Methods and reagents for analyzing protein-protein interfaces

Abstract

Methods and reagents useful for analyzing interfaces between target proteins are provided. [Solution] The present disclosure provides methods and reagents useful for analyzing the protein-protein 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 of the present disclosure, the target and / or presenter protein is an intracellular protein. In some embodiments, the target and / or presenter protein is a mammalian protein.

Description

[Technical Field]

[0001] The present invention relates to methods and reagents for analyzing protein-protein interfaces. [Background technology]

[0002] The vast majority of small molecule drugs act by binding to functionally important pockets on target proteins, thereby modulating their activity. For example, cholesterol-lowering drugs such as statins bind to the enzyme active site of HMG-CoA reductase, thereby preventing the enzyme from binding 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 case. Current estimates suggest that only about 10% of all human proteins are targetable by small molecules. The remaining 90% are currently considered difficult or challenging 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 great interest in discovering new molecular modalities that can modulate the function of such undruggable targets. Summary of the Invention

[0003] Small molecules are limited in their targeting ability because their interaction with targets is driven by adhesive forces, the strength of which is roughly proportional to the contact surface area.Due to their small size, the only way for small molecules to create sufficient intermolecular contact surface area to effectively interact with target proteins is to literally be incorporated by the protein.In fact, both a large amount of experimental and computational data supports the view that only proteins with hydrophobic "pockets" on their surface can bind small molecules.In this case, binding is possible through incorporation.

[0004] Nature has evolved 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 between small molecules and small display proteins. The combined surfaces of the small molecules and display proteins associate with 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.

[0005] The present inventors have developed compounds and conjugates useful for identifying presenter and target protein pairs and for exploring the interface between them for use in developing small molecules that can modulate these interactions.

[0006] 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 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.

[0007] In some aspects, the present disclosure provides compounds that can be used as cross-linking substrates. These compounds may contain 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 bound to the protein-binding moiety. In some embodiments, the compound contains only one cross-linking group.

[0008] Thus, in certain aspects, 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 to 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.

[0009] 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.

[0010] In some embodiments, the bridging group comprises a mixed disulfide, for example, the bridging group has the formula Ia:

[0011] [ka] The structure of where 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.

[0012] In some embodiments, R A is an optionally substituted C2-C9 heteroaryl (e.g., pyridyl). In some embodiments, the bridging group has the structure:

[0013] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0014] In some embodiments, R A is an optionally substituted C1-C6 heteroalkyl (e.g., N,N-dimethylethyl). In some embodiments, the bridging group has the structure:

[0015] [ka] Including, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound. In some embodiments, the bridging group has the structure:

[0016] [ka] Includes:

[0017] In some embodiments, R A is an optionally substituted C1-C6 alkyl (e.g., methyl). In some embodiments, the bridging group has the structure:

[0018] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0019] In some embodiments, the bridging group comprises a carbon-based bridging group (eg, a bridging group that forms a carbon-sulfide bond upon reaction with a thiol). In some embodiments, the bridging group comprises a maleimide, e.g., the bridging group is represented by Formula Ib, Ic, Id, or Ie:

[0020] [ka] The structure of where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; X A is -C(O)- or -SO2-, X B is -C(O)- or CR E R F and R B and R C are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C 10 Carbocyclyl, optionally substituted C6-C 10 Aryl, optionally substituted C-C 10arylC1-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 D is hydrogen, hydroxyl, 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; R E and R F 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl.

[0021] In some embodiments, the bridging group comprises a structure of formula Ib. A is —C(O)—. In some embodiments, X B is —C(O)—. In some embodiments, R B and R C is hydrogen or optionally substituted C1-C6 alkyl (e.g., methyl).

[0022] In some embodiments, the bridging group has the structure:

[0023] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0024] In some embodiments, the bridging group has the structure:

[0025] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0026] In some embodiments, the bridging group has the formula If, Ig, Ih, or Ii:

[0027] [ka] The structure of where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; X C is -C(O)- or -SO2-, X D does not exist, NR J R K , or OR L and RG , R H , and R I are independently hydrogen, nitrile, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, 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 J , R K , and R L are independently selected from the group consisting of: absent, 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl.

[0028] In some embodiments, the bridging group comprises a structure of formula If. In some embodiments, X D is absent. In some embodiments, R G , R H , and R Iis hydrogen. In some embodiments, X C is —C(O)—. In some embodiments, X C is -SO2-.

[0029] In some embodiments, the bridging group comprises a vinyl sulfone, for example, the bridging group has the structure:

[0030] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0031] In some embodiments, the bridging group comprises a vinyl sulfone, for example, the bridging group has the structure:

[0032] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0033] In some embodiments, the crosslinking group comprises a vinyl ketone, for example, the crosslinking group has the structure:

[0034] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0035] In some embodiments, the crosslinking group comprises a vinyl ketone, for example, the crosslinking group has the structure:

[0036] [ka] Includes where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0037] In some embodiments, the crosslinking group comprises a vinyl ketone, for example, the crosslinking group has the structure:

[0038] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0039] In some embodiments, the bridging group is an ynone, such as a group of formula Ij or Ik:

[0040] [ka] The structure of where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; X E does not exist, NR N R O , or OR P and R M is hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C 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 N , R O , and R Pare 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl.

[0041] In some embodiments, the crosslinking group comprises a vinyl ketone, for example, the crosslinking group has the structure:

[0042] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0043] In some embodiments, the bridging group has the formula Im or In:

[0044] [ka] The structure of where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; X F does not exist, NR S R T , or OR U and X G is absent or is -C(O)-, Y is a leaving group, R Q and R R 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; R S , R T , and R U are independently selected from the group consisting of: absent, 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl.

[0045] In some embodiments, Y is halogen (e.g., fluoro, chloro, bromo, or iodo), mesylate, tosylate, or triflate. In some embodiments, Y is nitrile. In some embodiments, X F and X G is absent. In some embodiments, R Q and R R is hydrogen. In some embodiments, the bridging group comprises an alkyl halide, e.g., an alkyl chloride, e.g., the bridging group has the structure:

[0046] [ka] Including, wherein 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, such as an alkyl chloride or alkyl fluoride, e.g., the bridging group has the structure:

[0047] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0048] In some embodiments, the bridging group comprises an epoxide, for example, the bridging group has the formula Io:

[0049] [ka] Contains the structure where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; R V , R W , and R Xare 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, optionally substituted C2-C9 heterocyclylC1-C6 alkyl.

[0050] In some embodiments, the bridging group has formula Ip:

[0051] [ka] The structure of where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; The dotted line represents an optional double bond, which is included if necessary to make the structure aromatic, and b is 0, 1, or 2; Y is a leaving group, R Y and R Z 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-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, optionally substituted C2-C9 heterocyclylC1-C6 alkyl; X H , X I , X J , X K , and X L Each of the NR AA , or CR AB and X H , X I , X J , X K , and X L At least five of them are NR AA , or CR AB and R AA is absent or is 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; R AB is hydrogen, nitrile, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C 10Carbocyclyl, 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.

[0052] In some embodiments, at least one R AB is an electron withdrawing group. In some embodiments, one to three R AB is an electron withdrawing group. In some embodiments, Y is nitrile. In some embodiments, Y is halogen (e.g., fluoro, chloro, bromo, or iodo), mesylate, tosylate, or triflate.

[0053] In some embodiments, the bridging group has the structure:

[0054] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0055] In some embodiments, the bridging group has the structure:

[0056] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0057] In some embodiments, the bridging group has the structure:

[0058] [ka] Including, where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

[0059] In some embodiments, the bridging group is an internal bridging group, for example, the bridging group is represented by formula Iq, Ir, or Is:

[0060] [ka] The structure of where the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound; X M is -C(O)- or -SO2-, X N does not exist, NR AE , or O, R AC and R AD are independently hydrogen, nitrile, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, 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 AEis 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 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl.

[0061] In some embodiments, X N is NR AE and R AE is hydrogen. In some embodiments, R AC and R AD is hydrogen. In some embodiments, X M is —C(O)—. In some embodiments, X M is -SO2-.

[0062] In some embodiments of any of the foregoing compounds, the site of the protein-binding moiety is capable of non-covalently interacting with the protein. In some embodiments of any of the foregoing compounds, the site of the protein-binding moiety is capable of covalently interacting with the protein.

[0063] In some aspects, the 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 through a linker.

[0064] In some aspects, the present disclosure provides a compound having the structure:

[0065] [ka]

[0066] [ka] The present invention provides a compound having the formula:

[0067] In some aspects, the present disclosure provides conjugates, methods for their synthesis, and uses thereof, which comprise a presenter protein binding moiety that can be covalently or non-covalently bound to a presenter protein conjugated to a target protein through a linker.

[0068] 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 site of the presenter protein binding moiety of the conjugate can interact non-covalently with the presenter protein. In some embodiments, the site of the presenter protein binding moiety of the conjugate can interact covalently with the presenter protein.

[0069] 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.

[0070] 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 providing (a) a compound comprising a presenter protein-binding moiety and a cross-linking group, (b) the target protein, and (c) the presenter protein, and reacting the compound with the target protein under conditions that allow for the production of the conjugate.

[0071] In some aspects, the present disclosure provides complexes, methods for their production, and uses thereof, including presenter proteins and conjugates comprising a presenter protein-binding moiety and a target protein.

[0072] Thus, in another aspect, the present disclosure provides (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, and (ii) a complex comprising the presenter protein.

[0073] In some aspects, the 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 combining a conjugate comprising a presenter protein-binding moiety conjugated to a target protein and the presenter protein under conditions that allow for the production of the complex.

[0074] In some aspects, the present disclosure provides methods for producing (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein and (ii) a complex comprising the presenter protein, the methods comprising providing (a) a compound comprising a presenter protein-binding moiety and a cross-linking group, (b) the target protein, and (c) the presenter protein, and reacting the compound with the target protein under conditions that allow for the formation of the complex.

[0075] In some embodiments of the aforementioned methods, the presenter protein binds to the compound in the absence of the target protein. In some embodiments of the aforementioned methods, the presenter protein does not substantially bind to the compound in the absence of the target protein. In some embodiments of the aforementioned methods, the compound and the target protein do not substantially react in the absence of the presenter protein. In some embodiments of the aforementioned methods, the compound and the target protein react in the absence of the presenter protein. In some embodiments of the aforementioned methods, the conditions do not include a reducing reagent. In some embodiments of the aforementioned methods, the conditions include an excess of presenter protein.

[0076] In some embodiments, detectable binding between the compound and the presenter protein is observed in the absence of the target protein. In some embodiments, however, 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, significant reaction (e.g., significant conjugate formation) between the crosslinking group and the target protein is not observed in the absence of the presenter protein. In some embodiments, however, 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 not present (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).

[0077] In some embodiments, the conjugation 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 described herein (e.g., a compound having a structure comprising 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 reaction of the cross-linking moiety with the target protein, such that a cross-link between the cross-linking moiety and the target protein is formed. In some embodiments, the cross-link is with a heteroatom within an amino acid (e.g., within an amino acid side chain) of the target protein. In some embodiments, the cross-link is with an -S- atom within a cysteine ​​in the target protein. In some embodiments, the target protein is a variant 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 higher) to the naturally occurring target protein, but differs by the substitution or addition of at least one amino acid in the cross-linking group that is susceptible to participation in cross-linking (e.g., the amino acid side chain contains a heteroatom that can participate in such cross-linking).

[0078] In some aspects, the present disclosure provides conjugates, methods for their synthesis, and uses thereof, which include a target protein binding moiety that can covalently or non-covalently bind to a target protein conjugated to a presenter protein through a linker.

[0079] 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 site of the target protein binding moiety of the conjugate is capable of non-covalently interacting with the target protein. In some embodiments, the site of the target protein binding moiety of the conjugate is capable of non-covalently interacting with the target protein. In some embodiments, the target protein binding moiety and the presenter protein are conjugated through a linker.

[0080] 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.

[0081] 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 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 for the production of the conjugate.

[0082] In some embodiments, detectable binding between the compound and the target protein is observed in the absence of the presenter protein. In some embodiments, however, 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, significant reaction (e.g., significant conjugate formation) between the crosslinking group and the presenter protein is not observed in the absence of the target protein. In some embodiments, however, 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 not present (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).

[0083] 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, a reaction (e.g., conjugate formation) between the crosslinking group and the target protein is not observed in the absence of the presenter protein. In some embodiments, however, a reaction between the crosslinking group and the target protein is observed even in the absence of the presenter protein. In some embodiments, the conjugate formation described herein is performed under conditions that do not include (e.g., are substantially free of) reducing reagents.

[0084] In some embodiments, the present invention provides a complex comprising (i) a presenter protein, (ii) a compound described herein (e.g., a compound having a structure comprising 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 reaction of the cross-linking moiety with the target protein, such that a cross-link between the cross-linking moiety and the target protein is formed. In some embodiments, the cross-link is with a heteroatom within an amino acid (e.g., within an amino acid side chain) of the target protein. In some embodiments, the cross-link is with an -S- atom within a cysteine ​​in the target protein. In some embodiments, the target protein is a variant of a naturally occurring target protein; in some such embodiments, the variant has an amino acid sequence that is highly similar (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or higher) to the naturally occurring target protein, but differs by the substitution or addition of at least one amino acid that is susceptible to participation 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).

[0085] In some aspects, the present disclosure provides complexes, methods for their production, and uses thereof, including conjugates comprising a target protein and a target protein-binding moiety conjugated to a presenter protein through a linker.

[0086] In some aspects, the present disclosure provides complexes 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 complexes are exposed to and / or maintained under conditions that allow reaction of the cross-linking moiety with the presenter protein, such that a cross-link between the cross-linking moiety and the presenter protein is formed. In some embodiments, the cross-link is with a heteroatom within an amino acid (e.g., within an amino acid side chain) of the presenter protein. In some embodiments, the cross-link is with an -S- atom within 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 higher) to the naturally occurring presenter protein, but differs by the substitution or addition of at least one amino acid that is susceptible to participation 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).

[0087] In some aspects, the 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 combining the conjugate comprising the target protein-binding moiety conjugated to the presenter protein and the target protein under conditions that allow for the production of the complex.

[0088] In some aspects, the present invention relates to methods for generating a complex comprising (i) a conjugate 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, methods are provided that include 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 include, for example, (i) combining (a) a compound (e.g., a compound having a structure that includes a target protein-binding moiety and a cross-linking group), (b) the target protein, and (c) a presenter protein with one another, 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 for the reaction of the cross-linking group with the presenter protein to generate the conjugate.

[0089] In some aspects, the present disclosure provides methods for producing (i) a conjugate comprising a target protein-binding moiety conjugated to a presenter protein and (ii) a complex comprising the 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.

[0090] In some such embodiments, the conditions are characterized by the compound, presenter protein, and / or target protein such that detectable binding between the compound and the target protein is observed in the absence of the presenter protein. In some embodiments, however, detectable binding between the compound and the target protein is not observed under the 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 the conditions in the absence of the target protein. In some embodiments, however, significant reaction between the crosslinking group and the presenter protein may be observed under the 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.

[0091] 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.

[0092] In some aspects, the present disclosure provides compounds comprising a presenter protein-binding moiety capable of non-covalently interacting with a presenter protein and a target protein-binding moiety capable of covalently or non-covalently interacting with a target protein. In some embodiments, the presenter protein-binding moiety and the target protein-binding moiety are attached via a linker.

[0093] Thus, in some embodiments, the present disclosure provides compounds of formula VII: ALB Formula VII having the structure wherein A is of formula VIIIa or VIIIb:

[0094] [ka] The structure of 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 an optionally substituted C-C 10 forming a carbocyclyl or an optionally substituted C2-C9 heterocyclyl; Each R3 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 an optionally substituted C3-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 provides a compound that is a target protein binding moiety.

[0095] In some embodiments of compounds of Formula VII, the target protein binding moiety, B, is capable of non-covalently interacting with the target protein. In some embodiments of compounds of Formula VII, the target protein binding moiety, B, is capable of covalently interacting 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.

[0096] In some aspects, the present disclosure provides ternary complexes, methods for their production, and uses thereof, which include a presenter protein, a target protein, and a compound comprising a presenter protein-binding moiety and a target protein-binding moiety.

[0097] 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 a presenter protein.

[0098] In some aspects, the present invention relates to methods for identifying and / or characterizing a conjugate described herein that is capable of forming a complex with a presenter protein (e.g., a compound having a structure comprising a presenter protein-binding moiety and a crosslinking group, conjugated to a target protein). In some embodiments, such methods comprise the steps of: (a) providing (i) such a conjugate (e.g., a compound having a structure comprising a presenter protein-binding moiety and a crosslinking group, conjugated to a target protein) and (ii) a presenter protein; (b) combining the conjugate and the 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 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.

[0099] Thus, in some aspects, the present disclosure provides methods for identifying and / or characterizing a conjugate capable of forming a complex with a presenter protein, 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 to allow complex formation if the conjugate is capable of forming a complex with the presenter protein, 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.

[0100] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful for identifying target proteins that are capable of forming a covalent bond to the compound in the presence of a presenter protein.

[0101] 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, wherein the compound comprises a presenter protein-binding moiety and a cross-linking moiety. The method comprises 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, 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 during complex formation to form a conjugate; wherein 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.

[0102] 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.

[0103] 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 (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 to allow complex formation, if the conjugate is capable of forming a complex with the presenter protein, and (c) determining whether the target protein binds to the presenter protein in the complex, wherein if the target protein binds to the presenter protein, the target protein is identified as binding to the presenter protein.

[0104] 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, 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 binds to the presenter protein in the complex, wherein if the target protein binds to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein.

[0105] 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, 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, wherein if the compound, target protein, and presenter protein form a complex, the target protein is identified as a target protein capable of forming a complex with the presenter protein.

[0106] In some aspects, the present disclosure provides a method for identifying and / or characterizing a target protein that binds to a presenter protein, 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 compound is capable of forming a complex with the presenter protein; and (c) determining whether the target protein binds to the presenter protein in the complex, wherein if the target protein binds to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein.

[0107] 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 the target proteins that form a complex with the presenter protein are identified as target proteins that can form a complex with the presenter protein.

[0108] 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 a target protein), and a presenter protein. In some embodiments, the method further comprises (d) identifying the target protein within a complex formed between one or more target proteins, a compound, and a 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 a presenter protein within a complex can be performed using a pull-down experiment, in which either the target protein or the presenter protein is labeled (e.g., the complex can be selectively pulled down in the presence of the target protein and / or the presenter protein that are not within the complex).

[0109] In some aspects, the present disclosure provides methods for identifying target proteins capable of forming a complex with a presenter protein by: (a) providing a presenter protein comprising (i) two or more target proteins, (ii) any of the aforementioned compounds, and (iii) an affinity tag; (b) combining the two or more target proteins, compounds, and presenter proteins under conditions that allow complex formation if the target proteins form a complex with the presenter protein; (c) selectively isolating one or more complexes of the target proteins, compounds, and presenter proteins formed in step (b); and (d) identifying the target proteins within 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 capable of forming a complex with the presenter protein.

[0110] In some embodiments, the determining step comprises 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 a target protein, a presenter protein, and a compound of the 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 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 a presenter protein within a complex can be performed using a pull-down experiment, in which either the target protein or the presenter protein is labeled (e.g., the complex can be selectively pulled down in the presence of the target protein and / or presenter protein that are not within the complex).

[0111] 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.

[0112] Thus, in another aspect, the present disclosure provides a method for identifying and / or characterizing a location on a target protein for forming a conjugate with a presenter protein-binding moiety, where the conjugate is capable of forming a complex with the presenter protein. The method comprises: (a) providing (i) a conjugate comprising a presenter protein-binding moiety conjugated to the 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), conjugating the presenter protein-binding moiety at a different location on the target protein, until the conjugate and the presenter protein form a complex; wherein a location on the target protein for forming a conjugate with the presenter protein-binding moiety capable of forming a complex with the presenter protein is identified when the conjugate and the presenter protein form a complex. In some embodiments, the presenter protein is a variant of a naturally occurring target protein.

[0113] In some aspects, the present disclosure provides methods for identifying and / or characterizing locations on a target protein for forming a conjugate with a presenter protein binding moiety, which conjugate is capable of forming a complex with a presenter protein. The method includes: (a) providing (i) a compound comprising a presenter protein-binding moiety and a crosslinking group, (ii) a target protein, and (iii) a presenter protein; (b) combining the compound 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), conjugating the presenter protein-binding moiety to a different location on the target protein, until the conjugate and presenter protein form a complex; wherein a location on the target protein for forming a conjugate with the presenter protein-binding moiety that is capable of complexing with the presenter protein is identified if the conjugate and presenter protein form a complex, thereby identifying a location on the target protein for forming a conjugate that is capable of complexing with the presenter protein. In some embodiments, the target protein is a variant of a naturally occurring target protein.

[0114] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful for identifying compounds that can form covalent bonds to target proteins in the presence of a presenter protein, hi some embodiments, the identified compounds selectively form covalent bonds with target proteins in the presence of the presenter protein.

[0115] 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) the presenter protein; and (b) determining whether the compound and the target protein form a covalent bond via the cross-linking group of the compound in the sample; 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.

[0116] In some aspects, the present disclosure provides methods for identifying and / or characterizing compounds capable of selectively and covalently binding to a target protein in the presence of a presenter protein, the methods comprising: (a) providing a first sample comprising (i) a compound comprising a presenter protein-binding moiety and a crosslinking group, (ii) a target protein, and (iii) a presenter protein, and a second sample comprising the same compound (i) comprising a presenter protein-binding moiety and a crosslinking group as in the first sample and the same target protein as in the first sample; and (b) determining the extent to which the compound and the target protein react in the first sample compared to the second sample, wherein the compound is identified as selectively and covalently binding to the target protein in the presence of the presenter protein if the compound and the target protein react more in the first sample than in the second sample.

[0117] 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-fold more in a first sample than in a second sample, hi 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.

[0118] In some embodiments, the compounds, conjugates, and complexes of the present invention may be useful for identifying conjugates comprising a target protein and a presenter protein-binding moiety that can form a complex with a presenter protein.

[0119] 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) a presenter protein, and (b) combining the conjugate and the presenter protein under conditions suitable to allow 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 a conjugate capable of forming a complex with the presenter protein.

[0120] 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.

[0121] 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.

[0122] Thus, in another aspect, the present disclosure provides a method for determining the structure of and / or evaluating one or more structural features of an interface within a complex comprising a presenter protein and a target 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) 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, 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 within the complex comprising the presenter protein and the target protein.

[0123] In some aspects, the present disclosure provides a method for determining the structure of and / or evaluating one or more structural features of an interface within a complex comprising a presenter protein and a target protein, 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 (e.g., in a vial) 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, and (c) determining a crystal structure of the complex, wherein the structure of the interface includes at least a portion of the crystal structure between the presenter protein and the target protein, thereby determining the structure of the interface within the complex comprising the presenter protein and the target protein.

[0124] In some aspects, the present disclosure provides a method for determining the structure of and / or evaluating one or more structural features of an interface within a complex comprising a presenter protein and a target 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) 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 within the complex comprising the presenter protein and the target protein.

[0125] In some aspects, the present disclosure provides methods for determining the structure of and / or evaluating one or more structural features of a protein-protein interface in a complex comprising a presenter protein and a target protein, the methods comprising the steps of (a) providing a crystal of any of the aforementioned complexes, and (b) determining the structure of the crystal, 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 protein-protein interface in the complex comprising the presenter protein and the target protein.

[0126] In some aspects, the present disclosure provides methods for identifying and / or characterizing compounds capable of modulating the biological activity of a target protein. The methods include (a) providing a structure of a protein-protein interface in a complex comprising a presenter protein and a 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 structure of a compound that can bind at the interface is determined by screening compounds comprising a presenter protein-binding moiety described herein for complex formation in the presence of the target protein and a presenter protein.

[0127] In some aspects, the present disclosure provides a method for obtaining X-ray crystallographic 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) a presenter protein, (b) combining the conjugate and the 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 the crystal structure of the complex, thereby obtaining X-ray crystallographic coordinates for the complex.

[0128] In some aspects, the present disclosure provides a method for obtaining X-ray crystallographic 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) combining the compound, the target protein, and the presenter protein under conditions suitable to allow complex formation, if the compound is capable of forming a complex with the presenter protein, and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystallographic coordinates for the complex.

[0129] 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) combining the compound, the target protein, and the presenter protein under conditions suitable to allow complex formation, if the compound is capable of forming a complex with the presenter protein, and (c) determining the crystal structure of the complex, thereby obtaining X-ray crystal coordinates for the complex.

[0130] In some aspects, the present disclosure provides methods for determining residues on a target protein involved in binding with a presenter protein. The methods comprise the steps of: (a) providing X-ray crystal coordinates of a complex obtained by the methods of the present invention; and (b) identifying residues on the target protein that include atoms within 4 Å of atoms on the presenter protein, thereby determining residues on the target protein that are involved in binding with the presenter protein. 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. The methods comprise the steps of: (a) providing X-ray crystal coordinates of a complex described herein obtained by the methods 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.

[0131] 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.

[0132] In some embodiments, the interface within the complex comprising the presenter protein and the target protein is or comprises a binding pocket. In some aspects, the present disclosure provides a composition comprising any of the aforementioned compounds, target proteins, and presenter proteins in solution.

[0133] In some aspects, the present disclosure provides pharmaceutical compositions comprising any of the compounds, conjugates, or complexes of the present invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in unit dosage form.

[0134] In some aspects, the present disclosure provides 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 comprise contacting the target protein with a modulating (e.g., positively or negatively 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.

[0135] 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 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 that allow the compound to form a complex with the presenter protein and allow the resulting complex to bind to the target protein, thereby modulating (e.g., positively or negatively modulating) the target protein.

[0136] 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 contacting the target protein with a conjugate of the invention comprising a target protein-binding moiety, thereby modulating the target protein.

[0137] In some aspects, the present disclosure provides methods for inhibiting prolyl isomerase activity. In some embodiments, such methods comprise contacting a cell expressing prolyl isomerase with a compound or composition herein under conditions that allow the formation of a complex between the compound and prolyl isomerase, thereby inhibiting prolyl isomerase activity.

[0138] In some aspects, the present disclosure provides methods for forming a presenter protein / compound complex in a cell. In some embodiments, such methods comprise contacting a cell expressing a presenter protein with a compound or composition of the invention under conditions that allow the formation of a complex between the compound and the presenter protein.

[0139] In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety is capable of binding to a protein encoded by any one of the genes listed in Table 1. In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety is a prolyl isomerase binding moiety. In some embodiments of any of the aforementioned 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 to FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52), a cyclophilin-binding moiety (e.g., the presenter protein-binding moiety can bind to 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 aforementioned methods, the presenter protein is known to bind to the presenter protein-binding moiety.

[0140] In some embodiments of any of the foregoing 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 foregoing compounds, conjugates, complexes, compositions, or methods, the FKBP-binding moiety has the formula IIa or IIb:

[0141] [ka] The structure of In the formula, Z 1 and Z 2 are each independently optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, or Z1 and Z 2 combine with the atoms to which they are attached to form an optionally substituted 10-40 membered macrocycle, and Z 1 or Z 2 at least one of comprises a point of attachment to a bridging 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 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 an optionally substituted C-C 10 forming a carbocyclyl or an optionally substituted C2-C9 heterocyclyl; Each R 3are 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 an optionally substituted C3-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.

[0142] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety has the structure:

[0143] [ka] Includes:

[0144] In some embodiments of any of the foregoing 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 foregoing compounds, conjugates, complexes, compositions, or methods, the cyclophilin-binding moiety has Formula III or IV:

[0145] [ka] The structure of In the 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 6 combine with the atoms to which they are attached to form an optionally substituted 10-40 membered 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 and R 7 are independently 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 10arylC1-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; 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.

[0146] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the cyclophilin binding moiety has formula IVa:

[0147] [ka] The structure of In the formula, 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).

[0148] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the presenter protein binding moiety has the structure:

[0149] [ka] Includes:

[0150] In some embodiments of any of the foregoing 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 foregoing compounds, conjugates, complexes, compositions, or methods, the target protein comprises an undruggable surface. In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the target protein does not have a conventional binding pocket.

[0151] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the target protein has been modified to replace at least one native amino acid with a reactive amino acid (e.g., a natural 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 foregoing compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the target protein has been modified to replace at least one native reactive amino acid (e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine) with a non-reactive amino acid (e.g., a natural 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 foregoing compounds, conjugates, complexes, compositions, or methods, the at least one native reactive amino acid is a solvent-exposed amino acid. In some embodiments of any of the aforementioned 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 aforementioned compounds, conjugates, complexes, compositions, or methods, the substitutions are conservative substitutions. In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the target protein contains only one solvent-exposed reactive amino acid.

[0152] In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the presenter protein is a protein encoded by any one of the genes listed in Table 1. In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the presenter protein is a prolyl isomerase. In some embodiments of any of the aforementioned 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.

[0153] In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein has been modified to replace at least one native amino acid with a reactive amino acid (e.g., a natural 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 aforementioned compounds, conjugates, complexes, compositions, or methods, the amino acid sequence of the presenter protein has been modified to replace at least one native reactive amino acid (e.g., cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine) with a non-reactive amino acid (e.g., a natural 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 aforementioned compounds, conjugates, complexes, compositions, or methods, the at least one native reactive amino acid is a solvent-exposed amino acid. In some embodiments of any of the aforementioned 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 aforementioned compounds, conjugates, complexes, compositions, or methods, the substitutions are conservative substitutions.

[0154] In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the linker is 1 to 20 atoms in length. In some embodiments of any of the aforementioned compounds, conjugates, complexes, compositions, or methods, the linker is 1.5 to 30 angstroms in length.

[0155] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the linker has 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 Formula V having the structure In the 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 optionally substituted C1-C2 alkyl, optionally substituted C1-C3 heteroalkyl, O, S, and NR N Selected from R N is hydrogen, optionally substituted C 1-4 Alkyl, optionally substituted C 2-4 Alkenyl, optionally substituted C 2-4 Alkynyl, optionally substituted C 2-6 Heterocyclyl, optionally substituted C 6-12 aryl, or optionally substituted C 1-7 Heteroalkyl, C 1 and C 2 are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; f, g, h, I, j, and k are each independently 0 or 1; and D is optionally substituted C 1-10 Alkyl, optionally substituted C 2-10 Alkenyl, optionally substituted C 2-10 Alkynyl, optionally substituted C 2-6 Heterocyclyl, optionally substituted C 6-12 Aryl, optionally substituted C-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 It is a chemical bond that connects

[0156] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the linker has the formula VI:

[0157] [ka] The structure of In the 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, respectively, independently, non-existent, 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 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl. In some embodiments, each R 9 , R 10 , and R11 are independently hydrogen, unsubstituted C1-C6 alkyl, unsubstituted C2-C6 alkenyl, unsubstituted C2-C6 alkynyl, unsubstituted aryl, C3-C7 carbocyclyl, unsubstituted C6-C 10 arylC1-C6 alkyl, and unsubstituted C3-C7 carbocyclylC1-C6 alkyl.

[0158] In some embodiments of any of the foregoing compounds, conjugates, complexes, compositions, or methods, the linker has the structure:

[0159] [ka] Includes: chemical terms Those of skill in the art will understand 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, the depicted structures can be understood to represent any such isomeric or isotopic forms individually or in combination.

[0160] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, e.g., 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 can be isolated as a mixture of isomers or as separated isomeric forms.

[0161] In some embodiments, one or more compounds presented herein may exist in various tautomeric forms. Unless expressly excluded, as may be clear from the context, reference to such a compound encompasses all such tautomeric forms. In some embodiments, a tautomeric form results 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 moieties with prototropic tautomeric forms 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 can be converted via acetal interconversion, for example, as shown in the following scheme:

[0162] [ka] This arises from interconversions exemplified by:

[0163] Those skilled in the art will understand 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 due to different numbers of neutrons in their 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 racemization rate of a chiral center.

[0164] As is known in the art, many chemical entities (particularly many organic molecules and / or many small molecules) can exist in a wide 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.

[0165] In some embodiments, the compounds described and / or illustrated herein may be provided and / or utilized in salt form. In certain embodiments, the compounds described and / or illustrated herein may be provided and / or utilized in the form of a hydrate or solvate.

[0166] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. The present disclosure specifically includes 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.

[0167] As used herein, phrases of the form "optionally substituted X" (e.g., optionally substituted alkyl) are intended to be equivalent to "X, wherein X is optionally substituted" (e.g., "alkyl, wherein said alkyl is optionally substituted"). It is not intended to imply that the feature "X" (e.g., alkyl) itself is optional.

[0168] The term "alkyl," as used herein, 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., straight-chained), and 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; (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 optionally substituted with an O-protecting group, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1-7Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) —COR optionally substituted with an O-protecting group 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) s3 OR' (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 R' is H or C 1-20 alkyl), and (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 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 1-6Alkyl, (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' (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 R' is H or C 1-20 alkyl), and (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 (19)-NRH’ C(O)R I’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1-6 alkyl; 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' (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 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), 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 (20)-NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1-6 alkyl; 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) C6-10 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' (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 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), 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 (21) amino-polyethylene glycols (wherein the alkyl group is an alkyl group), (22) amidines, and (23) silyl groups, such as trimethylsilyl, t-butyldimethylsilyl, and triisopropylsilyl. These groups may be optionally substituted with one, two, three, or, in the case of alkyl groups of two or more carbons, four substituents, independently selected from the group consisting of alkyl groups of two or more carbons, such as aryl groups, alkyl groups of two or more carbons ...

[0169] The terms "alkylene" and the prefix "alk-", as used herein, refer to a saturated divalent hydrocarbon group derived from a straight-chain or branched saturated hydrocarbon by removing two hydrogen atoms, and are exemplified by methylene, ethylene, isopropylene, and the like. x-y The terms "alkylene" and "Cx-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.

[0170] The term "alkenyl," as used herein, unless otherwise specified, represents a monovalent straight-chain or branched 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.

[0171] The term "alkynyl," as used herein, represents a monovalent linear or branched group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond, 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.

[0172] The term “amino” as used herein refers to —N(RN1 )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 combine 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 include 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, C 1-20 Alkyl (e.g., C 1-6alkyl), or C 6-10 (which may be aryl).

[0173] The term "amino acid," as used herein, 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, amino group, or 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, through 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, and 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 carboxy- and / or amino-terminal amino acids in a polypeptide, can contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids can be modified by methylation, amidation, acetylation, and / or substitution compared to the general structure. In some embodiments, such modifications can, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one containing an 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 an otherwise identical, unmodified amino acid. As may be clear from the context, in some embodiments, the term "amino acid" is used to refer to a free amino acid, and 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 group by a carbonyl group, where a side chain or amino group is attached to the carbonyl group. In some embodiments, an 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, selenocysteine, 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' (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 R' is H or C 1-20 alkyl), and (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 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 1-20Alkyl (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 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 R' is H or C 1-20 alkyl), and (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 (19)-NR H’ C(O)R I’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1-6 alkyl; 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' (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 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), 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 (20)-NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1-6 alkyl; 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' (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 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), 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 (21) Amino-polyethylene glycols (which are alkyl), and (22) amidines. In some embodiments, each of these groups can be further substituted as described herein.

[0174] The term "N-alkylated amino acid" as used herein refers to an amino acid containing an optionally C1-C6 alkyl 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.

[0175] 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; (1)C 1-7 Acyl (e.g., carboxaldehyde), (2) C 1-20 Alkyl (e.g., C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkylsulfinyl-C1-6 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., C 1-6 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 1-6 Alkyl, (c) C 6-10 aryl, and (d) C 1-6 ALC-C 6-10aryl), (19)—(CH) 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 The alkylene group of a C1-alkaryl or C1-alkheterocyclyl can be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkynyl, ...

[0176] An "arylalkyl" group, as used herein, represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted arylalkyl groups are those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1-6 ALC-C6-10 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" is a C 1-6 The alkylene refers to and the chemical structure to which it is attached is as defined herein.

[0177] The term "azido" refers to the group -N3, which may also be depicted as -N=N=N. The terms "carbocyclic" and "carbocyclyl" as used herein refer to an optionally substituted C ring in which the ring is formed by carbon atoms. 3-12 It refers to monocyclic, bicyclic, or tricyclic non-aromatic ring structures. Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloalkynyl groups.

[0178] A "carbocyclylalkyl" group, as used herein, represents a carbocyclic group, as defined herein, attached to the parent molecular group through 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 6-10 In 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" is a C 1-6 The alkylene means and the chemical structure to which it is attached is as defined herein.

[0179] 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.

[0180] The term "cyano" as used herein refers to a -CN group. The term "cycloalkyl," as used herein, unless otherwise specified, represents 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 those having (1) C 1-7 Acyl (e.g., carboxaldehyde), (2) C 1-20 Alkyl (e.g., C 1-6 Alkyl, C 1-6 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 6-10Aryl, (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., C 1-6 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 6-10 aryl, and (d) C 1-6 ALC-C6-10 (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 It can be optionally substituted with alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of C1-alkaryl or C1-alkheterocyclyl can be further substituted with an oxo group to obtain the respective aryloyl and (heterocyclyl)oyl substituents.

[0181] A "cycloalkylalkyl" group, as used herein, represents a cycloalkyl group, as defined herein, attached to the parent molecular group through 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.

[0182] The term "diastereomers," as used herein, means stereoisomers that are not mirror images of one another and are not superimposable with respect to one another. The term "enantiomer," as used herein, means 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%.

[0183] The term "halo," as used herein, refers to a halogen selected from bromine, chlorine, iodine, or fluorine. The term "heteroalkyl," as used herein, refers to an alkyl group, as defined herein, in which one or two of its constituent carbon atoms are replaced with 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. The terms "heteroalkenyl" and "heteroalkynyl," as used herein, refer to alkenyl and alkynyl groups, as defined herein, respectively, in which one or two of its constituent carbon atoms are replaced with 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.

[0184] The term "heteroaryl," as used herein, refers to the subset of heterocyclyl, as defined herein, that is aromatic; that is, 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, heteroaryls are substituted with 1, 2, 3, or 4 substituents as defined for heterocyclyl groups.

[0185] The term "heteroarylalkyl" refers to a heteroaryl group, as defined herein, attached to the parent molecular group through 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-C1-12 In some embodiments, alkylene and heteroaryl can each be further substituted with 1, 2, 3, or 4 substituents, as defined herein for that group. Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

[0186] The term "heterocyclyl," as used herein, unless otherwise specified, 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 0 to 2 double bonds, and 6- and 7-membered rings have 0 to 3 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, e.g., 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 carbon rings, such as aryl rings, cyclohexane rings, cyclohexene rings, cyclopentane rings, cyclopentene rings, 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. Examples of 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. zolidinyl, 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, and the like, including dihydro and tetrahydro forms thereof in which one or more double bonds are reduced and replaced with hydrogen. Still other exemplary heterocyclyls include 2,3,4,5-tetrahydro-2-oxo-oxazolyl, 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-imida ... 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl), 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-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-oxopyridinyl, 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., 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl), 1,6-dihydro-6-oxo-pyridazinyl (e.g., 1,6-dihydro-6-oxo-3-ethylpyridazinyl), 1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl), 2,3-dihydro-2-oxo-1H-indolyl (e.g., 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 (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 ( Examples include 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-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 those of the formula:

[0187] [ka] Also mentioned are groups During the ceremony, 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 described herein can be selected from the group consisting of: (1) C 1-7 Acyl (e.g., carboxaldehyde), (2) C 1-20 Alkyl (e.g., C 1-6 Alkyl, C 1-6 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 2-12Heteroaryl), (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)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 1-6 Alkyl, (c) C 6-10 aryl, and (d) C 1-6 ALC-C 6-10 aryl), (19)—(CH) 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-C1-12 Heterocyclyl (e.g., C 1-6 ALC-C 1-12 heteroaryl), (26) oxo, (27) (C 1-12 Heterocyclyl)imino, (28)C 2-20 Alkenyl, and (29)C 2-20 The alkylene group of a C1-alkaryl or C1-alkheterocyclyl can be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkynyl, ...

[0188] A "heterocyclylalkyl" group, as used herein, represents a heterocyclyl group, as defined herein, attached to the parent molecular group through 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 that group.

[0189] The term "hydrocarbon," as used herein, 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.

[0190] The term "isomer," as used herein, refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers and / or double bonds and, therefore, can exist as stereoisomers, e.g., double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present invention, the chemical structures depicted herein, and therefore the compounds of the invention, encompass all corresponding stereoisomers, i.e., both stereomerically pure (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) forms, as well as enantiomeric and stereoisomeric mixtures, 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 compound as a chiral salt complex, crystallization of the compound in a chiral solvent, etc. Enantiomers and stereoisomers can also be obtained from stereomerically- or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0191] The term "N-protected amino," as used herein, refers to an amino group, as defined herein, attached to one or two N-protecting groups, as defined herein. The term "N-protecting group," as used herein, refers to a group intended to protect an amino group against undesired reactions during synthetic procedures. Commonly used N-protecting groups are described in Greene, "Protective Groups in Organic Synthesis," 3 rd Edition(John Wiley & Sons, New York, 1999). N-protecting groups include acyl groups, aryloyl groups, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries, such as protected or deprotected D, L, or D,L-amino acids, such as arabinose. amine, leucine, phenylalanine, etc.; sulfonyl-containing groups, such as benzenesulfonyl, p-toluenesulfonyl, etc.; 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).

[0192] The term "nitro" as used herein refers to the group --NO.sub.2. The term "O-protecting group," as used herein, 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 described in Greene, "Protective Groups in Organic Synthesis," 3 rdEdition (John Wiley & Sons, New York, 1999). 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, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyl, and the like. 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, etc.; haloalkoxycarbonyl groups, such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, optionally substituted arylalkoxycarbonyl groups, such as 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, such as For example, phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p-methyl-phenoxycarbonyl, 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, ethyl, 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, tribenzylsilyl, triphenylsilyl, and diphenylmethylsilyl), carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, vinyl, allyl, nitrophenyl, benzyl, methoxybenzyl, 3,4-dimethoxybenzyl, and nitrobenzyl), carbonyl protecting groups (e.g., acetal and ketal groups, such as dimethyl acetal, 1,3-dioxolane, and the like, acylal groups, and dithiane groups, such as 1,3-dithiane, 1,3-dithiolane, etc.), carboxylic acid protecting groups (e.g., ester groups, such as methyl esters, benzyl esters, t-butyl esters, orthoesters, etc.), and oxazoline groups.

[0193] The term "oxo" as used herein refers to =O. The prefix "perfluoro," as used herein, refers to an alkyl group, as defined herein, in which each hydrogen radical bonded to the alkyl group has been replaced by a fluoride radical. For example, perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like.

[0194] The term "protected hydroxyl," as used herein, refers to an oxygen atom bonded to an O-protecting group. The term "spirocyclyl" as used herein refers to a C cyclic group, both termini of which are attached to the same carbon atom of a parent group to form a spirocyclic group. 2-7 Alkylene diradicals, and furthermore both ends of which are attached to the same atom, C 1-6 represents a heteroalkylene diradical. The heteroalkylene radical 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 diradical is attached. The spirocyclyl groups of the present invention can be optionally substituted with 1, 2, 3, or 4 substituents provided herein as optional substituents for cycloalkyl and / or heterocyclyl groups.

[0195] The term "stereoisomer," as used herein, refers to all possible different isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may possess, particularly all possible stereochemical and conformational isomeric forms, all diastereomers, enantiomers, and / or conformers of the basic molecular structure. 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.

[0196] The term "sulfonyl" as used herein refers to the group -S(O)2-. The term "thiol" as used herein 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 encompass the specified element or step, whether presented by itself or with one or more additional elements or steps, (iv) the terms "about" and "approximately" may be understood to allow for standard deviation, as would be understood by one of ordinary skill in the art, and (v) when ranges are provided, the endpoints are included.

[0197] 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 can be fixed at an excess of the ligand concentration to mimic physiological conditions. Alternatively, or in addition, in some embodiments, the binding partner concentration and / or the ligand concentration can be varied. In some such embodiments, affinity can be compared to a reference under equivalent conditions (e.g., concentrations).

[0198] As used herein, the terms "approximately" and "about," when appropriate in the relevant context, are each intended to encompass normal statistical variations that would be understood by one 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 of (greater than or less than) the stated value, respectively, unless otherwise stated or unless the context makes clear otherwise (e.g., where such number may exceed 100% of the possible values).

[0199] It will be understood that the term "binding," as used herein, typically refers to an association (e.g., non-covalent or covalent) between or among two or more entities. "Direct" binding necessarily involves physical contact between the entities or moieties, while indirect binding necessarily involves 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 the context of a more complex system (e.g., covalently bound or otherwise associated with a carrier entity and / or in a biological system or cell).

[0200] The affinity of a molecule X for 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. Specific exemplary and illustrative embodiments for measuring binding affinity are described below. D The term "dissociation equilibrium constant" as used herein is intended to refer to the dissociation equilibrium constant of a particular compound-protein or complex-protein interaction. Typically, the compounds of the present 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, approximately 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 a concentration of about 10 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, approximately 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or even lower dissociation equilibrium constant (K D ) which binds 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).

[0201] As used herein, the term "crosslinking group" refers to a group that contains a reactive functional group that can be chemically attached to a specific functional group (e.g., primary amines, sulfhydryls) on a protein or other molecule. A "moiety that can chemoselectively react with an amino acid," as used herein, refers to a moiety that contains a reactive functional group that can be chemically attached to a functional group (e.g., primary and secondary amines, sulfhydryls, alcohols, carboxyl groups, carbonyls, or triazole-forming functional groups such as azides or alkynes) of a natural or unnatural amino acid. 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).

[0202] As used herein, the term "complex" refers to a group of two or more compounds and / or proteins that are bound together through 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 invention bound to a presenter protein or target protein.

[0203] As used herein, the term "conjugate" refers to a compound formed by the joining (e.g., via a covalent bond-forming reaction) of two or more chemical compounds (e.g., a compound comprising a cross-linking group and a protein, such as a target protein or a presenter protein).

[0204] As used herein, the term "electron-withdrawing group" refers to a functional group that removes electron density from a π-system. Examples of electron-withdrawing groups include, but are not limited to, halides (e.g., fluoride, chloride, bromide, iodide), aldehydes, ketones, carboxylic acids, acyl chlorides, esters, amides, trihalides (e.g., trifluoromethyl, trichloromethyl), nitriles, sulfonates, and nitro groups.

[0205] As used herein, the term "leaving group" refers to a molecular fragment that departs with a pair of electrons upon heterolytic bond cleavage. Examples of leaving groups include, but are not limited to, halides (e.g., fluoride, chloride, bromide, iodide), carboxylate, tosylate, mesylate, perfluoroalkylsulfonates (e.g., triflate), nitrate, and phosphate.

[0206] 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. The term "presenter protein" refers to a protein that binds to a small molecule to form a complex that binds to and regulates 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 participation in a trimolecular complex does not substantially affect the biological function of the presenter protein within the cell and / or the viability or other characteristics of the cell). In certain embodiments, the presenter protein is a protein that has chaperone activity within the cell. In some embodiments, the presenter protein is a protein that 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 suspected to bind to and regulate the biological activity of the target protein.

[0207] The term "presenter protein binding moiety" refers to a group of atoms involved in binding to a presenter protein and a moiety attached thereto (e.g., within 20 atoms, e.g., within 15 atoms, within 10 atoms, within 5 atoms), and 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 presenter protein 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). 50, thereby inhibiting the peptidyl-prolyl isomerase activity of the presenter protein. It will be understood that a presenter protein binding moiety does not necessarily encompass all atoms in a compound that interact with a 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).

[0208] 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., 2, 3, 4, 5) 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, 3-fold, 4-fold, 5-fold, or 10-fold) for all members of the FKBP family.

[0209] 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), and the compound has a K, e.g., of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM). D 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). 50It will be understood that a protein binding moiety does not necessarily encompass all of the atoms in a compound that interact with a protein.

[0210] As used herein, the term "react" refers to a 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, such as the reaction between a reactive amino acid and a cross-linking group on a protein to form a covalent bond. The reaction may be measured by any method known in the art; for example, the formation of a reaction product may be determined by LC-MS or NMR.

[0211] 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 that have 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.

[0212] 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 a tangible medium. 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 the determination or characterization of the compound, individual, population, sample, sequence, or value of interest.

[0213] 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.

[0214] As used herein, the terms "specific binding" or "specific for" or "specific for" refer to an interaction between a binder and a target entity. As will be understood by one of skill in the art, an interaction may occur when it is favored in the presence of an alternative interaction, e.g., a K 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). DA binding agent is considered "specific" if it binds to the target entity at a specific binding site. In many embodiments, 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 can be assessed relative to the specificity of the binding agent for one or more other potential 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 non-specific binding agent.

[0215] The term "specific," when used in reference to a compound having activity, is understood by those skilled in the art to mean that the compound discriminates between potential target entities or conditions. For example, in some embodiments, a compound is said to "specifically" bind to a target if it preferentially binds to that target in the presence of one or more competing surrogate targets. 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 can be assessed relative to the specificity of a binding agent for one or more other potential 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 slower off-rate, increased affinity, reduced dissociation, and / or increased stability compared to competing surrogate target(s).

[0216] The term "substantially" refers to the qualitative condition of exhibiting a total or nearly total extent or degree of a characteristic or property 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 to perfection 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.

[0217] The term "does not substantially bind" to a particular protein, as used herein, refers to, for example, a target that does not bind to the target at all. -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 molecule can be represented by a molecule or a portion of a molecule having the following structure:

[0218] 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 trimolecular 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 a natural interaction with one or more natural presenter protein / natural small molecule complexes. In some embodiments, the target protein is characterized by a natural interaction with multiple different natural presenter protein / natural small molecule complexes; in some such embodiments, some or all of the complexes utilize the same presenter protein (and different small molecules). In some embodiments, the target protein does not substantially bind to a complex of cyclosporine, rapamycin, or FK506 and a presenter protein (e.g., FKBP). The target protein can be naturally occurring, e.g., wild-type. Alternatively, the target protein can be mutated from the wild-type protein but still maintain biological function, e.g., as an allelic variant, splice mutant, or 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 biological pathways associated with a disease, disorder, or condition.

[0219] In some embodiments, the target protein is a modified target protein. Modified target proteins can include conservative or non-conservative amino acid insertions, deletions, or substitutions in the protein sequence (e.g., D-amino acids, desamino acids) (e.g., in cases where such changes do not substantially alter the biological activity of the polypeptide). Specifically, 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., cysteines) are removed to reduce the number of potential conjugation sites on the protein. Amino acid substitutions can be conservative (i.e., replacing a residue with another residue of the same genus type or group) or non-conservative (i.e., replacing a residue with an amino acid of a different type). In addition, naturally occurring amino acids can be substituted with non-naturally occurring amino acids (i.e., non-naturally occurring conservative amino acid substitutions or non-naturally occurring non-conservative amino acid substitutions).

[0220] The term "target protein binding moiety" refers to the group of ring atoms 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, and the moiety attached thereto (e.g., within 20 atoms, e.g., within 15 atoms, within 10 atoms, within 5 atoms). 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.

[0221] 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 recognize 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.

[0222] The term "undruggable target" refers to a protein that is not a member of a protein family known to be targeted by drugs and / or does not have a binding site suitable for high-affinity binding to a small molecule. Methods for determining whether a target protein is undruggable are known in the art. For example, whether a target protein is undruggable can be determined using a structure-based algorithm such as that used by the program DOGSITESCORER® (Universität 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]

[0223] [Figure 1] 1 is an image illustrating SDS-PAGE analysis of KRASGTP / S39C lite / C2-FK506 conjugates: Lane 1: KRASGTP / S39C lite, Lane 2: KRASGTP / S39C lite / C2-FK506 reaction mixture, Lane 3: KRASGTP / S39C lite / C2-FK506 reaction mixture + 100 mM DTT. [Figure 2] 1 is an image illustrating SDS-PAGE analysis of KRASGTP / G12C lite / SFAX9DS conjugate. [Figure 3A] 1 is an image illustrating SEC and SDS-PAGE analysis of KRASGTP / S39C lite / C2Holt / FKBP12 complex formation. SEC purification profile. The blue dotted line indicates the peak corresponding to the elution of the KRASGTP / S39C lite / C2Holt / FKBP12 ternary complex. [Figure 3B] 1 is an image illustrating SEC and SDS-PAGE analysis of KRASGTP / S39C lite / C2Holt / FKBP12 complex formation. SDS-PAGE analysis of the SEC elution peak. The blue dotted line corresponds to the fractions collected for the KRASGTP / S39C lite / C2Holt / 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 PTP1BS187C lite and FKBP12 proteins and the PTP1BS187C lite / C3SLF / FKBP12 complex. [Figure 5B] 1 is an image illustrating the SEC profile and SDS-PAGE analysis of free PTP1BS187C lite and FKBP12 proteins and the PTP1BS187C lite / C3-SLF / FKBP12 complex. [Figure 6] 1 is an image illustrating the cross-linking efficiency of C3 and C4SLF by SDS-PAGE. [Figure 7] Figures 7A and 7B are images illustrating the crystal structure of the FKBP12-compound 1-KRASGTP / S39C complex. Figure 7A) 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) 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 9] Figures 9A and 9B are images illustrating the crystal structure of FKBP12-C3SLF-PTP1BS187C. Figure 9A illustrates that the crystal contains two complex molecules of FKBP12-C3SLF-PTP1BS187C in the asymmetric unit. Figure 9B illustrates that the buried surface area of ​​PTP1BS187C is 427 Å and that of C3SLF 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] 1 is an image illustrating the 2D 1H-15N TROSY-HSQC spectrum of KRASG12C-GTP. [Figure 17B] 1 is an image illustrating the addition of a stoichiometric amount of CYPA. [Figure 17C] Images illustrating KRAS and CYPA alone. DETAILED DESCRIPTION OF THE INVENTION

[0224] Small molecules are limited in their targeting ability because their interaction with targets is driven by adhesive forces, the strength of which is roughly proportional to the contact surface area.Due to their small size, the only way for small molecules to create sufficient intermolecular contact surface area to effectively interact with target proteins is to literally be incorporated by the protein.In fact, both a large amount of experimental and computational data supports the view that only proteins with hydrophobic "pockets" on their surface can bind small molecules.In this case, binding is possible through incorporation.

[0225] Nature has evolved 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 between small molecules and small display proteins. The combined surfaces of the small molecules and display proteins associate with 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.

[0226] Many important therapeutic targets exert their function through complexation with other proteins. The protein / protein interaction surface in many of these systems contains an inner core of hydrophobic side chains surrounded by a broad ring of polar residues. Hydrophobic residues contribute nearly all of the energetically favorable contacts, and this cluster has therefore been designated a "hot spot" for association in protein-protein interactions. Importantly, in the aforementioned complexes between naturally occurring small molecules and 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.

[0227] Nature has demonstrated the ability to reprogram the target specificity of presented small molecules—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, which interacts with an entirely different target, TorC1. To date, no method has been developed to reprogram the binding and regulatory capabilities of the presenter protein / ligand interface so that it can interact with and regulate other target proteins previously considered undruggable.

[0228] In addition, it is well established that some drug candidates do not function well to regulate the activity of both their intended target and other unintended proteins. 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 typically have unacceptable side effects of also modulating 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.

[0229] 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. 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 for 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 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. The present invention also relates to conjugates comprising a protein-binding moiety conjugated to a protein, for example, a presenter protein-binding moiety conjugated to a target protein or a target protein-binding moiety conjugated to a presenter protein.

[0230] The present invention relates to a compound of formula VII: ALB Formula VII Also relates to the compound wherein A is a group represented by formula VIII:

[0231] [ka] Includes the structure of

[0232] In some embodiments, the compounds of the present invention are

[0233] [ka]

[0234] [ka] 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, 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).

[0235] Exemplary crosslinking 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, Michael acceptor group (e.g., α,β-unsubstituted ketone or sulfone), epoxide, heteroaryl nitrile, and azide. Presenter protein binding site In some embodiments, the compounds of the present invention comprise a presenter protein binding moiety. In some embodiments, the presenter protein binding moiety comprises a group of atoms (e.g., 5-20 atoms, 5-10 atoms, 10-20 atoms) that participate in binding to the presenter protein, and may include any moiety attached thereto (e.g., within 20 atoms, e.g., within 15 atoms, within 10 atoms, within 5 atoms), and the compounds provided have a K 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). Dor specifically binds to the presenter protein 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). 50 In some embodiments, the presenter protein binding moiety does not encompass all atoms in the provided compounds 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.

[0236] In some embodiments, the presenter protein binding moiety comprises an N-acylproline moiety, an N-acyl-pipecolic acid moiety, an N-acyl 3-morpholino-carboxylic acid moiety, and / or an N-acylpiperdic 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-morpholino-carboxylic acid moiety. In some embodiments, the presenter protein binding moiety comprises an N-acylpiperdic acid moiety.

[0237] 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.

[0238] In some embodiments, the presenter protein binding moiety has Formulas II-IV:

[0239] [ka] It has a structure according to the following.

[0240] In some embodiments, the presenter protein binding moiety has the structure:

[0241] [ka]

[0242] [ka]

[0243] [ka]

[0244] [ka] or comprising or consisting of a stereoisomer thereof.

[0245] The presenter protein can be bound to an atom within the presenter protein binding moiety. Alternatively or additionally, the presenter protein can be bound to two or more atoms within the presenter protein binding moiety. In another alternative, the presenter protein can be bound to a substituent attached to one or more atoms within the presenter protein binding moiety. Furthermore, in some embodiments, the presenter protein can be bound to an atom within the presenter protein binding moiety and to a substituent attached to one or more atoms within the presenter protein binding moiety. In some embodiments, the presenter protein is bound to a group that mimics the natural ligand of the presenter protein, wherein the group that mimics the natural ligand of the presenter protein 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 is increased 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 exclusively, due to non-covalent interactions of the presenter protein with the presenter protein binding moiety. 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, 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). In some embodiments, the target protein binding moiety comprises a group of atoms (e.g., 5-20 atoms, 5-10 atoms, 10-20 atoms) that specifically binds to the target protein, and can include any moiety attached thereto (e.g., up to 20 atoms, up to 15 atoms, up to 10 atoms, up to 5 atoms). In some embodiments, the target protein binding moiety 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.

[0246] 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. In another alternative, the target protein can bind to a substituent attached to one or more atoms in the target protein binding moiety. In another alternative, the target protein can bind to an atom in the target protein binding moiety and to a substituent attached to one or more atoms in the target protein binding moiety. In another alternative, the target protein is bound to a group that mimics the target protein's natural ligand, where the group that mimics the target protein's natural ligand is attached to the target protein binding moiety. In yet another alternative, the target protein binds to a presenter protein, and the affinity of the target protein for the presenter protein in the binary complex is increased relative 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, due to non-covalent interactions of the target protein to the target protein binding moiety.

[0247] In some embodiments, the target protein binding moiety comprises a cross-linking group (eg, an internal cross-linking group). Linker The compounds of the invention comprise a linker (e.g., a moiety linker) that joins a protein-binding moiety (e.g., a presenter protein-binding moiety or a target protein-binding moiety) to a cross-linking 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, in its simplest form, is a bond, but can also provide a linear, cyclic, or branched molecular scaffold with pendant groups covalently linking the two moieties.

[0248] In some embodiments, at least one atom of the linker is involved in binding to the presenter protein and / or the target protein, while in certain embodiments, at least one atom of the linker is not involved in binding to the presenter protein and / or the target protein.

[0249] Thus, when included within compounds and / or conjugates as described herein, a 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 employed 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.

[0250] In some embodiments, such covalent linking of two (or more) moieties can be effected using a linker that contains a reactive moiety that can react with such a functional group present on either moiety. For example, an amine group on a moiety can react with a carboxyl group, or an activated derivative thereof, on the linker, resulting in the formation of an amide linking the two.

[0251] Examples of moieties capable of reacting with sulfhydryl groups include α-haloacetyl compounds of the XCH2CO- type (where X = Br, Cl, or I), which not only exhibit specific reactivity toward sulfhydryl groups but can also be used to modify imidazolyl, thioether, phenol, and amino groups, as described in Gurd, Methods Enzymol. 11:532 (1967). 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 (Traut et al., Biochemistry 12:3266 (1973)), which introduce thiol groups through conversion of amino groups, can be considered sulfhydryl reagents when linkage occurs through the formation of disulfide bridges.

[0252] Examples of reactive moieties that can react with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include: (i) α-haloacetyl compounds, which exhibit specificity for amino groups in the absence of reactive thiol groups and are of the XCHCO- (where X = Br, Cl, or I) type, as described, for example, in Wong Biochemistry 24:5337 (1979); (ii) N-maleimide derivatives, which can react with amino groups through a Michael-type reaction or through 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, such as those described in McKenzie et al., J. Protein Chem. 7:581 (1988); (v) aldehydes and ketones that can form Schiff bases with amino groups; the adducts formed are usually stabilized through reduction to give stable amines; (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 in Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening. (ix) squaric acid diethyl ester, which is 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. 28:463 (1993); Examples include:

[0253] Representative amino-reactive acylating agents include: (i) isocyanates and isothiocyanates, especially aromatic derivatives, which form stable urea and thiourea derivatives, respectively; (ii) sulfonyl chlorides, which are described in Herzig et al., Biopolymers 2:349 (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, such as those described in M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984: (vii) acyl azides, for example, the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974); (viii) imidoesters, which form stable amidines upon reaction with amino groups, as described, for example, in Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962), and (ix) Haloheteroaryl groups, such as halopyridine or halopyrimidine.

[0254] Aldehydes and ketones can react with amines to form Schiff bases, which can be advantageously stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, as described, for example, in Webb et al., Bioconjugate Chem. 1:96 (1990).

[0255] 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, that react through O-acylurea formation and subsequent amide bond formation are also available.

[0256] It should be understood that functional groups on either moiety may be converted to other functional groups, if desired, 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, or 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 or 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.

[0257] So-called zero-length linkers, which involve direct covalent joining of a reactive chemical group on one moiety to a reactive chemical group on another moiety without introducing an additional linker, can be used in accordance with the present invention if desired.

[0258] 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 within either moiety, resulting in a covalent linkage between the two. The reactive moieties within a linker can be the same (homobifunctional linker) or different (heterobifunctional linker, or, if several dissimilar reactive moieties are present, heteromultifunctional linker), providing a variety of potential reagents that can effect covalent attachment between two moieties.

[0259] The spacer elements in the linker are typically linear or branched chains, and 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.

[0260] In some examples, the linker is represented 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.

[0261] 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.

[0262] 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 Formula XIX having the structure 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 optionally substituted C1-C2 alkyl, optionally substituted C1-C3 heteroalkyl, O, S, and NR N Selected from R N is hydrogen, optionally substituted C 1-4 Alkyl, optionally substituted C 2-4 Alkenyl, optionally substituted C 2-4 Alkynyl, optionally substituted C 2-6 Heterocyclyl, optionally substituted C 6-12 aryl, or optionally substituted C 1-7 Heteroalkyl, 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; 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 -(B 1 ) a-(C 1 ) b -(B 2 ) c -(B 3 ) d -(C 2 ) e -(B 4 ) f -A 2 It is a chemical bond that connects protein Presenter Protein A presenter protein can bind to a small molecule to form a complex, and this complex can bind to and regulate 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 participation in a trimolecular complex does not substantially adversely affect the biological function of the presenter protein within the cell and / or the viability or other characteristics of the cell). In some embodiments, the presenter protein is more abundant than the 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, presenter proteins are proteins known to bind small molecules to form binary complexes that are known or suspected to bind to target proteins and regulate their biological activity. Immunophilins are a class of presenter proteins known to have these functions, including FKBPs and cyclophilins. In some embodiments, the reference presenter protein exhibits peptidyl prolyl isomerase activity; in some embodiments, the presenter protein exhibits activity comparable to the reference presenter protein.In certain embodiments, the presenter protein is a member of the FKBP family (e.g., FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36, FKBP38, FKBP51, FKBP52, FKBP60, FKBP65, and FKBP133), a member of the 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 and function as protein folding chaperones for proteins containing proline residues. Genes encoding proteins in this family include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and LOC541473.

[0263] The "cyclophilin family" is a family of proteins that bind to cyclosporine. Genes encoding proteins in this family include PPIA, PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1, PPIL2, PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary 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.

[0264] 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.

[0265] 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 the amino acid sequence of a reference presenter protein, ii) contains a site that shows significant identity to a corresponding site in the reference presenter protein, and / or iii) contains at least one characteristic sequence found in the presenter protein. In many embodiments, identity is considered to be "significant" by definition of a presenter protein if it is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In some embodiments, the sites showing significant identity are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 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 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 amino acids or more in length.

[0266] Representative presenter proteins are encoded by the genes listed in Table 1 or their homologs. In some embodiments, reference presenter proteins are encoded by the set of genes set forth 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 particular subsets of presenter proteins.

[0267] [Table 1] 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 states or symptoms of disease states. Therefore, modulating (inhibiting or increasing) their activity can achieve a desired therapeutic effect. Target proteins useful in the complexes and methods of the present invention include those that do not naturally associate with presenter proteins, e.g., those that have an affinity for presenter proteins 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 present invention. Alternatively, target proteins that do not naturally associate with presenter proteins have an affinity for compounds of the present 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. In another alternative, target proteins that do not naturally associate with presenter proteins have an affinity for binary complexes of cyclosporine, rapamycin, or FK506 with a presenter protein (e.g., FKBP) of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM. In yet another alternative, the target protein that does not naturally associate 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 and would not be used with the latter.

[0268] A target protein can be naturally occurring, e.g., wild-type, or it can differ from the wild-type protein but still maintain biological function, e.g., as an allelic variant, splice mutant, or biologically active fragment.

[0269] 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.

[0270] In some embodiments, target proteins of the invention are characterized by comprising one or more surface sites (e.g., planar surface sites) to which small molecules typically exhibit low or undetectable binding in the absence of presenter protein / compound complex formation. In some embodiments, target proteins comprise one or more surface sites (e.g., planar surface sites) to which particular small molecules (e.g., compounds) exhibit low or undetectable binding in the absence of presenter protein / compound complex formation (e.g., at least 2 / 3, 4 / 5, 6 / 7, 8 / 9, 10 / 10, 20 / 30, 40 / 50, 100 / 100, or less, of the binding observed for a presenter protein / compound complex containing the same compound). In some embodiments, the target protein has a surface (in some embodiments, the entire surface) characterized by one or more sites lacking any conventional binding pocket, e.g., a protein structural cavity or pocket with physiochemical and / or geometric properties comparable to those of a protein whose activity is regulated by one or more small molecules. In certain embodiments, the target protein has a conventional binding pocket and a site for protein-protein interaction. In some embodiments, the target protein is an undruggable target, e.g., the target protein is not a member of the protein family known to be targeted by drugs and / or does not have a binding site predicted to be suitable for binding to a small molecule (e.g., according to accepted understanding in the art, as described herein). In some embodiments, the protein contains at least one reactive cysteine.

[0271] The only way to do this is to use the GTP interface DIRAS1、DIRAS2、DIRAS3、ERAS、GEM、H RAS、KRAS、MRAS、NKIRAS1、NKIRAS2、NRAS、RALA、RALB、RAP1A、RAP1B、RAP2A RAP2B, RAP2C, RASD1, RASD2, RASL10A, RASL10B, RASL11A, RASL11B, RASL1 2, REM1, REM2, RERG, RERGL, RRAD, RRAS, RRAS2, RHOA, RHOB, RHOBTB1, RHOBT B2, RHOBTB3, RHOC, RHOD, RHOF, RHOG, RHOH, RHOJ, RHOQ, RHOU, RHOV, RND1, R ND2, RND3, RAC1, RAC2, RAC3, CDC42, RAB1A, RAB1B, RAB2, RAB3A, RAB3B, RAB 3C, RAB3D, RAB4A, RAB4B, RAB5A, RAB5B, RAB5C, RAB6A, RAB6B, RAB6C, RAB7A 、RAB7B、RAB7L1、RAB8A、RAB8B、RAB9、RAB9B、RABL2A、RABL2B、RABL4、RAB1 、RAB11A、RAB11B、RAB12、RAB13、RAB14、RAB15、RAB17、RAB18、RAB19、RAB2 、RAB21、RAB22A、RAB23、RAB24、RAB25、RAB26、RAB27A、RAB27B、RAB28、RAB2 B、RAB30、RAB31、RAB32、RAB33A、RAB33B、RAB34、RAB35、RAB36、RAB37、RAB3 8, RAB39, RAB39B, RAB40A, RAB40AL, RAB40B, RAB40C, RAB41, RAB42, RAB43 RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, ARF1, ARF3, ARF4, ARF5, ARF6, ARL1, AR L2, ARL3, ARL4, ARL5, ARL5C, ARL6, ARL7, ARL8, ARL9, ARL10A, ARL10B, ARL1 0C、ARL11、ARL13A、ARL13B、ARL14、ARL15、ARL16、ARL17、TRIM23、ARL4D、AR FRP1, 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, such as 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, GYF , HEAT, HECT, IQ, LRR, MBT, MH1, MH2, MIU, NZF, PAS, PB1, PDZ, PH, POLO-Box, PTB, PUF, PWWP, PX, RGS, RING, SAM, SC, SH2, SH3, SOCS, SPRY, START, SWIRM, TIR, TPR, TRAF, SNARE, TUBBY, TUDOR, UBA, UEV, UIM, VHL, VHS, WD40, WW, SH2, SH3, TRAF, bromodomain, or TPR. In some embodiments, the target protein is a heat shock protein, e.g., Hsp20, Hsp27, Hsp70, Hsp84,In certain embodiments, the target protein is alpha B-crystallin, TRAP-1, hsf1, or Hsp90. In certain embodiments, the target protein is an ion channel, such as 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, such as 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., cyclin D1, 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, A In some embodiments, the target protein is a phosphatase, such as WIP1, SHP2, SHP1, PRL-3, PTP1B, or STEP. In certain embodiments, the target protein is a ubiquitin or ubiquitin-like protein (e.g., NEDD8, ATG8 protein, SUMO protein, ISG15), an activating enzyme (E1's, e.g., UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, SAE1), a conjugation enzyme (E2's, e.g., UBE protein, ATG3, BIRC6), a ligation enzyme (E3's, 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, BHLHA1 5, BHLHB2, BHLBHB3, BHLHE22, BHLHE23, BHLHE41, CLOCK, FIGLA, HAS5, HES7, HEY1, HEY2, ID4, MAX, MESP1, MLX, MLXIP L, MNT, MSC, MYF6, NEUROD2, NEUROG2, NHLH1, OLIG1, OLIG2, OLIG3, SREBF2, TCF3, TCF4, TFAP4, TFE3, TFEB, TFEC, USF 1, ARF4, ATF7, BATF3, CEBPB, CEBPD, CEBPG, CREB3, 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,DLX2,DL X3, 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, HOXA10, 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, FOXO3, FOXB1, FOXC1, FOXC2, FOXD2, FOXD3, FOXG1, FOXI1, FOXJ2, FOXJ3, FOXK1, FOXL1, FOXO1, FOXO4, FOXO6, FOXP3, EOMES, MGA, NFAT5, NFATC1, NFKB1, NFKB2, TP63, RUNX2, RUNX3, T, TBR1, TBX1, TBX15, TBX19, TBX2, TBX20, T BX21, TBX4, TBX5, AR, ESR1, ESRRA, ESRRB, ESRRG, HNF4A, NR2C2, NR2E1, NR2F1, NR2F6, NR3C1, NR3C2, NR4A2, RARA, RARB, RA a transcription factor encoded by RG, RORA, RXRA, RXRB, RXRG, THRA, THRB, VDR, GATA3, GATA4, or GATA5, or C-myc, Max, Stat3, Stat4, Stat6, androgen receptor, C-Jun, C-Fox, N-Myc, L-Myc, MITF, Hif-1alpha, Hif-2alpha, Bcl6, E2F1, NF-kappaB, Stat5, or ER(coact). In certain embodiments, the target protein is selected from the group consisting of TrkA, P2Y14, 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, and Ane These include cytosine family members, BCOR, NCOR, beta-catenin, AAC11, PLD1, PLD2, Frizzled7, RaLP, 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, and Nrlp3. Protein variants Protein or polypeptide variants, as described herein, generally exhibit significant (e.g., 80% or greater, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to a reference polypeptide (e.g., a presenter protein or target protein described herein, such as, for example, a mammalian presenter protein or target protein), but may exhibit a limited number of specific amino acid sequences relative to the reference polypeptide. In certain embodiments, the variants share a relevant biological activity (e.g., binding to a particular compound or portion thereof) with the reference polypeptide; in some such embodiments, the variants exhibit such activity at a level that is about 50% or greater than that of the reference polypeptide and / or at a level that is no less than about 0.5-fold lower than that of the reference polypeptide.

[0272] 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 a greater number of cysteine ​​residues and / or has 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 described herein (e.g., of a presenter protein and / or of a target protein) can facilitate conjugation of such polypeptides, e.g., by disulfide bonding. In some embodiments, amino acid substitutions can be conservative (i.e., replacing a residue with another residue of the same genus type or group) or non-conservative (i.e., replacing a residue with an amino acid of a different type). In some embodiments, a naturally occurring amino acid can be substituted with 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.

[0273] Synthetically produced polypeptides can include substitutions of amino acids that are not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acids). Examples of non-naturally occurring 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 formula NH(CH) n Omega amino acids include COOH (where n is 2-6), neutral nonpolar amino acids such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine. Phenylglycine may replace Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar; cysteic acid is acidic; and ornithine is basic. Proline may be substituted with hydroxyproline, retaining the conformation that gives it its properties.

[0274] Analogs can be generated by substitutional mutagenesis and can 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, such as those referred to as "exemplary substitutions" in Table 2 or further described herein in relation to amino acid classes, are introduced and the products screened.

[0275] 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 charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulk of the side chain. Naturally occurring residues are grouped 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 / negative charge: aspartic acid (Asp), glutamic acid (Glu) (4) Basic: asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg) (5) Residues that affect chain orientation: glycine (Gly), proline (Pro), (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), histidine (His), (7)Polarity: Ser, Thr, Asn, Gln (8) Basic positive charge: Arg, Lys, His, and (9) Charge: Asp, Glu, Arg, Lys, His Other amino acid substitutions are listed in Table 2.

[0276] [Table 2] 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 can be conservative (i.e., replacing a residue with another residue of the same genus type or group) or non-conservative (i.e., replacing a residue with an amino acid of a different type). In addition, naturally occurring amino acids can be substituted with non-naturally occurring amino acids (i.e., non-naturally occurring conservative amino acid substitutions or non-naturally occurring non-conservative amino acid substitutions).

[0277] As known in the art, see, for example, Chin, JW, Expanding As described in "Reprogramming the Genetic Code of Cells and Animals," Annual Review of Biochemistry, Vol. 83:379-408, unnatural amino acids can be used in These side chains can be incorporated into proteins made in vitro. For example, in one system, the UAG amber (stop) codon was used to incorporate pyrrolysine via archaeal tRNA synthetase and tRNA, and 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-propynyloxy)carbonyl]-L-lysine, bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN), N-5-norbornen-2-yloxycarbonyl-L-lysine, N-tert-butyloxycarbonyl-L-lysine, and N-tert-butyloxycarbonyl-L-lysine. N-propynyl-L-lysine, N-2-azidoethyloxycarbonyl-L-lysine, N-thiaprolyl-L-lysine, N-D-cysteinyl-L-lysine, N-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. Complex In naturally occurring protein-protein interactions, binding events are typically driven primarily by hydrophobic residues on flat surface sites of the interacting proteins, in contrast to many small molecule-protein interactions, which are driven by interactions between small molecules within cavities or pockets on the protein. Generally, hydrophobic residues on flat surface sites of proteins form hydrophobic hotspots, where the majority of binding interactions between or within interacting proteins are van der Waals interactions. In some situations, small molecules may provide "portable hotspots" (or sites thereof), for example, by engaging in or generating hydrophobic interaction sites on a protein (e.g., a presenter protein) that would not exist in the absence of the small molecule. Aspects of the present disclosure are particularly applicable to such situations. For example, in some embodiments, compounds as described herein (and / or tagged forms thereof) form complexes with proteins (e.g., presenter protein / compound complexes) and participate in pseudoprotein-protein interactions (e.g., formation of a trimolecular complex with a target protein).

[0278] 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 a variety of 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 both a common structural organization and a resulting function that provides 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, binary complexes (e.g., between a compound and a presenter protein, or between a compound and a target protein) are mimicked by conjugating a presenter protein-binding moiety to the target protein, or a target protein-binding moiety to the presenter protein. The resulting conjugates of the present invention can then bind to the presenter protein or the target protein to form complexes that mimic the trimolecular 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 compounds of the present invention can be used, for example, to identify target proteins that can bind to the presenter protein. Purpose Target protein identification In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the 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.

[0279] The majority of 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 the 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 compounds that can simultaneously bind to both the presenter protein and the target protein.

[0280] 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 therapeutic agents capable of modulating the biological activity of the target protein.

[0281] In some embodiments, 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. compound design In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful in the design of compounds capable of modulating the biological activity of a target protein for use in the treatment of disease.

[0282] 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 that can promote 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 for constructing structures de novo and / or fragment-based drug design using methods such as fragment soaking of crystals of the complex of the present invention and determination of the resulting structure.

[0283] Compounds designed as described above can then be screened to determine their ability to modulate the biological activity of the target protein and, if desired, modified using medicinal chemistry techniques to generate therapeutically useful compounds. Identification of covalent small molecule therapeutics In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the present invention may be useful for identifying compounds that can modulate the biological activity of a target protein through covalent interactions.

[0284] For example, compounds of the present invention can be screened for their ability to covalently bind to a target protein in the presence and absence of a presenter protein to identify compounds that can selectively bind to the target protein only in the presence of the presenter protein. These compounds can then be tested for their ability to modulate the biological activity of the target protein and, if necessary, modified using medicinal chemistry techniques to generate therapeutically useful compounds. Determination of biochemical and / or biophysical properties In some embodiments, the compounds, conjugates, complexes, compositions, and / or methods of the invention may be useful in determining the biochemical and / or biophysical properties of a protein or complex.

[0285] 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. i , K. inact , and / or K i / K inact can be determined, for example, by mass spectrometry. 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 effects through their ability to modulate (e.g., positively or negatively 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) through interaction with a presenter protein and the target protein. kit In some embodiments, the present invention relates to kits for conveniently and effectively carrying out the methods of the present invention. Typically, pharmaceutical packs or kits include one or more containers filled with one or more of the components of the pharmaceutical compositions of the present invention. Such kits are particularly suitable for delivering solid oral dosage forms, such as tablets or capsules. Such kits preferably include several unit doses and may also include a card with the dosages listed in the order of their intended use. Optionally, for example, if the subject suffers from Alzheimer's disease, a memory aid can be provided, for example, in the form of numbers, letters, or other designations, or with a calendar insert designating the days within a treatment schedule on which the dosages can be administered. Alternatively, placebo doses or calcium dietary supplements, similar to or different from the dosages of the pharmaceutical compositions, can be included to provide a kit in which the dosages are to be taken daily. Optionally associated with such container(s) can be a notice in a form specified by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the regulatory agency 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 to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or therapy), the compounds will be formulated in a manner consistent with these parameters. An overview of such technology is provided in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams & Wilkins, (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0286] 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 administration by injection, inhalation, or direct contact with the 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.

[0287] Generally, for use in treatment, the compounds described herein can be used alone or in combination with one or more other active agents. Examples of other pharmaceutical agents to be combined with the compounds described herein would include pharmaceutical agents for the treatment of the same indication. Other examples of pharmaceutical agents that may be combined with the compounds described herein would include pharmaceutical agents for the treatment of different, but associated or related, symptoms or indications. Depending on the mode of administration, the compounds are formulated into suitable compositions to allow for easy delivery. Each compound in the combination therapy can be formulated in various ways known in the art. For example, the first and second active agents of the combination therapy can be formulated together or separately. Desirably, the first and second active agents are formulated together for simultaneous or near-simultaneous administration of the agents.

[0288] The compounds of the present invention can 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.

[0289] The formulation can be prepared in a manner suitable for systemic administration or local or topical administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, subcutaneous injection), or can 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 can also be administered in a liposome composition or as a microemulsion.

[0290] 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, such as, for example, sodium acetate, sorbitan monolaurate, and the like.

[0291] Various sustained release systems for drugs have also been devised, see, for example, U.S. Patent No. 5,624,677, which is incorporated herein by reference. Systemic administration can also include relatively non-invasive methods, such as suppositories, transdermal patches, transmucosal delivery, and intranasal administration. Oral administration is also suitable for the compounds of the present invention. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

[0292] Each compound of the combination therapy, as described herein, can be formulated in a variety of ways known in the art, for example, the first and second agents of the combination therapy can be formulated together or separately.

[0293] Individually or separately formulated active substances can be packaged together as a kit. Non-limiting examples include, but are not limited to, a kit containing two types of pills, a pill and a powder, a vial containing a suppository and a liquid, two types of topical creams, etc. The kit can 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. In addition, the unit dose kit can contain instructions for preparing and administering the composition. The kit can be manufactured as a single-use unit dose for one subject, as multiple uses for a specific subject (at a fixed dose or with varying potency of the individual compounds over the course of treatment), or the kit can contain multiple doses suitable for administration to multiple subjects ("bulk packaging"). The components of the kit can be assembled in a carton, blister pack, bottle, tube, etc.

[0294] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including 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), as well as lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0295] 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 may be contained on the inside of a tablet and a second compound on the outside, allowing a substantial portion of the second compound to be released before the release of the first compound.

[0296] Formulations for oral use can 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 can be prepared using the ingredients described above for tablets and capsules in a conventional manner, for example, using a mixer, fluidized bed equipment, or spray drying equipment.

[0297] 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 within a suitable matrix. Controlled release coatings include one or more of the coating materials described above and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In controlled-release matrix formulations, the matrix material may also 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.

[0298] Liquid forms into which the compounds and compositions of the present invention can be incorporated for oral administration include solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions, including edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0299] 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 and can be easily 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.

[0300] Administration of each drug in the combination therapy, as described herein, can be independently 1 to 4 times daily for 1 day to 1 year, or even for the life of the subject. Chronic, long-term administration may be required. [Example]

[0301] Example 1: Synthesis of certain cross-linking reagents Synthesis of acrylamide-containing cyclosporin analogues.

[0302] [ka] All reagents and solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. Fmoc-amino acids, HATU, HOAT, H-Ala-2-Cl-(Trt) resin (0.36 mmol / g), H-Leu-2-Cl-(Trt) resin (0.30 mmol / g), H-Phe-2-Cl-(Trt) resin (0.35 mmol / g), and H-Thr(tBu)-2-Cl-(Trt) resin (0.36 mmol / g) were purchased from GL Biochem (Shanghai) Ltd.

[0303] Coupling of linear peptides was carried out on an automated synthesizer using standard FmoC SPSS procedures. General Method A: Linear peptides were synthesized using a TETRAS™ synthesizer on a scale of 0.025 mmol of resin. The general protocol was as follows: 2×NMP, 30 s; 1×20% (vol / vol) piperidine in NMP, 15 min; 5×NMP, 30 s; a solution of amino acid (3 equivalents) in NMP was added to the vessel containing the resin, followed by the addition of solutions of HATU and DIEA in DMF, respectively, and coupling for 45 min; 3×NMP, 30 s. The double coupling strategy was applied to all amino acids. General Method B: Boc-7mer Addition The coupling was accomplished on a TETRAS™ synthesizer using the same general protocol as for amino acid coupling, except that the amount of Boc-7mer was 1.5 equivalents. Only one coupling was required. General Method C: Removal of the ivDde protecting group.

[0304] Removal of the ivDde protecting group on the Dap side chain was achieved on a TETRAS™ synthesizer. General protocol: A solution of 20% (vol / vol) hydrazine monohydrate in NMP was added to the vessel containing the resin. The vessel was shaken for 30 minutes. The resin was drained and rinsed with 5 x 5 mL (30 seconds) of NMP. General Method D: Addition of acrylic acid onto the side chain amino group of Dap.

[0305] Coupling was achieved on a TETRAS™ synthesizer by using the same general protocol as for amino acid coupling. A double coupling strategy was applied. General Method E: Deprotection of side chain protecting groups and final cleavage from the resin.

[0306] Global deprotection and cleavage from the resin were achieved with a TFA cocktail for 1-2 hours at room temperature. A cleavage cocktail (TFA / TIPS / HO, 95 / 2.5 / 2.5) or (TFA / DCM / TIPS, 40:60:1) can be used for the final cleavage. Most of the solvent was removed under reduced pressure, and the residue was concentrated in vacuo to remove traces of solvent. The resulting residue was used directly in the subsequent cyclization without further purification. General Method F: Cyclization of Linear Peptides The crude linear peptide was dissolved in dry DCM to a final concentration of 0.1 M. HATU (3 eq.), HOAt (3 eq.), and DIEA (6 eq.) were then added. The reaction mixture was stirred overnight and then monitored by ESI-LCMS. The solvent was concentrated under reduced pressure, and the residue was dissolved in NMP and purified by preparative HPLC. The cyclo-peptide was identified by ESI-LCMS.

[0307] Reverse-phase HPLC. An Accucore C18 column (2.6 μm, 2.1 mm × 50 mm) was used for analytical RP-HPLC at a flow rate of 1 mL / min. An Xselect Peptide CSH column (5 μm, 19 mm × 150 mm) was used for preparative RP-HPLC. Mobile phase A: water (0.1% formic acid), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 25% B to 95% B in 16 min. Synthesis of cyclo[7mer-NMALA-Dap-d-nmala-LEU]:

[0308] [ka] Assembly of the linear peptide chain was carried out using 850 mg of H-Leu-2-Cl-(Trt) resin (0.3 mmol / g, 0.025 mmol scale) using the general method described above. DN-methyl Ala, Dap, and LN-methyl Ala residues were added using general method A. The Boc-7mer was then assembled using general method B. Removal of the side-chain ivDde protecting group was achieved using general method C. Acrylic acid was then added to the free amino group of the side chain of Dap using general method D. After global deprotection and cleavage from the resin in one step (general method E), the crude linear peptide was subjected to cyclization (general method F). After final preparative HPLC, 17 mg of the final compound was obtained as a white solid (56.6% calculated by resin loading). ESI-MS: [M+1] + =1202, [M+Na] + =1224, [M / 2 + 1] + =602. Synthesis of cyclo[7mer-NMALA-Dap-d-nmala-PHE]:

[0309] [ka] 2.8 mg of the final compound was obtained as a white solid (9.1% calculated by resin loading) using a method similar to the synthesis of cyclo[7mer-NMALA-Dap-d-nmala-LEU]. ESI-MS: [M+1] + =1236, [M+Na] + =1258, [M / 2 + 1] + =619. Synthesis of cyclo[7mer-NMALA-Dap-d-nmala-THR]:

[0310] [ka] 3.4 mg of the final compound was obtained as a white solid (11.4% calculated by resin loading) using a method similar to the synthesis of cyclo[7mer-NMALA-Dap-d-nmala-LEU]. ESI-MS: [M+1] + =1190, [M+Na] + =1212, [M / 2 + 1] + =596. Synthesis of acrylamide-containing FKBP12 ligands

[0311] [ka] Boc-3mer All reagents and solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. Fmoc-amino acids, HATU, HOAT, 2-Cl-(Trt)-Cl resin (0.9 mmol / g based on active sites), and Ala-loaded 2-Cl-(Trt)-Cl resin (0.36 mmol / g) were purchased from GL Biochem (Shanghai) Ltd. The first amino acid is loaded onto the 2-Cl-(Trt)-Cl resin.

[0312] In an SPSS vessel, 5 g of resin was swelled in 50 mL of dry NMP for 40 minutes. The resin was drained and rinsed with DCM (5 × 30 mL) and then with 2% NMM / DCM (3 × 30 mL). A solution of Fmoc-Dap(ivDde)-OH (3.0 mmol, 1.6 g) with NMM (4 mmol, 400 mg) in 50 mL of DCM was added. The vessel was shaken overnight. Then, 2 mL of a 25% NMM / MeOH solution was added, and the vessel was shaken for an additional hour. The resin was drained and rinsed with DCM, NMP, MeOH, and EtOH (3 × 50 mL each). The resin was dried under vacuum at room temperature.

[0313] The loading was determined by Fmoc cleavage photometry (0.32 mmol / g). Coupling of linear peptides was carried out using the standard Fmoc SPSS procedure on an automated synthesizer.

[0314] General Method A: Linear peptides were synthesized using a TETRAS™ synthesizer on a scale of 0.025 mmol of resin. The general protocol was as follows: 2×NMP, 30 s; 1×20% (vol / vol) piperidine in NMP, 15 min; 5×NMP, 30 s; a solution of amino acid (3 equivalents) in NMP was added to the vessel containing the resin, followed by the addition of solutions of HATU and DIEA in DMF, respectively, and coupling for 45 min; 3×NMP, 30 s. The double coupling strategy was applied to all amino acids. General Method B: Boc-3mer addition The coupling was accomplished on a TETRAS™ synthesizer using the same general protocol as for amino acid coupling, except that the amount of Boc-3mer was 1.5 equivalents. Only one coupling was required. General Method C: Removal of the ivDde protecting group.

[0315] Removal of the ivDde protecting group on the Dap side chain was achieved on a TETRAS™ synthesizer. General protocol: A solution of 20% (vol / vol) hydrazine monohydrate in NMP was added to the vessel containing the resin. The vessel was shaken for 30 minutes. The resin was drained and rinsed with 5 x 5 mL (30 seconds) of NMP. General Method D: Addition of acrylic acid onto the side chain amino group of Dap.

[0316] Coupling was achieved on a TETRAS™ synthesizer by using the same general protocol as for amino acid coupling. A double coupling strategy was applied. General Method E: Deprotection of side chain protecting groups and final cleavage from the resin.

[0317] Global deprotection and cleavage from the resin were achieved with a TFA cocktail for 1-2 hours at room temperature. A cleavage cocktail (TFA / TIPS / HO, 95 / 2.5 / 2.5) or (TFA / DCM / TIPS, 40:60:1) can be used for the final cleavage. Most of the solvent was removed under reduced pressure, and the residue was concentrated in vacuo to remove traces of solvent. The resulting residue was used directly in the subsequent cyclization without further purification. General Method F: Cyclization of Linear Peptides The crude linear peptide was dissolved in dry DCM to a final concentration of 0.1 M. HATU (3 eq.), HOAt (3 eq.), and DIEA (6 eq.) were then added. The reaction mixture was stirred overnight and then monitored by ESI-LCMS. The solvent was concentrated under reduced pressure, and the residue was dissolved in NMP and purified by preparative HPLC. The cyclo-peptide was identified by ESI-LCMS.

[0318] Reverse-phase HPLC. An Accucore C18 column (2.6 μm, 2.1 mm × 50 mm) was used for analytical RP-HPLC at a flow rate of 1 mL / min. An Xselect Peptide CSH column (5 μm, 19 mm × 150 mm) was used for preparative RP-HPLC. Mobile phase A: water (0.1% formic acid), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 25% B to 95% B in 16 min. Synthesis of cyclo[diamine-Dap-ALA-ALA]:

[0319] [ka] Assembly of the linear peptide chain was carried out using 700 mg of H-Ala-2-Cl-(Trt) resin (0.025 mmol scale) using the general method described above. Ala and Dap residues were added using general method A. The Boc-3mer was then assembled using general method B. Removal of the side-chain ivDde protecting group was achieved by using general method C. Acrylic acid was then added to the free amino group of the Dap side chain using general method D. After global deprotection and cleavage from the resin in one step (general method E), the crude linear peptide was subjected to cyclization (general method F). After final preparative HPLC, 3.1 mg of the final compound was obtained as a white solid (14.5% calculated by resin loading). ESI-MS: [M+1] + =857, [M+Na] + =879. Synthesis of cyclo[diamine-ALA-ALA-Dap]

[0320] [ka] 2.2 mg of the final compound was obtained as a white solid (10.3% calculated by resin loading) using a method similar to the synthesis of cyclo[diamine-Dap-ALA-ALA]. ESI-MS: [M+1] + =857, [M+Na] + =879. Synthesis of cyclo[diamine-Dap-ALA]:

[0321] [ka] 2.7 mg of the final compound was obtained as a white solid (12.6% calculated by resin loading) using a method similar to the synthesis of cyclo[diamine-Dap-ALA-ALA]. ESI-MS: [M+1] + =786. Synthesis of sanglefehrin analogues:

[0322] [ka] Method A can be used to prepare compounds of formula IV as shown in Scheme 1 below.

[0323] [ka] In the formula, R 7 , R 8 , Z 5 , and Z 6 is as previously defined, and PG 1 is a suitable amine protecting group, including but not limited to Boc, Cbz, Alloc, and Fmoc, and X is either OH, Cl, F, or any other group suitable for displacement or activation followed by displacement.

[0324] In a typical procedure, the protected amine IV is reacted with a suitable reagent familiar to one skilled in the art to provide the protecting group PG 1 The isolated crude product is then treated with acylating agent Z. 5 C(O)X can be reacted in the presence of standard coupling agents known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperatures.

[0325] Alternatively, the deprotected amine can be reacted with an acylating reagent Z 5C(O)X, where X is a halogen, can be reacted directly with C(O)X in the presence of a base (including but not limited to pyridine, DIPEA, triethylamine, and NMM) in a suitable solvent (including but not limited to DMF, dichloromethane, DME, acetonitrile, and tetrahydrofuran) at a temperature ranging from -78°C to about 120°C, preferably from -20°C to 50°C.

[0326] Compounds of formula IVb in Scheme 1 can be prepared as shown in Scheme 2 below.

[0327] [ka] In the formula, R 7 , R 8 , and Z 6 is as previously defined, and PG 1 and PG 2 are each independently a suitable amine protecting group, including, but not limited to, Boc, Cbz, Alloc, and Fmoc.

[0328] In a typical procedure, the protected amine IVc is reacted with a suitable reagent familiar to one skilled in the art to provide the protecting group PG 2 is removed to generate the corresponding amine, which is then reacted with the appropriate amino acid in the presence of standard coupling agents known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). Combining the acid and amine coupling partners with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature provides compounds of formula IVb.

[0329] Compounds of formula IVc in Scheme 2 can be prepared as shown in Scheme 3 below.

[0330] [ka] In the formula, R 7 and Z 6 is as previously defined, and PG 2 are suitable amine protecting groups, including, but not limited to, Boc, Cbz, Alloc, and Fmoc.

[0331] In a typical procedure, protected amine IVd is reacted with piperazine acid derivative IVe in the presence of standard coupling agents known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature.

[0332] Alternatively, compounds of formula IVb can be synthesized from compounds of formula II-B as described in Scheme 4.

[0333] [ka] In the formula, R 7 , R 8 , and Z 6 is as previously defined, and PG 1 is a suitable amine protecting group, including, but not limited to, Boc, Cbz, Alloc, and Fmoc, and PG 3 is an alkyl or aryl group, including but not limited to methyl, ethyl, benzyl, allyl, phenyl, etc., which can be removed by methods known to those skilled in the art.

[0334] In a typical procedure, compound IVe is reacted at room temperature under hydrolysis conditions using a base (e.g., lithium hydroxide, sodium hydroxide, sodium carbonate) in a solvent system composed of organic (e.g., methanol, THF, dioxane) with or without water. Those skilled in the art will recognize that PG 3 Depending on the identity of the PG 3 It will be appreciated that removal of Z can also occur under hydrogenolysis conditions using a suitable catalyst, or under deallylation conditions using a palladium catalyst such as Pd(PPh3)4 and a base scavenger (e.g., piperidine, morpholine, piperidine). Following deprotection to give the resulting carboxylic acid, Z can be reacted with 6 can be introduced in the presence of standard coupling agents known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). Combining the carboxylic acid and coupling partner with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature provides product IVb.

[0335] Alternatively, compounds of formula IVb may be synthesized as shown in Scheme 5 below.

[0336] [ka] In the formula, R 7 , R 8 , and Z 6 is as previously defined, and PG 1 are suitable amine protecting groups, including, but not limited to, Boc, Cbz, Alloc, and Fmoc.

[0337] In a typical procedure, protected amine VI is reacted with reagent V in the presence of a standard coupling agent known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, NMM) at room temperature or slightly elevated temperature. Synthesis of intermediate I-8

[0338] [ka] To a room temperature solution of intermediate I-1 (2.00 g, 7.11 mmol) in DMF (13 mL) was added cesium carbonate (4.75 g, 14.58 mmol) and benzyl bromide (2.49 g, 14.58 mmol, 1.73 mL) at 25 °C. The reaction mixture was stirred for 2 h, then diluted with ethyl acetate (100 mL) and washed with brine (50 mL × 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude residue. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate → 20:1 to 10:1) to give intermediate I-2 (3.20 g, 96% yield) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ 7.45 - 7.30 (m, 10 H), 7.20 - 7.10 (m, 1 H), 6.95 - 6.85 (m, 1 H), 6.74 (s, 1 H), 6.70 - 6.60 (m, 1 H), 5.20 - 5.10 (m, 2 H), 4.99 (s, 2 H), 4.70 - 4.60 (m, 1 H), 3.15 - 3.05 (m, 2 H), 1.43 (s, 9 H). ESI-MS m / z=484.1 [M+Na] + ; Calculated molecular weight: 461.55.

[0339] To a solution of intermediate I-2 (3.20 g, 6.93 mmol) in tetrahydrofuran (15 mL) was added lithium hydroxide (1 M in water, 10 mL) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was adjusted to pH 6 using HCl (1 M in water) at 0 °C and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude residue. The crude product was dissolved in aqueous sodium bicarbonate (7 mL) and extracted with MTBE (100 mL × 3). The aqueous layer was adjusted to pH 6 using hydrochloric acid (1 M in HO) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate I-3 (2.27 g, 88% yield) as a colorless oil. 1 HNMR (400MHz, CDCl3) δ 7.45 - 7.30 (m, 5 H), 7.25 - 7.18 (m, 1 H), 6.90 - 6.85 (m, 1 H), 6.83 - 6.75 (m, 2 H), 5.05 (s, 2 H), 4.94 (d, J=8.00 Hz, 1H), 4.60 - 4.50 (m, 1 H), 3.20 - 3.12 (m, 1 H), 3.10 - 3.00 (m, 1 H), 1.43 (s, 9H). ESI-MS m / z = 394.3 [M+Na] + ; Calculated molecular weight: 371.43 To a solution of intermediate I-3 (888 mg, 2.39 mmol) in dichloromethane (15 mL) were added N-methylmorpholine (967 mg, 9.56 mmol), HOBt (65 mg, 478 mmol), (S)-methylhexahydropyridazine-3-carboxylate as a TFA salt (890 mg, 2.39 mmol), and EDCI (917 mg, 4.78 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 1 hour. The reaction mixture was diluted with dichloromethane (50 mL) and adjusted to pH 6 with 5% aqueous citric acid. The aqueous layer was extracted with dichloromethane (20 mL × 2). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography using silica gel (eluent: petroleum ether / ethyl acetate → 10:1, 5:1 to 3:1) to give intermediate I-4 (910 mg, 77% yield) as a colorless oil. 1 HNMR (400MHz, CDCl3) δ 7.50 - 7.35 (m, 5 H), 7.20 - 7.13 (m, 1 H), 6.90 - 6.80 (m, 3 H), 5.60 - 5.50 (m, 1 H), 5.30 - 5.20 (m, 1 H), 5.05 - 5.00 (m, 2 H), 4.40 - 4.30 (m, 1 H), 3.65 (s, 3 H), 3.55 (d, J=11.20 Hz, 1H), 3.00 - 2.93 (m, 1 H), 2.90 - 2.80 (m, 1 H), 2.75 - 2.65 (m, 1 H), 2.35 - 2.25 (m, 1 H), 1.80 - 1.72 (m, 2 H), 1.42 (s, 9 H). ESI-MS m / z = 520.1 [M+Na] + . Calculated molecular weight: 497.58 To a solution of intermediate I-4 (450 mg, 904 umol) in ethyl acetate (4 mL) was added hydrochloric acid in ethyl acetate (4 M, 8.00 mL) at 0° C. The reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was concentrated under reduced pressure to give the HCl salt of intermediate I-5 (390 mg, 100% yield) as a pale yellow solid, which was used in the next step without purification. ESI-MS m / z = 398.0 [M+H] + , 420.0 [M+Na] + , calculated molecular weight: 397.47.

[0340] To a solution of intermediate I-5 (195 mg, 899 μmol) in dichloromethane (5.00 mL), N-methylmorpholine (273 mg, 2.70 mmol), HOBt (24.29 mg, 179.75 μmol), (S)-methyl 1-((S)-2-amino-3-(3-(benzyloxy)phenyl)propanoyl)hexahydropyridazine-3-carboxylate HCl salt (390 mg, 899 μmol), and EDCI (345 mg, 1.80 mmol) were added at 0° C. The reaction mixture was stirred at 0° C. for 1 hour. The reaction mixture was diluted with dichloromethane (20 mL) and adjusted to pH 6 with 5% aqueous citric acid. The aqueous layer was extracted with dichloromethane (20 mL × 2). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate → 5:1, 2:1, 1:1) to give intermediate I-6 (750 mg, 70% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ 7.50 - 7.35 (m, 5 H), 7.23 - 7.15 (m, 1H), 6.90 - 6.80 (m, 3 H), 6.13 - 6.08 (m, 1 H), 5.83 - 5.73 (m, 1 H), 5.10 - 5.00 (m, 3 H), 4.30 - 4.20 (m, 1 H), 4.00 - 3.90 (m, 1 H), 3.65 (s, 3 H), 3.50 (d, J=11.20Hz, 1H), 3.05 - 2.97 (m, 1 H), 2.93 - 2.83 (m, 1 H), 2.80 - 2.70 (m, 1 H), 2.35 - 2.25 (m, 1 H), 2.15 - 2.10 (m, 1 H), 1.75 - 1.65 (m, 4 H), 1.45 (s, 9 H), 0.94 (d, J=6.80 Hz, 3H), 0.88 (d, J=6.80 Hz, 3H). ESI-MS m / z = 597.1 [M+H] + . Calculated molecular weight: 596.71.

[0341] To a solution of intermediate I-6 (3.00 g, 6.03 mmol) in methanol (300 mL) was added palladium on carbon (2 grams, 10% loading) under a nitrogen atmosphere. The suspension was degassed and purged with hydrogen gas, and the mixture was stirred under hydrogen (1 atm) at 20° C. for 3 hours. The mixture was filtered and concentrated under reduced pressure to give intermediate I-7 (2.3 g, 5.64 mmol, 94% yield) as a white solid. ESI-MS m / z = 430.1 [M+Na] + . Calculated molecular weight: 407.46.

[0342] To a mixture of intermediate I-7 (2.3 g, 5.64 mmol) in dioxane (10 mL) was added hydrochloric acid in dioxane (4 M, 30 mL) in one portion at 20 °C. The mixture was stirred at 20 °C for 3 h. The mixture was adjusted to pH 7-8 with saturated aqueous NaHCO3 and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give intermediate I-8 (1.3 g, 3.63 mmol, 64% yield, 86% purity) as a pale yellow solid. 1H NMR (400MHz, CDCl3) δ 7.15-7.02 (m, 1 H), 6.75-6.5 (m, 3 H), 4.90-4.77 (m, 1 H), 4.50-4.37 (m, 1 H), 4.00-3.90 (m, 1 H), 3.80-3.70 (m, 1 H), 3.68-3.65 (m, 3 H), 2.95-2.80 (m, 1 H), 2.77-2.62 (m, 2 H), 2.50-2.35 (m, 1 H), 1.97-1.85 (m, 1 H), 1.82-1.70 (m, 1 H), 1.60 - 1.45 (m, 1 H), 1.42-1.28 (m, 1H). ESI-MS m / z = 308.2 [M+Na] + . Calculated molecular weight: 307.34. Synthesis of intermediate I-11

[0343] [ka] To a solution of 2-(dimethylamino)ethanethiol I-9 (500 mg, 3.53 mmol) in methanol (10 mL) was added a solution of 1,2-di(pyridin-2-yl)disulfide (1.17 g, 5.3 mmol) in methanol (10 mL) at 0 °C. The mixture was stirred at 25 °C for 15 hours. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column (eluent: petroleum ether / ethyl acetate → 3:1, 1:1, then dichloromethane / ethyl acetate → 2:1, then dichloromethane / methanol → 10:1), which was combined with the previous batch to give intermediate I-10 (1.05 g, 58% yield) as a pale yellow solid. 1 H NMR (400MHz, CD3OD) δ 8.56 (d, J=4.0 Hz, 1 H), 7.85-7.75 (m, 1 H), 7.69 (d, J=8.0 Hz, 1 H), 7.36-7.26 (m, 1 H), 3.48-3.40 (m, 2 H), 3.28-3.20 (m, 2H), 2.93 (s, 6H). ESI-MS m / z = 214.9 [M+H] + . Calculated molecular weight: 214.35.

[0344] To a solution of 4-mercaptobutanoic acid (50 mg, 416 μmol) in methanol (1 mL) was added a solution of intermediate I-10 (125 mg, 499 μmol) in methanol (1.5 mL) at 25° C. The mixture was stirred at 25° C. for 15 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate → 2:1, 1:1, then dichloromethane / methanol → 10:1) to give intermediate I-11 (80 mg, 86% yield) as a white gum. 1 H NMR (400MHz, CD3OD) δ 3.55-3.45 (m, 2 H), 3.08-3.00 (m, 2 H), 2.93 (s, 6H), 2.83 (t, J=7.2 Hz, 2 H), 2.43 (t, J=7.2 Hz, 2 H), 2.05-1.94 (m, 2 H). Synthesis of intermediate I-15

[0345] [ka] To a solution of tert-butyl 2-mercaptoacetate I-12 (800 mg, 5.4 mmol) in N,N-dimethylformamide (15 mL) was added potassium carbonate (1.49 g, 10.8 mmol) and 1-bromo-2-chloroethane (2.32 g, 16.2 mmol). The mixture was stirred at 25 °C for 2 hours. The mixture was diluted with ethyl acetate (100 mL) and washed with water (50 mL × 3). The organic layer was dried over anhydrous sodium sulfate and concentrated to give intermediate I-13 (1.00 g, 79% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 3.64 - 3.71 (m, 2 H), 3.14 - 3.17 (m, 2 H), 2.95 - 3.01 (m, 2 H), 1.47 (s, 9 H).

[0346] To a solution of intermediate I-13 (1.00 g, 4.75 mmol) in dichloromethane (10 mL) was added a solution of m-CPBA (4.61 g, 21.4 mmol, 80% purity) in dichloromethane (10 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 2 h. The mixture was diluted with dichloromethane (80 mL) and washed with saturated aqueous sodium sulfite (50 mL) and saturated aqueous sodium bicarbonate (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude residue, which was purified by silica gel chromatography (eluent: petroleum ether / ethyl acetate → 5 / 1) to give intermediate I-14 (700 mg, 60% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 3.99 (s, 2 H), 3.90 - 3.95 (m, 2 H), 3.69 - 3.75 (m, 2 H), 1.50 - 1.53 (m, 9 H).

[0347] To a solution of intermediate I-14 (650 mg, 2.68 mmol) in dichloromethane (12 mL) was added trifluoroacetic acid (6.00 mL). The mixture was stirred at 45° C. for 2 hours. The mixture was then concentrated to give intermediate I-15 (360.00 mg, 72% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 4.34 (s, 2 H), 3.89 - 4.01 (m, 2 H), 3.71 - 3.81 (m, 2 H). Synthesis of Compound-1

[0348] [ka] To a solution of the HCl salt of intermediate I-8 (31 mg, 119 μmol) and intermediate I-11 (44 mg, 99 μmol) in dichloromethane (1.5 mL) was added HOBt (1.34 mg, 9.9 μmol), N-methylmorpholine (38.2 μL), and EDCI (27 mg, 139 μmol). The mixture was stirred at 25° C. for 1 h. The reaction mixture was diluted with dichloromethane (10 mL) and washed with water (5 mL). The aqueous layer was extracted with dichloromethane (5 mL × 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by reverse-phase HPLC (with HCl), repurified using reverse-phase HPLC (with HCOOH), and lyophilized to give compound-1 (5.8 mg, 9% yield) as a pale yellow oil. 1 H NMR (400MHz, MeOD) δ 8.53 (Broad singlet, 1 H), 7.12-7.04 (m, 1 H), 6.72-6.62 (m, 3H), 5.75 -5.65 (m, 1 H), 4.20-4.10 (m, 2H), 3.70 (s, 3H), 3.08- 3.00 (m, 2H), 2.95-2.76 (m, 5 H), 2.76-2.70 (m, 2 H), 2.60 (s, 6 H), 2.42-2.32 (m, 3 H), 2.10-1.98 (m, 3 H), 1.85-1.70 (m, 2 H), 1.55-1.40 (m, 2 H), 1.02-0.85 (m, 6 H). ESI-MS m / z = 612.1 [M+H] + , 634.5 [M+Na] + . Calculated molecular weight: 611.82. Synthesis of Compound-2 (SFAC4DS)

[0349] [ka] A solution of intermediate I-8 (27 mg, 60 μmol) in dichloromethane was treated with N,N-diisopropylethylamine (24 mg, 180 μmol), intermediate I-16 (14 mg, 60 μmol), HOBt (1.6 mg, 2 μmol), and finally EDCI (18 mg, 90 μmol). After stirring for 15 h, the solution was poured into water and ethyl acetate. The layers were separated, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under vacuum. Purification by reverse-phase HPLC (acetonitrile in water with 0.1% formic acid) and lyophilization afforded compound-2 (SFAC4DS) (16 mg, 42% yield) as a white solid. 1 H NMR (400MHz, CDCl3) 8.47 (d, 1H), 8.16 (broad singlet, 1H), 7.69-7.66 (m, 1H), 7.63-7.60 (m, 1H), 7.13 (apparent triplet, 1H), 7.09-7.05 (m, 1H), 5.85-5.79 (m, 1H), 4.72 (m, 1H), 4.31 (broad singlet, 1H), 3.70 (s, 3H), 3.42 (d, 1H), 3.00 (dd, 1H), 2.87-2.78 (m, 3H), 2.52-2.43 (m, 2H), 2.28 (broad singlet, 1H), 2.14 -2.04 (m, 3H), 1.83-1.73 (m, 2H), 1.62-143 (m, 5H), 0.95 (d, 3H), 0.90 (d, 3H). ESI-MS m / z = 617.9 [M+H] + . Calculated molecular weight: 617.78. Synthesis of Compound-3

[0350] [ka] To a solution of the HCl salt of intermediate I-15 (31.8 mg, 0.17 mmol), HOBt (2.3 mg, 0.017 mmol), and NMM (54 μL, 0.54 mmol) in DCM (0.75 mL) was added the HCl salt of I-8 (86 mg, 0.17 mmol) and EDCI (76 mg, 0.34 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with dichloromethane (2 mL), washed with citric acid (pH ∼3, 2 mL), saturated aqueous sodium bicarbonate solution (2 mL), and saturated aqueous sodium chloride solution (2 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 15 °C to give the product as a yellow oil. The residue was purified by preparative TLC on silica gel (eluent: DCM / MeOH → 10:1) followed by preparative HPLC (column: Phenomenex Gemini C18 250 × 50 10u; mobile phase: [water (0.225% FA)-ACN]; B%: 26%-56%, 11.2 min) to give compound-3 (8.5 mg, 8% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.02 (s, 1 H), 8.80 (s, 1 H), 8.42 (s, 1 H), 7.01 - 7.08 (m, 1 H), 6.82 (dd, J=16.3, 9.9 Hz, 1 H), 6.74 (d, J=7.5 Hz, 1 H), 6.64 - 6.70 (m, 2 H), 6.36 (d, J=16.5 Hz, 1 H), 6.12 - 6.16 (m, 2H), 4.81 - 4.85 (m, 1 H), 4.40 - 4.46 (m, 2 H), 4.21 - 4.25 (m, 1 H), 4.09 - 4.13 (m, 1 H), 3.57 (s, 3 H), 2.81 - 3.08 (m, 2 H), 2.63 - 2. 65 (m, 1 H), 2.15 - 2.19 (m, 1 H), 1.21 - 1.60 (m, 4 H), 0.88 - 0.92 (m, 6 H). ESI-MS m / z = 539.1 [M+H] + . Calculated molecular weight: 538.61. Synthesis of Compound-4

[0351] [ka] Compound-4 was prepared as described in the preparation of Compound-3 by using the HCl salt of intermediate I-8 (34 mg, 77 μmol) and 2-(3-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid I-17 (13 mg, 77 μmol) as starting materials. The resulting crude product was combined with the crude material previously synthesized and purified to give Compound-4 (29.0 mg, 51.28 μmol, 45% yield) as a white solid after lyophilization. 1 H NMR (400MHz, CDCl3) δ 8.55-8.40 (s, 1 H), 8.20-8.07 (m, 1 H), 7.10-6.97 (m, 2 H), 6.80-6.73 (m, 1 H), 6.73-6.63 (m, 1H), 6.63-6.50 (m, 1 H), 6.47-6.37 (m, 1 H), 5.90-5.75 (m, 1 H), 4.80-4.67 (m, 1 H), 4.35-4.15 (m, 3 H), 3.70-3.57 (m, 3 H), 3.45-3.30 (d, 1 H), 3.10-3.00 (m, 1H), 3.00-2.70 (m, 2 H), 2.20-2.00 (m, 5 H), 1.85-1.60 (m, 2 H), 1.60-1.45 (m, 1 H), 1.45-1.30 (m, 1 H), 1.05-0.80 (m, 6 H). ESI-MS m / z = 558.3 ​​[M+H] + ; Calculated molecular weight: 557.60. Synthesis of Compound-5

[0352] [ka] Compound-5 was prepared as described in the preparation of Compound-3 by using the HCl salt of intermediate I-8 (34 mg, 77 umol) and 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid I-18 (13 mg, 77 umol) as starting materials to give Compound-5. ESI-MS m / z = 544.0 [M+H] + ; Calculated molecular weight: 543.58. 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):

[0353] [ka] 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. After this period, amine 1 (Intermediate I-8) (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 of ethyl acetate. The pooled organic extracts were washed with 2 × 20 mL of brine, dried over saturated sodium sulfate, filtered, and concentrated in vacuo. The residue was purified using silica gel chromatography, eluting with dichloromethane:MeOH, 100:1 to 50:1, to give 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. Synthesis of SFAX9DS:

[0354] [ka] SFAX9DS was synthesized following a similar procedure described above for the synthesis of SFAX6. Synthesis of Compound-6

[0355] [ka] Compound-6 was prepared starting from intermediate I-19 to give Compound-6. ESI-MS m / z = 631.0 [M+H] + ; Calculated molecular weight: 630.82. Synthesis of Compounds of Formula IVf The synthesis of compounds of formula IVf can be synthesized as shown in Scheme 6 below.

[0356] [ka] In the formula, R 7 , R 8 , and Z 6 is as previously defined, and PG 1 is a suitable amine protecting group, including, but not limited to, Boc, Cbz, Alloc, and Fmoc, and PG 4 is an alkyl or aryl group, including but not limited to methyl, ethyl, benzyl, allyl, and phenyl, which can be removed by methods known to those skilled in the art.

[0357] In a typical procedure, compound IVd is reacted with reagent IVg in the presence of a standard coupling agent familiar to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). Acid and amine coupling partners are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature. After amide formation, the protecting group PG is removed. 4and then reacting the compound of formula IVe with PG 3 is removed using the conditions described for the removal of (Scheme 4).

[0358] [ka] Method B can be used to prepare compounds of formula IIa as shown in Scheme 7 below.

[0359] [ka] In the formula, R 1 , R 2 , R 3 , X 1 , X 2 , Z 1 and Z 2 is as previously defined.

[0360] In a typical procedure, carboxylic acid IIc is reacted with intermediate XI in the presence of a standard coupling agent known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). Acid IIc and the coupling partner are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, DCE, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or a slightly elevated temperature.

[0361] Compounds of formula IIc may be synthesized as shown in Scheme 8 below.

[0362] [ka] In the formula, R 1 , R 2 , R 3 , X 1 , and Z 2 is as previously defined, and PG5 is an alkyl or aryl group, including but not limited to methyl, ethyl, benzyl, allyl, and phenyl, which can be removed by methods known to those skilled in the art; LG 1 is a group that can be activated and displaced by the amine of compound IIe, for example, OH. Alternatively, LG 1 can be a suitable halogen compound (e.g., F, Cl, and Br) that can be displaced by a nucleophile.

[0363] In a typical procedure, IIe is converted into IIf(LG 1 = halogen compound) in the presence of a suitable base (including but not limited to pyridine, triethylamine, DIPEA, and NMM) in a suitable solvent (including but not limited to THF, DCM, and DMF) at a temperature range of -78°C to 120°C, but optimally -20°C to 50°C. 1 When is OH, XII is reacted with reagent IIf in the presence of a standard coupling agent known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature. Compound IIf (LG 1 Those skilled in the art will appreciate that the protecting groups PG (=halogenated compounds) can be readily prepared from the corresponding carboxylic acids by treatment with a suitable halogenating reagent. After amide formation between IIe and IIf, the protecting group PG 5 and then reacting with the compound of formula IVe, PG 3 was removed using the conditions described for the removal of (Scheme 4) to give compound IIc.

[0364] Method B-2 can be used to prepare compounds of formula IIa as shown in Scheme 9 below.

[0365] [ka] In the formula, R 1 , R 2 , R 3 , X 1 , X 2 , Z 1 and Z 2 is as previously defined. The reaction can be carried out under the conditions described for the coupling reaction between compound IIg and compound IIf (Scheme 8).

[0366] Compounds of formula IIg can be prepared as shown in Scheme 11 below.

[0367] [ka] In the formula, R 3 , X 1 , X 2 , Z 1 is as previously defined, and PG 6 are suitable amine protecting groups, including, but not limited to, Boc, Cbz, Alloc, and Fmoc.

[0368] In a typical procedure, carboxylic acid IIg is reacted with -X 2 -Z 1 The acid is reacted with an appropriate coupling partner containing the moiety in the presence of standard coupling agents known to those skilled in the art (e.g., EDC / HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU). The acid and coupling partner are combined with the coupling agent in an organic solvent (including, but not limited to, DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including, but not limited to, DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature. PG 6 is then removed by reacting the resulting intermediate with a suitable reagent familiar to those skilled in the art. Synthesis of Compound-7 (C3SLF)

[0369] [ka] To a solution of intermediate I-24 (18 mg, 0.085 mmol) in tetrahydrofuran (1.2 mL) was added N-methylmorpholine (10 μL, 0.085 mmol). The solution was cooled to −20° C., and then isobutyl chloroformate (11 μL, 0.085 mmol) was added dropwise. The solution was immediately warmed to 0° C. After stirring for 45 minutes at 0° C., the TFA salt of intermediate I-23 (37 mg, 0.057 mmol) was mixed with N-methylmorpholine (7 μL, 0.057 mmol) in anhydrous tetrahydrofuran (0.5 mL) and added dropwise over 5 minutes to the 0° C. solution of the mixed anhydride. The resulting solution was stirred for 1.5 hours at 0° C., at which point methanol (1 mL) was added, and the solution was immediately warmed to room temperature and stirred for 5 minutes. The solution was diluted with dichloromethane (75 mL) and saturated aqueous sodium bicarbonate (50 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2 x 30 mL). The organic layers were combined, washed with brine (20 mL), dried over magnesium sulfate, filtered, and the solvent removed in vacuo. Purification by silica gel chromatography (0-80% ethyl acetate in hexanes) afforded compound 7 (C3SLF) as a white foam (20 mg, 50% yield). 1 H NMR (400MHz, CDCl3) δ 9.49 (s, 1H), 8.55 (d, 1H), 7.77 (apparent triplet, 1H), 7.71-7.65 (m, 2H), 7.60-7.58 (m, 1H), 7.20-7.17 (m, 1H), 7.01 (d, 1H), 6.78-6.76 (m, 1H) 6.70-6.67 (m, 2H), 5.77 (dd, 1H), 5.33 (d, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.35 (d, 1H), 3.25 (t, 2H), 3.15 (dt, 1H), 2.92 (t, 2H), 2.64-2.50 (m, 2H), 2.38 (d, 1H), 2.29-2.15 (m, 1H), 2.10-2.00 (m, 1H), 1.78-1.63 (m, 3H), 1.53-1.37 (m, 3H), 1.22 (s, 6H), 0.89 (t, 3H, rotamer 1), 0.80 (t, 3H, rotamer 2). ESI-MS m / z = 722.0 [M+H] + , 744.0 [M+Na] + ; Calculated molecular weight: 721.93. Synthesis of Compound-8

[0370] [ka] To a solution of compound 7 (22 mg, 0.031 mmol) in dichloromethane (0.5 mL) was added triethylamine (62 μL, 0.55 mmol), followed by 2-(dimethylamino)ethanethiol (0.0500 mmol) (4.8 mg, 0.046 mmol). After stirring at room temperature for 30 min, the solution was poured into dichloromethane (30 mL) and saturated aqueous sodium bicarbonate (30 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (2 × 20 mL). The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under vacuum. Purification by silica gel chromatography (0–10% methanol in dichloromethane) gave an impure material, which was repurified by reverse-phase HPLC (acetonitrile in water with 0.1% formic acid) to give the formate salt of compound-8 (7.1 mg, 21% yield) as a white solid. 11H NMR (400 MHz, CDCl3) δ 12.53 (broad singlet, 1H), 9.50 (s, 1H), 8.00 (s, 1H), 7.75 (d, 1H), 7.68 (s, 1H), 7.29 (t, 1H), 7.02 (d, 1H), 6.79 - 6.77 (m, 1H), 6.70 - 6.67 (m, 2H), 5.76 (dd, 1H), 5.29 (d, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.36 - 3.27 (m, 2H), 3.23 - 3.11 (m, 4H), 2.90 (t, 2H), 2.79 - 2.71 (m, 8H), 2.62 - 2.50 (m, 2H), 2.55 (d, 1H), 2.28 - 2.17 (m, 1H), 2.12 - 2.03 (m, 1H), 1.72 - 1.61 (m, 3H), 1.49 - 1.36 (m, 3H), 1.22 (s, 3H), 1.21 (s, 3H), 0.88 (t, 3H, rotamer 1), 0.79 (t, 3H, rotamer 2). ESI - MS m / z = 716.1 [M + H] + ; Calculated molecular weight: 715.97. Synthesis of Compound - 9

[0371] [Chemical Structure Diagram] To a room temperature solution of intermediate I-23 (15 mg, 0.029 mmol), 3-(2,5-dioxopyrrol-1-yl)propanoic acid (5.8 mg, 0.034 mmol), and DMAP (0.4 mg, 0.003 mmol) in dichloromethane was added N,N'-dicyclohexylcarbodiimide (9.4 mg, 0.046 mmol). After stirring at room temperature for 15 hours, the resulting solid was filtered through a syringe filter and washed with dichloromethane. The solvent was removed under vacuum, and the resulting residue was taken up in ethyl acetate. After cooling to -20 °C, the suspension was filtered through a syringe filter and washed with cold ethyl acetate. The filtrate was diluted with ethyl acetate (30 mL) and water (30 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 20 mL). The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under vacuum. Purification by silica gel chromatography (0-90% ethyl acetate in hexanes) gave compound-9 (11 mg, 60% yield) as an oil. 1 H NMR (400MHz, CDCl3) δ 8.25 (broad singlet, 1H), 7.79 (d, 1H), 7.38 (s, 1H), 7.29 (t, 1H), 6.98 (d, 1H), 6.82 (s, 2H), 6.78-6.76 (m, 1H), 6.70-6.66 (m, 2H), 5.83 (dd, 1H), 5.37 (d, 1H), 4.43 (d, 1H), 4.37 (d, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.32 (d, 1H), 2.96 (t, 1H), 2.55 (t, 2H), 2.35 (d, 1H), 2.29-2.15 (m, 1H), 2.11-2.01 (m, 1H), 1.81-1.60 (m, 4H), 1.49-1.42 (m, 3H), 1.27 (s, 3H), 1.25 (s, 3H), 0.93 (t, 3H, rotamer 1), 0.82 (t, 3H, rotamer 2). ESI-MS m / z = 662.0 [M+H] +Calculated molecular weight: 661.75. 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):

[0372] [ka] 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 by 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. Example 2: Synthesis of certain conjugates General Protocol: This protocol describes a method for the formation of target protein-compound conjugates.

[0373] Reagents: Compounds (in-house) and mammalian target proteins (in-house) in 100% DMSO Equipment: Mini-PROTEAN TGX gel (Bio-Rad) Experimental protocol: A 1:2 molar ratio of target protein and compound 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. 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 adding 100 mM DTT to the reaction mixture, which reverts the conjugate to its constituent parts. A.KRAS GTP / S39C Formation of lite / C2-FK506 conjugate Reagents: C2-FK506 (in-house) in 100% DMSO, KRAS GTP / S39C lite (in-house; residues 1–169 containing G12V / S39C / C51S / C80L / C118S).

[0374] [ka] Equipment: Mini-PROTEAN TGX gel (Bio-Rad) Experimental protocol: 1:2 molar ratio of KRAS GTP / S39C lite and C2-FK506 in 12.5 mM HEPES, pH 7.4, 75 mM MgCl, containing 2% DMSO. The mixture is mixed together in NaCl buffer. 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. KRAS 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.

[0375] Results: C2-FK506 inhibits KRAS GTP / S39C It efficiently crosslinks with lite and is specific for cysteine ​​39 (Figure 1). B.KRASGTP / 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).

[0376] Equipment: 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. Cross-linking efficiency was assessed by SDS-PAGE gel. Wild-type CypA was also cross-linked with the compounds. Cysteine ​​52, the reactive cysteine ​​on CypA, was mutated to serine to abrogate presenter cross-linking.

[0377] Results: SFAX9DS is a KRAS GTP / G12C Efficiently crosslinks with lite proteins and CypA C52S does not cross-link to SFAX9DS (Fig. 2). Example 3: Formation of a specific complex General Protocol: This protocol describes two methods for the formation and isolation of complexes composed of a presenter protein, a compound, and a mammalian target protein.

[0378] Reagents: Compounds (in-house), presenter proteins (in-house), and mammalian target proteins (in-house) in 100% DMSO Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex75 (GE Healthcare, CV120mL).

[0379] 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) purification. 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 evaluated by SDS-PAGE to confirm the presence of the conjugate in the elution peak.

[0380] Experimental Protocol B: Crosslinking Reagent, Presenter Protein, and Target Protein A 1:2:2 molar ratio of compound, presenter protein, and target 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 complex is isolated by size-exclusion chromatography (SEC) purification. The reaction mixture is directly injected onto a Superdex 75 column (CV120 mL) 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 evaluated by SDS-PAGE to confirm the presence of the complex in the elution peak. A.KRAS GTP / S39C Formation of the lite / C2Holt / FKBP12 ternary complex Reagents: C2Holt (in-house) in 100% DMSO, KRAS GTP / S39C lite (in-house; residues 1–169 containing G12V / S39C / C51S / C80L / C118S), and FKBP12 (in-house).

[0381] [ka] Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex75 (GE Healthcare, CV120mL) Experimental protocol: 1:2:2 molar ratio of C2-Holt, FKBP12, and KRAS GTP / S39C The lyte and lyte 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 complex is isolated by size exclusion chromatography (SEC) purification. The reaction mixture is mixed with 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer containing 2% DMSO. The complex was injected directly onto a Superdex 75 column (CV 120 mL) pre-equilibrated with a buffer containing HEPES, pH 7.4, 75 mM NaCl. The complex eluted approximately 69 mL after injection, and the unreacted KRAS GTP / S39C KRAS lite and FKBP12 elute at approximately 75 mL and 87 mL, respectively, after injection. GTP / S39C Samples are further evaluated by SDS-PAGE to confirm the presence of lite and FKBP12.

[0382] Results: SEC profile and SDS-PAGE analysis of the elution peaks revealed KRAS GTP / S39C The formation of a lite / C2Holt / FKBP12 complex is confirmed (FIGS. 3A and 3B). B.KRAS GDP / S39C lite / SFAC4DS / CypA C52S Ternary complex formation Reagents: SFAC4DS (in-house) in 100% DMSO, KRAS GDP / S39C lite (in-house production; residues 1–169 containing G12V / S39C / C51S / C80L / C118S), and CypA C52S (Made in-house).

[0383] Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex75 (GE Healthcare, CV120mL) Experimental protocol: SFAC4DS, CypA in a 1:2:2 molar ratio C52S , and KRAS GDP / S39C The complex is 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 directly injected 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 Eluted at approximately 75 mL and 80 mL, respectively, after injection. KRAS in the elution peak GDP / S39C lite and CypA C52S Samples are further evaluated by SDS-PAGE to confirm the presence of

[0384] Results: SEC profile and SDS-PAGE analysis of the elution peaks revealed KRAS GDP / S39C lite / SFAC4DS / CypA C52S The formation of the complex is confirmed (Figure 4). C.PTP1B S187C Formation of the lite / C3SLF / FKBP12 ternary complex Reagents: C3SLF (in-house production), PTP1B in 100% DMSO E186C lite (in-house; residues 1–293 containing C32S / C92V / C121S / S187C), and FKBP12 (in-house).

[0385] Equipment: Mini-PROTEAN TGX gel (Bio-Rad), Superdex75 (GE Healthcare, CV120mL) Experimental protocol: 1:3:3 molar ratio of C3SLF, FKBP12, and PTP1B S187CThe PTP1B complex is 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 directly injected 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, and unreacted FKBP12 elutes at approximately 75 mL (dimer) and 90 mL (monomer), respectively, after injection. PTP1B in the elution peak S187C Samples are further evaluated 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 to determine their elution times.

[0386] Result: Free PTP1B S187C SEC profiles and SDS-PAGE analysis of the lite and FKBP12 proteins (Fig. 5A) revealed that free PTP1B S187C The SEC profile and SDS-PAGE analysis of the elution peak confirmed that PTP1B lite was eluted at approximately 64-65 ml. S187C The formation of the lite / C3SLF / FKBP12 complex was confirmed, eluting at approximately 61 ml (Figure 5B). Example 4: Conjugate formation in the presence of presenter protein but not in its absence This protocol describes methods to analyze cross-linking efficiency using mass spectrometry and gel shift assays in an attempt to assess presenter dependence of conjugate formation.

[0387] 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 AdvanceBio RP-mAb C4 column (2.1 × 100 mm, 3.5 μm) was employed, and the chromatograms were analyzed using an Agilent 6230 TOF-LC / MS with an autosampler and an Agilent A 1260 HPLC instrument was used. HPLC-grade acetonitrile and water (containing 1.0 mM ammonium formate and 1% by volume formic acid, 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.

[0388] All cross-linking reactions were carried out in 1.5 mL amber glass vials with 0.5 mL glass inserts. A water-soluble peptide (SEQ ID NO: 1: YQNLLVGRNRGEEILD) was employed as the 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 (which interferes with FKBP12) and cysteine ​​(which interferes with disulfide bond formation) residues were excluded. All reactions and standards were prepared in HEPES (pH 7.4, 1.0 mM MgCl) buffer.

[0389] Prior to every reaction, standard curves for individual components were obtained using a series of standards (an example of a standard curve analysis for FKBP12 is shown in Table 3 below). Data from the standard curves were used to plot μmol of protein sample versus the area ratio (sample:std), and a linear fit (y=mx+c) was used to obtain the slope and intercept. Slope and intercept values ​​for these standard curves were determined during evaluation of substrate and product concentrations before and during the course of the reaction. For all substrates / products, blank injections were performed following the initial injection to confirm 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 v Analysis was performed using B.07.0 software.

[0390] [Table 3] In a representative experiment assessing the presenter dependency of ligand crosslinking to a target protein, KRAS GTP / G12C KRAS and either C3 or C4SLF ligand were incubated in 12.5 mM HEPES, pH 7.4, 75 mM NaCl, 1 mM MgCl2, 3% DMSO at 2 μM KRAS, 10 μM FKBP12, and 10 μM C3 or C4SLF in the presence or absence of FKBP12 for 4 hours at room temperature. The amount of KRAS undergoing disulfide cross-linking with the ligand was analyzed using the method described above. As shown in Table 4, a 5- to 10-fold increase in cross-linking efficiency was observed in the presence of presenters.

[0391] [Table 4] In parallel with the mass spectrometry analysis, cross-linking reactions with C3 or C4SLF 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 cross-linking reactions were set up at higher concentrations (60 μM KRAS, 180 μM FKBP12, and 180 μM C3 or C4SLF), and the reactions were quenched with MMTS to terminate the reactions. Similar to the MS data, the ligand cross-linking efficiency was significantly boosted in the presence of FKBP12, but this was more pronounced for C4-SLF (Figure 6). 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-glucan, and the structure determination method for the crystal structure of this ternary complex. 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: Superdex75 (GE Healthcare) Experimental protocol: CHolt 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 / S39C The 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 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 an Advanced Photon Source (APS) and processed with the HKL program. Molecular replacement solutions were obtained using the program PHASER from the CCP4 suite, using the published structures of FKBP12 (PDB-ID 1FKD) and KRAS (PDB-ID 3GFT) as search models. Subsequent model building and refinement were performed according to standard protocols using the software packages CCP4 and COOT.

[0392] 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 / S39CThe electron density 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.

[0393] KRAS involved in C2Holt binding GTP / S39C The residues (cutoff distance of 4 Å) involved in KRAS binding to FKBP12 are Glu37, Cys39, Leu56, and Met67. GTP / S39C The 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.

[0394] The total buried surface area of ​​the complex is 1,947 Å 2 KRAS GTP / S39C The buried surface area of 2 Of these, 501 Å 2 contributed by FKBP12 (83%) and 99 Å 2 The buried surface 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 surface area of ​​C2Holt is 584Å 2 Of these, 132 Å 2 is KRAS GTP / S39C (23%) contributed 452 Å 2 FKBP12 (77%) contributes to KRAS GTP / S39CThe 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 binds to KRAS by design. GTP / S39C Forms minimal contact with (99 Å 2 ) but KRAS GTP / S39C It forms an H-bond with Glu37 of 1. Data collection and refinement statistics for the final structure are listed in Table 5 below. 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).

[0395] Device: Superdex75 (GE Healthcare) Experimental protocol: SFAC4DS and CypA C52S and KRAS at a 2:1 molar excess in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 1 mM MgCl, 2% 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, and 25% PEG3350. For data collection, the crystals were transferred to a solution containing mother liquor supplemented with additional PEG3350 to make it 40% PEG and then frozen in liquid nitrogen. Diffraction datasets were collected at the Advanced Light Source (ALS) and processed with the HKL program. Molecular replacement solutions were obtained using the program PHASER from the CCP4 suite, using the published structures of CypA (PDB-ID 1CWA) and KRAS (PDB-ID 3GFT) as search models. Subsequent model building and refinement was performed according to standard protocols using the software packages CCP4 and COOT.

[0396] 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. GDP / S39C The electron density 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.

[0397] KRAS involved in SFAC4DS binding GDP / S39C The residues (cutoff distance of 4 Å) are Glu3, Lys5, Cys39, Arg41, Leu52, Asp54, Ile55, and Leu56. CypA C52S KRAS involved in binding to GDP / S39CThe 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.

[0398] The total buried surface area of ​​this complex cannot be calculated due to partial structural disorder at the protein-protein interface. Excluding the region that is disordered for calculation, the buried surface 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 residues Arg55, Gln63, Asn102, and His126 of SFAC4DS. GDP / S39C 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. C.PTP1B S187C Crystal structure determination of the C3SLF / FKBP12 ternary complex Reagents: Ligand (C3SLF) (in-house), FKBP12 (in-house), PTP1B in 100% DMSO S187C lite (in-house production, residues 1–169 containing C32S / C92V / C121S / S187C).

[0399] Equipment: Superdex75 (GE Healthcare), Gryphon (Art Robbins Instruments) Experimental protocol: C3SLF and FKBP12 were mixed 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 for 36–72 h 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, 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 an Advanced Photon Source (APS) and processed with the XDS program. Molecular replacement solutions were obtained using the program PHASER from the CCP4 suite, using the published structures of FKBP12 (PDB-ID 2PPN) and PTP1B (PDB-ID 2NT7) as search models. Subsequent model building and refinement was performed according to standard protocols using the software packages CCP4 and COOT.

[0400] 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 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.

[0401] The total buried surface area of ​​the complex is 1,042 Å 2 PTP1B S187C The buried surface area of 2 The buried surface area of ​​C3SLF is 615Å 2 (Figure 9B). S187C The protein-protein interface between FKBP12 and FKBP13 is formed by both hydrophobic and polar interactions. D.MCL1 S245C Crystal structure determination of the C3SLF / FKBP52 ternary complex Reagents: Ligand (C3SLF) (in-house), FKBP52 (in-house, residues 1–140), MCL1 in 100% DMSO S245C lite (in-house production, residues 172–327 containing S245C / C286S).

[0402] Equipment: Superdex75 (GE Healthcare), Gryphon (Art Robbins Instruments) Experimental protocol: C3SLF and FKBP52 were incubated with MCL1 in a 3:1 molar excess in 12.5 mM HEPES, pH 7.4, 75 mM NaCl buffer, 2% DMSO. S245CThe complex was mixed with lysate and incubated for 24–48 h 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, 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 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 using an Advanced Photon Source (APS) and processed with the XDS program. Molecular replacement solutions were obtained using the program PHASER from the CCP4 suite, using the published structures of FKBP52 (PDB-ID 1N1A) and PTP1B (PDB-ID 3MK8) as search models. Subsequent model building and refinement was performed according to standard protocols using the software packages CCP4 and COOT.

[0403] Results: The crystals consisted of MCL1 in the asymmetric unit. S245C The complex contains one molecule of the C3SLF / FKBP52 complex (Figure 10). The resulting electron density revealed a clear bond between the two proteins, 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. The buried surface area of ​​the complex is 1,410 Å. 2 Approximately 60% of these proteins are FKBP52 (804Å 2 ) contributes, and approximately 40% is contributed by C3-SLF (606Å 2 ) contributes. Due to the limited resolution, detailed analysis of protein-protein and protein-ligand interactions has not been feasible.

[0404] [Table 5] Example 6: Determination of complex formation by TR-FRET TR-FRET technology (LANCE, Perkin Elmer) is a standard method for detecting the binary association of two fusion-tagged proteins, e.g., protein 1 / tag A and protein 2 / tag B, where A and B can be 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. CYPA-compound 3-KRAS by TR-FRET G12C-GTP Determination of complex formation Avi-tagged cyclophilin A and His-tagged KRAS G12C-GTP The chromatin was mixed with increasing concentrations of the ligand (compound 3) and incubated at room temp...

Claims

1. A compound comprising a protein-binding moiety and a cross-linking group.

2. The compound of claim 1 , wherein the bridging group is a moiety capable of chemoselectively reacting with an amino acid.

3. 3. The compound 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 of claim 3 , wherein the cross-linking group is a sulfhydryl-reactive cross-linking group.

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

6. The bridging group is represented by Formula I: 【Chemistry 1】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; a is 0, 1, or 2; R Ａ is optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Heteroalkyl, optionally substituted C ６ -C １０ aryl, or optionally substituted C ２ -C ９ The compound of claim 5 which is heteroaryl.

7. R Ａ is optionally substituted C ２ -C ９ The compound of claim 6 which is heteroaryl.

8. The optionally substituted C ２ -C ９ The compound of claim 7, wherein the heteroaryl is pyridyl.

9. The bridging group has the structure: 【Chemistry 2】 Including, 7. The compound of claim 6, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

10. R Ａ is optionally substituted C １ -C ６ The compound of claim 6 which is heteroalkyl.

11. The optionally substituted C １ -C ６ 11. The compound of claim 10, wherein the heteroalkyl is N,N-dimethyl-ethylene.

12. The bridging group has the structure: 【Transformation 3】 Including, 7. The compound of claim 6, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

13. The bridging group has the structure: 【Chemistry 4】 Including, 13. The compound of claim 12, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

14. R Ａ is optionally substituted C １ -C ６ The compound of claim 6, wherein the compound is alkyl.

15. The optionally substituted C １ -C ６ The compound of claim 10, wherein the alkyl is methyl.

16. 16. The compound of claim 14 or 15, wherein a is 2.

17. The bridging group has the structure: 【Transformation 5】 Including, 7. The compound of claim 6, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

18. The compound of any one of claims 1 to 4, wherein the bridging group comprises a maleimide.

19. The bridging group has formula Ib, Ic, Id, or Ie: 【Transformation 6】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; X Ａ is —C(O)— or —SO ２ - and X Ｂ is —C(O)— or CR Ｅ R Ｆ and R Ｂ and R Ｃ are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C １ -C ６ Alkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Alkynyl, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R Ｄ is hydrogen, hydroxyl, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R Ｅ and R Ｆ 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ 20. The compound of claim 18, wherein the compound is alkyl.

20. 20. The compound of claim 19, wherein the bridging group comprises the structure of formula Ib:

21. X Ａ The compound of claim 20, wherein is —C(O)—.

22. X Ｂ The compound according to claim 20 or 21, wherein is —C(O)—.

23. R Ｂ and R Ｃ is hydrogen or optionally substituted C １ -C ６ The compound of any one of claims 20 to 22, which is alkyl.

24. The bridging group has the structure: 【Transformation 7】 Including, 24. The compound of claim 23, wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound.

25. The bridging group has the structure: 【Transformation 8】 Including, 25. The compound of claim 24, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

26. The bridging group has the formula If, Ig, Ih, or Ii: 【Chemistry 9】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; X Ｃ is —C(O)— or —SO ２ - and X Ｄ But does not exist, NR Ｊ R Ｋ , or OR Ｌ and R Ｇ , R Ｈ , and R Ｉ are independently hydrogen, nitrile, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C １ -C ６ Alkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Alkynyl, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R Ｊ , R Ｋ , and R Ｌ are independently absent, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ The compound of any one of claims 1 to 4, which is alkyl.

27. 27. The compound of claim 26, wherein the bridging group comprises the structure of formula If.

28. X Ｄ 28. The compound of claim 26 or 27, wherein is absent.

29. R Ｇ , R Ｈ , and R Ｉ The compound of any one of claims 26 to 28, wherein is hydrogen.

30. X Ｃ But, -SO ２ The compound according to any one of claims 26 to 29, wherein

31. The compound of any one of claims 26 to 30, wherein the bridging group comprises a vinyl sulfone.

32. The bridging group has the structure: 【Chemistry 10】 Including, 27. The compound of claim 26, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

33. The bridging group has the structure: 【Chemistry 11】 Including, 33. The compound of claim 32, wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound.

34. X Ｃ The compound according to any one of claims 26 to 29, wherein is -C(O)-.

35. The compound of any one of claims 26 to 29, wherein the bridging group comprises a vinyl ketone.

36. The bridging group has the structure: 【Chemistry 12】 Including, 36. The compound of claim 35, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

37. The bridging group has the structure: 【Chemistry 13】 Including, 37. The compound of claim 36, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

38. The bridging group has the structure: 【Chemistry 14】 Including, 37. The compound of claim 36, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

39. 30. The compound of any one of claims 26 to 29, wherein the bridging group comprises an ynone.

40. The bridging group may be represented by formula Ij or Ik: 【Chemistry 15】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; X Ｅ But does not exist, NR Ｎ R Ｏ , or OR Ｐ and R Ｍ is hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C １ -C ６ Alkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Alkynyl, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R Ｎ , R Ｏ , and R Ｐ 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ 40. The compound of claim 39, which is alkyl.

41. 41. The compound of claim 40, wherein the bridging group comprises the structure of formula Ij.

42. The bridging group has the structure: 【Chemistry 16】 Including, 42. The compound of claim 40 or 41, wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound.

43. The bridging group has the formula Im or In: 【Chemistry 17】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; X Ｆ But does not exist, NR Ｓ R Ｔ , or OR Ｕ and X Ｇ is absent or is —C(O)—, Y is a leaving group; R Ｑ and R Ｒ 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R Ｓ , R Ｔ , and R Ｕ are independently absent, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ The compound of any one of claims 1 to 4, which is alkyl.

44. 44. The compound of claim 43, wherein Y is halogen.

45. 45. The compound of claim 44, wherein the halogen is chloride or fluoride.

46. 44. The compound of claim 43, wherein Y is a nitrile.

47. X Ｆ and X Ｇ The compound according to any one of claims 43 to 46, wherein is absent.

48. R Ｑ and R Ｒ The compound of any one of claims 43 to 47, wherein is hydrogen.

49. 44. The compound of claim 43, wherein the bridging group comprises an alkyl halide.

50. 50. The compound of claim 49, wherein the alkyl halide is an alkyl chloride or an alkyl fluoride.

51. The bridging group has the structure: [Chemistry 18] Including, 51. The compound of claim 50, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

52. The bridging group has the structure: 【Chemistry 19】 Including, 52. The compound of claim 51, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

53. The compound of any one of claims 1 to 4, wherein the bridging group comprises an epoxide.

54. The bridging group has the formula Io: 【Chemistry 20】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; R Ｖ , R Ｗ , and R Ｘ 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ Heterocyclyl, optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ 54. The compound of claim 53, which is alkyl.

55. The bridging group has the formula Ip: 【Chemistry 21】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; The dotted line represents an optional double bond that is included if necessary to make the structure aromatic; b is 0, 1, or 2; Y is a leaving group; R Ｙ and R Ｚ 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ Heterocyclyl, optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, X Ｈ , X Ｉ , X Ｊ , X Ｋ , and X Ｌ Each of the following may independently be absent: NR ＡＡ , or CR ＡＢ and X Ｈ , X Ｉ , X Ｊ , X Ｋ , and X Ｌ At least five of the ＡＡ , or CR ＡＢ and R ＡＡ is absent or is selected from the group consisting of 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R ＡＢ is hydrogen, nitrile, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C １ -C ６ Alkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Alkynyl, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ The compound of any one of claims 1 to 4, which is alkyl.

56. At least one R ＡＢ 56. The compound of claim 55, wherein is an electron withdrawing group.

57. 1 to 3 R ＡＢ 57. The compound of claim 56, wherein is an electron withdrawing group.

58. 58. The compound of any one of claims 55 to 57, wherein Y is a nitrile, halogen, mesylate, tosylate, or triflate.

59. The bridging group has the structure: 【Chemistry 22】 Including, 59. The compound of any one of claims 55-58, wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound.

60. The bridging group has the structure: 【Chemistry 23】 Including, 60. The compound of claim 59, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

61. The bridging group has the structure: 【Chemistry 24】 Including, 61. The compound of claim 60, wherein the wavy line illustrates the point of attachment of the bridging group to the remainder of the compound.

62. The compound of any one of claims 1 to 4, wherein the bridging group is an internal bridging group.

63. The bridging group has the formula Iq, Ir, or Is: 【Chemistry 25】 The structure of wherein the wavy line depicts the point of attachment of the bridging group to the remainder of the compound; X Ｍ is —C(O)— or —SO ２ - and X Ｎ But does not exist, NR ＡＥ , or O, R ＡＣ and R ＡＤ are independently hydrogen, nitrile, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C １ -C ６ Alkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Alkynyl, 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ is alkyl, R ＡＥ is 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 Heteroaryl, optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ Alkyl, optionally substituted C ２ -C ９ heterocyclyl, or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ 63. The compound of claim 62, which is alkyl.

64. X Ｎ But NR ＡＥ and R ＡＥ 64. The compound of claim 63, wherein is hydrogen.

65. R ＡＣ and R ＡＤ 65. The compound of claim 63 or 64, wherein is hydrogen.

66. X Ｍ The compound according to any one of claims 63 to 65, wherein is -C(O)-.

67. X Ｍ But, -SO ２ The compound according to any one of claims 63 to 65, wherein

68. 68. The compound of any one of claims 1 to 67, wherein the interaction between the protein-binding moiety and the protein is non-covalent.

69. 68. The compound of any one of claims 1 to 67, wherein the interaction between the protein-binding moiety and the protein is covalent.

70. A compound comprising a presenter protein-binding moiety and a cross-linking group.

71. 71. The compound of claim 70, wherein the presenter protein binding moiety is capable of binding to a protein encoded by any one of the genes listed in Table 1.

72. 71. The compound of claim 70, wherein the presenter protein binding moiety is a prolyl isomerase binding moiety.

73. 73. The compound of any one of claims 70 to 72, wherein the presenter protein binding moiety is an FKBP binding moiety, a cyclophilin binding moiety, or a PIN1 binding moiety.

74. 74. The compound of claim 73, wherein the presenter protein binding moiety is an FKBP binding moiety.

75. 75. The compound of claim 74, wherein the presenter protein binding moiety is capable of binding to FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

76. 76. The compound of claim 74 or 75, wherein the FKBP binding moiety is a selective FKBP binding moiety.

77. 76. The compound of claim 74 or 75, wherein the FKBP binding moiety is a non-selective FKBP binding moiety.

78. The FKBP binding moiety has formula IIa or IIb: 【Chemistry 26】 The structure of During the ceremony, Z １ and Z ２ are each independently an optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl or Z １ and Z ２ combine with the atoms to which they are attached to form an optionally substituted 10-40 membered macrocycle, and Z １ or Z ２ at least one of which comprises a point of attachment to said bridging 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, CH ２ ,O,S,SO,SO ２ , or NR ４ and 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 in combination with 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 in combination with optionally substituted C ３ -C １０ Carbocyclyl, optionally substituted C ６ -C １０ aryl or optionally substituted C ２ -C ９ forming a heteroaryl, 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 78. The compound of any one of claims 74 to 77, which is alkyl.

79. The presenter protein binding moiety has the structure: 【Chemistry 27】 79. The compound of claim 78, comprising:

80. 74. The compound of claim 73, wherein the presenter protein binding moiety is a cyclophilin binding moiety.

81. 81. The compound of claim 80, wherein the presenter protein binding moiety is capable of binding to PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

82. 82. The compound of claim 80 or 81, wherein the cyclophilin-binding moiety is a selective cyclophilin-binding moiety.

83. 82. The compound of claim 80 or 81, wherein the cyclophilin-binding moiety is a non-selective cyclophilin-binding moiety.

84. The cyclophilin binding moiety has Formula III or IV: 【Chemistry 28】 The structure of During the ceremony, Z ３ , Z ４ , Z ５ , and Z ６ are each independently selected from hydroxyl, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl or Z ３ and Z ４ Or Z ５ and Z ６ combine with the atoms to which they are attached to form an optionally substituted 10-40 membered macrocycle; Z ３ , Z ４ , Z ５ , Z ６ , or R ５ at least one of which comprises a point of attachment to said bridging group; e is 0, 1, 2, 3, or 4; R ５ is 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 is alkyl, R ６ is optionally substituted C １ -C ６ is alkyl, Each R ７ are independently selected from hydroxyl, cyano, 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 is alkyl, R ８ is 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 84. The compound of any one of claims 80 to 83, which is alkyl.

85. The cyclophilin binding moiety has formula IVa: 【Chemistry 29】 The structure of In the formula, each R ７ ' are independently selected from hydroxyl, cyano, 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 (e.g., optionally substituted C ２ -C ９ heteroaryl), or optionally substituted C ２ -C ９ Heterocyclyl C １ -C ６ Alkyl (e.g., optionally substituted C ２ -C ９ Heteroaryl C １ -C ６ 85. The compound of claim 84, wherein:

86. The presenter protein binding moiety has the structure: 【Transformation 30】 86. The compound of claim 84 or 85, comprising:

87. A compound comprising a target protein binding moiety and a cross-linking group.

88. 88. The compound of claim 87, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein with classical protein-protein interaction domains and motifs.

89. 89. The compound of claim 87 or 88, wherein the target protein comprises an undruggable surface.

90. 90. The compound of any one of claims 87 to 89, wherein the target protein does not have a conventional binding pocket.

91. 91. The compound of any one of claims 1 to 90, wherein the protein-binding moiety and the cross-linking group are joined through a linker.

92. 92. The compound of claim 91, wherein the linker is 1 to 20 atoms in length.

93. The linker has 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 Formula V having the structure In the 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 C １ -C ２ Alkyl, optionally substituted C １ -C ３ Heteroalkyl, O, S, and NR Ｎ and R Ｎ is hydrogen, optionally substituted C １－４ Alkyl, optionally substituted C ２－４ Alkenyl, optionally substituted C ２－４ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ aryl, or optionally substituted C １－７ Heteroalkyl, C １ and C ２ are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; f, g, h, I, j, and k are each independently 0 or 1; and D is optionally substituted C １－１０ Alkyl, optionally substituted C ２－１０ Alkenyl, optionally substituted C ２－１０ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ Aryl, optionally substituted 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 93. The compound of claim 91 or 92, wherein the chemical bond connecting

94. The linker has the formula VI: 【Chemistry 31】 The structure of In the 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 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 94. The compound of any one of claims 91 to 93, which is alkyl.

95. The linker has the structure: 【Chemistry 32】 95. The compound of claim 94, comprising:

96. structure: 【Chemistry 33-1】 【Chemistry 33-2】 A compound having the formula:

97. A conjugate comprising a presenter protein-binding moiety conjugated to a target protein.

98. 98. The conjugate of claim 97, wherein the site of the presenter protein binding portion of the conjugate is capable of non-covalently interacting with a presenter protein.

99. 98. The conjugate of claim 97, wherein the site of the presenter protein binding portion of the conjugate is capable of covalently interacting with a presenter protein.

100. 100. The conjugate of any one of claims 97 to 99, wherein the presenter protein binding moiety is capable of binding to a protein encoded by any one of the genes listed in Table 1.

101. 100. A conjugate according to any one of claims 97 to 99, wherein the presenter protein binding moiety is a prolyl isomerase binding moiety.

102. 102. The conjugate of any one of claims 97 to 101, wherein the presenter protein binding moiety is an FKBP binding moiety, a cyclophilin binding moiety, or a PIN1 binding moiety.

103. 103. The conjugate of claim 102, wherein the presenter protein binding moiety is an FKBP binding moiety.

104. 104. The conjugate of claim 103, wherein the presenter protein binding moiety is capable of binding to FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

105. 105. The conjugate of claim 103 or 104, wherein the FKBP binding moiety is a selective FKBP binding moiety.

106. 105. The conjugate of claim 103 or 104, wherein the FKBP binding moiety is a non-selective FKBP binding moiety.

107. The FKBP binding moiety has formula IIa or IIb: 【Transformation 34】 The structure of During the ceremony, Z １ and Z ２ are each independently an optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl or Z １ and Z ２ combine with the atoms to which they are attached to form an optionally substituted 10-30 membered macrocycle, and Z １ or Z ２ at least one of which comprises a point of attachment to said target protein; b and c are independently 0, 1, or 2; d is 0, 1, 2, 3, 4, 5, 6, or 7; X １ and X ２ are each independently absent, CH ２ ,O,S,SO,SO ２ , or NR １３ and 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 in combination with 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 in combination with optionally substituted C ３ -C １０ Carbocyclyl, optionally substituted C ６ -C １０ aryl or optionally substituted C ２ -C ９ The conjugate of any one of claims 103 to 106, which forms a heteroaryl.

108. The presenter protein binding moiety has the structure: 【Chemistry 35】 108. The conjugate of claim 107, comprising:

109. 103. The compound of claim 102, wherein the presenter protein binding moiety is a cyclophilin binding moiety.

110. 110. The compound of claim 109, wherein the presenter protein binding moiety is capable of binding to PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

111. 111. The compound of claim 109 or 110, wherein the cyclophilin binding moiety is a selective cyclophilin binding moiety.

112. 111. The compound of claim 109 or 110, wherein the cyclophilin-binding moiety is a non-selective cyclophilin-binding moiety.

113. The cyclophilin binding moiety has Formula III or IV: 【Transformation 36】 The structure of During the ceremony, Z ３ , Z ４ , Z ５ , and Z ６ are each independently selected from hydroxyl, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl or Z ３ and Z ４ Or Z ５ and Z ６ combine with the atoms to which they are attached to form an optionally substituted 10-40 membered macrocycle; Z ３ , Z ４ , Z ５ , Z ６ , or R ５ at least one of which comprises a point of attachment to said bridging group; d is 0, 1, 2, 3, or 4; R ５ is 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 is alkyl, R ６ is optionally substituted C １ -C ６ is alkyl, Each R ７ are independently selected from hydroxyl, cyano, 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 is alkyl, R ８ is 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 The compound of any one of claims 109 to 112, which is alkyl.

114. The presenter protein binding moiety has the structure: 【Chemistry 37】 114. The compound of claim 113, comprising:

115. 115. The conjugate of any one of claims 97 to 114, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeller, or a protein with classical protein-protein interaction domains and motifs.

116. The conjugate of any one of claims 97 to 115, wherein the target protein comprises an undruggable surface.

117. The conjugate of any one of claims 97 to 116, wherein the target protein does not have a conventional binding pocket.

118. 118. The conjugate of any one of claims 97 to 117, wherein the amino acid sequence of the target protein has been modified to replace at least one native amino acid with a reactive amino acid.

119. The conjugate of claim 118, wherein the reactive amino acid is a natural amino acid.

120. 120. The conjugate of claim 119, wherein the reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

121. The conjugate of claim 118, wherein the reactive amino acid is an unnatural amino acid.

122. 122. The conjugate of any one of claims 97 to 121, wherein the amino acid sequence of the target protein has been modified to replace at least one native reactive amino acid with a non-reactive amino acid.

123. 123. The conjugate of claim 122, wherein the at least one native reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

124. 124. The conjugate of claim 122 or 123, wherein the at least one native reactive amino acid is a solvent-exposed amino acid.

125. The conjugate of any one of claims 122 to 124, wherein the amino acid sequence of the target protein is modified to replace all reactive amino acids with non-reactive amino acids.

126. The conjugate of any one of claims 122 to 125, wherein the non-reactive amino acid is a natural amino acid.

127. The conjugate of any one of claims 118 to 126, wherein the substitution is a conservative substitution.

128. 128. The conjugate of any one of claims 121 to 127, wherein the reactive amino acid is substituted with serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine.

129. The conjugate of any one of claims 121 to 124, wherein the non-reactive amino acid is an unnatural amino acid.

130. 130. The conjugate of any one of claims 97 to 129, wherein the presenter protein binding moiety and the target protein are conjugated through a linker.

131. 131. The conjugate of claim 130, wherein the linker is 1 to 20 atoms in length.

132. The linker has the formula III: A １ -(B １ ) ｆ -(C １ ) ｇ -(B ２ ) ｈ -(D)-(B ３ ) ｉ -(C ２ ) ｊ -(B ４ ) ｋ -A ２ Formula V having the structure In the 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 C １ -C ２ Alkyl, optionally substituted C １ -C ３ Heteroalkyl, O, S, and NR Ｎ and R Ｎ is hydrogen, optionally substituted C １－４ Alkyl, optionally substituted C ２－４ Alkenyl, optionally substituted C ２－４ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ aryl, or optionally substituted C １－７ Heteroalkyl, C １ and C ２ are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; f, g, h, I, j, and k are each independently 0 or 1; and D is optionally substituted C １－１０ Alkyl, optionally substituted C ２－１０ Alkenyl, optionally substituted C ２－１０ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ Aryl, optionally substituted 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 ２ 132. The conjugate of claim 130 or 131, wherein the conjugate is a chemical bond linking

133. The linker has the formula IV: 【Transformation 38】 The structure of In the 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 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 The conjugate of any one of claims 130 to 132, which is alkyl.

134. The linker has the structure: 【Chemistry 39】 134. The conjugate of claim 133, comprising:

135. 1. A method 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 the production of said conjugate.

136. 1. A method for producing a conjugate comprising a presenter protein binding moiety conjugated to a target protein, comprising: (a) providing a compound comprising a presenter protein binding moiety and a cross-linking group, (b) a target protein, and (c) a presenter protein; and reacting said compound with said target protein under conditions that allow for the formation of said conjugate.

137. 137. The method of claim 136, wherein the presenter protein binds to the compound in the absence of the target protein.

138. 137. The method of claim 136, wherein the presenter protein does not substantially bind to the compound in the absence of the target protein.

139. 139. The method of any one of claims 136 to 138, wherein the compound and the target protein do not substantially react in the absence of the presenter protein.

140. 139. The method of any one of claims 136 to 138, wherein the compound and the target protein react in the absence of the presenter protein.

141. 141. The method of any one of claims 135 to 140, wherein the conditions do not include a reducing reagent.

142. A complex comprising (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein, and (ii) a presenter protein.

143. 143. The complex of claim 142, wherein the presenter protein is a protein encoded by any one of the genes listed in Table 1.

144. 143. The complex of claim 142, wherein the presenter protein is a prolyl isomerase.

145. 145. The complex of any one of claims 142 to 144, wherein the prolyl isomerase is a member of the FKBP family, a member of the cyclophilin family, or PIN1.

146. 146. The complex of claim 145, wherein the member of the FKBP family is FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

147. The complex of claim 145, wherein the member of the cyclophilin family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

148. 1. A method 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 combining a conjugate comprising a presenter protein binding moiety conjugated to a target protein and the presenter protein under conditions that allow the production of said complex.

149. A method for producing (i) a conjugate comprising a presenter protein-binding moiety conjugated to a target protein and (ii) a complex comprising a presenter protein, comprising: (a) a compound comprising a presenter protein-binding moiety and a cross-linking group; (b) a target protein; and (c) a presenter protein; reacting said compound with said target protein under conditions that allow for the formation of said complex.

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

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

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

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

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

155. 155. The method of any one of claims 148 to 154, wherein the condition comprises an excess of presenter protein.

156. A conjugate comprising a target protein binding moiety conjugated to a presenter protein.

157. 157. The conjugate of claim 156, wherein the site of the target protein binding portion of the conjugate is capable of non-covalently interacting with a target protein.

158. 157. The conjugate of claim 156, wherein the site of the target protein binding portion of the conjugate is capable of covalently interacting with a target protein.

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

160. 160. The conjugate of any one of claims 156 to 159, wherein the target protein comprises an undruggable surface.

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

162. 162. The conjugate of any one of claims 156 to 161, wherein the presenter protein is a protein encoded by any one of the genes listed in Table 1.

163. A conjugate according to any one of claims 156 to 161, wherein the presenter protein is a prolyl isomerase.

164. 164. The conjugate of any one of claims 156 to 163, wherein the presenter protein is a member of the FKBP family, a member of the cyclophilin family, or PIN1.

165. 165. The conjugate of claim 164, wherein the member of the FKBP family is FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

166. 165. The conjugate of claim 164, wherein the member of the cyclophilin family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

167. A conjugate according to any one of claims 156 to 166, wherein the amino acid sequence of the presenter protein has been modified to replace at least one amino acid with a reactive amino acid.

168. The conjugate of claim 167, wherein the reactive amino acid is a natural amino acid.

169. 169. The conjugate of claim 168, wherein the reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

170. The conjugate of claim 167, wherein the reactive amino acid is an unnatural amino acid.

171. 171. The conjugate of any one of claims 156 to 170, wherein the amino acid sequence of the presenter protein has been modified to replace at least one native reactive amino acid with a non-reactive amino acid.

172. 172. The conjugate of claim 171, wherein the at least one native reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

173. 173. The conjugate of claim 171 or 172, wherein said at least one native reactive amino acid is a solvent-exposed amino acid.

174. A conjugate according to any one of claims 171 to 173, wherein the amino acid sequence of the presenter protein is modified to replace all reactive amino acids with non-reactive amino acids.

175. The conjugate of any one of claims 171 to 174, wherein the non-reactive amino acid is a natural amino acid.

176. The conjugate of any one of claims 167 to 175, wherein the substitution is a conservative substitution.

177. 177. The conjugate of any one of claims 171 to 176, wherein the reactive amino acid is substituted with serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine.

178. The conjugate of any one of claims 171 to 174, wherein the non-reactive amino acid is an unnatural amino acid.

179. 179. The conjugate of any one of claims 156 to 178, wherein the target protein binding moiety and the presenter protein are joined through a linker.

180. 180. The conjugate of claim 179, wherein the linker is 1 to 20 atoms in length.

181. The linker has 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 Formula V having the structure In the 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 C １ -C ２ Alkyl, optionally substituted C １ -C ３ Heteroalkyl, O, S, and NR Ｎ and R Ｎ is hydrogen, optionally substituted C １－４ Alkyl, optionally substituted C ２－４ Alkenyl, optionally substituted C ２－４ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ aryl, or optionally substituted C １－７ Heteroalkyl, C １ and C ２ are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; f, g, h, I, j, and k are each independently 0 or 1; and D is optionally substituted C １－１０ Alkyl, optionally substituted C ２－１０ Alkenyl, optionally substituted C ２－１０ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ Aryl, optionally substituted 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 ２ 181. The conjugate of claim 179 or 180, wherein the chemical bond connecting

182. The linker has the formula IV: 【Chemistry 40】 The structure of In the 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 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 ６ 182. The conjugate of any one of claims 179 to 181, which is alkyl.

183. The linker has the structure: 【Chemistry 41】 183. The conjugate of claim 182, comprising:

184. 1. A method 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 the production of said conjugate.

185. 1. A method for producing a conjugate comprising a target protein binding moiety conjugated to a presenter protein, comprising: (a) providing a compound comprising a target protein binding moiety and a cross-linking group, (b) a presenter protein, and (c) a target protein; and reacting said compound with said presenter protein under conditions that allow the formation of said conjugate.

186. 186. The method of claim 185, wherein the target protein binds to the compound in the absence of the presenter protein.

187. 186. The method of claim 185, wherein the target protein does not substantially bind to the compound in the absence of the presenter protein.

188. 188. The method of any one of claims 185 to 187, wherein the compound and the presenter protein do not substantially react in the absence of the target protein.

189. 188. The method of any one of claims 185 to 187, wherein the compound and the presenter protein react in the absence of the target protein.

190. 190. The method of any one of claims 184 to 189, wherein the conditions do not include a reducing reagent.

191. A complex comprising (i) a conjugate comprising a target protein binding moiety conjugated to a presenter protein, and (ii) a target protein.

192. 192. The complex of claim 191, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein with classical protein-protein interaction domains and motifs.

193. 193. The complex of claim 191 or 192, wherein the target protein comprises an undruggable surface.

194. 194. The complex of any one of claims 191 to 193, wherein the target protein does not have a conventional binding pocket.

195. 1. A method 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 combining a conjugate comprising a target protein binding moiety conjugated to a presenter protein and the target protein under conditions that allow the production of said complex.

196. 1. A method for producing a complex comprising (i) a conjugate comprising a target protein binding moiety conjugated to a presenter protein and (ii) a target protein, comprising: (a) providing a compound comprising a target protein binding moiety and a cross-linking group, (b) a presenter protein, and (c) a target protein; and reacting said compound with said presenter protein under conditions that allow the formation of said complex.

197. 197. The method of claim 196, wherein the target protein binds to the compound in the absence of the presenter protein.

198. 197. The method of claim 196, wherein the target protein does not substantially bind to the compound in the absence of the presenter protein.

199. 199. The method of any one of claims 196 to 198, wherein the compound and the presenter protein do not substantially react in the absence of the target protein.

200. 200. The method of any one of claims 196 to 198, wherein the compound and the presenter protein react in the absence of the target protein.

201. 201. The method of any one of claims 195 to 200, wherein the conditions do not include a reducing reagent.

202. 202. The method of any one of claims 196-201, wherein the conditions comprise an excess of target protein.

203. Formula VII: A-L-B Formula VII having the structure wherein A is of formula VIIIa or VIIIb: 【Chemistry 42】 The structure of 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, CH ２ ,O,S,SO,SO ２ , or NR １３ and 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 in combination with 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 in combination with optionally substituted C ３ -C １０ Carbocyclyl, optionally substituted C ６ -C １０ Aryl, optionally substituted C ２ -C ９ heterocyclyl or optionally substituted C ２ -C ９ forming a heteroaryl, R ４ is optionally substituted C １ -C ６ is alkyl, L is an optional linker; The compound wherein B is a target protein binding moiety.

204. 204. The compound of claim 203, wherein the interaction between the target protein binding moiety and the target protein is non-covalent.

205. 204. The compound of claim 203, wherein the interaction between the target protein binding moiety and the target protein is covalent.

206. 206. The compound of any one of claims 203 to 205, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein with classical protein-protein interaction domains and motifs.

207. The compound of any one of claims 203 to 206, wherein the target protein comprises an undruggable surface.

208. 208. The compound of any one of claims 203 to 207, wherein the target protein does not have a conventional binding pocket.

209. A is a compound having the structure: 【Chemistry 43】 The compound of any one of claims 203 to 208, comprising:

210. 210. The compound of any one of claims 203 to 209, wherein the target protein binding moiety and the presenter protein are conjugated through a linker.

211. 211. The compound of claim 210, wherein the linker is 1 to 20 atoms in length.

212. The linker has 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 Formula V having the structure In the 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 C １ -C ２ Alkyl, optionally substituted C １ -C ３ Heteroalkyl, O, S, and NR Ｎ and R Ｎ is hydrogen, optionally substituted C １－４ Alkyl, optionally substituted C ２－４ Alkenyl, optionally substituted C ２－４ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ aryl, or optionally substituted C １－７ Heteroalkyl, C １ and C ２ are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; f, g, h, I, j, and k are each independently 0 or 1; and D is optionally substituted C １－１０ Alkyl, optionally substituted C ２－１０ Alkenyl, optionally substituted C ２－１０ Alkynyl, optionally substituted C ２－６ Heterocyclyl, optionally substituted C ６－１２ Aryl, optionally substituted 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 ２ 212. The compound of claim 210 or 211, wherein the chemical bond connecting

213. The linker has the formula IV: 【Chemistry 44】 The structure of In the 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 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 ６ 213. The compound of any one of claims 210 to 212, which is alkyl.

214. The linker has the structure: 【Chemistry 45】 The compound of claim 213, comprising:

215. A complex comprising (i) the compound of claim 203, (ii) a target protein, and (iii) a presenter protein.

216. 216. The complex of claim 215, wherein the target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein with classical protein-protein interaction domains and motifs.

217. 217. The complex of claim 215 or 216, wherein the target protein comprises an undruggable surface.

218. 218. The complex of any one of claims 215 to 217, wherein the target protein does not have a conventional binding pocket.

219. 219. The complex of any one of claims 215 to 218, wherein the presenter protein is a protein encoded by any one of the genes listed in Table 1.

220. The complex of any one of claims 215 to 218, wherein the presenter protein is a prolyl isomerase.

221. The compound of any one of claims 215 to 220, wherein the presenter protein is a member of the FKBP family, a member of the cyclophilin family, or PIN1.

222. 222. The compound of claim 221, wherein the member of the FKBP family is FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP51, or FKBP52.

223. The complex of claim 221, wherein the member of the cyclophilin family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

224. 1. A method for identifying a conjugate comprising a presenter protein binding moiety conjugated to a target protein capable of forming a complex with a presenter protein, the method comprising: (a) providing (i) a conjugate comprising a presenter protein binding moiety conjugated to a target protein and (ii) a presenter protein; (b) combining the conjugate and the 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 conjugate and the presenter protein form a complex; When the conjugate and the presenter protein form a complex, the conjugate is identified as a conjugate capable of forming a complex with the presenter protein; This identifies a conjugate comprising a presenter protein-binding moiety conjugated to a target protein capable of forming a complex with the presenter protein.

225. 1. A method for identifying a target protein that binds to a presenter protein, comprising: (a) providing (i) a conjugate comprising a presenter protein binding moiety conjugated to a target protein and (ii) a presenter protein; (b) combining the conjugate and the 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 binds to the presenter protein in the complex; the target protein is identified as binding to the presenter protein if the target protein binds to the presenter protein; This allows the target protein that binds to the presenter protein to be identified.

226. 1. A method for identifying 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 cross-linking moiety; (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 compound is capable of forming a complex with the presenter protein; and (c) determining whether the target protein and the compound react during the formation of the complex to form a conjugate; If the target protein and the compound react to form a conjugate during the formation of the complex, the target protein is identified as a target protein that can react with the compound in the presence of a presenter protein; This identifies target proteins that are capable of reacting with a compound comprising a presenter protein binding moiety and a cross-linking moiety in the presence of the presenter protein.

227. 1. A method for identifying a target protein that binds to a presenter protein, 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 to allow complex formation, if the compound is capable of forming a complex with the presenter protein; and (c) determining whether the target protein binds to the presenter protein in the complex; If the target protein binds to the presenter protein, the target protein is identified as a target protein that binds to a presenter protein; This allows the target protein that binds to the presenter protein to be identified.

228. 1. A method for identifying a target protein capable of forming a complex with a presenter protein, comprising: (a) providing (i) a compound of claim 181, (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 compound is capable of forming a complex with the presenter protein; and (c) determining whether the compound, the target protein, and the presenter protein form a complex; When the compound, the target protein, and the presenter protein form a complex, the target protein is identified as a target protein capable of forming a complex with the presenter protein; This identifies a target protein capable of forming a complex with a presenter protein.

229. 1. A method for identifying a target protein that binds to a presenter protein, comprising: (a) providing (i) a compound of claim 181, (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 compound is capable of forming a complex with the presenter protein; and (c) determining whether the target protein binds to the presenter protein in the complex; If the target protein binds to the presenter protein, the target protein is identified as a target protein that binds to the presenter protein; This allows the target protein that binds to the presenter protein to be identified.

230. 1. A method for identifying a location on a target protein for forming a conjugate with a presenter protein binding moiety, the conjugate being capable of forming a complex with a presenter protein, comprising: (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 under conditions suitable to allow complex formation, if the conjugate is capable of forming a complex with the presenter protein; (c) determining whether the conjugate and the presenter protein form a complex; and (d) repeating steps (a) to (c), conjugating the presenter protein binding moiety at different locations on the target protein, until the conjugate and the presenter protein form a complex; The method, wherein a location on the target protein for forming a conjugate with a presenter protein-binding moiety that can form a complex with a presenter protein is identified when the conjugate and the presenter protein form a complex, thereby identifying a location on the target protein for forming a conjugate that can form a complex with a presenter protein.

231. 1. A method for identifying a location on a target protein for forming a conjugate with a presenter protein binding moiety, the conjugate being capable of forming a complex with a presenter protein, comprising: (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 said compound with said target protein in the presence of said presenter protein under conditions that allow the formation of a conjugate comprising a presenter protein binding moiety conjugated to the target protein at a location; (c) determining whether the conjugate and the presenter protein form a complex; and (d) repeating steps (a) to (c), conjugating the presenter protein binding moiety at different locations on the target protein, until the conjugate and the presenter protein form a complex; a location on the target protein for forming a conjugate capable of complexing with a presenter protein is identified when said conjugate and said presenter protein form a complex; This method identifies locations on the target protein for forming conjugates with presenter protein-binding moieties that are capable of forming complexes with presenter proteins.

232. 232. The method of claim 230 or 231, wherein the amino acid sequence of the target protein has been modified to replace at least one amino acid with a reactive amino acid.

233. 233. The method of claim 232, wherein the reactive amino acid is a natural amino acid.

234. 234. The method of claim 233, wherein the reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

235. 233. The method of claim 232, wherein the reactive amino acid is an unnatural amino acid.

236. 236. The method of any one of claims 230-235, wherein the amino acid sequence of the target protein has been modified to replace at least one native reactive amino acid with a non-reactive amino acid.

237. 237. The method of claim 236, wherein the at least one native reactive amino acid is cysteine, lysine, tyrosine, aspartic acid, glutamic acid, or serine.

238. The method of claim 236 or 237, wherein the at least one native reactive amino acid is a solvent-exposed amino acid.

239. 239. The method of any one of claims 236 to 238, wherein the amino acid sequence of the target protein is modified to replace all reactive amino acids with non-reactive amino acids.

240. 240. The method of any one of claims 236 to 239, wherein the non-reactive amino acid is a natural amino acid.

241. 241. The method of any one of claims 230 to 240, wherein the substitution is a conservative substitution.

242. 242. The method of any one of claims 232-241, wherein the reactive amino acid is substituted with serine, valine, alanine, isoleucine, threonine, tyrosine, aspartic acid, glutamic acid, or leucine.

243. 243. The method of any one of claims 232-242, wherein the non-reactive amino acid is an unnatural amino acid.

244. 1. A method for identifying a compound capable of covalently binding to a target protein in the presence of a presenter protein, comprising: (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 through the cross-linking group of the compound in the sample; The method, wherein when the compound and the target protein react in the sample, the compound is identified as binding covalently to the target protein in the presence of a presenter protein.

245. 1. A method for identifying a compound capable of selectively and covalently binding to a target protein in the presence of a presenter protein, comprising: (a) providing a first sample comprising (i) a compound comprising a presenter protein binding moiety and a crosslinking group, (ii) a target protein, and (iii) a presenter protein, and a second sample comprising (i) the same compound comprising a presenter protein binding moiety and a crosslinking group as in the first sample, and (ii) the same target protein as in the first sample; (b) determining the extent to which the compound and the target protein react in the first sample compared to the second sample; The method, wherein 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 more in the first sample than in the second sample.

246. 246. The method of claim 245, wherein 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.

247. The method of claim 245 or 246, wherein 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 a first sample but do not substantially react in the second sample.

248. 1. A method for identifying a conjugate capable of forming a complex with a presenter protein, comprising: (a) providing (i) a conjugate comprising a presenter protein binding moiety conjugated to a target protein and (ii) a presenter protein; and (b) combining the conjugate and the presenter protein under conditions suitable to allow complex formation, if the conjugate is capable of forming a complex with the presenter protein; (c) determining whether the conjugate and the presenter protein form a complex; When the conjugate and the presenter protein form a complex, the conjugate is identified as being capable of forming a complex with the presenter protein; This identifies a conjugate capable of forming a complex with the presenter protein.

249. 1. A method for determining the structure of an interface within a complex comprising a presenter protein and a target protein, comprising: (a) providing (i) a conjugate comprising a presenter protein binding moiety conjugated to a target protein and (ii) a presenter protein; (b) combining the conjugate and the 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 the crystal structure of the complex; the interface structure includes at least a portion of the crystal structure between the presenter protein and the target protein; This method allows the structure of the interface within a complex containing the presenter protein and the target protein to be determined.

250. 1. A method for determining the structure of an interface within a complex comprising a presenter protein and a target protein, 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 to allow complex formation, if the compound is capable of forming a complex with the presenter protein; and (c) determining the crystal structure of the complex; The structure of the interface includes at least a portion of the crystal structure between the presenter protein and the target protein. This method allows the structure of the interface within a complex containing the presenter protein and the target protein to be determined.

251. 1. A method for determining the structure of an interface within a complex comprising a presenter protein and a target protein, comprising: (a) providing (i) a compound of claim 203, (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 compound is capable of forming a complex with the presenter protein; and (c) determining the crystal structure of the complex; the interface structure includes at least a portion of the crystal structure between the presenter protein and the target protein; This method allows the structure of the interface within a complex containing the presenter protein and the target protein to be determined.

252. 252. The method of any one of claims 249 to 251, wherein the interface in a complex comprising a presenter protein and a target protein is a binding pocket.

253. 1. A method for obtaining X-ray crystallographic coordinates for a complex, comprising: (a) providing (i) a conjugate comprising a presenter protein binding moiety conjugated to a target protein and (ii) a presenter protein; (b) combining the conjugate and the 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 the crystal structure of the complex; The method thereby provides X-ray crystallographic coordinates for the complex.

254. 1. A method for obtaining X-ray crystallographic coordinates for a complex, 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 to allow complex formation, if the compound is capable of forming a complex with the presenter protein; and (c) determining the crystal structure of the complex; The method thereby provides X-ray crystallographic coordinates for the complex.

255. 1. A method for obtaining X-ray crystallographic coordinates for a complex, comprising: (a) providing (i) a compound of claim 203, (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 compound is capable of forming a complex with the presenter protein; and (c) determining the crystal structure of the complex; The method thereby provides X-ray crystallographic coordinates for the complex.

256. 1. A method for determining residues on a target protein that are involved in binding to a presenter protein, comprising: (a) providing the X-ray crystal coordinates of a complex obtained by the method of any one of claims 253 to 255; (b) identifying residues of the target protein that include atoms within 4 Å of an atom on the presenter protein; This allows the residues on the target protein involved in binding to the presenter protein to be determined.

257. 224. A method for determining the biochemical and / or biophysical properties of a complex according to any one of claims 142 to 147, 191 to 194 or 215 to 223, comprising: (a) providing the X-ray crystal coordinates of the complex of any one of claims 142 to 147, 191 to 194, or 215 to 223, obtained by the method of any one of claims 253 to 255; (b) calculating the biochemical and / or biophysical properties of the complex; This method allows the biochemical and / or biophysical properties of the presenter protein / target protein complex to be determined.

258. The biochemical and / or biophysical properties include the free energy of binding of the complex, the K ｄ , the K of the complex ｉ , the K of the complex ｉｎａｃｔ and / or the K of said complex ｉ / K ｉｎａｃｔ The method of claim 257, comprising:

259. 259. The method of claim 258, wherein the biochemical and / or biophysical properties include the free energy of binding of the complex.

260. 260. The method of claim 259, wherein the free energy of binding of the complex is determined by isothermal titration calorimetry.

261. The biochemical and / or biophysical properties of the complex ｄ The method of claim 258, comprising:

262. The K ｄ The method of claim 261, wherein is determined by surface plasmon resonance.

263. The biochemical and / or biophysical properties of the complex ｉ , the K of the complex ｉｎａｃｔ and / or the K of said complex ｉ / K ｉｎａｃｔ The method of claim 258, comprising:

264. The K ｉ , the K of the complex ｉｎａｃｔ and / or the K of the complex ｉ / K ｉｎａｃｔ is determined by mass spectrometry.

265. A composition comprising a compound according to any one of claims 1 to 98 or 203 to 214 and a suitable carrier.

266. 216. A pharmaceutical composition comprising a compound of any one of claims 1-98 or 203-214 and a pharmaceutically acceptable carrier.

267. 267. A method of modulating a target protein, said method comprising contacting said target protein with a modulating amount of a compound of any one of claims 1-98 or 203-214 or a composition of claim 265 or 266.

268. 10. A method of modulating a target protein, comprising contacting a cell with an effective amount of a composition of any one of claims 1-98 or 203-214, or claim 265 or 266, thereby forming in the cell a complex of any one of claims 142-147, 191-194, or 215-223.

269. 184. A method of modulating a target protein, the method comprising contacting said target protein with a conjugate according to any one of claims 155 to 183.

270. 266. A method for inhibiting prolyl isomerase activity, comprising contacting a cell expressing said prolyl isomerase with a compound of any one of claims 1 to 98 or 203 to 214 or a composition of claim 265 or 266 under conditions that allow the formation of a complex between said compound and said prolyl isomerase, thereby inhibiting prolyl isomerase activity.

271. 266. A method for forming a complex according to any one of claims 142 to 147, 191 to 194 or 215 to 223 in a cell, the method comprising contacting a cell expressing a presenter protein with a compound according to any one of claims 1 to 98 or 203 to 214 or a composition according to claim 265 or 266 under conditions which allow the formation of a complex between said compound and said presenter protein.

272. 1. A method for identifying a target protein capable of forming a complex with a presenter protein, comprising: (a) providing a presenter protein comprising (i) one or more target proteins, (ii) a compound according to any one of claims 1 to 98, and (iii) a tag; (b) combining the one or more target proteins, the compound, and the presenter protein under conditions suitable to allow complex formation, if the target protein is capable of forming a complex with the presenter protein; and (c) determining whether the one or more target proteins form a complex with the compound and the presenter protein; The method, wherein the target protein that forms a complex with the compound and the presenter protein is identified as a target protein that can form a complex with a presenter protein.

273. 273. The method of claim 272, wherein the presenter protein comprises an affinity tag.

274. 274. The method of claim 272 or 273, wherein said determining step comprises utilizing the tag of said presenter protein to selectively isolate a target protein that is complexed with said presenter protein.

275. 275. The method of any one of claims 272 to 274, wherein the method further comprises (d) identifying one or more target proteins within a complex formed between the target protein(s) and the presenter protein.

276. 276. The method of claim 275, wherein said identifying the structure of said target protein comprises performing mass spectrometry on said complex.

277. 1. A method for identifying a target protein capable of forming a complex with a presenter protein, comprising: (a) providing (i) two or more target proteins, (ii) a compound according to any one of claims 1 to 98, and (iii) a presenter protein comprising an affinity tag; (b) combining the two or more target proteins, the compound, and the presenter protein under conditions suitable to allow complex formation, if the target proteins are capable of forming a complex with the presenter protein; (c) selectively isolating one or more complexes of the target protein, the compound, and the presenter protein formed in step (b); and (d) determining the structure of the target protein within the one or more complexes isolated in step (c) by mass spectrometry; This identifies a target protein capable of forming a complex with a presenter protein.

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