Detection of molecular interactions

Abstract

Methods for detecting molecular interactions, such as protein / protein interactions or small molecule / protein interactions, are described. That is, the present invention relates, in part, to cell-based systems for detecting various molecular interactions. In some embodiments, the present invention provides methods that allow for the testing of molecular interactions (e.g., protein / protein interactions, protein / small molecule interactions, and / or protein / protein interactions regulated by small molecules) that are not detectable using standard assays.

Description

Technical Field

[0001] The present invention relates, inter alia, to protein / protein interactions or protein / small molecule interactions, and / or the detection and identification of novel small molecules.

[0002] Cross-reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 949,023, filed on December 17, 2019. The entire disclosure of the priority application is incorporated herein by reference.

[0003] Description of the Text File Submitted Electronically The content of the text file submitted electronically that is appended to this specification is incorporated herein by reference in its entirety: a computer-readable form copy of the sequence listing (file name: "ORN-060PC_ST25.txt"; creation date: December 7, 2020; file size: 10,349 bytes).

Background Art

[0004] Molecular interactions, such as protein / protein interactions and protein / small molecule interactions, are crucial parts of many, if not all, biological processes. Several approaches have been developed to identify molecular interactions. For example, biochemical approaches include co-purification and immunoprecipitation. However, these techniques are cumbersome, do not enable large-scale, rapid screening, and require lysis, thus losing the normal cellular environment. Genetic approaches address some of these problems. For example, the yeast 2-hybrid method has shown outstanding usefulness. However, despite its widespread use, the yeast 2-hybrid system has several drawbacks; one such drawback is the need for nuclear translocation of the fusion protein. Another approach is phage display, which does not require nuclear translocation. However, the phage display approach is too unnatural; it requires exposing the protein to the phage surface, exposing the protein to an environment that is not physiologically appropriate, making it difficult to judge whether it is equivalent to interactions in living cells.

[0005] In other words, there is still a need for novel methods to detect molecular interactions. [Overview of the project]

[0006] Therefore, the present invention relates in part to a cell-based system for detecting various molecular interactions. In some embodiments, the present invention provides a method that enables testing of molecular interactions that are undetectable using standard assays (e.g., protein / protein, protein / small molecule, and / or protein / protein interactions regulated by small molecules). For example, in some embodiments, the method reduces or eliminates the occurrence of false positive or false negative signals.

[0007] In some embodiments, the method utilizes reversing the bait and play relative to known methods; for example, cytokine receptor-based interactive capture methods and methods such as those described herein, which enable improved molecular interaction detection.

[0008] For example, as described herein, "forward" mammalian protein / protein interaction traps (for MAPPIT, see Eyckerman et al., "Design and application of a cytokine-receptor-based interaction trap," Nat Cell Biol. 2001 Dec;3(12):1114-9; and Lievens et al., "Proteome-scale binary interactomics in human cells," Molecular & Cellular Proteomics 15.12 (2016): 3624-3639) may not work well in some cases. In various embodiments of the present invention, these problems are resolved, for example, by reversing the method design. For example, in various embodiments, the method enables the detection and / or discovery of interactions that are not clearly identified by the forward MAPIT method because, for example, when expressed in the forward MAPIT assay form (as described herein), the interaction partner is sequestered in the cell nucleus or / intracellular organelle. To give another example, in some embodiments, the method enables the detection of interactions that are not clearly identified by the forward MAPIT method because, when expressed in the forward MAPIT assay form, the interaction partner expressed in that assay form makes nonspecific contact with the cell membrane and / or nonspecific contact with the membrane-based construct used for this detection.

[0009] In various embodiments, the present invention relates to a method for detecting molecular interactions, wherein the method is: (a) To provide cells having ligand-based chimeric receptors, The chimeric receptor, (i) Extracellular portion of the ligand-binding domain derived from the first receptor, and (ii) The transmembrane domain and intracellular domain of the second receptor and the intracellular prey protein fused thereto, Includes, Here, the transmembrane domain and / or intracellular domain of the second receptor are transmembrane domains and intracellular domains that contain mutations that reduce or eliminate the recruitment of STAT (Signal Transducer and Activator of Transcription). To provide cells; (b) Expressing a bait protein fused to a receptor fragment in a cell, Here, the receptor fragment includes a functional STAT recruitment site. To express, and (c) detecting signals that suggest the presence of molecular interactions, Here, the bait protein, (i) They tend to localize in the cytoplasm rather than inside membrane-bound intracellular organelles, and / or (ii) Having a tendency to interact specifically with prey proteins rather than nonspecifically with the cell membrane and / or the non-prey portion of the chimeric receptor, Detecting a signal, This method detects molecular interactions. In this embodiment, the first receptor and the second receptor are the same receptor.

[0010] In some embodiments, the interaction between the prey protein and the bait protein triggers the recruitment of a receptor fragment to the intracellular domain of the second receptor fused to the first receptor, thereby restoring ligand-dependent receptor signaling and activating the STAT molecule. In some embodiments, the cell contains a STAT-responsive reporter gene. In some embodiments, the activated STAT molecule translocates to the nucleus and induces transcription of the STAT-responsive reporter gene, allowing for the detection of molecular interactions through this reporter gene signal.

[0011] In some embodiments, the molecular interaction is a protein / protein interaction. In some embodiments, the molecular interaction is a small molecule-mediated protein / protein interaction (for example, the method further includes introducing a small molecule that binds to a prey protein or bait protein). Specifically, in some embodiments, the molecular interaction is a protein / protein interaction mediated by the binding of a small molecule to a prey protein or bait protein. For example, the method may detect complex formation. In some embodiments, the small molecule induces the exposure of a hydrophobic surface of the prey protein or bait protein, enabling interaction with the prey protein or bait protein. In some embodiments, the small molecule is a molecular glue or a divalent hybrid ligand molecule (for example, PROTAC).

[0012] For example, in some embodiments, the interactions to be detected include, but are not limited to, immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, and related compounds; and / or compounds that bind to the same or similar site (pocket) of the cerebron (CRBN) protein, and in some embodiments, include an E3 ligase protein in contact with a compound that is a bait fused (or indirectly bound) to the receptor fragment of this "reverse" assay. [Brief explanation of the drawing]

[0013] [Figure 1] The concept of MAPPIT is presented. A bait protein ("B") is fused to the C-terminus of a chimeric receptor, which has, for example, the extracellular portion of a type I cytokine receptor ("CYT"), as well as the transmembrane and intracellular domains of a receptor created by mutation to impair STAT recruitment. When a prey protein ("P") fused to the receptor fragment containing the functional STAT recruitment site is co-expressed, the receptor complex functionally complements itself and restores (L) signaling upon cytokine ligand stimulation. The STAT molecule is activated and translocates to the nucleus, inducing transcription of a STAT-responsive reporter gene. Throughout this disclosure, this method is referred to as the "forward" assay. [Figure 2A] Figure 1 outlines the failure of the system, i.e., the occurrence of a false negative signal. Here, the bait fused to the membrane construct is inaccessible to the play, for example, because it is isolated in the nucleus and / or intracellular organelle (left panel). The right panel shows the inversion between the bait and play, releasing the play from isolation, thereby enabling the detection of molecular interactions (throughout this disclosure, this scheme will be referred to as the “reverse” assay). [Figure 2B] Figure 1 outlines another example of a further failure in the system, namely the occurrence of a false negative signal. Here, the prey interacts nonspecifically with the membrane construct (left panel). The right panel shows a reversal of the bait-prey interaction to weaken / reduce the nonspecific interaction (right panel; throughout this disclosure, this scheme will be referred to as the “reverse” assay). [Figure 3] An overview of another construct of the "reverse" assay is provided. In this construct, a bait that can interact with small molecules and / or prey associates with a scaffold protein. Shaded segments are scaffold proteins that are expressed independently in cells or as direct fusions with the bait protein. [Figure 4A]Evaluation of CRBN-binding compounds that recruit selected substrates using MAPPIT forward and / or reverse assay configurations. Recruitment induced by lenalidomide and CC-220, CRBN IMiD ligands from a known CRBN substrate panel described in the literature, was evaluated using MAPPIT. MAPPIT is a modification of a previously reported two-hybrid technology system (Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55; this reference is incorporated herein by reference in its entirety) and will be outlined in more detail in Example 1. More specifically, two configurations, referred to as the "forward" and "reverse" assay modes of the MAPPIT assay, were compared. The difference between these two configurations, as outlined more specifically in Example 1, is whether the bait protein, in this case CRBN, is fused to the MAPPIT chimeric transmembrane receptor (in the classical "forward" mode) or to the soluble gp130 receptor moiety (in the "reverse" mode). In both assay configurations, the activity of the test compound was evaluated while increasing the concentration of the test compound to monitor its ability to promote CRBN ligand-induced protein interactions (i.e., interactions with the indicated neosubstrate: IKZF1, IKZF3, IKZF2, SALL4, ZFP91, or CSNK1A (CK1a)) (dose-response test). As can be seen from the figure, in the case of IKZF1, compound-induced CRBN binding was detectable in both the forward and reverse assay configurations, but in all other cases, the interaction signal response was detectable only in the reverse assay configuration. [Figure 4B]Evaluation of CRBN-binding compounds that recruit selected substrates using MAPPIT forward and / or reverse assay configurations. Recruitment induced by lenalidomide and CC-220, CRBN IMiD ligands from a known CRBN substrate panel described in the literature, was evaluated using MAPPIT. MAPPIT is a modification of a previously reported two-hybrid technology system (Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55; this reference is incorporated herein by reference in its entirety) and will be outlined in more detail in Example 1. More specifically, two configurations, referred to as the "forward" and "reverse" assay modes of the MAPPIT assay, were compared. The difference between these two configurations, as outlined more specifically in Example 1, is whether the bait protein, in this case CRBN, is fused to the MAPPIT chimeric transmembrane receptor (in the classical "forward" mode) or to the soluble gp130 receptor moiety (in the "reverse" mode). In both assay configurations, the activity of the test compound was evaluated while increasing the concentration of the test compound to monitor its ability to promote CRBN ligand-induced protein interactions (i.e., interactions with the indicated neosubstrate: IKZF1, IKZF3, IKZF2, SALL4, ZFP91, or CSNK1A (CK1a)) (dose-response test). As can be seen from the figure, in the case of IKZF1, compound-induced CRBN binding was detectable in both the forward and reverse assay configurations, but in all other cases, the interaction signal response was detectable only in the reverse assay configuration. [Figure 4C]Evaluation of CRBN-binding compounds that recruit selected substrates using MAPPIT forward and / or reverse assay configurations. Recruitment induced by lenalidomide and CC-220, CRBN IMiD ligands from a known CRBN substrate panel described in the literature, was evaluated using MAPPIT. MAPPIT is a modification of a previously reported two-hybrid technology system (Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55; this reference is incorporated herein by reference in its entirety) and will be outlined in more detail in Example 1. More specifically, two configurations, referred to as the "forward" and "reverse" assay modes of the MAPPIT assay, were compared. The difference between these two configurations, as outlined more specifically in Example 1, is whether the bait protein, in this case CRBN, is fused to the MAPPIT chimeric transmembrane receptor (in the classical "forward" mode) or to the soluble gp130 receptor moiety (in the "reverse" mode). In both assay configurations, the activity of the test compound was evaluated while increasing the concentration of the test compound to monitor its ability to promote CRBN ligand-induced protein interactions (i.e., interactions with the indicated neosubstrate: IKZF1, IKZF3, IKZF2, SALL4, ZFP91, or CSNK1A (CK1a)) (dose-response test). As can be seen from the figure, in the case of IKZF1, compound-induced CRBN binding was detectable in both the forward and reverse assay configurations, but in all other cases, the interaction signal response was detectable only in the reverse assay configuration. [Figure 4D]Evaluation of CRBN-binding compounds that recruit selected substrates using MAPPIT forward and / or reverse assay configurations. Recruitment induced by lenalidomide and CC-220, CRBN IMiD ligands from a known CRBN substrate panel described in the literature, was evaluated using MAPPIT. MAPPIT is a modification of a previously reported two-hybrid technology system (Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55; this reference is incorporated herein by reference in its entirety) and will be outlined in more detail in Example 1. More specifically, two configurations, referred to as the "forward" and "reverse" assay modes of the MAPPIT assay, were compared. The difference between these two configurations, as outlined more specifically in Example 1, is whether the bait protein, in this case CRBN, is fused to the MAPPIT chimeric transmembrane receptor (in the classical "forward" mode) or to the soluble gp130 receptor moiety (in the "reverse" mode). In both assay configurations, the activity of the test compound was evaluated while increasing the concentration of the test compound to monitor its ability to promote CRBN ligand-induced protein interactions (i.e., interactions with the indicated neosubstrate: IKZF1, IKZF3, IKZF2, SALL4, ZFP91, or CSNK1A (CK1a)) (dose-response test). As can be seen from the figure, in the case of IKZF1, compound-induced CRBN binding was detectable in both the forward and reverse assay configurations, but in all other cases, the interaction signal response was detectable only in the reverse assay configuration. [Figure 4E]Evaluation of CRBN-binding compounds that recruit selected substrates using MAPPIT forward and / or reverse assay configurations. Recruitment induced by lenalidomide and CC-220, CRBN IMiD ligands from a known CRBN substrate panel described in the literature, was evaluated using MAPPIT. MAPPIT is a modification of a previously reported two-hybrid technology system (Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55; this reference is incorporated herein by reference in its entirety) and will be outlined in more detail in Example 1. More specifically, two configurations, referred to as the "forward" and "reverse" assay modes of the MAPPIT assay, were compared. The difference between these two configurations, as outlined more specifically in Example 1, is whether the bait protein, in this case CRBN, is fused to the MAPPIT chimeric transmembrane receptor (in the classical "forward" mode) or to the soluble gp130 receptor moiety (in the "reverse" mode). In both assay configurations, the activity of the test compound was evaluated while increasing the concentration of the test compound to monitor its ability to promote CRBN ligand-induced protein interactions (i.e., interactions with the indicated neosubstrate: IKZF1, IKZF3, IKZF2, SALL4, ZFP91, or CSNK1A (CK1a)) (dose-response test). As can be seen from the figure, in the case of IKZF1, compound-induced CRBN binding was detectable in both the forward and reverse assay configurations, but in all other cases, the interaction signal response was detectable only in the reverse assay configuration. [Figure 4F]Evaluation of CRBN-binding compounds that recruit selected substrates using MAPPIT forward and / or reverse assay configurations. Recruitment induced by lenalidomide and CC-220, CRBN IMiD ligands from a known CRBN substrate panel described in the literature, was evaluated using MAPPIT. MAPPIT is a modification of a previously reported two-hybrid technology system (Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55; this reference is incorporated herein by reference in its entirety) and will be outlined in more detail in Example 1. More specifically, two configurations, referred to as the "forward" and "reverse" assay modes of the MAPPIT assay, were compared. The difference between these two configurations, as outlined more specifically in Example 1, is whether the bait protein, in this case CRBN, is fused to the MAPPIT chimeric transmembrane receptor (in the classical "forward" mode) or to the soluble gp130 receptor moiety (in the "reverse" mode). In both assay configurations, the activity of the test compound was evaluated while increasing the concentration of the test compound to monitor its ability to promote CRBN ligand-induced protein interactions (i.e., interactions with the indicated neosubstrate: IKZF1, IKZF3, IKZF2, SALL4, ZFP91, or CSNK1A (CK1a)) (dose-response test). As can be seen from the figure, in the case of IKZF1, compound-induced CRBN binding was detectable in both the forward and reverse assay configurations, but in all other cases, the interaction signal response was detectable only in the reverse assay configuration. [Figure 5]Immunofluorescence staining has shown that the IKZF3 gp130 fusion construct localizes in the nucleus, which explains the lack of signal in the corresponding MAPPIT forward assay. As discussed in more detail in Example 1, one of the reasons for the inability to detect a specific interaction in the MAPPIT assay is that the gp130 domain fusion of the target protein used in the assay is not expressed in the intracellular cytoplasmic compartment and thus cannot form a complex with the membrane-bound bait protein. One example shown in FIGS. 4A-F is the interaction with IKZF3. In this case, compound-induced mobilization of CRBN was detectable only in the reverse mode and not in the forward assay configuration. In the latter forward assay configuration, the MAPPIT chimeric receptor fusion of CRBN was used in combination with the gp130-IKZF3 fusion protein. Here, immunofluorescence staining of the gp130-IKZF3 fusion protein was performed. The gp130-IKZF1 fusion protein was used as a control; the reason is that the CRBN-IKZF1 interaction was detectable in the forward mode. In the immunofluorescence image, for IKZF1, green staining indicating the gp130 fusion protein in the cytoplasm can be observed, while in the case of the IKZF3 fusion, its expression was almost exclusively limited to the nuclear compartment (visualized as blue staining). This explains the reason why the CRBN / IKZF3 interaction is assay-inable in the forward MAPPIT configuration and possible only in the reverse mode; in the reverse mode, IKZF3 is linked to the MAPPIT membrane receptor with its cytoplasmic exposure and thus available for interaction with the cytoplasmic CRBN bait protein. [Figure 6]MAPPIT binding analysis shows that the CSNK1A1-gp130 fusion protein exhibits strong, non-specific binding to the MAPPIT chimeric receptor construct, resulting in a high background reporter signal in the absence of molecular glue in the CRBN interaction assay. This explains the lack of compound / molecular glue-induced response specificity for this interaction in the forward assay configuration. As described in Example 1, another reason why the forward configuration of MAPPIT may not detect compound-inducible interactions for specific targets is that when cloned and expressed as a gp130 fusion protein, these targets exhibit high affinity for components of the MAPPIT chimeric receptor that are not bait proteins (such as the cytoplasmic tail of the leptin receptor or the receptor-binding JAK2 protein). An example of such a case is CSNK1A1 (or CK1a) shown in Figures 4A-F, in which case a specific signal of lenalidomide-inducible or CC-220-inducible is clearly shown only in the reverse MAPPIT configuration, and no such signal is obtained in the forward MAPPIT configuration. Here, we performed MAPPIT binding analysis to test the CSNK1A1 / gp130 fusion protein (CSNK1A1 fused to the N-terminus or C-terminus of the gp130 subdomain used in MAPPIT) for binding to MAPPIT chimeric receptor fusions or fusions of unrelated E. coli DHFR (dihydrofolate reductase) protein and CRBN bait. The luciferase reporter signal, which indicates the strength of the interaction, shows that the CSNK1A1-gp130 fusion interacts / binds to both receptor fusions. This suggests that this binding is not CRBN-specific, but rather that CSNK1A1 interacts with the chimeric receptor components themselves. In this configuration used to evaluate CRBN-binding proteins, the strong reporter signal obtained in the absence of specific compound-inducible interactions with CRBN masks the additional signal induced by the interaction of specific ply / gp130 fusions with CRBN, thus giving a behavior similar to that observed with CSNK1A1, making the forward assay configuration unsuitable for analyzing protein-CRBN interactions. [Figure 7]Analysis of compound-dependent CRBN-CSNK1A1 interaction by MAPPIT reverse configuration using an alternative CSNK1A1 chimeric receptor fusion protein. As discussed in Example 1, multiple receptor fusion constructs can be used in the MAPPIT assay. A typical fusion protein consists of the extracellular domain of the EPO receptor fused to the transmembrane and intracellular parts of a mutant leptin receptor; this is the construct used in FIGS. 4A-F, FIG. 6 and FIG. 8. However, the extracellular domain of the EPO receptor can be replaced by the extracellular domain of the leptin receptor, in which case an assay system activated by leptin rather than EPO is obtained. Here, the inventors used a CRBN gp130 fusion with an alternative CSNK1A1 receptor fusion and retested the lenalidomide-induced and CC-220-induced CRBN-CSNK1A1 interactions shown in FIGS. 4A-F; in this case, the extracellular domain of the leptin receptor was used instead of the extracellular domain of the EPO receptor, and the assay was activated with leptin. This result shows that detection of CSNK1A1 neo-substrate mobilization is possible even with this alternative receptor construct if it is in the reverse assay configuration. In each set of histograms, the leftmost bar represents 0 μM, the bar to its immediate right represents 0.1 μM, the bar to the immediate right of that represents 1 μM, and the rightmost bar represents 10 μM. [Figure 8A] Evaluation of compound-dependent FKBP1A (FKBP12) / target interactions by MAPPIT forward and reverse assay configurations. Similar to the analysis of FIGS. 4A-F for CRBN target interactions, the compound-dependent interactions of FKBP1A (FKBP12) with known target proteins were evaluated in MAPPIT forward and reverse configurations. As can be seen from the figure, rapamycin-induced mobilization of MTOR was detected in both the MAPPIT forward and reverse modes. Similarly, FK506-dependent binding of the catalytic PPP3CA subunit of calcineurin can also be monitored in both assay modes. It should be noted that in the case of calcineurin binding, co-expression of the PPP3R2 regulatory subunit significantly increases the signal window in both assay configurations; however, the degree is smaller in the reverse mode compared to the forward mode. [Figure 8B]Evaluation of compound-dependent FKBP1A (FKBP12) / target interactions using MAPPIT forward and reverse assay configurations. Similar to the analysis of CRBN target interactions in Figures 4A-F, compound-dependent interactions of FKBP1A (FKBP12) to known target proteins were evaluated using MAPPIT forward and reverse configurations. As can be seen from the figures, rapamycin-inducible recruitment of MTOR was detected in both MAPPIT forward and reverse modes. Similarly, FK506-dependent binding of the calcineurin-catalyzed PPP3CA subunit is also monitorable in both assay modes. Notably, in the case of calcineurin binding, co-expression of the PPP3R2 regulatory subunit significantly increases the signal window in both assay configurations; however, this is less pronounced in the reverse mode compared to the forward mode. [Figure 8C] Evaluation of compound-dependent FKBP1A (FKBP12) / target interactions using MAPPIT forward and reverse assay configurations. Similar to the analysis of CRBN target interactions in Figures 4A-F, compound-dependent interactions of FKBP1A (FKBP12) to known target proteins were evaluated using MAPPIT forward and reverse configurations. As can be seen from the figures, rapamycin-inducible recruitment of MTOR was detected in both MAPPIT forward and reverse modes. Similarly, FK506-dependent binding of the calcineurin-catalyzed PPP3CA subunit is also monitorable in both assay modes. Notably, in the case of calcineurin binding, co-expression of the PPP3R2 regulatory subunit significantly increases the signal window in both assay configurations; however, this is less pronounced in the reverse mode compared to the forward mode. [Figure 9]In the MAPPIT reverse configuration, inducible binding of the trimethoprim-lenalidomide hybrid ligand between CRBN and DHFR can be detected. A hybrid molecule consisting of DHFR ligand-trimethoprim (TMP) fused to lenalidomide, the CRBN ligand, via a PEG linker was used in the MAPPIT assay to induce DHFR recruitment to the CRBN bait. As shown in the figure, in the reverse assay configuration using CRBN expressed as a gp130 fusion and DHFR linked to the MAPPIT chimeric membrane receptor, a TMP-LEN dose-dependent signal can be observed. [Figure 10A]Utilization of the CRBN Reverse MAPIT Assay to Evaluate CRBN Binding of IMiD and Other Molecular Glues. Here, the reverse MAPIT TMP-LEN-dependent DHFR-CRBN binding assay shown in Figure 9 was used to evaluate the binding of CRBN molecular glues in a competitive setting. Cells transformed with appropriate cDNA encoding the transgene (DHFR and CRBN fusion protein) were used to generate a positive assay signal by adding the TMP-LEN hybrid ligand as shown in Figure 9. The ligand was set to 100% luciferase activity. In separate sample settings, cells were prepared in the same manner, but these cells were further incubated with the test compound whose interaction with CRBN was investigated. Binding to the CRBN fusion protein competes with the binding of the hybrid ligand to the same CRBN protein, thus inhibiting the assay signal by preventing the formation of the triple complex necessary for the generation of the assay signal. To determine the CRBN binding efficiency, as determined in this type of ligand competition experiment using live cells, the concentration of the test compound was increased and evaluated. As can be seen from the figure, known IMiD compounds (lenalidomide / LEN, pomalidomide / POM, CC-122, CC-220) effectively competed with lenalidomide hybrid ligands for binding to CRBN (dose-response curve for CRBN association assay signal inhibition). Similarly, other types of compounds also effectively competed at various levels of efficacy. Signal inhibition specificity is evaluated in a parallel experimental setting, in which the effect of the test compound is assessed in terms of inhibiting the signal generated by a control gp-130 fusion protein (CTRL) that directly binds to the DHFR receptor fusion protein (i.e., direct protein interaction) in the absence of the hybrid ligand. [Figure 10B]Utilization of the CRBN Reverse MAPIT Assay to Evaluate CRBN Binding of IMiD and Other Molecular Glues. Here, the reverse MAPIT TMP-LEN-dependent DHFR-CRBN binding assay shown in Figure 9 was used to evaluate the binding of CRBN molecular glues in a competitive setting. Cells transformed with appropriate cDNA encoding the transgene (DHFR and CRBN fusion protein) were used to generate a positive assay signal by adding the TMP-LEN hybrid ligand as shown in Figure 9. The ligand was set to 100% luciferase activity. In separate sample settings, cells were prepared in the same manner, but these cells were further incubated with the test compound whose interaction with CRBN was investigated. Binding to the CRBN fusion protein competes with the binding of the hybrid ligand to the same CRBN protein, thus inhibiting the assay signal by preventing the formation of the triple complex necessary for the generation of the assay signal. To determine the CRBN binding efficiency, as determined in this type of ligand competition experiment using live cells, the concentration of the test compound was increased and evaluated. As can be seen from the figure, known IMiD compounds (lenalidomide / LEN, pomalidomide / POM, CC-122, CC-220) effectively competed with lenalidomide hybrid ligands for binding to CRBN (dose-response curve for CRBN association assay signal inhibition). Similarly, other types of compounds also effectively competed at various levels of efficacy. Signal inhibition specificity is evaluated in a parallel experimental setting, in which the effect of the test compound is assessed in terms of inhibiting the signal generated by a control gp-130 fusion protein (CTRL) that directly binds to the DHFR receptor fusion protein (i.e., direct protein interaction) in the absence of the hybrid ligand. [Figure 11]Detection of TMP-FK506 inducible binding between FKBP1A (FKBP12) and DHFR using a reverse MAPIT assay configuration. The compound-inducible binding of DHFR to FKBP1A bait was tested using a hybrid molecule consisting of DHFR ligand-trimethoprim (TMP) fused to FKBP1A (FKBP12) ligand FK506 via a PEG linker; this test was performed using a reverse MAPIT assay configuration with FKBP1A as a gp130 fusion and DHFR linked to a chimeric membrane receptor. As can be seen in the figure, a clear dose-dependent MAPIT signal was observed, indicating that this reverse MAPIT assay configuration allows for the evaluation of FK506 hybrid ligand-inducible interactions between FKBP1A and DHFR. [Figure 12] In the MAPPIT reverse configuration, sulfonamide-inducible recruitment of RBM39 to DCAF15 is detectable, but not in the forward configuration. Similar to glue-inducible substrate recruitment to CRBN, compound-inducible substrate binding of other E3 ligases has also been reported (e.g., sulfonamide-dependent recruitment of RBM39 to DCAF15). Sulfonamide-inducible recruitment of RBM39 to DCAF15 was evaluated using MAPPIT in either the forward configuration (DCAF15 receptor fusion co-expressed with RBM39 gp130 fusion) or the reverse configuration (RBM39 receptor fusion combined with DCAF15 gp130 fusion). Different sulfonamides were evaluated: indislam (forward and reverse modes), as well as tasislam, chloroquinoxaline sulfonamide (CQS), and E7820 (reverse mode). As can be seen from the figure, dose-dependent luciferase signal increase was observed only in the reverse mode. [Figure 13A]By screening compound assemblies, we identified novel molecular glues that enable the recruitment of SALL4 to CRBN. Using a reverse MAPIT assay co-expressing CRBN gp130 fusion constructs and SALL4 chimeric receptor fusion constructs, we screened 96 IMiD and IMiD-like compound assemblies. In the first screening, we tested three different doses (low, medium, and high concentrations) of the compounds and determined the luciferase reporter signal. The curves shown in Figures 13A-C (left panel) show the luciferase signal frequency distribution for both compound-treated samples and DMSO-treated controls. The curve for compound-treated samples is bimodal, with the right-shifted peaks corresponding to compounds showing higher reporter signals than the DMSO-treated controls. For three compounds that showed responsiveness and therefore induced the recruitment of SALL4 to CRBN, confirmation of dose responsiveness (hit) is shown in the right panel. The signals corresponding to each test concentration in the first screening are shown as straight lines of the corresponding line type used in the dose-response curve (dotted, dashed, or solid). These sample curves demonstrate that the MAPPIT reverse approach can identify molecular glues over a wide efficacy range. [Figure 13B]By screening compound assemblies, we identified novel molecular glues that enable the recruitment of SALL4 to CRBN. Using a reverse MAPIT assay co-expressing CRBN gp130 fusion constructs and SALL4 chimeric receptor fusion constructs, we screened 96 IMiD and IMiD-like compound assemblies. In the first screening, we tested three different doses (low, medium, and high concentrations) of the compounds and determined the luciferase reporter signal. The curves shown in Figures 13A-C (left panel) show the luciferase signal frequency distribution for both compound-treated samples and DMSO-treated controls. The curve for compound-treated samples is bimodal, with the right-shifted peaks corresponding to compounds showing higher reporter signals than the DMSO-treated controls. For three compounds that showed responsiveness and therefore induced the recruitment of SALL4 to CRBN, confirmation of dose responsiveness (hit) is shown in the right panel. The signals corresponding to each test concentration in the first screening are shown as straight lines of the corresponding line type used in the dose-response curve (dotted, dashed, or solid). These sample curves demonstrate that the MAPPIT reverse approach can identify molecular glues over a wide efficacy range. [Figure 13C]By screening compound assemblies, we identified novel molecular glues that enable the recruitment of SALL4 to CRBN. Using a reverse MAPIT assay co-expressing CRBN gp130 fusion constructs and SALL4 chimeric receptor fusion constructs, we screened 96 IMiD and IMiD-like compound assemblies. In the first screening, we tested three different doses (low, medium, and high concentrations) of the compounds and determined the luciferase reporter signal. The curves shown in Figures 13A-C (left panel) show the luciferase signal frequency distribution for both compound-treated samples and DMSO-treated controls. The curve for compound-treated samples is bimodal, with the right-shifted peaks corresponding to compounds showing higher reporter signals than the DMSO-treated controls. For three compounds that showed responsiveness and therefore induced the recruitment of SALL4 to CRBN, confirmation of dose responsiveness (hit) is shown in the right panel. The signals corresponding to each test concentration in the first screening are shown as straight lines of the corresponding line type used in the dose-response curve (dotted, dashed, or solid). These sample curves demonstrate that the MAPPIT reverse approach can identify molecular glues over a wide efficacy range. [Figure 14]ORF cDNA library screening to identify novel molecular glue-inducible CRBN neosubstrates. Here, a MAPPIT reverse approach was used to screen a human ORF(eome) cDNA library for targets recruited to CRBN in response to CC-220, a known IMiD agent and CRBN ligand. Intracellular protein-small molecule interactions were assayed within cell clusters in array form. Each spot in the cell microarray corresponds to such a cell cluster expressing a single test ORF / protein candidate related to ligand-inducible (in this case, CC-220-inducible) interaction with CRBN. Positive interactions are measured as an increase in cell fluorescence. A dot plot of fluorescence intensity data from cell microarray screening across / related to numerous individual ORF / target protein candidates is shown. The X-axis represents the number of particles, and the Y-axis represents the integrated intensity of each cell cluster in the microarray. As can be seen from the figure, a significant induction of signal is observed for each ORF cDNA. For three ORF cDNAs that showed a response and were therefore shown to be proteins recruited to CRBN by the CC-220 molecular glue (indicated by arrows), dose-response curves were constructed to confirm the dose-dependent binding of CC-220 to CRBN. These examples demonstrate that this reverse MAPPIT screening approach enables the identification of novel molecular glue-inducible substrates of CRBN. [Figure 15A]Identification of rapamycin-inducible binding between FKBP proteins and MTOR using MAPPIT forward and reverse configurations. Regarding MTOR (FRB domain) recruitment, different members of the FKBP protein family (FKBP1A / FKBP12, FKBP3, FKBP4, and FKBP5) were evaluated using either a forward assay configuration (FKBP receptor fusion and MTOR gp130 fusion) or a reverse assay configuration (MTOR receptor fusion and FKBP gp130 fusion) in the MAPPIT assay. As shown in the figure, for each FKBP protein tested, a rapamycin-dependent signal was obtained in both the forward and reverse configurations, which were consistent with previously reported findings. In each set of histograms, the leftmost bar represents 0 nM rapamycin, the bar to its right represents 1 nM rapamycin, the bar to its right represents 10 nM rapamycin, and the rightmost bar represents 100 nM rapamycin. [Figure 15B] Identification of rapamycin-inducible binding between FKBP proteins and MTOR using MAPPIT forward and reverse configurations. Regarding MTOR (FRB domain) recruitment, different members of the FKBP protein family (FKBP1A / FKBP12, FKBP3, FKBP4, and FKBP5) were evaluated using either a forward assay configuration (FKBP receptor fusion and MTOR gp130 fusion) or a reverse assay configuration (MTOR receptor fusion and FKBP gp130 fusion) in the MAPPIT assay. As shown in the figure, for each FKBP protein tested, a rapamycin-dependent signal was obtained in both the forward and reverse configurations, which were consistent with previously reported findings. In each set of histograms, the leftmost bar represents 0 nM rapamycin, the bar to its right represents 1 nM rapamycin, the bar to its right represents 10 nM rapamycin, and the rightmost bar represents 100 nM rapamycin. [Modes for carrying out the invention]

[0014] This disclosure is partly based on the discovery of cell-based systems and methods that enable the testing of molecular interactions (e.g., protein / protein, protein / small molecule, and / or protein / protein interactions regulated by small molecules) that are undetectable using standard assays. In one aspect, the method enables a method for detecting molecular interactions, where the method is: (a) To provide cells containing ligand-based chimeric receptors, A chimeric receptor based on the ligand, (i) Extracellular portion of the ligand-binding domain derived from the first receptor, and (ii) The transmembrane domain and intracellular domain of the second receptor and the intracellular prey protein fused thereto, Herein, the transmembrane domain and / or intracellular domain of the second receptor include mutations that reduce or eliminate STAT recruitment. Transmembrane domain and intracellular domain, including, To provide cells; (b) Expressing a bait protein fused to a receptor fragment in a cell, Here, the receptor fragment includes a functional STAT recruitment site. To express, and (c) detecting signals that suggest the presence of molecular interactions, Here, the bait protein, (i) They tend to localize in the cytoplasm rather than inside membrane-bound intracellular organelles, and / or (ii) It has a tendency to interact specifically with prey proteins rather than nonspecifically with the non-prey portion of the cell membrane and / or chimeric receptor, Detecting a signal, Includes.

[0015] In some embodiments, the interaction between the prey protein and the bait protein triggers the recruitment of the receptor fragment to the intracellular domain of the second receptor fused to the first receptor, thereby restoring ligand-dependent receptor signaling and activating the STAT molecule. In some embodiments, the cell contains a STAT-responsive reporter gene. In some embodiments, the activated STAT molecule translocates to the nucleus and induces transcription of the STAT-responsive reporter gene, which may in some cases allow for the detection of molecular interactions through the reporter gene signaling.

[0016] In some cases, nonspecific interactions of the bait protein with any portion of the ligand-based chimeric receptor other than the prey protein may generate false signals or background noise in the signals obtained by the cell-based systems described herein. Therefore, in some embodiments, the bait protein specifically interacts with the prey protein or is involved in prey protein-mediated interactions. In some embodiments, the bait protein tends to interact with the prey protein more than with other portions of the ligand-dependent chimeric receptor. In other embodiments, the bait protein tends to interact with the prey protein more than with portions of the cell membrane or other cellular components. In some embodiments, the bait protein has a higher binding affinity to the prey protein than to the non-prey portion of the chimeric receptor. In some embodiments, the bait protein has a higher binding affinity to the prey protein compared to portions of the cell membrane.

[0017] If the bait protein is sequestered or captured in any part of the cell, it will be unable to interact with the prey protein, which can lead to a false negative signal in the cell-based systems described herein. Therefore, in some embodiments, the bait protein is free and available within the cell and can interact with the prey protein. In some embodiments, the bait protein is not substantially captured within the cell's intracellular organelles such as the nucleus, mitochondria, Golgi apparatus, or endoplasmic reticulum. In some embodiments, the bait protein is available within the cell's cytoplasm and can interact with the prey protein. In some embodiments, the bait protein does not substantially interact with the cell membrane. In some embodiments, the bait protein does not substantially interact with the non-prey portion of the chimeric receptor. In some embodiments, the bait protein does not substantially interact with the transmembrane domain and / or intracellular domain of the second receptor of the chimeric receptor.

[0018] In various embodiments, this bait protein is not fused to the ligand-gated chimeric receptor.

[0019] In various embodiments, when assayed with forward MAPPIT (i.e., bait is not bound to membrane proteins), this bait (in reverse mode, bait is bound to membrane proteins) is limited by being trapped inside membrane-bound intracellular organelles and / or by nonspecifically interacting with the cell membrane and / or the non-bait portion of the chimeric receptor. Furthermore, in the case of forward MAPPIT, bait may be poorly expressed as a receptor fusion protein and / or improperly folded and / or the interaction surface of bait may be obscured by fusion; therefore, in embodiments, these problems are solved in reverse MAPPIT by solubilizing (i.e., non-fusioning) the bait.

[0020] In various embodiments, when analyzed as a gp130 fusion in forward MAPPIT, the prey protein is undetectable or poorly detected. In various embodiments, molecular interactions (e.g., protein / protein interactions, or protein / protein interactions mediated by the binding of small molecules to prey or bait proteins, but not limited to these) are detected by this method but are undetectable or poorly detected when analyzed in forward MAPPIT.

[0021] In various embodiments, the bait protein is not fused to the transmembrane protein, thus allowing for the exposure of a partial surface of the protein structure that more closely resembles the protein exposure surface specific to physiological conditions. In other words, the non-fusion of the bait can more accurately reflect the state of the bait in natural conditions.

[0022] The present invention also includes, with respect to molecular interactions, the analysis of libraries of prey proteins, libraries of ligand-based chimeric receptors, and / or libraries of cells expressing libraries of ligand-based chimeric receptors. The present invention also includes the analysis of compound libraries. In embodiments, the bait binds to a compound, but this bait / compound complex may interact with prey. Thus, in embodiments, the method enables the detection and / or discovery of novel compound-mediated protein / protein interactions and / or novel protein / compound interactions. In embodiments, the method enables the detection and / or discovery of novel compounds that act as molecular glues. In embodiments, the method enables the detection and / or discovery of novel compounds that convert weak bait / prey interactions into strong bait / prey interactions.

[0023] For example, a playlibrary comprises at least two distinct types / kinds of intrinsic play proteins suitable for carrying out the method described herein.

[0024] The receptor library of the present invention comprises at least two ligand-based chimeric receptors, each of which is fused to a different type / kind of prey protein. The cell library comprises at least two different types / kinds of cells, each of which expresses one type / kind of chimeric protein, and each chimeric protein is fused to one or more types / kinds of prey proteins.

[0025] In some embodiments, the cell library comprises a first cell population / fraction and a second cell population / fraction, where in the first cell population, the chimeric receptor of the first population / fraction is fused to a first type of prey protein, and in the second cell population, the chimeric receptor of the second population / fraction is fused to a second type of prey protein. The number of cell populations included in the cell library is not limited. For example, in one embodiment, the cell library comprises two or more different cell populations, where each cell population has a different prey protein fused to a chimeric receptor, and in that respect differs from the other populations.

[0026] In some embodiments, the receptor library comprises a first population of chimeric receptors, each of which is a chimeric receptor fused to one type of prey protein, and a second population of chimeric receptors, each of which is a receptor fused to another type of prey protein. There is no limit to the number of different types of chimeric receptors that may be included in the receptor library. For example, in one embodiment, the receptor library comprises two or more different populations of chimeric receptors, where each receptor population has a different prey protein fused to the chimeric receptor, and in that respect differs from the other populations.

[0027] In various embodiments, the method relates to an open reading frame (ORF) library of prey proteins fused to chimeric receptors. In various embodiments, the method relates to a cell population having an ORF library of prey proteins fused to chimeric receptors. In embodiments, such an ORF library relating to prey proteins fused to chimeric receptors can be used for the purpose of testing single baits that are not fused to chimeric receptors.

[0028] In various embodiments, the method relates to an array-type configuration in which, for example, cDNAs encoding various bait proteins are spotted on a surface. In various embodiments, the method relates to a cell population-based approach in which, for example, a library of bait proteins is introduced into cells so that, on average, each cell expresses a single bait. In such embodiments, the encoding cDNA is identified and its interaction with the compound and / or bait is elucidated. In these embodiments, FACS or microfluidic separation is used for such identification.

[0029] In various embodiments, multiple prey proteins are analyzed in relation to molecular interactions with a single bait, where the bait is not fused to a ligand-dependent chimeric receptor.

[0030] In some embodiments, the molecular interaction is a protein / protein interaction. In some embodiments, both the bait and the play are proteins.

[0031] In some embodiments, the method further includes introducing a small molecule that binds to a prey protein or bait protein. In some embodiments, the molecular interaction is a protein / protein interaction mediated by the binding of the small molecule to the prey protein or bait protein. In these embodiments, both bait and prey are proteins.

[0032] In some embodiments, the molecular interaction is a combination of two or more protein / protein interactions mediated by the binding of a small molecule to a prey protein or bait protein. In some embodiments, the protein / protein interaction mediated by the binding of the small molecule to a prey protein or bait protein is a direct binding between the prey protein or bait protein and the small molecule at a protein / protein interface site.

[0033] In some embodiments, the protein / protein interaction mediated by the binding of the small molecule to the prey protein or bait protein is mediated by allosteric modifications on the protein surface of the prey protein or bait protein.

[0034] In some embodiments, the small molecule induces the exposure of the hydrophobic surface of the prey protein or bait protein, thereby enabling interaction with the prey protein. In some embodiments, the small molecule induces the exposure of the hydrophobic surface of the bait protein, thereby enabling interaction with the prey protein. In some embodiments, the small molecule induces the exposure of the hydrophobic surface of the prey protein, thereby enabling interaction with the bait protein.

[0035] In some embodiments, the small molecule is a molecular glue. In some embodiments, the small molecule is a divalent hybrid ligand molecule (e.g., PROTAC).

[0036] In some embodiments, the molecular interaction is complex formation.

[0037] In some embodiments, the molecular interaction is a small molecule / protein interaction.

[0038] In some embodiments, the prey protein is bound to a small molecule, which is linked via a linker to a second small molecule that binds to a bait protein.

[0039] In various embodiments, the first receptor and the second receptor are the same receptor.

[0040] In various embodiments, the first receptor and the second receptor are different receptors.

[0041] In various embodiments, the first receptor and / or the second receptor are multimerized receptors.

[0042] In some embodiments, the ligand-binding domain is derived from a cytokine receptor. In some embodiments, the ligand-binding domain is derived from a type 1 cytokine receptor (CR).

[0043] In some embodiments, the ligand-binding domain is derived from an erythropoietin receptor (EpoR) or leptin receptor. In some embodiments, the transmembrane and intracellular domains are derived from a mouse leptin receptor.

[0044] In some embodiments, the bait is heterogeneous to the first receptor and / or the second receptor fragment.

[0045] In some embodiments, the intracellular domain includes a JAK binding site.

[0046] In some embodiments, the intracellular domain includes glycoprotein 130 (gp130) or a fragment thereof.

[0047] In some embodiments, the receptor fragment comprises glycoprotein 130 (gp130) or a fragment thereof.

[0048] In some embodiments, the STAT is selected from STAT1 or STAT3.

[0049] In some embodiments, mutations that reduce or eliminate STAT recruitment occur at one or more tyrosine phosphorylation sites. In some embodiments, the transmembrane and intracellular domains are derived from the mouse leptin receptor, and the mutation is at one or more of the Y985, Y1077, and Y1138 locations. In some embodiments, the transmembrane and intracellular domains are derived from the mouse leptin receptor, and the mutations are Y985F, Y1077F, and Y1138F. In some embodiments, the transmembrane and intracellular domains have mutations functionally equivalent to Y985F, Y1077F, and Y1138F of the mouse leptin receptor. In some embodiments, a deletion of the transmembrane domain is provided, but JAK binding is retained.

[0050] The amino acid sequence of the mouse leptin receptor is as follows:

[0051] In some embodiments, the domain is derived from the mouse leptin receptor, specifically from amino acids 839-1162 of the mouse leptin receptor sequence.

[0052] In some embodiments, the bait protein includes a nuclear export sequence (NES). For example, in some embodiments, the bait protein is an intranuclear protein, but the NES makes it possible for it to exist in the cytoplasm (i.e., to come into contact with prey if prey is available). Thus, in some embodiments, even in the presence of a strong nuclear localization signal, the NES signaling facilitates interaction with prey by supporting the bait polypeptide's presence in the cytoplasm.

[0053] In some embodiments, the NES has 1 to 4 hydrophobic residues. In some embodiments, the hydrophobic residue is leucine. In some embodiments, the NES has the sequence LxxxLxxLxL, where L is a hydrophobic residue and x is any other amino acid. In some embodiments, the NES has the sequence LxxxLxxLxL, where L is leucine and x is any other amino acid.

[0054] In some embodiments, the NES contains amino acids 37–46 of a thermal stability inhibitor of cAMP-dependent protein kinase, which has been shown to neutralize potent nuclear localization signals (Wiley et al., (1999), J. Biol. Chem. 274:6381–6387; this reference is incorporated herein by reference in its entirety).

[0055] In some embodiments, the method enables the identification of novel interaction partners, e.g., substrates or neosubstrates of proteins that bind to a compound, wherein the protein has three tryptophan residues in a cage-like arrangement that can interact with the glutarimide ring of the compound, for example, by hydrogen bonding. In some embodiments, the interaction partner (e.g., neosubstrate) has a surface β-hairpin loop, which may have a configuration of three backbone hydrogen bond receptors followed by a glycine residue at the tip of the turn. In some embodiments, the interaction partner (e.g., neosubstrate) has a degron motif (see Meszaros et al., Sci Signal 2017:10, 470; this reference is incorporated herein by reference in its entirety).

[0056] In some embodiments, the bait is a protein having three tryptophan residues in a cage configuration that can interact with the glutarimide ring of the compound (such as an immunomodulatory agent or immunomodulatory imide agent (IMiD)) for example, by hydrogen bonding.

[0057] In some embodiments, the play (e.g., neosubstrate) may have a surface β-hairpin loop, the surface β-hairpin loop may have a configuration of three backbone hydrogen bond receptors followed by a glycine residue at the tip of the turn. In some embodiments, the play (e.g., neosubstrate) may have a degron motif (see Meszaros et al., Sci Signal 2017:10, 470; this reference is incorporated herein by reference in its entirety).

[0058] In some embodiments, the bait is a protein that modulates the ubiquitin-proteasome system. In some embodiments, the bait is an E3 ligase protein, or a protein that modulates the E3 ligase protein. In some embodiments, the bait is a karin ring ligase (CRL) protein, or a protein that modulates the CRL protein. In various embodiments, the bait is a CRL4 protein, or a protein that modulates the CRL4 protein. In some embodiments, the bait is a DDB1-CUL4-related factor (DCAF) protein, or a protein that modulates DCAF.

[0059] In some embodiments, the bait is one or more of the following: cereblon (CRBN), damaged DNA-binding protein 1 (DDB1), karin 4A (CUL4A), karin 1 regulator (ROC1), and von Hippel Lindau (VHL). In various embodiments, the bait is one or more of the following: cereblon (CRBN), damaged DNA-binding protein 1 (DDB1), karin 4A (CUL4A), karin 1 regulator (ROC1), and von Hippel Lindau (VHL). In various embodiments, the bait is one or more of the following: cereblon (CRBN), damaged DNA-binding protein 1 (DDB1), karin 4A (CUL4A), karin 1 regulator (ROC1), and von Hippel Lindau (VHL).

[0060] In some embodiments, the bait is or comprises an FK506-binding protein (FKBP) optionally selected from FKBP12, FKBP38, and FKBP52.

[0061] In some embodiments, the bait protein is fused to a receptor fragment. In some embodiments, the bait protein is fused to the N-terminus or C-terminus of the receptor fragment. In some embodiments, the bait protein is fused to gp130 or a fragment thereof. In some embodiments, the bait protein is fused to the N-terminus or C-terminus of gp130 or a fragment thereof.

[0062] In some embodiments, the bait is two or more proteins (e.g., two, three, four, or five proteins). In some embodiments, the bait comprises a first protein that is small in molecular weight and / or interacts with the bait, and the scaffold protein interacts with the first protein. In some embodiments, the method utilizes the system shown in Figure 3.

[0063] In some embodiments, the scaffold protein is fused to a receptor fragment. In some embodiments, the scaffold protein is fused to the N-terminus or C-terminus of the receptor fragment. In some embodiments, the scaffold protein is fused to gp130 or a fragment thereof. In some embodiments, the scaffold protein is fused to the N-terminus or C-terminus of gp130 or a fragment thereof.

[0064] In some embodiments, the scaffold protein is fused to a receptor fragment. In embodiments, the scaffold protein is fused to gp130 or a fragment thereof, and a protein capable of interacting with small molecules and / or bait interacts with the scaffold protein capable of interacting with play and / or small molecules.

[0065] In this embodiment, the bait is an E3 ligase substrate-binding subunit.

[0066] In this embodiment, the E3 ligase substrate-binding subunit is selected from proteins encoded by any of the following genes: AMFR, ANAPC11, APG16L, ARIH1, ARIH2, ARPC1A, ARPC1B, ASB2, ASB2, ATG16L1, BAF250, BARD1, BIRC2, BIRC3, BIRC4, BIRC7, BMI1, BRAP, BRCA1, bTrCP, CBL, CBLB, CBLC, CBLL1, CCIN, CCIN, CCNB1IP1, CRBN, CHFR, CHIP, CNOT4, COP1, CSA, DCAF1, DCAF10, DCAF11, DCAF12, DCAF13, DCAF14, DCAF15, DCAF16, DC AF17、DCAF19、DCAF2、DCAF3、DCAF4、DCAF5、DCAF6、DCAF7、DCAF8、DCAF9、Dda1、DDB2、DET1、DNAI2、DTX3、DZIP3、E6AP、EDD、EED、ENC1、EN1、FANCL、FBXL 1、FBXL10、FBXL11、FBXL12、FBXL13、FBXL14、FBXL15、FBXL16、FBXL17、FBXL18、FBXL19、FBXL20、FBXL21、FBXL22、FBXL3、FBXL4、FBXL5、FBXL7、FBXL8、FBXL XO1、FBXO10、FBXO11、FBXO12、FBXO13、FBXO14、FBXO15、FBXO16、FBXO17、FBXO18、FBXO19、FBXO2、FBXO20、FBXO21、FBXO22、FBXO3、FBXO4、FBXO5、FBXO6 FBXO7、FBXO8、FBXW1、FBXW10、FBXW11、FBXW12、FBXW5、FBXW7、FBXW8、FBXW9、FEM1A、FEM1B、FEM1C、GAN、GAN、GNB1、GNB2、GNB5、GRWD1、GTF2H2、GTF3C2、H ACE1、HECTD1、HECTD2、HECTD3、HERC1、HERC2、HERC3、HERC4、HERC5、HERC6、HLTF、HOIP、HUWE1、IBRDC2、IBRDC3、IFRG15、IPP、IPP、ITCH、IVNS1ABP、IVNS 1ABP、KATNB1、KBTBD10、KBTBD10、KBTBD11、KBTBD11、KBTBD12、KBTBD12、KBTBD13、KBTBD13、KBTBD2、KBTBD2、KBTBD3、KBTBD3、KBTBD4、KBTBD4、KBTBD5、KBTBD5、KBTBD6、KBTBD6、KBTBD7、KBTBD7、KBTBD8、KBTBD8、KCTD5、KEAP、KE AP1、KIAA0317、KIAA0614、KLHDC5、KLHL1、KLHL1、KLHL10、KLHL10、KLHL11、K LHL11、KLHL12、KLHL12、KLHL13、KLHL13、KLHL14、KLHL14、KLHL15、KLHL15、 KLHL17、KLHL17、KLHL18、KLHL18、KLHL2、KLHL2、KLHL20、KLHL21、KLHL21、KL HL22、KLHL22、KLHL23、KLHL23、KLHL24、KLHL24、KLHL25、KLHL25、KLHL26、K LHL26、KLHL28、KLHL28、KLHL29、KLHL29、KLHL3、KLHL3、KLHL30、KLHL30、KLH L31, KLHL31, KLHL32, KLHL32, KLHL33, KLHL33, KLHL34, KLHL34, KLHL35, KLHL35, KLHL36, KLHL36, KLHL38, KLHL38, KLHL4, KLHL4, KLHL5, KLHL5, KLHL6, KLHL6, KLHL7, KLHL7, KLHL8, KLHL8, KLHL9, KLHL9, LINCR, LNX1, LRR1, LRRC41, LRSAM1, LZTR1, LZTR1, MAGEA1, MAGE-A1, MAGEA2, MAGE-A2, MAGEA3, MAGE -A3、MAGEA6、MAGE-A6、MAGEB18、MAGE-B18、MAGEB2、MAGE-B2、MAGEC2、MAGE -C2、MALIN、MAP3K1、MARCH1、MARCH11、MARCH2、MARCH4、MARCH5、MARCH6、MAR CH7、MARCH8、MARCH9、MDM2、MDM4、MEX、MGRN1、MIB1、MIB2、MID1、MKRN1、MNA T1、MUF1、MULAN、MURF、MYCBP2、MYLIP、Nedd4、NEDD4L、NEDL1、NEDL2、NEURL、 NEURL2、NLE1、NUP43、OSTM1、PAFAH1B1、PARC、PARK2、PCGF1、PCGF2、PDZRN3、 PEX10、PEX7、PJA1、PJA2、POC1A、PPIL2、PRAME、PRPF19、PWP1、RACK1、RAD18、RAE1、RAG1、RBBP4、RBBP5、RBBP6、RBBP7、RBCK1、RBX1、RCHY1、RFFL、RFPL4A 、RFWD2、RING1、RNF103、RNF11、RNF111、RNF114、RNF12、RNF123、RNF125、RNF 128、RNF13、RNF130、RNF133、RNF135、RNF138、RNF139、RNF14、RNF144A、RNF 167、RNF168、RNF180、RNF181、RNF182、RNF185、RNF19、RNF2、RNF20、RNF20、R NF216、RNF25、RNF34、RNF4、RNF40、RNF41、RNF43、RNF43、RNF5、RNF6、RNF7、 RNF8、RNF85、RPTOR、SCAP、SH3RF1、SHPRH、SIAH1、SIAH2、SMU1、SMURF1、SMUR F2、SOCS1、SOCS3、SPOP、SPSB1、SPSB1、SPSB2、SPSB2、SPSB4、SPSB4、STXBP5 L、SYVN1、TAF5L、TBL1Y、THOC3、TLE1、TLE2、TLE3、TOPORS、TRAF2、TRAF6、TRA F7、TRAIP、TRIAD3、TRIM1、TRIM10、TRIM11、TRIM12、TRIM13、TRIM14、TRIM1 5、TRIM16、TRIM17、TRIM18、TRIM2、TRIM21、TRIM22、TRIM23、TRIM24、TRIM25 、TRIM26、TRIM27、TRIM28、TRIM29、TRIM29、TRIM3、TRIM31、TRIM32、TRIM33 、TRIM36、TRIM37、TRIM39、TRIM40、TRIM41、TRIM44、TRIM45、TRIM47、TRIM5、 TRIM50、TRIM52、TRIM54、TRIM55、TRIM58、TRIM59、TRIM62、TRIM65、TRIM66 、TRIM7、TRIM71、TRIM8、TRIM9、TRIP12、TRPC4AP、TSSC1、UBE3B、UBE3C、UBE4 A、UBE4B、UBOX5、UBR1、UBR2、UBR3、UBR4、UHRF1、UHRF2、VHL、VPS18、WDR12、W DR23、WDR26、WDR3、WDR31、WDR37、WDR39、WDR4、WDR47、WDR48、WDR5、WDR51B、WDR53, WDR57, WDR59, WDR5B, WDR61, WDR76, WDR77, WDR82, WDR83, WDR86, WSB1, WSB2, WWP1, WWP2, ZNF294, ZNF313, ZNF364, ZNRF1, ZNRF2, ZYG11A, ZYG11B, or ZYG11BL.

[0067] In this embodiment, the E3 ligase substrate-binding subunit is CRBN or VHL.

[0068] In this embodiment, the scaffold protein interacts with an E3 ligase substrate-binding subunit, and the complex between the scaffold protein and the E3 ligase substrate-binding subunit interacts with a small molecule, or the interaction is induced or mediated by a small molecule.

[0069] In this embodiment, the scaffolding is selected from the following: BIRC6, CUL3, DDB1, ELOB, ELOC, RBX1, SKP1, UBCH5A, UBE2A, UBE2B, UBE2B2, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2NL, UBE2O, UBE2Q1, UBE2Q2, UBE2QL, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V1, UBE2V2, and UBE2W.

[0070] In some embodiments, the scaffold protein is selected from damaged DNA-binding protein 1 (DDB1), karin 4A (CUL4A), and karin 1 regulator (ROC1).

[0071] In some embodiments, play is a substrate and / or neosubstrate of CRBN. In embodiments, the substrate and / or neosubstrate of CRBN includes a β-hairpin α-turn having an i-residue containing a hydrogen bond acceptor, such as an Asx or ST motif, which has hydrogen bonds between the side chain of i and the backbone NH of i+3, and between the carbonyl oxygen of the backbone of i and the backbone NH of i+4. In embodiments, the i+4 residue is glycine (including, but not limited to, GSPT1 and CK1a). In embodiments, the substrate and / or neosubstrate of CRBN has a β-hairpin α-turn containing residues i and i+3 which are cysteine ​​and residue i+4 which is glycine. These two Cys residues bind to the zinc ion to enhance the turn morphology (including, but not limited to, IKZF1, ZnF692, and all substrates reported in the following reference: "Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN," Sievers et al., Science Vol. 362, Issue 6414, DOI: 1 0.1126 / science.aat0572 (2018); this reference is incorporated herein by reference in its entirety). In embodiments, the substrate and / or neosubstrate of CRBN has a "pseudoloop" which is a β-hairpin β-turn containing glycine at the i+3 position. The turn structure can be enhanced by a hydrogen bond between the hydrogen bond acceptor of the i-1 side chain and the carbonyl of the i+3 glycine.

[0072] In some embodiments, the play is one or more of the following: Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), Eos (IKZF4), Pegasus (IKZF5), SALL4, CSNK1A, CK1a, and ZFP91. In various embodiments, the play is one or more of the following: Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), Eos (IKZF4), Pegasus (IKZF5), SALL4, CSNK1A, CK1a, and ZFP91. In some embodiments, the play is one or more of the following: Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), Eos (IKZF4), Pegasus (IKZF5), SALL4, CSNK1A, CK1a, and ZFP91.

[0073] In some embodiments, the small molecule or compound is an immunomodulator. In some embodiments, the compound is a glutamic acid derivative comprising a glutarimide ring and optionally comprising a phthalimide ring. In some embodiments, the phthalimide ring is chemically modified. In some embodiments, the glutamic acid derivative may be a synthetic derivative having properties according to the embodiments of this disclosure.

[0074] In some embodiments, the compound is a member of a group of compounds known as immunomodulatory agents or immunomodulatory imide agents (IMiDs).

[0075] In one embodiment, the compound contains an IMiD-like glutarimide ring, but is otherwise chemically different, and binds to the same or similar low-molecular-weight binding pocket as the glutaramide-IMiD of CRBN (the IMiD binding pocket of CRBN). In another embodiment, the compound can bind to the IMiD pocket of CRBN without containing a glutaramide ring. In yet another embodiment, the compound binds to CRBN but not to the IMiD pocket. In yet another embodiment, the compound binds to CRBN in a manner that is non-competitive with IMiD or IMiD-like compounds that bind in the IMiD pocket.

[0076] In some embodiments, the compound is: thalidomide, lenalidomide, pomalidomide, CC-220, CC-122, CC-885, or its derivatives, analogs, optical isomers or mixtures of optical isomers, or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, clathrates or polymorphs; or a compound that competitively binds to the same CRBN bait binding site as FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (CsA) or its derivatives or analogs; or a compound that competitively binds to the same FKBP bait binding site as FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (CsA) or its derivatives or analogs.

[0077] In this embodiment, the compound is selected from FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (CsA) or its derivatives or analogues; or from compounds that competitively bind to the same FKBP bait binding site as FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (CsA) or its derivatives or analogues.

[0078] In some embodiments, the compound is abadomide, endomid, iverdmid, lenalidomide, mitindomide, pomalidomide, and thalidomide, or derivatives, analogs, optical isomers or mixtures of optical isomers thereof, or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, clathrates, or polymorphs thereof.

[0079] In some embodiments, the method comprises one or more of CRBN, DDB1, CUL4A, ROC1, and VHL as a bait, and the bait is brought into contact with a compound described herein (e.g., a compound that binds to one or more of CRBN, DDB1, CUL4A, ROC1, and VHL, e.g., IMiD) to discover prey that interacts with the bait when the bait is modulated by the compound. For example, the method identifies prey that interacts with the bait when it is in contact with the compound. In some embodiments, the prey interacts with the bait and is therefore recruited and / or degraded. In such embodiments, the compound does not directly interact with the prey (e.g., in a complex between bait and prey, the compound does not have to come into contact with the prey, but is not limited thereto); however, it is not limited thereto.

[0080] In some embodiments, this method enables the identification of novel substrates or neosubstrates of CRBN.

[0081] In various embodiments, this method identifies novel molecular interactions. In various embodiments, this method identifies novel protein / protein interactions. In various embodiments, this method identifies novel protein / protein interactions mediated by binding to small molecule pre- or bait proteins.

[0082] In various embodiments, the method identifies molecular interactions that are undetectable or poorly detected when analyzed by forward MAPPIT. In various embodiments, the method identifies protein / protein interactions that are undetectable or poorly detected when analyzed by forward MAPPIT. In various embodiments, the method identifies protein / protein interactions mediated by the binding of small molecules to pre-proteins or bait proteins that are undetectable or poorly detected when analyzed by forward MAPPIT.

[0083] In various embodiments, the background signal in this method is lower than that observed in sequential MAPPIT. In various embodiments, the false positive signal is lower than that observed in sequential MAPPIT.

[0084] In various embodiments, the present invention provides the detection of molecular interactions by using both forward MAPPIT and the methods described herein. Thus, this combined method makes it possible to detect molecular interactions that would not be detected by using a single method, namely forward MAPPIT and the methods described herein.

[0085] In some embodiments, this method uses the system shown in Figure 2A (right panel).

[0086] In some embodiments, this method uses the system shown in Figure 3.

[0087] Examples Example 1: Comparison of MAPPIT forward and reverse assay configurations for detecting molecular glue-induced CRBN substrate interactions. In this example, to identify ligand-induced CRBN substrates or neosubstrates, we use a MAPPIT assay based on the method described by Lemmens et al., "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions," Methods Mol Biol. 2015;1278:447-55. Conventional MAPPIT assays have been used to monitor protein-protein interactions. A bait protein (protein A) is expressed as a fusion protein, in which protein A is genetically fused with an engineered modified intracellular receptor domain of the leptin receptor, and this intracellular receptor domain is itself fused to the extracellular domain of the erythropoietin (Epo) receptor. Binding of the Epo ligand to this Epo receptor component activates receptor-binding intracellular JAK2. However, activated JAK2 cannot activate the leptin receptor that triggers STAT3 binding and its phosphorylation; this is because a mutation has been introduced into the tyrosine residue that would normally be phosphorylated by activated JAK2. Instead, the interaction of protein B and protein A reconfigures the JAK2 phosphorylation-enabled STAT3 docking site; this causes protein B to fuse with the intracellular domain of the gp130 receptor (which will contain the appropriate tyrosine residue recognized by activated JAK2 kinase). In other words, the activation of the EPO-induced JAK2-STAT3 signaling pathway is reconfigured by the physical interaction of protein A and protein B or by the formation of a protein complex containing protein A and protein B. STAT3 activation can be monitored by introducing a STAT3-responsive reporter gene containing a gene encoding luciferase or a fluorescent marker (such as GFP or other types of fluorescent proteins (e.g., EGFP)). In this way, the MAPPIT assay provides a versatile assay for evaluating such recombinant protein / protein interactions in intact cells.

[0088] In this Example 1, we used a modified MAPPIT assay developed by the inventors, which is particularly used to determine CRBN ligand-induced protein interactions (i.e., it uses a specific CRBN bait protein to assay ligand-dependent induction of protein complex formation). In the conventional MAPPIT assay configuration previously reported (referred to as "forward" herein), the target bait protein (CRBN in this Example 1) is expressed as a fusion with a MAPPIT chimeric membrane receptor, and the interacting target protein is fused to an intracellular gp130 receptor fragment (in this Example 1, IKZF1, IKZF3, IKZF3, SALL4, ZFP91, or CSNK1A1). Here, we illustrate another assay mode, which we call the "reverse" assay configuration, in which the bait and bait fusion are reversed; that is, the CRBN bait is fused to a gp130 fragment, and the substrate protein is fused to a transmembrane chimeric receptor.

[0089] Compared to the classical forward mode, this reverse assay configuration offers several advantages. Firstly, target proteins that naturally localize to other cellular compartments rather than the cytoplasm are not accessible enough to interact with the membrane-bound bait (in this case, CRBN) when used as gp130 fusions. In this case, the problem can be solved by reversing the setup and forcing the target protein to localize to the cytoplasm by linking it to a MAPPIT chimeric transmembrane receptor. IKZF3 is one such example, which will be discussed in Example 2.

[0090] Another example where using a target / prey protein in a MAPPIT receptor fusion construct is more beneficial than using a gp130 fusion construct is when the target / prey protein as a gp130-fusion protein exhibits affinity for MAPPIT chimeric receptor components other than the interacting protein bait (e.g., the intracellular portion of the leptin receptor or JAK2). In this case, the aforementioned "adhesion" already generates a high reporter signal even in the absence of a specific bait / prey interaction, which can obscure further signal increases induced by specific protein / protein or compound-inducible bait / prey interactions. Such background signaling can be prevented by reversing the configuration and instead fusing the target / prey protein to the MAPPIT chimeric receptor. This is illustrated in Example 3 with regard to the compound-inducible interaction between CRBN and CSNK1A1(CK1a).

[0091] In Example 1, both forward and reverse MAPPIT assay configurations were evaluated for the detection of lenalidomide-inducible and CC-220-inducible binding of proteins to CRBN. HEK293T cells were transformed with plasmids encoding MAPPIT receptor fusions (pSEL; CRBN in forward mode, or all tested substrate / prey proteins in reverse mode), plasmids encoding MAPPIT gp130 fusions (CRBN in reverse mode, or all tested substrate / prey proteins in forward mode), and a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid), as described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886). For each of the target / prey proteins tested, a full-length protein was fused, with the exception of IKZF1, in which case isotype 7 was used. The MAPPIT receptor fusion used in Example 1 consists of a target protein (CRBN or target / prey protein) fused to an engineered, signal-deficient intracellular domain of the leptin receptor, the intracellular domain itself fused to the extracellular domain of the erythropoietin (EPO) receptor. To promote receptor / receptor-binding JAK2 activation, the EPO receptor extracellular domain and the leptin receptor extracellular domain (as used in Example 4) can be used interchangeably (activation by EPO or leptin, respectively). Twenty-four hours after transformation, cells were treated with erythropoietin (EPO) in or out of the indicated dose of the test compound. Luciferase activity was measured 24 hours after treatment with the test compound using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA).The data points represent the induction ratio of mean luciferase activity (triple sample) of cells treated with EPO + test compound compared to cells treated with EPO alone. The error bars represent the standard deviation. Curve fitting was performed using 4-parameter nonlinear regression with GRAPHPAD PRISM software. As can be seen from the figure, for most CRBN / (neo)substrate / prey interactions, only the inverse assay configuration was capable of detecting compound-inducible CRBN interactions.

[0092] Example 2: Immunofluorescence staining of the gp130 fusion revealed that the gp130-IKZF3 fusion protein was consistently localized in the nucleus, consistent with the signal deficiency in the CRBN-IKZF3 MAPPIT sequential assay configuration. In this study, the intracellular localization of the gp130 fusion protein was investigated by immunofluorescence staining. As described in Example 1, the reason why specific interactions were not detected when using the standard MAPPIT configuration is thought to be that the gp130-target fusion protein was not expressed in the cytoplasm. Therefore, HEK293T cells were seeded on poly-L-lysine coated glass slides and transformed after 24 hours with a plasmid encoding Flag-gp130-IKZF3 or Flag-gp130-IKZF1 (isotype 7). Furthermore, 24 hours after transformation, the cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and then stained with anti-Flag (SIGMA) primary antibody, followed by AlexaFluor488-labeled secondary antibody (THERMO Scientific). Simultaneously, the nuclei were stained with DAPI dye (SIGMA). These stained cell preparations were mounted in Vectashield mounting solution (VECTOR Laboratories) and imaged using a confocal microscope (OLYMPUS). In the image shown in Figure 5, the gp130 fusion protein and DAPI staining are shown in green and blue, respectively. The obtained microscopic images clearly show the difference in intracellular localization between IKZF1 gp130 fusion protein and IKZF3 gp130 fusion protein, i.e., their expression is localized to the cytoplasm or nucleus, respectively, which is consistent with the results obtained using the MAPPIT assay configuration, as discussed above.

[0093] Example 3: Nonspecific binding of the CSNK1A1-gp130 fusion to the common components of the MAPPIT chimeric receptor yields a high-background reporter signal, obscuring compound-dependent signal detection in the forward assay configuration. In this Example 3, protein-protein interactions were analyzed using a sequential MAPIT assay, as described by Lemmens et al. in Methods Mol Biol. 2015;1278:447-55, "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions." HEK293T cells were transformed with a plasmid encoding a MAPPIT receptor fusion (an EPO extracellular domain fused to an engineered modified intracellular domain of the leptin receptor; also used in Example 1) linked to either CRBN or E. coli DHFR (dihydrofolate reductase), along with a plasmid encoding a CSNK1A1-gp130 fusion (fused to either the N-terminus or C-terminus) and a reporter plasmid encoding a STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid). 24 hours after transformation, the cells were either treated with EPO or left untreated. Luciferase activity was measured 24 hours later using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA). The data points represent the induction ratio of the average luciferase activity of EPO-treated cells compared to untreated cells (triple sample). Error bars represent the standard deviation.The observed strong signal indicates a robust interaction between the CSNK1A1 / gp130 fusion protein and the chimeric receptor protein, independent of the properties of the bound bait protein, suggesting binding to transmembrane chimeric receptor components other than the bait protein. As discussed in Example 1, this adhesion interferes with the analysis of the interaction between CSNK1A1 and CRBN in the forward assay configuration. See Figure 6.

[0094] Example 4: Evaluation of alternative CSNK1A1 chimeric receptor fusion proteins in the MAPPIT reverse CRBN interaction assay. Here, a reverse MAPPIT interaction assay was used to detect lenalidomide-dependent and CC-220-dependent interactions between CRBN and CSNK1A1 neosubstrates. While similar to the assay discussed in Example 1, it differs in that it utilizes an alternative CSNK1A1 receptor fusion. As already mentioned in Example 1, an alternative receptor fusion in which the extracellular domain of EPO is replaced with the extracellular domain of the leptin receptor is available, but in this assay system, activation is performed with leptin instead of EPO. In this example, HEK293T cells were transformed with a plasmid encoding CSNK1A1 fused to a MAPPIT receptor fusion containing the extracellular domain of the leptin receptor (pCLG-CSNK1A1), a plasmid encoding CRBN fused to a partially gp130 domain, and a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid), as described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886). 24 hours after transformation, the cells were treated with leptin in or out of the indicated dose of the test compound. Luciferase activity was measured 24 hours after treatment with the test compound using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA). The data points represent the induction ratio (triple sample) of average luciferase activity between cells treated with leptin + test compound and cells treated with leptin alone. Error bars represent the standard deviation. The data in Figure 7 suggest that this alternative MAPPIT receptor fusion also enables the detection of molecular glue-dependent neosubstrate interactions with CRBN in a reverse assay configuration.

[0095] Example 5: Detection of compound-inducible FKBP1A (FKBP12) substrate interactions in forward and reverse MAPPIT assay configurations. In this example, forward and reverse MAPPIT assay configurations were compared for detecting compound-dependent interactions between FKBP1A (FKBP12) and MTOR and calcineurin subunits. The experimental setup followed the method described in Example 1, using plasmid constructs encoding the following MAPPIT receptor and gp130 fusions: In the forward mode, FKBP12 bait was fused to a MAPPIT chimeric receptor construct containing the extracellular domain of the EPO receptor (pSEL-FKBP1A), and the target protein was fused to a partial gp130 domain (MTOR FRB domain or PPP3CA); in the reverse mode assay, FKBP1A bait was fused to a partial gp130 domain, and MTOR(FRB) or PPP3CA was fused to the MAPPIT transmembrane receptor (pSEL-MTOR(FRB) and pSEL-PPP3CA). For calcineurin interactions, an additional assay configuration was used, in which a plasmid expressing unfused PPP3R2 was co-expressed in addition to the MAPPIT receptor and gp130 fusion. PPP3R2 encodes a calcineurin regulatory subunit and has been reported to enhance / promote FK506 macrolide-induced FKBP1A-calcineurin interactions. As described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886), HEK293T cells were transformed with plasmids encoding the indicated receptor, plasmids encoding gp130, and a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid). Twenty-four hours after transformation, cells were treated with EPO in or without the indicated dose of the test compound (rapamycin or FK506).Luciferase activity was measured 24 hours after treatment with the test compound using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA). The data points represent the induction ratio (triple sample) of mean luciferase activity in cells treated with EPO + test compound compared to cells treated with EPO alone. The error bars represent the standard deviation. Curve fitting was performed using 4-parameter nonlinear regression with GRAPHPAD PRISM software. The results shown in Figures 8A-C demonstrate that both forward and reverse assay modes can detect FKBP12 interactions.

[0096] Example 6: Evaluation of lenalidomide hybrid ligand inducible binding between CRBN and DHFR Here, we evaluated the binding between CRBN and DHFR (dihydrofolate reductase) induced by a hybrid molecule consisting of DHFR ligand trimethoprim (TMP) fused to the CRBN ligand lenalidomide (LEN) via a PEG linker, using the MAPPIT reverse assay mode. As described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886), HEK293T cells were co-transformed with a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid), a plasmid encoding a fusion construct of (E. coli) DHFR anchor protein linked to a chimeric MAPPIT-derived receptor containing the extracellular domain of the leptin receptor (pCLG-DHFR), and a gp130-CRBN bait fusion construct. Twenty-four hours after transformation, cells were treated with leptin in and out of the indicated concentration of TMP-LEN hybrid ligand, and luciferase activity was measured after another 24 hours. The dose-response curve shown in Figure 9 represents the induction ratio of mean luciferase activity (triple sample) of cells treated with leptin + test compound compared to cells treated with leptin alone. The error bars represent the standard deviation, and the curves were fitted using 4-parameter nonlinear regression with GRAPHPAD PRISM software. This example suggests that the binding between two proteins induced by a hybrid ligand can be evaluated using the MAPPIT reverse assay described herein.

[0097] Example 7: Characterization of molecular glue binding to CRBN using a CRBN reverse MAPPIT assay. The molecular glue binding to CRBN was evaluated using the MAPPIT assay described in Example 6; that is, the ability of the test compound to compete with the TMP-lenalidomide hybrid ligand for binding to CRBN in cells was determined. As described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886), HEK293T cells were transformed using a standard transformation method, similar to Example 6, with a plasmid encoding E. coli dihydrofolate reductase (DHFR) fused to the end of the intracellular domain of a mutant leptin receptor (pCLG-DHFR), a plasmid encoding CRBN play fused with the intracellular domain of gp130, or a plasmid encoding a gp130-REM2 control fusion of the DHFR fusion protein that can directly interact with the leptin receptor, and a STAT3-responsive pXP2d2-rPAPI luciferase reporter plasmid. Twenty-four hours after transformation, cells were treated with leptin to activate leptin receptor fusion proteins in and out of the indicated dose of the test compound, and 300 nM of the TMP-lenalidomide fusion compound (a hybrid ligand in which trimethoprim interacts with DHFR and lenalidomide interacts with CRBN) was added. Twenty-four hours after compound treatment, luciferase activity induced by the formation of a triple complex containing DHFR-TMP-lenalidomide-CRBN, and the resulting STAT3 signaling activation, was measured using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA).The data points in Figures 10A-B represent the mean luciferase activity (triple sample) of cells treated with leptin + test compound or leptin + hybrid ligand + test compound (CRBN) relative to the REM2 control (CTRL) (for both cases, the signal obtained in the absence of the added test compound is set as 100% luciferase activity on the y-axis). The error bars represent the standard deviation. Curve fitting was performed using 4-parameter nonlinear regression with GRAPHPAD PRISM software. As can be seen from Figures 10A-B, known IMiD compounds such as lenalidomide (LEN), pomalidomide (POM), CC-122, and CC-220 specifically and dose-dependently inhibit the activation of hybrid ligand-induced luciferase reporters. This reflects the effectiveness of competition for binding to CRBN and inhibiting the binding of the hybrid ligand to CRBN (i.e., inhibition of the assay signal). Furthermore, several other compounds (Cmpd1, cmpd2, cmpd3, and cmpd4) were also evaluated in this assay and were found to specifically inhibit TMP-LEN-induced luciferase signaling at various levels of efficacy (affinity ranging from micromolar (Cmpd1) to nanomolar (Cmpd4)).

[0098] Example 8: Detection of TMP-FK506 hybrid ligand-inducible binding between FKBP1A (FKBP12) and DHFR by reverse MAPPIT The binding between FKBP1A (FKBP12) and DHFR (dihydrofolate reductase), induced by a hybrid molecule consisting of DHFR ligand / trimethoprim (TMP) fused to FKBP1A ligand FK506 via a PEG linker, was tested using the MAPPIT reverse assay mode. As described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886), HEK293T cells were co-transformed with a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid), a plasmid encoding a fusion construct of (E. coli) DHFR anchor protein linked to a chimeric MAPPIT-derived receptor containing the extracellular domain of the leptin receptor (pCLG-DHFR), and a gp130-FKBP1A bait fusion construct. Twenty-four hours after transformation, cells were treated with leptin in and out of the indicated concentration of TMP-FK506 hybrid ligand, and luciferase activity was measured after a further 24 hours. The dose-response curve shown in Figure 11 represents the induction ratio of mean luciferase activity (triple sample) between cells treated with leptin + test compound and cells treated with leptin alone. Error bars represent the standard deviation, and the curves were fitted using 4-parameter nonlinear regression with GRAPHPAD PRISM software. These data also suggest that the inverse MAPPIT assay mode enables the detection of hybrid ligand-induced protein recruitment to FKBP1A.

[0099] Example 9: Evaluation of sulfonamide-inducible binding of RBM39 to DCAF15 using MAPPIT forward and reverse configurations. In this example, we developed MAPPIT forward and reverse assays that enable the evaluation of sulfonamides that induce binding between DCAF15 and RBM39. DCAF15 is an E3 ligase substrate receptor that has been shown to recruit RBM39 as a substrate for subsequent ubiquitination, and this recruitment is dependent on sulfonamides such as indislam, tasislam, chloroquinoxaline sulfonamide (CQS), and E7820. The experimental setup followed the method described above, using plasmid constructs encoding the following MAPPIT receptor and gp130 fusions: In forward mode, DCAF15 bait was fused to a MAPPIT chimeric receptor construct containing the leptin receptor extracellular domain (pCLL-DCAF15), and the RBM39 protein was fused to a partial gp130 domain; in reverse mode, DCAF15 bait was fused to a partial gp130 domain, and RBM39 was fused to the MAPPIT transmembrane receptor (pCLG-RBM39). HEK293T cells were transformed with plasmids encoding the indicated receptor, plasmids encoding gp130, and a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid), as described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886). Twenty-four hours after transformation, cells were treated with leptin in or out of the indicated dose of the test compound (indislam, tasislam, CQS, or E7820). Twenty-four hours after treatment with the test compound, luciferase activity was measured using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA). The data points represent the induction ratio of average luciferase activity (triple sample) between cells treated with leptin + test compound and cells treated with leptin alone.The error bars represent the standard deviation. Curve fitting was performed using 4-parameter nonlinear regression with GRAPHPAD PRISM software. The results shown in Figure 12 suggest that sulfonamide-inducible DCAF15 / RBM39 binding is detectable only in reverse assay mode.

[0100] Example 10: Compound screening for novel molecular glue-induced SALL4 mobilization to CRBN In this example, to identify compounds that induce SALL4 recruitment to CRBN, a reverse MAPPIT CRBN-SALL4 recruitment assay, also described in Example 1, was used to screen assemblies of 96 different IMiDs and IMiD-like molecular glues in microtiter plate form. HEK293T cells were co-transformed with a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid), a plasmid encoding a fusion construct of SALL4 fused to a chimeric MAPPIT membrane receptor containing the extracellular domain of the EPO receptor (pSEL-SALL4), and a gp130-CRBN bait fusion construct. Twenty-four hours after transformation, cells were treated with EPO and the compound (or DMSO as a negative control). Three concentrations were used for each compound (labeled "low," "medium," and "high" in Figures 13A-C): 0.8, 4, and 20 μM or 0.2, 1, and 5 μM, depending on the previously evaluated cytotoxicity level of the compound. Each compound concentration was tested twice. Twenty-four hours after compound treatment, luciferase activity was measured using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA). The graphs in Figures 13A-C (left panel) show the frequency distribution of the mean luciferase signal (raw data) for both the compound-treated samples and the DMSO-treated control, with each graph corresponding to data for one of the three compound concentrations (low, medium, and high) tested.The right-shifted portion of the bimodal distribution corresponding to the compound-treated samples represents compounds with higher signals than the background, and therefore these compounds induce SALL4 recruitment to CRBN. Three compounds that show a reporter signal higher than the background at one or more of the three test concentrations are marked with a line (dotted, dashed, or solid) on the luciferase signal; the corresponding dose-response curves are shown in the right panel. These dose-response curves were obtained using the same assay settings and protocols as used in the first screening, but here a dose range of nine points at the indicated concentrations was tested. Here, the data points represent the induction ratio of mean luciferase activity (triple sample) of cells treated with EPO + test compound compared to cells treated with EPO alone. Error bars represent the standard deviation, and curve fitting was performed using 4-parameter nonlinear regression with GRAPHPAD PRISM software. In summary, this embodiment suggests that the MAPPIT reverse assay described here can be used for compound assembly screening to identify known and novel molecular glue-inducible substrate recruitments to CRBN. Figures 13A-C illustrate compound screening specifically for glue-inducible SALL4 recruitment to CRBN, but this approach can be applied to screening other potential substrates.

[0101] Example 11: Identification of novel molecular glue-inducible CRBN substrates using a reverse MAPPIT ORF cDNA library screening approach To identify ligand-induced CRBN substrates or neosubstrates, we performed MAPPIT cell microarray screening using the method described in Lievens et al. ("Proteome-scale binary interactomics in human cells," Molecular & Cellular Proteomics 1 5.12 (2016): 3624-3639). Briefly, HEK293T cells were transformed with a CRBN bait expression plasmid encoding a gp130-CRBN fusion construct. These transformed cells were then added to a microarray screening plate containing a MAPPIT chimeric membrane receptor fusion expression plasmid assembly encompassing over 15,000 ORFs. Each spot in the microarray contained a different chimeric receptor ORF fusion expression plasmid as well as a reporter plasmid encoding a STAT3-responsive fluorescent protein. Therefore, since Gp130-CRBN bait-transformed cells that reach and adhere to these spots are all transformed with receptor ORF play plasmids and reporter plasmids, cells on each different microarray spot are tested with different CRBN-ORF combinations. Twenty-four hours after transformation, cells were specifically stimulated with erythropoietin in or out of the presence of CRBN ligand CC-220 (final concentration, 1 μM), and the reporter signal (GFP-like fluorescent reporter) was measured after 48 hours. The fluorescence intensity data were analyzed in a manner previously reported to create a volcano plot; as shown in Figure 14, this plot shows the q value calculated based on the cumulative fluorescence intensity (Y axis) of the cell clusters in each microarray, against the ratio of the median number of fluorescent particles (X axis) of the corresponding cell clusters. To confirm the dose response using an inverse MAPPIT assay setup, three ORF cDNAs showing strong signals (indicated by arrows on the dot plot in Figure 14) were selected. HEK293T cells were co-transformed with a gp130-CRBN fusion plasmid along with the corresponding receptor ORF plasmid and luciferase reporter plasmid.Twenty-four hours after transformation, cells were treated with EPO in or without the indicated concentration of CC-220, and luciferase activity was determined after a further 24 hours. The dose-response curves represent the induction ratio (triple sample) of mean luciferase activity in cells treated with EPO + test compound compared to cells treated with EPO alone. Error bars represent the standard deviation, and the curves were fitted using GRAPHPAD PRISM software with 4-parameter nonlinear regression. As exemplified for CC-220 in this example, this example suggests that the inverse MAPPIT assay described herein can be used to screen ORF cDNA assemblies for identifying known and novel molecular glue-inducible CRBN substrates.

[0102] Example 12: Detection of rapamycin-inducible recruitment of MTOR to FKBP protein using MAPPIT forward and reverse configurations. In this example, we developed MAPPIT forward and reverse assays to monitor rapamycin-inducible binding between MTOR and members of the FKBP protein family (specifically FKBP1A (FKBP12), FKBP3, FKBP4, and FKBP5). In forward mode, FKBP cDNA (pSEL-FKBPx) was cloned as a MAPPIT receptor fusion containing the extracellular domain of the EPO receptor, and MTOR (FRB domain) was cloned as a gp130 fusion; in reverse mode, FKBP cDNA was fused to gp130, and MTOR was cloned as a receptor fusion (pSEL-MTOR). HEK293T cells were co-transformed with a combination of an FKBP fusion construct, an MTOR fusion plasmid, and a reporter plasmid encoding STAT3-responsive luciferase (pXP2d2-rPAPI luciferase reporter plasmid) as described in the literature (Lievens et al., "Array MAPPIT: high-throughput interactome analysis in mammalian cells," Journal of Proteome Research 8.2 (2009): 877-886). Twenty-four hours after transformation, cells were treated with EPO in or without the indicated dose of rapamycin. Twenty-four hours after treatment with the test compound, luciferase activity was measured using a luciferase assay system kit (PROMEGA, Madison, WI) and an Ensight plate reader (PERKIN ELMER LIFE SCIENCES, Waltham, MA). The data points represent the induction ratio of mean luciferase activity (triple sample) of cells treated with EPO + test compound compared to cells treated with EPO or leptin alone. The error bars represent the standard deviation. As can be seen from Figures 15A-B, for each FKBP-MTOR interaction, rapamycin-inducible reporter signals were obtained in both forward and reverse MAPPIT assay configurations that reproduced the published data on these interactions.

Claims

1. A method for detecting molecular interactions, wherein the method is: (a) To provide cells containing a ligand-dependent chimeric receptor protein, wherein the ligand-dependent chimeric receptor protein is: (i) Extracellular portion of the ligand-binding domain derived from the first receptor, and (ii) The transmembrane domain and intracellular domain of the second receptor and the intracellular prey protein fused thereto, Includes, Here, the transmembrane domain and / or intracellular domain of the second receptor include mutations that reduce or eliminate the recruitment of STAT (Signal Transducer and Activator of Transcription), The transmembrane domain and the intracellular domain, To provide cells; (b) Expressing a bait protein fused to a receptor fragment in a cell, Here, the receptor fragment includes a functional STAT recruitment site. To express, and (c) Detecting signals that suggest the presence of molecular interactions, Here, the bait protein is not captured within the intracellular organelle of the cell, does not interact with the cell membrane, and does not interact with the transmembrane domain and / or intracellular domain of the second receptor of the chimeric receptor. The interaction between the prey protein and the bait protein recruits the receptor fragment fused to the bait protein to the transmembrane chimeric receptor protein, thereby restoring ligand-dependent transmembrane chimeric receptor signaling and activating the STAT molecule. Detecting a signal, including, method.

2. The method according to claim 1, wherein the cell comprises a STAT-responsive reporter gene, and / or, the activated STAT molecule translocates into the nucleus and induces transcription of a STAT-responsive reporter gene, thereby enabling the detection of molecular interactions by this reporter gene signal.

3. A method according to claim 1 or 2, wherein the bait protein associates with the scaffold protein, and / or, the molecular interaction is a protein / protein interaction, and / or the method further comprises introducing a small molecule that binds to the prey protein or bait protein, and / or the molecular interaction is a protein / protein interaction mediated by the binding of the small molecule to the prey protein or bait protein. and / or, the molecular interaction is a combination of two or more protein / protein interactions mediated by the binding of the small molecule to the prey protein or bait protein. A method wherein the protein / protein interaction mediated by the binding of the small molecule to the prey protein or bait protein is a direct binding between the prey protein or bait protein and the small molecule at the site of the protein / protein interface.

4. The method according to claim 3, wherein the protein / protein interaction mediated by the binding of the small molecule to the prey protein or bait protein is mediated by allosteric modification of the protein surface of the prey protein or bait protein, and / or induce exposure of the hydrophobic surface of the prey protein or bait protein, which allows the small molecule to interact with the prey protein or bait protein. and / or the small molecule induces hydrophobic surface exposure of the bait protein, thereby enabling interaction with the prey protein, or the small molecule induces hydrophobic surface exposure of the prey protein, thereby enabling interaction with the bait protein, and / or a method wherein the low molecular weight is a molecular glue.

5. A method according to claim 1, wherein the molecular interaction is complex formation.

6. A method according to claim 1, wherein the molecular interaction is a small molecule / protein interaction.

7. A method according to claim 1, wherein the prey protein is bound to a small molecule, the small molecule is linked to a second small molecule via a linker, and the second small molecule is bound to the bait protein.

8. A method according to claim 1, wherein the bait protein is bound to a small molecule, the small molecule is linked to a second small molecule via a linker, and the second small molecule is bound to the bait protein.

9. A method according to any one of claims 1 to 8, wherein the first receptor and the second receptor are the same receptor or different receptors. and / or the first receptor and / or the second receptor are multimerized receptors, A method wherein the ligand-binding domain is derived from a cytokine receptor, and / or the ligand-binding domain is derived from a type 1 cytokine receptor (CR), or from an erythropoietin receptor (EpoR) or leptin receptor (LR), and / or the transmembrane domain and intracellular domain are derived from a mouse leptin receptor (LR).

10. A method according to any one of claims 1 to 9, wherein the bait is heterogeneous to the first receptor and / or second receptor fragment, and / or, the intracellular domain comprises a receptor fragment containing a JAK binding site and / or gp130, and / or, the STAT is selected from STAT1 or STAT3, and / or a method wherein the mutation that reduces or eliminates STAT recruitment occurs at one or more tyrosine phosphorylation sites.

11. A method according to any one of claims 1 to 10, wherein the transmembrane domain and intracellular domain are derived from a mouse leptin receptor (LR), and the mutation is at one or more of the positions Y985, Y1077, and Y1138 relative to SEQ ID NO:

1. and / or the transmembrane domain and intracellular domain have mutations functionally equivalent to those of mouse leptin receptor (LR) Y985F, Y1077F, and Y1138F for SEQ ID NO: 1, and / or the bait protein comprises a nuclear export sequence (NES) having 1 to 4 hydrophobic residues, and / or, the NES having the sequence LxxxLxxxLxL, where L is leucine and x is any other amino acid.

12. A method according to any one of claims 1 to 11, wherein the bait is an E3 ligase substrate-binding subunit selected from cereblon (CRBN) and von Hippel Lindau (VHL) (VHL), and / or the bait is associated with a scaffold protein selected from damaged DNA-binding protein 1 (DDB1), karin 4A (CUL4A), and karin 1 regulator (ROC1), and / or, prior to interaction with the prey protein, the bait is in contact with a compound, and / or, the compound comprises a glutarimide ring and a phthalimide ring, and / or, the compound is selected from thalidomide, lenalidomide, pomalidomide, CC-220, CC-122, and CC-885.

13. A method according to any one of claims 1 to 12, wherein the method comprises assaying a plurality of cells, wherein the plurality of cells comprises a ligand-gated chimeric receptor, and the ligand-gated chimeric receptor is: (i) Extracellular portion of the ligand-binding domain derived from the first receptor, and (ii) The transmembrane domain and intracellular domain of the second receptor and the intracellular prey protein fused thereto, Methods that include...

14. A method according to claim 13, wherein a single bait protein is expressed in each cell, or a single bait protein is assayed for molecular interactions with multiple prey proteins.

15. A method according to any one of claims 1 to 14, wherein the method identifies a novel protein / protein interaction, and / or, if the method further comprises introducing a small molecule that binds to the prey protein or bait protein, the method for identifying a novel protein / protein interaction mediated by the binding of the small molecule to the prey protein or bait protein.

16. A method according to claim 15, wherein the method identifies a small molecule compound that induces, mediates or stabilizes a protein / protein interaction comprising the prey protein and the bait protein, and / or the small molecule compound is a molecular glue or hybrid ligand.

17. A method according to any one of claims 1 to 11 or 13 to 16, wherein the bait is an FK506-binding protein (FKBP), and / or the FK506-binding protein (FKBP) is selected from FKBP12, FKBP38 and FKBP52, or the compound is selected from FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (CsA); or compounds that competitively bind to the same FKBP bait binding site as FK506 (tacrolimus), rapamycin (sirolimus), and cyclosporine A (CsA).

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