Methods for identifying compounds that 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 are associated with a disease or disorder.

Description

Background Art

[0001] Related Applications This application claims the benefit and priority of U.S. Application No. 18 / 339,429, filed on June 22, 2023, the content of which is incorporated herein by reference in its entirety.

[0002] Background A molecular glue is a chemical compound that induces the proximity of one or more proteins to each other. Molecular glue compounds enhance the affinity of one or more proteins for each other, thus causing the proteins to interact. Depending on the particular proteins, the interaction can affect the function of one or both proteins. Functional consequences can include, but are not limited to, changes in protein stability, changes in protein levels, changes in protein post-translational modifications, changes in protein localization, and / or changes in protein activity.

[0003] In the context of proteolysis, a molecular glue enhances the binding of a protein of interest (POI) to an E3 ligase (E3), which then ultimately leads to the ubiquitin proteasome system (UPS)-mediated degradation of the POI. Molecular glues are difficult to discover for several reasons. First, they often show little or no binding to either the individual POI or E3. Complexes are often only observed in the presence of the molecular glue, POI, and E3. Even when an adhesive compound shows measurable binding to either protein, what matters is the ternary complex, and most binary binding events do not lead to ternary complex formation. Thus, all three components, the POI, E3, and molecular glue, need to be present in the screen. Second, the requirement for the presence of all three components in the screening assay mixture poses a major challenge. This is 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 it is necessary to screen a large number of compounds, often exceeding several hundred thousand, testing many individual E3 ligases in combination with numerous compounds becomes difficult. Thus, there remains a need for novel methods for identifying molecular adhesives. [Overview of the Initiative]

[0005] In some embodiments, the Disclosure provides a method for identifying a first protein, a second protein, compounds that target the first and second proteins, or any combination thereof.

[0006] In some embodiments, the Disclosure provides a first protein identified by the method of the Disclosure.

[0007] In some embodiments, the Disclosure provides a second protein identified by the method of the Disclosure.

[0008] In some embodiments, the Disclosure provides a combination of a first protein and a second protein identified by the method of the Disclosure.

[0009] In some embodiments, the Disclosure provides a first protein, a second protein, and combinations of compounds that target the first and second proteins, as identified by the method of the Disclosure.

[0010] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure.

[0011] In some embodiments, the Disclosure provides a method for degrading a first protein in a subject, and includes administering a compound identified by the method of the Disclosure to the subject.

[0012] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in degrading a first protein in a subject.

[0013] In some embodiments, the Disclosure provides the use of compounds identified herein in the manufacture of pharmaceuticals for the degradation of a first protein in a subject.

[0014] In certain embodiments, the Disclosure provides a method for treating and / or preventing a disease or disorder associated with a first protein in a subject, which includes administering to the subject a therapeutically effective dose of a compound identified by the method of the Disclosure.

[0015] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in treating and / or preventing diseases or disorders associated with a first protein in a subject.

[0016] In some embodiments, the Disclosure provides the use of compounds identified by the methods of the Disclosure in the manufacture of a pharmaceutical product for the treatment and / or prevention of a disease or disorder associated with a first protein in a subject.

[0017] In some embodiments, the Disclosure provides a method for degrading a second protein in a subject, which includes administering a compound identified by the method of the Disclosure to the subject.

[0018] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in degrading a second protein in a subject.

[0019] In some embodiments, the Disclosure provides the use of the compounds identified herein in the manufacture of pharmaceuticals for the degradation of a second protein in a subject.

[0020] In certain embodiments, the Disclosure provides a method for treating and / or preventing a disease or disorder associated with a second protein in a subject, which includes administering to the subject a therapeutically effective dose of a compound identified by the method of the Disclosure.

[0021] In some embodiments, the present disclosure provides a compound identified by the methods of the present disclosure for use in treating and / or preventing a disease or disorder associated with a second protein in a subject.

[0022] In some embodiments, the present disclosure provides the use of a compound identified by the methods of the present disclosure in the manufacture of a medicament for treating and / or preventing a disease or disorder 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 belongs. In this specification, the singular forms also include the plural unless the context clearly dictates otherwise. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials are described below. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, this specification, including definitions, will control. Also, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict between the chemical structure and the name of a compound disclosed herein, the chemical structure will control.

[0024] Other features and advantages of the present disclosure will be apparent from the following detailed description and the claims. **Brief Description of the Drawings**

[0025] [Figure 1A] FIG. 1A is a graph showing the simulated time course and dose-dependence of fluorescence signal generation for GFP11-RBM39 and GFP10-DCAF15 (Combination A). [Figure 1B] FIG. 1B is a graph showing the simulated time course and dose-dependence of fluorescence signal generation for RBM39-GFP11 and GFP10-DCAF15 (Combination B). [Figure 1C]Figure 1C is a graph showing the simulated time course and dose-dependence of fluorescence signal generation for GFP11-RBM39 and DCAF15-GFP10 (combination C). [Figure 1D] Figure 1D is a graph showing the simulated time course and dose-dependence of fluorescence signal generation for RBM39-GFP11 and DCAF15-GFP10 (combination D). [Figure 2A] Figure 2A is a graph showing the simulated effect of GFP-booster on fluorescence signal intensity for combination A and combination C. [Figure 2B] Figure 2B is a graph showing the simulated effect of GFP-booster on fluorescence signal intensity for combination B and combination D. [Figure 3] Figure 3 is a simulated graph showing the potential detection of test compounds from MALDI MS experiments based on the simulated MS signals between indisulam and lenalidomide. [Figure 4] Figure 4 is a simulated graph showing the potential detection of E3 from MALDI MS experiments. [Figure 5] Figure 5 is a graph with experimental data showing the time course of fluorescence signal generation for IKZF2-CRBN adhesive compound 1, IKZF2-CRBN adhesive compound 2, IKZF2-CRBN adhesive compound 3, and IKZF2-CRBN non-adhesive compounds chloroquine and indisulam. [Figure 6] Figure 6 is a graph with experimental data showing the dose response of fluorescence signal generation for four different RBM39-DCAF15 adhesive compounds. The initial rate (k1) is shown as the ratio to that for DMSO-treated samples (S / B) of signal generation.

Mode for Carrying Out the Invention

[0026] Detailed Description This disclosure relates to a method for identifying compounds for inducing protein-protein interactions. The disclosure also relates to the compounds identified in this method, as well as their use, for example, in degrading POIs and / or treating or preventing diseases or disorders. The methods of this disclosure are applicable to a variety of fields, including, but are not limited to, human therapy, agriculture, and animal health.

[0027] Method of Disclosure In some embodiments, the Disclosure provides a method for identifying a first protein, a second protein, compounds that target the first and second proteins, or any combination thereof.

[0028] In some embodiments, this disclosure provides methods applicable to human treatment, agriculture, and / or animal health. In some embodiments, the methods are applicable to human treatment. In some embodiments, the methods are applicable to humans. In some embodiments, the methods are applicable to agriculture. In some embodiments, the methods are applicable to agricultural entities (e.g., seeds, saplings, leaves, flowers, plants, etc.). In some embodiments, the methods are applicable to animal health. In some embodiments, the methods are applicable to animals.

[0029] In some embodiments, the method involves identifying a first protein (e.g., POI) that is targeted by a compound containing a second protein.

[0030] In some embodiments, the method involves identifying a second protein that is targeted by a compound associated with a first protein.

[0031] In some embodiments, the method involves identifying a combination of a first protein and a second protein that are targeted by the compound.

[0032] In some embodiments, the method involves identifying a first protein, a second protein, and combinations of compounds that target the first and second proteins.

[0033] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided such that the assay mixture is configured to generate or enhance an assay signal when proximity is induced between the first protein or its fragment and the second protein or its fragment.

[0034] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The first tag fragment and the second tag fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and a compound; (ii-a) Contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) detecting and / or isolating the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0035] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The assay mixture provides a first tag fragment and a second tag fragment that induce proximity between the first protein or a fragment thereof and the second protein or a fragment thereof; the first tag fragment and the second tag fragment generate or enhance the assay signal.

[0036] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and the compound; and (ii) Identifying a first protein, a second protein, a compound, or any combination thereof associated with the complex.

[0037] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The assay mixture yields a complex comprising a first protein or fragment thereof, a second protein or fragment thereof, and a compound; the first tag fragment and the second tag fragment provide the conditions necessary to generate or enhance the assay signal; (ii-a) Contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) detecting and / or isolating the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0038] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided such that the assay mixture generates or enhances an assay signal when proximity is induced between the first protein or its fragment and the second protein or its fragment; and (ii) Identifying a first protein, a second protein, a compound, or any combination thereof that is associated with (e.g., causes) the induction of proximity, The identified compounds are capable of degrading the first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0039] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof covalently attached to the first tag GFP fragment; (b) A second protein or fragment thereof covalently attached to a second I GFP fragment; (c) Detector GFP fragments complementary to the first tagged GFP fragment and the second tagged GFP fragment; and (d) containing candidate compounds, The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are provided, configured to generate or enhance an assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and the compound; and (ii) Identifying a first protein, a second protein, a compound, or any combination thereof associated with the complex, the identified compound being capable of modulating a protein-protein interaction (PPI) between the first protein and the second protein.

[0040] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof covalently attached to the first tag GFP fragment; (b) A second protein or fragment thereof covalently attached to a second I GFP fragment; (c) Detector GFP fragments complementary to the first tagged GFP fragment and the second tagged GFP fragment; and (d) containing candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and the compound; (ii-a) Contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) Detecting and / or isolating the immobilized complex, thereby identifying the first protein, the second protein, a compound, or any combination thereof, the identified compound being capable of modulating a protein-protein interaction (PPI) between the first protein and the second protein.

[0041] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof covalently attached to the first tag GFP fragment; (b) A second protein or fragment thereof covalently attached to a second I GFP fragment; (c) Detector GFP fragments complementary to the first tagged GFP fragment and the second tagged GFP fragment; and (d) containing candidate compounds, The first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are provided such that the assay mixture generates or enhances an assay signal when proximity is induced between the first protein or fragment and the second protein or fragment; and (ii) Identifying a first protein, a second protein, a compound, or any combination thereof associated with the induction of proximity The identified compound is capable of causing the degradation of the first protein in the presence of the second protein.

[0042] In some embodiments, proximity induction is associated with a compound (e.g., caused by).

[0043] In some embodiments, the assay mixture further comprises a detector fragment complementary to the first tag fragment and the second tag fragment.

[0044] In some embodiments, the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance an assay signal when the assay mixture induces proximity between the first protein or its fragment and the second protein or its fragment.

[0045] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment; (c) Detector fragments complementary to the first tag fragment and the second tag fragment; and (d) containing candidate compounds, The first tag fragment, the second tag fragment, and the detector fragment are provided such that the assay mixture is configured to generate or enhance an assay signal when proximity is induced between the first protein or its fragment and the second protein or its fragment.

[0046] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment; (c) Detector fragments complementary to the first tag fragment and the second tag fragment; and (d) containing candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The first tag fragment, the second tag fragment, and the detector fragment are provided, configured to generate or enhance an assay signal when the assay mixture induces proximity between the first protein or its fragment and the second protein or its fragment; (ii-a) Contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) detecting and / or isolating the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0047] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment; (c) Detector fragments complementary to the first tag fragment and the second tag fragment; and (d) containing candidate compounds, The first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance an assay signal when the assay mixture induces proximity between the first protein or its fragment and the second protein or its fragment; and The invention provides that the identified compound is capable of causing the degradation of a first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0048] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment; (c) Detector fragments complementary to the first tag fragment and the second tag fragment; and (d) containing candidate compounds, The assay mixture is provided such that it induces proximity between a first protein or fragment thereof and a second protein or fragment thereof; and the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance an assay signal.

[0049] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment; (c) Detector fragments complementary to the first tag fragment and the second tag fragment; and (d) containing candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The assay mixture is provided such that it induces proximity between a first protein or fragment thereof and a second protein or fragment thereof; and 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 an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) detecting and / or isolating the immobilized complex, thereby identifying the first protein, the second protein, the compound, or any combination thereof.

[0050] In some embodiments, the method is (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment; (c) Detector fragments complementary to the first tag fragment and the second tag fragment; and (d) containing candidate compounds, The assay mixture induces proximity between a first protein or fragment and a second protein or fragment; the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance the assay signal; and The invention provides that the identified compound is capable of causing the degradation of a first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0051] In some embodiments, the assay mixture comprises several different first proteins or fragments thereof.

[0052] In some embodiments, the assay mixture brings together a complex such that the assay signal is generated or enhanced.

[0053] In some embodiments, the assay mixture induces proximity between protein molecules so that an assay signal is generated or enhanced.

[0054] 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.

[0055] In some embodiments, the assay mixture contains 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.

[0056] In some embodiments, the assay mixture contains 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.

[0057] In some embodiments, the assay mixture contains 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.

[0058] In some embodiments, the first protein is an isolated first protein.

[0059] In some embodiments, the assay mixture comprises several different second proteins or fragments thereof.

[0060] 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, or at least 1000 different second proteins or fragments thereof.

[0061] 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, or at least 700 different second proteins or fragments thereof.

[0062] In some embodiments, the assay mixture contains 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.

[0063] 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.

[0064] In some embodiments, the assay mixture contains several different candidate compounds.

[0065] 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, or at least 1000 different candidate compounds.

[0066] In some embodiments, the candidate compounds are small molecules (e.g., <5000Da, <4500Da, <4000Da, <3500Da, <3000Da, <2500Da, <2000Da, <1500Da, <1000Da, <900Da, <800Da, <700Da, <600Da, <500Da, or <400Da).

[0067] In some embodiments, none of the candidate compounds are water.

[0068] In some embodiments, none of the candidate compounds are buffer salts.

[0069] In some embodiments, none of the candidate compounds are proteins or their fragments. In some embodiments, none of the candidate compounds are proteins. In some embodiments, none of the candidate compounds are protein fragments.

[0070] In some embodiments, none of the candidate compounds are GFP proteins or fragments thereof. In some embodiments, none of the candidate compounds are GFP proteins. In some embodiments, none of the candidate compounds are GFP protein fragments.

[0071] In some embodiments, none of the candidate compounds contain GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, or GFP11, or any combination thereof.

[0072] In some embodiments, none of the candidate compounds are GFP1-9.

[0073] In some embodiments, the assay mixture contains about 50 to about 250 different candidate compounds.

[0074] In some embodiments, the assay mixture contains about 250 to about 2500 different candidate compounds.

[0075] In some embodiments, the assay mixture contains more than approximately 2500 different candidate compounds.

[0076] In some embodiments, the first tag fragment and the second tag fragment are configured to generate or enhance an assay signal when the assay mixture yields a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and a compound.

[0077] In some embodiments, the assay mixture yields a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound; the first tag fragment and the second tag fragment generate or enhance the assay signal.

[0078] In some embodiments, the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance an assay signal when the assay mixture yields a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and a compound.

[0079] In some embodiments, the assay mixture yields a complex comprising a first protein or fragment thereof, a second protein or fragment thereof, and a compound; the first tag fragment, the second tag fragment, and the detector fragment are arranged to generate or enhance the assay signal.

[0080] In some embodiments, the assay mixture yields a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound.

[0081] In some embodiments, the assay signal is light emission.

[0082] In some embodiments, the assay signal is fluorescence, fluorescence polarization, time-resolved fluorescence (TRF), homogeneous time-resolved fluorescence, emission, or UV / Vis.

[0083] In some embodiments, the assay signal is fluorescence, luminescence, phosphorescence, or scintillation.

[0084] In some embodiments, the assay signal is fluorescence.

[0085] In some embodiments, the assay signal is a radioactivity-based signal, such as a scintillation proximity assay (SPA) signal.

[0086] In some embodiments, the assay signal is a radiometric signal.

[0087] In some embodiments, the assay signal is a change in color.

[0088] In some embodiments, step (i) further includes detecting the generated or enhanced assay signal (e.g., fluorescence).

[0089] In some embodiments, the method of the disclosure is performed more than once. In some embodiments, the method of the disclosure is performed once, twice, three times, four times, or five times. In some embodiments, the method of the disclosure is performed multiple times. In some embodiments, the method of the disclosure is performed multiple times, wherein the method is performed using a new well each time.

[0090] Tags and detector fragments In some embodiments, the first tag fragment and the second tag fragment are configured to form a signal protein when the assay mixture induces proximity between the first protein or its fragment and the second protein or its fragment.

[0091] In some embodiments, the assay mixture induces proximity between a first protein or fragment thereof and a second protein or fragment thereof; causing the first tag fragment and the second tag fragment to generate or enhance the assay signal.

[0092] 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 proximity between the first protein or its fragment and the second protein or its fragment.

[0093] In some embodiments, the assay mixture induces proximity between a first protein or fragment thereof and a second protein or fragment thereof; the first tag fragment, the second tag fragment, and the detector fragment generate or enhance the assay signal.

[0094] In some embodiments, the signal protein is a fluorescent protein, such as a manipulated fluorescent protein.

[0095] In some embodiments, the signal 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 variant thereof.

[0096] In some embodiments, the signal protein is GFP.

[0097] In some embodiments, the signaling protein is, for example, a GFP variant (BFPms1) that preferentially binds to Zn(II) and Cu(II) over Ca(II).

[0098] In some embodiments, the signal protein is a fluorescent protein (which may be of different natural origins) having a beta-barrel structure composed of 11-stranded beta sheets.

[0099] In some embodiments, the signal protein is mCherry (which is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs)). In some embodiments, the signal protein is mRFP1, mStrawberry, mOrange, or dTomato.

[0100] In some embodiments, the first tag fragment is a first tag green fluorescent protein (GFP) fragment.

[0101] 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).

[0102] In some embodiments, the detector fragment is a detector GFP fragment (e.g., a second tagged GFP fragment and a detector GFP fragment different from the first tagged GFP fragment).

[0103] In some embodiments, the first tagged GFP fragment and the second tagged GFP fragment are configured to form GFP when the assay mixture induces proximity between the first protein or fragment and the second protein or fragment (e.g., induced by a candidate compound).

[0104] In some embodiments, the assay mixture induces proximity between a first protein or fragment thereof and a second protein or fragment thereof; so that the first tagged GFP fragment and the second tagged GFP fragment are configured to form GFP.

[0105] In some embodiments, the first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are configured to form GFP when the assay mixture induces proximity between the first protein or fragment and the second protein or fragment (e.g., induced by a candidate compound).

[0106] In some embodiments, the assay mixture induces proximity between a first protein or fragment thereof and a second protein or fragment thereof; so that the first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment are configured to form GFP.

[0107] 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 Annu RevBiophys 6(48):19-44 (2019) (incorporated herein by reference).

[0108] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP1 and GFP2.

[0109] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP2 and GFP3.

[0110] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP5 and GFP6.

[0111] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP7 and GFP8.

[0112] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP8 and GFP9.

[0113] In some embodiments, the first tag GFP fragment and the second tag GFP fragment are independently selected from GFP9 and GFP10.

[0114] In some embodiments, the first tag GFP fragment is GFP10, and the second tag GFP fragment is GFP11.

[0115] In some embodiments, the first tag GFP fragment is GFP11, and the second tag GFP fragment is GFP10.

[0116] In some morphologies, the detector GFP fragments include GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, and GFP9.

[0117] In some embodiments, the detector GFP fragment further includes a chromophore.

[0118] In some embodiments, the first tagged GFP fragment, the second tagged GFP fragment, and the detector fragment are selected from the combinations listed in Table 1 below.

[0119] [Table 1]

[0120] Targeted protein In some embodiments, the first protein is the protein of interest (POI).

[0121] In some embodiments, the fragment of the first protein includes a binding site for the second protein.

[0122] In some embodiments, the fragment of the first protein includes at least a portion of the binding site of the first protein to the identified compound.

[0123] In some embodiments, the fragment of the first protein includes a binding site for the first protein to the identified compound.

[0124] In some embodiments, the first protein fragment includes a binding site for the target protein.

[0125] In some embodiments, the POI is a kinase, phosphatase, glycosylase, deglycosylase, methylase, demethylase, smoylase, dubiquitinase, acetyltransferase, deacetylase, fatty acyltransferase, protease, isomerase, or arginase.

[0126] In some embodiments, the POI is a cytoskeletal protein or a protein with scaffolding function that holds other proteins together in a complex.

[0127] 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 related enzyme (e.g., adenylyl cyclase), RNA polymerase, RNA splicing enzyme, transporter, DNA helicase, or DNA endonuclease.

[0128] In some embodiments, POI is a hydroxylase, dehydrogenase, reductase, oxidase, oxygenase, oxidoreductase, carboxylase, decarboxylase, lyase, aldolase, desaturase, mutase, epimerase, isomerase, racemase, esterase, amidase, deaminase, aminotransferase, hydratase, superoxide dismutase, ligase, carbonic anhydrase, nucleotide transferase, glycosyltransferase, or glycosidase.

[0129] In some embodiments, the POI is a ubiquitin ligase (i.e., an E3 ligase).

[0130] In some embodiments, the POI is a protein associated with a disease or disorder. In some embodiments, the protein has no known function in the mechanism of the disease or disorder.

[0131] In some embodiments, the first protein (e.g., POI) is associated with a disease or disorder.

[0132] In some embodiments, the presence or activity of a first protein (e.g., POI) is associated with a disease or disorder.

[0133] In some embodiments, a specific post-translational modification of the first protein is associated with a disease or disorder.

[0134] In some embodiments, the degradation of a first protein (e.g., POI) results in the treatment or prevention of a disease or disorder.

[0135] In some embodiments, the second protein can induce the degradation of the first protein under conditions of proximity between the first and second proteins (e.g., in cellular conditions, or when other components, such as the proteasome mechanism, are provided in a biochemical system).

[0136] In some embodiments, the second protein induces the degradation of the first protein under conditions of proximity between the first and second proteins (e.g., in a cellular setting, or when other components, such as the proteasome mechanism, are provided in a biochemical system).

[0137] In some embodiments, the second protein is a ubiquitin ligase (i.e., E3 ligase).

[0138] In some embodiments, the fragment of the second protein includes a binding site for the first protein.

[0139] 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.

[0140] In some embodiments, the fragment of the second protein includes a binding site for the second protein to the identified compound.

[0141] In some embodiments, the second protein fragment contains a ubiquitin ligase binding site.

[0142] In some embodiments, the second protein fragment contains a binding site for E3 ligase.

[0143] 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, cereblon (CRBN), CRBN / DDB1, DCAF15, DCAF15 / DDB1, VHL, and VHL / EloB / EloC.

[0144] In some embodiments, the binding site of the E3 ligase includes an adapter protein component. In some embodiments, the adapter protein component includes, but is not limited to, DDB1.

[0145] In some embodiments, the second protein is the protein of interest (POI).

[0146] In some embodiments, the second protein is a kinase, phosphatase, glycosylase, deglycosylase, methylase, demethylase, smoylase, dubiquitinase, acetyltransferase, deacetylase, lipid acyltransferase, protease, isomerase, or arginase.

[0147] In some embodiments, the second protein is a protease, a proteasome, a component of a proteasome complex, an autophage receptor, or a component of an autophage receptor complex.

[0148] In some embodiments, the second protein is a scaffolding protein that holds other proteins together in a complex.

[0149] In some embodiments, the second protein is a metabolic enzyme, a transcription factor, a cell surface receptor (e.g., GPCR or receptor tyrosine kinase), an ion channel, a membrane-embedded or related enzyme (e.g., adenylyl cyclase), an RNA splicing enzyme, a transporter, a DNA helicase, or a DNA endonuclease.

[0150] In some embodiments, the second protein is a hydroxylase, dehydrogenase, reductase, oxidase, oxygenase, oxidoreductase, carboxylase, decarboxylase, lyase, aldolase, desaturase, mutase, epimerase, isomerase, racemase, esterase, amidase, deaminase, aminotransferase, hydratase, superoxide dismutase, ligase, carbonic anhydrous enzyme, nucleotide transferase, glycosyltransferase, or glycosidase.

[0151] In some embodiments, the second protein is a heat shock protein.

[0152] In some embodiments, the second protein is associated with a disease or disorder.

[0153] In some embodiments, the presence or activity of a second protein is associated with a disease or disorder.

[0154] In some embodiments, the post-translational modification of the second protein is associated with a disease or disorder.

[0155] In some embodiments, the degradation of a second protein results in the treatment or prevention of a disease or disorder.

[0156] In some embodiments, the first protein is POI, and the second protein is ubiquitin ligase.

[0157] In some embodiments, the first protein is a ubiquitin ligase, and the second protein is a POI.

[0158] In some embodiments, both the first and second proteins are the same protein (e.g., POI or ubiquitin ligase). For example, the first and second proteins may homodimerize in the presence of the compound of interest. In another example, the first and second proteins are kinases that may homodimerize or heterodimerize in the presence of the compound of interest, and they may be subjected to cross-phosphorylation and activation. In yet another example, the first and second proteins are E3 ligases that may homodimerize or heterodimerize in the presence of the compound of interest, and they may be subjected to cross-ubiquitination and degradation.

[0159] Characterization of the formed complex In some embodiments, the assay mixture yields a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a compound.

[0160] In some embodiments, the first protein or a fragment thereof, or the second protein or a fragment thereof, is covalently attached to the affinity component.

[0161] In some embodiments, a first protein or fragment thereof, or a second protein or fragment thereof, together with affinity components, form a fusion protein (e.g., glutathione-S-transferase (GST) or maltose-binding protein (MBP)).

[0162] In some embodiments, the method is (ii-a) The method comprises contacting the complex with an immobilized affinity binder that targets an affinity component (or fusion protein), thereby forming an immobilized complex.

[0163] In some embodiments, step (ii-a) includes contacting the assay mixture with a plate coated with an affinity binder.

[0164] In some embodiments, step (ii-a) includes contacting the assay mixture with beads coated with an affinity binder.

[0165] In some embodiments, the affinity components and affinity binders are selected from the combinations listed in Table 2 below, but are not limited.

[0166] [Table 2]

[0167] In some embodiments, the affinity component is biotin, desthiobiotin, or a derivative thereof.

[0168] In some embodiments, the affinity binder is streptavidin, avidin, neutraavidin, or a derivative thereof.

[0169] In some embodiments, the affinity component is biotin or a derivative thereof, and the affinity binder is streptavidin or a derivative thereof.

[0170] In some embodiments, step (ii-a) further includes incubating the mixture with the immobilized affinity binder for a time ranging from about 1 minute to about 90 minutes (e.g., about 30 minutes to about 60 minutes).

[0171] In some embodiments, step (i) further comprises the assay mixture comprising a GFP booster, and step (ii-a) comprising incubating the mixture with an immobilized affinity binder for a time ranging from 1 to 30 minutes.

[0172] In some embodiments, the method is (ii-b) Further comprising detecting and / or isolating the immobilized complex and thereby identifying the compound.

[0173] In some embodiments, step (ii-b) includes detecting the immobilized complex and thereby identifying the compound.

[0174] In some embodiments, step (ii-b) includes isolating the immobilized complex and thereby identifying the compound.

[0175] In some embodiments, step (ii-b) includes detecting and isolating the immobilized complex, thereby identifying the compound.

[0176] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) one or more times, thereby identifying the compounds, the first protein or fragment thereof, and / or the second protein or fragment thereof in the complex.

[0177] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) one or more times to identify the compounds in the complex.

[0178] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) one or more times to identify the first protein or fragment thereof in the complex.

[0179] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) one or more times to identify a second protein or fragment thereof in the complex.

[0180] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) one or more times, thereby identifying the compounds in the complex, the first protein or a fragment thereof, and the second protein or a fragment thereof.

[0181] 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 in step (i)).

[0182] In some embodiments, step (ii) or step (ii-b) includes repeating step (i) using a first protein or a fragment thereof, a second protein, and / or a candidate compound.

[0183] In some embodiments, individual first proteins, second proteins, and candidate compounds in an assay mixture with generated or enhanced assay signals are tested in a manner that uniquely and distinctly identifies specific combinations of first proteins, second proteins, and candidate compounds that form complexes in the assay mixture. For example, individual combinations of first and second proteins may be tested in each sample using the same pool of multiple candidate compounds to identify combinations of first and second proteins. Then, individual candidate compounds may be tested using the identified specific combinations of first and second proteins. Alternatively, individual candidate compounds may be tested in each sample using the same multiple first proteins and multiple second proteins to identify candidate compounds. And specific candidate compounds can be tested in each sample using unique combinations of first and second proteins. Other methods may also be used, as long as they can distinctly identify unique combinations of first proteins, second proteins, and candidate compounds.

[0184] In some embodiments, step (ii-b) includes characterizing the composite using mass spectrometry (MS).

[0185] In some embodiments, step (ii-b) includes reducing, denaturing, alkylating, and / or digesting the immobilized complex to form a mixture of peptides, and characterizing the mixture of peptides using MS to identify a first protein or fragment thereof and / or a second protein or fragment thereof in the complex.

[0186] In some embodiments, the peptide mixture further comprises candidate compounds, and candidate compounds in the complex are further identified by MS characterization.

[0187] In some embodiments, step (ii-b) includes isolating the complex and characterizing the complex using MS.

[0188] In some embodiments, step (ii-b) includes isolating the complex, dissociating a first protein or fragment thereof and / or a second protein or fragment thereof from the complex, and characterizing the dissociated proteins using MS to identify the first protein or fragment thereof and / or the second protein or fragment thereof in the complex.

[0189] In some embodiments, step (ii-b) includes isolating the complex, dissociating a first protein or a fragment thereof and / or a second protein or a fragment thereof from the complex, and characterizing the dissociated proteins using MS to identify the first protein or a fragment thereof in the complex.

[0190] In some embodiments, step (ii-b) includes isolating the complex, dissociating a first protein or fragment thereof and / or a second protein or fragment thereof from the complex, and characterizing the dissociated proteins using MS to identify the second protein or fragment thereof in the complex.

[0191] In some embodiments, step (ii-b) includes isolating the complex, dissociating a first protein or fragment thereof and / or a second protein or fragment thereof from the complex, and characterizing the dissociated proteins using MS to identify the first protein or fragment thereof and the second protein or fragment thereof in the complex.

[0192] In some embodiments, step (ii-b) further comprises dissociating the candidate compounds from the complex, and further identifying the candidate compounds in the complex by MS characterization.

[0193] In some embodiments, MS characterization is based on a “bottom-up mass spectrometry-based proteomics” method. In such a method, proteins captured in incubation may be reduced and alkylated under denaturing conditions, followed by digestion into smaller peptides using a sequence-specific protease before analysis of the sample by mass spectrometer. Various options may be available for this procedure. For example, high concentrations of urea or other chaotropic agents may be used for protein denaturation. Dithiothreitol, beta-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine) may be used to reduce disulfide bonds in the protein. Iodoacetamide, iodoacetic acid, or iodoethanol may be used as alkylating agents to covalently modify free sulfhydryl groups on the protein after the disulfide bonds have been reduced. Trypsin may be used as a sequence-specific protease to digest proteins into peptides for the purpose of mass spectrometry-based protein analysis. 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 by standard proteomics methods. Such methods may be based on comparing the acquired m / z (mass-to-charge ratio) values ​​of the peptide and their MS2 fragmentation spectra with theoretical data obtained from in silico digestion of protein sequences in protein databases using a computer program (also known as a "search engine") for suitable species. For mass spectrometry data acquisition, in-line separation of peptides on liquid chromatography (LC) may be combined with different data acquisition modes by a mass spectrometer. Data-dependent MS2 acquisition (DDA) and / or data-independent acquisition (DIA) may be suitable for such purposes. Any other data acquisition methods may also be used, insofar as a clear identification of the unique peptide sequence can be established. Direct data acquisition of peptides may be performed without LC separation, particularly when the sample composition is relatively simple.Matrix-assisted laser desorption / ionization (MALDI) time-of-flight instruments may be suitable for such purposes. The identity of proteins in a sample can be estimated by comparing the amino acid sequence of the peptide with the amino acid sequence of the intact protein in a protein database. During the digestion of the protein in the complex, candidate compounds trapped in the complex may also be released into the digestion solution. The identity of candidate compounds can also be obtained from the same sample and / or the same experiment by comparing the molecular weight of the compounds in the assay mixture with the expected molecular weight of the candidate compounds estimated from the m / z values ​​of the MS1 spectrum. If necessary, the MS2 fragmentation spectrum of the compound may also be used to aid in compound identification.

[0194] In some embodiments, MS characterization is based on a “top-down” method (e.g., without protein digestion, if the proteins in the complex can be released into solution). In this method, the intact protein mass obtained by the mass spectrometer may be compared to the expected mass of the individual proteins used in this experiment. To release the intact protein from the complex, a short peptide sequence may be introduced between the first or second protein and the first or second tag fragment (e.g., GFP10 or GFP11 tag fragment), which may be cleaved by a highly sequence-specific protease. For example, the short peptide sequence may be a TEV cleavage sequence, ENLYFQS(G,A). The TEV protease may be able to cleave between Q and S in a highly sequence-specific manner. Thus, the entire protein can be released from the complex bound to the plate, for example, by incubating the plate with a solution containing the TEV protease. Once the first or second protein has been cleaved from the tag fragment, the two proteins can be dissociated from the ternary complex by acidification of the sample. Measuring the intact mass of dissociated proteins can lead to the identification of specific proteins within the ternary complex.

[0195] In some embodiments, the method is (ii-c) Further comprising subjecting the identified compound to a validation assay.

[0196] In some embodiments, the validation assay is configured to test the ability of a compound to induce proximity between a first protein or fragment thereof and a second protein or fragment thereof.

[0197] 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).

[0198] In some embodiments, in step (ii-c), the identified compound induces proximity between the first protein or fragment and the second protein or fragment in the validation assay.

[0199] In some embodiments, in step (ii-c), the identified compound leads to the degradation of the first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase) in a validation assay.

[0200] Identified first protein, second protein, compound, and combination In some embodiments, the Disclosure provides a first protein identified by the method of the Disclosure.

[0201] In some embodiments, the Disclosure provides a second protein identified by the method of the Disclosure.

[0202] In some embodiments, the Disclosure provides a combination of a first protein and a second protein identified by the method of the Disclosure.

[0203] In some embodiments, the Disclosure provides a first protein, a second protein, and combinations of compounds that target the first and second proteins, as identified by the method of the Disclosure.

[0204] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure.

[0205] In some embodiments, the identified compounds are small molecules (e.g., <5000Da, <4500Da, <4000Da, <3500Da, <3000Da, <2500Da, <2000Da, <1500Da, <1000Da, <900Da, <800Da, <700Da, <600Da, <500Da, or <400Da).

[0206] In some embodiments, the identified compound is not water.

[0207] In some embodiments, the identified compound is not a buffer salt.

[0208] In some embodiments, the identified compound is not a protein or a fragment thereof. In some embodiments, the identified compound is not a protein. In some embodiments, the identified compound is not a protein fragment.

[0209] In some embodiments, the identified compound is not a GFP protein or a fragment thereof. In some embodiments, the identified compound is not a GFP protein. In some embodiments, the identified compound is not a GFP protein fragment.

[0210] In some embodiments, the identified compounds do not contain GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, or GFP11, or any combination thereof.

[0211] In some embodiments, the identified compounds are not GFP1-9.

[0212] In some embodiments, the identified compound is a binder compound.

[0213] In some embodiments, the identified compounds (e.g., binder compounds) can modulate protein-protein interactions (PPIs) between a first protein or fragment and a second protein or fragment.

[0214] In some embodiments, the identified compounds (e.g., binder compounds) modulate (increase or decrease) protein-protein interactions (PPIs) between a first protein and a second protein.

[0215] In some embodiments, the identified compounds (e.g., binding compounds) increase the protein-protein interaction (PPI) between a first protein and a second protein, or stabilize the PPI between the two proteins.

[0216] In some embodiments, the identified compounds (e.g., binding compounds) reduce the protein-protein interaction (PPI) between the first and second proteins, or destabilize the PPI between the two proteins.

[0217] In some embodiments, the PPI results in changes in protein stability (e.g., changes in the stability of the first protein and / or changes in the stability of the second protein).

[0218] In some embodiments, the PPI results in changes at the protein level (e.g., changes at the level of the first protein and / or changes at the level of the second protein).

[0219] In some embodiments, the PPI results in changes in the post-translational modifications of proteins (e.g., changes in the post-translational modifications of the first protein and / or the second protein).

[0220] In some embodiments, the PPI results in changes in protein localization (e.g., changes in the localization of the first protein and / or changes in the localization of the second protein).

[0221] In some embodiments, the PPI causes a change in protein activity (e.g., a change in the activity of the first protein and / or a change in the activity of the second protein).

[0222] In some embodiments, the PPI results in the degradation of the first or second protein.

[0223] In some embodiments, PPIs result in increased stability of intracellular proteins or protection against protein degradation.

[0224] In some embodiments, the PPI results in post-translational modification of the first or second protein.

[0225] In some embodiments, the PPI results in the removal of existing post-translational modifications of the first or second protein.

[0226] In some embodiments, PPIs result in alterations in the intracellular localization of the first or second protein.

[0227] In some embodiments, the PPI here results in the modulation of the activity of the first or second protein.

[0228] In some embodiments, the identified compound (e.g., a binder compound) can cause the degradation of a first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0229] In some embodiments, the identified compound (e.g., a binder compound) causes degradation of the first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0230] In some embodiments, the identified compound (e.g., a binder compound) can cause post-translational modification of the first protein (e.g., POI) in the presence of a second protein (e.g., an E3 ligase).

[0231] In some embodiments, the identified compound (e.g., a binder compound) causes post-translational modification of the first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0232] In some embodiments, the identified compounds (e.g., binder compounds) can regulate the activity of a first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0233] In some embodiments, the identified compound (e.g., a binder compound) modulates the activity of a first protein (e.g., POI) in the presence of a second protein (e.g., E3 ligase).

[0234] In some embodiments, the identified compounds (e.g., binder compounds) can cause changes in protein stability (e.g., of POI), changes at the protein level (e.g., of POI), changes in post-translational modification (e.g., of POI), changes in protein localization (e.g., of POI), and / or changes in protein activity (e.g., of POI).

[0235] In some embodiments, the identified compounds (e.g., binder compounds) can alter the protein stability (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) can alter the protein level (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) can alter the post-translational modification (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) can alter the protein localization (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) can alter the protein activity (e.g., of POI).

[0236] In some embodiments, the identified compound (e.g., a binder compound) causes changes in protein stability (e.g., of a POI), changes at the protein level (e.g., of a POI), changes in post-translational modification (e.g., of a POI), changes in protein localization (e.g., of a POI), and / or changes in protein activity (e.g., of a POI).

[0237] In some embodiments, the identified compounds (e.g., binder compounds) cause changes in protein stability (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) cause changes at the protein level (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) cause changes in post-translational modification (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) cause changes in protein localization (e.g., of POI). In some embodiments, the identified compounds (e.g., binder compounds) cause changes in protein activity (e.g., of POI).

[0238] Method for synthesizing identified compounds In some embodiments, the Disclosure provides a method for preparing the compounds identified in the methods of the Disclosure.

[0239] The compounds of this disclosure can be prepared by any suitable technique known in the art. Those skilled in organic synthesis will understand that the functionalities present on various parts of the molecule must be compatible with the compound and reaction conditions used. The resulting compounds can be isolated and purified using techniques well known in the art.

[0240] Conveniently, the reaction of the compounds takes place in the presence of a suitable solvent, which is preferably inert under the respective reaction conditions. Examples of suitable solvents are, 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, cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE), or dioxane; and glyco The solvents include ethers such as ethylene glycol monomethyl ether or monoethyl ether, or ethylene glycol dimethyl ether (diglym); ketones such as acetone, methyl isobutyl ketone (MIBK), or butanone; amides such as acetamide, dimethylacetamide, dimethylformamide (DMF), or N-methylpyrrolidinone (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 such solvents, or mixtures with water.

[0241] As will be understood by those skilled in the art of organic synthesis, the compounds are readily accessible through various synthetic routes, some of which are illustrated in the accompanying examples. Those skilled in the art will readily recognize which types of compounds and reaction conditions to use to obtain the compounds of this disclosure, and how they apply and adapt in any particular case—wherever necessary or useful. Furthermore, some of the compounds can be readily synthesized by reacting other compounds of this disclosure under appropriate conditions, by applying standard synthetic methods such as reduction, oxidation, addition, or substitution reactions, to convert one particular functional group present in the compounds of this disclosure, or a suitable precursor molecule thereof, into another; these methods are well known to those skilled in the art. Similarly, those skilled in the art will apply synthetic protecting (or protecting) groups whenever necessary or useful; suitable protecting groups and methods for introducing and removing them are well known to those skilled in the art of chemical synthesis, for example, PGMWuts, T.W. Greene, “Greene's Protect ti This is described in more detail in "Organic Synthesis Groups," 4th edition (2006) (John Wiley & Sons).

[0242] Validation assay for identified compounds Compounds identified by the methods described above, once produced, can be validated using various assays known to those skilled in the art to determine whether the compounds possess biological activity (e.g., inducing proximity between a first protein or fragment and a second protein or fragment). For example, compounds can be characterized by conventional assays, including but not limited to those described below, to determine whether they possess predictive activity, binding activity, and / or binding specificity.

[0243] Furthermore, high-throughput screening can be used to accelerate analysis using such assays. As a result, it is possible to rapidly screen for the activity of the molecules described herein using techniques known in the art. General methodologies for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker; and U.S. Patent No. 5,763,263. High-throughput assays may be one or more different assay techniques, including, but are not limited to, those described below.

[0244] Various in vitro or in vivo biological assays may be suitable for detecting the effects of a compound. These in vitro or in vivo biological assays may include, but are not limited to, enzyme activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell survival assays, and assays described herein.

[0245] In some embodiments, the validation assay is a biological assay that validates the formation of a ternary complex. In some embodiments, the validation assay measures the proximity between the first protein and the second protein or a fragment thereof in a compound-dependent manner. In some embodiments, the validation assay may include, but is not limited to, AlphaALISA, TR-FRET, fluorescence polarization assays, surface plasmon resonance (SPR), and mass photometry. In some embodiments, the validation assay includes a pull-down of the ternary complex and its quantification by mass spectrometry.

[0246] In some embodiments, the validation assay is an intracellular biological assay. In some embodiments, the intracellular biological assay comprises a nanoBRET system. In some embodiments, the nanoBRET system measures the formation of ternary complexes inside cells. In some embodiments, the nanoBRET system further comprises detecting the generation or enhancement of a luminescence signal upon formation of a ternary complex comprising the interaction of a first protein and a second protein, or fragments thereof, in the presence of a compound. In some embodiments, the intracellular biological assay comprises a fluorescence resonance energy transfer (FRET) system. In some embodiments, the FRET system measures the formation of ternary complexes inside cells. In some embodiments, the FRET system further comprises detecting the generation or enhancement of a signal upon formation of a ternary complex comprising the interaction of a first protein and a second protein, or fragments thereof, in the presence of a compound, wherein the first protein and the second protein, or fragments thereof, are fused to a fluorescence donor and / or a fluorescence acceptor.

[0247] In some embodiments, the validation assay is a cell-based degradation assay. In some embodiments, the cell-based degradation assay can include, but is not limited to, Western blot, intracellular Western assay, HiBit assay, fluorescence reporter assay, and mass spectrometry proteomics.

[0248] Without wishing to be bound by theory, compound-dependent ubiquitination, post-translational modification, changes in enzyme activity, or cellular degradation of the POI in a cell-based degradation validation assay do not directly measure the formation of a ternary complex comprising a first protein and a second protein, or fragments thereof, in the presence of a compound, but require the formation of a ternary complex and can thus be used to detect ternary complex formation.

[0249] Pharmaceutical composition In some embodiments, the Disclosure provides a pharmaceutical composition comprising a compound identified by the method of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0250] As used herein, the term “composition” is intended to encompass products containing specified components in specified amounts, as well as any products directly or indirectly resulting from combinations of specified components in specified amounts. The compounds of this disclosure can be formulated for oral administration in forms such as tablets, capsules (each of which may be a sustained-release or time-release formulation), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds of this disclosure 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 pharmaceutical technology.

[0251] Any suitable solubility-enhancing compound can be used. Examples of solubility-enhancing compounds include cyclodextrins, such as hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulfated-β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, branched-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin, as well as mixtures thereof, selected from the group.

[0252] 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 salts, disodium edetate, trisodium edetate, and tetrasodium edetate, and mixtures thereof.

[0253] 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, benzethonium chloride, cetylpyridinium chloride, benzyl bromide, phenylmercury nitrate, phenylmercury acetate, phenylmercury neodecanoate, melthiolate, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl-p-hydroxybenzoic acid, propylaminopropyl biguanide, and butyl-p-hydroxybenzoic acid, as well as mixtures thereof.

[0254] The aqueous solvent may also contain a tonic compound to adjust the tonicity (osmotic pressure). The tonic compound can be selected from the group consisting of glycols (e.g., propylene glycol, diethylene glycol, triethylene glycol, etc.), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, as well as mixtures thereof. The aqueous solvent may also contain a viscous / suspension compound. Suitable viscous / suspension compounds include those selected from the group consisting of cellulose derivatives, e.g., methylcellulose, ethylcellulose, hydroxyethylcellulose, polyethylene glycol (e.g., polyethylene glycol 300, polyethylene glycol 400, etc.), carboxymethylcellulose, hydroxypropylmethylcellulose, and crosslinked acrylic acid polymers (carbomers), e.g., polymers of acrylic acid crosslinked with polyalkenyl ethers or divinyl glycol (Carbopol—e.g., Carbopol 934, Carbopol 934P, Carbopol 971, Carbopol 974, and Carbopol 974P, etc.), as well as mixtures thereof. The aqueous solvent may also contain a buffer compound to stabilize the pH. When used, buffers are selected from the group consisting of phosphate buffers (e.g., sodium dihydrogen phosphate and disodium hydrogen phosphate), borate buffers (e.g., boric acid or salts thereof including disodium tetraborate), citrate buffers (e.g., citric acid or salts thereof including sodium citrate), and ε-aminocaproic acid, as well as mixtures thereof.

[0255] The composition may further contain wetting compounds. Suitable classes of wetting compounds include those selected from the group consisting of polyoxypropylene-polyoxyethylene block copolymers (poloxamers), polyethoxylated ethers of castor oil, polyoxyethylene-sorbitan esters (polysorbates), polymers of oxyethylated octylphenol (tyroxapole), polyoxyl 40-stearic acid, fatty acid glycol esters, fatty acid glyceryl esters, sucrose fatty acid esters, and polyoxyethylene fatty acid esters, as well as mixtures thereof.

[0256] The compositions of this disclosure may be obtained by conventional procedures using conventional pharmaceutical excipients that are well known in the art. Thus, compositions intended for oral use may include, for example, one or more coloring, sweetening, flavoring, and / or preservative compounds.

[0257] Methods for using identified compounds In some embodiments, the Disclosure provides a method for degrading a first protein in a subject, and includes administering a compound identified by the method of the Disclosure to the subject.

[0258] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in degrading a first protein in a subject.

[0259] In some embodiments, the Disclosure provides the use of compounds identified herein in the manufacture of pharmaceuticals for the degradation of a first protein in a subject.

[0260] In certain embodiments, the Disclosure provides a method for treating and / or preventing a disease or disorder associated with a first protein in a subject, which includes administering to the subject a therapeutically effective dose of a compound identified by the method of the Disclosure.

[0261] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in treating and / or preventing diseases or disorders associated with a first protein in a subject.

[0262] In some embodiments, the Disclosure provides the use of compounds identified by the methods of the Disclosure in the manufacture of a pharmaceutical product for the treatment and / or prevention of a disease or disorder associated with a first protein in a subject.

[0263] In some embodiments, this disclosure provides methods for treating and / or preventing diseases or disorders associated with human therapy, agriculture, and / or animal health. In some embodiments, the methods for treating and / or preventing diseases or disorders are associated with human therapy. In some embodiments, the methods for treating and / or preventing diseases or disorders are for humans. In some embodiments, the methods for treating and / or preventing diseases or disorders are associated with agriculture. In some embodiments, the methods for treating and / or preventing diseases or disorders are for agricultural entities (e.g., seeds, saplings, leaves, flowers, plants, etc.). In some embodiments, the methods for treating and / or preventing diseases or disorders are associated with animal health. In some embodiments, the methods for treating and / or preventing diseases or disorders are for animals.

[0264] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in human therapeutics, agriculture, and / or animal health. In some embodiments, the compounds are for use in human therapeutics. In some embodiments, the compounds are for use in humans. In some embodiments, the compounds are for use in agriculture. In some embodiments, the compounds are for use in agricultural entities (e.g., seeds, saplings, leaves, flowers, plants, etc.). In some embodiments, the compounds are for use in animal health. In some embodiments, the compounds are for use in animals.

[0265] In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for human therapy, agriculture, and / or animal health. In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for human therapy. In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for humans. In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for agriculture. In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for agricultural entities (e.g., seeds, saplings, leaves, flowers, plants, etc.). In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for animal health. In some embodiments, the disclosure provides for the use of a compound identified by the methods of the disclosure in the manufacture of a medicament for animals.

[0266] In some embodiments, the disease or disorder is associated with a first protein (e.g., POI).

[0267] In some embodiments, the disease or disorder is mediated by a first protein (e.g., POI).

[0268] In some embodiments, the disclosure provides a method of degrading a second protein in a subject, comprising administering to the subject a compound identified by the methods of the disclosure.

[0269] In some embodiments, the disclosure provides a compound identified by the methods of the disclosure for use in degrading a second protein in a subject.

[0270] In some embodiments, the disclosure provides for the use of a compound identified by the disclosure in the manufacture of a medicament for degrading a second protein in a subject.

[0271] In certain embodiments, the Disclosure provides a method for treating and / or preventing a disease or disorder associated with a second protein in a subject, which includes administering to the subject a therapeutically effective dose of a compound identified by the method of the Disclosure.

[0272] In some embodiments, the Disclosure provides compounds identified by the methods of the Disclosure for use in treating and / or preventing diseases or disorders associated with a second protein in a subject.

[0273] In some embodiments, the Disclosure provides the use of compounds identified by the methods of the Disclosure in the manufacture of a pharmaceutical product for treating and / or preventing a disease or disorder associated with a second protein in a subject.

[0274] In some embodiments, the disease or disorder is associated with a second protein (e.g., E3 ligase).

[0275] In some embodiments, the disease or disorder is mediated by a second protein.

[0276] In some embodiments, the subject is a cell.

[0277] In some embodiments, the subject is an animal.

[0278] In some embodiments, the subject is agricultural entities (e.g., seeds, saplings, leaves, flowers, plants, etc.).

[0279] In some embodiments, the subject is a mammal.

[0280] In some embodiments, the subject is a human.

[0281] In some embodiments, the disease or disorder is cancer.

[0282] In some embodiments, cancers include prostate cancer (small cell carcinoma, neuroendocrine tumor, transitional cell carcinoma, sarcoma), breast cancer (intraductal carcinoma, 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, cholangiocarcinoma), endometrial cancer, salivary gland cancer, leukemia, NUT midline carcinoma, multiple myeloma, lung cancer (small cell lung cancer, non-small cell lung cancer), neuroblastoma, and cervical cancer (squamous cell carcinoma). The following are selected from cancers: adenocarcinoma, esophageal cancer, colorectal cancer, brain cancer (glioma), glioblastoma, Banayan-Zonana syndrome, Cowden disease, Lhermit-Dacross 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.

[0283] In some embodiments, the disease or disorder is a benign proliferative disorder.

[0284] In some embodiments, benign proliferative disorders are selected from benign soft tissue tumors, bone tumors, brain and spinal cord tumors, eyelid and orbital tumors, granulomas, lipomas, meningiomas, multiple endocrine neoplasias, nasal polyps, pituitary tumors, prolactinomas, pseudobrain tumors, seborrheic keratosis, gastric polyps, thyroid nodules, pancreatic cystic tumors, hemangiomas, vocal cord nodules / polyps, and cysts, Castleman disease, chronic pyodermapiloidal cystosis, dermatofibromas, follicular cysts, pyogenic granulomas, and juvenile polyposis syndrome.

[0285] In some embodiments, the disease or disorder is an immunological disease or disorder.

[0286] In some embodiments, the immunological disease or disorder includes T-cell-mediated inflammatory diseases and B-cell-mediated inflammatory diseases. In some embodiments, the immunological disease or disorder is 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's disease, celiac disease-sprue (glumine-sensitive enteropathy), dermatomyositis, Graves' disease, Hashimoto's disease, multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes mellitus, inflammatory bowel disease, chronic inflammatory demyelinating polyneuropathy, and ankylosing spondylitis.

[0287] In some embodiments, the disease or disorder is a neurological disease or disorder.

[0288] In some embodiments, neurological disorders or conditions are selected from a list that includes, but is not limited to, acute spinal cord injury, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia, Bell's palsy, brain tumors, cerebral aneurysms, dementia, epilepsy and seizures, Guillain-Barré syndrome, Huntington's disease, headaches, head trauma, hydrocephalus, lumbar disc disease (herniated disc), meningitis, multiple sclerosis, muscular dystrophy, neurocutaneous syndromes, Parkinson's disease, stroke (cerebral seizure), cluster headaches, tension headaches, migraines, encephalitis, sepsis, myasthenia gravis, muscular dystrophy, and neuromuscular diseases.

[0289] Exemplary Embodiments Exemplary embodiment A1. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and the compound; and (ii) A method comprising identifying a first protein, a second protein, a compound, or any combination thereof associated with a complex.

[0290] Exemplary embodiment A2. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The first tag fragment and the second tag fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and a compound; (ii-a) Contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) A method comprising detecting and / or isolating an immobilized complex and thereby identifying a first protein, a second protein, a compound, or any combination thereof.

[0291] Exemplary embodiment A3. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided such that the assay mixture generates or enhances an assay signal when proximity is induced between the first protein or its fragment and the second protein or its fragment; and (ii) Identifying a first protein, a second protein, a compound, or any combination thereof associated with the induction of proximity A method by which an identified compound can cause the degradation of a first protein (e.g., POI) in the presence of a second protein.

[0292] Exemplary embodiment A4. One of the exemplary embodiments A1 to A3, wherein the assay mixture induces proximity such that an assay signal is generated or enhanced.

[0293] Exemplary embodiment A5. A method of any one of the exemplary embodiments A1 to A4, wherein the assay mixture further comprises a detector fragment complementary to a first tag fragment and a second tag fragment.

[0294] Exemplary embodiment A6. One of the exemplary embodiments A1 to A5, wherein the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance an assay signal when the assay mixture induces proximity between the first protein or its fragment and the second protein or its fragment.

[0295] Exemplary embodiment A7. One of the exemplary embodiments A1 to A6, wherein the first tag fragment, the second tag fragment, and the detector fragment are configured to generate or enhance an assay signal when the assay mixture yields a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and a compound.

[0296] Exemplary embodiment A8. A method of any one of the exemplary embodiments A1 to A7, wherein the assay mixture comprises several different first proteins or fragments thereof.

[0297] Exemplary embodiment A9. A method of any one of the exemplary embodiments A1 to A8, wherein the assay mixture comprises several different second proteins or fragments thereof.

[0298] Exemplary embodiment A10. A method from any one of the exemplary embodiments A1 to A9, wherein the assay mixture contains several different candidate compounds.

[0299] Exemplary embodiment A11. One of the exemplary embodiments A1 to A10, wherein the assay mixture contains about 50 to about 250 different candidate compounds.

[0300] Exemplary embodiment A12. A method in any one of the exemplary embodiments A1 to A11, wherein the assay mixture contains approximately 250 to approximately 2500 different candidate compounds.

[0301] Exemplary embodiment A13. One of the exemplary embodiments A1 to A12, wherein the assay mixture contains more than approximately 2500 different candidate compounds.

[0302] Exemplary embodiment A14. One of the exemplary embodiments A1 to A13, wherein the assay mixture yields a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a candidate compound.

[0303] Exemplary embodiment A15. One of the exemplary embodiments A1 to A14 of the assay method, wherein the assay signal is fluorescence.

[0304] Exemplary embodiment A16. One of the exemplary embodiments A1 to A15, wherein the first tag fragment is the first tag GFP fragment.

[0305] Exemplary embodiment A17. One of the exemplary embodiments A1 to A16, wherein the second tag fragment is a second tag GFP fragment.

[0306] Exemplary embodiment A18. One of the exemplary embodiments A1 to A17, wherein the detector fragment is a detector GFP fragment.

[0307] Exemplary embodiment A19. One of the exemplary embodiments A1 to A18, wherein a first tagged GFP fragment, a second tagged GFP fragment, and a detector GFP fragment are configured to form GFP when the assay mixture induces proximity between the first protein or fragment and the second protein or fragment.

[0308] Exemplary embodiment A20. A method from any one of the exemplary embodiments A1 to A19, wherein the first tagged GFP fragment and the second tagged GFP fragment independently include one or more of GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, and GFP11.

[0309] Exemplary embodiment A21. One of the exemplary embodiments A1 to A20, wherein the first tagged GFP fragment and the second tagged GFP fragment independently comprise one or more GFP10 and GFP11.

[0310] Exemplary embodiment A22. A method from any one of the exemplary embodiments A1 to A21, wherein the first tagged GFP fragment comprises GFP10 and the second tagged GFP fragment comprises GFP11.

[0311] Exemplary embodiment A23. A method from any one of the exemplary embodiments A1 to A22, wherein the detector GFP fragment is selected from GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, and GFP9.

[0312] Exemplary embodiment A24. A method according to any one of the exemplary embodiments A1 to A23, wherein the first protein is the protein of interest (POI).

[0313] Exemplary embodiment A25. One of the exemplary embodiments A1 to A24, wherein the second protein is a ubiquitin ligase.

[0314] Exemplary embodiment A26. A method of any one of the exemplary embodiments A1 to A25, further comprising step (i) detecting the generated or enhanced assay signal.

[0315] Exemplary embodiment A27. One of the exemplary embodiments A1 to A26 of the assay method, wherein the assay signal is fluorescence.

[0316] Exemplary embodiment A28. A method from any one of the exemplary embodiments A1 to A27, wherein a first protein or a fragment thereof, or a second protein or a fragment thereof, is covalently attached to an affinity component.

[0317] Exemplary embodiment A29. A method from any one of the exemplary embodiments A1 to A28, wherein the affinity component is biotin.

[0318] Exemplary embodiment A30. The method is (ii-a) Any one of the exemplary embodiments A1 to A29, further comprising contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex.

[0319] Exemplary embodiment A31. A method of any one of the exemplary embodiments A1 to A30, wherein step (ii-a) includes contacting the assay mixture with a plate coated with an affinity binder.

[0320] Exemplary embodiment A32. A method according to any one of the exemplary embodiments A1 to A31, wherein the affinity binder is streptavidin.

[0321] Exemplary embodiment A33. A method from any one of the exemplary embodiments A1 to A32, further comprising step (ii-a) incubating the assay mixture in a plate for a time ranging from 1 minute to 90 minutes.

[0322] Exemplary embodiment A34. A method of any one of the exemplary embodiments A1 to A33, wherein in step (i), the assay mixture further comprises a GFP booster, and hereby, step (ii-a) comprises incubating the assay mixture with a plate for a time ranging from 1 to 30 minutes.

[0323] Exemplary embodiment A35. The method is (ii-b) Any one of the exemplary embodiments A1 to A34, further comprising detecting and / or isolating the immobilized complex and thereby identifying the compound.

[0324] Exemplary embodiment A36. A method of any one of the exemplary embodiments A1 to A35, wherein step (ii) or step (ii-b) is to repeat step (i) one or more times using 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, first proteins or fragments thereof, and / or second proteins or fragments thereof in the complex.

[0325] Exemplary embodiment A37. A method from any one of the exemplary embodiments A1 to A36, wherein step (ii) or step (ii-b) is to repeat step (i) one or more times using 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 compounds in the complex.

[0326] Exemplary embodiment A38. A method from any one of the exemplary embodiments A1 to A37, wherein step (ii) or step (ii-b) is a repeat of step (i) one or more times using 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 obtaining the first protein or fragments thereof in the complex.

[0327] Exemplary embodiment A39. A method from any one of the exemplary embodiments A1 to A38, wherein step (ii) or step (ii-b) is to repeat step (i) one or more times using 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 fragment in the complex.

[0328] Exemplary embodiment A40. A method from any one of the exemplary embodiments A1 to A39, wherein step (ii-b) comprises characterizing the complex using mass spectrometry (MS).

[0329] Exemplary embodiment A41. A method from any one of the exemplary embodiments A1 to A40, 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 using MS to identify a first protein or fragment thereof and / or a second protein or fragment thereof in the complex.

[0330] Exemplary embodiment A42. A method of any one of the exemplary embodiments A1 to A41, 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 using MS to identify the first protein or a fragment thereof in the complex.

[0331] Exemplary embodiment A43. A method of any one of the exemplary embodiments A1 to A42, wherein step (ii-b) comprises reducing, denaturing, alkylating, and / or digesting the immobilized complex to form a peptide mixture, and characterizing the peptide mixture using MS to identify a second protein or fragment thereof in the complex.

[0332] Exemplary embodiment A44. A method of any one of the exemplary embodiments A1 to A43, 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 using MS to identify a first protein or fragment thereof and a second protein or fragment thereof in the complex.

[0333] Exemplary embodiment A45. A method from any one of the exemplary embodiments A1 to A44, wherein the peptide mixture further contains compounds, and the compounds in the complex are further identified by MS characterization.

[0334] Exemplary embodiment A46. A method from any one of the exemplary embodiments A1 to A45, wherein step (ii-b) comprises isolating the complex, dissociating a first protein or fragment thereof and / or a second protein or fragment thereof from the complex, and characterizing the dissociated proteins using MS to identify the first protein or fragment thereof and / or the second protein or fragment thereof in the complex.

[0335] Exemplary embodiment A47. A method from any one of the exemplary embodiments A1 to A46, further comprising step (ii-b) dissociating a candidate compound from the immobilized complex, and further identifying the candidate compound in the complex by MS characterization.

[0336] Exemplary embodiment A48. One of the exemplary embodiments A1 to A47, wherein the identified compound modulates the protein-protein interaction (PPI) between a first protein and a second protein.

[0337] Exemplary embodiment A49. A method in any one of the exemplary embodiments A1 to A48, wherein the PPI results in the degradation of a first or second protein.

[0338] Exemplary embodiment A50. One of the exemplary embodiments A1 to A49, wherein the PPI results in the stabilization of the first or second protein.

[0339] Exemplary embodiment A51. A method, one of the exemplary embodiments A1 to A50, wherein the identified compound causes degradation of the first protein in the presence of the second protein, or degradation of the second protein in the presence of the first protein.

[0340] Exemplary embodiment A52. A method, one of the exemplary embodiments A1 to A51, in which the PPI results in a post-translational modification of the first or second protein.

[0341] Exemplary embodiment A53. A method in any one of the exemplary embodiments A1 to A52, wherein the PPI results in the removal of existing post-translational modifications of the first or second protein.

[0342] Exemplary embodiment A54. One of the exemplary embodiments A1 to A53, wherein the PPI results in the modulation of the activity of the first or second protein.

[0343] Exemplary embodiment A55. One of the exemplary embodiments A1 to A54, wherein the PPI brings about a change in the intracellular localization of the first or second protein.

[0344] Exemplary embodiment A56. One of the exemplary embodiments A1 to A55, wherein the second protein is associated with a disease or disorder.

[0345] Exemplary embodiment A57. A method which is performed one or more times, one of the exemplary embodiments A1 to A56.

[0346] Exemplary embodiment A58. One of the exemplary embodiments A1 to A57, wherein the method is carried out using a new well at each time interval.

[0347] Exemplary embodiment A59. A first protein identified by any one of the exemplary embodiments A1 to A58.

[0348] Exemplary embodiment A60. A second protein identified by any one of the exemplary embodiments A1 to A58.

[0349] Exemplary embodiment A61. A combination of a first protein and a second protein, identified by any one of the exemplary embodiments A1 to A58.

[0350] Exemplary embodiment A62. A combination of a first protein, a second protein, and compounds targeting the first and second proteins, as identified by any one of the exemplary embodiments A1 to A58.

[0351] Exemplary embodiment A63. A compound identified by any one of the exemplary embodiments A1 to A58.

[0352] Exemplary embodiment A64. A pharmaceutical composition comprising a compound identified by any one of the exemplary embodiments A1 to A58.

[0353] Exemplary embodiment A65. A method for degrading a first protein or a second protein in a subject, comprising administering to the subject a compound identified by any one of the exemplary embodiments A1 to A58.

[0354] Exemplary embodiment A66. A method for regulating the PPI between a first protein and a second protein in a subject, comprising administering to the subject a compound identified by any one of the exemplary embodiments A1 to A58.

[0355] Exemplary embodiment A67. A compound identified by any one of the exemplary embodiments A1 to A58 for use in degrading a first or second protein in a subject.

[0356] Exemplary embodiment A68. Compounds identified by any one of the exemplary embodiments A1 to A58 for use in regulating the PPI between a first protein and a second protein in a subject.

[0357] Exemplary embodiment A69. Use of a compound identified by any one of the exemplary embodiments A1 to A58 in the manufacture of a pharmaceutical product for degrading a first or second protein in a subject.

[0358] Exemplary embodiment A70. Use of a compound identified by any one of the exemplary embodiments A1 to A58 in the manufacture of a pharmaceutical product for modulating the PPI between a first protein and a second protein in a subject.

[0359] Exemplary embodiment A71. A method for treating and / or preventing a disease or disorder associated with a first or second protein in a subject, comprising administering to the subject a therapeutically effective amount of a compound identified by any one of the exemplary embodiments A1 to A58.

[0360] Exemplary embodiment A72. Compounds identified by any one of the exemplary embodiments A1 to A58 for use in treating and / or preventing diseases or disorders associated with a first or second protein in a subject.

[0361] Exemplary embodiment A73. Use of a compound identified by any one of the exemplary embodiments A1 to A58 in the manufacture of a pharmaceutical product for treating and / or preventing a disease or disorder associated with a first or second protein in a subject.

[0362] Exemplary embodiment B1. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and the compound; and (ii) A method comprising identifying a first protein, a second protein, a compound, or any combination thereof associated with a complex.

[0363] Exemplary embodiment B2. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, A first protein or fragment thereof, and / or a second protein or fragment thereof, are covalently attached to the affinity component; and The first tag fragment and the second tag fragment are provided, configured to generate or enhance the assay signal when the assay mixture yields a complex containing the first protein or its fragment, the second protein or its fragment, and a compound; (ii-a) Contacting the complex with an immobilized affinity binder that targets affinity components, thereby forming an immobilized complex; and (ii-b) A method comprising detecting and / or isolating an immobilized complex and thereby identifying a first protein, a second protein, a compound, or any combination thereof.

[0364] Exemplary embodiment B3. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, the assay mixture is (a) The first protein or a fragment thereof, covalently attached to the first tag fragment; (b) A second protein or fragment thereof covalently attached to the second tag fragment, wherein the second tag fragment is complementary to the first tag fragment; and (c) Includes candidate compounds, The first tag fragment and the second tag fragment are provided such that the assay mixture generates or enhances an assay signal when proximity is induced between the first protein or its fragment and the second protein or its fragment; and (ii) Identifying a first protein, a second protein, a compound, or any combination thereof associated with the induction of proximity A method by which an identified compound can cause the degradation of a first protein in the presence of a second protein.

[0365] Exemplary embodiment B4. The method of exemplary embodiment B1, wherein the assay mixture further comprises a detector fragment complementary to the first tag fragment and the second tag fragment.

[0366] Exemplary embodiment B5. The method of exemplary embodiment B1, wherein the assay mixture comprises several different first proteins or fragments thereof.

[0367] Exemplary embodiment B6. The method of exemplary embodiment B1, wherein the assay mixture comprises several different second proteins or fragments thereof.

[0368] Exemplary embodiment B7. The method of exemplary embodiment B1, wherein the assay mixture contains several different candidate compounds.

[0369] Exemplary embodiment B8. An exemplary embodiment of the method in B1, wherein the assay mixture yields a complex comprising a first protein or a fragment thereof, a second protein or a fragment thereof, and a candidate compound.

[0370] Exemplary embodiment B9. The method of exemplary embodiment B1, in which the assay signal is fluorescence.

[0371] Exemplary embodiment B10. The method of exemplary embodiment B4, wherein the first tag fragment is a first tag GFP fragment.

[0372] Exemplary embodiment B11. A method of exemplary embodiment B10, wherein the second tag fragment is a second tag GFP fragment.

[0373] Exemplary embodiment B12. An exemplary embodiment of the method of B11, wherein the detector fragment is a detector GFP fragment.

[0374] Exemplary embodiment B13. The method of exemplary embodiment B12, wherein the first tagged GFP fragment and the second tagged GFP fragment independently include one or more of GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, GFP9, GFP10, and GFP11.

[0375] Exemplary embodiment B14. An exemplary embodiment of the method of B12, wherein the first tagged GFP fragment and the second tagged GFP fragment independently comprise one or more GFP10 and GFP11.

[0376] Exemplary embodiment B15. The method of exemplary embodiment B12, wherein the first tagged GFP fragment comprises GFP10 and the second tagged GFP fragment comprises GFP11.

[0377] Exemplary embodiment B16. The method of exemplary embodiment B12, wherein the detector GFP fragment is selected from GFP1, GFP2, GFP3, GFP4, GFP5, GFP6, GFP7, GFP8, and GFP9.

[0378] Exemplary embodiment B17. The method of exemplary embodiment B1, wherein the first protein is the protein of interest (POI).

[0379] Exemplary embodiment B18. The method of exemplary embodiment B1, wherein the second protein is a ubiquitin ligase.

[0380] Exemplary embodiment B19. An exemplary embodiment of the method of B1 further comprising step (i) detecting the generated or enhanced assay signal.

[0381] Exemplary embodiment B20. An exemplary embodiment of the method in Embodiment B1, in which the identified compound modulates the protein-protein interaction (PPI) between the first protein and the second protein.

[0382] Exemplary embodiment B21. An exemplary embodiment of the method of B1, wherein the identified compound causes degradation of the first protein in the presence of the second protein, or degradation of the second protein in the presence of the first protein.

[0383] Exemplary embodiment B22. A first protein identified by the method of exemplary embodiment B1.

[0384] Exemplary embodiment B23. A second protein identified by the method of exemplary embodiment B1.

[0385] Exemplary embodiment B24. A combination of a first protein and a second protein identified by the method of exemplary embodiment B1.

[0386] Exemplary embodiment B25. A combination of a first protein, a second protein, and compounds targeting the first and second proteins, as identified by the method of exemplary embodiment B1.

[0387] Exemplary embodiment B26. Compounds identified by the method of exemplary embodiment B1.

[0388] Exemplary embodiment B27. A pharmaceutical composition comprising a compound identified by the method of exemplary embodiment B1.

[0389] Exemplary embodiment B28. A method for degrading a first protein or a second protein in a subject, comprising administering to the subject a compound identified by the method of exemplary embodiment B1.

[0390] Exemplary embodiment B29. A method for regulating the PPI between a first protein and a second protein in a subject, comprising administering to the subject a compound identified by the method of exemplary embodiment B1.

[0391] Exemplary embodiment B30. A method for treating and / or preventing a disease or disorder associated with a first or second protein in a subject, comprising administering to the subject a therapeutically effective amount of a compound identified by the method of exemplary embodiment B1.

[0392] definition Unless otherwise stated, the following terms used in this specification and in the claims have the meanings set forth below.

[0393] Unless otherwise indicated, where the amounts of a first protein or fragment thereof, a second protein or fragment thereof, and candidate compounds in an assay mixture are referred to, it is understood that this disclosure intends to describe the number of different structures rather than the molecular weights of the first protein or fragment thereof, the second protein or fragment thereof, and candidate compounds. For example, if the assay mixture contains “first protein”, then the first protein present in the assay mixture may have the same or different structures. If the assay mixture contains “multiple first proteins”, then the first proteins present in the assay mixture may have at least two different structures. In another example, if the assay mixture contains “second protein”, then the second protein present in the assay mixture may have the same or different structures. If the assay mixture contains “multiple second proteins”, then the second proteins present in the assay mixture may have at least two different structures. In yet another example, if the assay mixture contains “candidate compounds”, then all candidate compounds present in the assay mixture may have the same or different structures. If the assay mixture contains "multiple candidate compounds," then the candidate compounds present in the assay mixture have at least two different structures.

[0394] As used herein, the term “immobilized” means that the attached portion is attached to a solid surface by various means (e.g., covalent attachment or high-affinity non-covalent attachment) in such a manner that it does not substantially diffuse into the solution (e.g., incubation solution).

[0395] As used herein, the term “binding site” refers to a fragment or portion of a protein that can bind to (e.g., interact with) a compound, such as the compounds identified in the methods of this disclosure, or to a protein. In one embodiment, the protein binding site includes a domain of the protein. In some embodiments, the protein binding site includes an engineered domain of the protein.

[0396] As used herein, the term “binding compound” refers to a compound capable of targeting one or more targets (e.g., one or more proteins). In some embodiments, the compound can induce proximity between one or more targets (e.g., one or more proteins). In some embodiments, the compound can cause an effect (e.g., degradation, change in activity, or post-translational modification) on one or more targets (e.g., one or more POIs) in the presence of another one or more targets (e.g., E3 ligases).

[0397] As used herein, the term “targeting” refers to the ability, effect, or action of associating with a target (e.g., a protein) by a reference element (e.g., a binder compound). In some embodiments, the reference element (e.g., a binder compound) is capable of, or does associate with, a target (e.g., a protein) via covalent and / or non-covalent attachment (e.g., high-affinity non-covalent attachment). In some embodiments, the association between the reference element (e.g., a binder compound) and the target (e.g., a protein) causes an effect on the target (e.g., a POI) (e.g., degradation, change in activity, or post-translational modification), for example, in the presence of another target (e.g., an E3 ligase or other protein).

[0398] As used herein, the term “fragment” refers to a portion of a reference element. For example, a protein fragment refers to a portion of a protein. In some embodiments, a protein fragment is a portion comprising one or more components of a protein (e.g., one or more components having a functional and / or structural role in the protein).

[0399] As used herein, the term “configured” means any means of constructing or configuring a reference element (e.g., physical, chemical, or biological) such that the element is capable of performing its reference function.

[0400] As used herein, the term “complex” refers to a group of related elements (e.g., compounds, proteins, and / or protein fragments) by various means (e.g., covalent and / or non-covalent attachments). In some embodiments, elements in a complex are associated through proximity, for example, compound-induced proximity between two or more proteins or protein fragments.

[0401] As used herein, the term “affinity binder” refers to an element (e.g., a molecule, peptide, or protein moiety) that can associate with affinity components by various means (e.g., covalent attachment or high-affinity non-covalent attachment). In some embodiments, the affinity binder is an immobilized binder that forms an immobilized complex upon contact with a complex containing affinity components (e.g., via association between the affinity binder and the affinity components).

[0402] As used herein, the expressions "one or more A, B, or C," "one or more A, B, or C," "one or more 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," and similar expressions are used interchangeably and all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof, unless otherwise indicated.

[0403] Throughout the description, when a compound is described as having, including, or comprising certain components, it should be understood that the composition is also intended to consist of or be essentially composed of the listed components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process is also intended to consist of or be essentially composed of the listed process steps. Furthermore, it should be understood that the order of the steps, or the order in which certain actions are performed, is not important as long as the invention remains operational. Moreover, two or more steps or actions can be performed simultaneously.

[0404] Unless otherwise stated, any description of a method of treatment or prevention should be understood to include the use of compounds to provide treatment or prevention as described herein. Unless otherwise stated, any description of a method of treatment or prevention should be further understood to include the use of compounds to prepare a pharmaceutical product for the treatment or prevention of such condition. Treatment or prevention includes treatment or prevention in human or non-human animals, including rodents and other disease models.

[0405] The “subjects” to which the administration is intended are not limited to humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus macaques, rhesus macaques), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is human. In certain embodiments, the subject is a non-human animal. In some embodiments, the subject is an agricultural entity (e.g., seeds, saplings, leaves, flowers, plants, etc.).

[0406] As used herein, the terms “to treat” or “to cure” describe the management and care of a patient for the purpose of combating a disease, condition, or disorder, and include the administration of the compounds of this disclosure, or pharmaceutically acceptable salts, polymorphs, or solvates thereof, for the purpose of reducing the symptoms or complications of the disease, condition, or disorder, or for the purpose of eliminating the disease, condition, or disorder. The term “to treat” may also include the treatment of cell or animal models in vitro. References to “to treat” or “to cure” should be understood to include the reduction of established symptoms of a condition. "Treating" or "treating" a condition, disorder, or state therefore includes: (1) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or state occurring in a person who has or is predisposed to having such condition, disorder, or state, but has not yet experienced or exhibited any clinical or potential symptoms of such condition, disorder, or state; (2) inhibiting the condition, disorder, or state, i.e., stopping, reducing, or delaying the progression of the disease or its relapse (in the case of maintenance therapy) or at least one clinical or potential symptom of the disease; or (3) reducing or alleviating the disease, i.e., causing regression of at least one clinical or potential symptom of the condition, disorder, or state.

[0407] As used herein, the terms “prevent” or “prevent” mean reducing or eliminating the onset of symptoms or complications of such disease, condition, or disorder.

[0408] As used herein, the term “pharmaceutical composition” refers to a formulation comprising the compounds of the Disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dose form. A unit dose form is any of a variety of forms, including, for example, capsules, IV bags, tablets, a single pump on an aerosol inhaler, or vials. The amount of the active ingredient (e.g., a formulation of the disclosed compound or a salt, hydrate, solvate, or isomer thereof) in a unit dose of the composition is an effective amount and varies according to the specific treatment to which it is administered. Those skilled in the art will understand that it is sometimes necessary to make routine variations in the dosage depending on the patient’s age and condition. The dosage also depends on the route of administration. A variety of routes are intended, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, subarachnoid, intranasal, and similar. Dosage forms for topical or transdermal administration of the compounds of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservative, buffer, or propellant.

[0409] As used herein, the term “pharmaceutically acceptable” means, within the bounds of sound medical judgment, a compound, anion, cation, material, composition, carrier, and / or dosage form that is appropriate for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, corresponding to a reasonable benefit-risk ratio.

[0410] As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is generally safe, non-toxic, and useful in preparing a pharmaceutical composition that is not biologically or otherwise undesirable, and includes excipients that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, “pharmaceutically acceptable excipient” includes both one and more than one such excipient.

[0411] As used herein, the term “therapeutic effective dose” refers to the amount of a pharmaceutical compound required to treat, alleviate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact effective dose for a subject depends on the subject's weight, size, and health; the nature and severity of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. A therapeutic effective dose for a given situation can be determined by routine experiments within the clinician’s skill and judgment.

[0412] Pharmaceutical compositions comprising the active compounds of this disclosure may be manufactured in a manner generally known, for example, by conventional mixing, dissolution, granulation, sugar-coated tablet manufacturing, powdering, emulsification, encapsulation, or lyophilization processes. The pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and / or adjuvants that facilitate the treatment of the active compounds into pharmaceutically usable preparations. Of course, the suitable formulation depends on the chosen route of administration.

[0413] It should be understood that all of the compounds of this disclosure that can further form salts are also intended to be within the scope of the claimed disclosure.

[0414] As used herein, the term “pharmaceutically acceptable salt” refers to a derivative of the compound of the present disclosure, wherein the parent compound is modified by the preparation of an acid or a base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, basic residues, such as mineral or organic salts of amines, acidic residues, such as alkali or organic salts of carboxylic acids, and the like. pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, EDTA, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycolylarsanic acid, hexylresorcinic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, and milk. This includes acids, lactobionic acid, lauryl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, napsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, subacetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and those derived from inorganic and organic acids selected from commonly occurring amino acids, such as glycine, alanine, phenylalanine, and arginine.

[0415] In some embodiments, pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, magnesium salts, diethylamine salts, choline salts, meglumine salts, benzathine salts, trometamic acid salts, ammonia salts, arginine salts, or lysine salts.

[0416] Other examples of pharmaceutically acceptable 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]-octo-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The disclosure also includes salts formed when an acidic proton present in the parent compound is substituted with a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion; or when it is coordinated with an organic base, such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In salt form, it is understood that the ratio of the compound to the salt cation or anion can be 1:1, or any other ratio, such as 3:1, 2:1, 1:2, or 1:3.

[0417] All references to pharmaceutically acceptable salts should be understood to include the solubilated form (solvate) or crystalline form (polymorph) of the same salt as defined herein.

[0418] The compound, or a pharmaceutically acceptable salt thereof, is administered orally, nasally, percutaneously, pulmonaryly, by inhalation, buccally, 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.

[0419] All percentages and ratios used herein are by weight unless otherwise indicated. Other features and advantages of this disclosure are evident from different examples. The provided examples illustrate different components and methodologies 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 use other components and methodologies useful in practicing this disclosure.

[0420] All publications and patent documents referenced herein are incorporated herein by reference as if each such publication or document were specifically and individually indicated to be incorporated herein by reference. No reference to publications and patent documents is intended as an acknowledgment that either is relevant prior art, nor does it constitute any acknowledgment of their content or date. Although the present invention has been described herein by written description, those skilled in the art will recognize that the present invention can be practiced in various embodiments, and that the preceding description and examples below are for illustrative purposes only and not limitations on the claims that follow.

[0421] Appropriate pharmaceutically acceptable prodrugs of the compounds disclosed herein are deemed appropriate for administration to the human or animal body without undesirable pharmacological activity and without excessive toxicity, based on reasonable medical judgment. Various forms of prodrugs are described in the following documents, for example: a) Methods in Enzymology, Vol. 42, pp. 309-396, edited by KWidder, et al. (Academic Press, 1985); b) Design of Pro-drugs, edited by HBundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and HBundgaard, Chapter 5 “Design and Application of Pro-drugs”, by HBundgaard, pp. 113-191 (1991); d) HBundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) HBundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) NKakeya, et al. al., ChemPharmBull., 32, 692 (1984); g) THiguchi and VStella, “Pro-Drugs as Novel Delivery Systems”, ACS Symposium Series, Volume 14; and h) ERoche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.

[0422] A suitable pharmaceutically acceptable prodrug of a compound disclosed herein having a hydroxyl group is, for example, an in vivo cleavable ester or ether thereof. An in vivo cleavable ester or ether of any one of the compounds of the formulas disclosed herein containing a hydroxyl group is, for example, a pharmaceutically acceptable ester or ether that is cleaved in the body of a human or animal to produce a parent hydroxyl compound. Suitable pharmaceutically acceptable ester-forming groups for a hydroxyl group include inorganic esters, such as phosphate esters (including phosphoramido cyclic esters). A more suitable pharmaceutically acceptable ester-forming group for a hydroxyl group is C1-C 10 Alkanoyl groups, such as acetyl groups, benzoyl groups, phenylacetyl groups, and substituted benzoyl and phenylacetyl groups, C1-C 10 Alkoxycarbonyl groups include, for example, ethoxycarbonyl groups, N,N-(C1-C6 alkyl)2-carbamoyl groups, 2-dialkylaminoacetyl groups, and 2-carboxyacetyl groups. Examples of ring substituents on phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazine-1-ylmethyl, and 4-(C1-C4 alkyl)piperazine-1-ylmethyl. Suitable pharmaceutically acceptable ether-forming groups for hydroxyl groups include α-acyloxyalkyl groups, for example, acetoxymethyl groups and pivaloyloxymethyl groups.

[0423] Suitable pharmaceutically acceptable prodrugs of the compounds disclosed herein that have a carboxyl group include, for example, amides formed with their in vivo cleavable amides, such as amines, such as ammonia; C1-C4 alkylamines, such as methylamine; (C1-C4 alkyl)2 amines, such as dimethylamine, N-ethyl-N-methylamine, or diethylamine; C1-C4 alkoxy C1-C4 alkylamines, such as 2-methoxyethylamine; phenyl C1-C4 alkylamines, such as benzylamine; and amino acids, such as glycine or its esters.

[0424] A suitable pharmaceutically acceptable prodrug of a compound disclosed herein having an amino group is, for example, an in vivo cleavable amide derivative thereof. A suitable pharmaceutically acceptable amide from an amino group is, for example, a C1-C 10 The amides are formed with alkanoyl groups, such as acetyl groups, benzoyl groups, phenylacetyl groups, and substituted benzoyl and phenylacetyl groups. Examples of ring substituents on phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazine-1-ylmethyl, and 4-(C1-C4 alkyl)piperazine-1-ylmethyl. [Examples]

[0425] The following embodiments are provided for illustrative purposes only and should not be construed as limiting the invention.

[0426] Example 1. Designing an exemplary method. Several exemplary methods are designed. The detailed design of these methods is summarized below.

[0427] Example Method 1: The adhesive screening assay mixture contains the following components in a physiological buffer: GFP1-9, a protein of interest (POI) tagged with another short stretch of amino acid sequence from GFP, typically corresponding to GFP11, an E3 ligase of choice tagged with a short stretch of amino acid sequence from green fluorescent protein (GFP), typically corresponding to GFP10, and the compound of interest as a potential molecular adhesive. The POI can be tagged with GFP10 instead of GFP11. In this case, the E3 ligase is tagged with GFP11. Full-length GFP consists of 11 distinct stretches of amino acid residues that form a barrel-shaped structure. GFP1-9 contains the first nine of these distinct stretches. GFP10 represents the stretch of amino acid residue corresponding to the 10th of the 11 stretches in full-length GFP. Similarly, GFP11 represents the 11th of the 11 distinct stretches of amino acids in full-length GFP. There are multiple variations of GFP, which are either of natural origin or genetically engineered. Despite some variations in optimal fluorescence wavelengths, all of these proteins have a similar barrel-shaped structure with 11 distinct stretches of amino acid residues. In this disclosure, GFP refers to any one of their variant forms. Two stretches used to tag the first protein (e.g., POI) and the second protein (e.g., E3 ligase) can be tagged using other distinct stretches of amino acids, insofar as the two stretches used to tag the first and second proteins, respectively, are adjacent to each other in a fully formed barrel-shaped structure. In this case, GFP1-9 are replaced with a continuous stretch of amino acid residues from GFP, except for the two stretches used to tag the two proteins, in such a way that the three parts of GFP can be assembled to form a barrel-shaped mature GFP. In a typical screening assay, up to 10 or more POIs, each with a GFP11 (or GFP10) tag, can be tested simultaneously in the same assay mixture.Similarly, up to 10 or more E3 ligases, each tagged with GFP10 (or GFP11 if the POI is tagged with GFP10), can be tested simultaneously in the same assay mixture. GFP1-9 do not require any specific tag and are typically used at higher concentrations than either the individual E3 or POI. The compounds of interest can be tested one at a time. To increase screen throughput, the compounds of interest are typically pooled together in a mixture of 100-200 compounds per pool, using a stock solution dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. If 100 compounds are pooled in the mixture starting from the original 10 mM stock solution, the pooled solution will have a concentration of 100 μM for each individual compound. If the original compound stock solution is available at a higher concentration, then higher throughput can be achieved by pooling more compounds in each pool. Alternatively, compounds can be tested at higher concentrations while maintaining the same throughput.

[0428] In addition to tagging POI and E3 using GFP-derived amino acid sequences, either POI or E3, but not both, can be modified with biotin as a handle for capturing these proteins via high-affinity interactions between biotin and streptavidin, avidin, or their variants. Protein biotinylation can be carried out by any number of well-established methods, including biochemical conjugations using biotin analogs with reactive functional groups that form covalent bonds with certain amino acid side chains or free N-terminuses or C-terminuses. Other frequently used methods involve expressing the protein as a hybrid protein containing a stretch of amino acid residues, often referred to as an "Avi tag." The Avi tag can be biotinylated intracellularly during protein synthesis or enzymatically after protein purification. Other types of tags can be used instead of biotin tags if the same tag is not found in any of the other components of the assay mixture and a corresponding high-affinity capture reagent is available to bind the tag.

[0429] When a ternary complex is formed between any combination of POI, E3, and adhesive molecules, GFP11 and GFP10 tags are brought into close proximity on the POI and E3. 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 the solution. The binding of GFP1-9 to GFP11 and GFP10 for the formation of barrel-shaped GFP complexes is not actually reversible under physiological conditions, thus stabilizing the ternary complex of POI, E3, and adhesive molecules. The appearance of fully mature GFP can be monitored by measuring the generation of green fluorescence in solution. This is typically done by repeated measurements of green fluorescence over time from 8 hours to overnight. Significantly higher fluorescence from certain wells compared to the majority of wells in the plate indicates the presence of ternary complexes in those wells. These wells are considered to be activators.

[0430] At the end of an incubation period sufficient to allow for ternary complex formation and subsequent assembly of GFP on the ternary complex, the assay mixture can be transferred to a plate pre-coated with streptavidin to capture the biotin-tagged proteins. The streptavidin coating on the plate should be sufficiently high to capture all proteins using biotinylated proteins. If other types of tags are used instead of biotin, the plates should be pre-coated with a suitable high-affinity agent instead of streptavidin, which can capture the tags. After incubation of the assay mixture in the streptavidin-coated plate, the plate is washed with a buffer solution for typically 30 minutes to 1 hour to remove any unbound proteins and compounds. If the biotin tag is placed on the POI, most of the E3 protein and GFP1-9 are washed away, except for those recruited to the POI as part of a ternary complex with the adhesive compound. If the biotin tag is placed on the E3, the reverse is true for the POI protein and GFP1-9. To further enhance the fluorescence signal from fully assembled GFP, a solution containing a GFP booster reagent may be added to each well. The GFP booster is an affinity conjugate to GFP labeled with a fluorescent dye. The dye has the same fluorescence wavelength as GFP, with a higher quantum yield. In this case, the GFP booster is in the form of an antibody against fully assembled GFP, conjugated with a chemical fluorophore having the same excitation / emission wavelength as GFP. For example, Alexa Fluor 488 conjugated GFP nanobodies from Chromotek can be used for this purpose. Due to the high specificity of this reagent for fully assembled GFP above GFP1-9, GFP10, or GFP11, only wells containing fully assembled GFP will bind to the GFP booster. Unbound GFP booster will be washed away. To further increase the specificity of the GFP booster for fully assembled GFP, the incubation time of the GFP booster solution should be limited to less than 30 minutes.Due to the higher affinity of the GFP booster for fully assembled GFP compared to GFP1-9, GFP10, or GFP11, a shorter incubation time ensures that the GFP booster binds to the plate only if fully assembled GFP is captured on the plate. Alternatively, the GFP booster 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 due to the specificity of the GFP booster for full-length GFP. Once the plate is incubated with the GFP booster reagent and washed to remove unbound GFP booster, the plate is sealed with a clear film, and fluorescence is measured from individual wells in the plate. Other types of affinity methods can be used for capturing the ternary complex, including the use of anti-GFP antibodies to capture fully assembled GFP. Affinity agents can also be conjugated or coated onto any solid-phase surface, such as magnetic beads, agarose beads, or other types of beads.

[0431] The screening method can be performed multiple times. For example, in an initial high-throughput screen, fluorescence is detected in active wells containing a pooled chemistry diversity library (100-200 compounds per well) using one or more POIs and one or more E3s. A second screen is then performed, repeating the initial screen in new wells with the same pooled compounds, POIs, and E3s. Performing the screen a second time increases the confidence that any detected initial signals are repeatable and not due to noise or other ambient factors, without the need to be constrained by theory.

[0432] Due to the multiplicative nature of the screening method, the identities of individual POIs, E3s, and adhesive compounds in the activator are not known at this stage. Two different methods are used for this identification purpose: deconvolution and MS-based identification, which are described below.

[0433] In deconvolution methods, individual POIs, E3s, and potential adhesive compounds in activators are tested in a way that uniquely and clearly identifies which particular combination of POIs, E3s, and potential adhesive compounds led to the formation of the ternary complex in the identified activator. 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 exact combinations of POIs and E3s. Individual compounds are then tested using the specific POI and E3 pairs identified in the previous step. Several other methods can be used, as long as they can clearly identify the unique combinations of POIs, E3s, and adhesive compounds. For example, the same protein mixture can be tested against individual compounds in an activator pool for the identification of the active adhesive molecule, followed by testing of the specific individual combinations of POIs and E3s with the same active adhesive molecule.

[0434] In MS-based identification, components in the ternary complex of the activator are identified by a standard "bottom-up mass spectrometry-based proteomics" method. In this method, the protein captured in the well is typically reduced and alkylated under denaturing conditions, followed by digestion into smaller peptides using a sequence-specific protease before analysis of the sample by mass spectrometer. Several options are available for this well-established procedure. For example, high concentrations of urea, guanidine hydrocholoride, acid-unstable ionic surfactants, such as RapiGest or other chaotropic agents, are used for the purpose of protein denaturation. Dithiothreitol, beta-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine) are often used for the purpose of reducing disulfide bonds in proteins. Iodoacetamide, iodoacetic acid, or iodoethanol are some of the commonly used alkylating agents that, once the disulfide bonds are reduced, covalently modify the free sulfhydryl groups on the protein. Trypsin is the most commonly used sequence-specific protease for digesting proteins into peptides for mass spectrometry-based protein analysis purposes, due to its high degree of 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 for this purpose. The amino acid sequence of the digested peptide is identified by standard proteomics methods. Briefly, this method is based on comparing the obtained m / z (mass-to-charge ratio) values ​​of the peptides and their MS2 fragmentation spectra with theoretical data obtained from in silico digestion of protein sequences in protein databases for suitable species. Numerous publications are available for this method, and various software, often referred to as search engines, are readily available from multiple sources.For mass spectrometry data acquisition, in-line separation of peptides on liquid chromatography (LC) is often combined with different data acquisition modes using a mass spectrometer. Data-dependent MS2 acquisition (DDA) is a 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 clear identification of the unique peptide sequence can be established. Direct data acquisition can be performed without LC separation of peptides, especially when the sample composition is relatively simple. Matrix-assisted laser desorption / ionization (MALDI) time-of-flight instruments are one such example. Direct injection of digested samples through an electrospray ionization interface is another example. Protein identity in a sample can be estimated by comparing the amino acid sequence of the peptide with the amino acid sequence of the intact protein in a protein database. During the digestion of proteins in a ternary complex, molecular adhesive compounds trapped in the ternary complex are released into the digestion solution. The identity of adhesive compounds can also be obtained from the same sample, and often from the same experiment, by comparing the precise molecular weight of the compounds in the pool for the activator with the expected molecular weight of the compounds estimated from the m / z values ​​of the MS1 spectrum. Alternatively, protein identity information can be obtained directly using a “top-down” method that does not involve protein digestion, if the proteins in the ternary complex can be released into solution. In this method, the intact protein mass obtained by mass spectrometry is compared with the expected mass of the individual proteins used in this experiment. One such method of “releasing” intact proteins from GFP is to introduce 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. One such example 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 causes the 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 TEV protease, followed by acidification.

[0435] The deconvolution and MS-based identification methods described above are not mutually exclusive and can be used in combination for the highest level of confidence regarding the identity of individual components within the ternary complex within each hit. It should also be noted that MS-based methods do not provide identity for specific affinity-tagged proteins (biotinylated E3 or biotinylated POI) that were part of the ternary complex, as all biotinylated proteins are captured from all samples, regardless of the presence or absence of ternary complex formation.

[0436] Example 2. Results using an exemplary method Results for Exemplary Method 1a In one version of the assay, the adhesive screening assay mixture is assembled in 10 μL of assay buffer (50 mM Hepes buffer, pH 7.5, 150 mM KCl, 1 mM TCEP) with 1 μg of GFP1-9 in complex with DDB1 and DDA1, 0.1 μg of GFP11-tagged RBM39, and 0.1 μg of GFP10-tagged DCAF15 (hereinafter referred to as DCAF15, as DCAF15 is always used as a complex with DDB1 and DDA1). Two different versions of GFP11-tagged RBM39 are tested using GFP11 fused to the N-terminus and C-terminus of RBM39 (GFP11-RBM39 and RBM39-GFP11, respectively). Similarly, two different versions of GFP10-tagged DCAF15 are tested: GFP10 fused to the N-terminus of DCAF15 (GFP10-DCAF15) and GFP10 fused to the C-terminus of DCAF15 (DCAF15-GFP10). For the primary screen, these four different proteins are combined and added to a plate containing the compound pool. Once the active pool is identified, the same assay mixture is tested against each individual compound in the active pool, and the identified compounds are then tested against different pairs of E3 and POI for deconvolution. In this example, four different assay mixtures are prepared, each using different combinations of GFP11-tagged RBM39 and GFP10-tagged DCAF15 (see Table A).

[0437] [Table A]

[0438] In addition to GFP11, the RBM39 protein has a biotin tag on the opposite side of GFP11 via labeling with an Avi tag sequence. These different assay mixtures are added to a 384-well assay plate containing 100 nL of equimolar mixtures of the test compounds at various concentrations in DMSO (0 nM, 10 nM, 100 nM, 1 μM, 1 μM, and 100 μM, respectively). Two different test compounds are used in this experiment: indislam (MedKoo, 201540) as a positive control and lenalidomide (aablocks, AA002FDI) as a negative control. The assay plate is sealed with clear tape (Eppendorf 0030127838), mixed by vortexing and spinning for a short time, and the liquid is collected at the bottom of the plate before incubation at 4°C with shaking. Green fluorescence is measured from assay plates after incubation at 2, 6, 18, 24, 48, and 72 hours using a fluorescence plate reader with excitation / emission wavelengths of 488 / 509 nm. Figures 1A–1D show simulation results using the various combinations outlined above. In Figures 1A, 1B, and 1D, the dose-dependent signal development plateaus at earlier time points with higher compound concentrations. Lower compound concentrations lead to later signal development and lower plateau levels. Figure 1B represents combinations that show no activity.

[0439] At the end of the 72-hour incubation, the contents of the assay plate are transferred to a capture plate containing 2 μL of GFP-Booster (Chromotek gb2AF488-50), pre-coated with streptavidin (Greiner, catalog no. 781995) and diluted 1:200 in assay buffer. After a 30-minute incubation at room temperature to capture biotin-tagged RBM39, the plate is inverted onto a collection plate and briefly spun at 1500 rpm to remove the contents, followed by the addition of 20 μL of assay buffer. This process is repeated three times to remove unbound proteins from the plate. The addition of assay buffer is omitted after the third washing step, and green fluorescence is measured again as described above after the addition of 20 μL of ammonium bicarbonate solution (simulation results in Figures 2A-2B). Mass spectrometry analysis of all samples is taken without delay as described below.

[0440] Add 5 μL of reducing agent (in a 50 mM ammonium bicarbonate solution containing 25 mM DTT, 0.05% RapiGest (Waters, 186008740)) to each well in the capture plate, and seal the plate using heat-seal tape. Allow the capture plate to cool to room temperature and incubate at 80°C for 5 minutes before transferring the contents to a new 384-well plate containing 5 μL of 75 mM iodoacetamide in 50 mM ammonium bicarbonate. Incubate the plate at room temperature in the dark for 45 minutes with shaking. At the end of incubation, transfer 2 μL of the contents of each well to a new blank 384-well plate and mix with 1 μL of MALDI matrix for mass spectrometry of the captured adhesive compound (simulation results in Figure 3). To the remaining sample, add 5 μL of trypsin solution (MS grade 90057 from ThermoFisher, diluted to 0.05 μg / μL in 50 mM ammonium bicarbonate), seal the plate, and incubate overnight at 37°C. At the end of incubation, transfer 2 μL of digested sample to another 384-well plate and mix with 1 μL of MALDI matrix (listed below) for mass spectrometry of the digested peptides of the captured proteins (simulation results in Figure 4).

[0441] The MALDI matrix contains 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 is incubated at 55°C for 1 hour to promote the hydrolysis of RapiGest in the sample before spotting 2 μL of the final sample onto an AnchorChip target plate (Bruker, 8280790), and then dried before analysis using a timsTOF mass spectrometer (Bruker) according to the manufacturer's protocol.

[0442] Results for Exemplary Method 1b In another version of the assay, the adhesive screening assay mixture was assembled in 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-beta-glucopyranoside, 5% glycerol) in a clear-bottom 1536-well plate (Greiner, Cat # 789866) with 0.3 μg of GFP1-9, 0.05 μg of GFP11-tagged IKZF2, and 0.3 μg of GFP10-tagged CRBN in a complex with DDB1 (hereafter referred to as CRBN, because CRBN was always used as a complex with DDB1). Various buffer solutions provided consistent signals in this assay (0.1–1 mg / mL BSA, 0–10% glycerol, 0–1 mM octyl-beta-glucopyranoside). As in previous versions, two different versions of GFP11-tagged IKZF2 were tested using GFP11 fused to the N-terminus and C-terminus of IKZF2 (GFP11-IKZF2 and IKZF2-GFP11, respectively). Similarly, two different versions of GFP10-tagged CRBN were tested: GFP10 fused to the N-terminus of CRBN (GFP10-CRBN) and GFP10 fused to the C-terminus of CRBN (CRBN-GFP10). The concentrations correspond to 5 μM GFP (1–9), 1.25 μM of each IKZF2 construct, and 0.5 μM of each of the two CRBN constructs. The concentrations of IKZF2 and CRBN proteins were optimized for the best signal relative to background by titrating the proteins against each other.

[0443] For the primary screening, these five different proteins (GFP1-9, GFP11-IKZF2, IKZF2-GFP11, GFP10-CRBN, and CRBN-GFP10) were combined in assay buffer and added to plates containing different compound pools. During deconvolution, the assay mixture was tested against each individual compound, and the identified compounds were then tested against different pairs of POI and E3.

[0444] When protein mixtures were added to compounds known as established adhesives for IKZF2-CRBN (IKZF2-CRBN adhesive compound 1, IKZF2-CRBN adhesive compound 2, IKZF2-CRBN adhesive 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 adhesives because the signals from other compounds (chloroquinoxaline, indisram) were indistinguishable from the DMSO control. These compounds are known adhesives for RBM39 and DCAF15 protein pairs and are known to lack adhesive activity for IKZF2-CRBN protein pairs (Figure 5).

[0445] Results for Exemplary Method 1c In another version of the assay, the adhesive screening assay mixture was assembled in a manner similar to exemplary method 1b, using RBM39 and DCAF15 proteins instead of IKZF2 and CRBN proteins.

[0446] When a protein mixture containing 1.25 μM RBM39 tagged with GFP11 in a complex with DDB1&DDA1 (hereafter referred to as DCAF15, because DCAF15 has always been used as a complex with DDB1&DDA1), 0.5 μM DCAF15 tagged with GFP10, and 5 μM GFP1-9 was added to a compound known as an established adhesive for RBM39:DCAF15 (NSC-339004, Tasislam, E7820, and E7070), the GFP signal increased in a compound dose-dependent manner compared to the DMSO control. The fluorescence signal was measured at multiple time points over a 16-hour incubation. The initial rate (k1) was obtained by linearly fitting the data during the first 8 hours. The ratio of the k1 value to that of the DMSO-treated sample (S / B) is shown for each concentration of different compounds (Figure 6).

[0447] Equivalents Details of one or more embodiments of this disclosure are shown in the accompanying description above. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described herein. Other features, purposes, and advantages of this disclosure will be apparent from the description and the claims. In this specification and the accompanying claims, singular nouns include plural nouns unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference.

[0448] The foregoing description is provided for illustrative purposes only and is not intended to limit this disclosure to the exact form disclosed, but rather to the claims appended herein.

Claims

1. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, wherein the assay mixture is (a) The first protein or a fragment thereof covalently attached to the first tag GFP fragment; (b) A second protein or fragment thereof covalently attached to the second tag GFP fragment; (c) A detector GFP fragment complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compound Includes, When the assay mixture yields a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound, the first tag GFP fragment, the second tag GFP fragment, and the detector GFP fragment are configured to generate or enhance the assay signal. To provide; and (ii) Identifying the first protein, the second protein, the compound, or any combination thereof associated with the complex, wherein the identified compound is capable of regulating the protein-protein interaction (PPI) between the first protein and the second protein. Methods that include...

2. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, wherein the assay mixture is (a) The first protein or a fragment thereof covalently attached to the first tag GFP fragment; (b) A second protein or fragment thereof covalently attached to the second tag GFP fragment; (c) A detector GFP fragment complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compound Includes, The first protein or fragment thereof, and / or the second protein or fragment thereof, are covalently attached to the affinity component; and When the assay mixture yields a complex comprising the first protein or a fragment thereof, the second protein or a fragment thereof, and the compound, the first tag GFP fragment, the second tag GFP fragment, and the detector GFP fragment are configured to generate or enhance the assay signal. To provide; (ii-a) Contacting the complex with an immobilized affinity binder that targets the affinity component, thereby forming an immobilized complex; and (ii-b) Detecting and / or isolating the immobilized complex and thereby identifying the first protein, the second protein, the compound, or any combination thereof, wherein the identified compound is capable of modulating the protein-protein interaction (PPI) between the first protein and the second protein. Methods that include...

3. A method for identifying a first protein, a second protein, a compound that targets the first protein and the second protein, or any combination thereof, (i) To provide an assay mixture, wherein the assay mixture is (a) The first protein or a fragment thereof covalently attached to the first tag GFP fragment; (b) A second protein or fragment thereof covalently attached to the second tag GFP fragment; (c) A detector GFP fragment complementary to the first tag GFP fragment and the second tag GFP fragment; and (d) Candidate compound Includes, The assay mixture is configured such that when it induces proximity between the first protein or a fragment thereof and the second protein or a fragment thereof, the first tagged GFP fragment, the second tagged GFP fragment, and the detector GFP fragment generate or enhance the assay signal. To provide; and (ii) Identifying the first protein, the second protein, the compound, or any combination thereof that is associated with the induction of proximity. Includes, The identified compound is capable of causing degradation of the first protein in the presence of the second protein. method.

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

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

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

7. The method according to any one of the claims, wherein the assay mixture yields 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 claims, wherein the assay signal is fluorescent.

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

10. The method according to any one of the claims, wherein the first tag GFP fragment and the second tag GFP fragment independently comprise one or more GFP 10 and GFP 11.

11. The method according to any one of the claims, wherein the first tag GFP fragment comprises GFP 10 and the second tag GFP fragment comprises GFP 11.

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

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

15. The method according to any one of the claims, further comprising step (i) detecting the generated or enhanced assay signal.

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

17. A first protein identified by the method described in any one of the preceding claims.

18. A second protein identified by the method described in any one of the preceding claims.

19. A combination of a first protein and a second protein identified by the method according to any one of the claims above.

20. A combination of a first protein, a second protein, and a compound that targets the first protein and the second protein, as identified by the method of any one of the claims.

21. A compound identified by the method described in any one of the above claims.

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

23. A pharmaceutical composition comprising a compound identified by the method described in any one of the above claims.

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

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

26. The method according to claim 25, wherein the PPI results in changes in protein stability, changes at the protein level, changes in post-translational modification of proteins, changes in protein localization, and / or changes in protein activity.

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

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