N2 , SO2R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, and each R N2 is H, C1~20 Alkyl (e.g., C 1~6 alkyl), or C 6~10 aryl).

[0136] As used herein, the term "amino acid" refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group of -COH or a sulfo group of -SOH), where the amino acid is attached to the parent molecular group by the side chain, the amino group, or the acid group (e.g., the side chain). As used herein, the term "amino acid" in its broadest sense refers to 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 HN-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. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including those at the carboxy and / or amino termini in a polypeptide, may contain structural modifications relative to the above general structure. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, and / or substitution relative to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one containing the otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one containing the otherwise identical unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" is used to refer to a free amino acid. In some embodiments, it is used to refer to an amino acid residue of a polypeptide. In some embodiments, an amino acid is attached to a parent molecular moiety by a carbonyl group, with the side chain or amino group attached 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. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocystine, serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid group is (1)C. 1~6 Alkoxy, (2)C 1~6 alkylsulfinyl, (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH) or substituted amino (i.e., —N(R N1 )2(wherein, R N1 is as defined for amino), (4) C 6~10 Aryl C 1~6 Alkoxy, (5) Azido, (6) Halo, (7) (C 2~9 heterocyclyl)oxy, (8) hydroxyl, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1~7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) -CO2R A’ (In the formula, R A’ is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3OR', where s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (15) —C(O)NR B’ R C’ (In the formula, R B’ and R C’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (16)-SO2R D’ (In the formula, R D’ is (a)C 1~6 Alkyl, (b) C 6~10 Aryl, (c) C 1~6 ALC-C 6~10 (17) -SO2NR E’ R F’ (In the formula, R E’ and R F’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (18)—C(O)R G’ (In the formula, R G’is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (19)-NR H’ C(O)R I’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R I’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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 (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (20)-NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R K’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 0 to 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(h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (21) an amino-polyethylene glycol having a carbon number of 1, 2, 3, or in the case of an amino acid group of 2 or more carbons, 4 substituents independently selected from the group consisting of (21) an amidine; and (21) an amino-polyethylene glycol having a carbon number of 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of (21) an amidine. In some embodiments, any of these groups may be optionally substituted with one, two, three, or four substituents independently selected from the group consisting of (21) an amino-polyethylene glycol having a carbon number of 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of (21) an amidine. Each 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 optionally substituted on the nitrogen of the amino acid that forms the peptide bond. N-alkylated amino acids include, but are not limited to, N-methyl amino acids such as N-methyl-alanine, N-methyl-threonine, N-methyl-phenylalanine, N-methyl-aspartic acid, N-methyl-valine, N-methyl-leucine, N-methyl-glycine, N-methyl-isoleucine, N(α)-methyl-lysine, N(α)-methyl-asparagine, and N(α)-methyl-glutamine.

[0138] The term "aryl" as used herein refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having one or two aromatic rings, and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and includes (1)C 1~7 Acyl (e.g., carboxaldehyde), (2) C1~20 Alkyl (e.g., C 1~6 Alkyl, alkoxy-C 1~6 Alkyl, C 1~6 Alkylsulfinyl-C 1~6 Alkyl, Amino-C 1~6 Alkyl, azido-C 1~6 Alkyl, (carboxaldehyde)-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 (e.g., C 1~6 alkoxy, for example perfluoroalkoxy), (4) C 1~6 Alkylsulfinyl, (5)C 6~10 Aryl, (6) Amino, (7) C 1~6 ALC-C 6~10 Aryl, (8) Azide, (9) C 3~8 Cycloalkyl, (10)C 1~6 ALC-C 3~8 Cycloalkyl, (11) halo, (12) C 1~12 Heterocyclyl (e.g., C 1~12 Heteroaryl), (13)(C 1~12 (heterocyclyl)oxy, (14) hydroxyl, (15) nitro, (16) C 1~20 Thioalkoxy (e.g., thioalkoxy), (17)-(CH2) q CO2R A’ (wherein q is an integer of 0 to 4, and R A’ is (a) alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 ALC-C 6~10 aryl), (18)-(CH2) q CONR B’ R C’ (wherein q is an integer of 0 to 4, and R B’ and R C’ is (a) hydrogen, (b) C 1~6Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (19)—(CH2) q SO2R D’ (wherein q is an integer of 0 to 4, and R D’ is (a) alkyl, (b) C 6~10 aryl, and (c) alk-C 6~10 aryl), (20)-(CH2) q SO2NR E’ R F’ (wherein q is an integer of 0 to 4, and R E’ and R F’ are (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 (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 ALC-C 1~12 Heterocyclyl (e.g., C 1~6 ALC-C 1~12 Heteroaryl), (26)C 2~20 Alkenyl, and (27)C 2~20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to provide an aryloyl and (heterocyclyl)oyl substituent, respectively.

[0139] As used herein, an "arylalkyl" group represents an aryl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted arylalkyl groups include those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 ALC-C 6~10 Aryl, C 1~10 ALC-C 6~10 Aryl, or C 1~20 ALC-C 6~10 In some embodiments, alkylene and aryl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for that group. Other groups preceded by the prefix "alk-" are similarly defined, where "alk-" refers to any group selected from C, C's ... 1~6 The alkylene means and the chemical structure to which it is attached is as defined herein.

[0140] The term "azido" refers to the group -N3, which may also be depicted as -N=N=N. As used herein, the terms "carbocyclic" and "carbocyclyl" refer to an optionally substituted C 3~12 It refers to monocyclic, bicyclic, or tricyclic non-aromatic ring structures. Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloalkynyl groups.

[0141] As used herein, a "carbocyclylalkyl" group represents a carbocyclic group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted carbocyclylalkyl groups include those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 ALC-C 6~10 Carbocyclyl, C 1~10 ALC-C 6~10 Carbocyclyl, or C 1~20 ALC-C 6~10In some embodiments, alkylene and carbocyclyl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for that group. Other groups preceded by the prefix "alk-" are similarly defined, where "alk-" refers to any group selected from the group consisting of C, C- ... 1~6 The alkylene means and the chemical structure to which it is attached is as defined herein.

[0142] The term "carbonyl" as used herein refers to a C(O) group, which may also be depicted as C=O. The term "carboxy" as used herein means -CO2H.

[0143] As used herein, the term "cyano" refers to a -CN group. The term "cycloalkyl," as used herein, unless otherwise specified, refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of 3 to 8 carbons, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycloheptyl, and the like. When a cycloalkyl group contains one carbon-carbon double bond, the cycloalkyl group can be referred to as a "cycloalkenyl" group. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. Cycloalkyl groups of the present invention include (1) C 1~7 acyl (e.g., carboxaldehyde), (2) C1-20 alkyl (e.g., alkyl, alkoxy-C1-6 alkyl, alkylsulfinyl-C1-6 alkyl, amino-C1-6 alkyl, azido-C1-6 alkyl, (carboxaldehyde)-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 (e.g., C 1~6 alkoxy, for example perfluoroalkoxy), (4) C 1~6 Alkylsulfinyl, (5)C 6~10Aryl, (6) Amino, (7) C 1~6 ALC-C 6~10 Aryl, (8) Azide, (9) C 3~8 Cycloalkyl, (10)C 1~6 ALC-C 3~8 Cycloalkyl, (11) halo, (12) C 1~12 Heterocyclyl (e.g., 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’ (wherein q is an integer of 0 to 4, and R A’ is (a)C 1~6 Alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 ALC-C 6~10 aryl), (18)-(CH2) q CONR B’ R C’ (wherein q is an integer of 0 to 4, and R B’ and R C’ is (a) hydrogen, (b) C 6~10 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (19)—(CH2) q SO2R D’ (wherein q is an integer of 0 to 4, and R D’ is (a)C 6~10 Alkyl, (b) C 6~10 aryl, and (c) C 1~6 ALC-C 6~10 aryl), (20)-(CH2) q SO2NR E’ R F’ (wherein q is an integer of 0 to 4, and R E’ and R F’ are (a) hydrogen, (b) C 6~10 Alkyl, (c) C 6~10aryl, and (d) C 1~6 ALC-C 6~10 (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 ALC-C 1~12 Heterocyclyl (e.g., C 1~6 ALC-C 1~12 Heteroaryl), (26) oxo, (27) C 2~20 Alkenyl, and (28)C 2~20 and (heterocyclyl)oyl.

[0144] As used herein, a "cycloalkylalkyl" group refers to a cycloalkyl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein (e.g., an alkylene group of 1 to 4, 1 to 6, 1 to 10, or 1 to 20 carbons). In some embodiments, the alkylene and cycloalkyl can each be further substituted with 1, 2, 3, or 4 substituents, as defined herein for that group.

[0145] As used herein, the term "diastereomers" means stereoisomers that are not mirror images of one another and are not superimposable with respect to one another. As used herein, the term "enantiomer" refers to each individual optically active form of a compound of the present invention having an optical purity or enantiomeric excess (as determined by standard methods in 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] The term "halo" as used herein refers to a halogen selected from bromine, chlorine, iodine, or fluorine. As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or two of the constituent carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, heteroalkyl groups can be further substituted with one, two, three, or four substituents as described herein for alkyl groups. As used herein, the terms "heteroalkenyl" and "heteroalkynyl" refer to alkenyl and alkynyl groups, as defined herein, respectively, in which one or two of the constituent carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, heteroalkenyl and heteroalkynyl groups can be further substituted with one, two, three, or four substituents as described herein for alkyl groups.

[0147] As used herein, the term "heteroaryl" refers to the subset of heterocyclyl, as defined herein, that is aromatic; i.e., they contain 4n+2 pi-electrons in a single ring or polycyclic ring system. Exemplary unsubstituted heteroaryl groups are 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) carbons. In certain embodiments, heteroaryl is substituted with 1, 2, 3, or 4 substituents as defined for heterocyclyl groups.

[0148] The term "heteroarylalkyl" means a heteroaryl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted heteroarylalkyl groups include those having 2 to 32 carbons (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 carbons, e.g., C 1~6 ALC-C 1~12 Heteroaryl, C 1~10 ALC-C 1~12 Heteroaryl, or C 1~20 ALC-C 1~12 In some embodiments, alkylene and heteroaryl can each be further substituted with 1, 2, 3, or 4 substituents, as defined herein for each group. Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

[0149] As used herein, unless otherwise specified, the term "heterocyclyl" refers to a 5-, 6-, or 7-membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Five-membered rings have two zero to double bonds, and 6- and 7-membered rings have three zero to double bonds. Exemplary unsubstituted heterocyclyl groups are 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) carbons. The term "heterocyclyl" also refers to heterocyclic compounds having bridged polycyclic ring structures in which one or more carbons and / or heteroatoms bridge two non-adjacent members of a single ring, such as a quinuclidinyl group. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocycles are fused to one, two, or three carbocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or other monocyclic heterocycles, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, etc. Examples of fused heterocyclyls include tropane and 1,2,3,5,8,8a-hexahydroindolizine, including dihydro and tetrahydro forms in which one or more double bonds have been reduced and replaced with hydrogen. Heterocyclic compounds include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, and isothiazolyl. 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), purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, etc. Still other exemplary heterocyclyls include 2,3,4,5-tetrahydro-2-oxooxazolyl, 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), 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g., 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl). 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1H-imidazolyl), 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-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-methylpyrimidin-1-yl), 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g., 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'-spiropropane-1H-indol-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-benzimidazolyl, aryl (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-benzothiazinyl, 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-purinyl (e.g., 1,2,3 ,6-tetrahydro-1,3-dimethyl-2,6-dioxo-1H-purinyl), 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g., 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl), 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-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and 2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups also include groups of the formula:

[0150] [ka]

[0151] (In the formula, E' is selected from the group consisting of -N- and -CH-; F' is selected from the group consisting of -N=CH-, -NH-CH2-, -NH-C(O)-, -NH-, -CH=N-, -CH2-NH-, -C(O)-NH-, -CH=CH-, -CH2-, -CH2CH2-, -CHO-, -OCH2-, -O-, and -S-; and G' is selected from the group consisting of -CH- and -N-. Any of the heterocyclyl groups listed herein can be selected from the group consisting of: (1) C 1~7 Acyl (e.g., carboxaldehyde), (2) C 1~20 Alkyl (e.g., alkyl, alkoxy-C 1~6 Alkyl, alkylsulfinyl-C 1~6 Alkyl, Amino-C 1~6 Alkyl, azido-C 1~6 Alkyl, (carboxaldehyde)-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 (e.g., C 1~6 alkoxy, for example perfluoroalkoxy), (4) C 1~6 Alkylsulfinyl, (5)C 6~10 Aryl, (6) Amino, (7) C 1~6 ALC-C 6~10 Aryl, (8) Azide, (9) C 3~8 Cycloalkyl, (10)C 1~6 ALC-C 3~8 Cycloalkyl, (11) halo, (12) C 1~12 Heterocyclyl (e.g., C2-12 heteroaryl), (13) (C 1~12(heterocyclyl)oxy, (14) hydroxyl, (15) nitro, (16) C 1~20 Thioalkoxy (e.g., C 1~6 Thioalkoxy), (17)-(CH2) q CO2R A’ (wherein q is an integer of 0 to 4, and R A’ is (a) alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 ALC-C 6~10 aryl), (18)-(CH2) q CONR B’ R C’ (wherein q is an integer of 0 to 4, and R B’ and R C’ is (a) hydrogen, (b) alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (19)—(CH2) q SO2R D’ (wherein q is an integer of 0 to 4, and R D’ is (a)C 1~6 Alkyl, (b) C 6~10 aryl, and (c) C 1~6 ALC-C 6~10 aryl), (20)-(CH2) q SO2NR E’ R F’ (wherein q is an integer of 0 to 4, and R E’ and R F’ are (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 (21) thiol; (22) C 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24) arylalkoxy, (25) C 1~6 ALC-C 1~12 Heterocyclyl (e.g., C 1~6 ALC-C 1~12heteroaryl), (26) oxo, (27) (C 1~12 Heterocyclyl)imino, (28)C 2~20 Alkenyl, and (29)C 2~20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to provide an aryloyl and (heterocyclyl)oyl substituent, respectively.

[0152] As used herein, a "heterocyclylalkyl" group refers to a heterocyclyl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted heterocyclylalkyl groups include those having 2 to 32 carbons (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 carbons, e.g., C 1~6 ALC-C 1~12 Heterocyclyl, C 1~10 ALC-C 1~12 Heterocyclyl, or C 1~20 ALC-C 1~12 In some embodiments, the alkylene and heterocyclyl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the respective group.

[0153] As used herein, the term "hydrocarbon" refers to a group consisting solely of carbon and hydrogen atoms. The term "hydroxyl, as used herein, refers to an -OH group. In some embodiments, the hydroxyl group can be substituted with 1, 2, 3, or 4 substituents (e.g., O-protecting groups) as defined herein for alkyl.

[0154] As used herein, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention may possess one or more chiral centers and / or double bonds and may therefore exist as stereoisomers, such as double bond isomers (i.e., E / Z geometric isomers) and diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present invention, the chemical structures depicted herein, i.e., compounds of the invention, encompass all corresponding stereoisomers, i.e., stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) as well as mixtures of enantiomers or stereoisomers (e.g., racemates). Enantiomeric and stereoisomeric mixtures of the compounds of the present invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallization of the compounds as chiral salt complexes, crystallization of the compounds in chiral solvents, etc. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0155] The term "N-protected amino" as used herein means an amino group, as defined herein, bound 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 against undesired reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference.N-protecting groups include acyl, aryloyl, 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, e.g., sulfonyl-containing groups, such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate-forming groups, such as 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 , methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc., alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, etc., and silyl groups such as trimethylsilyl, etc. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0156] The term "nitro" as used herein refers to the group --NO.sub.2. As used herein, the term "O-protecting group" refers to a group intended to protect an oxygen-containing (e.g., phenol, hydroxyl, or carbonyl) group from undesired reactions during synthetic procedures. Commonly used O-protecting groups are those described in Greene, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, 2002). Wiley & Sons, New York, 1999) (incorporated herein by reference). Exemplary O-protecting groups include acyl, aryloyl, 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-isopropylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, fluoroacetyl ... arylcarbonyl groups such as phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl; alkylcarbonyl groups such as acyl, acetyl, propionyl, pivaloyl, and the like; optionally substituted arylcarbonyl groups such as benzoyl; silyl groups such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), and the like; ester-forming groups, such as methyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, trityl, etc.; alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbon ... alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyl groups, such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,optionally substituted arylalkoxycarbonyl 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 the like, and optionally substituted aryloxycarbonyl 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 (e.g., trityl, methylthiomethyl, methoxymethyl, benzyloxymethyl, siloxymethyl, etc.), , 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, tribenzyl, protecting groups (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 and ketal groups, e.g., dimethyl acetal, 1,3-dioxolane, and the like, acylal groups, and dithiane groups, e.g., 1,3-dithiane, 1,3-dithiolane, etc.), carboxylic acid protecting groups (e.g., ester groups, e.g., methyl ester, benzyl ester, t-butyl ester, orthoester, etc.), and oxazoline groups.

[0157] The term "oxo" as used herein refers to =O. The prefix "perfluoro" as used herein refers to any group, as defined herein, in which each hydrogen radical bonded to an alkyl group has been replaced with a fluoride group. For example, perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like.

[0158] As used herein, the term "protected hydroxyl" means an oxygen atom bonded to an O-protecting group. As used herein, the term "spirocyclyl" refers to a C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 -C 11 -C 12 -C 13 -C 14 -C 15 -C 16 -C 17 -C 18 -C 19 -C 20 -C 21 -C 22 -C 23 -C 24 -C 2~7 It represents a divalent alkylene group, as well as a divalent heteroalkylene group having both termini bonded to the same atom. The heteroalkylene groups 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 groups of the present invention can be optionally substituted with 1, 2, 3, or 4 substituents provided herein as optional substituents for the cycloalkyl and / or heterocyclyl groups.

[0159] As used herein, the term "stereoisomer" refers to all different possible isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may exist in, particularly all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers, and / or conformers. Some compounds of the present invention may exist in different tautomeric forms, and all of these tautomeric forms are included within the scope of the present invention.

[0160] The term "sulfonyl" as used herein refers to a -S(O)2- group. As used herein, the term "thiol" refers to a -SH group. definition In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to include the itemized ingredient or step, whether presented by itself or with one or more additional ingredients or steps, and (iv) the terms "about" and "approximately" may be understood to allow for standard deviation as understood by one of ordinary skill in the art, and (v) when ranges are provided, the endpoints are included.

[0161] As 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 a variety of ways. In some embodiments, affinity is measured by a quantitative assay. 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 the ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under equivalent conditions (e.g., concentrations).

[0162] As used herein, the terms "approximately" and "about," where appropriate in the relevant context, are intended to encompass normal statistical variations understood by those of ordinary skill in the art. In certain embodiments, the terms "approximately" or "about" refer to a range of values ​​that falls within 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 than) the stated value, unless otherwise specified or otherwise obvious from the context (e.g., when such number exceeds 100% of the possible values).

[0163] It will be understood that the term "binding," as used herein, typically refers to an association (e.g., non-covalent or covalent) between two or more entities. "Direct" binding includes physical contact between the entities or moieties, while indirect binding includes physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied in isolation or in more complex systems (e.g., covalently bound or otherwise associated with a carrier entity and / or a biological system or cell).

[0164] The affinity between a molecule X and its partner Y is generally expressed as the dissociation constant (K D ) Affinity can be measured by conventional methods known in the art, including those described herein. Certain exemplary and representative embodiments for measuring binding affinity are described below. As used herein, "K D The term "" is intended to mean the dissociation equilibrium constant of a particular compound-protein or complex-protein interaction. Typically, the compounds of the invention have a dissociation equilibrium constant of about 10, as determined, for example, by surface plasmon resonance (SPR) techniques using a presenter protein as the analyte and the compound as the ligand. -6 Less than M, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M, or even lower dissociation equilibrium constant (K D The presenter protein / compound complex of the present invention binds to the presenter protein at about 10 s when determined, for example, by surface plasmon resonance (SPR) technology using the target protein as the analyte and the complex as the ligand. -6 Less than M, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M, or even lower dissociation equilibrium constant (K D ) to bind to 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).

[0165] As used herein, the term "crosslinking group" refers to a group containing a reactive functional group that can chemically attach to a specific functional group (e.g., primary amines, sulfhydryls) on a protein or other molecule. A "moiety capable of chemoselective reaction with an amino acid," as used herein, refers to a moiety containing a reactive functional group that can chemically attach to a functional group of a natural or unnatural amino acid (e.g., primary and secondary amines, sulfhydryls, alcohols, carboxyl groups, carbonyls, or triazole-forming functional groups such as 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 such as NHS esters, imidoesters, 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, such as hydrophobic effect interactions, electrostatic interactions, van der Waals interactions, or π-effect interactions). Examples of complexes are "presenter protein / conjugate complexes" and "target protein / conjugate complexes" that comprise a conjugate of the present invention bound to a presenter protein or target protein. As used herein, the term "conjugate" refers to a compound formed by the joining (e.g., by a covalent bond-forming reaction) of two or more compounds (e.g., a compound comprising a crosslinking group and a protein, such as a target protein or a presenter protein).

[0167] As used herein, an atom "involved in a bond" is either within 4 Å of the entity to which it is bonded or is connected to an atom that is 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 and modulates the activity of 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, the presenter protein is a relatively abundant protein (e.g., the presenter protein is sufficiently abundant that its participation in a tripartite complex does not substantially affect the biological role of the presenter protein in the cell and / or the viability or other attributes of the cell). 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 natural interaction partners in the cell. In certain embodiments, the presenter protein is known to bind to a small molecule to form a binary complex that is known or predicted to bind to and modulate the biological activity of the target protein.

[0169] The term "presenter protein binding moiety" refers to a compound that specifically binds to said presenter protein, e.g., 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). 50and refers to a group of atoms and their bound moieties (e.g., atoms within 20 atoms, e.g., atoms within 15 atoms, atoms within 10 atoms, atoms within 5 atoms) that are involved in binding to the presenter protein so as to inhibit the peptidyl-prolyl isomerase activity of the presenter protein. It will be understood that a presenter protein-binding moiety does not necessarily encompass all of the atoms in a compound that interact with the presenter protein. It will also be understood that one or more atoms of a presenter protein-binding moiety can be within a target protein-binding moiety (e.g., a eukaryotic target protein-binding moiety, such as a mammalian target protein-binding moiety or a fungal target protein-binding moiety, or a prokaryotic target protein-binding moiety, such as a bacterial target protein-binding moiety).

[0170] As used herein, "FKBP binding moiety" refers to a presenter protein binding moiety that is selective for a presenter protein in the FKBP family of proteins (e.g., FKBP12, FKBP12.6, FKBPP13, FKBP25, FKBP51, or FKBP52). As used herein, a "selective FKBP binding moiety" refers to a binding moiety that is specific for one or more (e.g., two, three, four, five) members of the FKBP family over all other members of the FKBP family. As used herein, a "non-selective FKBP binding moiety" refers to a binding moiety that has comparable affinity (within 2-fold, within 3-fold, within 4-fold, within 5-fold, within 10-fold) for all members of the FKBP family.

[0171] The term "protein binding moiety" refers to a group of atoms involved in binding to a protein (e.g., a presenter protein or a target protein) and a moiety attached thereto (e.g., within 20 atoms, e.g., within 15 atoms, within 10 atoms, within 5 atoms), wherein the compound has a K of, e.g., 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 or specifically binds to the protein with an IC of, e.g., 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 It is understood that a protein binding moiety does not necessarily encompass all atoms in a compound that interact with a protein.

[0172] As used herein, the term "react" refers to the process in which atoms of the same or different elements rearrange themselves to form a new substance.For example, the formation of a covalent bond between two atoms, for example, the reaction between a reactive amino acid on a protein and a crosslinking group to form a covalent bond.Reaction can be measured by any method known in the art, for example, the formation of reaction products 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 cross-linking group). Examples of reactive amino acids include cysteine, lysine, serine, and amino acids with an azide on the side chain. A "non-reactive 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 non-reactive amino acids include valine, alanine, isoleucine, theronine, and leucine.

[0174] The term "reference" is often used herein to describe a standard or control compound, individual, population, sample, sequence, or value with which a compound, individual, population, sample, sequence, or value of interest is compared. In some embodiments, the reference compound, individual, population, sample, sequence, or value is examined and / or determined substantially simultaneously with the examination or determination of the compound, individual, population, sample, sequence, or value of interest. In some embodiments, the reference compound, individual, population, sample, sequence, or value is a historical reference, optionally embodied in tangible media. Typically, as will be understood by one of skill in the art, the reference compound, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent to those utilized in determining or characterizing the compound, individual, population, 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, a solvent-exposed amino acid is an amino acid that, when substituted, does not substantially alter the three-dimensional structure of the protein.

[0176] As used herein, the terms "specific binding" or "specific" refer to an interaction between a binder and a target entity. As will be understood by those skilled in the art, an interaction is considered "specific" if it favors binding with a KD of, for example, 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) in the presence of alternative interactions. In many embodiments, the specific interaction depends on the presence of a particular structural feature of the target entity (e.g., an epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of a binder for one or more other possible target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binder. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding agent.

[0177] The term "specific," when used in reference to a compound having activity, will be understood by those skilled in the art to mean that the compound distinguishes between potential target entities or states. 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 surrogate targets. In many embodiments, specific interaction depends on the presence of a particular structural feature (e.g., an epitope, cleft, binding site) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of a binding agent for one or more other possible target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding agent. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding agent. In some embodiments, an agent or entity does not detectably bind to a competing surrogate target under conditions of binding to its target entity. In some embodiments, a binding agent binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to a competing surrogate target.

[0178] The term "substantially" refers to the qualitative condition of exhibiting the entire or nearly entire extent or degree of a property or characteristic of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, reach an end state and / or proceed perfectly or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0179] As used herein, the term "does not substantially bind" to a particular protein means, for example, that the target -4 M or more, alternatively 10 -5 M or more, alternatively 10 -6 M or more, alternatively 10-7 M or more, alternatively 10 -8 M or more, alternatively 10 -9 M or more, alternatively 10 -10 M or more, alternatively 10 -11 M or more, alternatively 10 -12 K over M D or 10 -4 M~10 -12 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 K within M D The term can be represented by a molecule or portion of a molecule having the formula:

[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 a small molecule. In some embodiments, the target protein is a naturally occurring protein. In some such embodiments, the target protein is naturally found in a particular mammalian cell (e.g., a mammalian target protein), fungal cell (e.g., a fungal target protein), bacterial cell (e.g., a bacterial target protein), or plant cell (e.g., a plant target protein). In some embodiments, the target protein is characterized by native interactions with one or more native presenter protein / native small molecule complexes. In some embodiments, the target protein is characterized by native interactions with multiple different native presenter protein / native 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 complexes of cyclosporine, rapamycin, or FK506 with a presenter protein (e.g., FKBP). The target protein can be naturally occurring, e.g., wild-type. Alternatively, the target protein can differ from the wild-type protein but still maintain a biological function, e.g., an allelic variant, a splice mutant, or a biologically active fragment. Exemplary mammalian target proteins are 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 a biological pathway associated with a disease, disorder, or pathology.

[0181] In some embodiments, the target protein is a modified target protein. Modified target proteins can include conservative or non-conservative insertions, deletions, or substitutions of amino acids in the protein sequence (e.g., D-amino acids, desamino acids) (e.g., where 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 carboxy terminus of any of the polypeptides of the present invention can facilitate conjugation of these proteins, for example, via 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 can be conservative (i.e., where the residue is replaced with another of the same general type or group) or non-conservative (i.e., where the residue is replaced with an amino acid of a different type). Furthermore, non-naturally occurring amino acids (i.e., non-naturally occurring conservative amino acid substitutions or non-naturally occurring non-conservative amino acid substitutions) can be substituted for natural amino acids. The term "target protein binding moiety" refers to a group of ring atoms and their attached moieties (e.g., atoms within 20 atoms, e.g., atoms within 15 atoms, atoms within 10 atoms, atoms within 5 atoms) that are involved in binding to 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) when the compound is in complex with a presenter protein. It will be understood that the target protein binding moiety does not necessarily encompass all of the atoms in a compound that interact with the target protein. It will also be understood that one or more atoms of the presenter protein binding moiety may also be present in the target protein binding moiety.

[0182] The term "conventional binding pocket" refers to a cavity or pocket in a protein structure that has physiochemical and / or geometric properties comparable to those of a protein whose activity is modulated by one or more small molecules. In some embodiments, a conventional binding pocket is a protein having a 1000 A 3 A conventional binding pocket is a well-defined pocket with a volume greater than 1000 kJ / mol. 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 not to have 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 a drug target and / or does not have a suitable binding site 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 that used by the program DOGSITESCORER® (University of Hamburg, Hamburg, Germany), which evaluates druggability based on calculated parameters for the binding pocket on the protein, including volume, surface area, lipophilic surface area, depth, and / or hydrophobicity ratio. [Brief explanation of the drawings]

[0184] [Figure 1] 1 is an image illustrating SDS-PAGE analysis of KRASGTP / S39Clite / C2-FK506 conjugates. 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]1 is an image illustrating SDS-PAGE analysis of KRASGTP / G12Clite / SFAX9DS conjugate. [Figure 3A] 1 is an image illustrating 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] 1 is an image illustrating SEC and SDS-PAGE analysis of KRASGTP / S39Clite / C2-Holt / FKBP12 complex formation. SDS-PAGE analysis of the SEC elution peak. The blue dotted line corresponds to the fractions collected for the KRASGTP / S39Clite / C2-Holt / FKBP12 elution peak. [Figure 4] 1 is an image illustrating the SEC profile and SDS-PAGE analysis of the elution peak confirming the formation of the KRASGDP / S39Clite / SFAC4DS / CypAC52S complex. [Figure 5A] 1 is an image illustrating the SEC profile and SDS-PAGE analysis of free PTP1BS187Clite and FKBP12 proteins and the PTP1BS187Clite / C3-SLF / FKBP12 complex. [Figure 5B] 1 is an image illustrating the SEC profile and SDS-PAGE analysis of free PTP1BS187Clite and FKBP12 proteins and the PTP1BS187Clite / C3-SLF / FKBP12 complex. [Figure 6] 1 is an image illustrating the cross-linking efficiency of C3- and C4-SLF by SDS-PAGE. [Figure 7A] 1 is an image illustrating the crystal structure of the FKBP12-Compound 1-KRASGTP / S39C complex. Ribbon representation showing FKBP12, KRASGTP / S39C, and the ligand. Fo-Fc electron density at 3σ is shown for the ligand in a close-up view. [Figure 7B]1 is an image illustrating the crystal structure of the FKBP12-Compound 1-KRASGTP / S39C complex. It is a representation of the surface of the complex, with atoms within 4 Å of the ligand or partner protein colored red. [Figure 8] 1 is an image illustrating the crystal structure of CypAC52S-SFAC4DS-KRASGDP / S39C. [Figure 9A] 1 is an image illustrating the crystal structure of FKBP12-C3SLF-PTP1BS187C, illustrating that the crystal contains two complex molecules of FKBP12-C3SLF-PTP1BS187C in the asymmetric unit. [Figure 9B] 1 is an image illustrating the crystal structure of FKBP12-C3SLF-PTP1BS187C, illustrating that the buried area of ​​PTP1BS187C is 427 Å and that of C3-SLF is 615 Å. [Figure 10] 1 is an image illustrating the crystal structure of MCL1S245C / C3SLF / FKBP52. [Figure 11] 1 is an image illustrating the binding curve of W21487-dependent complex formation of the CYPA-W21487-KRASG12C-GTP ternary complex. [Figure 12] 1 is an image illustrating the binding curve of W21487-dependent complex formation of the CYPA-W21487-KRASG12C-GTP ternary complex. [Figure 13] 1 is an image illustrating ITC measurements of the binding of FKBP12-Compound 1 and FKBP12-Compound 2 binary complexes to CEP250. [Figure 14-1] 1 is an image illustrating SPR sensorgrams for FKBP12 / Compound 1 binding to CEP25011.4 and CEP25029.2. [Figure 14-2] 1 is an image illustrating SPR sensorgrams for FKBP12 / Compound 1 binding to CEP25011.4 and CEP25029.2. [Figure 15]1 is an image illustrating a sensogram and steady-state fitting curve for CYPA / Compound 3 binding to KRASG12C-GTP. [Figure 16] 1 is an image illustrating the fluorescence polarization curve for CypA:C3DS:KRAS complex formation. [Figure 17A] 17A is an image illustrating the 2D1H-15N TROSY-HSQC spectrum of KRASG12C-GTP (FIG. 17A). [Figure 17B] 1 is an image illustrating the addition of a stoichiometric amount of CYPA. [Figure 17C] 1 is an image illustrating KRAS and CYPA alone. DETAILED DESCRIPTION OF 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 surface area of ​​contact. Due to their small size, the only way for small molecules to create sufficient intermolecular surface area for effective interaction with target proteins is by directly enveloping the protein. In fact, a large body of both experimental and computational data supports the view that only proteins with hydrophobic "pockets" on their surfaces can bind small molecules. In this case, binding is enabled by envelopment.

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

[0187] Many important therapeutic targets exert their function through complexation with other proteins. In many of these systems, protein / protein interaction surfaces contain an inner core of hydrophobic side chains surrounded by a broad ring of polar residues. Because hydrophobic residues contribute nearly all of the energetically favorable contacts, this cluster has been represented as a "hot spot" for protein-protein interaction engagement. Importantly, in the above-listed complexes of naturally occurring small molecules with small, displayed proteins, the small molecule provides a cluster of hydrophobic features similar to the hot spot, while the protein provides a ring of primarily polar residues. In other words, the displayed small molecule systems mimic the surface architecture commonly 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 an entirely different target, TorC1. To date, no method has been developed to reprogram the binding and modulation capabilities of presenter protein / ligand interfaces to interact with and modulate other target proteins previously considered undruggable.

[0189] Additionally, it is widely recognized that some drug candidates fail because they modulate the activity of both their intended target and other unintended proteins in the same way. This problem is particularly challenging when the drug-binding site of the target protein is similar to that of a non-target protein. The 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 unacceptable side effects that also modulate the insulin receptor. However, structural dissimilarity exists between these two proteins in the region surrounding the ATP-binding pocket. Despite this knowledge, there is currently no method to take advantage of these differences to develop drugs that are more specific to IGF-1R than IR.

[0190] The present disclosure provides methods and reagents useful for analyzing protein-protein interfaces, for example, the interface between a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin 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 a complex with a presenter protein and a small molecule. In some embodiments, these methods and reagents may be useful in identifying compounds that can inhibit or activate a target protein by forming a complex with a presenter protein and the 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 cross-linking group. The invention also features conjugates 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] The present invention also provides a compound of formula VII, ALB (Formula VII) wherein A is a structure of formula VIII

[0192] [ka]

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

[0194] [ka]

[0195] JPEG0007812813000021.jpg59170

[0196] is. crosslinking group In some embodiments, the compounds of the present invention contain a crosslinking group. A crosslinking group refers to a group containing a reactive functional group that can chemically attach to a specific functional group (e.g., primary amine, sulfhydryl) on a protein or other molecule. 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, such as 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 cross-linking groups include 2'-pyridyl disulfide, 4'-pyridyl disulfide iodoacetyl, maleimide, thioester, alkyl disulfide, alkylamine disulfide, nitrobenzoic acid disulfide, anhydride, NHS ester, aldehyde, alkyl chloride, alkyne, and azide.

[0198] Presenter protein binding site In some embodiments, the compounds of the invention comprise a presenter protein binding moiety, which in some embodiments is capable of binding to said presenter protein such that the provided compounds specifically bind to said presenter protein, e.g., 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). 50and may include a group of atoms (e.g., 5-20 atoms, 5-10 atoms, 10-20 atoms) and their bound moieties (e.g., atoms within the 20 atoms, e.g., atoms within the 15 atoms, atoms within the 10 atoms, atoms within the 5 atoms) involved in binding to the presenter protein so as to inhibit the peptidyl-prolyl isomerase activity of the presenter protein. In some embodiments, the presenter protein binding moiety does not encompass 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 moiety do not interact with the presenter protein.

[0199] In some embodiments, the presenter protein binding moiety comprises an N-acylproline moiety, an N-acyl-pipecolic acid moiety, an N-acyl3-morpholinocarboxylic acid moiety, and / or an N-acylpiperazine acid moiety (e.g., either nitrogen atom is acylated). In certain embodiments, the presenter protein binding moiety comprises an N-acyl-pipecolic 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of Tyr27, Phe37, Asp38, Arg41, Phe47, Gln54, Glu55, Val56, Ile57, Trp60, Ala82, Try83, His88, Ile92, and / or Phe100 of FKBP12. In some embodiments, at least one atom of the presenter protein binding moiety is involved in binding to at least one (e.g., 2, 3, or 4) of Arg41, Gln54, Glu55, and / or Ala82 of FKBP12.

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

[0202] [ka]

[0203] It has the structure shown below. In some embodiments, the presenter protein binding moiety has the structure

[0204] [ka]

[0205] JPEG0007812813000024.jpg209170

[0206] JPEG0007812813000025.jpg230170

[0207] JPEG0007812813000026.jpg138170

[0208] or comprising or consisting of a stereoisomer thereof. The presenter protein can bind to an atom in the presenter protein binding moiety. Alternatively or additionally, the presenter protein can bind to two or more atoms in the presenter protein binding moiety. In another alternative, the presenter protein can bind to a substituent attached to one or more atoms in the presenter protein binding moiety. Furthermore, in some embodiments, the presenter protein can bind to an atom in the presenter protein binding moiety and to a substituent attached to one or more atoms in the presenter protein binding moiety. In some embodiments, the presenter protein is bound to a group that mimics the presenter protein's natural ligand, wherein the group that mimics the presenter protein's natural ligand is attached to the presenter protein binding moiety. 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 relative to the affinity of the presenter protein for the presenter protein in the absence of the complex. Binding in such instances is typically, but not limited to, through non-covalent interactions of the presenter protein to the presenter protein binding moiety.

[0209] Target protein binding moiety In some embodiments, compounds of the invention comprise 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 comprises a group of atoms (e.g., 5-20 atoms, 5-10 atoms, 10-20 atoms) and can include any moiety attached thereto that specifically binds to the target protein (e.g., within 20 atoms, within 15 atoms, within 10 atoms, within 5 atoms). In some embodiments, the target protein binding moiety comprises 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 moiety. Alternatively or additionally, the target protein can bind to two or more atoms in the target protein binding moiety. Alternatively, the target protein can bind to a substituent bound to one or more atoms in the target protein binding moiety. Alternatively, the target protein can bind to an atom in the target protein binding moiety and a substituent bound to one or more atoms in the target protein binding moiety. Alternatively, the target protein is bound to a group that mimics the target protein's natural ligand, and the group that mimics the target protein's natural ligand is bound to the target protein binding moiety. Alternatively, the target protein is bound to a presenter protein, and the affinity of the target protein for the presenter protein in the binary complex is increased compared to the affinity of the target protein for the presenter protein in the absence of the complex. Binding in these examples is typically, but not exclusively, via non-covalent interactions of the target protein to the target protein binding moiety. Linker The compounds of the invention comprise a linker (e.g., a partial linker) that joins a protein-binding moiety (e.g., a presenter protein-binding moiety or a target protein-binding moiety) to a crosslinking group, or a linker that joins a protein-binding moiety to a protein (e.g., a presenter protein or a target protein). The linker component of the invention is, in its simplest form, a bond, but can also provide a linear, cyclic, or branched molecular backbone with pendant groups covalently linking the two moieties.

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

[0212] Thus, when included in compounds and / or conjugates as described herein, the linker achieves linkage of two (or more) moieties by covalent means involving bond formation with one or more functional groups located on either moiety. 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, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.

[0213] In some embodiments, covalent attachment of two or more moieties may be achieved using a linker that contains a reactive moiety capable of reacting with such functional groups present in both moieties, for example, an amine group on a moiety may react with a carboxyl group or an activated derivative thereof on a linker, thus forming an amide linking the two.

[0214] Examples of moieties capable of reacting with sulfhydryl groups include α-haloacetyl compounds of the XCH2CO- type (where X = Br, Cl, or I). These compounds not only exhibit specific reactivity toward sulfhydryl groups, as described by Gurd, Methods Enzymol. 11, 532 (1967), but can also be used to modify imidazolyl, thioether, phenolic, and amino groups. N-Maleimide derivatives are also considered selective for sulfhydryl groups, but may also be useful for coupling with amino groups under certain conditions. Reagents such as 2-iminothiolane, which introduces a thiol group by conversion of an amino group (Traut et al., Biochemistry 12, 3266 (1973)), can be considered sulfhydryl reagents when coupling is achieved by disulfide bridge formation.

[0215] Examples of reactive moieties capable of reacting with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include: (i) α-haloacetyl compounds, which are specific for amino groups in the absence of reactive thiol groups and are of the XCHCO- type (where X = Br, Cl, or I), as described, for example, in Wong, Biochemistry, 24, 5337, 1979; (ii) N-maleimide derivatives, which can react with amino groups via a Michael-type reaction or via acylation by addition to a ring carbonyl group, as described, for example, in Smyth et al., J. Am. Chem. Soc., 82, 4600 (1960) and Biochem. J., 91, 589 (1964); (iii) aryl halides, e.g., reactive nitrohaloaromatic compounds; (iv) alkyl halides, for example, McKenzie et al., J. Protein Chem., Vol. 7, p. 581, 1988; (v) aldehydes and ketones capable of forming Schiff bases with amino groups, the adducts formed usually affording stable amines upon reduction; (vi) epoxide derivatives, such as epichlorohydrin and bisoxiranes, which can react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) chlorine-containing derivatives of s-triazines, which are highly reactive towards nucleophiles such as amino, sulfhydryl and hydroxyl groups; (viii) Aziridines based on the s-triazine compounds detailed above, such as those described by Ross in J. Adv. Cancer Res., Vol. 2, p. 1, 1954, which react with nucleophiles such as amino groups by ring-opening. (ix) squaric acid diethyl ester, as described in Tietze, Chem. Ber., 124, 1215, 1991, and (x) α-haloalkyl ethers, which are more reactive alkylating agents than conventional alkyl halides due to the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem., Vol. 28, p. 463, 1993; Examples include:

[0216] Representative amino-reactive acylating agents include: (i) isocyanates and isothiocyanates, especially aromatic derivatives, which form stable urea and thiourea derivatives, respectively; (ii) sulfonyl chlorides, as described by Herzig et al., Biopolymers, Vol. 2, p. 349, 1964; (iii) acid halides, (iv) active esters, such as nitrophenyl esters or N-hydroxysuccinimidyl esters; (v) acid anhydrides, such as mixed, symmetric, or N-carboxy anhydrides; (vi) Other useful reagents for amide bond formation, e.g., M. Bodansky, Principles of Peptide Synthesis Synthesis, Springer-Verlag, 1984, (vii) acyl azides, the azide group of which is generated from a preformed hydrazide derivative using sodium nitrite as described by Wetz et al., Anal. Biochem., 58, 347, 1974; (viii) imidoesters, which react with amino groups to form stable amidines, as described, for example, by Hunter and Ludwig, J. Am. Chem. Soc., 84, 3491 (1962), and (ix) a haloheteroaryl group, such as a halopyridine or halopyrimidine; Examples include:

[0217] Aldehydes and ketones can react with amines to form Schiff bases, which can be advantageously stabilized by reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to generate 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, which react with high specificity to produce ester groups, as described, for example, in Herriot, Adv. Protein Chem., 3, 169, 1947. Carboxyl-modifying reagents such as carbodiimides are also available, which react via O-acylurea formation and subsequent amide bond formation.

[0219] It will be appreciated that, if desired, functional groups on either moiety may be converted to other functional groups prior to reaction, for example to impart additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic acid anhydrides, conversion of amines to thiols using reagents such as N-acetylhomocysteine ​​thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, and thiol-containing succinimidyl derivatives, conversion of thiols to carboxyls using reagents such as α-haloacetates, conversion of thiols to amines using reagents such as ethyleneimine and 2-bromoethylamine, conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines, and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to thiols using sodium acetate.

[0220] If desired, so-called zero-length linkers may be used in accordance with the present invention, which involve a direct covalent bond between a reactive chemical group on one moiety and a reactive chemical group on another moiety without the introduction of additional linking material.

[0221] More commonly, however, a linker comprises two or more reactive moieties connected by a spacer element, as described above. The presence of such a spacer allows the bifunctional linker to react with a specific functional group in either moiety to form a covalent bond between the two. The reactive moieties in the linker can be the same (homo-bifunctional linker) or different (heterobifunctional linker, or, if several dissimilar reactive moieties are present, a multifunctional linker), providing a variety of possible reagents that can form a covalent bond between two moieties.

[0222] The spacer element in the linker typically consists of a straight or branched chain, 1~10 Alkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, C 2~ 6Heterocyclyl, C 6~12 Aryl, C 7~14 Alkaril, C 3~10 Alkheterocyclyl, C2-C 100 Polyethylene glycol, or C 1~10 It may include heteroalkyl.

[0223] In some cases, the linker is described by Formula V: Examples of homobifunctional linkers useful in 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 polycaprolactone diol.

[0224] In some embodiments, a linker is a bond or a linear chain of up to 10 atoms independently selected from carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, each atom in the chain 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, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and any two atoms in the chain together with the substituents attached thereto can form a ring, which can be further substituted and / or fused to one or more optionally substituted carbocyclic, heterocyclic, aryl, or heteroaryl rings.

[0225] In some embodiments, the linker has 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 is the bond between the linker and the presenter protein binding moiety, and A 2 is the bond between the mammalian target interacting moiety and the linker, and B 1 , B 2 , B 3 , and B 4 are each independently an optionally substituted C1-C2 alkyl, an optionally substituted C1-C3 heteroalkyl, O, S, and NR N Selected from R N is hydrogen, optionally substituted C1~4 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, C 1 and C 2 are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; a, b, c, d, e, and f 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 C2-6 heterocyclyl, optionally substituted C 6~12 Aryl, optionally substituted C-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 the structure of (a chemical bond that connects and).

[0226] protein Presenter Protein A presenter protein can bind to a small molecule to form a complex, and this complex can bind to and modulate the activity of 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, 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 association with a tripartite complex does not substantially adversely affect the biological role of the presenter protein in the cell and / or the viability or other attributes of the cell). In some embodiments, the presenter protein is a more abundant target protein. In certain embodiments, the presenter protein is a protein that has chaperone activity within the cell. In some embodiments, the presenter protein has multiple natural interaction partners within the cell. In certain embodiments, the presenter protein is known to bind to a small molecule to form a binary complex that is known or predicted to bind to a target protein and modulate its biological activity. Immunophilins are a class of presenter proteins known to have this function, including FKBPs and cyclophilins. In some embodiments, the reference presenter protein exhibits peptidylprolyl isomerase activity. In some embodiments, the presenter protein exhibits activity comparable to that of 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 cyclophilin 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 with prolyl isomerase activity that function as protein folding chaperones for proteins containing proline residues. Genes encoding proteins of this family include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and LOC541473.

[0227] The "cyclophilin family" is a family of proteins that bind to cyclosporine. Genes encoding proteins of this family include PPIA, PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1, PPIL2, PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary cyclophilins 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 allelic variant or splice variant of an FKBP or cyclophilin disclosed herein. In some embodiments, a presenter protein is a polypeptide whose amino acid sequence i) shows significant identity to that of a reference presenter protein, ii) contains a portion that shows significant identity to a corresponding portion of the reference presenter protein, and / or iii) contains at least one characteristic sequence found in a presenter protein. In many embodiments, identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more is considered "significant" for the purposes of defining a presenter protein. In some embodiments, the portion showing significant identity is 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24 and having 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 listed in Table 1 or their homologs. In some embodiments, the reference presenter protein is encoded by the set of genes shown in Table 1. Moreover, those skilled in the art can readily refer to Table 1 to identify sequences characteristic of presenter proteins in general and / or specific subsets of presenter proteins.

[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 symptoms of disease pathology. Therefore, by modulating (inhibiting or increasing) their activity, a desired therapeutic effect can be achieved. Target proteins useful in the complexes and methods of the invention include those that are not naturally associated with a presenter protein, e.g., those that have an affinity for the presenter protein of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM, in the absence of a binary complex with a compound of the invention. Alternatively, target proteins that are not naturally associated with a presenter protein are those that have an affinity for the compound of the invention of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM, in the absence of a binary complex. Alternatively, the target protein not naturally associated with the presenter protein has an affinity of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM for a binary complex of the presenter protein with cyclosporine, rapamycin, or FK506 (e.g., FKBP). Alternatively, the target protein not naturally associated with the presenter protein is other than calcineurin or mTOR. The selection of a suitable target protein for the complexes and methods of the present invention may depend on the presenter protein. For example, a target protein with low affinity for cyclophilin may have high affinity for FKBP, in which case it would not be used with the latter.

[0233] A target protein can be naturally occurring, e.g., wild-type. Alternatively, a target protein can differ from the wild-type protein but still maintain a biological function, e.g., an allelic variant, a splice mutant, or a 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 of a dimeric complex.

[0235] In some embodiments, target proteins of the invention comprise one or more surface sites (e.g., flat surface sites) that are characterized by low or undetectable binding of small molecules to the site in the absence of presenter protein / compound complex formation. In some embodiments, target proteins comprise 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., at most 2 / 3, 4 / 5, 6 / 7, 8 / 9, 10 / 10, 20 / 30, 40 / 50, 100 / 100, or less binding of 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 that lack any conventional binding pocket, e.g., lacks protein structural cavities or pockets with physiochemical and / or geometric properties comparable to proteins whose activity is modulated by one or more small molecules. 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 undruggable target, e.g., the target protein is not a member of a protein family known to be a drug target and / or does not have a binding site expected to be suitable for binding to a small molecule (e.g., according to art-accepted understanding 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, RHOT1In 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, e.g., CNRASGEF, RASGEFlA, RASGRF2, RASGRPl, RASGRP4, SOSl, RALGDS, RGLl, RGL2, RGR, ARHGEFlO, ASEF / ARHGEF4, ASEF2, DBS, ECT2, GEF-Hl, LARG, NETl, OBSCURIN, P-REXl, P-REX2, PDZ-RHOGEF, TEM4, TIAMl, TRIO, VAVl, VAV2, VAV3, DOCKl, DOCK2, DOCK3, DOCK4, DOCK8, DOCKlO, 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 In some embodiments, the target protein is a heat shock protein, e.g., Hsp20, Hsp27, Hsp70, Hsp84, Hsp85, Hsp86, Hsp87, Hsp88, Hsp89, Hsp80, Hsp81, Hsp82, Hsp83, Hsp84, Hsp85, Hsp86, Hsp87, Hsp88, Hsp89 ...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, syntaxin1a, FYCO1, or CEP250. In certain embodiments, the target protein is a kinase, e.g., 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 , 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, e.g., WIP1, SHP2, SHP1, PRL-3, PTP1B, or STEP. In certain embodiments, the target protein is a ubiquitin or ubiquitin-like protein (e.g., NEDD8, ATG8 protein, SUMO protein, ISG15), an activating enzyme (E1, e.g., UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, SAE1), a conjugation enzyme (E2, e.g., UBE protein, ATG3, BIRC6), a ligation enzyme (E3, e.g., BMI-1,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, e.g., 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, RARB, RARG, RORA, RXRA, RXRB, RXRG, THRA, THRB, VDR, GATA3, GATA4, or GATA5, or a transcription factor encoded by 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 protein is 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, RALGAPB , 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 variants Protein or polypeptide variants as described herein generally exhibit significant (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) identity 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 that contains a limited number of specific amino acid changes (e.g., conservative or non-conservative insertions, deletions, or substitutions) and / or contains one or more amino acid variants or analogs (e.g., D-amino acids, desamino acids) relative to the reference polypeptide. In certain embodiments, the variant shares a relevant biological activity (e.g., binding to a specific compound or portion thereof) with the reference polypeptide. In some such embodiments, the variant exhibits such activity at a level that is about 50% or more of the activity of the reference polypeptide and / or that is about 0.5-fold or less of the activity of the reference polypeptide.

[0238] In some embodiments, a variant polypeptide has an amino acid sequence that differs from that of a reference polypeptide at least (or only) in that the variant has multiple 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 carboxy terminus of any of the polypeptides (e.g., presenter proteins and / or target proteins) as described herein can facilitate conjugation of such polypeptides, for example, by disulfide bonds.

[0239] In some embodiments, amino acid substitutions can be conservative (i.e., where the residue is replaced with another of the same general type or group) or non-conservative (i.e., where the residue is replaced with an amino acid of another type). In some embodiments, a naturally occurring amino acid may be substituted for a non-naturally occurring amino acid (i.e., a non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution), or vice versa.

[0240] Synthetically produced polypeptides can include substitutions of amino acids that are not naturally encoded by DNA (e.g., non-naturally occurring 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, amino acids with the chemical formula NH(CH). n Omega-amino acids with COOH (where n is 2-6) include neutral nonpolar amino acids such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine. Phenylglycine can substitute for Trp, Tyr, or Phe. Citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline can be substituted with hydroxyproline to retain conformational properties.

[0241] Analogs can be generated by substitutional mutagenesis and retain the structure (e.g., local or global structure) of the original protein. Examples of substitutions identified as "conservative substitutions" are shown in Table 2. If such substitutions result in undesirable changes, other types of substitutions, designated "exemplary substitutions" in Table 2 or as further described herein in relation to amino acid classes, are introduced and the products screened.

[0242] Substantial modifications in function or immunological identity are achieved by selecting substitutions that differ significantly in (a) the structure of the protein backbone in the region of the substitution, e.g., as a sheet or helical conformation, (b) the molecular alteration or hydrophobicity at the target site, or (c) their effect on maintaining the bulk of the side chain. Naturally occurring residues are divided into groups based on common 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 influence 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, protein or polypeptide variants may include the addition of one or more reactive amino acid residues (e.g., cysteines) to the protein (e.g., at the amino or carboxy terminus of any of the proteins described herein), which can facilitate conjugation of these proteins, for example, via disulfide bonds. In some embodiments, one or more reactive amino acids (e.g., cysteines) may be removed to reduce the number of potential conjugation sites on the protein. Amino acid substitutions may be conservative (i.e., where the residue is replaced with another of the same general type or group) or non-conservative (i.e., where the residue is replaced with an amino acid of another type). Additionally, natural amino acids can be substituted for non-natural amino acids (i.e., non-naturally occurring conservative amino acid substitutions or non-naturally occurring non-conservative amino acid substitutions).

[0245] As is known in the art, see, for example, Chin, JW, Expanding and Reprogramming the Genetic Code of Cells and Animals. Reprogramming the Genetic Code of Cells As described in "Analysis of Proteins and Animals," Annual Review of Biochemistry, Vol. 83, pp. 379-408, unnatural amino acids can be incorporated into proteins made in vitro. For example, in one system, the UAG amber (stop) codon has been used to incorporate pyrrolysine 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 unnatural amino acids that have been described in the art include cyclopropene, trans-cyclooctene, bicyclo[6.1.0]nonyne-lysine, coumarin, p-azidophenylalanine, N6-[(2-propynloxy)carbonyl]-L-lysine, bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN), N-5-norbornen-2-yloxycarbonyl-L-lysine, N-tert-butyl oxycarbonyl-l-lysine, N-2-azidoethyloxycarbonyl-l-lysine, N-L-thiaprolyl-L-lysine, N-D-cysteinyl-L-lysine, N-L-cysteinyl-L-lysine, N-[(2-propynyloxy)carbonyl]-L-lysine, N-[(2-azidoethoxy)carbonyl]-L-lysine, benzophenone, 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine, and cyclooctyne.

[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 driven largely by hydrophobic residues on the flat surface regions of the interacting proteins. Generally, hydrophobic residues on the flat surface regions of proteins form hydrophobic hot spots, where the majority of binding interactions between or within interacting proteins are van der Waals interactions. In some situations, a small molecule may provide a "mobile hot spot" (or a portion thereof), for example, by engaging or generating a hydrophobic interaction site on a protein (e.g., a presenter protein), which is not present in the absence of the small molecule. Aspects of the present disclosure are particularly applicable to such situations. For example, in some embodiments, a compound as described herein (and / or a tagged form thereof) forms a complex with a protein (e.g., a presenter protein / compound complex) and participates in a pseudoprotein-protein interaction (e.g., forming a tripartite complex with a target protein).

[0247] Many mammalian proteins can bind to any of several different partners, and in some cases, these alternative binding interactions contribute to the protein's biological activity. Many of these proteins display identical residues in different structural contexts, adapting to the inherent variability of hotspot protein regions. More specifically, protein-protein interactions can be mediated by a class of natural products produced by a select group of fungal and bacterial species. These molecules exhibit a common structural organization and consequently function, providing the ability to modulate protein-protein interactions. These molecules contain a highly conserved presenter protein-binding portion and a target protein-interacting portion that exhibits a high degree of variability among different natural products. The presenter protein-binding portion confers specificity for the presenter protein, allowing the molecule to bind to the presenter protein and form a complex. The mammalian target protein-binding portion confers specificity for the target protein, allowing the binary complex to bind to the target protein and typically modulate its activity (e.g., positively or negatively). In the present invention, a binary complex (e.g., between a compound and a presenter protein, or between a compound and a target protein) is mimicked by conjugating a presenter protein-binding moiety to the target protein, or a target protein-binding moiety to the presenter protein. The resulting conjugate of the present invention can then bind to the presenter protein or the 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 a presenter protein-binding moiety to the target protein, the compound of the present invention can be used, for example, to identify target proteins that can bind to the presenter protein.

[0248] use Target protein identification 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 a complex with a presenter protein (e.g., in the presence of a small molecule). A target protein may be identified by forming a conjugate comprising a presenter protein-binding moiety conjugated to a targeting moiety, and determining whether the conjugate forms a complex with the presenter protein.

[0249] Most target proteins known in the art to form ternary complexes with presenter proteins and small molecules were fortunately identified during the determination of the mechanism of action of small molecules. The present method allows for the rational identification of target proteins that can form complexes with presenter proteins in the presence of small molecules by covalently conjugating a presenter protein-binding moiety to the target molecule and allowing the complex to form prior to 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 the presenter protein and the identified target protein can then be carried out to identify potential therapeutics that can modulate the biological activity of the target protein.

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

[0252] Compound Design In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful for the design of compounds capable of modulating the biological activity of a target protein for use in the treatment of disease.

[0253] For example, the formation of a complex between the presenter protein and the 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 of the complex and crystal structure determination. Once the crystal structure of the complex of the present invention has been determined, small molecules capable of promoting 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 de novo construction of structures, e.g., fragment soaking of crystals of the complex of the present invention, and fragment-based drug design using the structure thus obtained.

[0254] Compounds designed as described above may then be screened to determine their ability to modulate the biological activity of the target protein and may be modified, if necessary, using medicinal chemistry techniques to generate therapeutically useful compounds.

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

[0256] For example, compounds of the present invention may 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 the target protein only in the presence of the presenter protein. These compounds may then be tested for their ability to modulate the biological activity of the target protein and modified, if necessary, using medicinal chemistry techniques to generate therapeutically useful compounds.

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

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

[0259] Treatment of a disease or disorder The compounds, conjugates, and complexes described herein may be useful in methods of treating diseases or disorders associated with the target proteins described herein, and, without being bound by theory, are believed to exert their desired effect through interaction with the presenter protein and the target protein by virtue of their ability to modulate (e.g., positively or negatively modulate) the activity of the target protein (e.g., a eukaryotic target protein, e.g., a mammalian target protein or a fungal target protein or a prokaryotic target protein, e.g., a bacterial target protein).

[0260] kit In some embodiments, the present invention relates to kits for conveniently and effectively practicing the methods of the present invention. Typically, pharmaceutical packs or kits include one or more containers filled with one or more components of the pharmaceutical compositions of the present invention. Such kits are particularly suitable for the delivery of solid oral formulations, such as tablets or capsules. Such kits may preferably include a card containing several unit doses and indicating the dosages in the order of their intended use. Optionally, for example, if the subject is suffering from Alzheimer's disease, a memory aid can be provided, e.g., in the form of numbers, letters, or other designations, or by designating a treatment schedule with days on a calendar by which administration may occur. Alternatively, a kit can be provided that includes placebo doses (or calcium dietary supplements) similar to or different from the dosages of the pharmaceutical composition, so that a dosage is taken daily. Optionally, associated with such containers can be a notice in the form specified by a government agency regulating the manufacture, use, or sale of pharmaceuticals. The notice reflects the regulatory agency's approval for manufacture, use, or sale for human administration. Pharmaceutical Composition For use in treating human and animal subjects, the compounds and conjugates of the present invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject being treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or therapy), the compounds are formulated in a manner consistent with these parameters. Overviews of such techniques can be found in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, 2005; and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York (each of which is 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 compositions may be provided in a dosage form suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, ​​intrathecal, epidural, otic, or ocular administration, or for injection, inhalation, or direct contact with nasal, urogenital, genital, or oral mucosa. Thus, pharmaceutical compositions 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, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

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

[0263] The compounds of the present invention may be prepared and used as pharmaceutical compositions comprising an effective amount of a 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] The formulation may be prepared in a manner suitable for systemic or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, subcutaneous injection), or may be prepared for transdermal, transmucosal, or oral administration. The formulation generally includes a diluent, and in some cases, an adjuvant, a buffer, a preservative, etc. The compound may also be administered as a liposome composition or microemulsion.

[0265] For injection, the preparations can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid preparations suitable for dissolving or suspending in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol, etc. Such compositions may also contain various amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, for example, sodium acetate, sorbitan monolaurate, and the like.

[0266] Various sustained release systems for drugs have also been devised, see, for example, U.S. Patent No. 5,624,677 (incorporated herein by reference). Systemic administration can also include relatively non-invasive methods, such as using 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 of the combination therapy described herein may be formulated in a variety of ways known in the art, for example, the first and second agents of the combination therapy may be formulated together or separately.

[0268] Individually or separately formulated agents can be packaged together as a kit. Examples include, but are not limited to, a kit containing two pills, a pill plus a powder, a suppository plus a liquid in a vial, two topical creams, etc. The kit may include optional components to aid in administering the unit dose to a subject, such as a vial for reconstituting a powder form, a syringe for injection, a customized IV delivery system, an inhaler, etc. Additionally, a unit dose kit may contain instructions for preparing and administering the composition. The kit may be manufactured as a single-use unit dose for one subject, as multiple uses for a particular subject (either at a fixed dose or with varying potencies of individual compounds over the course of treatment), or the kit may contain multiple doses suitable for administration to multiple subjects ("bulk packaging"). The kit components may be assembled into cartons, blister packs, bottles, tubes, etc.

[0269] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such excipients can be, 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, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), and lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

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

[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 oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets may be prepared using the above-listed ingredients in tablets and capsules in a conventional manner, for example, in a mixer, fluid bed apparatus, or spray drying apparatus.

[0272] Dissolution or diffusion controlled release can be achieved by applying a suitable coating to tablets, capsules, pellets, or granules of the compound or by incorporating the compound in a suitable matrix, such as 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, ethyl cellulose, 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. Additionally, 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 preparations into which the compounds and compositions of the present invention may be incorporated for oral administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions, including edible oils such as Mentha oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar pharmaceutical vehicles.

[0274] Generally, when administered to humans, the oral dosage of any of the compounds of the combination of the present invention will depend on the properties of the compound, but can be readily determined by one skilled in the art. Typically, such dosages will usually be about 0.001 mg to 2000 mg per day, preferably about 1 mg to 1000 mg per day, and more preferably about 5 mg to 500 mg per day. Doses of up to 200 mg per day may be necessary.

[0275] Administration of each agent of the combination therapy described herein can independently be four times daily for one day to one year, or even for the life of the subject. Chronic long-term administration may be required. [Example]

[0276] Example 1: Synthesis of specific cross-linking reagents Synthesis of (R)-3-(3,4-dimethoxyphenyl)-1-(3-(3-(pyridin-2-yldisulfanyl)propanamido)phenyl)propyl (S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate (C3-SLF):

[0277] [ka]

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

[0279] Synthesis of (R)-3-(3,4-dimethoxyphenyl)-1-(3-(4-(pyridin-2-yldisulfanyl)butanamido)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 equiv.), disulfide 2 (79 mg, 343 μmol, 2 equiv.), and diisopropylethylamine (149 μL, 111 mg, 858 μmol, 5 equiv.) in DMF (3 mL) was added HATU (130 mg, 343 μmol, 2 equiv.), and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with water and extracted with ethyl acetate (3×). The organic extract was washed with water, saturated sodium chloride, dried over magnesium sulfate, and evaporated. The residue was purified on silica gel gradient elution (20% ethyl acetate:80% heptane → 100% ethyl acetate) to give the title compound C4-SLF (98 mg, 77%). MS (ESI) calculated = 736.3 (M+H), observed = 736.3.

[0282] Synthesis of methyl (S)-1-((S)-3-(3-hydroxyphenyl)-2-((S)-3-methyl-2-(4-(pyridin-2-yldisulfanyl)butanamido)butanamido)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) was added triethylamine (16.5 μL, 118 μmol, 2.4 equiv.), followed by acid chloride 2 (13.8 mg, 59.2 μmol, 1.2 equiv.). The reaction mixture was stirred at room temperature for 14 h, then concentrated, and the residue was purified by preparative TLC (dichloromethane:MeOH:NH4OH, 20:1:0.1) to give 14.0 mg (47%) of the product as a colorless foam. f = 0.59 (dichloromethane:MeOH:NH4OH, 10:1:0.1). MS(ESI) calculated = 618.2 (M+H), observed = 618.2.

[0285] Synthesis of methyl (S)-1-((S)-3-(3-hydroxyphenyl)-2-((S)-3-methyl-2-(3-(2-(pyridin-2-yldisulfanyl)ethoxy)propanamido)butanamido)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 equiv.) were mixed in 3 mL of acetonitrile, and the resulting suspension was stirred at room temperature for 15 min. Following this period, amine 1 (110 mg, 0.270 mmol, 1.00 equiv.) was added, followed by triethylamine (113 μL, 0.810 mmol, 3.00 equiv.), and the mixture was stirred at room temperature for 18 h. The mixture was then treated with 20 mL of saturated sodium bicarbonate and extracted with 2 × 30 mL portions of ethyl acetate. The pooled organic extracts were washed with 2 × 20 mL portions of brine, dried over saturated sodium sulfate, filtered, and concentrated under vacuum. The residue was purified using silica gel chromatography eluting with dichloromethane:MeOH, 100:1 to 50:1, to afford 70 mg (40%) of the product as a colorless oil. f = 0.31 (dichloromethane:MeOH, 20:1). MS(ESI) calculated = 648.2 (M+H), observed = 648.2.

[0288] Synthesis of N-(4-((2S,11R,14S,17S,20S,23S,26S)-26-ethyl-23-((1R,2R,E)-1-hydroxy-2-methylhex-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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)butyl)-4-(pyridin-2-yldisulfanyl)butanamide (CsA3))

[0289] [ka]

[0290] To a solution of amine 1 (100 mg, 90.5 μmol) and carboxylic acid 2 (31 mg, 135.2 μmol, 1.5 equiv.) in NMP (6 mL) was added HATU (51 mg, 134.1 μmol, 1.48 equiv.) and DIPEA (70 μL, 401.9 μmol, 4.4 equiv.). The reaction was stirred at room temperature for 1 h, then diluted with water and extracted with three 30 mL portions of ethyl acetate. The organic extract was washed with saturated sodium chloride solution and concentrated under vacuum. The crude material was purified by reverse-phase chromatography on C18 media, 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 = 658.9 (M + 2H), observed = 659.0.

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

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

[0293] B.KRAS GTP / G12C Formation of lite / SFAX9DS conjugate Reagents: SFAX9DS (in-house) in 100% DMSO, KRAS GTP / G12C lite (in-house production; residues 1–169 containing G12C / C51S / C80L / C118S). Apparatus: Mini-PROTEAN TGX gel (Bio-Rad) Experimental protocol: 1:2 molar ratio of KRAS 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 was incubated at 37°C for 30 minutes, followed by overnight incubation at room temperature. Crosslinking efficiency was assessed by SDS-PAGE gel. Wild-type CypA was also crosslinked with the compounds. Cysteine ​​52, the reactive cysteine ​​on CypA, was mutated to serine to abrogate presenter crosslinking. Results: SFAX9DS is a KRAS GTP / G12CEfficiently crosslinks with lite proteins and CypA C52S does not cross-link to SFAX9DS (Fig. 2).

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

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

[0296] Experimental Protocol B: Crosslinking reagents, presenter proteins and target proteins A 1:2:2 molar ratio of compound, presenter protein, and target protein is mixed together in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction is incubated at 37°C for 30 minutes, followed by overnight incubation at room temperature. The pure complex is isolated by size-exclusion chromatography (SEC). The reaction mixture is injected directly onto a Superdex 75 column (120 mL CV) pre-equilibrated 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 protein and presenter protein. Samples are 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 (in-house) in 100% DMSO, KRAS GTP / S39C lite (made in-house; residues 1–169 containing G12V / S39C / C51S / C80L / C118S), and FKBP12 (made in-house). Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare), CV120mL) Experimental protocol: 1:2:2 molar ratio of C2-Holt, FKBP12, and KRAS GTP / S39CThe complexes are mixed together in a 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction is incubated at 37°C for 30 minutes, followed by overnight incubation at room temperature. The pure complex is isolated by size-exclusion chromatography (SEC) purification. The reaction mixture is injected directly onto a Superdex 75 column (CV 120 mL) pre-equilibrated with a buffer containing 12.5 mM HEPES, pH 7.4, 75 mM NaCl. The complex elutes at approximately 69 mL after injection, revealing the presence of unreacted KRAS. GTP / S39C KRAS lite and FKBP12 eluted at approximately 75 mL and 87 mL, respectively, after injection. GTP / S39C Samples are also assessed by SDS-PAGE to confirm the presence of lite and FKBP12. Results: SEC profile and SDS-PAGE analysis of the elution peaks showed that 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 Ternary complex formation Reagents: SFAC4DS (in-house) in 100% DMSO, KRAS GDP / S39C lite (in-house; residues 1–169 containing G12V / S39C / C51S / C80L / C118S), and CypA C52S (Made in-house). Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare), CV120mL) Experimental protocol: SFAC4DS, CypA in a 1:2:2 molar ratio C52S , and KRAS GDP / S39CThe complexes are mixed together in a 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The reaction is incubated at 37°C for 30 minutes, followed by overnight incubation at room temperature. The pure complex is isolated by size-exclusion chromatography (SEC) purification. The reaction mixture is injected directly onto a Superdex 75 column (CV 120 mL) pre-equilibrated with a buffer containing 12.5 mM HEPES, pH 7.4, 75 mM NaCl. The complex elutes at approximately 69 mL after injection, revealing the presence of unreacted KRAS. GDP / S39C lite and CypA C52S The KRAS peaks eluted at approximately 75 mL and 80 mL, respectively. GDP / S39C lite and CypA C52S Samples are also assessed by SDS-PAGE to confirm the presence of . Results: SEC profile and SDS-PAGE analysis of the elution peaks revealed KRAS 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 (in-house production), PTP1B in 100% DMSO E186C lite (in-house; residues 1–293 containing C32S / C92V / C121S / S187C), and FKBP12 (in-house). Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex 75 (GE Healthcare) Healthcare), CV120mL) Experimental protocol: 1:3:3 molar ratio of C3-SLF, FKBP12, and PTP1B S187CThe PTP1B complexes are mixed together in a 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 4% DMSO. The reaction is incubated at 37°C for 30 minutes, followed by overnight incubation at room temperature. The pure complex is isolated by size-exclusion chromatography (SEC) purification. The reaction mixture is injected directly onto a Superdex 75 column (CV 120 mL) pre-equilibrated with a buffer containing 12.5 mM HEPES, pH 7.4, 75 mM NaCl. The complex elutes at approximately 62 mL after injection, while unreacted FKBP12 elutes at approximately 75 mL (dimer) and 90 mL (monomer), respectively. PTP1B in the elution peak S187C Samples are also assessed by SDS-PAGE to confirm the presence of free PTP1B and FKBP12. S187C The lite and FKBP12 mixtures are subjected to a Superdex 75 column under the same conditions and their elution times are determined. Results: Free PTP1B eluted at approximately 64-65 ml S187C Check for free PTP1B S187C SEC profiles and SDS-PAGE analysis of the elution peaks of PTP1B and FKBP12 proteins (Figure 5A). S187C The formation of a lite / C3-SLF / FKBP12 complex was confirmed, which eluted at approximately 61 ml (FIG. 5B).

[0300] Example 4: Conjugation in the presence of a presenter protein, but not in its absence This protocol describes how to analyze crosslinking efficiency using mass spectrometry and gel shift assays in an attempt to assess the presenter dependence of conjugate formation. Reagents: Compounds (in-house), FKBP12 (in-house), KRAS in 100% DMSO GTP / G12C (in-house production, residues 1–169). Experimental Protocol: To follow the kinetics of the disulfide cross-linking reaction, an Agilent 6230 TOF-LC / MS and an Agilent 1260 HPLC instrument equipped with an autosampler were used, using an AdvanceBio RP-mAb C4 column (2.1 × 100 mm, 3.5 μm). HPLC-grade acetonitrile and water (containing 1.0 mM ammonium formate and 1% formic acid by volume, respectively) were used as the mobile phase with the following ramp: 0.6 ml / min flow rate, 95:5 water:acetonitrile, ramping from 0.0 to 13.0 min, to 5:95 water:acetonitrile, ramping from 13.0 to 17.0 min. Total time: 17.0 min.

[0301] All cross-linking 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 is not critical, the selection of amino acid residues was crucial to avoid interference in the cross-linking assay. Therefore, proline (interfering with FKBP12) and cysteine ​​(interfering with disulfide bond formation) residues were excluded. All reactions and standards were prepared in HEPES (pH 7.4, 1.0 mM MgCl) buffer.

[0302] Prior to every reaction, standard curves were generated for individual components using a series of standards (an example of a standard curve analysis for FKBP12 is shown in Table 3 below). Using data from the standard curves, μmol of protein sample was plotted against the area ratio (sample:std), and a linear fit (y = mx + c) was used to obtain the slope and intercept. The slope and intercept values ​​for these standard curves were interpreted during evaluation of substrate and product concentrations before and during the course of the reaction. 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 could be adjusted to include an appropriate number of blank injections to remove residual components, if any. MS spectra were analyzed using Agilent MassHunter vB.07.0 software.

[0303] [Table 3]

[0304] In a representative experiment assessing the presenter dependency of ligand crosslinking to a target protein, KRAS GTP / G12C The C3- or C4-SLF ligands were incubated at 2 μM KRAS, 10 μM FKBP12, and 10 μM C3- or C4-SLF in 12.5 mM HEPES, pH 7.4, 75 mM NaCl, 1 mM MgCl2, and 3% DMSO for 4 hours at room temperature, in the presence or absence of FKBP12. The amount of KRAS disulfide-crosslinked to the ligand was analyzed using the method described above. As shown in Table 4, a 5- to 10-fold increase in crosslinking efficiency was observed in the presence of presenters.

[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 in the presence or absence of FKBP12 under the same experimental conditions as above, except that the crosslinking reactions were set up at higher concentrations (60 μM KRAS, 180 μM FKBP12, and 180 μM C3- or C4-SLF). These were quenched with MMTS to terminate the reaction. Similar to the MS data, the ligand crosslinking efficiency was significantly boosted in the presence of FKBP12, although this was more pronounced for C4-SLF (Figure 6).

[0307] Example 5: Determination of presenter protein / target protein interface structure by X-ray analysis This protocol is for FKBP12-C2Holt-KRAS GTP / S39C The present invention describes the crystallization of the ternary complex of α- and β-glucan, and the structure determination method for the crystal structure of this ternary complex.

[0308] A. FKBP12-C2Holt-KRAS GTP / S39C Crystal structure determination of the ternary complex Reagents: Ligand (C2Holt) (in-house), FKBP12 (in-house), KRAS in 100% DMSO GTP / S39C lite (in-house production, residues 1–169 containing G12V / S39C / C51S / C80L / C118S). Device: Superdex 75 (GE Healthcare) Experimental protocol: C-Holt and FKBP12 were incubated with KRAS at 3:1 and 1.5:1 molar excess in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 1 mM MgCl, 2% DMSO. GTP / S39CThe mixture was added to lite and incubated overnight at 20°C or for 36–72 hours at 4°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 15–20 mg / ml) was subjected to crystallization screening at 20°C using the sitting-drop vapor diffusion method. Crystals were grown in a well solution containing 0.1 M MES, pH 6.5, and 20–22% PEG 20,000. For data collection, the crystals were transferred to a solution containing mother liquor supplemented with 15% glycerol and then frozen in liquid nitrogen. Diffraction datasets were collected at the Advanced Synchrotron Radiation Facility (ASR). The data were collected at the APS (Abstract Photon Source) and processed with the HKL program. Molecular replacement solutions were obtained using the program PHASER in the CCP4 suite, using the published structures of FKBP12 (PDB-ID1FKD) and KRAS (PDB-ID3GFT) as search models. Subsequent model building and refinement were performed by standard protocols with the software packages CCP4 and COOT.

[0309] Result:FKBP12-C2Holt-KRAS GTP / S39C Overall structure of the crystal: FKBP12 and KRAS in the asymmetric unit. GTP / S39C The model contains one heterodimer of FKBP12 from residues Met1 to Glu108 and KRAS (Figure 7). GTP / S39C The electron density thus obtained indicates a clear binding mode, including the orientation and conformation of the ligand. Continuous electron density was observed for the disulfide resulting from the cysteine ​​of the protein and the sulfur from the ligand.

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

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

[0312] B.KRAS GDP / S39C / SFAC4DS / CypA C52S Crystal structure determination of the ternary complex Reagents: Ligand (SFAC4DS) (in-house production), CypA in 100% DMSO C52S (In-house), KRAS GDP / S39C lite (in-house production, residues 1–169 containing G12V / S39C / C51S / C80L / C118S). Device: Superdex 75 (GE Healthcare) Experimental protocol: SFAC4DS and CypA C52S KRAS was diluted in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 1 mM MgCl, 2% DMSO at a 2:1 molar excess and 2:1 DMSO. GDP / S39CThe mixture was added to lite and 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 the sitting-drop vapor diffusion method. Crystals were grown in a well solution containing 0.1 M bis-Tris, pH 6.5, 25% PEG3350. For data collection, 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 data sets were collected at the Advanced Light Source (ALS) and processed with the HKL program. Molecular replacement solutions were obtained using the program PHASER in the CCP4 suite, using the published structures of CypA (PDB-ID1CWA) and KRAS (PDB-ID3GFT) as search models. Subsequent model building and refinement were performed by standard protocols with the software packages CCP4 and COOT.

[0313] Result: CypA C52S -SFAC4DS-KRAS GDP / S39C The overall structure of the crystal shows CypA in the asymmetric unit. C52S and KRAS GDP / S39C The model contains one heterodimer of CypA from Met1 to Glu165 and KRAS from Met1 to Glu165 (Figure 8). GDP / S39C The electron density thus obtained indicates a clear binding mode, including the orientation and conformation of the ligand. Continuous electron density was observed for the disulfide resulting from the cysteine ​​of the protein and the sulfur from the ligand.

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

[0315] The total buried area of ​​this complex cannot be calculated due to partial structural disorder at the protein-protein interface. Excluding the disordered regions for calculation, the buried area at the protein-protein interface is 1,350 Å. 2 Of these, more than 30% is SFAC4DS (443Å 2 ) contributes to the 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 contributed by both hydrophobic and polar interactions. The carbonyl and NH groups of the ligand and CypA C52S There are six H-bonds between SFAC4DS and residues Arg55, Gln63, Asn102, and His126 of KRAS. GDP / S39C Although it forms minimal direct contact with KRAS GDP / S39C It forms one H-bond with Arg41 of . 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-house), FKBP12 (in-house), PTP1B in 100% DMSO S187C lite (in-house production, residues 1–169 containing C32S / C92V / C121S / S187C). Devices: Superdex 75 (GE Healthcare), Gryphon (Art Robbins Instruments) Experimental protocol: C3SLF and FKBP12 were diluted with PTP1B in a 3:1 molar excess in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 4% DMSO. S187C The mixture was added to lite and incubated at 4°C for 36–72 h. 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 the sitting-drop vapor diffusion method. Crystals were grown in a well solution containing 0.2 M magnesium acetate and 20% w / v PEG3350. For data collection, 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 replacement solutions were obtained using the program PHASER in the CCP4 suite, using the published structures of FKBP12 (PDB-ID2PPN) and PTP1B (PDB-ID2NT7) as search models. Subsequent model building and refinement was performed by standard protocols involving the software packages CCP4 and COOT.

[0317] Result:FKBP12-C3SLF-PTP1B S187C Overall structure of the crystal: FKBP12-C3SLF-PTP1B in the asymmetric unit.S187C The model contains two complex molecules (Figure 9A). The model consists of residues Gly2 to Glu108 of FKBP12 and PTP1B. S187C The electron density thus obtained indicates a clear binding mode, including the orientation and conformation of the ligand. Continuous electron density was observed for the disulfide resulting from the cysteine ​​of the protein and the sulfur from the ligand.

[0318] The total buried area of ​​the complex is 1,042 Å 2 PTP1B S187C The buried area is 427Å 2 The buried area of ​​C3-SLF is 615 Å 2 (Figure 9B). S187C The protein-protein interface between FKBP12 and FKBP13 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-house), FKBP52 (in-house, residues 1–140), MCL1 in 100% DMSO S245C lite (in-house production, residues 172–327 containing S245C / C286S). Devices: Superdex 75 (GE Healthcare), Gryphon (Art Robbins Instruments) Experimental protocol: C3SLF and FKBP52 were incubated in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 2% DMSO at a 3:1 molar excess with MCL1. S245CThe mixture was mixed with lysate 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 the sitting-drop vapor diffusion method. Crystals were grown in a well solution containing 2.1 M malic acid. For data collection, the crystals were transferred to a solution containing mother liquor supplemented with 20% glycerol and then flash-frozen in liquid nitrogen. A 3.0 Å resolution diffraction dataset was measured at the Advanced Photon Source (APS) and processed with the XDS program. Molecular replacement solutions were obtained using the program PHASER in the CCP4 suite, using the published structures of FKBP52 (PDB-ID1N1A) and PTP1B (PDB-ID3MK8) as search models. Subsequent model building and refinement was performed by standard protocols involving the software packages CCP4 and COOT.

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

[0321] [Table 5]

[0322] Example 6: Determination of complex formation by TR-FRET TR-FRET technology (LANCE, PerkinElmer) 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 can be any of glutathione-S-transferase (GST), hexahistidine (His6), FLAG, biotin-avi, Myc, and hemagglutinin (HA)). In this example, this technology is used to measure compound-promoted association of a presenter protein with a target protein. The presenter protein / Tag A and target protein / Tag B mixtures are added to a 384-well assay plate containing compounds of the 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, emission = 665 / 615 nm. Compounds that promote ternary complex formation are identified as eliciting an increase in the TR-FRET ratio relative to DMSO control wells.

[0323] CYPA-compound 3-KRAS by TR-FRET G12C-GTP Determination of complex formation Avi-tagged cyclophilin A and His-tagged KRAS G12C-GTPThe presenter was mixed with increasing concentrations of the ligand (compound 3) and incubated for 15 minutes at room temperature to allow the formation of the ternary complex. A premix 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 (PerkinElmer, Ex 320 nm, Em 665 / 615 nm). A counter screen without the presenter and target protein was also performed to exclude the contribution of the compound alone.

[0324] Reagents and equipment His6-KRAS G12C-GTP (in-house; residues 1–169); 1.2 mM in PBS buffer, pH 7.4 Avi-CYPA (in-house production; residues 1–165); 556 μM in PBS buffer, pH 7.4 Anti-His Eu-W1024 (Perkin Elmer) Streptavidin APC (Perkin Elmer) Ligand (W21487), 10 mM in 100% DMSO EnVision (Perkin Elmer) Combi Multidrop Liquid Dispenser with 8-Channel Low-Volume Cassettes 384-well ProxiPlate (black) Experimental protocol 1. Using Mosquito, dispense 100 nL / well of compound (varying concentrations in DMSO) into a 384-well black ProxiPlate to create an Assay Ready Plate (ARP).

[0325] Make 2x 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. Make 2x PRE-MIX A: 100 nM His6-KRas G12C-GTP(1-169) and 1000 nM Avi-CypA(1-165) in 1x assay buffer.

[0327] 4. Using the MutiDrop Combi, dispense 2x PRE-MIX A into the ARP, 5 μl / well. Incubate at room temperature for 15 minutes.

[0328] 5. Make 2x PRE-MIX B: 10 nM anti-His Eu-W1024 and 40 nM SA APC. 6. Using a MutiDrop Combi, dispense 2x PRE-MIX B into the ARP, 5 μl / well. Shake briefly on the Combi and incubate for 60 minutes at room temperature.

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

[0330] Results: The binding curve (FIG. 11) showed a binding affinity of CYPA-Compound 3-KRAS with a calculated EC50 value of 2.1 μM. G12C-GTP Compound 3-dependent complex formation of the ternary complex is shown. Example 7: Determining complex formation by an amplified luminescent proximity homogeneous assay AlphaScreen technology (PerkinElmer) 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 can be any of glutathione-S-transferase (GST), hexahistidine (His6), FLAG, biotin-avi, Myc, and hemagglutinin (HA)). In this example, this technology is used to measure compound-promoted association of a presenter protein with a target protein. A mixture of presenter protein / Tag A and target protein / Tag B is added to a 384-well assay plate containing a compound of the invention and incubated for 15 minutes. A mixture of anti-fusion tag A or B AlphaScreen donor beads and anti-fusion tag A or B AlphaScreen acceptor beads is added, and the reaction is incubated for 240 minutes. AlphaScreen signals are read on an EnVision microplate reader (Perkin Elmer) using excitation = 680 nm, emission = 585 nm. Compounds that promote ternary complex formation are identified as those that elicit an increase in AlphaScreen signal relative to DMSO control wells.

[0331] CYPA-Cmp3-KRAS by Alpha-LISA G12C-GTP Determination of complex formation Avi-tagged cyclophilin A and His-tagged KRAS G12C-GTPThe beads were mixed with increasing concentrations of the ligand (compound 3) and incubated for 60 minutes at room temperature to allow for the formation of the ternary complex. A premix of nickel-chelating donor beads and streptavidin acceptor beads was then added and incubated for 60 minutes. AlphaLISA signals were read on an EnVision microplate reader (PerkinElmer, Ex 680 nm, Em 615 nm). A counter screen without the presenter and target protein was also performed to exclude the contribution of the compound alone.

[0332] Reagents and equipment: His6-KRAS G12C-GTP (in-house; residues 1–169); 1.2 mM in PBS buffer, pH 7.4 Avi-CYPA (in-house production; residues 1–165); 556 μM in PBS buffer, pH 7.4 Nickel chelate donor beads (Perkin Elmer) Streptavidin acceptor beads (Perkin Elmer) Ligand (W21487), 10 mM in 100% DMSO EnVision (Perkin Elmer) Combi Multidrop Liquid Dispenser with 8-Channel Low-Volume Cassettes AlphaPlate-384 Plate (White) Experimental Protocol: 1. Using Mosquito, dispense 100 nL / well of compound (varying concentrations in DMSO) into a 384-well black ProxiPlate to create an Assay Ready Plate (ARP).

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

[0334] 4. Using the MutiDrop Combi, dispense 2x PRE-MIX A into the ARP, 5 μl / well. Incubate at room temperature for 60 minutes.

[0335] 5.2x PRE-MIX B: Make 30 μg / ml streptavidin acceptor beads and 30 μg / ml nickel chelate donor beads. 6. Using a MutiDrop Combi, dispense 2x PRE-MIX B into the ARP, 5 μl / well. Shake briefly on the Combi and incubate for 60 minutes at room temperature.

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

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

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

[0339] Determination of the kinetics and thermodynamics of binding between FKBP12-compound 1 and CEP250 by ITC Reagents: Compound 1 and Compound 2 (in-house) in 100% DMSO, protein buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer + 1% DMSO), FKBP12 (in-house), CEP250 29.4 (in-house production, residues 1982–2231) and CEP250 11.4 (in-house production, residues 2134–2231).

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

[0341] [Table 6]

[0342] Experimental protocol: FKBP12 stock solution is diluted to 10 μM in assay buffer (1% DMSO final). Compounds are added to FKBP12 to a concentration of 20 μM (1% DMSO final). After a 5-10 minute preincubation period, the binary complex is loaded into the reaction cell of the ITC instrument. CEP250 protein stock is diluted to 50 μM in assay buffer, and 20 μM compound is added (1% DMSO final). This is then loaded into the injection syringe. A control experiment is also performed in the absence of compound to determine operational artifacts and heat associated with dilution of the titrant during injection from the syringe into the reaction cell. More detailed experimental parameters are shown in Table 7 below.

[0343] [Table 7]

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

[0345] 2) "mRawlTC": Integrates all peaks by adjusting the integrated peaks and baseline. 3) "ΔH" - Data management: Remove bad data (injection #1 and other artifacts) and subtract a line (background subtraction).

[0346] 4) "ΔH" - Model fitting: Select one set of site models and perform fitting using the Levenberg-Marquardt algorithm until χ no longer decreases, then click "done" to finish (the parameters N, Ka, and ΔH are calculated based on the fitting).

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

[0348] [Table 8]

[0349] Example 9: Determination of the binding kinetics between the conjugate and the protein by surface plasmon resonance Surface plasmon resonance (SPR) is a biophysical technique used to measure the kinetics associated with the binary interaction of two proteins, or a protein 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. Increasing concentrations of the second component (analyte) are then injected over the activated surface for a fixed period of time. An increase in the SPR signal (expressed in resonance units, RU) during the association phase and a decrease in the SPR signal during the dissociation phase indicate an interaction and can be fitted to a binding model to determine the associated K D , K. a , K. dIn this example, this method is used to measure the kinetics of binding of a 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 a surface, or (ii) the presenter protein is immobilized on a chip via a fusion tag and the conjugate is injected onto the surface.

[0350] Determination of the binding kinetics between FKBP12-compound 1 and CEP250 by SPR This protocol studies the kinetics (K) of binding of CEP250 (analyte) to immobilized FKBP12-compound 1 binary complex (ligand). D , K. a , K. d Surface plasmon resonance (SPR) is used as a method for determining the surface plasmon resonance (SPR).

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

[0352] Device: BIACORE™ X100 (GE Healthcare) Equipment: NTA sensor chip (GE Healthcare BR-1000-34) Experimental Protocol: Experiments are 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 two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for nonspecific interactions of the analyte with the sensor chip. Various concentrations of CEP250 (ranging from 1 nM to 1 μM), serially diluted in the same assay buffer containing 1 μM Compound 1 (final 1% DMSO), are injected over the FKBP12 and reference surfaces at a flow rate of 10 μl / min. The surfaces are regenerated between analyte injections with 350 mM EDTA.

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

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

[0355] Example 10: Determining the kinetics of binding between conjugates and proteins by biolayer interferometry Biolayer interferometry (BLI) is a biophysical technique used to measure the kinetics associated with the binary interaction of two proteins, or a protein to a ligand. Typically, one component of a binary interaction pair is immobilized on a biosensor chip via a fusion tag. Increasing concentrations of the second component (analyte) are then injected over the biosensor chip for a fixed period of time. An increase in the BLI signal (optical thickness, expressed in nm) during the association phase and a decrease in the BLI signal during the dissociation phase indicate an interaction and are fit to a binding model to determine the associated K D , K. a , K. d In this example, this method is used to measure the kinetics of binding of a 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 a 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 binding between This protocol involves the binding of KRAS to an immobilized CYPA-compound 3 binary complex (ligand). G12C-GTP The dissociation constant (K) for the binding of (analyte) D Biolayer interferometry (BLI) is used as a method to determine the surface area of ​​the polymer.

[0357] Reagents: Compound 3 (in-house) in 100% DMSO, ForteBio kinetics buffer (ForteBio, Menlo Park, CA), assay buffer (kinetics buffer, 1% DMSO, 2 μM Compound 3), Avi-tagged CYPA (in-house), KRAS G12C-GTP (residues 1 to 169) (manufactured in-house). Apparatus: Octet Red 96 instrument (ForteBio, Menlo Park, CA) Equipment: Streptavidin (SA) biosensor (ForteBio) Experimental protocol: Streptavidin (SA) biosensors were coated at 25°C in a solution containing 10 μM Avi-CYPA protein to a loading signal of 0.6 nm. Protein loading demonstrated long-term stability and the absence of baseline drift. Ternary complex formation was observed with KRAS starting at 200 μM in a 1:2 dilution series. G12C-GTP The effect of the protein concentration on the binding activity was evaluated in a dose-response experiment. For the negative control, the Avi-CYPA protein-coated sensor was immersed in a well containing only the screening buffer (supplemented with 2 μM of Compound 3). Corrected binding response sensograms were recorded and analyzed.

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

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

[0360] Example 11: Proteomics of FKBP12-binding target proteins for cross-linking reagents Identification reagents: compounds (in-house produced) in 100% DMSO, N-terminal biotin-FKBP12 (in-house produced), HEK293T cell lysate (in-house produced).

[0361] Experimental Protocol: HEK293T cell lysates were prepared using sonication on ice (4, 10-second pulses at 20% power) using 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 lysate was first cleared by centrifugation, and the resulting supernatant was passed through a 0.2 mm syringe filter on ice. N-terminally biotinylated FKBP12 was added to 500 ml of lysate to a final concentration of 4 mM and mixed by pipetting. Compounds were then added to a final concentration of 10 mM, and the reaction was mixed by pipetting. 60 mL of 50% slurry agarose-streptavidin resin (pre-equilibrated in lysis buffer) was added, and the reaction was allowed to proceed for 1 hour at 4°C with gentle rocking. After incubation, the resin was gently pelleted and washed four times with 1 mL of lysis buffer on ice by addition, centrifugation, and aspiration, followed by an additional four washes with 1 mL of lysis buffer without detergent in the same manner. Retained proteins were 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 for 2 hours at 37°C for protein digestion. The sample was then diluted to 0.8 M urea with 100 mM HEPES, pH 8.0, trypsin was added, and the sample was digested for an additional 16 hours at 37°C. After digestion was complete, samples were prepared for LC-MS / MS analysis using a C18 SPE filter onto which the sample was loaded, washed, eluted, dried in a speed vac, and finally suspended in 10 ml of 5% acetonitrile, 5% formic acid 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.Peptide sequences were assigned using the Sequest algorithm, and identified proteins are compared to a control sample (DMSO only) to identify candidate target proteins.

[0362] Results: Using the above protocol, over 100 target proteins have been identified that can bind to presenter proteins in the presence of crosslinking compounds. The 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: Determining the binding between the conjugate and the protein by fluorescence polarization The technique of fluorescence polarization (FP) is based on the observation that when a fluorescently labeled molecule is excited by polarized light, it emits light with a degree of polarization that is inversely proportional to the molecular rotation. Small molecules rotate rapidly during the excited state and, upon emission, have a low polarization value. Large complexes formed by binding of a labeled molecule to a second molecule rotate slightly during the excited state and therefore have a high polarization value. This property of fluorescence can be used to measure the interaction of labeled ligands with larger proteins and provides the basis for direct and competitive binding assays. In this example, this method is used to measure the binding of a 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 (in-house) in 100% DMSO, protein buffer (12.5 mM HEPES, pH 7.4, 1 mM MgCl), assay buffer (25 mM HEPES, pH 7.3, 0.002% Tween 20, 0.1% BSA, 10 mM NaCl, 1 mM MgCl), CYPA (in-house), Mant-GMP-PNP-loaded KRAS (residues 1-169).

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

[0366] FP (polarization units x 10^-3) = Signal(parallel) - Signal(perpendicular) / [Signal(parallel) + Signal(perpendicular)] Results: A representative curve is shown in Figure 16, and a table listing the EC50 (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 indicates the effect of the ligand C3DS on enhancing the binding between CYPA and KRAS.

[0367] [Table 9]

[0368] Example 13: Determination of binding between conjugate and protein by nuclear magnetic resonance Nuclear magnetic resonance (NMR) spectroscopy is a technique used to elucidate the three-dimensional structure 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 (2D NMR) is the most sensitive. 1 H- 15The most sensitive method for such studies is the addition of increasing concentrations of ligand to a protein NMR sample, and the 2D TROSY-HSQC spectra (NMR spectra) and identification of critical residues involved in ligand (drug) binding. 1 H- 15 The acquisition of N-TROSY-HSQC provides highly resolved atomic-level residue perturbation information, called chemical shift perturbation (CSP), which directly provides more accurate information for identifying ligand-binding sites than any other available biophysical technique. This approach allows for the study of weak, intermediate, and strong affinity ligand binding to proteins or binary protein complexes, and this information can be directly linked to existing structural, dynamic, and kinetic information. In this example, this method is used to demonstrate the binding (e.g., non-covalent or covalent) of a compound (drug) or conjugate of the present invention to a presenter protein.

[0369] Determination of KRAS(G12C)-Cyclophilin-Compound 3 Binding in the Ternary Complex by Solution MR Spectroscopy Reagents: Compound 3 (in-house) 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 KRAS NMR samples (100 μM DSS in 93% H2O and 7% D2O), assay buffer (protein buffer in DMSO (≤5%) + increasing equivalents of drug), GMP- 15 N-KRAS(G12C)-16 (N-His, residues 1–169, in-house), unlabeled (UL) cyclophilin (CYPA; residues 1–165) (in-house).

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

[0371] NMR data processing and analysis: 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 The N-KRAS(G12C)-16 stock solution was used to prepare NMR samples at 0.18 mM in 600 μl (including NMR additives). DSS was used as an internal standard for chemical shift referencing (at 0.0 ppm). 1 H peak). 15 N KRAS 2D 1 H- 15 N TROSY-HSQC spectra were collected (data size, 2048 x 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 min. The final NMR sample volume was kept at 600 μl. The binary complex ( 15 N-KRAS+UL-CYPA) 2D 1 H- 15 N TROSY-HSQC spectra were collected (data size 2048 × 128) while keeping other acquisition parameters the same (KRAS 1 H- 15 Only N correlation cross-peaks are visible in the spectrum). A 20 mM stock solution of compound 3 in 100% DMSO was used for the NMR titration. Compound 3 was added sequentially to the NMR sample to form the binary complex ( 15In the NMR samples of N-KRAS + UL-CYPA, 0.5 equivalents, 1.0 equivalents, 2.5 equivalents, and 5.0 equivalents (relative to that of the 15N-KRAS concentration) were obtained. At each stage, the sample volume was maintained at 600 μl while maintaining the same acquisition parameters. At each stage of compound 3 addition, 2D 1 H- 15 N TROSY-HSQC spectra were acquired to investigate the chemical shift perturbations (CSPs) of KRAS residues. All spectra were overlaid on top of each other. The effective CSPs at each Compound 3 titration point were determined using the chemical shift differences of each KRAS residue in the ternary complex (KRAS + CYPA + Compound 3) relative to the binary complex (KRAS + CYPA). Subsequently, the weighted average chemical shifts (Δδ) of each KRAS residue were calculated. 加重 ) is determined using the following formula: Δδ 加重 =[(Δ 1 H) 2 +(Δ 15 N / 5) 2 ] 1 / 2 Δδ more than 1 standard deviation from the grand mean 加重 Residues that elicit a .DELTA. are considered significantly perturbed and are used in binding site mapping. In separate titration experiments, we calculated the 2D .DELTA. for the binary complex (KRAS+CYPA) by sequentially adding equivalent amounts of DMSO (to achieve equivalent solvent concentrations as in the above experiment) and subtracting the contribution from DMSO addition. 1 H- 15 In a second control experiment, we collected a series of 2D TROSY-HSQC spectra of 15N-KRAS titrated with compound 3 at different equivalents (in the absence of CYPA). 1 H- 15 N TROSY-HSQC spectra were collected.

[0373] Valid CSPs are tabulated and analyzed. Drug-binding residues of KRAS (in the presence of CYPA) are mapped onto the protein surface. The dissociation constant, K D Determine. Experimental and processing parameters: Spectral data size: 2048 (1H dimension) x 128 (15N dimension) 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 in the indirect dimension. The data set was extrapolated by zero-filling once in each dimension before Fourier transformation.

[0374] Result: KRAS G12C-GTP The 2D1H-15N TROSY-HSQC spectrum of is shown in Figure 17A. Adding a stoichiometric amount of CYPA had no effect on the KRAS amide backbone crosspeak (Figure 17B), indicating that KRAS and CYPA do not directly interact. Titration of W21487 into a 1:1 sample of CYPA:KRAS elicits distinct chemical shifts indicative of a direct interaction with KRAS (Figure 17C).

[0375] Example 14. Determining the binding between a conjugate and a protein by microscale thermophoresis Microscale thermophoresis (MST) is a technique for characterizing biomolecular interactions by correlating changes in conformation to the molecular properties of molecules, such as size and mobility, in a directional temperature gradient. The generation of the gradient is induced by an infrared laser. Biomolecular movement is often characterized by labeling the molecules with covalently attached fluorophores or even changing fluorescence. In this example, this method is used to measure the binding of compounds or conjugates of the present invention to presenter proteins and establish ternary complex formation with target proteins, 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 binding between conjugates and proteins by second harmonic generation techniques Second harmonic generation (SHG) is an optical phenomenon that can be used to measure conformational changes in aqueous solution in real time. SHG signal intensity is sensitive to the average angular orientation of the dyes that label proteins attached to a surface, and the magnitude of the signal change directly correlates with the amount of angular change. Different conformations can be classified by the magnitude of the signal change upon binding, the signal relative to the baseline (a more perpendicular orientation relative to the surface-generated positive signal change, and vice versa), and the kinetics. In this example, this method is used to measure the binding of a compound or conjugate of the present invention to a presenter protein and establish ternary complex formation with a target protein, where (i) the dye-labeled conjugate is immobilized on a surface via a fusion tag and the presenter protein is injected over the surface, or (ii) the dye-labeled presenter protein is immobilized on a surface via a fusion tag and the conjugate is injected over the surface.

[0377] Example 16. Determination of binding between conjugates and proteins by differential scanning fluorimetry Differential scanning fluorimetry (DSF) allows the determination of protein melting temperatures (T m Fluorescence intensity is a solution-based biophysical technique used to measure T. In a typical experiment, a 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, giving the T m is calculated from the negative derivative minimum 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, this method is used to measure the thermal shift (e.g., non-covalent or covalent binding) of a compound or conjugate of the 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 by nanoDSF NanoDSF changes tryptophan or tyrosine fluorescence of proteins using m It is an advanced DSF method for measuring T. In a typical experiment, a 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 can be calculated from the change in tryptophan fluorescence intensity or from the rate of tryptophan emission at 330 nm and 350 nm, which accounts for the shift in tryptophan emission due to unfolding. For a target protein, ΔT in the presence of a small molecule is m can be measured to assess whether a small molecule binds to and stabilizes a protein. In this example, this method is used to measure the thermal shift (e.g., non-covalent or covalent binding) of a compound or conjugate of the 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 complex formation by differential light scattering Dynamic light scattering (DLS) is an established biophysical method used to measure time-dependent fluctuations in scattering intensity undergoing random Brownian motion. Diffusion coefficient and particle size information can be obtained from analyzing these fluctuations. More specifically, this method provides the ability to measure size characteristics, including radius and molecular weight, of proteins in aqueous solution. In this example, this method is used to measure the change in radius or molecular weight of (i) a presenter protein upon binding of a conjugate of the invention, or (ii) a conjugate of the invention upon binding to a presenter protein.

[0380] Example 19. Determination of binding between conjugates and proteins by sonic acoustic techniques Surface acoustic wave (SAW) technology is a biophysical method used for real-time detection of binding-induced conformational changes by monitoring the shift in the phase of a surface acoustic wave traveling along a biosensor. It can be used to measure the kinetics associated with the binary interaction of two proteins or proteins to a ligand. Typically, one component of the binary interaction pair is immobilized on the biosensor via a fusion tag. Increasing concentrations of the second component (analyte) are then injected over the biosensor for a fixed period of time. The increase in signal during the association phase (measured by a change in wave phase or amplitude) and the decrease in signal during the dissociation phase indicate the interaction and can be fitted to a binding model to determine the associated K D , K. a , K. d In this example, this method is used to measure the kinetics of binding of a conjugate of the 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 over the surface, or (ii) the presenter protein.

[0381] Example 20: Determination of complex formation by small angle X-ray scattering Small-angle X-ray scattering (SAXS) is a solution-based method used to determine protein structure in terms of average particle size and shape. It can deliver structural information in the 1-25 nm resolution range and repeat distance in partially ordered systems up to 150 nm in size. Ultra-small-angle scattering (USAS) can resolve 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 transfer (s = 4π sinθ / λ, where 2θ is the angle between the incident and scattered radiation). The scattering from the solvent alone is subtracted from the solution intensity. The X-ray scattering curve (intensity versus scattering angle) is then used to generate a low-resolution model of the protein or protein complex. In this example, the method is used to identify the presence of a ternary complex (e.g., non-covalent or covalent binding) of a compound or conjugate of the present invention to a presenter protein.

[0382] Other embodiments While the present disclosure has been described in conjunction with its detailed description, it should be understood that the above description illustrates the scope of the disclosure, but is not intended to limit the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments in accordance with the present invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims. In the claims, articles such as "a," "an," and "the" can mean one or more unless indicated to the contrary or otherwise clear from the context. Claims or descriptions containing "or" between one or more members of a group are considered to be satisfied when one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0383] It is also noted that the term "comprising" is intended to be open, allowing but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is thus also included and disclosed.

[0384] When ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can contemplate any specific value or subrange within the stated range in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Furthermore, it should be understood that any particular embodiment of the invention that falls within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are deemed known to those of ordinary skill in the art, such embodiments may be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the compositions 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 related to the existence of prior art or not.

Claims

1. A compound or pharmaceutically acceptable salt comprising a presenter protein binding moiety and a cross-linking group, wherein the presenter protein binding moiety has the structure of Formula IIa or IIb 【Chemistry 1】 Including, During the ceremony, Z １ and Z ２ each independently represents an optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ heteroalkyl or Z １ and Z ２ together with the atoms to which they are attached form an optionally substituted 10-40 member macrocycle, and Z １ or Z ２ at least one of comprises a point of attachment to said bridging group; b is 0, 1, or 2; c is 0 or 1; and d is 0, 1, 2, 3, 4, 5, 6, or 7; X1 is CH2; X 2 is absent, CH 2 , O, S, SO, SO 2 , or NR 4 ; Each R １ and R ２ are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ alkenyl, optionally substituted C ２ ~C ６ Alkynyl, optionally substituted C １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, optionally substituted C ２ ~C ６ Heteroalkynyl, optionally substituted C ３ ~C １０ Carbocyclyl, optionally substituted C ６ ~C １０ Aryl, optionally substituted C ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heterocyclyl, optionally substituted C ２ ~C ９ Heterocyclyl C １ ~C ６ alkyl or R １ and R ２ combine with the carbon atom to which they are attached to form C=O, or R １ and R ２ are taken together to form an optionally substituted C ３ ~C １０ Carbocyclyl or optionally substituted C ２ ~C ９ forming a heterocyclyl, Each R ３ are independently selected from hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ alkenyl, optionally substituted C ２ ~C ６ Alkynyl, optionally substituted C １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, optionally substituted C ２ ~C ６ Heteroalkynyl, optionally substituted C ３ ~C １０ Carbocyclyl, optionally substituted C ６ ~C １０ Aryl, optionally substituted C ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ heterocyclyl, or optionally substituted C ２ ~C ９ Heterocyclyl C １ ~C ６ alkyl or two R ８ are taken together to form an optionally substituted C ３ ~C １０ Carbocyclyl, optionally substituted C ６ ~C １０ forming an aryl, Each R ４ are independently hydrogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ alkenyl, optionally substituted C ２ ~C ６ alkynyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, optionally substituted C ６ ~C １０ Aryl C １ ~C ６ Alkyl, and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ is alkyl, A compound or a pharmaceutically acceptable salt thereof, wherein said bridging group is capable of undergoing a chemoselective reaction with an amino acid.

2. The bridging group is (a) mixed disulfides, (b) maleimide, (c) vinyl sulfone, (d) a vinyl ketone, or (e) alkyl halide 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, comprising:

3. The compound or pharmaceutically acceptable salt of claim 1 or 2, wherein the bridging group is a sulfhydryl-reactive bridging group, an amino-reactive bridging group, a carboxyl-reactive bridging group, a carbonyl-reactive bridging group, or a triazole-forming bridging group.

4. The compound or pharmaceutically acceptable salt of claim 3, wherein the bridging group is a sulfhydryl-reactive bridging group.

5. The bridging group has the structure of Formula I 【Chemistry 2】 Including, wherein the wavy line indicates the point of attachment of the bridging group to the remainder of the compound; a is 0, 1, or 2; R Ａ is an 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 5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, which is heteroaryl.

6. The bridging group has the structure 【Transformation 3】 Including, 6. The compound or pharmaceutically acceptable salt of claim 5, wherein the wavy line indicates the point of attachment of the bridging group to the remainder of the compound.

7. The presenter protein binding moiety has the structure 【Chemistry 4】 7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, having the formula:

8. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the presenter protein binding moiety and the cross-linking group are joined via a linker.

9. The linker of claim 8, wherein the linker has a structure of 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 and wherein A 1 is the bond between the linker and the presenter protein binding moiety, A 2 is the bond between the bridging group and the linker, B 1 , B 2 , B 3 , and B 4 are each independently selected from optionally substituted C 1 -C 2 alkyl, optionally substituted C 1 -C 3 heteroalkyl, O, S, and NR N , where RN 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; 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 C 2 -C 10 polyethylene glycol, or optionally substituted C 1-10 heteroalkyl; or A 1 -(B 1 ) f -(C 1 ) g -(B 2 ) h -(B 3 ) i -(C 2 ) j -(B 4 ) k -A 2 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein:

10. The linker of claim 1, wherein the linker has the structure of Formula VI: 【Transformation 5】 and wherein A 1 is the bond between the linker and the presenter protein binding moiety; A 2 is the bond between the bridging group and the linker; l is 0, 1, 2, or 3; m is 0 or 1; n is 0, 1, or 2; X 3 , X 4 , and X 5 are each independently absent, O, S, —C≡C—, CR 9 R 10 , or NR 11 ; 10. The compound or pharmaceutically acceptable salt of claim 8 or 9, wherein each R 9 , R 10 , and R 11 is independently hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted aryl, C 3 -C 7 carbocyclyl, optionally substituted C 6 -C 10 arylC 1 -C 6 alkyl, and optionally substituted C 3 -C 7 carbocyclylC 1 -C 6 alkyl.

11. The linker of claim 1, wherein the linker has the structure 【Transformation 6】 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, comprising:

12. The compound of claim 1, 【Chemistry 7-1】 【Chemistry 7-2】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

13. The compound of claim 12, 【Transformation 8】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

14. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt according to any one of claims 1 to 13, for use in a therapeutic method for modulating a target protein, the method comprising contacting the target protein with a modulating amount of the compound or pharmaceutically acceptable salt.

16. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, for use in a therapeutic method for inhibiting the activity of prolyl isomerase, comprising contacting a cell expressing said prolyl isomerase with said compound or a pharmaceutically acceptable salt thereof under conditions allowing the formation of a complex between said compound and prolyl isomerase, thereby inhibiting the activity of prolyl isomerase.

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