WO2023028508A2

WO2023028508A2 - Myc:trrap inhibitors and uses thereof

Info

Publication number: WO2023028508A2
Application number: PCT/US2022/075378
Authority: WO
Inventors: Edmond J. Feris; Michael D. Cole
Original assignee: Trustees Of Dartmouth College
Priority date: 2021-08-25
Filing date: 2022-08-24
Publication date: 2023-03-02
Also published as: WO2023028508A3

Abstract

The present disclosure relates to inhibitors of the MYC:TRRAP interaction and their use in the prevention or treatment of cancer and disclosed. Disclosed compounds include 3,9-Dihydroxy-4-prenyl-[6aR;11aR]pterocarpan, N-[4-(diethylamino)benzyl]-1-(methylsulfonyl)-3-(pyridin-3-yl)-1H-1,2,4-triazol-5-amine, and N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide.

Description

MYC:TRRAP INHIBITORS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This patent application claims priority to U.S. Provisional Patent Application No. 63/317,365, filed on March 7, 2022 and U.S. Provisional Patent Application No. 63/236,845, filed on August 25, 2021, the entire contents of which are fully incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[002] This invention was made with government support under R01 CA055248 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[003] The present disclosure provides inhibitors of the MYC:TRRAP interaction, particularly small molecule MYC:TRRAP inhibitors, and their use to treat various conditions, disorders, and/or diseases, including proliferative disorders such as cancer.

BACKGROUND OF THE INVENTION

[004] The transcription factor MYC is the most frequently amplified gene in human cancer and is overexpressed because of mutations in an array of oncogenic signaling pathways. The fact that many cancer cells cannot survive without MYC - a phenomenon termed “MYC addiction” - provides a compelling case for the development of MYC-specific targeted therapies. As such, MYC has been recognized as the 'most wanted’ target in cancer for decades, but most efforts have met with inescapable off-target toxicity.

[005] Since MYC has no inherent enzymatic activity, it has been inaccurately described as an “undruggable” target. However, it does have a functional DNA-binding domain and a transactivation domain (TAD). The DNA-binding domain requires a protein-protein interaction (PPI) with its obligate partner MAX. Several labs have attempted to find small molecules that inhibit MYC:MAX with limited success. It is informative to point out the timeline of MYC:MAX inhibitor research. The first MYC:MAX inhibitor was re-ported in 2002, and it functioned at 50-100 pM. Improved analogs of this inhibitor (~25 pM) have been reported and other inhibitors have been reported more recently. However, the specificity of these inhibitors for MYC: MAX is often unclear.

[006] TRRAP is a well-characterized MYC cofactor and is an essential component of various histone acetyltransferase (HAT) complexes. The identification of TRRAP as an essential MYC cofactor established a link to HAT complexes containing GCN5 and TIP60 and provided an important mechanistic insight into MYC function. The MYC:TRRAP interaction occurs at a precise motif in the TAD of MYC known as the MYC Homology Box 2 (MB2). The importance of MB2 in MYC-driven tumorigenesis is well established in cellular assays and animal models, presumably because it is necessary for the MYC:TRRAP interaction. Therefore, the TAD of MYC, MB2, and MYC:TRRAP are required for MYC-driven transactivation and cancer promotion.

[007] Despite attempts at inhibiting MYC expression, its direct interaction with DNA and its obligate partner MAX, or any indirect MYC effectors, no clinically useful drug has emerged in nearly 20 years due to poor accessibility and specificity. Thus, novel targeted therapies that aim at correcting dysregulated transcriptional programs in cancer are an unmet medical need. The present application provides a radical new strategy to inhibit MYC function by disrupting its essential interaction with TRRAP using small molecules and further provides first-in-class inhibitors of the MYC:TRRAP interaction.

SUMMARY OF THE INVENTION

[008] The present disclosure relates to compounds and methods for inhibiting MYC function by disrupting the MYC:TRRAP interaction.

[009] In one aspect, this disclosure provides a small molecule inhibitor of the MYC:TRRAP interaction. In certain embodiments, the inhibitor is selected from the list of compounds in Table A. In certain embodiments, the inhibitor is an analog or derivative of a compound listed in Table A. For example, the inhibitor can have chemical similarity to one of the compounds listed in T able A. Exemplary compounds may be identified through chemical similarity searching. The present disclosure relates to the use of such compounds as MYC:TRRAP inhibitors.

[010] In another aspect, this disclosure provides methods for inhibiting the MYC:TRRAP interaction. The methods comprise contacting a system comprising a MYC protein or a fragment thereof and a TRRAP protein or a fragment thereof with a small molecule inhibitor disclosed herein. In certain embodiments, the contacting occurs in vivo. In certain embodiments, the contacting occurs in vitro.

[011] In yet another aspect, this disclosure provides methods for preventing or treating conditions, diseases, or disorders treatable or preventable by modulation of the MYC:TRRAP interaction. The methods comprise administering to a subject in need thereof a prophylactically or therapeutically effective amount of a small molecule inhibitor disclosed herein. One exemplary disease is cancer, which includes, but is not limited to, breast cancer, lung cancer, colon cancer, leukemia and lymphoma. Thus, in certain embodiments, this disclosure provides methods for preventing or treating cancer. Other conditions, diseases, or disorders that may be treated or prevented with a small molecule inhibitor disclosed herein include aging (e.g., extending the health span of healthy individuals) and male reproduction (e.g., as a male birth control pill).

[012] The disclosure also relates to pharmaceutical compositions comprising small molecule MYC:TRRAP inhibitors and the use of such pharmaceutical compositions as human and/or animal therapeutics and medicines.

[013] In certain embodiments, the small molecule MYC:TRRAP inhibitor binds to MYC. In some such embodiments, the small molecule MYC:TRRAP inhibitor directly disrupts the TRRAP binding site (e.g., MB2).

[014] In certain embodiments, the small molecule MYC:TRRAP inhibitor binds to TRRAP. In some such embodiments, the small molecule MYC:TRRAP inhibitor binds to TRRAP as a MB2 mimetic or an allosteric modulator.

[015] The compounds, pharmaceutical compositions comprising the compounds, and methods for treating or preventing conditions, disorders, and/or diseases by administering the compounds are further described herein.

[016] These and other objects of the invention are described in the following paragraphs. These objects should not be deemed to narrow the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[017] For a better understanding of the invention, reference may be made to embodiments shown in the following drawings. The components in the drawings are not necessarily to scale and related elements may be omitted, or in some instances proportions may have been exaggerated, so as to emphasize and clearly illustrate the novel features described herein. In addition, system components can be variously arranged, as known in the art.

[018] FIG. 1A is a Western blot analysis of a co-immunoprecipitation experiment were the indicated regions of TRRAP were cloned into a CMV-FLAG expression vector and transfected into HEK293T cells. Proteins were co-expressed with PYO-tagged full-length MYC and then MYC was IPed with anti-PYO beads. Co-IP was assessed by western blot with anti-FLAG. The most critical binding domain is within residues 2033-2088, consistent with previous results (Feris et al., PLoS ONE 14, e0225784 (2019), the entire contents of which are fully incorporated herein by reference), and the minimal domain TRRAP 2033-2283 is established.

[019] FIG. 1B is a Western blot analysis of a co-immunoprecipitation experiment were the indicated regions of TRRAP were cloned into a CMV-FLAG expression vector and transfected into HEK293T cells. Endogenous MYC was IPed with C-33 beads (Santa Cruz Biotechnology). The TRRAP and FLAG blots show TRRAP 2033-2283 can compete off the endogenous native MYC:TRRAP complex in cells, but the same domain of TRRAP lacking the critical region 2033- 2088 does not show the same level of competition.

[020] FIG. 2A is a schematic representation of MYC 1-190 and TRRAP 2033-2283 fused to each subunit of the NanoLuc® Binary Technology (NanoBiT) system.

[021] FIG. 2B is a bar graph depicting luminescence measurements of cells transfected with TRRAP 2033-2283-LgBiT and SmBiT-MYC 1-190 with and without MB2 and alone. The LgBiT subunit was co-transfected in excess with SmBiT-MYC 1-190 with and without MB2 and alone. The panel on the left show measurements carried out in cells while the right one shows measurements in cellular extracts. A 10-fold decrease is observed with the deletion of MB2. MYC binding to TRRAP is MB2 dependent, validating in vivo immunoprecipitation experiments with native proteins.

[022] FIG. 3 is a graph of luminescence measurements of cells transfected with TRRAP 2033- 2283-LgBiT and SmBiT-MYC 1-190 in the presence of 10 pM each of the 50,000 NIBR compounds.

[023] FIG. 4 is a table listing thirty-three MYC:TRRAP inhibitors.

[024] FIG. 5 depicts data from a MYC NaLTSA assay. Left Panel: quantification of western blot analysis of NaLTSA experiments of the native MYC protein. The curve indicates the melting temperatures for the native MYC protein. NaLTSA measurements were carried out using the native MYC protein fused to NL enzyme as a reporter. MYC-NL expression for these measurements was -10% of the endogenous MYC levels. Right Panel: establishment of the MYC-NL luminescence signal at MYC endogenous levels. Western blot of the endogenous MYC protein and MYC-NL fusion protein and luminescence measurements of the same cells with decreasing amounts of MYC-NL exogenously introduced.

[025] FIG. 6 shows exemplary data that was used to evaluate compounds for binding to either MYC or TRRAP using NanoLuc Thermal Shift Assays (NaLTSA).

[026] FIG. 7 shows the structures of 9 selected compounds grouped into three categories: MYC specific, TRRAP specific or undetermined and additionally shows CETSA data for two exemplary compounds, Compound 1 (CID 46880035) and Compound 7 (CID 6501132).

[027] FIG. 8 shows data for Compound 25 from the NanoLuc® Binary Technology (NanoBiT) system, a P493-6 cell viability assay, a Growth in Low Attachment (GILA) assay, a NanoLuc Thermal Shift Assay (NaLTSA), and a co-IP assay DESCRIPTION OF THE INVENTION

[028] This detailed description is intended only to acquaint others skilled in the art with the present invention, its principles, and its practical application so that others skilled in the art may adapt and apply the invention in its numerous forms, as they may be best suited to the requirements of a particular use. This description and its specific examples are intended for purposes of illustration only. This invention, therefore, is not limited to the embodiments described in this patent application and may be variously modified.

[029] Using a platform for identifying small molecules as inhibitors of the MYC:TRRAP interaction (see PCT/US2020/062870, the entire contents of which are fully incorporated herein by reference), first- in-class inhibitors of the MYC:TRRAP interaction, with activity in the low nanomolar to micromolar range, were identified and provided herein. The robust and high- throughput amenable platform was used to identify inhibitors of the MYC:TRRAP interaction. In particular, using luminescence complementation and the minimal MYC and TRRAP interacting domains, a PPI assay was developed that results in an active luciferase enzyme when MYC:TRRAP complexes form. The NanoLuc® Binary Technology (Nano-BiT®), a split version of the NanoLuc® luciferase (a 19.1 kDa protein that produces an ATP-independent glow-type luminescence with half-life > 2 h; Promega Corporation) intended for measurement of PPIs in live cells was employed. Unlike co-IPs and other binding assays, the NanoBiT® system allows for quantifiable measurements without cell lysis. With high sensitivity and broad dynamic range, bioluminescent methods have proven useful for many applications, including binding assays and drug discovery.

[030] A. DEFINITIONS

[031] As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated:

[032] The term “about” as used herein means approximately, and in most cases within 10% of the stated value.

[033] The term “MYC” as used herein refers to the MYC transcription factor protein, transcript (mRNA), and/or gene expressing said protein from human (NCBI GenelD No. 4609) or from any other mammalian species, including all isoforms and allelic variants thereof. MYC is also known as MRTL, MYCC, bHLHe39, and c-MYC.

[034] The term “TRRAP” as used herein refers to the “Transformation/Transcription Domain- Associated Protein” protein, transcript (mRNA), and/or gene expressing said protein from human (NCBI GenelD No. 8295) or from any other mammalian species, including all isoforms and allelic variants thereof. TRRAP is also known as DEDDFA, PAF350/400, PAF400, STAF40, TR-AP, and Tra1.

[035] The term “MAX” as used herein refers to the “MYC-associated factor X” protein, transcript (mRNA), and/or gene expression said protein from human (NCBI GenelD No. 4149) or from any other mammalian species, including all isoforms and allelic variants thereof. MAX is also known as bHLHd4.

[036] The term “pharmaceutically acceptable” is used adjectivally to mean that the modified noun is appropriate for use as a pharmaceutical product for human use or as a part of a pharmaceutical product for human use.

[037] The terms “treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a condition, disorder, or disease and/or the attendant symptoms thereof.

[038] B. COMPOUNDS

[039] In one aspect, compounds disclosed herein possess biological activity, for example, as a inhibitors of the MYC:TRRAP interaction. In certain embodiments, a compound disclosed herein may be transformed by methods well known to those skilled in the art of synthetic organic chemistry into a derivative compound that possesses biological activity, for example, as an inhibitor of the MYC:TRRAP interaction.

[040] In certain aspects, the present disclosure relates to a compound or a pharmaceutically acceptable salt thereof for use as an inhibitor of the MYC:TRRAP interaction. In some embodiments, the compound is selected from the list of compounds in Table A. In other embodiments, the compound is an analog or derivative of a compound listed in Table A.

[041] In some such embodiments, the small molecule inhibitor of the MYC:TRRAP interaction is a compound listed in Table A. In some such embodiments, the small molecule inhibitor of the MYC:TRRAP interaction is Compound 1 , Compound 7, or Compound 25.

[042] In some such embodiments, the small molecule inhibitor of the MYC:TRRAP interaction is a pharmaceutically acceptable salt of a compound listed in Table A. In some such embodiments, the small molecule inhibitor of the MYC:TRRAP interaction is a pharmaceutically acceptable salt of Compound 1 , Compound 7, or Compound 25. [043] Table A. Exemplary Compounds as Inhibitors of MYC:TRRAP Interaction

[044] In certain embodiments, a small molecule inhibitor of the MYC:TRRAP interaction is an analog or derivative of a compound listed in Table A. For example, such analogs or derivatives may be identified through chemical similarity searching. In some such embodiments, the small molecule inhibitor of the MYC:TRRAP interaction is an analog or derivative of Compound 1 , Compound 7, or Compound 25.

[045] An expanded similarity set of ~1400 has been identified. These compounds can be used as MYC:TRRAP inhibitors.

[046] In certain embodiments, the small molecule inhibitor of the MYC:TRRAP interaction may be synthesized by methods known to those of ordinary skill in the art. In some such embodiments, the small molecule inhibitor of the MYC:TRRAP interaction is an analog and/or derivative of a compound listed in Table A. Methods for derivatizing small molecule organic compounds are known to those of ordinary skill in the art. For example, halogenation and alkylation reactions are known to those of ordinary skill in the art.

[047] Compound 1 (PubChem CID 46880035) has a molecular formula of C20H20O4 and can be referred to as 3,9-Dihydroxy-4-prenyl-[6aR;11aR]pterocarpan:

[048] Compound 7 (PubChem CID 6501132) has a molecular formula of C19H24N6O2S and can be referred to as N-[4-(diethylamino)benzyl]-1-(methylsulfonyl)-3-(pyridin-3-yl)-1 H-1 ,2,4-triazol-5- amine:

[049] Compound 25 (PubChem CID 5081913) has a molecular formula of C15H8CIF6NO2 and can be referred to as N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide:

[050] C. METHODS OF USE

[051] In at least one aspect, the present disclosure includes a method for treating or preventing a MYC-dependent condition, disorder, and/or disease in a subject in need of such treatment or prevention. In certain embodiments, the MYC-dependent condition, disorder, and/or disease is a proliferative disease. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”). In particular, exemplary proliferative diseases that may be treated or prevented include breast cancer, lung cancer, colon cancer, leukemia, and lymphoma. In other such embodiments, the MYC-dependent condition, disorder, and/or disease is male fertility or the health span of an individual.

[052] Thus, one aspect of the present disclosure includes a method for treating cancer. The method comprises administering to a patient in need thereof a therapeutically effective amount of a compound described herein (including, but not limited to, compound listed in Table A or an analog or derivative thereof) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound (or pharmaceutically acceptable salt thereof) is administered orally.

[053] In certain embodiments, the cancer is a MYC-dependent cancer.

[054] In at least one aspect, the present disclosure includes a compound disclosed herein or a pharmaceutically acceptable salt thereof for use in a method for treating or preventing a MYC- dependent condition, disorder, and/or disease in a subject in need of such treatment or prevention. In certain embodiments, the MYC-dependent condition, disorder, and/or disease is a proliferative disease. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”). In particular, exemplary proliferative diseases that may be treated or prevented include breast cancer, lung cancer, colon cancer, leukemia, and lymphoma.

[055] Thus, the present disclosure includes a compound disclosed herein (including, but not limited to, compound listed in Table A or an analog or derivative thereof) or a pharmaceutically acceptable salt thereof for use in a method for treating a cancer, particularly breast cancer, lung cancer, colon cancer, leukemia, and lymphoma.

[056] Another aspect of the present disclosure includes a method for treating or preventing a disease or condition treatable or preventable by selectively modulating the MYC:TRRAP interaction in a subject in need of such treatment or prevention.

[057] Yet another aspect of the present disclosure includes a method for inhibiting the MYC:TRRAP interaction in a cell (e.g., tumor cell). In certain embodiments, the cell is present in a subject (e.g., a subject afflicted with cancer). In certain embodiments, the cell is characterized by overexpression and/or aberrant activity of MYC. [058] In certain embodiments, for any of the aforementioned aspects, the subject or patient is a mammal. In some such embodiments, the mammal is a human.

[059] In certain embodiments, for any of the aforementioned aspects, the subject or patient has been diagnosed with a cancer or is suspected of having a cancer. In some such embodiments, the cancer is associated with and/or characterized by overexpression and/or aberrant activity of MYC. In some such embodiments, the cancer is a MYC-dependent cancer. In some such embodiments, the cancer is treatable or preventable by selectively modulating the MYC:TRRAP interaction.

[060] In certain embodiments, for any of the aforementioned aspects, the methods comprise selecting a subject or patient. In some such embodiments, the subject or patient is selected on the basis of having, or being suspected of having, a cancer associated with and/or characterized by overexpression and/or aberrant activity of MYC. In some such embodiments, the subject or patient is selected on the basis of having, or being suspected of having, a MYC-dependent cancer. In some such embodiments, the subject or patient is selected on the basis of having, or being suspected of having, a cancer that is treatable or preventable by selectively modulating the MYC:TRRAP interaction. In some such embodiments, the subject or patient is selected on the basis of having tissue or cells (e.g., tumor cells) that overexpress or aberrantly express MYC.

[061] MYC expression can be assessed using methods for detecting and visualizing a biological target, including immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in situ hybridization (ISH), and others. An exemplary method for assessing MYC expression in a biological sample involves obtaining a biological sample from a subject or patient and contacting the biological sample with a compound or an agent capable of detecting MYC such that the presence of MYC is detected in the biological sample. One exemplary agent for detecting MYC is a nucleic acid probe capable of hybridizing to MYC mRNA (or cDNA copies thereof) contained in the biological sample. In some such embodiments, the nucleic acid probe is a labeled with a detectable label. Another exemplary agent for detecting MYC is an anti-MYC antibody capable of binding to MYC contained in the biological sample. In some such embodiments, the antibody is labeled with a detectable label. A detectable label may give off a color, a fluorescent signal, or radioactivity, for example. The methods for assessing MYC expression may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one nucleic acid probe or antibody reagent described herein, which may be conveniently used, for example, in clinical settings to diagnose a patient exhibiting symptoms or family history of a cancer associated with and/or characterized by overexpression and/or aberrant activity of MYC. [062] In certain embodiments, for any of the aforementioned aspects, the methods comprise administering to the subject a therapeutically effective amount of a compound described herein (including, but not limited to, compound listed in Table A or an analog or derivative thereof) or a pharmaceutically acceptable salt thereof as single agent or in combination with another therapeutic agent.

[063] The preferred total daily dose of the compound or salt (administered in single or divided doses) is typically from about 0.001 to about 100 mg/kg, more preferably from about 0.001 to about 30 mg/kg, and even more preferably from about 0.01 to about 10 mg/kg (i.e., mg of the compound or salt per kg body weight). In certain embodiments, dosage unit compositions contain such amounts or submultiples thereof to make up the daily dose. In many instances, the administration of the compound or salt will be repeated a plurality of times. In certain embodiments, multiple doses per day typically may be used to increase the total daily dose, if desired.

[064] Factors affecting the preferred dosage regimen include the type, age, weight, sex, diet, and condition of the patient; the severity of the pathological condition; the route of administration; pharmacological considerations, such as the activity, efficacy, pharmacokinetic, and toxicology profiles of the particular compound or salt used; whether a drug delivery system is utilized; and whether the compound or salt is administered as part of a drug combination. Thus, the dosage regimen actually employed can vary widely, and therefore, can derive from the preferred dosage regimen set forth above.

[065] D. COMPOSITIONS

[066] In at least one aspect, the present disclosure includes compositions comprising a compound described herein (including, but not limited to, compound listed in Table A or an analog or derivative thereof) or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises one or more conventional pharmaceutically acceptable excipients.

[067] Pharmaceutical compositions disclosed herein comprise a compound disclosed herein or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition is an oral dosage form, preferably a solid oral dosage form (e.g., a tablet). In some such embodiments, the solid oral dosage form may comprise pharmaceutically acceptable excipients such as excipients that function as binders, glidants, lubricants, and fillers. Thus, a solid oral dosage form comprising a compound disclosed herein ora pharmaceutically acceptable salt thereof further optionally comprises one or more conventional pharmaceutically acceptable excipients [068] In at least one aspect, the present disclosure includes a pharmaceutical composition for treating a MYC-dependent condition, disorder, and/or disease such as cancer, the composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[069] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the compositions and methods of the invention described herein may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein.

[070] The compounds, compositions, and methods described herein will be better understood by reference to the following examples, which are included as an illustration of and not a limitation upon the scope of the invention.

[071] E. EXAMPLES

[072] EXAMPLE 1. TRRAP 2033-2283 is the minimal MYC-binding domain.

[073] Previous work showed that MYC 1-190 is sufficient for a stable MYC:TRRAP interaction (Feris et al., 2019). TRRAP 1997-2401 binds to MYC and TRRAP 1997-2088 is required but not sufficient to sustain the MYC:TRRAP interaction in co-IP assays (Feris et al., 2019). Deletion studies showed that a minimal domain of TRRAP (2033-2283) is sufficient for protein complex formation in co-IP experiments (Fig. 1A).

[074] To validate this mapping, co-IP experiments were performed to test if TRRAP 2033-2283 is essential in the native MYC:TRRAP complex. A competition experiment between the TRRAP 2033-2283 domain and native MYC:TRRAP complexes showed that overexpression of the critical TRRAP domain inhibited formation of native MYC:TRRAP complexes (Fig. 1 B). This effect was attenuated when the core internal binding region of TRRAP (2033-2088) was not present (Fig. 1 B, lane 3).

[075] EXAMPLE 2. MYC:TRRAP luminescence complementation platform is MB2- dependent

[076] Defining the minimal domains that form the MYC:TRRAP complex allowed for the development an assay for PPI using the NanoBiT® PPI system, which fuses the Large Binary Technology (LgBiT) and Small Binary Technology (SmBiT) subunits to different interacting proteins. Briefly, residues 2033-2283 of TRRAP and 1-190 of MYC were respectively cloned into CMV-driven vectors containing the LgBiT (18kDa) and SmBiT (11aa) subunits to monitor the MYC:TRRAP PPI. Upon protein dimerization of MYC:TRRAP, the NanoBiT® subunits complement and form a highly active luciferase enzyme (Fig. 2A). [077] Next, MYC:TRRAP complex formation was measured using luminescence complementation. The minimal interacting domains described above gave a robust luminescence, whereas deletion of Myc Homology Box 2 (MB2, amino acids 128-144) reduced luminescence by 90% both in cells and in cellular extracts (Fig. 2B compare bars 1 and 2). Thus, this luciferase assay appeared to recapitulate the in vivo MYC:TRRAP interaction (Feris et al., 2019; McMahon et al., 1998). The LgBiT subunit alone was co-transfected in 100-fold excess with both MYC and MYC AMB2, which caused the SmBiT subunit in those constructs to interact independently with the LgBiT subunit. This provided a measure of equal expression for both MYC and MYC AMB2 constructs (Fig. 2B lanes 4 and 5).

[078] Two additional LgBiT-TRRAP constructs were tested for luminescence. To confirm the importance of the TRRAP 2033-2088 domain, luminescence was measured with TRRAP 2033- 2283 compared to a similar construct lacking the MYC binding region, TRRAP 2088-2283. Like MB2, the absence of TRRAP 2033-2088 diminished binding, consistent with co-IP experiments (Feris et al., 2019). Furthermore, fusion of LgBiT to the N-terminus of full-length TRRAP did not produce measurable luminescence when co-transfected with an N-terminal fusion of SmBiT with full length MYC or MYC 1-190 (data not shown). It is possible that the N-terminus of TRRAP is too far from the MYC interacting domain to allow for complementation of the luciferase subunits.

[079] EXAMPLE 3. Identification of MYC:TRRAP inhibitors in high throughput

[080] The assay was adapted and optimized for use in the robotic facilities at Novartis Institute for Biomedical Research (NIBR) in 1536-well format. The assay proved to be consistent and robust, and that it could be adapted for high-throughput screening with an average z’ score of 0.75 in cells and 0.9 in cellular extracts.

[081] Next, 50,000 non-proprietary compounds provided by NIBR were assayed. All measurements in this primary screen were performed in duplicate and measured in cells. 397 compounds (0.8% primary hit rate) showed inhibitory activity that was three standard deviations from the vehicle controls (Fig. 3), and these were validated with measurements in 8-point half-log concentration curves in cells and in cellular extracts (in vitro).

[082] All primary hits were subsequently counter-screened against the MYC AMB2:TRRAP interaction and the luciferase fragments interaction alone (Halo-SmBiT: LgBiT) in cellular extracts to eliminate false positives. Thirty-three compounds were identified that inhibit the MYC:TRRAP interaction (>1 pM), but not Halo-SmBiT:LgBiT luciferase alone (Table A; Fig. 4). All assays beyond the primary screen were performed in triplicate and used to calculate IC50 values.

[083] The top 33 selected inhibitors were used for a hit expansion through chemical similarity searching 250,000 compounds available at NIBR. [084] EXAMPLE 4. Determination of the molecular targets of MYC:TRRAP inhibitors

[085] The top 33 selected inhibitors were evaluated for binding to either MYC or TRRAP using Cellular Thermal Shift Assays (CETSA). Certain selected inhibitors were evaluated for binding to either MYC or TRRAP using NanoLuc Thermal Shift Assays (NaLTSA) developed by Promega.

[086] Briefly, CETSA measures the thermal stability of proteins with and without binding of small molecules. Molina et al., Science, 341 :84-87 (2013). If a small molecule binds, the thermal stability (i.e., denaturation temperature) can shift up or down by a few degrees. No change is expected if the small molecule does not bind.

[087] Using a fusion to NanoLuc (NL) luciferase and luminescence measurements to determine the amount of soluble protein remaining after temperature incubations and protein precipitations provides a much more quantifiable and reproducible assay that can detect smaller shifts in melting temperatures. Dart et al., ACS Med Chem Lett, 9:546-551 (2018). NaLTSA uses a fusion of the NL enzyme with the protein of interest (e.g., MYC or TRRAP) to determine relative protein concentration by measuring luciferase activity. If the protein of interest denatures and precipitates at increasing temperatures, the luciferase activity is reduced proportionately.

[088] Native full-length MYC and TRRAP proteins were fused to a NanoLuc® (Promega) luciferase construct used as a reporter to perform NaLTSA. Constructs with both MYC and TRRAP were created and used to carry out initial verification measurements (Fig. 5). This NaLTSA system allowed for optimization of the system for high-throughput screening in a 384-well format. NL luciferase remains active and soluble at <63°C, well above the melting temperature for native MYC (45°C) or TRRAP (48°C).

[089] Native MYC-NL and TRRAP-NL proteins were evaluated as in Fig. 5, where the compounds were added to the media for 1 h. Then, aliquots of cells in suspension were subjected to precise thermal treatment. Denatured proteins aggregate and are precipitated by centrifugation at each temperature point. Luminescence measurements of MYC-NL and TRRAP-NL determined the ratio of denatured to non-denatured protein and generate a thermal stability curve of each protein +/- compound. Shifting of these curves suggests if either of these two proteins is the binding partner for each compound. A change in the thermal stability of a protein is indicated by a compound-induced shift of its melting curve, which is apparent comparing the melting points derived under vehicle- and compound-incubated conditions.

[090] Figure 6 shows an example of how the data was evaluated. The top row of graphs shows a compound that could destabilize the melting temperature of MYC but not TRRAP. The middle row shows one that could stabilize the melting temperature of TRRAP and not MYC. Finally, the third row shows one that could disrupt both, as an example of a non-specific inhibitor. [091] Compounds that did not have any effect on the luminescence signal at 25C were selected and then compounds that had a shift in either MYC or TRRAP melting temperature but not both were selected to identify the top compounds that are specific either MYC or TRRAP.

[092] Figure 7 shows the structures of 9 selected compounds grouped into three categories: MYC specific, TRRAP specific or undetermined. Figure 7 additionally shows CETSA data for two exemplary compounds, Compound 1 (CID 46880035) and Compound 7 (CID 6501132).

[093] Table B shows a summary of 11 selected compounds that are specific either MYC or TRRAP.

[094] EXAMPLE 5. Additional Assays.

[095] P493-6 Cell Viability. The P493-6 Human Lymphoblastoid Cell Line expresses c-myc under control of a tetracycline-inducible promoter. P493-6 cell viability was assessed in the presence/absence of compound and/or the presence/absence of doxycycline.

[096] GILA Assay. A Growth in Low Attachment (GILA) assay was developed as a quantifiable measure of anchorage-independent growth which is a marker of cellular transformation. (Izar et al., Curr Protoc Mol Biol. 116:28.8.1-28.8.12, 2016). MCF-10A cells were engineered with constitutive MYC overexpression. The protocol for GILA measurements was adapted from Izar et al., 2016. Briefly, after selection, 100 pL of cells were seeded at 10⁴ cells/mL in ultra-low attachment 96-well plates from Corning®. After a 7-day incubation with or without MYC:TRRAP inhibitors in both low adherence and adherence plates, cell proliferation was quantified using CellTiter-Glo® 2.0 Cell Viability Assay according to manufacturer’s instructions. All measurements were taken on a SpectraMax i3 instrument (Molecular Devices). [097] Co-immunoprecipitation. Immunoprecipitations were performed using anti-PYO (Covance), or anti-MYC (C33 Santa Cruz Biotechnology) agarose pre-conjugated beads at 4°C for 16-20 h. Co-immunoprecipitation was analyzed by western blots with the following antibodies: MYC (sc-764 Santa Cruz Biotechnology) or MYC (9402 Cell Signaling Technology), TRRAP (A301-132A Bethel Laboratories), MAX (sc-8011 Santa Cruz Biotechnology), and FLAG (F7425 Millipore Sigma).

[098] Figure 8 and Table C show a summary of selected compounds.

[099] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations, or methods, or any combination of such changes and modifications of use of the invention, may be made without departing from the spirit and scope thereof.

[0100] All references (patent and non-patent) cited above are incorporated by reference into this patent application. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art (or prior art at all). Applicant reserves the right to challenge the accuracy and pertinence of the cited references.

Claims

What is claimed is:

1. A method for preventing or treating a cancer in a subject in need thereof, the method comprising administering to the subject a prophylactical ly or therapeutically effective amount of an inhibitor of the MYC:TRRAP interaction, wherein the inhibitor is compound listed in Table A or an analog or derivative thereof or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 , wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

22

3. The method of claim 1 , wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

4. The method of any one of the preceding claims, wherein the subject is a human.

5. The method of any one of the preceding claims, wherein the subject has been diagnosed with a cancer or is suspected of having a cancer.

6. The method of any one of the preceding claims, wherein the subject is at risk for developing cancer, optionally because of a genetic risk factor, previous cancer, and/or expression of a biomarker correlated to cancer.

7. The method of any one of the preceding claims, wherein the cancer is breast cancer, lung cancer, colon cancer, leukemia, or lymphoma.

8. The method of any one of the preceding claims, wherein the inhibitor is conjugated to a protein (e.g., antibody) or small molecule, optionally via a linker.

25

9. A pharmaceutical composition for use in inhibiting the MYC:TRRAP interaction comprising (i) an inhibitor of the MYC:TRRAP interaction, wherein the inhibitor is a compound listed in Table A or an analog or derivative thereof or a pharmaceutically acceptable salt thereof and (ii) a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein the inhibitor is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

26

11. The pharmaceutical composition of claim 9, wherein the inhibitor is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

12. An inhibitor of the MYC:TRRAP interaction for use in the prevention or treatment of cancer, wherein the inhibitor is a compound listed in Table A or an analog or derivative thereof or a pharmaceutically acceptable salt thereof.

13. The inhibitor of claim 12, wherein the inhibitor is selected from the group consisting of:

analogs thereof, derivatives thereof, and pharmaceutically acceptable salts thereof.

14. The inhibitor of claim 12, wherein the inhibitor is selected from the group consisting

and pharmaceutically acceptable salts thereof.

31