Methods for identifying compounds used to induce protein-protein interactions

Abstract

This disclosure relates to a method for identifying compounds that target a first protein and a second protein, wherein the first protein and / or the second protein is associated with a disease or condition.

Description

Related applications

[0001] This application claims the benefit and priority of U.S. Application No. 18 / 339,429, filed June 22, 2023, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0002] Molecular glues are chemical compounds that induce one or more proteins to approach each other. Molecular glue compounds enhance the affinity between one or more proteins, thereby causing protein-protein interactions. Depending on the specific protein, these interactions can affect the function of one or both proteins. Functional consequences may include, but are not limited to: changes in protein stability, changes in protein levels, changes in post-translational modifications, changes in protein localization, and / or changes in protein activity.

[0003] In the context of protein degradation, molecular glue enhances the binding of the protein of interest (POI) to the E3 ligase (E3), which in turn leads to POI degradation mediated by the ubiquitin-proteasome system (UPS). Molecular glue is difficult to detect for several reasons. First, it often shows little or no binding to either a single POI or E3. Complexes are typically only observed in the presence of molecular glue, POI, and E3. Even when the glue compound shows measurable binding to either protein, the ternary complex is important, and most binary binding events do not lead to ternary complex formation. Therefore, the presence of all three components—POI, E3, and molecular glue—is required during screening. Second, the requirement for the presence of all three components in the screening assay mixture presents a significant challenge because it is extremely difficult to predict which of the numerous (>600) E3 ligases in the human proteome are likely to form a ternary complex with a given POI.

[0004] Because screening a vast number of compounds (often exceeding hundreds of thousands) is required, testing combinations of many individual E3 ligases with a large number of compounds becomes challenging. Therefore, novel methods for identifying molecular gels are still needed. Summary of the Invention

[0005] In some aspects, this disclosure provides a method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof.

[0006] In some respects, this disclosure provides for the identification of a first protein by the methods of this disclosure.

[0007] In some respects, this disclosure provides for the identification of a second protein by the methods of this disclosure.

[0008] In some respects, this disclosure provides a combination of a first protein and a second protein identified by the methods of this disclosure.

[0009] In some aspects, this disclosure provides combinations of a first protein, a second protein, and compounds targeting the first and second proteins identified by the methods of this disclosure.

[0010] In some respects, this disclosure provides compounds identified by the methods of this disclosure.

[0011] In some aspects, this disclosure provides a method for degrading a first protein in a subject, the method comprising administering to the subject a compound identified by the method of this disclosure.

[0012] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for degrading a first protein in a subject.

[0013] In some respects, this disclosure provides for the use of compounds identified by this disclosure in the preparation of a medicament for degrading a first protein in a subject.

[0014] In some aspects, this disclosure provides methods for treating and / or preventing diseases or conditions associated with a first protein in a subject, the methods comprising administering to the subject a therapeutically effective amount of a compound identified by the methods of this disclosure.

[0015] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for the treatment and / or prevention of a disease or condition associated with a first protein in a subject.

[0016] In some respects, this disclosure provides for the use of compounds identified by the methods of this disclosure in the preparation of medicaments for treating and / or preventing diseases or conditions associated with a first protein in a subject.

[0017] In some aspects, this disclosure provides a method for degrading a second protein in a subject, the method comprising administering to the subject a compound identified by the method of this disclosure.

[0018] In some respects, this disclosure provides a compound identified by the methods of this disclosure for degrading a second protein in a subject.

[0019] In some respects, this disclosure provides for the use of compounds identified by this disclosure in the preparation of medicaments for degrading a second protein in a subject.

[0020] In some aspects, this disclosure provides methods for treating and / or preventing diseases or conditions associated with a second protein in a subject, the methods comprising administering to the subject a therapeutically effective amount of a compound identified by the methods of this disclosure.

[0021] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for the treatment and / or prevention of diseases or conditions associated with a second protein in a subject.

[0022] In some respects, this disclosure provides for the use of compounds identified by the methods of this disclosure in the preparation of medicaments for treating and / or preventing diseases or conditions associated with a second protein in a subject.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification, the singular form includes the plural unless the context clearly requires otherwise. Although similar or equivalent methods and materials described herein may be used to practice or test this disclosure, suitable methods and materials are described below. References cited herein are not considered prior art to the claimed invention. In case of conflict, this specification, including the definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be restrictive. In case of conflict between the chemical structures and names of compounds disclosed herein, the chemical structures shall prevail.

[0024] Other features and advantages of this disclosure will become apparent from the following detailed description and claims. Attached Figure Description

[0025] Figure 1A This is a graph showing the simulated time process and dose dependence of fluorescence signal generation for GFP11-RBM39 and GFP10-DCAF15 (combination A).

[0026] Figure 1B This is a graph showing the simulated time process and dose dependence of fluorescence signal generation of RBM39-GFP11 and GFP10-DCAF15 (combination B).

[0027] Figure 1C This is a graph showing the simulated time process and dose dependence of fluorescence signal generation for GFP11-RBM39 and DCAF15-GFP10 (combination C).

[0028] Figure 1D This is a graph showing the simulated time process and dose dependence of fluorescence signal generation for RBM39-GFP11 and DCAF15-GFP10 (combination D).

[0029] Figure 2A This is a graph showing the simulated effect of GFP enhancer on the fluorescence signal intensity of combinations A and C.

[0030] Figure 2B This is a graph showing the simulated effect of GFP enhancer on the fluorescence signal intensity of combination B and combination D.

[0031] Figure 3 This is a simulation diagram illustrating the potential detection of test compounds from a MALDI MS experiment based on simulated MS signals between indisulam and lenalidomide.

[0032] Figure 4 This is a simulation plot showing the potential detection of E3 from the MALDI MS experiment.

[0033] Figure 5 This is a graph showing the time-series of fluorescence signal generation for IKZF2-CRBN gel compound 1, IKZF2-CRBN gel compound 2, IKZF2-CRBN gel compound 3, and the IKZF2-CRBN non-gel compound chloroquine oxalool and indexsulfanilamide.

[0034] Figure 6 This is a graph showing the dose-response data for the fluorescence signal generation of four different RBM39-DCAF15 gel compounds. The initial rate of signal generation (k1) is shown as the ratio of the DMSO-treated sample (S / B). Detailed Implementation

[0035] This disclosure relates to methods for identifying compounds used to induce protein-protein interactions. This disclosure also relates to compounds identified in said methods, and the use of said compounds, for example, in the degradation of POIs and / or in the treatment or prevention of diseases or conditions. The methods of this disclosure are applicable to a variety of fields, including but not limited to human therapy, agriculture, and animal health.

[0036] The method disclosed herein In some aspects, this disclosure provides a method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof.

[0037] In some aspects, this disclosure provides a method applicable to human therapy, agriculture, and / or animal health. In some embodiments, the method is applicable to human therapy. In some embodiments, the method is applicable to humans. In some embodiments, the method is applicable to agriculture. In some embodiments, the method is applicable to agricultural entities (e.g., seeds, seedlings, leaves, flowers, plants, etc.). In some embodiments, the method is applicable to animal health. In some embodiments, the method is applicable to animals.

[0038] In some embodiments, the method is a method for identifying a first protein (e.g., a POI) that is targeted by the compound and the second protein.

[0039] In some embodiments, the method is a method for identifying a second protein that is targeted by the compound and the first protein.

[0040] In some embodiments, the method is a method for identifying a combination of a first protein and a second protein targeted by a compound.

[0041] In some embodiments, the method is a method for identifying a combination of a first protein, a second protein, and compounds that target the first protein and the second protein.

[0042] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a assay signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0043] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The first and second tag fragments are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0044] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The assay mixture induces a close proximity between the first protein or a fragment thereof and the second protein or a fragment thereof, thereby causing the first tag fragment and the second tag fragment to generate or enhance the assay signal.

[0045] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof associated with the complex.

[0046] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The assay mixture generates a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; such that the first tag fragment and the second tag fragment generate or enhance the assay signal; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0047] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a assay signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; and (ii) Identify any combination of the first protein, the second protein, and the compound that is associated with (e.g., causes) the induced proximity; The identified compound is capable of causing degradation of the first protein (e.g., POI) in the presence of the second protein (e.g., E3 ligase).

[0048] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag GFP fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second I GFP fragment; (c) a detector GFP fragment, said detector GFP fragment being complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compounds, The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof associated with the complex, wherein the identified compound is capable of modulating protein-protein interactions (PPIs) between the first protein and the second protein.

[0049] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag GFP fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second I GFP fragment; (c) a detector GFP fragment, said detector GFP fragment being complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The first tag GFP fragment, the second tag GFP fragment, and the detector GFP fragment are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof, wherein the identified compound is capable of modulating protein-protein interactions (PPIs) between the first protein and the second protein.

[0050] In some embodiments, the method includes: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag GFP fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second I GFP fragment; (c) a detector GFP fragment, said detector GFP fragment being complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compounds, The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are configured to generate or enhance a assay signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof that are associated with the induced proximity. The identified compound can cause the degradation of the first protein in the presence of the second protein.

[0051] In some embodiments, the induced proximity is associated with (e.g., caused by) the compound.

[0052] In some embodiments, the assay mixture further comprises a detector fragment complementary to the first tag fragment and the second tag fragment.

[0053] In some embodiments, the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0054] In some embodiments, the method includes: (i) Provide the following assay mixture: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag fragment; (c) a detector segment, said detector segment being complementary to the first tag segment and the second tag segment; and (d) Candidate compounds, The first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0055] In some embodiments, the method includes: (i) Provide the following assay mixture: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag fragment; (c) a detector segment, said detector segment being complementary to the first tag segment and the second tag segment; and (d) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0056] In some embodiments, the method includes: (i) Provide the following assay mixture: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag fragment; (c) a detector segment, said detector segment being complementary to the first tag segment and the second tag segment; and (d) Candidate compounds, The first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; and The identified compound is capable of causing degradation of the first protein (e.g., POI) in the presence of the second protein (e.g., E3 ligase).

[0057] In some embodiments, the method includes: (i) Provide the following assay mixture: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag fragment; (c) a detector segment, said detector segment being complementary to the first tag segment and the second tag segment; and (d) Candidate compounds, The assay mixture induces a close proximity between the first protein or a fragment thereof and the second protein or a fragment thereof, such that the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance the assay signal.

[0058] In some embodiments, the method includes: (i) Provide the following assay mixture: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag fragment; (c) a detector segment, said detector segment being complementary to the first tag segment and the second tag segment; and (d) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof, such that the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance the assay signal; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0059] In some embodiments, the method includes: (i) Provide the following assay mixture: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag fragment; (c) a detector segment, said detector segment being complementary to the first tag segment and the second tag segment; and (d) Candidate compounds, The assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; such that the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance the assay signal; and The identified compound is capable of causing degradation of the first protein (e.g., POI) in the presence of the second protein (e.g., E3 ligase).

[0060] In some embodiments, the assay mixture contains a variety of different first proteins or fragments thereof.

[0061] In some embodiments, the measurement mixture generates a complex, thereby generating or enhancing the measurement signal.

[0062] In some embodiments, the assay mixture induces proximity between protein molecules, thereby generating or enhancing the assay signal.

[0063] In some embodiments, the assay mixture comprises at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 different first proteins or fragments thereof.

[0064] In some embodiments, the assay mixture comprises about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 different first proteins or fragments thereof.

[0065] In some embodiments, the assay mixture comprises about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, or about 600 to about 700 different first proteins or fragments thereof.

[0066] In some embodiments, the assay mixture comprises about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, or about 600 to about 1000 different first proteins or fragments thereof.

[0067] In some embodiments, the first protein is a separated first protein.

[0068] In some embodiments, the assay mixture contains a variety of different second proteins or fragments thereof.

[0069] In some embodiments, the assay mixture contains at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or at least 1000 different second proteins or fragments thereof.

[0070] In some embodiments, the assay mixture contains at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or at least 700 different second proteins or fragments thereof.

[0071] In some embodiments, the assay mixture comprises about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 different second proteins or fragments thereof.

[0072] In some embodiments, the assay mixture contains about 2 to about 5, about 5 to about 10, or about 10 to about 20 different second proteins or fragments thereof.

[0073] In some embodiments, the mixture being measured contains a variety of different candidate compounds.

[0074] In some embodiments, the mixture being measured contains at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or at least 1000 different candidate compounds.

[0075] In some embodiments, candidate compounds are small molecules (e.g., <5000 Da, <4500 Da, <4000 Da, <3500 Da, <3000 Da, <2500 Da, <2000 Da, <1500 Da, <1000 Da, <900 Da, <800 Da, <700 Da, <600 Da, <500 Da, or <400 Da).

[0076] In some embodiments, none of the candidate compounds is water.

[0077] In some embodiments, none of the candidate compounds are buffer salts.

[0078] In some embodiments, none of the candidate compounds are proteins or fragments thereof.

[0079] In some embodiments, none of the candidate compounds is GFP protein or a fragment thereof.

[0080] In some embodiments, no candidate compound contains GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, or GFP11, or any combination thereof.

[0081] In some embodiments, none of the candidate compounds are GFP1-9.

[0082] In some embodiments, the mixture being measured contains about 50 to about 250 different candidate compounds.

[0083] In some embodiments, the mixture being measured contains about 250 to about 2,500 different candidate compounds.

[0084] In some embodiments, the assay mixture contains more than about 2,500 different candidate compounds.

[0085] In some embodiments, the first tag fragment and the second tag fragment are configured to generate or enhance the assay signal when the assay mixture produces a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound.

[0086] In some embodiments, the assay mixture generates a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound; such that the first and second tag fragments generate or enhance the assay signal.

[0087] In some embodiments, the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound.

[0088] In some embodiments, the assay mixture generates a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound; such that the first tag fragment, the second tag fragment, and the detector fragment generate or enhance the assay signal.

[0089] In some embodiments, the mixture is measured to produce a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound.

[0090] In some embodiments, the measured signal is light emission.

[0091] In some embodiments, the measurement signal is fluorescence, fluorescence polarization, time-resolved fluorescence (TRF), homogeneous time-resolved fluorescence, luminescence, or UV / Vis.

[0092] In some embodiments, the measurement signal is fluorescence, luminescence, phosphorescence, or scintillation.

[0093] In some embodiments, the measured signal is fluorescence.

[0094] In some embodiments, the measurement signal is based on a radioactive signal, such as a scintillation proximity (SPA) signal.

[0095] In some embodiments, the measured signal is a radiation signal.

[0096] In some embodiments, the measured signal is a change in color.

[0097] In some embodiments, step (i) further includes detecting the generated or enhanced measurement signal (e.g., fluorescence).

[0098] In some embodiments, the methods of this disclosure are performed once or multiple times. In some embodiments, the methods of this disclosure are performed once, twice, three times, four times, or five times. In some aspects, the methods of this disclosure are performed multiple times. In some aspects, the methods of this disclosure are performed multiple times, wherein the method is performed using a new hole each time.

[0099] Label and detector fragments In some embodiments, the first tag fragment and the second tag fragment are configured to form a signaling protein when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0100] In some embodiments, the assay mixture induces a close proximity between the first protein or a fragment thereof and the second protein or a fragment thereof, thereby causing the first and second tagged fragments to generate or enhance the assay signal.

[0101] In some embodiments, the first tag fragment, the second tag fragment, and the detector fragment are configured to form a signal protein when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0102] In some embodiments, the assay mixture induces a close proximity between the first protein or a fragment thereof and the second protein or a fragment thereof, thereby causing the first tag fragment, the second tag fragment, and the detector fragment to generate or enhance the assay signal.

[0103] In some embodiments, the signaling protein is a fluorescent protein, such as an engineered fluorescent protein.

[0104] In some embodiments, the signaling protein is green fluorescent protein (GFP), enhanced GFP (EGFP), superfolder GFP (sfGFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet, mTurquoise2), yellow fluorescent protein (e.g., YFP, Citrine, Venus, Ypet), redox-sensitive GFP (roGFP), or a mutant thereof.

[0105] In some embodiments, the signaling protein is GFP.

[0106] In some embodiments, the signaling protein is a GFP mutant (BFPms1), for example, preferentially binding to Zn(II) and Cu(II) rather than Ca(II).

[0107] In some embodiments, the signaling protein is a fluorescent protein having a β-barrel structure consisting of 11-chain β-sheets (which may be derived from various natural sources).

[0108] In some embodiments, the signaling protein is mCherry (a member of the mFruit family of monomeric red fluorescent protein (mRFP)). In some embodiments, the signaling protein is mRFP1, mStrawberry, mOrange, or dTomato.

[0109] In some embodiments, the first tag fragment is a first tag green fluorescent protein (GFP) fragment.

[0110] In some embodiments, the second tag fragment is a second tag GFP fragment (e.g., a second tag GFP fragment that is different from the first tag GFP fragment).

[0111] In some embodiments, the detector fragment is a detector GFP fragment (e.g., a detector GFP fragment that is different from the second tag GFP fragment and the first tag GFP fragment).

[0112] In some embodiments, the first-tagged GFP fragment and the second-tagged GFP fragment are configured to form GFP when the assay mixture induces an induced proximity between the first protein or a fragment thereof and the second protein or a fragment thereof (e.g., induced by a candidate compound).

[0113] In some embodiments, the determination of the mixture induces a close proximity between a first protein or a fragment thereof and a second protein or a fragment thereof, such that a first-tagged GFP fragment and a second-tagged GFP fragment are configured to form GFP.

[0114] In some embodiments, the first tag GFP fragment, the second tag GFP fragment, and the detector GFP fragment are configured to form GFP when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof (e.g., induced by a candidate compound).

[0115] In some embodiments, the determination of the mixture induces an approach between a first protein or a fragment thereof and a second protein or a fragment thereof, such that a first-tagged GFP fragment, a second-tagged GFP fragment, and a detector GFP fragment are configured to form GFP.

[0116] In some embodiments, the first-tagged GFP fragment and the second-tagged GFP fragment are independently selected from GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, and GFP11. In some embodiments, the first-tagged GFP fragment and the second-tagged GFP fragment are selected from combinations described in the Annual Review of Biophysics. Annu. Rev. Biophys. )》 6(48):19-44 (2019) (incorporated into this paper by reference).

[0117] In some embodiments, the first tagged GFP fragment and the second tagged GFP fragment are independently selected from GFP1 and GFP2.

[0118] In some embodiments, the first tagged GFP fragment and the second tagged GFP fragment are independently selected from GFP2 and GFP3.

[0119] In some embodiments, the first tagged GFP fragment and the second tagged GFP fragment are independently selected from GFP5 and GFP6.

[0120] In some embodiments, the first tagged GFP fragment and the second tagged GFP fragment are independently selected from GFP7 and GFP8.

[0121] In some embodiments, the first tagged GFP fragment and the second tagged GFP fragment are independently selected from GFP8 and GFP9.

[0122] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP9 and GFP10.

[0123] In some embodiments, the first tag GFP fragment is GFP10, and the second tag GFP fragment is GFP11.

[0124] In some embodiments, the first tag GFP fragment is GFP11, and the second tag GFP fragment is GFP10.

[0125] In some embodiments, the detector GFP fragment includes GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, and GFP9.

[0126] In some embodiments, the detector GFP fragment further includes a chromophore.

[0127] In some embodiments, the first tag GFP fragment, the second tag GFP fragment, and the detector fragment are selected from the combinations described in Table 1 below.

[0128] Table 1 Targeted protein In some embodiments, the first protein is the protein of interest (POI).

[0129] In some embodiments, a fragment of the first protein contains a binding site for the second protein.

[0130] In some embodiments, a fragment of the first protein includes at least a portion of the binding site of the first protein to the identified compound.

[0131] In some embodiments, a fragment of the first protein includes a binding site for the first protein to the identified compound.

[0132] In some embodiments, a fragment of the first protein contains a binding site for the protein of interest.

[0133] In some embodiments, POI is a kinase, phosphatase, glycosylationase, deglycosylationase, methylationase, demethylationase, sumoylase, deubiquitinase, acetyltransferase, deacetylase, fatty acyltransferase, protease, isomerase, or arginase.

[0134] In some embodiments, the POI is a cytoskeletal protein or a protein that has a scaffold function to aggregate other proteins together into a complex.

[0135] In some embodiments, the POI is a metabolic enzyme, transcription factor, cell surface receptor (e.g., GPCR or receptor tyrosine kinase), ion channel, membrane-embedded or associated enzyme (e.g., adenylate cyclase), RNA polymerase, RNA splicease, transport protein, DNA helicase, or DNA endonuclease.

[0136] In some embodiments, POI is a hydroxylase, dehydrogenase, reductase, oxidase, oxygenase, oxidoreductase, carboxylase, decarboxylase, lyase, aldolase, desaturase, mutase, epimerase, isomerase, racemic enzyme, esterase, amidase, deaminase, aminotransferase, hydratase, superoxide dismutase, ligase, carbonic anhydrase, nucleotide transferase, glycosyltransferase, or glycosidase.

[0137] In some embodiments, POI is a ubiquitin ligase (i.e., an E3 ligase).

[0138] In some embodiments, the POI is a protein associated with a disease or condition. In some embodiments, the protein does not have any known function in the mechanism of the disease or condition.

[0139] In some embodiments, the first protein (e.g., POI) is associated with a disease or condition.

[0140] In some embodiments, the presence or activity of a first protein (e.g., POI) is associated with a disease or condition.

[0141] In some embodiments, certain post-translational modifications of the first protein are associated with a disease or condition.

[0142] In some embodiments, degradation of the first protein (e.g., POI) enables the treatment or prevention of a disease or condition.

[0143] In some embodiments, the second protein can induce the degradation of the first protein upon induced proximity between the first and second proteins (e.g., in a cellular environment or when other components are provided in a biochemical system, such as a proteasome mechanism).

[0144] In some embodiments, the second protein induces the degradation of the first protein upon induced proximity between the first and second proteins (e.g., in a cellular environment or when other components are provided in a biochemical system, such as a proteasome mechanism).

[0145] In some embodiments, the second protein is a ubiquitin ligase (i.e., an E3 ligase).

[0146] In some embodiments, a fragment of the second protein contains a binding site for the first protein.

[0147] In some embodiments, the fragment of the second protein includes at least a portion of the binding site of the second protein to the identified compound.

[0148] In some embodiments, a fragment of the second protein includes a binding site for the second protein to the identified compound.

[0149] In some embodiments, a fragment of the second protein contains a binding site for a ubiquitin ligase.

[0150] In some embodiments, the fragment of the second protein contains a binding site for the E3 ligase.

[0151] In some embodiments, the binding site of the E3 ligase includes a substrate receptor component. In some embodiments, the substrate receptor component includes, but is not limited to, human cerebellar protein (CRBN), CRBN / DDB1, DCAF15, DCAF15 / DDB1, VHL, and VHL / EloB / EloC.

[0152] In some embodiments, the binding site of the E3 ligase comprises an adaptor protein component. In some embodiments, the adaptor protein component comprises, but is not limited to, DDB1.

[0153] In some embodiments, the second protein is the protein of interest (POI).

[0154] In some embodiments, the second protein is a kinase, phosphatase, glycosylationase, deglycosylationase, methylationase, demethylationase, sumoylase, deubiquitinase, acetyltransferase, deacetylase, fatty acyltransferase, protease, isomerase, or arginase.

[0155] In some embodiments, the second protein is a protease, a proteasome, a component of a proteasome complex, an autophagy receptor, or a component of an autophagy receptor complex.

[0156] In some embodiments, the second protein is a protein that has a scaffold function to aggregate other proteins together into the complex.

[0157] In some embodiments, the second protein is a metabolic enzyme, transcription factor, cell surface receptor (e.g., GPCR or receptor tyrosine kinase), ion channel, membrane-embedded or associated enzyme (e.g., adenylate cyclase), RNA splicease, transport protein, DNA helicase, or DNA endonuclease.

[0158] In some embodiments, the second protein is a hydroxylase, dehydrogenase, reductase, oxidase, oxygenase, oxidoreductase, carboxylase, decarboxylase, lyase, aldolase, desaturase, mutase, epimerase, isomerase, racemic enzyme, esterase, amidase, deaminase, aminotransferase, hydratase, superoxide dismutase, ligase, carbonic anhydrase, nucleotide transferase, glycosyltransferase, or glycosidase.

[0159] In some embodiments, the second protein is a heat shock protein.

[0160] In some embodiments, the second protein is associated with a disease or condition.

[0161] In some embodiments, the presence or activity of the second protein is associated with a disease or condition.

[0162] In some embodiments, post-translational modifications of the second protein are associated with a disease or condition.

[0163] In some embodiments, the degradation of the second protein enables the treatment or prevention of diseases or conditions.

[0164] In some embodiments, the first protein is POI and the second protein is ubiquitin ligase.

[0165] In some embodiments, the first protein is a ubiquitin ligase and the second protein is a POI.

[0166] In some embodiments, both the first and second proteins are the same protein (e.g., a POI or a ubiquitin ligase). For example, the first and second proteins can homodimerize in the presence of the compound of interest. In another example, the first and second proteins are each a kinase that can homodimerize or heterodimerize in the presence of the compound of interest, which can lead to cross-phosphorylation and activation. In yet another example, the first and second proteins are each E3 ligases that can homodimerize or heterodimerize in the presence of the compound of interest, which can lead to cross-ubiquitination and degradation.

[0167] Characterization of the formed composite In some embodiments, the mixture is measured to produce a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound.

[0168] In some embodiments, the first protein or a fragment thereof, or the second protein or a fragment thereof, is covalently linked to the affinity component.

[0169] In some embodiments, the first protein or a fragment thereof or the second protein or a fragment thereof together with the affinity component form a fusion protein (e.g., glutathione S-transferase (GST) or maltose-binding protein (MBP)).

[0170] In some embodiments, the method further includes: (ii-a) Contact the complex with a fixed affinity conjugate of the target affinity component (or fusion protein), thereby forming a fixed complex.

[0171] In some embodiments, step (ii-a) includes contacting the assay mixture with a plate coated with an affinity binder.

[0172] In some embodiments, step (ii-a) includes contacting the assay mixture with beads coated with an affinity binder.

[0173] In some embodiments, the affinity component and affinity conjugate are selected from, but not limited to, the combinations described in Table 2 below.

[0174] Table 2 In some embodiments, the affinity component is biotin, desulfurized biotin, or a derivative thereof.

[0175] In some embodiments, the affinity conjugate is streptavidin, avidin, neutral avidin, or a derivative thereof.

[0176] In some embodiments, the affinity component is biotin or a derivative thereof, and the affinity conjugate is streptavidin or a derivative thereof.

[0177] In some embodiments, step (ii-a) further comprises incubating the mixture with the immobilized affinity conjugate for a period of time ranging from about 1 minute to about 90 minutes (e.g., from about 30 minutes to about 60 minutes).

[0178] In some embodiments, in step (i), the mixture is determined to further contain a GFP enhancer, and step (ii-a) includes incubating the mixture with the immobilized affinity conjugate for a period of time ranging from 1 minute to 30 minutes.

[0179] In some embodiments, the method further includes: (ii-b) Detection and / or isolation of the immobilized complex, thereby identifying the compound.

[0180] In some embodiments, step (ii-b) includes detecting the immobilized complex, thereby identifying the compound.

[0181] In some embodiments, step (ii-b) comprises isolating the immobilized complex, thereby identifying the compound.

[0182] In some embodiments, step (ii-b) includes detecting and isolating the immobilized complex, thereby identifying the compound.

[0183] In some embodiments, step (ii) or step (ii-b) comprises repeating step (i) once or more, thereby identifying the compound, the first protein or a fragment thereof, and / or the second protein or a fragment thereof in the complex.

[0184] In some embodiments, step (ii) or step (ii-b) comprises repeating step (i) once or more, thereby identifying the compounds in the complex.

[0185] In some embodiments, step (ii) or step (ii-b) comprises repeating step (i) once or more, thereby identifying a first protein or a fragment thereof in the complex.

[0186] In some embodiments, step (ii) or step (ii-b) comprises repeating step (i) once or more, thereby identifying a second protein or a fragment thereof in the complex.

[0187] In some embodiments, step (ii) or step (ii-b) comprises repeating step (i) once or more, thereby identifying the compound, the first protein or a fragment thereof, and the second protein or a fragment thereof in the complex.

[0188] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) with fewer different first proteins or fragments thereof, fewer different second proteins or fragments thereof, and / or fewer different candidate compounds (e.g., compared to a previous occurrence of step (i)).

[0189] In some embodiments, step (ii) or step (ii-b) comprises repeating step (i) with a first protein or a fragment thereof, a second protein and / or a candidate compound.

[0190] In some embodiments, the individual first protein, second protein, and candidate compound in the assay mixture are tested in such a manner that a specific combination of the first protein, second protein, and candidate compound forming a complex in the assay mixture is uniquely and definitively identified. For example, the same pool of multiple candidate compounds can be used to test individual combinations of the first protein and second protein in each sample to identify combinations of the first protein and second protein. And then, the identified specific combination of the first protein and second protein can be used to test individual candidate compounds. Alternatively, the same multiple first proteins and multiple second proteins can be used to test individual candidate compounds in each sample to identify candidate compounds. And a unique combination of the first protein and second protein can be used to test specific candidate compounds in each sample. Other methods can also be used, as long as the unique combination of the first protein, second protein, and candidate compound can be definitively identified.

[0191] In some embodiments, step (ii-b) includes characterizing the complex using mass spectrometry (MS).

[0192] In some embodiments, step (ii-b) comprises reducing, denaturing, alkylating and / or digesting the immobilized complex to form a mixture of peptides, and characterizing the mixture of peptides by MS, thereby identifying a first protein or fragment thereof and / or a second protein or fragment thereof in the complex.

[0193] In some embodiments, the mixture of peptides further comprises candidate compounds, and MS characterization further identifies the candidate compounds in the complex.

[0194] In some embodiments, step (ii-b) includes isolating the complex and characterizing the complex using MS.

[0195] In some embodiments, step (ii-b) comprises separating the complex by dissociating the first protein or a fragment thereof and / or the second protein or a fragment thereof from the complex, and characterizing the dissociated proteins by MS, thereby identifying the first protein or a fragment thereof and / or the second protein or a fragment thereof in the complex.

[0196] In some embodiments, step (ii-b) comprises separating the complex by dissociating the first protein or a fragment thereof and / or the second protein or a fragment thereof from the complex, and characterizing the dissociated proteins by MS, thereby identifying the first protein or a fragment thereof in the complex.

[0197] In some embodiments, step (ii-b) comprises separating the complex by dissociating the first protein or a fragment thereof and / or the second protein or a fragment thereof from the complex, and characterizing the dissociated proteins by MS, thereby identifying the second protein or a fragment thereof in the complex.

[0198] In some embodiments, step (ii-b) comprises separating the complex by dissociating the first protein or a fragment thereof and / or the second protein or a fragment thereof from the complex, and characterizing the dissociated proteins by MS, thereby identifying the first protein or a fragment thereof and the second protein or a fragment thereof in the complex.

[0199] In some embodiments, step (ii-b) further comprises dissociating the candidate compound from the complex, and MS characterization further identifying the candidate compound in the complex.

[0200] In some embodiments, MS characterization is based on a "bottom-up mass spectrometry-based proteomics" approach. In such approaches, proteins captured during incubation can be reduced and alkylated under denaturing conditions before analysis of the sample by mass spectrometry, followed by digestion into smaller peptides using a sequence-specific protease. Various options may be available for this procedure. For example, high concentrations of urea or other ionizing agents can be used for protein denaturation. Dithiothreitol, β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine) can be used to reduce disulfide bonds in proteins. Iodoacetamide, iodoacetic acid, or iodoethanol can be used as alkylating agents, which covalently modify free thiol groups on the protein once the disulfide bonds are reduced. For the purpose of mass spectrometry-based protein analysis, trypsin can be used as a sequence-specific protease to digest proteins into peptides. Other proteases that can be used for this purpose include Lys-C and chymotrypsin. The amino acid sequence of the digested peptide can then be identified using standard proteomics methods. Such methods can be based on comparing the obtained m / z (mass-to-charge ratio) values ​​of the peptide and its MS2 fragmented spectra with theoretical data obtained by computer digestion of protein sequences from protein databases of the appropriate species using a computer program (also known as a "search engine"). For mass spectrometry data acquisition, online separation of peptides on liquid chromatography (LC) can be combined with different data acquisition modes via the mass spectrometer. Data-dependent MS2 acquisition (DDA) and / or data-independent acquisition (DIA) can be suitable for this purpose. Any other data acquisition method can also be used, as long as a clear identification of a unique peptide sequence can be established. Direct data acquisition can be performed without LC separation of the peptide, especially when the sample composition is relatively simple. Matrix-assisted laser desorption / ionization (MALDI) time-of-flight instruments can be suitable for this purpose. The identity of proteins in the sample can be deduced by comparing the amino acid sequence of the peptide with the amino acid sequence of the complete protein in a protein database. During the digestion of proteins in the complex, candidate compounds captured in the complex can also be released into the digestion solution. The identity of candidate compounds can also be obtained from the same sample and / or from the same experiment by comparing the molecular weight of the compound in the mixture with the expected molecular weight of the candidate compound derived from the m / z values ​​of the MS1 spectrum. If necessary, the MS2 fragmented spectrum of the compound can also be used to assist in the identification of the compound.

[0201] In some embodiments, MS characterization is based on a "top-down" approach (e.g., if the proteins in the complex can be released into solution, the proteins are not digested). In this approach, the mass of the intact protein obtained by mass spectrometry can be compared with the expected mass of the individual proteins used in this experiment. To release the intact protein from the complex, a short peptide sequence that can be cleaved by a highly sequence-specific protease can be introduced between the first or second protein and the first or second tag fragment (e.g., a GFP10 tag fragment or a GFP11 tag fragment). For example, the short peptide sequence could be the TEV cleavage sequence ENLYFQS(G,A). The TEV protease can cleave between Q and S in a highly sequence-specific manner. Thus, the entire protein can be released from the plate-bound complex, for example, by incubating the plate with a solution containing the TEV protease. Once the first or second protein is cleaved from the tag fragment, acidification of the sample can dissociate both proteins from the ternary complex. Measuring the intact mass of the dissociated protein allows for the identification of specific proteins within the ternary complex.

[0202] In some embodiments, the method further includes: (ii-c) To subject the identified compound to a verification assay.

[0203] In some embodiments, the validation assay is configured to test the ability of a compound to induce proximity between a first protein or a fragment thereof and a second protein or a fragment thereof.

[0204] In some embodiments, the validation assay is configured to test the ability of a compound to induce the degradation of a first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0205] In some embodiments, in steps (ii-c), the compound identified in the validation assay causes an induced proximity between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0206] In some embodiments, in step (ii-c), during the validation assay, the compound identified in the presence of a second protein (e.g., E3 ligase) causes degradation of the first protein (e.g., POI).

[0207] The first protein, second protein, compound, and combination identified In some respects, this disclosure provides for the identification of a first protein by the methods of this disclosure.

[0208] In some respects, this disclosure provides for the identification of a second protein by the methods of this disclosure.

[0209] In some respects, this disclosure provides a combination of a first protein and a second protein identified by the methods of this disclosure.

[0210] In some aspects, this disclosure provides combinations of a first protein, a second protein, and compounds targeting the first and second proteins identified by the methods of this disclosure.

[0211] In some respects, this disclosure provides compounds identified by the methods of this disclosure.

[0212] In some embodiments, the identified compound is a small molecule (e.g., <5000 Da, <4500 Da, <4000 Da, <3500 Da, <3000 Da, <2500 Da, <2000 Da, <1500 Da, <1000 Da, <900 Da, <800 Da, <700 Da, <600 Da, <500 Da, or <400 Da).

[0213] In some embodiments, the identified compound is not water.

[0214] In some embodiments, the identified compound is not a buffer salt.

[0215] In some embodiments, the identified compound is not a protein or a fragment thereof.

[0216] In some embodiments, the identified compound is not GFP protein or a fragment thereof. In some embodiments, the identified compound is not GFP protein. In some embodiments, the identified compound is not a fragment of GFP protein.

[0217] In some embodiments, the identified compound does not contain GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, or GFP11, or any combination thereof.

[0218] In some embodiments, the identified compound is not GFP1-9.

[0219] In some embodiments, the identified compound is a complex compound.

[0220] In some embodiments, the identified compound (e.g., a conjugate compound) is capable of modulating protein-protein interactions (PPIs) between a first protein or a fragment thereof and a second protein or a fragment thereof.

[0221] In some embodiments, the identified compound (e.g., a conjugate compound) modulates (increases or decreases) the protein-protein interaction (PPI) between the first and second proteins.

[0222] In some embodiments, the identified compound (e.g., a conjugate compound) increases the protein-protein interaction (PPI) between the first and second proteins or stabilizes the PPI between the two proteins.

[0223] In some embodiments, the identified compound (e.g., a conjugate compound) reduces the protein-protein interaction (PPI) between the first and second proteins or destabilizes the PPI between the two proteins.

[0224] In some embodiments, PPIs cause changes in protein stability (e.g., changes in the stability of a first protein and / or a second protein).

[0225] In some embodiments, PPIs cause changes in protein levels (e.g., changes in the level of a first protein and / or a second protein).

[0226] In some embodiments, PPIs cause changes in the post-translational modifications of proteins (e.g., changes in the post-translational modifications of a first protein and / or a second protein).

[0227] In some embodiments, PPIs cause changes in protein localization (e.g., changes in the localization of a first protein and / or a second protein).

[0228] In some embodiments, PPIs cause changes in protein activity (e.g., changes in the activity of a first protein and / or a second protein).

[0229] In some embodiments, PPIs cause degradation of the first or second protein.

[0230] In some embodiments, PPIs cause increased protein stability within cells or prevent protein hydrolysis.

[0231] In some embodiments, PPIs induce post-translational modifications of the first or second protein.

[0232] In some embodiments, PPI causes the removal of a pre-existing post-translational modification of the first or second protein.

[0233] In some embodiments, PPIs cause changes in the subcellular localization of the first or second protein.

[0234] In some embodiments, PPIs induce regulation of the activity of a first or second protein.

[0235] In some embodiments, the identified compound (e.g., a conjugate compound) is capable of causing degradation of the first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0236] In some embodiments, the identified compound (e.g., a conjugate compound) causes degradation of the first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0237] In some embodiments, the identified compound (e.g., a conjugate compound) is capable of inducing post-translational modification of a first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0238] In some embodiments, the identified compound (e.g., a conjugate compound) induces post-translational modification of the first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0239] In some embodiments, the identified compound (e.g., a conjugate compound) is able to induce regulation of the activity of the first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0240] In some embodiments, the identified compound (e.g., a conjugate compound) induces regulation of the activity of the first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0241] In some embodiments, the identified compound (e.g., conjugate compound) can cause changes in protein stability (e.g., POI), protein level (e.g., POI), protein post-translational modification (e.g., POI), protein localization (e.g., POI), and / or protein activity (e.g., POI).

[0242] In some embodiments, the identified compound (e.g., a conjugate compound) can induce changes in protein stability (e.g., point of interest (POI)). In some embodiments, the identified compound (e.g., a conjugate compound) can induce changes in protein level (e.g., POI). In some embodiments, the identified compound (e.g., a conjugate compound) can induce changes in protein post-translational modifications (e.g., POI). In some embodiments, the identified compound (e.g., a conjugate compound) can induce changes in protein localization (e.g., POI). In some embodiments, the identified compound (e.g., a conjugate compound) can induce changes in protein activity (e.g., POI).

[0243] In some embodiments, the identified compound (e.g., conjugate compound) causes changes in protein stability (e.g., POI), protein level (e.g., POI), protein post-translational modification (e.g., POI), protein localization (e.g., POI), and / or protein activity (e.g., POI).

[0244] In some embodiments, the identified compound (e.g., a conjugate compound) causes changes in protein stability (e.g., point of interest (POI)). In some embodiments, the identified compound (e.g., a conjugate compound) causes changes in protein level (e.g., POI). In some embodiments, the identified compound (e.g., a conjugate compound) causes changes in protein post-translational modifications (e.g., POI). In some embodiments, the identified compound (e.g., a conjugate compound) causes changes in protein localization (e.g., POI). In some embodiments, the identified compound (e.g., a conjugate compound) causes changes in protein activity (e.g., POI).

[0245] Methods for synthesizing the identified compounds In some aspects, this disclosure provides a method for preparing compounds identified in the methods of this disclosure.

[0246] The compounds disclosed herein can be prepared by any suitable technique known in the art. Those skilled in organic synthesis will understand that the functionality present at each part of the molecule must be compatible with the compound used and the reaction conditions. The resulting compounds can be isolated and purified using techniques well known in the art.

[0247] Conveniently, the reaction of the compounds is carried out in the presence of a suitable solvent, which is preferably inert under the appropriate reaction conditions. Examples of suitable solvents include, but are not limited to: hydrocarbons, such as hexane, petroleum ether, benzene, toluene, or xylene; chlorinated hydrocarbons, such as trichloroethylene, 1,2-dichloroethane, tetrachloromethane, chloroform, or dichloromethane; alcohols, such as methanol, ethanol, isopropanol, n-propanol, n-butanol, or tert-butanol; ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentylmethyl ether (CPME), methyl tert-butyl ether (MTBE), or dioxane; ethyl... Glycol ethers, such as ethylene glycol monomethyl or monoethyl ether or ethylene glycol dimethyl ether (diethylene glycol dimethyl ether); ketones, such as acetone, methyl isobutyl ketone (MIBK) or butanone; amides, such as acetamide, dimethylacetamide, dimethylformamide (DMF) or N-methylpyrrolidone (NMP); nitriles, such as acetonitrile; sulfoxides, such as dimethyl sulfoxide (DMSO); nitro compounds, such as nitromethane or nitrobenzene; esters, such as ethyl acetate or methyl acetate; or mixtures of the solvents or mixtures with water.

[0248] As those skilled in the art of organic synthesis will understand, the compounds can be readily obtained through a variety of synthetic routes, some of which are illustrated in the appended examples. Those skilled in the art will readily recognize which types of compounds and reaction conditions to use, and how to apply and adjust the reagents and reaction conditions in any particular case, when necessary or useful, to obtain the compounds of this disclosure. Furthermore, some of the compounds can be readily synthesized by reacting other compounds of this disclosure under suitable conditions, for example by applying standard synthetic methods (such as reduction, oxidation, addition, or substitution reactions) to convert a particular functional group present in the compounds of this disclosure or a suitable precursor molecule thereto into another functional group; these methods are well known to those skilled in the art. Similarly, those skilled in the art will apply synthetic protecting (or protective) groups when necessary or useful; suitable protecting groups and methods for their introduction and removal are well known to those skilled in the art of chemical synthesis and are described in more detail, for example, in PGM Wuts, TW Greene, “Greene's Protecting Groups in Organic Synthesis”. ti "Ve Groups in Organic Synthesis", 4th edition (2006) (John Wiley & Sons).

[0249] Verification assay of the identified compounds Once a compound identified by the methods described above is generated, it can be verified using a variety of assays known to those skilled in the art to determine whether the compound has biological activity (e.g., to induce proximity between a first protein or a fragment thereof and a second protein or a fragment thereof). For example, the compound can be characterized by conventional assays, including but not limited to those described below, to determine whether the compound has the predicted activity, binding activity, and / or binding specificity.

[0250] Furthermore, high-throughput screening can be used to accelerate analyses using such assays. Therefore, the activity of molecules described herein can be rapidly screened using techniques known in the art. General methods for performing high-throughput screening are described, for example, in the following literature: Devlin (1998) "High-throughput Screening (… High Throughput Screening (See also Marcel Dekker, Inc.); and U.S. Patent No. 5,763,263. High-throughput assays can be performed using one or more different assay techniques, including but not limited to the assay techniques described below.

[0251] Various in vitro or in vivo bioassays can be adapted to detect the effects of compounds. These in vitro or in vivo bioassays may include, but are not limited to, enzyme activity assays, electrophoretic mobility variation assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.

[0252] In some embodiments, the validation assay is a bioassay that validates the formation of the ternary complex. In some embodiments, the validation assay measures the proximity between the first protein and the second protein or fragments thereof in a compound-dependent manner. In some embodiments, the validation assay may include, but is not limited to, AlphALISA, TR-FRET, fluorescence polarization assay, surface plasmon resonance (SPR), and mass spectrometry. In some embodiments, the validation assay includes pull-down of the ternary complex and its quantification by mass spectrometry.

[0253] In some embodiments, the validation assay is an intracellular bioassay. In some embodiments, the intracellular bioassay includes a nanoBRET system. In some embodiments, the nanoBRET system measures the formation of an intracellular ternary complex. In some embodiments, the nanoBRET system further includes detecting the generation or enhancement of a luminescent signal during the formation of the ternary complex in the presence of a compound, the ternary complex comprising the interaction of a first protein and a second protein or fragments thereof. In some embodiments, the intracellular bioassay includes a fluorescence resonance energy transfer (FRET) system. In some embodiments, the FRET system measures the formation of an intracellular ternary complex. In some embodiments, the FRET system further includes detecting the generation or enhancement of a signal during the formation of the ternary complex in the presence of a compound, the ternary complex comprising the interaction of a first protein and a second protein or fragments thereof, wherein the first protein and the second protein or fragments thereof are fused with a fluorescent donor and / or a fluorescent acceptor.

[0254] In some embodiments, the validation assay is based on cell degradation assays. In some embodiments, cell-based degradation assays may include, but are not limited to, Western blotting, intracellular Western blotting assays, HiBit assays, fluorescent reporter gene assays, and mass spectrometry proteomics.

[0255] To avoid being bound by theory, the compound-dependent ubiquitination, post-translational modification, enzymatic activity changes, or cellular degradation of POIs in cell degradation validation assays do not directly measure the formation of ternary complexes containing the first and second proteins or fragments thereof in the presence of compounds. Instead, the formation of ternary complexes is required, and therefore can be used to detect ternary complex formation.

[0256] Pharmaceutical Composition In some aspects, this disclosure provides a pharmaceutical composition comprising a compound identified by the methods of this disclosure or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0257] As used herein, the term "composition" is intended to cover products containing specified amounts of specified ingredients, and any products directly or indirectly produced by combinations of specified amounts of specified ingredients.

[0258] The compounds disclosed herein can be formulated into forms for oral administration, such as tablets, capsules (each of which includes sustained-release or delayed-release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds disclosed herein can also be formulated for intravenous (bolus or infusion), intraperitoneal, topical, subcutaneous, intramuscular, or transdermal (e.g., patch) administration, all using forms well known to those skilled in the art of pharmacy.

[0259] Any suitable solubilizing compound may be used. Examples of solubilizing compounds include cyclodextrins, such as those selected from the group consisting of: hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, random methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonium)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulfated-β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulfonyl ether, branched-chain-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, random methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin and mixtures thereof.

[0260] Any suitable chelating compound can be used. Examples of suitable chelating compounds include those selected from the group consisting of: ethylenediaminetetraacetic acid and its metal salt, disodium edetate, trisodium edetate, and tetrasodium edetate and mixtures thereof.

[0261] Any suitable preservative can be used. Examples of preservatives include those selected from the group consisting of: quaternary ammonium salts, such as benzalkonium halide (preferably benzalkonium chloride), chlorhexidine gluconate, benzyl chloride, hexadecylpyridine chloride, benzyl bromide, phenylmercuric nitrate, phenylmercuric acetate, phenylmercuric neodecanoate, thimerosal, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethylparaben, polyurethane biguanide, and butylparaben and mixtures thereof.

[0262] Aqueous mediators may also include tension compounds to adjust tension (osmotic pressure). Tensile compounds may be selected from the group consisting of: ethylene glycols (such as propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextran, glycerin, mannitol, potassium chloride, sodium chloride, and mixtures thereof. Aqueous mediators may also contain viscosity compounds / suspension compounds. Suitable viscosity compounds / suspension compounds include those selected from the group consisting of: cellulose derivatives, such as methylcellulose, ethylcellulose, hydroxyethylcellulose, polyethylene glycols (such as polyethylene glycol 300, polyethylene glycol 400), carboxymethylcellulose, hydroxypropyl methylcellulose, and crosslinked acrylic polymers (carbomers), such as polymers of acrylic acid crosslinked with polyolefin ethers or divinyl glycol (carbomers - such as carbomer 934, carbomer 934P, carbomer 971, carbomer 974, and carbomer 974P) and mixtures thereof. Aqueous mediators may also contain buffering compounds to stabilize pH. When used, the buffer solution is selected from the group consisting of: phosphate buffers (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), borate buffers (such as boric acid or its salts, including disodium tetraborate), citrate buffers (such as citrate or its salts, including sodium citrate), and ε-aminocaproic acid and mixtures thereof.

[0263] The composition may further comprise a wetting compound. Suitable classes of wetting compounds include those selected from the group consisting of: polyoxypropylene-polyoxyethylene block copolymers (poloxamer), polyethoxylated ethers of castor oil, polyoxyethylene-modified sorbitan esters (polysorbates), polymers of oxyethylene-modified octylphenol (Tyloxapol), polyethylene glycol 40 stearate, ethylene glycol esters of fatty acids, glyceryl esters of fatty acids, sucrose fatty acid esters, and polyoxyethylene fatty acid esters and mixtures thereof.

[0264] The compositions disclosed herein can be obtained using conventional procedures employing conventional pharmaceutical excipients well known in the art. Therefore, compositions intended for oral use may contain, for example, one or more coloring compounds, sweetening compounds, flavoring compounds, and / or preservative compounds.

[0265] Methods using the identified compounds In some aspects, this disclosure provides a method for degrading a first protein in a subject, the method comprising administering to the subject a compound identified by the method of this disclosure.

[0266] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for degrading a first protein in a subject.

[0267] In some respects, this disclosure provides for the use of compounds identified by this disclosure in the preparation of a medicament for degrading a first protein in a subject.

[0268] In some aspects, this disclosure provides methods for treating and / or preventing diseases or conditions associated with a first protein in a subject, the methods comprising administering to the subject a therapeutically effective amount of a compound identified by the methods of this disclosure.

[0269] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for the treatment and / or prevention of a disease or condition associated with a first protein in a subject.

[0270] In some respects, this disclosure provides for the use of compounds identified by the methods of this disclosure in the preparation of medicaments for treating and / or preventing diseases or conditions associated with a first protein in a subject.

[0271] In some aspects, this disclosure provides a method for treating and / or preventing diseases or conditions associated with human therapies, agriculture, and / or animal health. In some embodiments, the method for treating and / or preventing a disease or condition is associated with human therapies. In some embodiments, the method for treating and / or preventing a disease or condition is directed at a human. In some embodiments, the method for treating and / or preventing a disease or condition is associated with agriculture. In some embodiments, the method for treating and / or preventing a disease or condition is used on agricultural entities (e.g., seeds, seedlings, leaves, flowers, plants, etc.). In some embodiments, the method for treating and / or preventing a disease or condition is associated with animal health. In some embodiments, the method for treating and / or preventing a disease or condition is directed at an animal.

[0272] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for use in human therapy, agriculture, and / or animal health. In some embodiments, the compound is used for human therapy. In some embodiments, the compound is used for humans. In some embodiments, the compound is used for agriculture. In some embodiments, the compound is used for agricultural entities (e.g., seeds, seedlings, leaves, flowers, plants, etc.). In some embodiments, the compound is used for animal health. In some embodiments, the compound is used for animals.

[0273] In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for human therapy, agriculture, and / or animal health. In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for human therapy. In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for human use. In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for agricultural purposes. In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for agricultural entities (e.g., seeds, seedlings, leaves, flowers, plants, etc.). In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for animal health. In some aspects, this disclosure provides the use of compounds identified by the methods of this disclosure in the preparation of medicaments for animal use.

[0274] In some embodiments, a disease or symptom is associated with a first protein (e.g., POI).

[0275] In some embodiments, the disease or condition is mediated by a first protein (e.g., POI).

[0276] In some aspects, this disclosure provides a method for degrading a second protein in a subject, the method comprising administering to the subject a compound identified by the method of this disclosure.

[0277] In some respects, this disclosure provides a compound identified by the methods of this disclosure for degrading a second protein in a subject.

[0278] In some respects, this disclosure provides for the use of compounds identified by this disclosure in the preparation of medicaments for degrading a second protein in a subject.

[0279] In some aspects, this disclosure provides methods for treating and / or preventing diseases or conditions associated with a second protein in a subject, the methods comprising administering to the subject a therapeutically effective amount of a compound identified by the methods of this disclosure.

[0280] In some aspects, this disclosure provides a compound identified by the methods of this disclosure for the treatment and / or prevention of diseases or conditions associated with a second protein in a subject.

[0281] In some respects, this disclosure provides for the use of compounds identified by the methods of this disclosure in the preparation of medicaments for treating and / or preventing diseases or conditions associated with a second protein in a subject.

[0282] In some embodiments, the disease or condition is associated with a second protein (e.g., E3 ligase).

[0283] In some embodiments, the disease or condition is mediated by a second protein.

[0284] In some embodiments, the subject is a cell.

[0285] In some embodiments, the subjects are animals.

[0286] In some embodiments, the subject is an agricultural entity (e.g., a seed, seedling, leaf, flower, plant, etc.).

[0287] In some embodiments, the subjects are mammals.

[0288] In some embodiments, the subject is a human being.

[0289] In some embodiments, the disease or condition is cancer.

[0290] In some embodiments, the cancer is selected from prostate cancer (small cell carcinoma, neuroendocrine tumor, transitional cell carcinoma, sarcoma), breast cancer (ductal carcinoma in situ, invasive breast cancer, triple-negative breast cancer (TNBC), inflammatory breast cancer, Paget's disease of the breast, angiosarcoma, phyllodes tumor), ovarian cancer (epithelial ovarian cancer, germ cell tumor, stromal cell tumor), bladder cancer (urothelial carcinoma, squamous cell carcinoma, adenocarcinoma), gastric cancer (adenocarcinoma, primary gastric lymphoma, gastrointestinal stromal tumor, and neuroendocrine carcinoid tumor), pancreatic cancer (adenocarcinoma and neuroendocrine tumor), liver cancer (hepatocellular carcinoma, bile duct carcinoma), endometrial cancer, salivary gland cancer, leukemia, NUT-midline carcinoma, multiple myeloma, lung cancer (small cell lung cancer, non-small cell lung cancer), neuroblastoma, cervical cancer (squamous cell carcinoma, adenocarcinoma), esophageal cancer, colorectal cancer, brain (glioma), glioblastoma, Bannayan-Zonana syndrome. This includes diseases such as syndrome, Cowden disease, Lhermitte-Duclos disease, Wilms' tumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer (primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, and melanoma), head and neck cancer, lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma), skin cancer (basal cell carcinoma, squamous cell carcinoma), bone marrow cancer (melanoma, lymphoma, myeloma), kidney cancer (renal cell carcinoma, urothelial carcinoma, Wilms' tumor), sarcoma, bone cancer (osteosarcoma, Ewing's sarcoma, chondrosarcoma, fibrosarcoma, giant cell tumor of bone, chordoma, multiple myeloma), and thyroid cancer.

[0291] In some embodiments, the disease or condition is a benign proliferative condition.

[0292] In some embodiments, benign proliferative conditions are selected from benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granulomas, lipomas, meningiomas, multiple endocrine tumors, nasal polyps, pituitary tumors, prolactinomas, pseudotumors of the brain, seborrheic keratosis, gastric polyps, thyroid nodules, cystic tumors of the pancreas, hemangiomas, vocal cord nodules, polyps and cysts, Castleman disease, chronic pilonidal disease, dermatofibromas, pilocystic follicles, pyogenic granulomas, and juvenile polyposis syndrome.

[0293] In some embodiments, the disease or condition is an immune disease or condition.

[0294] In some embodiments, immune diseases or conditions include T-cell-mediated inflammatory diseases and B-cell-mediated inflammatory diseases. In some embodiments, immune diseases or conditions are selected from Crohn's disease, ulcerative colitis, lupus, cystic fibrosis, childhood asthma, adult asthma, allergic diseases, chronic obstructive pulmonary disease, psoriasis, atherosclerosis, acute and chronic inflammation, Addison disease, celiac-ostomy (gluten-sensitive bowel disease), dermatomyositis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type I diabetes, inflammatory bowel disease, chronic inflammatory demyelinating polyneuropathy, and ankylosing spondylitis.

[0295] In some embodiments, the disease or condition is a neurological disease or condition.

[0296] In some embodiments, neurological diseases or conditions are selected from a list including, but not limited to, acute spinal cord injury, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia, Bell's palsy, brain tumor, cerebral aneurysm, dementia, epilepsy and seizures, Guillain-Barré syndrome, Huntington's disease, headache, head injury, hydrocephalus, lumbar disc herniation, meningitis, multiple sclerosis, muscular dystrophy, neurocutaneous syndrome, Parkinson's disease, stroke, cluster headache, tension headache, migraine, encephalitis, sepsis, myasthenia gravis, muscular dystrophy, and neuromuscular diseases.

[0297] Exemplary embodiments Exemplary Example A1. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof associated with the complex.

[0298] Exemplary Example A2. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The first and second tag fragments are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0299] Exemplary Example A3. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a assay signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof that are associated with the induced proximity; The identified compound is capable of causing degradation of the first protein (e.g., POI) in the presence of the second protein.

[0300] Exemplary Example A4. The method according to any of the foregoing exemplary embodiments, wherein the measuring mixture induces an approach, thereby generating or enhancing the measuring signal.

[0301] Exemplary Example A5. The method according to any one of the foregoing exemplary embodiments, wherein the measuring mixture further comprises a detector segment complementary to the first tag segment and the second tag segment.

[0302] Exemplary Example A6. The method according to any one of the foregoing exemplary embodiments, wherein the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture causes induced proximity between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0303] Exemplary Example A7. The method according to any one of the foregoing exemplary embodiments, wherein the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance a measurement signal when the assay mixture generates a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound.

[0304] Exemplary Example A8. The method according to any one of the foregoing exemplary embodiments, wherein the assay mixture comprises a variety of different first proteins or fragments thereof.

[0305] Exemplary Example A9. The method according to any one of the foregoing exemplary embodiments, wherein the assay mixture contains a variety of different second proteins or fragments thereof.

[0306] Exemplary Example A10. The method according to any one of the foregoing exemplary embodiments, wherein the determination mixture comprises a variety of different candidate compounds.

[0307] Exemplary Example A11. The method according to any one of the foregoing exemplary embodiments, wherein the determination mixture comprises about 50 to about 250 different candidate compounds.

[0308] Exemplary Example A12. The method according to any one of the foregoing exemplary embodiments, wherein the determination mixture comprises about 250 to about 2,500 different candidate compounds.

[0309] Exemplary Example A13. The method according to any one of the foregoing exemplary embodiments, wherein the determination mixture contains more than about 2,500 different candidate compounds.

[0310] Exemplary Example A14. The method according to any one of the foregoing exemplary embodiments, wherein the determination mixture produces the complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the candidate compound.

[0311] Exemplary Example A15. The method according to any one of the foregoing exemplary embodiments, wherein the measurement signal is fluorescence.

[0312] Exemplary Example A16. The method according to any one of the foregoing exemplary embodiments, wherein the first tag fragment is a first tag GFP fragment.

[0313] Exemplary Example A17. The method according to any one of the foregoing exemplary embodiments, wherein the second tag fragment is a second tag GFP fragment.

[0314] Exemplary Example A18. The method according to any one of the foregoing exemplary embodiments, wherein the detector fragment is a detector GFP fragment.

[0315] Exemplary Example A19. The method according to any one of the foregoing exemplary embodiments, wherein the first tag GFP fragment, the second tag GFP fragment, and the detector GFP fragment are configured to form GFP when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof.

[0316] Exemplary Example A20. The method according to any one of the foregoing exemplary embodiments, wherein the first tag GFP fragment and the second tag GFP fragment independently comprise one or more of the following: GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10 and GFP11.

[0317] Exemplary Example A21. The method according to any one of the foregoing exemplary embodiments, wherein the first tagged GFP fragment and the second tagged GFP fragment independently comprise one or more of GFP10 and GFP11.

[0318] Exemplary Example A22. The method according to any one of the foregoing exemplary embodiments, wherein the first tag GFP fragment comprises GFP10 and the second tag GFP fragment comprises GFP11.

[0319] Exemplary Example A23. The method according to any one of the foregoing exemplary embodiments, wherein the detector GFP fragment is selected from GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8 and GFP9.

[0320] Exemplary Example A24. The method according to any one of the foregoing exemplary embodiments, wherein the first protein is the protein of interest (POI).

[0321] Exemplary Example A25. The method according to any one of the foregoing exemplary embodiments, wherein the second protein is a ubiquitin ligase.

[0322] Exemplary Example A26. The method according to any one of the foregoing exemplary embodiments, wherein step (i) further comprises detecting the generated or enhanced measurement signal.

[0323] Exemplary Example A27. The method according to any one of the foregoing exemplary embodiments, wherein the measurement signal is fluorescence.

[0324] Exemplary Example A28. The method according to any one of the foregoing exemplary embodiments, wherein the first protein or a fragment thereof or the second protein or a fragment thereof is covalently linked to an affinity component.

[0325] Exemplary Example A29. The method according to any one of the foregoing exemplary embodiments, wherein the affinity component is biotin.

[0326] Exemplary Example A30. The method according to any one of the foregoing exemplary embodiments, wherein the method further comprises: (ii-a) Contact the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex.

[0327] Exemplary Example A31. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-a) comprises contacting the assay mixture with a plate coated with the affinity binder.

[0328] Exemplary Example A32. The method according to any one of the foregoing exemplary embodiments, wherein the affinity conjugate is streptavidin.

[0329] Exemplary Example A33. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-a) further comprises incubating the assay mixture together with the plate for a time ranging from 1 minute to 90 minutes.

[0330] Exemplary Example A34. The method according to any one of the foregoing exemplary embodiments, wherein in step (i), the assay mixture further comprises a GFP enhancer, and wherein step (ii-a) comprises incubating the assay mixture together with the plate for a period of time ranging from 1 minute to 30 minutes.

[0331] Exemplary Example A35. The method according to any one of the foregoing exemplary embodiments, wherein the method further comprises: (ii-b) Detection and / or isolation of the immobilized complex, thereby identifying the compound.

[0332] Exemplary Example A36. The method according to any one of the foregoing exemplary embodiments, wherein step (ii) or step (ii-b) comprises repeating step (i) once or more with fewer different first proteins or fragments thereof, fewer different second proteins or fragments thereof, and / or fewer different candidate compounds compared to a previous occurrence of step (i), thereby identifying the compounds, the first protein or fragments thereof, and / or the second protein or fragments thereof in the complex.

[0333] Exemplary Example A37. The method according to any one of the foregoing exemplary embodiments, wherein step (ii) or step (ii-b) comprises repeating step (i) once or more with fewer different first proteins or fragments thereof, fewer different second proteins or fragments thereof, and / or fewer different candidate compounds compared to a previous occurrence of step (i), thereby identifying the compounds in the complex.

[0334] Exemplary Example A38. The method according to any one of the foregoing exemplary embodiments, wherein step (ii) or step (ii-b) comprises repeating step (i) once or more with fewer different first proteins or fragments thereof, fewer different second proteins or fragments thereof, and / or fewer different candidate compounds compared to a previous occurrence of step (i), thereby the first protein or fragments thereof in the complex.

[0335] Exemplary Example A39. The method according to any one of the foregoing exemplary embodiments, wherein step (ii) or step (ii-b) comprises repeating step (i) once or more with fewer different first proteins or fragments thereof, fewer different second proteins or fragments thereof, and / or fewer different candidate compounds compared to a previous occurrence of step (i), thereby identifying the second protein or fragments thereof in the complex.

[0336] Exemplary Example A40. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) comprises characterizing the complex by mass spectrometry (MS).

[0337] Exemplary Example A41. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) comprises reducing, denaturing, alkylating and / or digesting the immobilized complex to form a mixture of peptides, and characterizing the mixture of peptides by MS, thereby identifying the first protein or a fragment thereof and / or the second protein or a fragment thereof in the complex.

[0338] Exemplary Example A42. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) comprises reducing, denaturing, alkylating and / or digesting the immobilized complex to form a mixture of peptides, and characterizing the mixture of peptides by MS, thereby identifying the first protein or a fragment thereof in the complex.

[0339] Exemplary Example A43. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) comprises reducing, denaturing, alkylating and / or digesting the immobilized complex to form a mixture of peptides, and characterizing the mixture of peptides by MS, thereby identifying the second protein or a fragment thereof in the complex.

[0340] Exemplary Example A44. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) comprises reducing, denaturing, alkylating and / or digesting the immobilized complex to form a mixture of peptides, and characterizing the mixture of peptides by MS, thereby identifying the first protein or a fragment thereof and the second protein or a fragment thereof in the complex.

[0341] Exemplary Example A45. The method according to any one of the foregoing exemplary embodiments, wherein the mixture of peptides further comprises the compound, and the MS characterization further identifies the compound in the complex.

[0342] Exemplary Example A46. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) comprises separating the complex by dissociating the first protein or a fragment thereof and / or the second protein or a fragment thereof from the complex, and characterizing the dissociated proteins by MS, thereby identifying the first protein or a fragment thereof and / or the second protein or a fragment thereof in the complex.

[0343] Exemplary Example A47. The method according to any one of the foregoing exemplary embodiments, wherein step (ii-b) further comprises dissociating the candidate compound from the immobilized complex, and the MS characterization further identifies the candidate compound in the complex.

[0344] Exemplary Example A48. The method according to any one of the foregoing exemplary embodiments, wherein the identified compound modulates the protein-protein interaction (PPI) between the first protein and the second protein.

[0345] Exemplary Example A49. The method according to any one of the foregoing exemplary embodiments, wherein the PPI causes the degradation of the first protein or the second protein.

[0346] Exemplary Example A50. The method according to any one of the foregoing exemplary embodiments, wherein the PPI causes the first protein or the second protein to stabilize.

[0347] Exemplary Example A51. The method according to any one of the foregoing exemplary embodiments, wherein the identified compound causes degradation of the first protein in the presence of the second protein or causes degradation of the second protein in the presence of the first protein.

[0348] Exemplary Example A52. The method according to any of the foregoing exemplary embodiments, wherein the PPI causes post-translational modification of the first protein or the second protein.

[0349] Exemplary Example A53. The method according to any one of the foregoing exemplary embodiments, wherein the PPI causes the removal of a pre-existing post-translational modification of the first protein or the second protein.

[0350] Exemplary Example A54. The method according to any one of the foregoing exemplary embodiments, wherein the PPI causes the activity of the first protein or the second protein to be modulated.

[0351] Exemplary Example A55. The method according to any one of the foregoing exemplary embodiments, wherein the PPI causes a change in the subcellular localization of the first protein or the second protein.

[0352] Exemplary Example A56. The method according to any one of the foregoing exemplary embodiments, wherein the second protein is associated with a disease or condition.

[0353] Exemplary Example No. A57. The method according to any one of the foregoing exemplary embodiments, wherein the method is performed once or multiple times.

[0354] Exemplary Example A58. The method according to any one of the foregoing exemplary embodiments, wherein the method is performed using a new hole each time.

[0355] Exemplary Example No. A59. A first protein, which is identified by the method according to any one of the foregoing exemplary embodiments.

[0356] Exemplary Example A60. A second protein, which is identified by the method according to any one of the foregoing exemplary embodiments.

[0357] Exemplary Example A61. A combination of a first protein and a second protein, said combination being identified by a method according to any one of the foregoing exemplary embodiments.

[0358] Exemplary Example A62. A combination of a first protein, a second protein, and a compound targeting the first protein and the second protein, said combination being identified by a method according to any one of the foregoing exemplary embodiments.

[0359] Exemplary Example No. A63. A compound identified by a method according to any one of the foregoing exemplary embodiments.

[0360] Exemplary Example No. A64. A pharmaceutical composition comprising a compound identified by the method according to any one of the foregoing exemplary embodiments.

[0361] Exemplary Example A65. A method for degrading a first or second protein in a subject, the method comprising administering to the subject a compound identified by a method according to any one of the foregoing exemplary embodiments.

[0362] Exemplary Example A66. A method for regulating the PPI between a first protein and a second protein in a subject, the method comprising administering to the subject a compound identified by a method according to any one of the foregoing exemplary embodiments.

[0363] Exemplary Example No. A67. A compound for degrading a first or second protein in a subject, identified by the method according to any one of the foregoing exemplary embodiments.

[0364] Exemplary Example No. A68. A compound for regulating the PPI between a first protein and a second protein in a subject, identified by the method according to any one of the foregoing exemplary embodiments.

[0365] Exemplary Example A69. Use of a compound identified by the method according to any one of the foregoing exemplary embodiments in the preparation of a medicament for degrading a first or second protein in a subject.

[0366] Exemplary Example A70. Use of a compound identified by the method according to any one of the foregoing exemplary embodiments in the preparation of a medicament for regulating the PPI between a first protein and a second protein in a subject.

[0367] Exemplary Example A71. A method for treating and / or preventing a disease or condition associated with a first or second protein in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound identified by the method according to any one of the foregoing exemplary embodiments.

[0368] Exemplary Example No. A72. A compound identified by the method according to any one of the foregoing exemplary embodiments for treating and / or preventing diseases or conditions associated with a first or second protein in a subject.

[0369] Exemplary Example No. A73. Use of a compound identified by the method according to any one of the foregoing exemplary embodiments in the preparation of a medicament for treating and / or preventing a disease or condition associated with a first or second protein in a subject.

[0370] Exemplary Example B1. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof associated with the complex.

[0371] Exemplary Example B2. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The first and second tag fragments are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; (ii-a) Contacting the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; and (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0372] Exemplary Example B3. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag fragment; (b) a second protein or a fragment thereof, the second protein or a fragment thereof being covalently linked to a second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Candidate compounds, The first and second tag fragments are configured to generate or enhance a assay signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof that are associated with the induced proximity; The identified compound can cause the degradation of the first protein in the presence of the second protein.

[0373] Exemplary Example B4. The method according to Exemplary Example B1, wherein the measuring mixture further comprises a detector segment complementary to the first tag segment and the second tag segment.

[0374] Exemplary Example B5. The method according to Exemplary Example B1, wherein the assay mixture contains a variety of different first proteins or fragments thereof.

[0375] Exemplary Example B6. The method according to Exemplary Example B1, wherein the assay mixture contains a variety of different second proteins or fragments thereof.

[0376] Exemplary Example B7. The method according to Exemplary Example B1, wherein the determination mixture contains a variety of different candidate compounds.

[0377] Exemplary Example B8. The method according to Exemplary Example B1, wherein the determination mixture produces the complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the candidate compound.

[0378] Exemplary Example B9. The method according to Exemplary Example B1, wherein the measurement signal is fluorescence.

[0379] Exemplary Example B10. The method according to Exemplary Example B4, wherein the first tag fragment is a first tag GFP fragment.

[0380] Exemplary Example B11. The method according to Exemplary Example B10, wherein the second tag fragment is a second tag GFP fragment.

[0381] Exemplary Example B12. The method according to Exemplary Example B11, wherein the detector fragment is a detector GFP fragment.

[0382] Exemplary Example B13. The method according to Exemplary Example B12, wherein the first tag GFP fragment and the second tag GFP fragment independently comprise one or more of the following: GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10 and GFP11.

[0383] Exemplary Example B14. The method according to Exemplary Example B12, wherein the first tagged GFP fragment and the second tagged GFP fragment independently comprise one or more of GFP10 and GFP11.

[0384] Exemplary Example B15. The method according to Exemplary Example B12, wherein the first tag GFP fragment comprises GFP10 and the second tag GFP fragment comprises GFP11.

[0385] Exemplary Example B16. The method according to Exemplary Example B12, wherein the detector GFP fragment is selected from GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8 and GFP9.

[0386] Exemplary Example B17. The method according to Exemplary Example B1, wherein the first protein is the protein of interest (POI).

[0387] Exemplary Example B18. The method according to Exemplary Example B1, wherein the second protein is a ubiquitin ligase.

[0388] Exemplary Example B19. The method according to Exemplary Example B1, wherein step (i) further comprises detecting the generated or enhanced measurement signal.

[0389] Exemplary Example B20. The method according to Exemplary Example B1, wherein the identified compound modulates the protein-protein interaction (PPI) between the first protein and the second protein.

[0390] Exemplary Example B21. The method according to Exemplary Example B1, wherein the identified compound causes degradation of the first protein in the presence of the second protein or causes degradation of the second protein in the presence of the first protein.

[0391] Exemplary Example B22. A first protein, which is identified by the method according to Exemplary Example B1.

[0392] Exemplary Example B23. A second protein, which is identified by the method according to Exemplary Example B1.

[0393] Exemplary Example B24. A combination of a first protein and a second protein, said combination being identified by the method according to Exemplary Example B1.

[0394] Exemplary Example B25. A combination of a first protein, a second protein, and a compound targeting the first protein and the second protein, said combination being identified by the method according to Exemplary Example B1.

[0395] Exemplary Example B26. A compound identified by the method according to Exemplary Example B1.

[0396] Exemplary Example B27. A pharmaceutical composition comprising a compound identified by the method according to Exemplary Example B1.

[0397] Exemplary Example B28. A method for degrading a first or second protein in a subject, the method comprising administering to the subject a compound identified by the method according to Exemplary Example B1.

[0398] Exemplary Example B29. A method for regulating the PPI between a first protein and a second protein in a subject, the method comprising administering to the subject a compound identified by the method according to Exemplary Example B1.

[0399] Exemplary Example B30. A method for treating and / or preventing a disease or condition associated with a first or second protein in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound identified by the method according to Exemplary Example B1.

[0400] definition Unless otherwise stated, the following terms used in the specification and claims have the meanings set forth below.

[0401] It should be understood that, unless otherwise stated, when referring to the amounts of a first protein or fragment thereof, a second protein or fragment thereof, and candidate compounds in a mixture, this disclosure is intended to describe the number of different structures of the first protein or fragment thereof, the second protein or fragment thereof, and candidate compounds, rather than their molecular weight. For example, when the mixture contains a “first protein,” the first proteins present in the mixture may have the same structure or different structures. When the mixture contains “multiple first proteins,” the first proteins present in the mixture have at least two different structures. Similarly, when the mixture contains a “second protein,” the second proteins present in the mixture may have the same structure or different structures. When the mixture contains “multiple second proteins,” the second proteins present in the mixture have at least two different structures. Furthermore, when the mixture contains a “candidate compound,” the candidate compounds present in the mixture may all have the same structure or different structures. When the mixture contains “multiple candidate compounds,” the candidate compounds present in the mixture have at least two different structures.

[0402] As used herein, the term "fixed" refers to a connection to a solid surface by various methods (e.g., covalent bonding or high-affinity non-covalent bonding) such that the connected portion does not substantially diffuse into the solution (e.g., incubation solution).

[0403] As used herein, the term "binding site" refers to a fragment or portion of a protein that is capable of binding to (e.g., interacting with) a compound, such as those identified in the methods of this disclosure, or a protein. In one embodiment, the protein binding site comprises a protein domain. In some embodiments, the protein binding site comprises an engineered protein domain.

[0404] As used herein, the term "conjugate compound" refers to a compound capable of targeting one or more targets (e.g., one or more proteins). In some embodiments, the compound is capable of inducing proximity between one or more targets (e.g., one or more proteins). In some embodiments, the compound is capable of eliciting the effects (e.g., degradation, changes in activity, or post-translational modifications) of one or more targets (e.g., one or more POIs) in the presence of another target (e.g., an E3 ligase).

[0405] As used herein, the term “targeting” refers to the ability, effect, or action of an element (e.g., a conjugate compound) to associate with a target (e.g., a protein). In some embodiments, the element (e.g., a conjugate compound) is capable of associating with or relating to a target (e.g., a protein) via covalent and / or non-covalent linkages (e.g., high-affinity non-covalent linkages). In some embodiments, such as in the presence of another target (e.g., an E3 ligase or other protein), the association between the element (e.g., a conjugate compound) and the target (e.g., a protein) causes the effect of the target (e.g., a point of interest) (e.g., degradation, changes in activity, or post-translational modification).

[0406] As used herein, the term "fragment" refers to a portion of the element mentioned. For example, a fragment of a protein refers to a portion of a protein. In some embodiments, a fragment of a protein is a portion of one or more components comprising the protein (e.g., one or more components having the functional and / or structural role of the protein).

[0407] As used herein, the term “configured to” refers to any method (e.g., physical, chemical, or biological) in which the element is constructed or configured, enabling the element to perform the function mentioned.

[0408] As used herein, the term "complex" refers to a group of associating elements (e.g., compounds, proteins, and / or protein fragments) through various methods (e.g., covalent and / or non-covalent linkages). In some embodiments, the elements in a complex are associated by proximity (e.g., proximity induced by a compound between two or more proteins or protein fragments).

[0409] As used herein, the term "affinity conjugate" refers to an element (e.g., a molecule, peptide, or protein moiety) capable of associating with an affinity component by various means (e.g., covalent linkage or high-affinity non-covalent linkage). In some embodiments, the affinity conjugate is a fixed conjugate that forms a fixed complex upon contact with a complex containing an affinity component (e.g., through association between the affinity conjugate and the affinity component).

[0410] As used herein, the expressions “one or more of A, B or C”, “one or more A, B or C”, “one or more of A, B and C”, “one or more A, B and C”, “selected from the group consisting of A, B and C”, “selected from A, B and C”, etc., are used interchangeably and all refer to the group consisting of A, B and / or C, that is, one or more A, one or more B, one or more C or any combination thereof, unless otherwise stated.

[0411] It should be understood that throughout the description, when a compound is described as having, including, or comprising a specific component, the composition is also considered to consist substantially of or comprise only of the described component. Similarly, when a method or process is described as having, including, or comprising specific process steps, the process is also considered to consist substantially of or comprise only of the described processing steps. Furthermore, it should be understood that the order of steps or the sequence of certain actions is irrelevant as long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0412] It should be understood that, unless otherwise stated, any description of a treatment or prevention method includes the use of compounds to provide such treatment or prevention as described herein. It should be further understood that, unless otherwise stated, any description of a treatment or prevention method includes the use of compounds to prepare a medicament for the treatment or prevention of such conditions. Treatment or prevention includes treatment or prevention in humans or non-human animals, including rodents and other disease models.

[0413] The “subject” to which this treatment is intended includes, but is not limited to, humans (i.e., men or women of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, such as mammals, like primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is an agricultural entity (e.g., seeds, seedlings, leaves, flowers, plants, etc.).

[0414] As used herein, the terms "treating" or "treatment" describe the management and care of a patient for the purpose of combating a disease, symptom, or condition, and include the administration of compounds of this disclosure or their pharmaceutically acceptable salts, polymorphs, or solvates to alleviate or eliminate symptoms or complications of the disease, symptom, or condition. The term "treatment" may also include treatment in in vitro cell or animal models. It should be understood that references to "treating" or "treatment" include the relief of identified symptoms of a condition. Therefore, “treating” or “treatment” of a state, condition or symptom includes: (1) preventing or delaying the onset of clinical symptoms of a state, condition or symptom that develops in a person who may have or is susceptible to the state, condition or symptom but has not yet experienced or shown clinical or subclinical symptoms of the state, condition or symptom; (2) suppressing the state, condition or symptom, i.e., preventing, reducing or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one of its clinical or subclinical symptoms; or (3) alleviating or slowing the disease, i.e. causing the disappearance of at least one of the state, condition or symptom or its clinical or subclinical symptoms.

[0415] As used herein, the term “preventing” or “prevention” describes the reduction or elimination of the onset of symptoms or complications of such diseases, conditions, or ailments.

[0416] As used herein, the term "pharmaceutical composition" refers to a formulation containing a compound of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. Unit dosage form is any of a variety of forms, including, for example, capsules, IV bags, tablets, single pumps on an aerosol inhaler, or vials. The amount of active ingredient (e.g., a formulation of the disclosed compound or its salts, hydrates, solvates, or isomers) in a unit dose of the composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will understand that it is sometimes necessary to routinely change the dosage according to the patient's age and condition. The dosage will also depend on the route of administration. A variety of routes are considered, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, oral, sublingual, intrapleural, intrathecal, intranasal, etc. Dosage forms for topical or transdermal application of the compounds of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives, buffers, or propellants.

[0417] As used herein, the term “pharmaceutically acceptable” means those compounds, anions, cations, materials, compositions, carriers, and / or dosage forms that are suitable for use in human and animal tissues to the extent of correct medical judgment without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0418] As used herein, the term "pharmaceuticalally acceptable excipient" means an excipient that can be used to prepare pharmaceutical compositions that are generally safe and non-toxic and are biologically or otherwise desirable and include excipients acceptable for veterinary and human pharmaceutical use. "Pharmaceuticalally acceptable excipient" as used in the specification and claims includes one or more such excipients.

[0419] As used herein, the term "therapeutic effective amount" refers to the amount of a pharmaceutical compound used to treat, improve, or prevent an identified disease or symptom, or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's weight, body size, and health status; the nature and severity of the symptom; and the chosen treatment or combination of treatments. The therapeutic effective amount for a given situation can be determined through routine laboratory testing within the skill and judgment of a clinician.

[0420] Pharmaceutical compositions containing the active compounds of this disclosure can be prepared in commonly known ways, such as by conventional mixing, dissolving, granulation, pelleting, grinding, emulsification, encapsulation, embedding, or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, said carriers containing excipients and / or adjuvants that facilitate the processing of the active compounds into pharmaceutically usable formulations. Of course, suitable formulations depend on the chosen route of administration.

[0421] It should be understood that all these forms are also considered within the scope of the claimed disclosure for compounds of this disclosure that are capable of further forming salts.

[0422] As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the compounds disclosed herein, wherein the parent compound is modified by preparing an acid or its basic salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, and basic or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, non-toxic salts derived from inorganic and organic acids selected from: 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetate, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, glycolamide arsenoic acid, hexylresorcinol acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid. Hydroxymaleic acid, hydroxynaphthylcarboxylic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauryl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, aminosulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid; and common amino acids, such as glycine, alanine, phenylalanine, arginine, etc.

[0423] In some embodiments, pharmaceutically acceptable salts are sodium, potassium, calcium, magnesium, diethylamine, choline, meglumine, benzathine penicillin, tromethamine, ammonium, arginine, or lysine.

[0424] Other pharmaceutically acceptable examples of salts include hexanoic acid, cyclopentanepropionic acid, pyruvate, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, mucoconic acid, etc. This disclosure also covers salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, alkaline earth ion, or aluminum ion) or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc. In salt form, it should be understood that the ratio of the cation or anion of the compound to the salt can be 1:1, or any ratio other than 1:1, such as 3:1, 2:1, 1:2, or 1:3.

[0425] It should be understood that all references to pharmaceutically acceptable salts include the same salt in solvent-added forms (solvents) or crystalline forms (polymorphs) as defined herein.

[0426] The compound or a pharmaceutically acceptable salt thereof may be administered orally, nasally, dermally, pulmonaryly, by inhalation, sublingually, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and parenterally. In one embodiment, the compound is administered orally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0427] Unless otherwise stated, all percentages and ratios used herein are by weight. Other features and advantages of this disclosure will be apparent from various examples. The examples provided illustrate different components and methods useful in practicing this disclosure. The examples do not limit the claimed disclosure. Based on this disclosure, those skilled in the art can identify and employ other components and methods that can be used in practicing this disclosure.

[0428] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document were specifically and individually indicated to be incorporated herein by reference. Reference to publications and patent documents does not imply an admission that any publication or patent document is relevant prior art, nor does it constitute any admission of the content or date of said publication or patent document. The invention has now been described in writing, and those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and the following examples are for illustrative purposes and not for limiting the scope of the following claims.

[0429] The suitable pharmaceutically acceptable prodrugs of the compounds disclosed herein are prodrugs that are appropriate for administration to humans or animals without undesirable pharmacological activity and without excessive toxicity, based on reasonable medical judgment. Various forms of prodrugs have been described in documents such as: a) *Methods in Enzymology*, Vol. 42, pp. 309-396, edited by K. Widder et al. (Academic Press, 1985); b) *Design of Prodrugs*, edited by H. Bundgaard (Elsevier, 1985); c) *Textbook of Drug Design and Development*, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5, “Design and Application of Prodrugs”, by H. Bundgaard, pp. 113-191 (1991); d) H. Bundgaard, *Advanced Drug Delivery Reviews*, 8, 1-38 (1992); e) H. Bundgaard et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya et al., Chem. Pharm. Bull., 32, 692 (1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, ACS Symposium Series, Vol. 14; and h) E. Roche (ed.), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.

[0430] Suitable pharmaceutically acceptable prodrugs of the compounds disclosed herein that contain a hydroxyl group are, for example, in vivo cleavable esters or ethers. In vivo cleavable esters or ethers of any of the compounds of the formula containing a hydroxyl group disclosed herein are, for example, pharmaceutically acceptable esters or ethers that are cleaved in the human or animal body to produce a parent hydroxyl compound. Suitable pharmaceutically acceptable ester-forming groups for the hydroxyl group include inorganic esters, such as phosphate esters (including cyclic aminophosphate esters). Other suitable pharmaceutically acceptable ester-forming groups for the hydroxyl group include: C1-C... 10 Alkyl groups, such as acetyl, benzoyl, phenylacetyl, and substituted benzoyl and phenylacetyl groups; C1-C 10 Alkoxycarbonyl groups, such as ethoxycarbonyl, N,N-(C1-C6 alkyl)2-carbamoyl, 2-dialkylaminoacetyl, and 2-carboxyacetyl. Examples of cyclic substituents on phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinemethyl, piperazine-1-ylmethyl, and 4-(C1-C4 alkyl)piperazine-1-ylmethyl. Suitable pharmaceutically acceptable ether-forming groups for the hydroxyl group include α-acyloxyalkyl groups, such as acetoxymethyl and neopentyloxymethyl.

[0431] Suitable pharmaceutically acceptable prodrugs of the compounds disclosed herein that have a carboxyl group are, for example, cleavable amides in vivo, such as amines like ammonia; C1-C4 alkylamines like methylamine; (C1-C4 alkyl)2amines like dimethylamine; N-ethyl-N-methylamine or diethylamine; C1-C4 alkoxy-C1-C4 alkylamines like 2-methoxyethylamine; phenyl-C1-C4 alkylamines like benzylamine; and amides formed from amino acids such as glycine or its esters.

[0432] Suitable pharmaceutically acceptable prodrugs of the compounds disclosed herein that contain an amino group are, for example, their in vivo cleavable amide derivatives. Suitable pharmaceutically acceptable amides derived from an amino group include, for example, those using C1-C... 10 Alkyl groups, such as acetyl, benzoyl, phenylacetyl, and amides formed by substituted benzoyl and phenylacetyl groups. Examples of cyclic substituents on phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinemethyl, piperazine-1-ylmethyl, and 4-(C1-C4 alkyl)piperazine-1-ylmethyl.

[0433] Example The following examples are provided for illustrative purposes only and should not be construed as limiting the invention.

[0434] Example 1. Design an exemplary method Several exemplary methods have been designed. A detailed design overview of the methods is as follows.

[0435] Exemplary method 1: The gel screening assay mixture in physiological buffer contains the following components: GFP1-9, the protein of interest (POI) labeled with another short amino acid sequence from GFP, typically corresponding to GFP11, a selected E3 ligase labeled with a short amino acid sequence from green fluorescent protein (GFP), typically corresponding to GFP10, and the compound of interest as a potential molecular gel. The POI can be labeled with GFP10 instead of GFP11. In this case, the E3 ligase is labeled with GFP11. Full-length GFP consists of 11 distinct amino acid residue segments forming a barrel structure. GFP1-9 contains the first 9 of those distinct segments. GFP10 indicates the amino acid residue segment corresponding to the 10th of the 11 segments in full-length GFP. Similarly, GFP11 indicates the 11th of the 11 distinct amino acid segments in full-length GFP. Various variants of GFP exist, both naturally occurring and genetically engineered. Although there are some variations in the optimal fluorescence wavelength, these proteins all possess a similar barrel structure with 11 distinct amino acid residue segments. In this disclosure, GFP refers to any of those variant forms. Other different amino acid segments can be used to label the first protein (e.g., POI) and the second protein (e.g., E3 ligase), provided that the two segments used to label the first and second proteins are adjacent to each other in the fully formed barrel structure. In this case, GFP1-9 will be replaced by consecutive amino acid residue segments from GFP (excluding the two segments used to label the two proteins) such that the three parts of GFP can be assembled to form a barrel-shaped mature GFP. In a typical screening assay, multiple POIs, each tagged with GFP11 (or GFP10), can be tested simultaneously in the same assay mixture (up to 10 or more). Similarly, multiple E3 ligases, each tagged with GFP10 (or GFP11 when the POI is tagged with GFP10), can be tested simultaneously in the same assay mixture (up to 10 or more). GFP1-9 does not require any specific tag and is typically used at higher concentrations than any single E3 or POI. One compound of interest can be tested at a time. To increase screening throughput, compounds of interest are typically pooled together in a mixture of 100-200 compounds per pool using a stock solution, usually dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. When pooling 100 compounds from the initial 10 mM stock solution into the mixture, the concentration of each individual compound in the pooled solution is 100 μM. Higher throughput can be achieved by pooling more compounds into each pool when a higher concentration of the initial compound stock solution is available. Alternatively, compounds can be tested at higher concentrations while maintaining the same throughput.

[0436] In addition to tagging POI and E3 with GFP-derived amino acid sequences, POI or E3 (but not both) can be modified with biotin as a handle to capture these proteins via a high-affinity interaction between biotin and streptavidin, avidin, or variants thereof. Biotinylation of proteins can be accomplished by any number of recognized methods, including biochemical conjugation using biotin analogs having reactive functional groups that form covalent bonds with certain amino acid side chains or free N-termini or C-termini. Other common methods include expressing proteins as hybrid proteins containing a segment of amino acid residues commonly referred to as an "Avi tag." The Avi tag can be enzymatically biotinylated intracellularly during protein synthesis or after protein purification. If the same tag is not found in any other component of the assay mixture, and a corresponding high-affinity capture reagent is available for tag binding, other types of tags can be used instead of biotin tags.

[0437] When a ternary complex is formed in any combination of POI, E3, and gel molecules, the GFP11 and GFP10 tags on POI and E3 are brought close together. Under these conditions, the assembly of mature GFP consisting of GFP1-9, GFP11, and GFP10 can occur at a dramatically accelerated rate compared to the random assembly of these three components in solution, which can be detected by the appearance of green fluorescence in solution. The binding of GFP1-9 with GFP11 and GFP10 to form a barrel-shaped GFP complex is virtually irreversible under physiological conditions and thus stabilizes the ternary complex of POI, E3, and gel molecules. The presence of fully mature GFP can be monitored by measuring the development of green fluorescence in solution. This is done by repeatedly measuring the change in green fluorescence over time (typically 8 hours to overnight). Significantly higher fluorescence from certain wells compared to most wells in the plate indicates the presence of the ternary complex in these wells. These wells are considered to contain the active material.

[0438] At the end of an incubation period sufficient to allow for ternary complex formation and subsequent GFP assembly on the ternary complex, the assay mixture can be transferred to a plate pre-coated with streptavidin to capture biotinylated proteins. The capacity of the streptavidin coated on the plate should be high enough to capture all proteins with biotinylated proteins. When using other types of tags instead of biotin, the plate should be pre-coated with an appropriate high-affinity agent instead of streptavidin capable of capturing the tag. After incubating the assay mixture in a streptavidin-coated plate, typically for 30 minutes to one hour, the plate is washed with a buffer solution to remove any unbound proteins and compounds. When biotin tags are placed on POIs, most E3 proteins and GFP1-9 are washed away, except for those recruited to the POI as part of the ternary complex with the gel compound. The opposite is true when biotin tags are placed on E3s. To further enhance the fluorescence signal from fully assembled GFP, a solution containing a GFP enhancer can be added to each well. The GFP enhancer is an affinity conjugate of GFP labeled with a fluorescent dye. The dye has the same fluorescence wavelength as GFP but a higher quantum yield. In this case, the GFP enhancer is an antibody form targeting fully assembled GFP conjugated to a chemifluorophore with the same excitation / emission wavelength as GFP. For example, GFP nanobodies conjugated with Alexa Fluor 488 from Chromotek can be used for this purpose. Because this reagent has high specificity for fully assembled GFP relative to GFP1-9, GFP10, or GFP11, only those wells containing fully assembled GFP bind to the GFP enhancer. Unbound GFP enhancer is washed away. To further improve the specificity of the GFP enhancer for fully assembled GFP, the incubation time of the GFP enhancer solution is limited to less than 30 minutes. Because the GFP enhancer has a higher affinity for fully assembled GFP compared to GFP1-9, GFP10, or GFP11, the shorter incubation time ensures that the GFP enhancer binds to the plate only when fully assembled GFP is captured on the plate. Alternatively, the GFP enhancer can be added directly to the assay mixture at the end of incubation to form the ternary complex and subsequent assembly of full-length GFP, but before transfer to a streptavidin-coated plate. Similar results are expected because the GFP enhancer is specific for full-length GFP. Once the plate is incubated with the GFP enhancer and washed to remove unbound GFP enhancer, the plate is sealed with a clear membrane, and fluorescence is measured from individual wells in the plate. Other types of affinity methods can be used to capture the ternary complex, including using anti-GFP antibodies to capture fully assembled GFP. Additionally, the affinity agent can be conjugated or coated onto any solid-phase surface, such as magnetic beads, agarose beads, or other types of beads.

[0439] The screening method can be performed multiple times. For example, in an initial high-throughput screening, fluorescence is detected in active wells containing a chemically diverse library (100-200 compounds / well) pooled with one or more POIs and one or more E3s. Subsequently, a second screening is performed, repeating the same pooled compounds, POIs, and E3s from the initial screening in new wells. Not wanting to be bound by theory, performing the second screening increases the confidence that any initial signals detected are reproducible and not due to noise or other environmental factors.

[0440] Due to the multifaceted nature of screening methods, the identity of individual POIs, E3s, and gel compounds among active substances is unknown at this stage. Two different methods are used for this identification purpose: deconvolution and MS-based identification, which will be explained below.

[0441] In deconvolution methods, individual POIs, E3s, and potential gel compounds in an active substance are tested in a way that uniquely and definitively identifies which specific combinations of POIs, E3s, and potential gel compounds lead to the formation of a ternary complex in the identified active substance. In one example, all individual combinations of POIs and E3s are tested in each sample using the same pool of compounds to first identify the correct combinations of POIs and E3s. Then, individual compounds are tested using specific pairs of POIs and E3s identified in the previous step. A variety of other methods can be used, as long as the unique combinations of POIs, E3s, and gel compounds can be definitively identified. For example, the same protein mixture can be tested against individual compounds in the active pool to identify active gel molecules, followed by testing for specific combinations of POIs and E3s with the same active gel molecules.

[0442] In MS-based identification, components in ternary complexes of active substances are identified using the standard "bottom-up mass spectrometry-based proteomics" method. In this method, proteins captured in wells are typically reduced and alkylated under denaturing conditions before analysis by mass spectrometry, followed by digestion into smaller peptides using a sequence-specific protease. Several options are available for this accepted procedure. For example, high concentrations of urea, guanidine hydrochloride, acid-labile ionic detergents such as RapiGest, or other ionizing agents are used for protein denaturation. Dithiothreitol, β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine) are commonly used to reduce disulfide bonds in proteins. Iodoacetamide, iodoacetic acid, or iodoethanol are some commonly used alkylating agents that covalently modify free thiol groups on proteins once disulfide bonds are reduced. For mass spectrometry-based protein analysis, trypsin is the most commonly used sequence-specific protease for digesting proteins into peptides due to its high sequence specificity for protein cleavage. Other proteases commonly used for this purpose include Lys-C, Arg-C, and chymotrypsin. A mixture of Lys-C and trypsin, or the sequential use of these two proteases, is also a common choice. The amino acid sequence of the digested peptide is identified using standard proteomics methods. In short, this method is based on comparing the obtained m / z (mass-to-charge ratio) values ​​of the peptide and its MS2 fragmented spectra with theoretical data obtained from computer digestion of protein sequences from protein databases of the appropriate species. Many publications are available for this method, and various software programs, often referred to as search engines, are readily available from multiple sources. For mass spectrometry data acquisition, online separation of peptides on liquid chromatography (LC) is typically combined with different data acquisition modes via the mass spectrometer. Data-dependent MS2 acquisition (DDA) is the conventional method for this purpose, and data-independent acquisition (DIA) methods are also used. Any other data acquisition method can be used, as long as a definitive identification of a unique peptide sequence can be established. Direct data acquisition can be performed without LC separation of the peptide, especially when the sample composition is relatively simple. Matrix-assisted laser desorption / ionization (MALDI) time-of-flight instruments are one example. Direct delivery of digested samples via an electrospray ionization interface is another. Protein identity within a sample can be deduced by comparing the amino acid sequence of a peptide with the amino acid sequence of a complete protein in a protein database. During the digestion of proteins in a ternary complex, molecular gel compounds trapped within the ternary complex are released into the digestion solution. The identity of these gel compounds can also be obtained from the same sample, and typically from the same experiment, by comparing the precise molecular weight of the compound in the active substance pool with the expected molecular weight of the compound deduced from the m / z value of the MS1 spectrum.Alternatively, if the protein in the ternary complex can be released into solution, protein identity information can be obtained directly using a top-down approach without digesting the protein. In this method, the mass of the intact protein obtained by mass spectrometry is compared to the expected mass of the individual protein used in this experiment. One such method for “releasing” the intact protein from GFP involves introducing a short peptide sequence between the E3 or POI protein and the GFP10 or GFP11 tag, which can be cleaved by a highly sequence-specific protease. An example of this is the TEV cleavage sequence ENLYFQS(G / A). The TEV protease cleaves between Q and S in a highly sequence-specific manner. Acidification of the sample will cause dissociation of the ternary complex. Thus, the entire protein can be released from the ternary complex bound to the plate by incubating the plate with a solution containing the TEV protease, followed by acidification.

[0443] The deconvolution methods and MS-based identification methods described above are not mutually exclusive and can be used in combination to obtain the highest confidence level of identity for individual components within the ternary complex within a single hit. It should also be noted that MS-based methods do not provide identity for proteins with specific affinity tags (biotinylated E3 or biotinylated POI) that are part of the ternary complex, because all biotinylated proteins will be captured from all samples, regardless of whether ternary complex formation is present.

[0444] Example 2. Result of the Exemplary Method Results of Exemplary Method 1a In one version of the assay, the gel screening assay mixture was assembled in 10 µL of assay buffer (50 mM Hepes buffer (pH 7.5), 150 mM KCl, 1 mM TCEP) containing 1 µg GFP1-9, 0.1 µg GFP11-labeled RBM39, and 0.1 µg GFP10-labeled DCAF15 complexed with DDB1 and DDA1 (hereinafter referred to as DCAF15, as DCAF15 is always used as the complex with DDB1 and DDA1). Two different versions of GFP11-labeled RBM39 were tested, in which GFP11 was fused to the N-terminus and C-terminus of RBM39 (GFP11-RBM39 and RBM39-GFP11, respectively). Similarly, two different versions of GFP10-labeled DCAF15 were tested: GFP10 fused to the N-terminus of DCAF15 (GFP10-DCAF15) and GFP10 fused to the C-terminus of DCAF15 (DCAF15-GFP10). For primary screening, these four different proteins were combined and added to a plate containing a pool of compounds. Once the active pool was identified, the same assay mixture was tested for each individual compound within the active pool, and then the confirmed compounds were tested for deconvolution against different E3 and POI pairs. In this example, four different assay mixtures were prepared, each using different combinations of GFP11-labeled RBM39 and GFP10-labeled DCAF15 (see Table A).

[0445] Table A In addition to GFP11, the RBM39 protein also has a biotin tag on the opposite side of GFP11 via an Avi tag sequence. These different assay mixtures were added to 384-well assay plates containing 100 nL equimolar mixtures of the test compounds in DMSO at various concentrations (0 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM, respectively). Two different test compounds were used in this experiment: indexsulfanilamide (MedKoo, 201540) as a positive control and lenalidomide (aablocks, AA002FDI) as a negative control. The assay plates were sealed with transparent tape (Eppendorf, 0030127838), mixed by vortexing, and briefly rotated with shaking before incubation at 4°C to collect liquid at the bottom of the plate. After incubation for 2, 6, 18, 24, 48, and 72 hours, green fluorescence was measured from the assay plate using a fluorescent plate reader with excitation / emission wavelengths of 488 / 509 nm. Figure 1A-1DSimulation results with various combinations outlined above are shown. Figure 1A , Figure 1B and Figure 1D In this study, the dose-dependent development of the signal plateaued earlier at higher compound concentrations. Lower compound concentrations resulted in slower signal development and lower plateau levels. Figure 1B This indicates a combination that does not exhibit any activity.

[0446] At the end of the 72-hour incubation, the contents of the assay plate were transferred to a capture plate pre-coated with streptavidin (Greiner, catalog number 781995) and containing 2 µL of GFP enhancer (Chromotek gb2AF488-50) diluted 1:200 in assay buffer. After incubation at room temperature for 30 minutes to capture biotin-labeled RBM39, the plate was inverted onto a collection plate and briefly rotated at 1500 rpm to remove the contents, followed by the addition of 20 µL of assay buffer. This process was repeated three times to remove unbound proteins from the plate. The addition of assay buffer was omitted after the third washing step, and green fluorescence was measured again after the addition of 20 µL of ammonium bicarbonate solution as described above (simulated results are available in [reference needed]). Figure 2A-2B (In the following text, mass spectrometry-based analysis is performed on all samples immediately.)

[0447] Add 5 µL of reducing agent (25 mM DTT in 50 mM ammonium bicarbonate containing 0.05% RapiGest (Waters, 186008740)) to each well of the capture plate and seal the plate with heat-sealing tape. Incubate the capture plate at 80 °C for 5 min, then cool to room temperature and transfer the contents to a new 384-well plate containing 5 µL of 50 mM ammonium bicarbonate containing 75 mM iodoacetamide. Incubate the plate in the dark with shaking at room temperature for 45 min. At the end of the incubation, transfer 2 µL of the contents from each well to a new blank 384-well plate and mix with 1 µL of MALDI matrix for mass spectrometric analysis of the captured gel compounds (simulation results are available in [link to relevant documentation]). Figure 3 Add 5 µL of trypsin solution (Thermo Fisher MS grade, 90057, diluted to 0.05 µg / µL in 50 mM ammonium bicarbonate) to the remaining sample, seal the plate, and incubate overnight at 37°C. At the end of incubation, transfer 2 µL of the digested sample to another 384-well plate and mix with 1 µL of MALDI matrix (described below) for mass spectrometry analysis of the digested peptides of the captured protein. Figure 4 ).

[0448] The MALDI matrix contained a saturated solution of α-cyano-4-hydroxycinnamic acid in TA30 solvent (30:70 [v:v] acetonitrile: water with 0.1% TFA). The sample mixed with the MALDI matrix solution was incubated at 55°C for 1 hour to promote the hydrolysis of RapiGest in the sample. Then, 2 µL of the final sample was spotted onto an AnchorChip target plate (Bruker, 8280790) and dried before analysis using a timsTOF mass spectrometer (Bruker) according to the manufacturer's protocol.

[0449] Results of Exemplary Method 1b In another version of the assay, the gel screening assay mixture was assembled in a 2 µL volume of assay buffer (50 mM HEPES buffer (pH 7.5), 150 mM KCl, 4 mM DTT, 0.1 mg / mL BSA, 0.25 mM octyl-β-glucopyranoside, 5% glycerol) in a clear-bottom 1536-well plate (Grena, catalog number 789866). 0.3 µg GFP1-9, 0.05 µg GFP11-labeled IKZF2, and 0.3 µg GFP10-labeled CRBN were conjugated with DDB1 (hereinafter referred to as CRBN, as CRBN is always used as the conjugate with DDB1). In this assay, various variations in the buffer provided a consistent signal (0.1–1 mg / mL BSA, 0–10% glycerol, 0–1 mM octyl-β-glucopyranoside). Similar to previous versions, two different versions of GFP11-labeled IKZF2 were tested, with GFP11 fused to the N-terminus and C-terminus of IKZF2 (GFP11-IKZF2 and IKZF2-GFP11, respectively). Similarly, two different versions of GFP10-labeled CRBN were tested: GFP10 fused to the N-terminus of CRBN (GFP10-CRBN) and GFP10 fused to the C-terminus of CRBN (CRBN-GFP10). Concentrations corresponded to 5 µM GFP (1–9), 1.25 µM for each IKZF2 construct, and 0.5 µM for each of the two CRBN constructs. The concentrations of IKZF2 and CRBN proteins were optimized by titrating the proteins against each other to obtain the best signal-to-background ratio.

[0450] For initial screening, the five different proteins (GFP1-9, GFP11-IKZF2, IKZF2-GFP11, GFP10-CRBN, and CRBN-GFP10) were combined in assay buffer and added to a plate containing different compound pools. During deconvolution, the assay mixture was tested for each individual compound, and then the confirmed compounds were tested for different POIs and E3 pairs.

[0451] When protein mixtures were added to compounds known for gel formation against IKZF2-CRBN (IKZF2-CRBN gel compound 1, IKZF2-CRBN gel compound 2, and IKZF2-CRBN gel compound 3), the GFP signal increased over time compared to the DMSO control. Figure 5 The signals generated in these experiments were specific to these IKZF2-CRBN gels because the signals from other compounds (chloroquineoxorline, indexsulfanilamide) were indistinguishable from the DMSO control. These compounds are known gels for protein pairs RBM39 and DCAF15 and are known to have no gel activity against IKZF2-CRBN protein pairs. Figure 5 ).

[0452] Result of Exemplary Method 1c In another version of the assay, the gel screening assay mixture is assembled in a manner similar to that of Exemplary Method 1b, wherein RBM39 and DCAF15 proteins are used in place of IKZF2 and CRBN proteins.

[0453] When a protein mixture containing 1.25 µM RBM39 labeled with GFP11, 0.5 µM DCAF15 labeled with GFP10 (hereinafter referred to as DCAF15, as DCAF15 is always used as a complex with DDB1 and DDA1) and 5 µM GFP1-9 was added to compounds known for constructing gels against RBM39:DCAF15 (NSC-339004, Tasisulam, E7820, and E7070), the GFP signal increased in a compound dose-dependent manner compared to the DMSO control. Fluorescence signals were measured at multiple time points over 16 hours of incubation. Data from the first 8 hours were fitted to a straight line to obtain the initial rate (k1). The ratio (S / B) of the k1 value at each concentration of different compounds to the k1 value of the DMSO-treated sample is shown. Figure 6 ).

[0454] equivalent Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are now described. Other features, objects, and advantages of this disclosure will be apparent from this specification and the claims. In the specification and appended claims, the singular form also includes plural references unless the context clearly requires otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in this specification are incorporated herein by reference.

[0455] The foregoing description is presented for illustrative purposes only and is not intended to limit this disclosure to the precise form disclosed, but is limited by the appended claims.

Claims

1. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag GFP fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag GFP fragment; (c) a detector GFP fragment, said detector GFP fragment being complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compounds, The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof associated with the complex, wherein the identified compound is capable of modulating protein-protein interactions (PPIs) between the first protein and the second protein.

2. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag GFP fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag GFP fragment; (c) a detector GFP fragment, said detector GFP fragment being complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compounds, Wherein the first protein or a fragment thereof and / or the second protein or a fragment thereof are covalently linked to the affinity component; and The first tag GFP fragment, the second tag GFP fragment, and the detector GFP fragment are configured to generate or enhance a measurement signal when the assay mixture produces a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound; (ii-a) Contact the complex with a fixed affinity conjugate targeting the affinity component, thereby forming a fixed complex; as well as (ii-b) Detect and / or isolate the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof, wherein the identified compound is capable of modulating protein-protein interactions (PPIs) between the first protein and the second protein.

3. A method for identifying a first protein, a second protein, a compound targeting the first protein and the second protein, or any combination thereof, the method comprising: (i) Providing a assay mixture, the assay mixture comprising: (a) A first protein or a fragment thereof, wherein the first protein or a fragment thereof is covalently linked to a first tag GFP fragment; (b) A second protein or a fragment thereof, wherein the second protein or a fragment thereof is covalently linked to a second tag GFP fragment; (c) a detector GFP fragment, said detector GFP fragment being complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compounds, The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are configured to generate or enhance a assay signal when the assay mixture induces an approach between the first protein or a fragment thereof and the second protein or a fragment thereof; and (ii) Identify the first protein, the second protein, the compound, or any combination thereof that are associated with the induced proximity. The identified compound can cause the degradation of the first protein in the presence of the second protein.

4. The method according to any one of the preceding claims, wherein the assay mixture comprises a plurality of different first proteins or fragments thereof.

5. The method according to any one of the preceding claims, wherein the assay mixture comprises a variety of different second proteins or fragments thereof.

6. The method according to any one of the preceding claims, wherein the assay mixture comprises a variety of different candidate compounds.

7. The method according to any one of the preceding claims, wherein the assay mixture produces the complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the candidate compound.

8. The method according to any one of the preceding claims, wherein the measurement signal is fluorescence.

9. The method according to any one of the preceding claims, wherein the first tag GFP fragment and the second tag GFP fragment independently comprise one or more of the following: GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10 and GFP11.

10. The method according to any one of the preceding claims, wherein the first tag GFP fragment and the second tag GFP fragment independently comprise one or more of GFP10 and GFP11.

11. The method according to any one of the preceding claims, wherein the first tag GFP fragment comprises GFP10 and the second tag GFP fragment comprises GFP11.

12. The method according to any one of the preceding claims, wherein the detector GFP fragment is selected from GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8 and GFP9.

13. The method according to any one of the preceding claims, wherein the first protein is the protein of interest (POI).

14. The method according to any one of the preceding claims, wherein the second protein is a ubiquitin ligase.

15. The method according to any one of the preceding claims, wherein step (i) further comprises detecting the generated or enhanced measurement signal.

16. The method according to any one of the preceding claims, wherein the identified compound causes degradation of the first protein in the presence of the second protein or causes degradation of the second protein in the presence of the first protein.

17. A first protein, identified by the method according to any one of the preceding claims.

18. A second protein, identified by the method according to any one of the preceding claims.

19. A combination of a first protein and a second protein, said combination being identified by the method according to any one of the preceding claims.

20. A combination of a first protein, a second protein, and a compound targeting the first protein and the second protein, said combination being identified by the method according to any one of the preceding claims.

21. A compound identified by the method according to any one of the preceding claims.

22. The compound of claim 21, for use in human therapy, agriculture and / or animal health.

23. A pharmaceutical composition comprising a compound identified by the method according to any one of the preceding claims.

24. A method for degrading a first or second protein in a subject, the method comprising administering to the subject a compound identified by the method according to any one of the preceding claims.

25. A method for regulating the PPI between a first protein and a second protein in a subject, the method comprising administering to the subject a compound identified by the method according to any one of the preceding claims.

26. The method of claim 25, wherein the PPI causes changes in protein stability, protein level, protein post-translational modifications, protein localization, and / or protein activity.

27. A method for treating and / or preventing a disease or condition associated with a first or second protein in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound identified by the method according to any one of the preceding claims.

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