Regulation of protein degradation

Abstract

Methods are provided for evaluating the effectiveness of an agent in treating a disease or disorder, comprising determining whether the agent causes or inhibits the direct or indirect recruitment and / or ubiquitination and / or degradation of argininosuccinate synthase 1 (ASS1).

Description

Technical Field

[0001] Methods for screening compounds and / or evaluating the efficacy of compounds in the treatment of diseases or disorders are provided based on the presence or absence of cereblon (CRBN) or the level of interaction with argininosuccinate synthase 1 (ASS1).

[0002] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 949,021, filed on December 17, 2019, the entire content of which is incorporated herein by reference.

Background Art

[0003] Targeted proteolysis by methods that eliminate target proteins intracellularly by routing them to the proteasome or targeted protein inhibition by methods that trap target proteins in higher - order protein complexes is an attractive new area in drug discovery. Molecular glues and bivalent derivatives (also known as proteolysis - targeting chimeras, or PROTACs) have the potential to inhibit and / or degrade targets, including at sub - stoichiometric concentrations, in ways that were impossible with conventional inhibitors and that previously did not lead to new drug development, representing attractive new methods in therapy. Molecular glues and PROTACs bind to a specific protein inside the cell and induce the formation of molecular complexes that inhibit or degrade disease - target proteins.

[0004] Although the potential of these agents is great, they are hampered by certain tendencies of agents such as engaging with targets that are not therapeutically relevant (off - targets), where such engagement either retains the risk of drug side - effects and toxicity and / or reduces the effective engagement of therapeutically relevant targets (e.g., due to competitive engagement of any off - target). There remains a need to develop agents without such tendencies.

Summary of the Invention

[0005] Accordingly, in various embodiments, the present invention provides the discovery of therapeutic agents that promote, induce, enhance, and / or stabilize interactions of small molecule proteins (or small molecule protein complexes), and / or lack or substantially reduce target tendencies. In various embodiments, the present invention provides methods for identifying compounds that are more effective for more selective modulation of disease-related targets and / or are acceptable for disease treatment, without irrelevant, risky, or harmful interactions, such as off-target cross-reactivity and deviation.

[0006] In embodiments, the present invention relates to a method for identifying compounds that are not burdensome by directly or indirectly interacting with and / or inducing direct or indirect interactions with argininosuccinate synthase 1 (ASS1). For example, in embodiments, the present invention provides a selection of compounds that have reduced, decreased, or substantially no activity or ability to induce, induce, enhance or stabilize the recruitment of ASS1 to a protein complex, and / or consequently inhibit ASS1 and / or promote the ubiquitination and / or degradation of ASS1. In embodiments, the present invention provides a selection of compounds that have reduced, decreased, or substantially no activity or ability to induce, induce, enhance or stabilize the direct binding of ASS1 to CRBN.

[0007] In various embodiments, the present invention relates to a method for obtaining a test compound having the ability to bind to cereblon (CRBN), contacting the test compound with CRBN in the presence of ASS1, assaying for one or more of the recruitment of ASS1 to CRBN, enhanced binding of ASS1 to the CRBN / test drug complex, ubiquitination of ASS1 and / or degradation of ASS1, and identifying candidate compounds by classifying the test compound as a candidate compound if reduced, decreased, or substantially absent recruitment of ASS1 to CRBN, enhanced binding to the CRBN / test drug complex, and / or ubiquitination of ASS1 and / or degradation of ASS1 are detected.

[0008] In various embodiments, the present invention relates to a method for obtaining a test compound having the ability to bind to cereblon (CRBN), contacting the test compound with CRBN in the presence of ASS1, assaying for the direct binding of ASS1 and CRBN, and identifying candidate compounds by classifying the test compound as a candidate compound when a reduction, decrease, or substantially absence of direct binding of ASS1 to CRBN is detected.

[0009] In other embodiments, the present invention relates to a method for producing a candidate composition by identifying a candidate compound and formulating a candidate composition for therapeutic use, wherein the identification of a candidate compound is by obtaining a test compound having the ability to bind to CRBN; contacting the test compound with CRBN in the presence of ASS1; assaying for the recruitment, enhanced binding, ubiquitination and / or degradation of ASS1 to the CRBN / test drug complex; and classifying the test compound as a candidate compound if a reduction, decrease, or substantially absence of recruitment, enhanced binding of ASS1 to the CRBN / test drug complex, and / or ubiquitination and / or degradation of ASS1 is detected.

[0010] In other embodiments, the present invention relates to a method for producing a candidate composition by identifying a candidate compound and formulating a candidate composition for therapeutic use, wherein the identification of a candidate compound is achieved by obtaining a test compound having the ability to bind to CRBN; contacting the test compound with CRBN in the presence of ASS1; assaying the direct binding of ASS1 to the CRBN / test drug complex; and classifying the test compound as a candidate compound if a reduction, decrease, or substantially absence of ASS1 binding to the CRBN / test drug complex is detected.

[0011] In embodiments, the method further includes the step of assaying for the recruitment, ubiquitination, and / or degradation of a CRBN substrate or neosubstrate other than ASS1 (e.g., degron motifs, e.g., IKZF1, Helios, IKZF2, Aeolus, IKZF3, IOS, IKZF4, Pegasus, IKZF5, SALL4, CSNK1A, CK1a, and / or ZFP91).

[0012] In embodiments, the classification is based on the ability of the test compound to shift the ratio of ASS1 recruitment, binding to CRBN, ubiquitination, and / or degradation to the recruitment, binding to CRBN, ubiquitination, and / or degradation of CRBN substrates or neosubstrates other than ASS1.

[0013] In the embodiment, the candidate compound exhibits reduced side effects in subjects receiving the candidate compound compared to one of thalidomide, lenalidomide, and pomalidomide.

[0014] In various embodiments, the test compound or candidate compound is a component of a proteolytic chimera (PROTAC). In various embodiments, the PROTAC comprises (i) a CRBN binder, such as the test compound or candidate compound described herein, e.g., the molecular glue compound described above, and (ii) a compound that can bind to a target protein different from the protein bound by the test compound (e.g., a CRBN substrate or protein that would become a neosubstrate upon recruitment to the test compound / CRBN complex), wherein (i) and (ii) are covalently linked via a linker.

[0015] In various embodiments, the test compound or candidate compound is a therapeutic compound. [Brief explanation of the drawing]

[0016] [Figure 1]Identification of recombinant ASS1 as a molecular glue-induced CRBN neosubstrate. ASS1 was identified by screening a human ORF(eome) cDNA library for targets recruited to CRBN in response to CC220, a known IMiD drug, and a CRBN ligand, using MAPPIT, a variation of the two-hybrid technology system previously described (Lemmens, et al. "MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions", Methods Mol Biol. 2015;1278:447-55 and Lievens, et al. "Array MAPPIT: high-throughput interactome analysis in mammalian cells", J Proteome Res. 2009 Feb;8(2):877-86, the entire content of which is incorporated herein by reference) and outlined in more detail in Example 1. Protein interactions in cells were assayed within cell clusters presented in array format. Each spot in the cell microarray corresponds to a cluster of cells expressing a single ORF / protein candidate being tested for ligand-induced (in this case, CC220-induced) interaction with CRBN. Positive interactions were read as an increase in cell fluorescence. Shown are dot plots of fluorescence intensity data from the cell microarray screen for / across numerous individual ORF / target protein candidates. The X-axis represents the number of particles, and the Y-axis represents the integrated intensity for each cell cluster in the microarray. As shown and suggested, a significant induction of signal is observed in the cell array coordinates representing the ASS1 ORF. Induction of signal for IKZF1, a known CC220-induced CRBN interacting substance, is also shown for reference. [Figure 2]Identification of endogenous ASS1 as a molecular glue-inducible CRBN neosubstrate. Using the “protein trap” technique known as ViroTrap and previously described (Eyckerman, et al. “Trapping mammalian protein complexes in viral particles,” Nature Communications 7:11416 (2016), the entire content of which is incorporated herein by reference) and outlined in more detail in Example 2, endogenous ASS1 was identified as a lenalidomide-inducible interacting substance of CRBNs. The binding of ASS1 to CRBNs in response to lenalidomide was detected by identifying the ASS1 trypsin peptide from virus-like particle-containing cellular CRBNs recruited to such particles (including any associated proteins or multiple proteins) during the particle budding process. Therefore, what is shown is a volcano plot of trypsin peptide identity for peptides isolated from CRBNs, including virus-like particles isolated from cells exposed to lenalidomide (LEN) and DMSO control media using the ViroTrap procedure (to identify LEN-induced CRBN interacting substances). The relative magnification changes in the trypsin peptide signal corresponding to ASS1 (log10 p-value) and the ASS1 trypsin peptide signal (X-axis) in the presence of lenalidomide (LEN) or DMSO control media identify endogenous ASS1 as a LEN-induced CRBN interacting substance. [Figure 3A-B]Ligand-induced CRBN-ASS1 interactions in living cells, as evaluated by co-immunoprecipitation analysis. In this study, we examined the ability of Flag-tagged ASS1 (or Flag-tagged gp130-ASS1 fusion protein, as identified in the assay described in Figure 1) and HA-tagged CRBN to interact in living cells during transfection and expression of each construct in HEK293T cells, in or out of the presence of the CRBN lMiD ligand CC220. We also investigated the ability of HA-tagged CRBN to interact with the known CRBN neosubstrate IKZF3 in response to CC220 (using IKZF3 expressed as Flag-tagged IKZF3 fusion protein). Figure 3A shows the results obtained from co-immunoprecipitation using an anti-Flag antibody, followed by Western blot analysis of the immunoprecipitation samples, and elution from beads using the Flag peptide. Western blotting was performed using an anti-HA antibody to measure the degree of immunoprecipitated CRBN in each sample (expected to vary depending on CC220 exposure), and using an anti-Flag antibody to determine the degree of immunoprecipitated Flag-ASS1, Flag-gp130-ASS1, or Flag-IKZF3 in each sample (expected to be the same). As shown in Figure 3A (upper panel), HA-CRBN was observed in the ASS1 immunoprecipitate only in the presence of CC220, which is consistent with the findings outlined in Figures 1 and 2 that ASS1 is a ligand-induced neosubstrate of CRBN—i.e., a target recruited to CRBN in response to CC220 binding to CRBN. Figure 3A (lower panel) shows that the same amount of Flag-tagged ASS1 or Flag-tagged IKZF3 was present in the anti-Flag co-immunoprecipitate across all relevant samples. Figure 3B shows that, in all samples subjected to immunoprecipitation analysis shown in Figure 3A, the relative expression of each protein was similar across various samples obtained from cells transfected with various constructs, as shown in the upper panel for HA-CRBN and in the lower panel for Flag-ASS1, Flag-gp130-ASS1, or Flag-IKZF3. The results are consistent with those shown in Figure 3A (lower panel). [Figure 4] Ligand-induced recruitment of ASS1 to CRBNs has been associated with ASS1 degradation in living cells. In this study, we investigated whether ligand-induced recruitment of ASS1 to CRBNs may result in subsequent ASS1 degradation (as induced by interaction with CRBN E3 ligase). HEK293 cells were co-transfected with cDNA constructs encoding Flag-tagged ASS1 and HA-tagged CRBN and exposed to increasing concentrations of CC220 for 24 hours. Samples were generated for Western blot analysis, and steady-state levels of ASS1 were determined across different experimental conditions, evaluated using an anti-Flag antibody. As shown, we observed loss of ASS1 expression (but not actin control protein expression) in a dose-dependent manner, specifically in response to CC220 exposure. These results indicate that ASS1 is a neosubstrate of CRBN and that its ligand-induced interaction with CRBN triggers its proteasomal degradation, as has been observed for some other known CRBN neosubstrates such as IKZF1 / 3. [Figure 5A-L]Discovery and characterization of compounds that bind to CRBN but do not effectively recruit the CRBN neosubstrate ASS1 compared to known CRBN IMiD ligands, such as lenalidomide / LEN and CC220, or other CRBN ligands (names in Roman numerals on the left). In this study, we show that we can identify CRBN ligands that bind to CRBN with high potency even within the IMiD ligand binding pocket, as suggested by competitive experiments, but do not recruit ASS1. The following experimental setup was used to first evaluate the CRBN binding efficiency of compounds in living cells: HEK293 cells were transfected with appropriate cDNA encoding the transgene (encoding the DHFR and CRBN fusion protein) using a MAPPIT-like assay, as described in Figure 1 and the relevant Methods section of Examples 1 and 5, to form a ternary protein / compound complex containing the DHFR fusion protein, trimethoprim-lenalidomide hybrid ligand (trimethoprim is the ligand for DHFR), and CRBN-gp130 fusion protein (CRBN binds the ligand lenalidomide)—thus generating a positive assay signal as a result of DHFR-Trim-Len-CRBN complex formation. Complex formation results in activation of the STAT-reactive luciferase reporter gene. In Figures 5A–5C, the signal is set as 100% luciferase activity. In a different sample setup, cells were prepared in the same manner but further co-incubated with test compounds whose interaction with CRBN has been investigated. Binding to the CRBN fusion protein competes with the binding of the hybrid ligand to the same CRBN protein, and therefore inhibits the assay signal by preventing the formation of the ternary complex required to generate the assay signal. CRBN binding efficiency was determined by evaluating increasing concentrations of the test compound, as determined in this type of ligand competition experiment in live cells. As shown, a known IMiD compound (lenalidomide / LEN, CC220) efficiently competed with the lenalidomide hybrid ligand for binding to CRBN (dose-response curve for CRBN-related assay signal inhibition). Similarly, a range of other compounds effectively compete.The specificity of signal inhibition is assessed by a parallel experimental setup in which the effect of the test compound on inhibiting the signal generated by a control gp130 fusion protein (CTRL) that directly binds to the DHFR fusion protein in the absence of a hybrid ligand (i.e., direct protein interaction). In summary, the results shown in Figures 5A–5C identify various compounds as potent CRBN binders. In Figures 5D–5G and 5H–5L, we determined which of these CRBN-binding compounds is an effective IKZF1 and / or ASS1 neosubstrate recruiter (respectively). In this experimental setup, cells were transfected with constructs encoding the CRBN fusion protein and either the IKZF1 or ASS1 fusion protein. To monitor the ability to promote CRBN ligand-induced protein interaction—i.e., the recruitment of IKZF1 or ASS1 neosubstrates—the activity of the test compound was assessed by increasing the concentration of the test compound (dose-response study). As shown, known IMiD compounds (LEN, CC220), like some other compounds, promote the recruitment of both IKZF1 (Figures 5D-5G) and ASS1 (Figures 5H-5L). In contrast, the two compounds shown here (v, vi), which are as effective in CRBN binding as the other compounds (Figures 5A-5C, competition curves), do not recruit IKZF1 (Figures 5D-5G) and ASS1 (Figures 5H-5L). This indicates that CRBN ligands lacking ASS1 neosubstrate recruitment activity can be identified and characterized for their differential protein recruitment activity. Compounds with reduced ability to recruit ASS1 to other substrates (e.g., IKZF1) compared to LEN and CC220 have also been observed. [Figure 6]Figure 6 shows that molecular glue-induced CRBN-ASS1 interaction can be detected using an alternative MAPPIT assay configuration that applies DDB1 receptor fusion. An alternative CRBN substrate binding assay was tested in which DDB1 was fused to a MAPPIT chimeric receptor construct (pSEL-DDB1), and an unfused CRBN bait protein was co-expressed with either IKZF1 (gp130-IKZF1) or ASS1 (gp130-ASS1) substrate gp130 fusion protein. In the absence of CRBN co-expression ("no CRBN"), lenalidomide (LEN)-induced signaling could not be observed. However, when the unfused CRBN expression construct was co-transfected, LEN-dependent signals were obtained for both IKZF1 and ASS1 interactions. [Modes for carrying out the invention]

[0017] This invention is partly based on the finding that certain CRBN-binding compounds also recruit, promote, enhance, and / or stabilize the binding of CRBN and ASS1, and / or induce the recruitment of ASS1 to CRBN, ubiquitination of ASS1, and / or degradation of ASS1. Without wishing to be bound by theory, these interactions of CRBN-binding compounds with ASS1 may represent an off-target tendency that reduces or depletes the ability of CRBN-binding compounds to mediate CRBN-based interactions with more therapeutically relevant substrates or neosubstrates of CRBN. Accordingly, in various embodiments, this invention provides a method for identifying CRBN-binding compounds that have substantially no ASS1-mediated effect.

[0018] In various embodiments, the method enables insights into interaction CRBN-based networks that highlight the potential for therapeutic effects and ASS1 or other target-related tendencies of various compounds, thereby enabling the discovery and construction of novel or improved compounds that do not exhibit, or exhibit reduced, cross-reactivity and tendencies common to IMiDs such as thalidomide, lenalidomide, and pomalidomide, which are currently marketed drugs for the treatment of diseases.

[0019] Methods for identifying and / or screening compounds In embodiments, methods for screening compounds for the treatment of a disease or disorder and / or evaluating the efficacy of CRBN-binding compounds are provided based on the presence or level of direct or indirect interaction with ASS1. In embodiments, methods are provided for discovering agents that bind to or interact with CRBN but do not cause, induce, enhance, and / or stabilize direct or indirect recruitment of ASS to CRBN, and / or cause ubiquitination and / or degradation of ASS1.

[0020] In various embodiments, the present invention relates to a method for obtaining a test compound having the ability to bind to CRBN, contacting the test compound with CRBN in the presence of ASS1, assaying for one or more of the following: recruitment to CRBN, enhanced binding of ASS1 to the CRBN / test drug complex, ubiquitination of ASS1 and / or degradation of ASS1, and a method for identifying candidate compounds by classifying the test compound as a candidate compound if a reduction, decrease, or substantially no change is detected in the recruitment to CRBN, binding of ASS1 to the CRBN / test drug complex, ubiquitination of ASS1 and / or degradation of ASS1.

[0021] In various aspects, the present invention relates to a method for identifying candidate compounds by obtaining a test compound having the ability to bind to CRBN, contacting the test compound with CRBN in the presence of ASS1, assaying for direct binding of ASS1 to the CRBN / test agent complex, and classifying the test compound as a candidate compound if a reduction, decrease, or substantial lack of change in the direct binding of ASS1 to the CRBN / test agent complex is detected.

[0022] In other aspects, the present invention relates to a method for producing a candidate composition by identifying a candidate compound and formulating a candidate composition for use in therapy, wherein the identification of the candidate compound comprises obtaining a test compound having the ability to bind to CRBN; contacting the test compound with CRBN in the presence of ASS1; assaying for one or more of recruitment of ASS1 to CRBN, enhanced binding of ASS1 to the CRBN / test agent complex, ubiquitination of ASS1 and / or degradation of ASS1; and classifying the test compound as a candidate compound if a reduction, decrease, or substantial lack of recruitment of ASS1 to CRBN, enhanced binding of ASS1 to the CRBN / test agent complex, ubiquitination of ASS1 and / or degradation of ASS1 is detected.

[0023] In other aspects, the present invention relates to a method for producing a candidate composition by identifying a candidate compound and formulating a candidate composition for use in therapy, wherein the identification of the candidate compound comprises obtaining a test compound having the ability to bind to CRBN; contacting the test compound with CRBN in the presence of ASS1; assaying for direct binding of ASS1 to the CRBN / test agent complex; and classifying the test compound as a candidate compound if a reduction, decrease, or substantial lack of direct binding of ASS1 to the CRBN / target agent complex is detected.

[0024] In embodiments, the present invention relates to a method for developing agents that affect protein complex formation, ubiquitination, and / or degradation. For example, in various embodiments, the present invention relates to a method for developing agents that affect protein complex formation, and / or degradation by shifting the activity and / or influence of CRBN toward therapeutic pathways that support the recruitment and / or degradation of therapeutically relevant CRBN substrates or neosubstrates (e.g., degron motifs, e.g., IKZF1, Helios, IKZF2, Aeolus, IKZF3, IKZF4, Pegasus, IKZF5, CSNK1A, CK1a, and / or ZFP91), and toward non-therapeutic pathways (e.g., off-target) that do not support the recruitment and / or degradation of, for example, ASS1 and / or other off-target proteins, e.g., SALL4, but not limited.

[0025] In some embodiments, the recruitment and / or ubiquitination and / or degradation of substrates or neosubstrates of ASS1 and / or non-ASS1 CRBNs is assessed by measuring the levels of protein or nucleic acid (e.g., RNA levels) of the substrates or neosubstrates of ASS1 and / or non-ASS1 CRBNs. In some embodiments, the recruitment and / or ubiquitination and / or degradation of substrates or neosubstrates of ASS1 and / or non-ASS1 CRBNs are assessed relative to a reference (and / or each other). In embodiments, the reduction in relative ASS1 recruitment and / or ubiquitination and / or degradation is, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. In embodiments, the relative decrease in the recruitment, and / or ubiquitination, and / or degradation of ASS1 is at least 2x, 3x, 4x, 5x, 7x, 10x, 15x, or 20x. In embodiments, the relative increase in the recruitment, and / or ubiquitination, and / or degradation of CRBN substrates or neosubstrates other than ASS1 is, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In embodiments, the relative increase in the recruitment, and / or ubiquitination, and / or degradation of non-ASS1 CRBN substrates or neosubstrates is at least 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold, 15-fold, or 20-fold.

[0026] In some embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to CRBN, allowing binding or interaction between CRBN and CRBN substrates or neosubstrates other than ASS1, but not substantially binding or interaction between CRBN and ASS1. In embodiments, the compound, test compound, candidate compound, or therapeutic compound mediates binding or interaction between CRBN and CRBN substrates or neosubstrates other than ASS1, but not substantially mediates binding or interaction between CRBN and ASS1.

[0027] In various embodiments, the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of substrates or neosubstrates of CRBNs other than ASS1 are about 5 times, 10 times, or about 100 times, or about 1,000 times, or about 10,000 times, or about 100,000 times higher than that of ASS1.

[0028] In various embodiments, the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of substrates or neosubstrates of CRBNs other than ASS1 is about 5 to 10 times, or about 5 to about 25 times, than the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1. Approximately 5 to 50 times, or approximately 5 to 100 times, or approximately 5 to 250 times, or approximately 5 to 500 times, or approximately 5 to 1,000 times, or approximately 5 to 3,000 times, or approximately 5 to 5,000 times, or approximately 5 to 10,000 times, or approximately 5 to 30,000 times, or approximately 5 to 50,000 times, or approximately 5 to 100,000 times, or 10 to 50 times, or approximately 10 times Approximately 100 times, or approximately 10 to approximately 250 times, or approximately 10 to approximately 500 times, or approximately 10 to approximately 1,000 times, or approximately 10 to approximately 3,000 times, or approximately 10 to approximately 5,000 times, or approximately 10 to approximately 10,000 times, or approximately 10 to approximately 30,000 times, or approximately 10 to approximately 50,000 times, or approximately 10 to approximately 100,000 times, or approximately 100 to approximately 1,000 times, or approximately 100 to It is approximately 3,000 times, or approximately 100 to 5,000 times, or approximately 100 to 10,000 times, or approximately 100 to 30,000 times, or approximately 100 to 50,000 times, or approximately 100 to 100,000 times, or approximately 1,000 to 10,000 times, or approximately 1,000 to 30,000 times, or approximately 1,000 to 50,000 times, or approximately 1,000 to 100,000 times higher.

[0029] In various embodiments, the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of substrates or neosubstrates of CRBNs other than ASS1, mediated by the compound, test compound, candidate compound, or therapeutic compound, is about 10 to about 50 times, or about 10 to about 100 times, or about 10 to about 250 times, or about 10 to about 500 times, or about 10 to about 1,000 times, or about 10 to about 3,000 times, or approximately 10 to approximately 5,000 times, or approximately 10 to approximately 10,000 times, or approximately 10 to approximately 30,000 times, or approximately 10 to approximately 50,000 times, or approximately 10 to approximately 100,000 times, or approximately 100 to approximately 1,000 times, or approximately 100 to approximately 3,000 times, or approximately 100 to approximately 5,000 times, or approximately 10 It is 0 to approximately 10,000 times higher, or approximately 100 to approximately 30,000 times higher, or approximately 100 to approximately 50,000 times higher, or approximately 100 to approximately 100,000 times higher, or approximately 1,000 to approximately 10,000 times higher, or approximately 1,000 to approximately 30,000 times higher, or approximately 1,000 to approximately 50,000 times higher, or approximately 1,000 to approximately 100,000 times higher.

[0030] In some embodiments, the affinity (with respect to CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 mediated by a compound, test compound, candidate compound, or therapeutic compound is assayed against the affinity (with respect to CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 by a reference compound.

[0031] In various embodiments, the reference compound is thalidomide, lenalidomide, pomalidomide, CC-220, or CC-122.

[0032] In some embodiments, the affinity (with respect to CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1, mediated by a compound, test compound, candidate compound, or therapeutic compound, is assayed against the affinity (with respect to CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 in its ground state.

[0033] In some embodiments, the affinity (for CRBNs) and / or recruitment (by CRBNs) and / or ubiquitination and / or degradation of ASS1, mediated by a compound, test compound, candidate compound, or therapeutic compound, is assayed for the affinity (for CRBNs) and / or recruitment (by CRBNs) and / or ubiquitination and / or degradation of substrates or neosubstrates of CRBNs other than ASS1, mediated by a compound, test compound, candidate compound, or therapeutic compound.

[0034] In some embodiments, the method provides (a) assaying the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 mediated by a compound, test compound, candidate compound, or therapeutic compound against the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of a substrate or neosubstrate of a CRBN other than ASS1, mediated by a compound, test compound, candidate compound, or therapeutic compound; and (b) comparing the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 mediated by a reference compound against the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of a substrate or neosubstrate of a CRBN other than ASS1, mediated by a reference compound.

[0035] In various embodiments, the reference compound is thalidomide, lenalidomide, pomalidomide, CC-220, or CC-122.

[0036] In some embodiments, the method provides (a) assaying the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 mediated by a compound, test compound, candidate compound, or therapeutic compound against the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of a substrate or neosubstrate of a CRBN other than ASS1 mediated by a compound, test compound, candidate compound, or therapeutic compound; and (b) comparing the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of ASS1 mediated by lenalidomide against the affinity (for CRBN) and / or recruitment (by CRBN) and / or ubiquitination and / or degradation of a substrate or neosubstrate of a CRBN other than ASS1 mediated by lenalidomide.

[0037] In some embodiments, a method for identifying candidate compounds is provided, comprising contacting cells with a test compound having the ability to bind to CRBN using cells expressing CRBN; assaying for ASS1 recruitment and / or ubiquitination and / or degradation; and classifying the test compound as a candidate compound if a reduction, decrease, or substantially absence of ASS1 recruitment and / or ubiquitination and / or degradation is detected. In embodiments, cells are further assayed for recruitment and / or ubiquitination and / or degradation of CRBN substrates or neosubstrates other than ASS1.

[0038] In embodiments, the methods described herein further include assaying for the recruitment and / or ubiquitination and / or degradation of substrates or neosubstrates of ASS1 and / or CRBNs other than ASS1.

[0039] In embodiments, a degradation assay, a ubiquitination assay, or a proteomics experiment is used to assay recruitment, ubiquitination, or degradation. For example, see Kim, Sung Ah, et al. "A novel cereblon modulator for targeted protein degradation." European Journal of Medicinal Chemistry 166(2019):65-74; U.S. Patent Application Publication No. 2019 / 0017998, which is incorporated in its entirety by reference.

[0040] Cerebron (CRBN) In embodiments, CRBN refers to a human CRBN protein (e.g., human CRBN isoform 1 (GenBank acceptance number NP_057386); or human CRBN isoform 2 (GenBank acceptance number NP_001166953), the whole of which is incorporated herein by reference, respectively) and a polypeptide comprising the amino acid sequence of any CRBN, such as an associated polypeptide including its SNP variant. The associated CRBN polypeptide includes allelic variants (e.g., SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, which in certain embodiments are sufficient to retain CRBN activity and / or generate an anti-CRBN immune response.

[0041] In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that bind CRBN. In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that induce a change in the conformation of CRBN (e.g., within the binding pocket of CRBN) or otherwise modify the properties of the CRBN surface (e.g., on the adjacent region of the protein), wherein the change or modification of the CRBN conformation results in ubiquitination of the neosubstrate.

[0042] ASS1 In embodiments, ASS1 refers to the human ASS1 protein (e.g., GenBank acceptance number AAH21676.1, the whole of which is incorporated herein by reference) and polypeptides comprising any amino acid sequence of ASS1, such as related polypeptides including its SNP variants. Related ASS1 polypeptides include allelic variants (e.g., SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, which, in certain embodiments, retain ASS1 activity.

[0043] ASS1 is a urea cycle enzyme that, with the help of argininosuccinate lyase (ASL), represents the rate-limiting step in the arginine biosynthesis pathway by converting citrulline to arginine. More specifically, ASS1 catalyzes the condensation of citrulline and aspartate to form argininosuccinate, the direct precursor of arginine. ASS1 is widely found in the liver as part of the urea cycle and is recognized as a ubiquitous enzyme in other tissues as well. Haines, et al., "Argininosuccinate synthase: at the center of arginine metabolism", International Journal of Biochemistry and Molecular Biology 2.1(2011):8-23.

[0044] ASS1 is ubiquitously expressed in various tissues, with the most abundant expression in the liver and kidneys. Yu, et al., Preparation of recombinant argininosuccinate synthetase and argininosuccinate lyase: expression of the enzymes in rat tissues, J Biochem. 1995 May;117(5):952-7.

[0045] ASS1 deficiency is associated with abnormal T cell differentiation and function, resulting in primary immune dysfunction.

[0046] Compounds and / or therapeutic compounds and / or candidate compounds and / or test compounds In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that bind CRBN. In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that induce a change in CRBN conformation (e.g., within the binding pocket of CRBN) or otherwise modify the properties of the CRBN surface (e.g., on the facultative region of a protein). In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that induce a change in CRBN conformation (e.g., within the binding pocket of CRBN) or otherwise modify the properties of the CRBN surface (e.g., on the facultative region of a protein), wherein the change or modification of the CRBN conformation results in the binding of CRBN and substrates and / or neosubstrates of non-ASS1 CRBN, and / or causes ubiquitination of substrates and / or neosubstrates of non-ASS1 CRBN and / or degradation of substrates and / or neosubstrates of non-ASS1 CRBN.

[0047] In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that bind to CRBN but weakly or substantially not to ASS1. In embodiments, the present invention relates to compounds, test compounds, candidate compounds, or therapeutic compounds that induce a change in CRBN conformation (e.g., within the CMA-binding pocket of CRBN) or otherwise alter the properties of the CRBN surface (e.g., on the adjacent region of the protein), such that the change or alteration of CRBN conformation results in ubiquitination and / or degradation of the substrate and / or neosubstrate of CRBN that does not bind to ASS1 but weakly or substantially not to ASS1.

[0048] In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to CRBN but weakly or substantially does not bind to the substrate and / or neosubstrate of CRBN that is not ASS1. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to CRBN with an affinity of about 1 μM or higher. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to CRBN with an affinity of about 500 nM, or about 300 nM, about 100 nM, about 30 nM, about 10 nM, or about 1 nM. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to CRBN with an affinity of about 500 nM, or about 300 nM, about 100 nM, about 30 nM, about 10 nM, or about 1 nM, but binds to non-ASS1 CRBN substrates and / or neosubstrates with an affinity of at least 1 μM, or at least 3 μM, or at least 10 μM, or at least 30 μM, or at least 100 μM, or at least 300 μM, or at least 1000 μM. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to ASS1 weakly or substantially not. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to ASS1 with an affinity of at least 1 μM, or at least 3 μM, or at least 10 μM, or at least 30 μM, or at least 100 μM, or at least 300 μM, or at least 1000 μM. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds ASS1 with an affinity of at least 1 μM, or at least 3 μM, or at least 10 μM, or at least 30 μM, or at least 1000 μM, but binds CRBN with an affinity of about 500 nM, or about 300 nM, about 100 nM, about 30 nM, about 10 nM, or about 1 nM.

[0049] In various embodiments, the binding K of a compound, test compound, candidate compound, or therapeutic compound for CRBN is used. DAlternatively, EC50 is the binding K of a compound, test compound, candidate compound, or therapeutic compound for ASS1. D Or substantially lower than EC50 (i.e., the binding of CRBN is substantially tighter than the binding for ASS1).

[0050] In various embodiments, the affinity of the compound, test compound, candidate compound, or therapeutic compound for CRBN is about 10 times, or about 100 times, or about 1,000 times, or about 10,000 times, or about 100,000 times, higher than the affinity of the compound, test compound, candidate compound, or therapeutic compound for ASS1.

[0051] In various embodiments, the affinity of a compound, test compound, candidate compound, or therapeutic compound for CRBN is about 10 to 50 times, or about 10 to 100 times, or about 10 to 250 times, or about 10 to 500 times, or about 10 to 1,000 times, or about 10 to 3,000 times, or about 10 to 5,000 times, or about 10 to 10,000 times, or about 10 to 30,000 times, or about 10 to Approximately 50,000 times, or approximately 10 to approximately 100,000 times, or approximately 100 to approximately 1,000 times, or approximately 100 to approximately 3,000 times, or approximately 100 to approximately 5,000 times, or approximately 100 to approximately 10,000 times, or approximately 100 to approximately 30,000 times, or approximately 100 to approximately 50,000 times, or approximately 100 to approximately 100,000 times, or approximately 1,000 to approximately 10,000 times, or approximately 1,000 to approximately 30,000 times, or approximately 1,000 to approximately 50,000 times, or approximately 1,000 to approximately 100,000 times lower.

[0052] In various embodiments, the compound, test compound, candidate compound, or therapeutic compound binds CRBN about 10 times, 100 times, 1,000 times, 10,000 times, or 100,000 times more tightly than it binds ASS1.

[0053] In various embodiments, the compound, test compound, candidate compound, or therapeutic compound binds to ASS1 by approximately 10 to 50 times, or approximately 10 to 100 times, or approximately 10 to 250 times, or approximately 10 to 500 times, or approximately 10 to 1,000 times, or approximately 10 to 3,000 times, or approximately 10 to 5,000 times, or approximately 10 to 10,000 times, or approximately 10 to 30,000 times, or approximately 10 to 50,000 times, or It binds to CRBN approximately 10 to 100,000 times, or approximately 100 to 1,000 times, or approximately 100 to 3,000 times, or approximately 100 to 5,000 times, or approximately 100 to 10,000 times, or approximately 100 to 30,000 times, or approximately 100 to 50,000 times, or approximately 100 to 100,000 times, or approximately 1,000 to 10,000 times, or approximately 1,000 to 30,000 times, or approximately 1,000 to 50,000 times, or approximately 1,000 to 100,000 times more firmly.

[0054] In embodiments, the compound, test compound, candidate compound, or therapeutic compound is a molecular glue. In embodiments, the compound, test compound, candidate compound, or therapeutic compound comprises a glutarimide ring and a phthalimide ring, one or both of which are optionally chemically modified. In embodiments, the glutarimide ring of the compound, test compound, candidate compound, or therapeutic compound can hydrogen bond with the cage of three tryptophan residues in the CRBN. In embodiments, the compound, test compound, candidate compound, or therapeutic compound induces exposure of the hydrophobic surface of the CRBN, enabling interaction with the neosubstrate.

[0055] In the embodiments, the compound, test compound, candidate compound, or therapeutic compound is an immunomodulator or immunomodulatory drug (IMiD). In the embodiments, the compound, test compound, candidate compound, or therapeutic compound is a compound containing an IMiD-like glutarimide ring; otherwise, it has a different chemical structure and binds to the same small molecule binding pocket (IMiD binding pocket in CRBN) as glutarimide-IMiD in CRBN. In the embodiments, the compound, test compound, candidate compound, or therapeutic compound is a compound that does not contain a glutarimide ring and can bind CRBN within the IMiD pocket. In the embodiments, the compound, test compound, candidate compound, or therapeutic compound is a compound that binds CRBN but is not within the IMiD pocket.

[0056] Thalidomide, known as an immunomodulatory agent (IMiD), and its analogous derivatives, lenalidomide and pomalidomide, are used to treat a variety of clinical conditions, including multiple myeloma, lymphoma, and other hematological diseases. Without being limited to any particular theory, the immunomodulatory agents used in this invention may be potent co-stimulators of T cells, enhancing cell proliferation in a dose-dependent manner. The immunomodulatory agents of this invention may also have a greater co-stimulatory effect on CD8+ T cell subsets than on CD4+ T cell subsets. In addition, the immunomodulatory agents possess anti-inflammatory properties and co-stimulate T cells.

[0057] In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds CRBN but not ASS1. In embodiments, the compound, test compound, candidate compound, or therapeutic compound binds CRBN and a substrate and / or neosubstrate of CRBN that is not ASS1. In embodiments, the compound, test compound, candidate compound, or therapeutic compound can simultaneously bind CRBN and one or more substrates and / or neosubstrates of CRBN that is not ASS1 and CRBN.

[0058] In the embodiments, the compound, test compound, candidate compound, or therapeutic compound is heterobifunctional or a component of a heterobifunctional compound.

[0059] In embodiments, the compound, test compound, candidate compound, or therapeutic compound is a proteolytic chimera (PROTAC). In various embodiments, the test compound or candidate compound is a component of a proteolytic chimera (PROTAC).

[0060] In various embodiments, the present invention relates to the discovery or identification of compounds suitable for inclusion in PROTACs (e.g., as components of PROTACs) that, for example, can bind to CRBN but weakly or substantially do not bind to ASS1.

[0061] In various embodiments, the present invention relates to the discovery or identification of compounds suitable for inclusion in PROTACs (e.g., as components of PROTACs) that can bind to CRBNs but have the characteristic of weakly binding to or substantially not binding to ASS1 and / or non-ASS1 CRBN substrates and / or neosubstrates.

[0062] In one embodiment, PROTAC incorporates an intracellular target protein and a ligand for an E3 ubiquitin ligase recruiting group, which are linked by a linker of appropriate length to combine the target protein and ubiquitination mechanism, thereby inducing ubiquitination of the protein of interest and subsequent degradation in the proteasome.

[0063] In various embodiments, PROTAC comprises (i) a CRBN binder, such as a test compound or candidate compound as described herein, e.g., the molecular glue compound described above, and (ii) a compound that can bind to a target protein different from the protein bound by the test compound (e.g., a CRBN substrate or protein that would become a neosubstrate upon recruitment to the test compound / CRBN complex), wherein (i) and (ii) are covalently linked via a linker.

[0064] In the embodiment, the PROTAC further includes a portion capable of binding a substrate and / or neosubstrate of a CRBN other than ASS1.

[0065] In embodiments, PROTAC further comprises a linker. In embodiments, the linker is of a length appropriate to bring together the target protein (e.g., substrates and / or neosubstrates of CRBNs other than ASS1) and the ubiquitination mechanism, thereby inducing ubiquitination of the protein of interest and subsequent degradation in the proteasome.

[0066] In embodiments, the PROTAC includes (i) a compound, test compound, candidate compound, or therapeutic compound, and (ii) a portion that can bind a non-ASS1 CRBN substrate and / or neosubstrate to the linker.

[0067] In various embodiments, the present invention relates to PROTACs that include (a) compounds, test compounds, candidate compounds, or therapeutic compounds that bind to CRBN but weakly or substantially do not bind to ASS1, and (b) compounds that bind to substrates and / or neosubstrates of CRBN that are not ASS1.

[0068] In embodiments, a compound, test compound, candidate compound, or therapeutic compound includes (i) a compound, test compound, candidate compound, or therapeutic compound that binds CRBN, and (ii) a portion that can bind substrates and / or neosubstrates of ASS1 and / or non-ASS1 CRBNs, covalently bonded to a linker.

[0069] In embodiments, the compound, test compound, candidate compound, or therapeutic compound is heterobifunctional and can be conjugated via click chemistry. Click chemistry describes reactions that are stereospecific, simple to perform, and can be carried out in readily removable or mild solvents, producing only byproducts that can be removed without chromatography, in high yield, with a wide range of characteristics (Rostovtsev et al. (2002) A Stepwise Huisgen Cycloaddition Process: Copper(l)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. 41:2596-2599). Click chemistry has been carried out in many different forms and has a wide range of applications in both chemistry and biology. Subclasses of click reactions include reactants that are inert to the surrounding biological environment. Such click reactions are called bioorthogonal (Sletten et al. (2009) Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of ​​Functionality. Angew. Chem. Int. Ed. 48: 6974-6998). Bioorthogonal reactant pairs suitable for bioorthogonal click chemistry are a group of molecules having the following properties: (1) They react with each other but do not significantly cross-react or interact with cellular biochemical systems in the intracellular environment; (2) They, as well as their products and by-products, are stable and non-toxic in a physiological setting; and (3) Their reactions are highly specific and rapid. In embodiments, compounds, test compounds, candidate compounds, or therapeutic compounds are heterobifunctional and can be conjugated via bioorthogonal click chemistry.

[0070] In embodiments, the compound, test compound, candidate compound, or therapeutic compound is a clickable proteolytic chimera (CLIPTAC). Such a CLIPTAC comprises, in embodiments, (a) a first portion containing a ligand for an intracellular target protein (e.g., ASS1 and / or a substrate and / or neosubstrate of a non-ASS1 CRBN); (b) a second portion containing a ligand for an E3 ubiquitin ligase; and (c) a linker portion covalently coupling the first and second portions, the linker comprising a covalent bond produced by a bioorthogonal click reaction between compatible pairs of reactive portions.

[0071] In embodiments, the compound, test compound, candidate compound, or therapeutic compound is an intracellular click-forming protein degradation-inducing chimera (CLIPTAC).

[0072] Determination of other substrates / ratios In the embodiment, the non-ASS1 CRBN substrate and / or neosubstrate is, for example, a protein substrate of an E3 ubiquitin ligase complex containing CRBN, or a downstream substrate thereof.

[0073] In embodiments, the methods described herein further include assaying for the recruitment and / or ubiquitination and / or degradation of substrates and / or neosubstrates of non-ASS1 CRBNs. In embodiments, the substrates and / or neosubstrates of non-ASS1 CRBNs include a degron motif.

[0074] In embodiments, the substrate and / or neosubstrate of a non-ASS1 CRBN includes a b hairpin a turn having an i residue holding a side chain having a hydrogen bond acceptor such as an Asx or ST motif, which has hydrogen bonds between the i side chain and the i+3 NH backbone and between the i carbonyl oxygen and the i+4 NH backbone. In embodiments, the i+4 residue is glycine (non-limiting examples include GSPT1, CK1a).

[0075] In embodiments, CRBN substrates and / or neosubstrates other than ASS1 have a b hairpin a turn with residues i and i+3, which is cysteine, and i+4, which is glycine. The two Cys residues bind to the zinc ion and strengthen the shape of the turn (non-limiting examples include IKZF1, ZnF692, and all substrates reported in “Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN”, Sievers t al, Science Vol.362, Issue 6414, DOI:10.1126 / science.aat0572(2018), which is incorporated in its entirety by reference).

[0076] In embodiments, substrates and / or neosubstrates of CRBNs other than ASS1 have a “pseudoloop”, a b hairpin b turn, holding glycine at the i+3 position. The turn structure may be strengthened by a hydrogen bond between the hydrogen bond acceptor of the i-1 side chain and the carbonyl of glycine at i+3 (a non-limiting example is CDC7).

[0077] In the embodiment, the substrate and / or neosubstrate of a CRBN other than ASS1 is selected from Ikaros (IKZF1), Helios (IKZF2), Aeolus (IKZF3), Eos (IKZF4), Pegasus (IKZF5), CSNK1A, CK1a, and ZFP91.

[0078] In embodiments, the methods described herein further include assaying for the recruitment and / or degradation of ASS1.

[0079] In various embodiments, the method enables the determination of ASS1 levels compared to substrates and / or neosubstrates of CRBNs that are not ASS1.

[0080] In various embodiments, the reduction, decrease, or substantially absence of ASS1 recruitment and / or degradation is related to the amount of non-ASS1 substrate and / or neosubstrate recruitment and / or degradation of CRBN.

[0081] In embodiments, the classification described herein is based on the ability of the test compound to shift the ratio of recruitment, binding to CRBN, ubiquitination of ASS1, and / or degradation of ASS1, with respect to recruitment by CRBN, binding to CRBN, ubiquitination of different substrates and / or neosubstrates of CRBN other than ASS1, and / or degradation of ASS1.

[0082] For example, in one embodiment, the shift is in the ratio of ASS1 recruitment to the recruitment of non-ASS1 CRBN substrates and / or neosubstrates of CRBN; the shift is in the ratio of ASS1 recruitment to the binding of non-ASS1 CRBN substrates and / or neosubstrates to CRBN; the shift is in the ratio of ASS1 recruitment to the ubiquitination of non-ASS1 CRBN substrates and / or neosubstrates; or the shift is in the ratio of ASS1 recruitment to the degradation of non-ASS1 CRBN substrates and / or neosubstrates.

[0083] For example, in one embodiment, the shift is in the ratio of ASS1 binding to CRBN to the recruitment of substrates and / or neosubstrates of CRBNs other than ASS1, the shift is in the ratio of ASS1 binding to CRBN to the binding of substrates and / or neosubstrates of CRBNs other than ASS1, the shift is in the ratio of ASS1 binding to CRBN to the ubiquitination of substrates and / or neosubstrates of CRBNs other than ASS1, or the shift is in the ratio of ASS1 binding to CRBN to the degradation of substrates and / or neosubstrates of CRBNs other than ASS1.

[0084] As another example, in one embodiment, the shift is in the ratio of ubiquitination of ASS1 to the recruitment of substrates and / or neosubstrates of non-ASS1 CRBNs, the shift is in the ratio of ubiquitination of ASS1 to the binding of substrates and / or neosubstrates of non-ASS1 CRBNs to CRBNs, the shift is in the ratio of ubiquitination of ASS1 to the ubiquitination of substrates and / or neosubstrates of non-ASS1 CRBNs, or the shift is in the ratio of ubiquitination of ASS1 to the degradation of substrates and / or neosubstrates of non-ASS1 CRBNs.

[0085] As a further example, in one embodiment, the shift is in the ratio of ASS1 degradation to the recruitment of substrates and / or neosubstrates of non-ASS1 CRBNs, the shift is in the ratio of ASS1 degradation to the binding of substrates and / or neosubstrates of non-ASS1 CRBNs to CRBNs, the shift is in the ratio of ASS1 degradation to the ubiquitination of substrates and / or neosubstrates of non-ASS1 CRBNs, or the shift is in the ratio of ASS1 degradation to the degradation of substrates and / or neosubstrates of non-ASS1 CRBNs.

[0086] In various embodiments, a compound or test compound is classified as a candidate compound or therapeutic compound based on its ability to support the recruitment, ubiquitination, and / or degradation of substrates and / or neosubstrates of non-ASS1 CRBNs compared to the recruitment, ubiquitination, and / or degradation of ASS1.

[0087] In various embodiments, the reduction, decrease, or substantially absence of ASS1 recruitment and / or degradation is related to the amount of ASS1 recruitment, ubiquitination, and / or degradation in a reference sample lacking the compound or test compound.

[0088] In various embodiments, the reduction, decrease, or substantially absence of ASS1 recruitment and / or degradation is related to the amount of ASS1 recruitment, ubiquitination, and / or degradation in the ground state.

[0089] In the embodiment, degradation is ubiquitin-dependent.

[0090] Examples of diseases The compounds, test compounds, candidate compounds, or therapeutic compounds described herein can be formulated for the treatment of various types of cancer.

[0091] In embodiments, a method is provided for producing candidate compounds for cancer therapy by obtaining a test compound having the ability to bind to CRBN, contacting the test compound with CRBN in the presence of ASS1, assaying for the recruitment and / or degradation of ASS1, and identifying candidate compounds by detecting a reduction, decrease, or substantially absence of ASS1 recruitment and / or degradation, and classifying the test compound as a candidate compound when formulating a candidate compound for use in cancer.

[0092] In embodiments, a method is provided for producing therapeutic compounds for cancer therapy by identifying therapeutic compounds and formulating therapeutic compositions for therapeutic use, wherein the identification of therapeutic compounds is by obtaining a test compound having the ability to bind to CRBN; contacting the test compound with CRBN in the presence of ASS1; assaying for the recruitment and / or degradation of ASS1; and detecting a reduction, decrease, or substantially absence of ASS1 recruitment and / or degradation, thereby classifying the test compound as a therapeutic compound for formulation as a therapeutic compound for use in cancer.

[0093] In an embodiment, cancer includes basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; neoplasm in situ; kidney cancer or renal cancer; pharyngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer). Alveolar lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancers of the urinary system; vulvar cancer; Hodgkin lymphoma and non-Hodgkin lymphoma. Furthermore, lymphomas including B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-incisional nuclear cell NHL; giant tumor lesion NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis, edema (such as those associated with brain tumors), and Meegs syndrome.

[0094] In some embodiments, cancer is leukemia or lymphoma. Examples of leukemia or lymphoma include, but are not limited to, low-grade and intermediate-grade non-Hodgkin lymphoma (NHL), relapsed Hodgkin's disease, high-grade resistant Hodgkin's disease, lymphoid-dominant subtypes of Hodgkin lymphoma, precursor B-cell lymphoblastic leukemia / lymphoma, mature B-cell neoplasm, B-cell chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), low-grade, intermediate-grade and high-grade follicular lymphoma (F). This includes leukemia or lymphoma selected from B-cell lymphomas, including FL (L), cutaneous follicular lymphoma, marginal zone B-cell lymphoma, MALT-type marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytoma myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, multiple myeloma, and B-cell lymphomas including anaplastic large cell lymphoma (ALCL), and non-Hodgkin lymphoma (NHL).

[0095] In embodiments, the cancer is a hematological malignancy and may be selected from multiple myeloma and 5q deletion-associated myelodysplastic syndrome (del(5q)MDS).

[0096] In some embodiments, the cancer is multiple myeloma.

[0097] Side effect profiling and / or reduction In various embodiments, this method enables the identification of improved compounds that may help reduce or prevent various side effects, such as IMiD, which is associated with lenalidomide. Drug-induced liver injury is a known serious problem that can develop after the use of many drugs. Drug-induced liver injury was estimated to occur between 10 and 15 times per 10,000 to 100,000 people exposed to prescription drugs each year. See Sgro et al., Hepatology 2002;36(2):451. ASS1 is present in immune tissues and T cells and has been suggested to play a role in T cell function. See Tarasenko et al., Impaired T cell function in argininosuccinate synthetase deficiency. Journal of Leukocyte Biology. 2015;97(2):273-278. Studies have shown that defects in the ASS1 enzyme in T cells are associated with abnormal T cell differentiation and function, resulting in primary immunodeficiency.

[0098] Despite their therapeutic benefits, thalidomide, lenalidomide, and pomalidomide are known to be associated with complications, such as renal and hepatic toxicity. Both thalidomide and lenalidomide have been linked to cases of acute liver injury, which can be severe and lead to death due to acute liver failure. Renal failure, for example, is a serious potential complication of lenalidomide therapy in multiple myeloma. See Kreiniz et al. 2016. Acute Renal Failure Associated with Lenalidomide Treatment in Multiple Myeloma: A Rare Occurrence? Anticancer Res. 36(6):2889-2892. In one study, 66% of patients with AL amyloidosis showed worsening renal function during lenalidomide treatment, and renal impairment was severe in 32% of patients. Specter et al. (2011). Kidney dysfunction during lenalidomide treatment for AL amyloidosis. Nephrology Dialysis Transplantation, 26(3):881-886. Nephrotoxicity reported in patients with multiple myeloma and other cancers is now recognized as a potential complication of lenalidomide and pomalidomide treatment. Wanchoo et al. (2017). Renal Toxicities of Novel Agents Used for Treatment of Multiple Myeloma. Clinical Journal of the American Society of Nephrology:CJASN, 12(1):176-189.

[0099] In embodiments, the present invention relates to a method for producing candidate compounds and / or therapeutic compounds in a manner that reduces or eliminates potential side effects of a drug. In embodiments, candidate compounds and / or therapeutic compounds are identified by determining whether the compound binds to or interacts with CRBN to alter the ratio of therapeutically relevant downstream activity of CRBN to downstream activity that induces side effects of CRBN, wherein the therapeutically relevant downstream activity of CRBN includes recruitment and / or degradation of CRBN substrates and / or neosubstrates other than ASS1 (e.g., those containing degron motifs, e.g., IKZF1, Helios (IKZF2), Aeolus (IKZF3), Eos (IKZF4), Pegasus (IKZF5), CSNK1A, CK1a, and / or ZFP91), and the downstream activity that induces side effects of CRBN includes reduction of cleaved recruitment and / or degradation of ASS1. In other words, the drug supports the therapeutic downstream effects of CRBN, but does not support the non-therapeutic downstream effects of CRBN.

[0100] In some embodiments, the method includes matching ASS1 as an indicator of whether a compound has safety concerns, such as renal or hepatic safety concerns, or effects on T cells (e.g., but not limited to T cell dysfunction). For example, detection of ASS1 degradation may suggest that the compound is plagued by safety concerns.

[0101] In some embodiments, the method relates to the treatment of a disease in a manner with few or no side effects. For example, the treatment method, in embodiments, includes selecting a therapeutic CRBN-binding compound based on an ASS1 profile that supports fewer side effects (e.g., reduced cleavage recruitment and / or degradation of ASS1).

[0102] In the embodiments, the compound, test compound, candidate compound, and / or therapeutic compound demonstrate a reduction in side effects in subjects who received the compound, test compound, candidate compound, and / or therapeutic compound compared to other CRBN-binding compounds. In the embodiments, the compound, test compound, candidate compound, and / or therapeutic compound demonstrate a reduction in side effects in subjects who received the compound, test compound, candidate compound, and / or therapeutic compound compared to one of thalidomide, lenalidomide, and pomalidomide.

[0103] In embodiments, side effects include decreased or impaired liver function and / or decreased or impaired kidney function. In embodiments, side effects include T-cell effects (e.g., impaired T-cell function).

[0104] Promotion of ASS1 degradation / ASS1-dependent cancer ASS1 is overexpressed in various human cancers, including lung cancer, colorectal cancer, gastric cancer, and ovarian cancer. Delage et al. (2010). Arginine deprivation and argininosuccinate synthetase expression in the treatment of cancer. Int. J. Cancer 126(12):2762-2772. For example, in recent years, ASS1 has been shown to be an upregulated target in primary human colorectal tumors, and pharmacological inhibition or genetic resection of ASS1 has been shown to impair the pathogenicity of colorectal cancer. Bateman et al. (2017). Argininosuccinate Synthase 1 is a Metabolic Regulator of Colorectal Cancer Pathogenicity. ACS Chem Biol. 12(4):905-911. Therefore, inhibition of ASS1 may be included in the treatment of colorectal cancer.

[0105] Without being constrained by theory, ASS1 contributes to the invasion and progression of gastric cancer by regulating autophagy. Furthermore, ASS1 is a pathogenic metabolic regulator of colorectal cancer. Therefore, in various embodiments, the present invention relates to the identification of compounds that promote the recruitment and / or degradation of ASS1.

[0106] In various embodiments, the present invention relates to a method for identifying candidate compounds by obtaining a test compound having the ability to bind to CRBN; contacting the test compound with CRBN in the presence of ASS1; assaying the recruitment and / or degradation of ASS1; and classifying the test compound as a candidate compound if high or increased recruitment and / or degradation of ASS1 is detected.

[0107] In various embodiments, the present invention relates to a method for producing a candidate composition, comprising identifying a candidate compound and formulating a candidate composition for therapeutic use, wherein the identification involves obtaining a test compound having the ability to bind to CRBN, contacting the test compound with CRBN in the presence of ASS1, assaying for the recruitment and / or degradation of ASS1, and classifying the test compound as a candidate compound if high or increased recruitment and / or degradation of ASS1 is detected.

[0108] In embodiments, the high or increased recruitment and / or degradation of ASS1 is related to the recruitment and / or degradation of the substrate and / or neosubstrate of the non-ASS1 CRBN when the substrate and / or neosubstrate of the non-ASS1 CRBN is selected from Ikaros (IKZF1), Helios (IKZF2), Aeolus (IKZF3), Eos (IKZF4), Pegasus (IKZF5), CSNK1A, CK1a, and ZFP91 in the presence of the CRBN in contact with the test compound.

[0109] In the embodiment, high or increased recruitment and / or degradation of ASS1 is related to the amount of recruitment and / or degradation of ASS1 in a reference sample without the test compound.

[0110] In the embodiments, candidate compounds identified when high or increased recruitment and / or degradation of ASS1 is detected are suitable for use in treating cancers that are dependent on ASS1, such as gastric cancer or colorectal cancer.

[0111] Examples Example 1: Discovery of recombinant argininosuccinate synthase 1 (ASS1) as a substrate / neosubstrate directly recruited to cereblon by molecular glue. To identify ligand-induced CRBN substrates, or neo-substrates, MAPPIT cell microarray screens were performed using the procedure described in Lievens, et al. "Proteome-scale binary interactomics in human cells." Molecular & Cellular Proteomics 15.12(2016):3624-3639. Conventional MAPPIT assays are used to monitor protein-protein interactions. The bait protein (protein A) is expressed as a fusion protein that is genetically fused to an engineered intracellular receptor domain of the leptin receptor, which itself is fused to the extracellular domain of the erythropoietin (Epo) receptor. Binding of the Epo ligand to the EpoR component results in the activation of receptor-associated intracellular JAK2. However, the activated JAK2 is unable to activate the leptin receptor to induce STAT3 binding and its phosphorylation because its tyrosine residue, normally phosphorylated by activated JAK2, is mutated. The rearrangement of the JAK2 phosphorylation-enabled STAT3 docking site is instead created through the interaction of protein B with protein A, thereby fusing protein B to the cytoplasmic domain of the gp130 receptor (now holding the appropriate tyrosine residue recognized by activated JAK2 kinase). Thus, the physical interaction of protein A with protein B is rearranged, and EPO induces activation of the JAK2-STAT3 signaling pathway. STAT3 activation can be monitored by introducing a STAT3-reactive reporter gene, including a gene encoding a luciferase or a gene encoding a fluorescent marker such as GFP or some other type of fluorescent protein (e.g., EGFP). Thus, the MAPPIT assay provides a versatile assay for evaluating such recombinant protein-protein interactions in intact cells.This method can be used to screen cDNA libraries encoding protein B fusion proteins (i.e., protein B-gp130 fusion proteins) and identify any protein that can interact with protein A bait (fused to EpoR-LepR fusion proteins).

[0112] In this Example 1, we used a derivative of the MAPPIT assay, which we specifically developed to assay for ligand-dependent induction of protein complex formation, including a protein B fusion protein (protein B-gp130 fusion protein), using a specific CRBN bait protein (fused to a mutant leptin receptor in this system) to determine CRBN ligand-induced protein interactions. In this way, we were able to screen a cDNA library for ligand-dependent candidate CRBN neosubstrates. This assay has also been used to characterize such interactions (or lack thereof) in other examples in this document (Example 5).

[0113] In short, HEK293T cells were transfected with a CRBN bait expression plasmid (pSEL-CRBN) and added to a microarray screening plate containing a prey expression plasmid collection covering 15K ORFs. 24 hours after transfection, cells were differentially stimulated with erythropoietin with or without CRBN ligand CC-220 (10 μM), and the reporter signal (GFP-like fluorescent reporter) was read at 48 hours. Fluorescence intensity data were analyzed as previously reported to create the dot plot shown in Figure 1. The top-ranked hits included Ikaros (IKZF1), a known CC-220-induced CRBN substrate. In this study, as shown, we also identified argininosuccinate synthase 1 (ASS1) as a novel CC220-induced CRBN neosubstrate. ASS1 has also been identified in this way with other compounds such as lenalidomide, another type of IMiD that binds to CRBN (commercially known as the well-known anticancer drug Revlimid™).

[0114] Example 2: Discovery of endogenous argininosuccinate synthase 1 (ASS1) as a substrate / neo-substrate recruited to cereblon by molecular glue. In this study, we set out to confirm that IMiDs such as lenalidomide can induce the recruitment of endogenous ASS1 to CRBN. For this purpose, we used a previously described method known as ViroTrap (Titeca, et al. "Analyzing trapped protein complexes by Virotrap and SFINX". Nature Protocols 12.5(2017):881). Using this method, CRBN is expressed as a fusion protein with the viral protein HIV-GAG. When HIV-GAG is expressed intracellularly, such as in HEK293, it can induce the formation of virus-like particles that sprout from the cell. Within the particle, the HIV-GAG protein is directed toward the center of the particle. When fused with CRBN, CRBN is also presented in the inner core of the particle. In that event, the GAG-CRBN fusion interacts with intracellular proteins, and such proteins are recruited / trapped into the virus-like particle along with the GAG-CRBN. Thus, endogenous proteins that interact with CRBN can be identified in the presence or absence of CRBN ligands. This is achieved by standard mass spectrometry of particle lysis and trypsin digestion of the sample. Subtractive analysis then identifies proteins that specifically associate with particles in response to CRBN ligands such as lenalidomide. Using this method, we found that lenalidomide induces the association of endogenous ASS1 with CRBN, i.e., ASS1 trypsin digest identified ASS1 as a protein recruited to viral particles in a lenalidomide-dependent manner. In short, HEK293T cells were co-transfected with a gag-CRBN bait expression plasmid and a Flag-VSV-G coding plasmid (for particle purification from cell medium) and incubated for 24 hours. Cells were then treated with 10 μM lenalidomide or DMSO as a negative control. 24 hours after compound addition, virus-like particles (VLPs) were isolated from the cell supernatant using magnetic beads coated with anti-Flag.After elution from the beads using Flag peptide, the purified VLPs were lysed, and the protein contents were digested overnight with trypsin. Peptide samples were analyzed by LC-MS / MS, data from triplicate samples were processed using the MaxQuant software package, and volcano plots (Figure 2) were generated using the Perseus tool. ASS1 was identified among the top hits with low p values, showing a high signal ratio for the lenalidomide vs. DMSO control sample.

[0115] Example 3: Evaluation of ligand-induced CRBN-ASS1 interaction in living cells by co-immunoprecipitation analysis In this study, we evaluated the ability of the CRBN ligand CC220 to induce interaction with ASS1 when expressed in HEK293 cells via co-immunoprecipitation of the protein complex. Consistent with the findings in Examples 1 and 2, this alternative approach showed that CC220 specifically induces the recruitment of ASS1 to the CRBN-CC220 complex. Similar observations were made for the known CRBN neosubstrate IKZF3, which was lined up as a positive control. Briefly, plasmids encoding (i) FLAG-tagged gp130-ASS1 (the version used in the MAPPIT assay in Example 1), (ii) FLAG-tagged ASS1, or (iii) FLAG-tagged IKZF3 were transiently co-transfected with HA-tagged-CRBN in HEK293T cells. 24 hours after transfection, cells were further treated with different doses (0 μM, 1 μM, and 3 μM) of CC-220 for another 24 hours. The following day, the cells were lysed, and 1 / 10 of the lysate was set aside for expression analysis of FLAG-tagged fusion protein and HA-tagged CRBN (shown in Figure 3B). Co-IP using anti-FLAG Ab (Sigma) and Dynabeads streptavidin T1 (Invitrogen) was performed on the remaining 9 / 10 of the lysate (Figure 3A). After immunoprecipitation, the bound FLAG protein and any associated proteins were eluted from the beads by elution using FLAG peptide (Sigma). The presence of HA-CRBN (Figure 3A, upper panel), i.e., its recruitment to the FLAG-ASS1 immunoprecipitate, was investigated by SDS-PAGE and Western blot analysis of eluted samples using anti-HA Ab (Roche). FLAG-tagged ASS1 was detected using FLAG Ab (Sigma), respectively (Figure 3A, lower panel). The expression of all proteins in cell lysates was investigated by SDS-PAGE and Western blot analysis of expression lysates using anti-FLAG and anti-HA Ab (Sigma or Roche, respectively), as shown in Figure 3B.

[0116] Example 4: Ligand-induced recruitment of ASS1 to CRBN is associated with the degradation of ASS1 in living cells. In this study, we investigated whether CC-220 CRBN ligand-induced recruitment of ASS1 to CRBN can lead to subsequent ASS1 degradation (induced by interaction with CRBN E3 ligase). For this analysis, cells were transfected with FLAG-tagged ASS1 and HA-tagged CRBN coding constructs, and any changes in the steady-state level of ASS1 in response to cell incubation with CC220 for a specified time were evaluated by Western blot analysis. The study shows that the steady-state level of ASS1 is indeed reduced in a dose-response manner in response to CC220, confirming that ligand-induced recruitment of ASS1 to CRBN induces ASS1 proteolysis. Briefly, plasmids encoding FLAG-ASS1 and HA-CRBN were co-transfected into HEK293T cells. Twenty-four hours after transfection, cells were either left untreated or treated with different doses of CC-220 (0.3 μM, 1 μM, 3 μM, 10 μM, or 30 μM) for an additional 24 hours. The cells were then lysed, and fractions of the lysate were analyzed via SDS-PAGE and Western blotting using anti-FLAG antibody (Sigma). Western blotting was performed using anti-actin antibody (Sigma) as a loading control.

[0117] Example 5: Discovery and characterization of compounds that bind to CRBN but do not effectively recruit the CRBN neosubstrate ASS1 compared to known CRBN IMiD ligands such as lenalidomide / LEN and CC220, or other CRBN ligands. In this study, we conducted a comparative study of a range of compounds that bind to CRBN and their ability to differentially recruit ASS1 neosubstrates. We found in our study that all known IMiDs, such as lenalidomide and CC220 (see Examples 1-4), induce the recruitment of ASS1 to CRBN, and that this is associated with the degradation of ASS1 in cells (Example 4). Given that ASS1 is an important cellular protein and that loss of its expression has been shown to be associated with defects in cellular metabolism and survival, such as in T cells of the immune system, we raised the question of whether it was possible to identify and characterize compounds that bind to CRBN but do not recruit ASS1. In summary, this study demonstrates that we were able to confirm that CRBN binders competing for lenalidomide's binding to CRBN are potent (equal to or even potent than lenalidomide) CRBN binders in cells, but do not induce ASS1 recruitment, in contrast to lenalidomide. This highlights the opportunity for differential screening and characterization in the development of CRBN ligands that evade the tendency for ASS1 recruitment. Details of the study are shown in Figures 5A–5L, where we recognize the potential of the compounds in a) binding to CRBN in cells and b) inducing the recruitment of ASS1 or IKZF1 to CRBN.

[0118] CRBN binding was evaluated by determining the ability of the test compound to compete with lenalidomide hybrid ligands for binding to CRBN in cells using the MAPPIT-like assay described in Example 1. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and incubated at 37°C and 8% CO2. Cells were transfected using the standard transfection method described (Lievens, et al. "Array MAPPIT: high-throughput interactome analysis in mammalian cells"). This method used the standard transfection method described in the journal (Lievens, et al. "Array MAPPIT: high-throughput interactome analysis in mammalian cells"). Journal of Proteome Research 8.2(2009):877-886) with plasmids encoding E. coli dihydrofolate reductase (DHFR) fused to the tail of the cytoplasmic domain of the mutated leptin receptor (pCLG-eDHFR), plasmids encoding the CRBN prey fused to the gp130 cytoplasmic domain (pMG1-CRBN), plasmids encoding the REM control prey that can directly interact with the leptin receptor of the DHFR fusion protein (pMG1-REM2), and the STAT3-reactive pXP2d2-rPAPI-luciferase reporter plasmid. Twenty-four hours after transfection, cells were treated with leptin to activate the leptin receptor fusion protein, and a 300 nM trimethoprim-lenalidomide fusion compound (hybrid ligand, trimethoprim interacts with DHFR and lenalidomide interacts with CRBN) was added in or without the indicated dose of the test compound. Luciferase activity induced by the formation of a ternary complex containing DHFR-trimethoprim-lenalidomide-CRBN, and the resulting activation of STAT3 signaling, were measured 24 hours after compound treatment using a luciferase assay system kit (Promega, Madison, WI) with an Ensight plate reader (PerkinElmer Life Sciences, Waltham, MA).The data points in Figures 5A–5C represent the mean luciferase activity of triplicate samples derived from cells treated with leptin + test compound or leptin + hybrid ligand + test compound (CRBN) for the REM2 control (CTRL), compared to samples treated with leptin (CTRL) or leptin + hybrid ligand (CRBN) alone (for both cases, the signal obtained without the added test compound is set to 100% of luciferase activity on the y-axis). Error bars represent the standard deviation. Curves were fitted using 4-parameter nonlinear regression in GRAPHPAD PRISM software. As shown in Figures 5A–5C, known IMiD compounds such as lenalidomide and CC220 specifically inhibit hybrid ligand-induced luciferase reporter activation in a dose-dependent manner. This reflects effective competition for binding to CRBN and prevention of binding of the hybrid ligand to CRBN (and thus inhibition of the assay signal).

[0119] Figures 5D–5G and 5H–5L address the question of whether any of the test compounds characterized in Figures 5A–5C effectively bind to CRBN in cells, act as molecular glue, or induce the recruitment of ASS1 or IKZF to CRBN. For this analysis, HEK293T cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and incubated at 37°C and 8% CO2. Cells were transfected with a plasmid (pSEL-CRBN) encoding a CRBN bait fused to the cytoplasmic domain of a leptin receptor, which itself is fused to the extracellular domain of an erythropoietin (EPO) receptor. The extracellular EPO receptor domain can be used interchangeably with the extracellular leptin receptor domain (as used in Figures 5A–5C) to promote receptor / receptor-associated JAK2 activation (using EPO or leptin, respectively). In addition, cells were transfected with plasmids encoding IKZF1 (isoform 7) fused to the gp130 cytoplasmic domain (pMG1-IKZF1(iso7)) or ASS1 fused to the gp130 cytoplasmic domain (pMG1-ASS1), and a STAT3-reactive luciferase-coding reporter plasmid (pXP2d2-rPAPI-luciferase reporter plasmid) as described (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 transfection, cells were treated with erythropoietin (EPO) in or without the indicated doses of the test compound. Luciferase activity was measured 24 hours after compound treatment using a luciferase assay system kit (Promega, Madison, WI) with an Ensight plate reader (PerkinElmer Life Sciences, Waltham, MA). Data points show the induction ratio of mean luciferase activity in triplicate samples from EPO+ test compound-treated cells versus EPO-only treated cells. Error bars represent the standard deviation.We fitted the curves using four-parameter nonlinear regression in GRAPHPAD PRISM software. As shown in both Figures 5D–5G and 5H–5L, we were able to identify compounds that effectively bound to CRBN but did not induce ASS1 recruitment compared to known IMiDs, including lenalidomide and CC220.

[0120] Table 1 summarizes the results of Figures 5A–5L. The potency / efficacy of CRBN-binding compounds, calculated as IC50 from competitive analysis in Figures 5A–5C (dose-response curves), is shown. Maximum luciferase reporter induction achieved at any dose in either the IKZF1 or ASS1 recruitment study is also shown. Compounds "v" and "vi," which are CRBN binders as potent as LEN, do not recruit the ASS1 (or IKZF1) target at high concentrations of 30 micromolar. As shown, different recruitment patterns can be observed, and the lack of ASS1 recruitment was selectively monitored in competitive analysis for the evolution of compounds with a tendency toward dialed-out target recruitment (i.e., ASS1 recruitment).

[0121] [Table 1]

[0122] Example 6: Detection of compound-induced CRBN-ASS1 interaction using a DDB1 MAPPIT receptor fusion construct Since it can be advantageous to test compound-induced CRBN-substrate interactions using an unfused version of CRBN bait, we developed a MAPPIT-derived assay that fuses DDB1 to the MAPPIT chimeric receptor construct rather than CRBN. DDB1 is an adapter protein that connects CRBN to the core E3 ubiquitin ligase complex scaffold subunit CUL4A or CUL4B (karin 4A or karin 4B). HEK293T cells were transfected with a plasmid encoding DDB1 (pSEL-DDB1) anchored to a MAPPIT receptor fusion containing the extracellular domain of the EPO receptor, a plasmid encoding a CRBN substrate protein (IKZF1 isoform 7 or ASS1) fused to a partial gp130 domain, and a STAT3-reactive luciferase-coding reporter plasmid (pXP2d2-rPAPI-luciferase reporter plasmid) as described (Lievens, et al. "Array MAPPIT: high-throughput interactome analysis in mammalian cells". Journal of Proteome Research 8.2(2009):877-886). In addition, cells were also co-transfected with different amounts of unfused CRBN expression constructs. 24 hours after transfection, cells were treated with EPO, either without or with the indicated doses of lenalidomide (LEN). Luciferase activity was measured 24 hours after compound treatment using a luciferase assay system kit (Promega, Madison, WI) with an Ensight plate reader (PerkinElmer Life Sciences, Waltham, MA). Data points show the induction ratio of mean luciferase activity in triplicate samples from EPO+ test compound-treated cells versus EPO-only treated cells. Error bars represent the standard deviation. As shown in Figure 6, a potent lenalidomide-dependent MAPPIT signal is obtained from both IKZF1 and ASS1 interactions, but only in the presence of co-expressed unfused CRBNs does the signal appear to be mediated by the binding of the substrate gp130 fusion protein to the CRBN.

[0123] Equivalents Although the present invention has been described in relation to its particular embodiments, further modifications are possible, and it will be understood that this application is intended to cover any modifications, uses, or adaptations of the present invention, including any deviations from this disclosure that are generally known or customary in the art in which the invention relates, in accordance with the principles of the invention, and that these are described in the appended claims and, to that extent, applicable to the essential features described herein.

[0124] Those skilled in the art will be able to identify or confirm numerous equivalents to the specific embodiments described herein by means of routine experiments. Such equivalents are intended to be included within the scope of the following claims.

[0125] Built-in by reference All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0126] The publications discussed herein were provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention does not grant prior rights to such publications on the grounds of prior art.

[0127] As used herein, all headings are for organizational purposes only and are not intended to limit this disclosure in any way. The contents of any individual section may be equally applicable to all sections.

Claims

1. A method for identifying candidate compounds: (a) To obtain a test compound that has the ability to bind to cereblon (CRBN); (b) Contacting the test compound with CRBN in the presence of argininosuccinate synthase 1 (ASS1); (c) Assaying for direct or indirect recruitment, ubiquitination, or degradation of ASS1; and (d) Classifying the test compound as a candidate compound if reduced, slight, or absent direct or indirect recruitment, ubiquitination, or degradation of ASS1 is detected. Includes, The reduction, slight, or absence of direct or indirect recruitment, ubiquitination, or degradation of ASS1 compared to the direct or indirect recruitment, ubiquitination, or degradation of substrates or neosubstrates of non-ASS1 CRBNs, or The reduction, slight decrease, or absence of direct or indirect recruitment, ubiquitination, or degradation of ASS1 is compared to the amount, or baseline level, of direct or indirect recruitment, ubiquitination, or degradation of ASS1 in a reference sample without the test compound. method.

2. A method for manufacturing a therapeutic composition: (a) (i) Obtain a test compound that has the ability to bind to cereblon (CRBN); (ii) Contacting the test compound with CRBN in the presence of argininosuccinate synthase 1 (ASS1); (iii) Assaying for the direct or indirect recruitment, ubiquitination, or degradation of ASS1; and (iv) Classifying the test compound as a candidate compound if reduced, slight, or absent direct or indirect recruitment, ubiquitination, or degradation of ASS1 is detected; and (b) Formulating the candidate compound for therapeutic use. Includes, The reduction, slight, or absence of direct or indirect recruitment, ubiquitination, or degradation of ASS1 compared to the direct or indirect recruitment, ubiquitination, or degradation of substrates or neosubstrates of non-ASS1 CRBNs, or The reduction, slight decrease, or absence of direct or indirect recruitment, ubiquitination, or degradation of ASS1 is compared to the amount, or baseline level, of direct or indirect recruitment, ubiquitination, or degradation of ASS1 in a reference sample without the test compound. method.

3. The method according to claim 1 or 2, wherein the recruitment of ASS1 is the direct bonding of ASS1 to CRBN.

4. Further comprising assaying the direct or indirect recruitment, ubiquitination, or degradation of non-ASS1 CRBN substrates or neosubstrates, The substrate or neosubstrate of a CRBN that is not ASS1, (i) Degron motif, (ii) A surface β-hairpin loop having three skeletal hydrogen bond receptors at the apex of the turn followed by a glycine residue, or (iii) Saved Cys ２ -His ２ (C ２ H ２ )motif including, The method according to any one of claims 1 to 3.

5. The candidate compound exhibits reduced side effects in subjects receiving the candidate compound compared to one of thalidomide, lenalidomide, and pomalidomide, or The aforementioned side effects include decreased or impaired liver function, or decreased or impaired kidney function, or decreased or impaired T cell function, or The aforementioned test compound or candidate compound binds to CRBN, but only slightly to ASS1, or does not bind at all. The method according to any one of claims 1 to 4.

6. The method according to any one of claims 4 to 5, wherein the test compound or candidate compound binds to CRBN but only slightly to or does not bind to the substrate or neosubstrate of CRBN that is not ASS1.

7. The aforementioned test compound or candidate compound binds CRBN with an affinity of 1 μM or higher, or The aforementioned test compound or candidate compound binds CRBN with affinity of 500 nM, 300 nM, 100 nM, 30 nM, 10 nM, or 1 nM. The method according to any one of claims 1 to 6.

8. The aforementioned test compound or candidate compound is a molecular glue, or The aforementioned test compound or candidate compound contains a glutarimide ring and a phthalimide ring, The method according to any one of claims 1 to 7.

9. The method according to any one of claims 1 to 5, wherein the test compound or candidate compound binds to CRBN but does not bind to ASS1.

10. The method according to any one of claims 4 to 5, wherein the test compound or candidate compound binds to the substrate or neosubstrate of CRBN and CRBN other than ASS1.

11. The aforementioned test compound or candidate compound is heterobifunctional. The method according to claim 9 or 10.

12. The aforementioned test compound or candidate compound is a proteolytic chimera (PROTAC) or a component of PROTAC. The method according to any one of claims 9 to 11.

13. The method according to any one of claims 9 to 12, wherein the test compound or candidate compound is heterobifunctional and can be conjugated via click chemistry.

14. The method according to any one of claims 9 to 12, wherein the degradation is ubiquitin-dependent.

15. The assay further comprises assaying for the direct or indirect recruitment, ubiquitination, or degradation of a non-ASS1 CRBN substrate or neosubstrate, wherein the non-ASS1 CRBN substrate or neosubstrate is selected from Ikaros (IKZF1), Helios (IKZF2), Aeolus (IKZF3), Eos (IKZF4), Pegasus (IKZF5), CSNK1A, CK1a, and ZFP91; and The classification is based on the ability of the test compound to shift the ratio of the direct or indirect recruitment, ubiquitination, or degradation of ASS1 to the direct or indirect recruitment, ubiquitination, or degradation of the substrate and / or neosubstrate of a non-ASS1 CRBN, The method according to claim 1.

16. The test compound or candidate compound binds to CRBN, but only slightly or not at all to ASS1, or the test compound or candidate compound binds to CRBN with an affinity of 1 μM or higher. The method according to claim 15.

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