Harnessing the TAF1 acetyltransferase for targeted acetylation of the tumor suppressor p53

Abstract

Pharmacological reactivation of the tumor suppressor p53 remains a key challenge for the treatment of cancer. We previously reported Acetylation Targeting Chimera (AceTAC), a novel technology that hijacks lysine acetyltransferases p300 / CBP to acetylate the p53Y220C mutant. However, p300 / CBP are the only acetyltransferases harnessed for AceTAC development to date. In this study, we demonstrate for the first time that the TAF1 acetyltransferase can be recruited to acetylate p53Y220C. We discovered a novel TAFlrecruiting AceTAC, MS172, which effectively acetylated p53Y220C lysine 382 in a concentration-, time- and TAF1 -dependent manner via inducing the ternary complex formation between p53Y220C and TAF1. Notably, MS172 suppressed the proliferation in multiple p53Y220C-harboring cancer cell lines more potently than our previously reported p300 / CBPrecruiting p53Y220C AceTAC MS78 with little toxicity in p53 WT and normal cells. Additionally, MS 172 is bioavailable in mice and suitable for in vivo efficacy studies.

Description

Attorney Docket No. 27527-0233WO1HARNESSING THE TAF1 ACETYLTRANSFERASE FOR TARGETED ACETYLATION OF THE TUMOR SUPPRESSOR P53CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application No.63 / 733.331, filed December 12, 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named 27527-0233WOl_SL_ST26.xml. The XML file, created on December 8, 2025 is 2,845 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Protein acetylation is one of the most important post-translational modifications (PTMs) that regulates fundamental biological functions such as cell cycle and chromatin remodeling in cells.1Lysine acetylation involves the lysine acetyltransferase (KAT)-catalyzed transfer of the acetyl group from the cofactor acetyl-CoA to the e-amino group of lysine residues, leading to the regulation of distinct protein activity and functions.2A number of KATs have been identified and they can share substrate specificity and be divided into three main families: (1) GCN5, (2) p300 and (3) MYST.3On the other hand, lysine acetylation can be reversed by histone deacetylases (HDACs), which include Zn2+-dependent deacetylases and NAD+-dependent deacetylases.3Dysregulated protein lysine acetylation can lead to the pathogenesis of multiple diseases such as cancer, inflammation and neurodegenerative disorders.4Thus, the development of a selective and targeted protein acetylation (TPA) strategy is a potential therapeutic approach to thwart the progression of diseases such as cancer.

[0004] Previously, we reported a novel technology and modality, termed Acetylation Targeting Chimera (AceTAC), for inducing targeted protein acetylation.5AceTAC is a heterobifunctional small molecule that induces selective acetylation of a protein-of-interest (POI) by hijacking the p300 / CBP acetyltransferases.5We discovered MS78, the first-in-class AceTAC, which links a selective small-molecule binder of p53Y220C to a selective small-Attorney Docket No. 27527-0233WO1molecule ligand of p300 / CBP.5MS78 induced p53Y220C acetylation in a concentration- and time-dependent manner and had superior antiproliferation activities compared to the parent p53Y220C binder.5Importantly, AceTAC is the first small molecule-based TPA modality that does not require any genetic manipulation to induce protein acetylation.6We also performed structure-activity relationship (SAR) studies on p300 / CBP-recruiting p53Y220C AceTACs.7Recently, it was reported that PCAF / GCN5 can be hijacked for protein acetylation using a chemogenetic approach.8However, this approach requires the genetic tagging of specific proteins,8making it difficult to be developed into cancer therapies.

[0005] In this study, we demonstrated that TATA-binding protein-associated factor 1 (TAF1), which is the biggest subunit of the multi-protein Transcription Factor II D (TFIID) complex, can be utilized as a KAT for targeted protein acetylation.9,10TAF1 is known to initiate the transcriptional preinitiation complex (PIC) formation through the recognition of the core promoter region of genes and also interacts with MYC to regulate gene transcription.11,12Full-length TAF1 possesses six distinct domains including an N-terminal kinase domain, an acetyltransferase domain, followed by ubiquitin-activating / conjugating domain (E1 / E2), two tandem bromodomains categorized as TAF1-BD1 and TAF1-BD2, and a C-terminal kinase domain.10,13However, whether TAF 1 can be hij acked for targeted protein acetylation has never been shown. We discovered the first-in-class TAF 1 -recruiting p53Y220C AceTAC, MS172, which effectively acetylates p53Y220C without changing p53 protein levels. We validated the MS172-induced ternary complex formation between p53Y220C and TAF1 via both biochemical and cellular assays. Furthermore, we demonstrated that MS172 is selective for TAF1-BD2 over several other bromodomain-containing acetyltransferases. MS 172 also exhibits a superior anti-proliferative effect compared to the parent p53Y220C binder, TAF1 bromodomain binder and p300 / CBP-recruiting AceTAC, MS78, in multiple cancer cell lines harboring the p53Y220C mutation. Lastly, we discovered that the p53Y220C acetylation induced by MS 172 upregulates metallothionine protein functions using RNA-seq and RT-qPCR studies. Overall, we showed that the TAF1 acetyltransferase can be harnessed for inducing targeted protein acetylation.Design and synthesis of TA Fl -recruiting p53Y220C-targeting AceTACs.

[0006] Similar to our previous AceTAC design, we analyzed the co-crystal structure of p53Y220C in complex with PK9328 (PDB ID: 6GGF), a previously reported p53Y220C smallmolecule stabilizer, and utilized the solvent-exposed methylamine group as a suitable exitAttorney Docket No. 27527-0233WO1vector for attaching linkers (Fig. 1 A).5,7,14To recruit TAF1 for inducing acety lation without significant loss of its KAT activity we chose to utilize a small-molecule binder of the TAF1 bromodomain, but not small-molecule inhibitors ofTAFl acetyltransferase.15There are several reported TAF1 bromodomain binders including: benzoisoquinolinedi one-based compounds, such as BAY-299,16naphthyridones, such as compound 23,17and dual TAF1 and ataxia telangiectasia and Rad3 related (ATR) kinase inhibitor, AZD6738.18In this study, we utilized GNE-371, which contains a pyrrolopyridone core and is a potent and selective TAF1-BD2 binder with a KD of 1 nM and over 2,000-fold selectivity against BRD419The co-crystal structure of TAF1-BD2 in complex with GNE-371 (PDB ID: 6DF7) revealed that the benzimidazole ring forms a hydrogen bond interaction with the gatekeeper Tyrl589 residue and the morpholine moiety of GNE-371 is solvent-exposed (Fig. IB).19We replaced the solvent-exposed morpholine group with the piperazine group which serves as a handle for linker installation. We designed and synthesized a series of putative TAF1 -recruiting p53Y220C AceTACs (1 - 12) using either an alkylene linker or polyethylene glycol (PEG) linker (Fig. 1C-D, Schemes SI-2).BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0008] FIG. 1. Design and evaluation of TAF1 -recruiting p53Y220C-targeting AceTACs. (A) Co-crystal structure of the p53Y220C-PK9328 complex (PDB ID: 6GGF).14Left: the cross-section of the p53Y220C binding pocket (in gray) occupied by PK9328 (in magenta). The solvent-exposed region of PK9328 is highlighted by the red dashed cycle. Right: the chemical structure of PK9328. (B) Co-crystal structure of the TAF1 (BD2)-GNE-371 complex (PDB ID: 6DF7).19Left: the cross-section of the TAF1-BD2 binding pocket (in gray) occupied by GNE-371 (in magenta). The solvent-exposed region of GNE-371 is highlighted by the red dashed cycle. Right: the chemical structure of GNE-371. (C) Chemical structures of alkylene linker-based p53Y220C AceTAC compounds 1-8. (D) Chemical structures of PEG linkerbased p53Y220C AceTAC compounds 9-12. (E) Representative western blot (WB) results of PK9328, compounds 1-12 and GNE-371 in H1299-p53Y220C stable cells treated with the indicated compound at 5 pM for 8 h (from two independent experiments). Total cell lysate wasAttorney Docket No. 27527-0233WO1used for WB and vinculin was used as a loading control. (F) Quantification of the fold change of the p53K382ac level (p53K382ac level over total p53 protein level) for the WB results shown in panel E and its biological repeat.

[0009] To evaluate these putative TAF1 -recruiting p53Y220C AceTACs, we utilized an NCI-H1299 cell line which is endogenously p53-null to stably express the FLAG tagged p53Y220C mutant, termed H1299-p53Y220C. We observed that both PK9328 and GNE-371 at 5 pM (8 h treatment) did not significantly induce acetylation of p53Y220C lysine 382 residue (p53K382ac) in H1299-p53Y220C cell line (Fig. 1E-F). On the other hand, compound 2 (MS 172) at 5 pM (8 h treatment) induced the most significant increase in the p53K382ac level (~ 10-fold) among the 12 AceTAC compounds tested (Fig. 1E-F). Notably, TAF1 -based p53Y220C AceTACs with a longer alkylene linker (such as 8-carbon linker) or a PEG linker did not induce significant p53Y220C K382 acetylation (Fig. 1E-F). Overall, we identified MS 172 as the most effective TAF1 -recruiting p53Y220C AceTAC from this SAR study and selected this compound for further characterization.

[0010] FIG. 2. MS172 induces p53Y220C acetylation in a concentration- and timedependent manner in BxPC3 cells. (A) Chemical structure of MS172. (B) WB results of the p53K382ac level in BxPC3 cells treated with PK9328, GNE-371 or MS 172 at 0, 100, 300 or 1000 nM for 24 h. Results shown are representative of three independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. (C) Left: WB results of the p53K382ac level in BxPC3 cells treated with MS172 at 0, 3, 10, 30, 100, 300 or 1000 nM for 24 h. Results shown are representative of three independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. Right: quantification of the fold change of the p53K382ac level shown on the left and its biological repeats. Results shown are the mean values ± SD from three independent experiments. (D) Total p53 lysine acetylation in BxPC3 cells treated with PK9328 or MS 172 at 100, 300 or 1000 nM for 24 h. Ap53 mouse monoclonal antibody (DO-1) was coated onto the microwells of an ELISA plate. After incubation with total cell lysates for 2 h, p53 was captured by the coated antibody. Following extensive washing, acetylated-lysine rabbit monoclonal antibody was added to detect the total acetylated lysine on p53. An anti-rabbit IgG with horse-radish-peroxidase (HRP) linked antibody was then used to recognize the bound detection antibody. Vehicle treatment was used to normalize the fold change of total p53 lysine acetylation level. Results shown are the mean values ± SD from three independent experiments. *P < 0.05, ***P < 0.001. (E) WB results of the p53K382ac level in BxPC3 cells treated with 1000 nM of MS172 at the indicated timeAttorney Docket No. 27527-0233WO1point. Results shown are representative of two independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. (F) Quantification of the fold change of the p53K382ac level from the WB results shown in panel E and its biological repeat. Vehicle treatment from each specific time point was used to normalize the fold change of the p53K382ac level.

[0011] FIG. 3: MS172 induces ternary' complex formation between p53Y220C and TAF1. Thermal shift assay (TSA) results of p53Y220C-DBD (A) and TAF1-BD2 (B) induced by MS 172. Results shown are the mean values ± SD from three biological repeats. (C) FRET-based biochemical assay results for GNE-371- and MS172-mediated ternary complex formation between GST-TAF1-BD2 and p53Y220C-DBD-His. Briefly, anti-GST and anti-His HTRF antibody pairs bind to GST-tagged TAF1-BD2 and His-tagged p53Y220C-DBD. A HTRF signal is generated only if the compound binds and the ternary complex is formed and signal is normalized to the DMSO treatment. Results shown are the mean values ± SD from two biological repeats. (D) Representative WB results of MS172-mediated p53Y220C-TAFl interaction via p53-FLAG pull-down in H1299-p53-null and H1299-p53Y220C cells treated with MS 172 at 0, 1 or 10 pM for 24 h. Results shown are representative of two independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. (E) Representative WB results of p53-FLAG pull-down after treatment of H1299-p53Y220C cells with DMSO or MS 172 at 10 pM for 24 h with either control shRNA or TAF 1 shRNA. Results shown are representative of two independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. (F) WB results of p53-FLAG pulldown after treatment of H1299-p53Y220C cells with GNE-371 alone at 10 pM, MS172 alone at 3 pM, or GNE-371 pretreatment at 10 pM for 4 h, followed by MS 172 treatment at 3 pM for 20 h. Results shown are representative of two independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control.

[0012] FIG. 4. MS172 effectively acetylates p53Y220C and inhibits the growth in p53Y220C-harboring cancer cell lines and is non-toxic in p53 WT cells. WB results of PK9328, GNE-371, MS78 and MS172 in (A) BxPC3, (B) NUGC3 and (C) Huh7 cells treated with the indicated compound at 0, 100, 300 or 1000 nM for 24 h. Results shown are representative of two independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. (D) Total p53 lysine acetylation in BxPC3, NUGC3 and Huh7 cells treated with PK9328, GNE-371, MS78 or MS172 at 1000 nM for 24 h. Vehicle treatment was used to normalize the fold change of the total p53 lysine acety lation level. Results show n are the meanAttorney Docket No. 27527-0233WO1values ± SD from three independent experiments. *P < 0.05, **P < 0.01. (E) Cell viability of PK9328. GNE-371, MS78 and MS172 in BxPC3, NUGC3, and Huh7 cells. The cells were treated with DMSO or the indicated compound for 72 h. The mean values ± SD from three biological experiments (each in technical triplicates) are shown. GraphPad Prism 8 was used in analysis of raw data. (F) WB results of PK9328, GNE-371 and MS172 in U2OS cells treated with the indicated compound at 0, 100, 300 or 1000 nM for 24 h. Results shown are representative of two independent experiments. Total cell lysate was used for WB and vinculin was used as a loading control. (G) Cell viability of PK9328, GNE-371 and MS172 in U2OS cells. The cells were treated with the indicated compound for 72 h. The mean value ± SD for each concentration (in technical triplicates from two biological experiments) is shown in the curves. GraphPad Prism 8 was used in analysis of raw data.

[0013] FIG. 5: MS172-mediated p53Y220C acetylation induces regulation of metallothionine proteins in BxPC3 cells. (A) Heatmap enrichment in high confidence p53-target genes. BxPC3 cells were treated with DMSO, PK9328 or MS 172 at 5 pM for 4 h in triplicates. (B) Volcano plot of differential gene expression (DGE) of upregulated proteins and pathways upon MS 172 treatment compared to PK9328 treatment after normalization with DMSO treatment. (C) Enrichment plot of significant genes upregulated from the 343 high-confidence p53-target genes (q-value < 0.01, normalized enrichment score (NES) = 1.49) in BxPC3 cells treated with 5 pM of MS 172 compared to 5 pM of PK9328 treatment for 4 h in triplicates. (D) KEGG pathway analysis of upregulated pathways upon 5 pM of MS 172 treatment compared to 5 pM of PK9328 for 4 h in triplicates. (E) RT-qPCR of MT1X, MT1N, ZNF391, and ZNF778 expression in BxPC3 cells treated with DMSO, PK.9328 or MS 172 at 0.2, 1 or 5 pM for 4 h from three independent experiments. The mRNA expression for each gene was first normalized to internal GAPDH and then calculated relative to the DMSO control. *P < 0.05, **P < 0.01. ***P < 0.001.

[0014] FIG. 6. Sequence comparison of TAF1 and other bromodomain-containing proteins. The sequence of TAF1-BD2 is compared to BRD4-BD2, CBP-BD and PCAF-BD. (*) denotes positions with fully conserved residues, (:) denotes conservation of residues with strongly similar properties, and (.) denotes conservation of residues with weakly similar properties.

[0015] FIG. 7. TSA results of MS172 at 100 pM against other bromodomaincontaining proteins. Results shown are the mean values from two independent experiments.Attorney Docket No. 27527-0233WO1

[0016] FIG. 8. MS172 does not induce an interaction between p53Y220C and p300 acetyltransferase. Representative WB results of p53-FLAG pull-down after treatment of H1299-p53Y220C cells with DMSO, MS78 or MS172 at 10 pM for 24 h. Total cell lysate was used for WB and vinculin was used as a loading control. The results shown are representative of two independent experiments.

[0017] FIG. 9. MS172 does not significantly inhibit the growth in PNT2 normal prostate cells. PNT2 cells were treated with the indicated compound at the indicated concentration for 72 h. The mean value ± SD for each concentration point (in technical triplicates from three biological experiments) is shown. GraphPad Prism 8 was used in analysis of raw data.

[0018] FIG. 10. MS172 is bioavailable in an in vivo mouse pharmacokinetic study.Plasma concentrations of MS 172 over 8 h in Swiss albino mice were determined following a single 50 mg / kg intraperitoneal (IP) injection. Experiments were performed in biological triplicates. Each point represents the mean concentration ± SEM.

[0019] FIG. 11. Novel mechanism of action of MS 172. RT-qPCR results of MT1X, MT1N, ZNF391, and ZNF778 expression in NUGC3, HCT116 and NCI-H1299 cells treated with DMSO, PK9328 or MS172 at 5 pM for 4 h from two independent experiments. The mRNA expression for each gene was first normalized to internal GAPDH and then calculated relative to the DMSO control.

[0020] FIG. 12. ’H NMR spectrum of MS 172.

[0021] FIG. 13.13C NMR spectrum of MS 172.

[0022] FIG. 14. LC-MS NMR spectrum of MS 172.DETAILED DESCRIPTIONPharmaceutically acceptable isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents). Specifically, an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number,Attorney Docket No. 27527-0233WO1but an atomic mass different from the atomic mass usually found in nature. Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e.g,2H,3H,13C,14C,15N,17O,18O,32P,35S,18F, and36C1.

[0023] Isotopic variations (e.g, isotopic variations containing2H) can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations (particularly those containing a radioactive isotope) can be used in drug or substrate tissue distribution studies. The radioactive isotopes tritium (3H) and carbon- 14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0024] Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated. A solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g, D2O in place of H2O, -acetone in place of acetone, or rfe-DMSO in place of DMSO).

[0025] Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate fluorinated variations of those reagents). Specifically, a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g, increased in vivo half-life or reduced dosage requirements.Definition of Terms

[0026] As used herein, the terms “comprising'’ and “including’" are used in their open, non-limiting sense.

[0027] " Pharmaceutically acceptable salt" includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0028] " Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwiseAttorney Docket No. 27527-0233WO1undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and di carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., " Pharmaceutical Salts," Journal of Pharmaceutical Science, 66:1-19 (1997), which is hereby incorporated by reference in its entirety)- Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

[0029] " Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropyl amine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N, N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine,Attorney Docket No. 27527-0233WO1ethylenediamine, A-methylgl ucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, / V-ethyl piperidine, poly amine resins and the like. See Berge et al., supra.Pharmaceutical Compositions

[0030] In some aspects, the compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.

[0031] In some aspects, the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment. For example, in some instances, pharmaceutical compositions disclosed herein can be combined with one or more (eg., one, two, three, four, five, or less than ten) compounds.

[0032] In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the compounds or its delivery form.

[0033] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle. As used herein, the phrase ‘'pharmaceutically acceptable7’ refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable carrier, adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Exemplar}' conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0034] In particular, pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery' systems (SEDDS) such as d-a- tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric deliveryAttorney Docket No. 27527-0233WO1matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylenepoly oxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0035] As used herein, the compounds disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A ‘‘pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g. carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof.Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger’s Medicinal Chemistry and Drug Discovery, 5thEdition, Vol. 1:Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.

[0036] The compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivative thereof.

[0037] In particular, pharmaceutically acceptable salts of the compounds disclosed herein include, e.g., those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate.Attorney Docket No. 27527-0233WO1butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate. hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate. Salts derived from appropriate bases include, e.g, alkali metal (e.g., sodium), alkaline earth metal (e g., magnesium), ammonium salts. The invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization.

[0038] In some aspects, the pharmaceutical compositions disclosed herein can include an effective amount of one or more compounds. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. In some aspects, pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment in amounts effective for causing an intended effect or physiological outcome.

[0039] In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.Administration of Pharmaceutical Compositions

[0040] The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http: / / www.fda.gov7Drugs / DevelopmentApprovalProcess / FormsSubmissionRequirements / ElectronicSubmissions / DataStandardsManualmonographs). In particular, the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques.Attorney Docket No. 27527-0233WO1

[0041] For example, the pharmaceutical compositions disclosed herein can be administered, e.g, topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.

[0042] For example, pharmaceutical compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.

[0043] For example, the pharmaceutical compositions of this invention can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

[0044] For example, the pharmaceutical compositions of this invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.

[0045] For example, the pharmaceutical compositions of this invention can be administered by injection (e.g, as a solution or powder). Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employedAttorney Docket No. 27527-0233WO1as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, e.g., olive oil or castor oil, especially in their poly oxy ethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

[0046] In some aspects, an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001. 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100. 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg / kg / day, or according to the requirements of the particular pharmaceutical composition.

[0047] When the pharmaceutical compositions disclosed herein include a combination of a compound of the formulae described herein and one or more additional compounds, both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention.Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.

[0048] In some aspects, the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration.Methods of Treatment

[0049] The methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect. Typically, the compounds or compositions of the invention will be administered from about 1 to about 6Attorney Docket No. 27527-0233WO1times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Alternatively, such preparations can contain from about 20% to about 80% active compound.

[0050] In some aspects, the present disclosure provides methods for using a composition comprising a compound, including pharmaceutical compositions (indicated below as X') disclosed herein in the following methods: Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein. Use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment ofY.

[0051] In some aspects, the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g, a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment. In some aspects, the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required.

[0052] In some aspects, subject selection can include obtaining a sample from a subject (e.g, a candidate subject) and testing the sample for an indication that the subject is suitable for selection. In some aspects, the subject can be confirmed or identified, e.g., by a health care professional, as having had or having a condition or disease. In some aspects, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority' of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g. relative to other subjects (e.g. the majority of subjects) with the same condition or disease). In some aspects, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response. In some aspects, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidateAttorney Docket No. 27527-0233WO1subject and a second party can test the sample. In some aspects, subjects can be selected or referred by a medical practitioner (e.g., a general practitioner). In some aspects, subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.

[0053] In some aspects, methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject is suffering. In some aspects, methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.

[0054] The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).

[0055] The terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form. For example, the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.

[0056] The terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present invention.

[0057] The terms “prevent,” “preventing,” and “prevention.” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention.Attorney Docket No. 27527-0233WO1

[0058] As used herein, the term “preventing a disease” in a subject means for example, to stop the development of one or more symptoms of a disease in a subject before they occur or are detectable, e.g., by the patient or the patient’s doctor. Preferably, the disease does not develop at all, i.e., no symptoms of the disease are detectable. However, it can also mean delaying or slowing of the development of one or more symptoms of the disease.Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0059] Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0060] An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. Moreover, treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.

[0061] Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected. Upon improvement of a patient’s condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary’. Subsequently, the dosage or frequency of administration, or both, can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.Attorney Docket No. 27527-0233WO1MS172 induces p53Y220C acetylation in a concentration- and time -dependent manner in endogenously p53Y220C-expressing cells.

[0062] We assessed the effect of MS172 on inducing p53Y220C K382 acetylation in the endogenously p53Y220C-expressing BxPC3 (p53Y220C / -) cell line. Compared to PK9328 and GNE-371, MS172 induced significant p53Y220C K382 acetylation, even at 300 nM, in BxPC3 cells (Fig. 2A-B). We determined that the p53K382ac induced by MS 172 in BxPC3 cells is concentration-dependent with an ACE50(the concentration at which 50% of p53Y220C is acetylated) of 182 ± 20 nM (with the acetylation level induced by MS172 at 1000 nM as the maximum response) (Fig. 2C). To assess whether MS 172 induces total p53 acetylation, we employed an enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total acetylated p53. Compared to PK9328, MS172 significantly induced total p53 lysine acetylation, even at 100 nM (Fig. 2D). In addition, MS172 induced p53K382 acetylation in a time-dependent manner. It increased the p53K382ac level by about 2-fold at 4 h and by about 10-fold at 24 h (Fig. 2E-F). Overall, these results demonstrate that MS 172 effectively induces p53Y220C acetylation in a concentration- and time-dependent manner in p53Y220C-harboring cells.MS172 induces ternary complex formation between p53Y220C and TAF1

[0063] To determine whether MS 172 induces a ternary complex formation between p53Y220C and TAF1, we first assessed the target engagement profile of MS 172. We expressed and purified both p53Y220C-DBD and TAF1-BD2 and performed a protein thermal shift assay to determine the binding and stabilization effect of MS 172 (Fig. 3A-B). We observed a concentration-dependent thermal stability shift (ΔTM) of both proteins by MS 172 with a ΔTM of 3.5 and 5.8°C for p53Y220C-DBD and TAF1-BD2, respectively, at 50 pM (Fig. 3A-B). To further assess selectivity against other homologous bromodomain-containing proteins, we also expressed and purified BRD4-BD2, CBP-BD and PCAF-BD (Fig. SI). Even at a higher concentration (100 pM), we found that MS 172 did not stabilize bromodomains of BRD4, CBP and PCAF (Fig. 7). In summary. MS 172 stabilized p53Y220C and bound to TAF1 selectively over several other bromodomain-containing proteins.

[0064] Next, we performed a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) biochemical assay to assess the protein-protein interaction between p53Y220C-DBD and TAF1-BD2 in the presence of MS172 (Fig. 3C). We observed that MS172 induced ternary complex formation between p53Y220C-DBD and TAF1-BD2 while GNE-371 did notAttorney Docket No. 27527-0233WO1(Fig. 3C). We also performed immunoprecipitation (IP) experiments to pull down FLAG-tagged p53Y220C to assess the ternary complex formation between p53Y220C and TAF1 intracellularly (Fig. 3D). As expected, MS172 induced an interaction between p53Y220C and TAF1 and induced p53K382ac in a concentration-dependent manner in H1299-p53Y220C cells, whereas no interaction was observed in H1299-p53-null cells (Fig. 3D). To further confirm that p53Y220C K382 acetylation depends on TAF1, we knocked down TAF1 using shRNA in the H1299-p53Y220C cell line and monitored the p53Y220C-TAFl interaction and p53Y220C K382 acetylation induced by MS172 (Fig. 3E). Upon treatment with MS172, there was an induction of the p53K382ac level in control shRNA-treated cells concurrent with the interaction between p53Y220C and TAF1 (Fig. 3E). On the other hand, the interaction between p53Y220C and TAF1 and p53Y220C K382 acetylation in theTAFl shRNA-treated cells were significantly diminished (Fig. 3E), thereby confirming that the MS172-mediated p53Y200C K382 acetylation is dependent onTAFl interaction. Finally, w e performed a competition rescue experiment to further validate the ternary complex formation between p53Y220C and TAF1 (Fig. 3F). By pretreating H1299-p53Y220C cells with 10 pM of GNE-371 for 4 hours, the p53Y220C-TAF1 interaction was significantly abolished and the p53K382ac level induced by MS 172 was significantly reduced (Fig. 3F). To further confirm that MS 172 does not recruit p300, we performed a FLAG-tag pulldown experiment with MS78 as a positive control. As expected. MS78 induced a ternary complex interaction between p53Y220C and p300 acetyltransferase, whereas MS 172 did not induce any ternary complex formation between p53Y220C and p300 acetyltransferase (Fig. 8). Altogether, we confirmed that MS172-induced p53Y220C K382 acetylation depends on the ternary complex formation between p53Y220C and TAF1 acetyltransferase by utilizing biochemical, FLAG-IP, knockdown and rescue experiments in isogenic cell lines.MS 172 effectively inhibits the proliferation in p53Y220C-harboring cancer cells and is more potent than the p300 / CBP-recruiting AceTAC MS78,

[0065] Next, we compared the effects of MS172 to the p300 / CBP-recruiting AceTAC MS78 on inducing p53Y220C acetylation and their anti-proliferative activity in multiple p53Y220C-harboring cancer cell lines (BxPC3 (p53Y220C / -), NUGC3 (p53Y220C / +), and Huh7 (p53Y220C / -)). First, we observed that MS 172 induced p53Y220C K382 acetylation more effectively than MS78 in BxPC3, NUGC3 and Huh7 cells (Fig. 4A-C). As expected, both PK9328 and GNE-371 did not increase the p53K382ac level compared to the baseline at the concentrations tested (Fig. 4A-C). We also assessed the total p53 lysine acetylation induced byAttorney Docket No. 27527-0233WO1MS172 and saw a significant upregulation compared to PK9328, GNE-371 and MS78 (Fig.4D). To examine the correlation between p53Y220C acetylation and anti-proliferative activity, we performed cell viability experiments for MS172, MS78, PK9328 and GNE-371 in all three cell lines (Fig. 4E). As expected, MS172 displayed the most potent anti-proliferative activity and is more potent than MS78 in all three p53 Y220C -harboring cell lines (Fig. 4F, Table SI). Altogether, MS172 is more effective than MS78 in both inducing p53Y220C acetylation and suppressing the proliferation in multiple p53Y220C -harboring cancer cell lines.

[0066] We then tested the cytotoxicity profile of MS 172 in both p53 wildtype (WT) cell line and normal cells. MS 172 did not induce any significant p53 K382 acetylation at the concentrations tested in U2OS, ap53 WT cell line, similar to PK9328 and GNE-371 (Fig. 4F). Additionally, MS 172 as well as PK9328 and GNE-37 did not display significant cell growth inhibition effect in U2OS cells (Fig. 4G). We also assessed the effect MS 172 on the growth of normal prostate PNT2 cells and did not observe significant growth inhibition (Fig. 9). Finally, we evaluated the in vivo mouse pharmacokinetic (PK) properties of MS 172 (Fig. 10). We determined the plasma concentrations of MS 172 in Swiss albino mice following a single intraperitoneal (IP) administration of 50 mg / kg. The plasma concentrations of MS 172 were about 0.7 pM for the first 2 hours and maintained above 0.5 pM for 8 hours post the IP inj ection (Fig. 10). We did not observe significant toxicity ofMS172 in vivo and the compound was well tolerated in the treated mice. Future studies to assess in vivo efficacy of MS172 are warranted. Taken together, these results indicate that MS 172 is non-toxic in p53 WT cancer cells and normal cells and has sufficient mouse PK properties to be used as a chemical biology tool to investigate the role of p53Y220C acetylation in vivo.Novel regulation of metallothionine proteins by MS 172 in BxPC3 cells.

[0067] Finally, we evaluated the downstream signaling difference between MS 172 and the parent p53Y220C stabilizer PK9328. While we previously assessed the effect of MS78 in H1299-p53Y220C cells,5we did not evaluate (1) the effect of p53Y220C AceTAC in a cell line that endogenously expresses p53Y220C, and (2) which genes are regulated first after p53Y220C acetylation. Thus, we performed an RNA-sequencing (RNA-seq) study in BxPC3 cells treated with DMSO, PK9328 or MS172 at 5 pM for 4 h, a relatively early time point (Fig.5). Interestingly, while PK9328 induced the upregulation of ty pical p53-regulated target genes such as BAX, CHEK1 and CDKN1A. it also induced the expression of NFKB1, KRAS and MDM2 genes at this early time point (Fig. 5A), which is consistent with previous RNA-seq andAttorney Docket No. 27527-0233WO1RT-qPCR results of p53Y220C stabilizers.20,21’22On the other hand, at this early time point, MS 172 treatment significantly upregulated MT IX, MT IN, PLK1 and DKK1 genes (Fig. 5A). Particularly, we compared the gene expression profile of MS 172 to PK9328 (after normalization with DMSO-treated samples) and observed a significant upregulation of these metallothionine proteins while a downregulation of zinc-finger containing proteins (log2fold change >2, p- value <0.05) (Fig. 5B). It was reported previously that WT p53 can bind metallothionine proteins and inactivation of WT p53 leads to a downregulation of metal response elements (MREs).23,24However, to the best of our knowledge, the regulation of metallothionine proteins by p53Y220C has not been previously reported. Furthermore, unbiased analysis using differential gene expression (DGE) and enrichment in KEGG pathways revealed that MS 172 treatment led to the upregulation of proteins involved in oxidative phosphorylation compared to PK.9328 (q-value = 0.005, normalized enrichment score (NES) = 1.49) (Fig. 5C-D). These results are consistent with previous studies which showed that metallothionine proteins are involved in oxidative stress and inhibition of metallothionine proteins can increase cancer cell growth by regulating zinc-finger proteins.25,26To validate the RNA-seq results, we performed a subsequent RT-qPCR study in BxPC3 cells treated with DMSO, PK9328 or MS 172 at different concentrations (Fig. 5E). We observed a concentrationdependent upregulation of MT1X and MT1N genes and a simultaneous downregulation of ZNF391 and ZNF778 genes at 4 h time point induced by MS172, while PK9328 treatment led to no changes compared to the DMSO control. We also determined whether these novel pathways are regulated in other p53Y220C-harboring cancer cell lines as well as p53-WT and p53-null cell lines (Fig. 11). MS 172 indeed induced the expression of MT1X and MT1N genes while suppressing ZNF391 and ZNF778 genes at 4 h time point in NUGC3 cell line, but not in p53-WT (HCT116) and p53-null (NCI-H1299) cell lines (Fig 11). It should be noted while these short-treatment RNA-seq and RT-qPCR studies revealed a novel mechanism of action for MS 172, additional studies are warranted to assess how the changes in these gene pathways at an early time point lead to differential gene expression at later time points compared to the parent p53Y220C stabilizer. Moreover, further studies to determine whether p53Y220C acetylation by AceTAC leads to novel DNA-binding by p53Y220C are also warranted. In summary, we observed a novel regulation of metallothionine proteins by MS172-induced p53Y220C acetylation compared to the parent p53Y220C stabilizer.

[0068] In this study, we discovered and characterized the first-in-class TAF1 -recruiting p53Y220C AceTAC, MS 172. MS 172 effectively induced p53Y220C K382 acetylation andAttorney Docket No. 27527-0233WO1increased total p53Y2220C acetylation in a concentration- and time-dependent manner. We validated that the p53Y220C K382 acetylation induced by MS 172 depends on the ternary complex formation between p53Y220C and TAF1 using a battery of biochemical, immunoprecipitation, knockdown and rescue experiments. We also determined that MS 172 effectively induced p53Y220C K382 acetylation and inhibited the proliferation in multiple p53Y220C-harboring cancer cell lines. Notably, MS 172 is more potent than the parent p53Y220C binder PK.9328. the TAF1 bromodomain binder GNE-371. and our previously reported p300 / CBP-recruiting p53Y220C AceTAC MS78 in inducing p53Y220C K382 acetylation and total p53 acetylation in multiple p53Y220C-harboring cancer cell lines. Importantly, our TAF1 -recruiting p53Y220C AceTAC MS 172 is effective in suppressing the tumor cell growth in multiple cancer cell lines including pancreatic (BxPC3), gastric (NUGC3) and liver (Huh7), and is more potent than PK9328, GNE-371 and MS78, providing a potential therapeutic approach for the treatment of p53Y220C-based solid cancers. Furthermore, MS 172 is non-toxic in p53 WT and normal cells and is bioavailable in mice via IP administration, thus making it suitable for in vivo efficacy studies. Lastly, we uncovered novel upregulation of metallothionein proteins mediated by MS172-induced p53Y220C acetylation using RNA-seq and RT-qPCR studies.

[0069] While our proof-of-concept study shows potential therapeutic utilities of TAF1-recruiting p53Y220C AceTAC, it should be noted that the lead compound from this proof-of-concept study, MS28, which displayed superior antiproliferative effects than the parent p53Y200C stabilizer, is not a drug candidate. Further optimization of MS28, which is a useful tool compound, is needed to develop a clinical candidate for translating this therapeutic approach in the clinic. It is likely that p53Y220C AceTAC will provide a complementary therapeutic approach to Rezatapopt (PC14586), a p53Y220C small-molecule stabilizer in clinical development, which has shown some efficacy in p53Y220C- harboring solid cancers.27Currently, AceTAC targeting p53 is limited to p53Y220C, due to the lack of bona fide smallmolecule binders for WT p53 or other p53 mutants. However, in the future, upon development of bona fide small-molecule binders for WT p53 or other p53 mutants by the scientific community, novel AceTACs targeting WT p53 or other p53 mutants will be developed, which could augment p53-acetylation mediated tumor suppression for the treatment of cancer.

[0070] Overall, we have demonstrated that the TAF 1 acetyltransferase can be harnessed for AceTAC development, thus expanding the very limited repertoire of the KATs that can be hijacked for inducing targeted protein acetylation. We also provide MS 172, a valuable chemicalAttorney Docket No. 27527-0233WO1biology tool and a potential therapeutic, to the scientific community to further investigate the role of p53Y220C acetylation in cancer.Table 1: GI50 values of MS172, MS78, PK9328 and GNE-371 in BxPC3, NUGC3 and Huh7 cells.BxPC3 NUGC3Huh7Compound Average GI50(µM) Average GI50(µM) Average GI50(µM)PK9328 10.8 ± 2.1 14.3 ± 1.2 13.0 ± 1.5 GNE-371 >15 >15 >15 MS78 3.22 ± 0.9 2.81 ± 0.3 2.49 ± 0.6MS172 1.33 ± 0.2 1.01 ±0.4 0.45 ± 0.2

[0071] Cell viability results of MS172, MS78, PK9328, and GNE-371 in BxPC3. NUGC3 and Huh7 cells. The cells were treated with the indicated compound for 72 h. The mean Gbo values ± SD from three biological experiments (in technical triplicates) are shown.Materials & MethodsCell Lines. Tissue Culture and Transfection.

[0072] NCI-H1299 (CRL-5803), BxPC3 (CRL-1687) and U2OS (HTB-96) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). In addition, NUGC-3 (JCRB0822) and Huh7 (JCRB0403) were obtained from the Japan Health Science Research Resources Bank (JCRB, Japan). Normal prostate epithelium PNT2 cell line (95012613) was purchased from Sigma-Aldrich (Burlington, Massachusetts). NCI-H1299, BxPC3, NUGC-3 and PNT2 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific Inc., Waltham. Massachusetts) supplemented with 10% heat-inactivated fetal bovine serum Gibco™ (FBS) (Life Technologies, Grand Island, NY) and 1% Gibco™ Penicillin / Streptomycin (Life Technologies, Grand Island, NY). Huh7 cells were cultured in DMEM Medium (Thermo Fisher Scientific Inc., Waltham, Massachusetts) supplemented with 10% heat-inactivated FBS and 1% Penicillin / Streptomycin. U2OS cells were cultured in McCoy's 5A (Modified) Medium (Thermo Fisher Scientific Inc., Waltham, Massachusetts) supplemented with 10% heat-inactivated FBS and 1% Penicillin / Streptomycin. All cells were incubated at 37 °C in a standard humidified incubator containing 5% CO2and 95% O2.Attorney Docket No. 27527-0233WO1

[0073] For shRNA transfection, control and TAF1 shRNA(sc-106597-SH) were purchased from Santa Cruz Biotechnology. Briefly, H1299-p53Y220C cells were seeded on 6-well plates at a density of 3 × 105cells / well (~75–80% confluency). Cells were transfected the following day with Lipofectamine 3000 transfection reagent (L3000008, ThermoFisher Scientific) and harvested 48 h after transfection. For the construction of the FLAG-p53Y220C stable cell line, NCI-H1299 cells were transfected with sequence verified pcDNA3.1(+)-N-DYK vectors containing specific ORFs (Genscript, Piscataway, NJ) and selected with G418 for 14 days. Antibodies and Immunoblotting.

[0074] Total cell lysate was used for western blots, as previously described.1The following primary antibodies were used in the study: Vinculin (Cell Signaling Technology [CST], 13901), p53 (CST, 2527), acetyl-p53 (lys-382) (CST, 2525), acetylated-lysine antibody (CST, 9441), TAF1 (D6J8B) (CST, 12781), p53 polyclonal antibody (Proteintech, 10442-1-AP). Blots were imaged using fluorescence-labeled secondary antibodies on LI-COR Odyssey CLx Imaging Systems.Protein expression and purification.

[0075] A stabilized version of the human p53 DBD (residues 94-312; M133L / V203A / N239Y / N268D) with an additional Y220C mutation was expressed and purified as previously described.2TAF1-BD2, BRD4-BD2, CBP-BD and PCAF-BD were expressed and purified as previously described.3Total p53 Acetylation ELISA Assay

[0076] PathScan® Acetylated p53 Sandwich ELISA Kit (Cat# 7236) was purchased from CST and used according to manufacturer’s instructions. Briefly, cell lysate after compound treatment was used. Absorbance signals were obtained with Infinite F PLEX plate reader (TECAN, Morrisville, NC) at 450 nm with 550 nm as reference wavelength after adding stop solution. GraphPad Prism 8 was used in the analysis of the total p53 lysine acetylation from three independent experiments.Thermal Shift Assay.

[0077] Thermal shift assay was performed in 100-µl 96-well PCR plates. First 10× SYPRO® Orange dye (S6650, ThermoFisher Scientific) was prepared in IxHEPES buffer. PK9328, GNE-371 or MS172 was diluted to the target concentration (50 µM) and titrated in aAttorney Docket No. 27527-0233WO12-fold serial dilution in the buffer. In 96-well PCR plates, 20 µL of 2.5 µM protein, 20 µL of drug titrant, and 10× SYPRO® Orange were combined and centrifuged to bring solutions to the bottom of the well and remove bubbles. The plates were then sealed with optically clear adhesive sheet and subjected to Applied Biosystems ViiA7 real-time PCR instrument for spectrometry. Fluorescence counts versus temperature for each experimental condition were plotted, and the melting temperature was determined by the greatest first derivative of the plot.FLAG-immunoprecipitation

[0078] Endogenous co-IP experiments were performed as described previously.1Briefly, 2 ×106cells were seeded in a 10-cm dish, and treated with 0, 1 or 10 pM of MS 172 for 24 h. Cells were then washed with ice-cold PBS twice and centrifuged at 200 xg for 3 min to get cell pellets which were then lysed with 300 pL of lysis buffer (20 mM HEPES, pH 7.4. 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and EDTA-free phosphatase and protease inhibitor) for 30 min on ice, and centrifuged at 14,000g for 10 min to get supernatant as cell lysate. The ANTI-FLAG® M2 affinity gel (Product #A2220, Sigma- Aldrich, Inc.) was used for the purification of FLAG-p53Y220C, according to the manufacturer’s guidelines. Following incubation of M2 affinity gel, protein was eluted using 3X FLAG® Peptide (Product #F4799, Sigma-Aldrich, Inc.) and supernatant was resuspended in 30 pL of 1 x SDS-PAGE Laemmli buffer for boiling. The protein samples were then tested by WB.Cell Viability Assay

[0079] The cell viability assay was performed as previously described.1Briefly, 5×104cells were seeded per well into 96-well microplates. After 24 h, cells were treated with 3-fold serially diluted compounds in triplicate for 72 h. Cell viability was evaluated using WST-8 reagent (CK04, Dojindo). Absorbance signals were obtained with Infinite F PLEX plate reader (TECAN, Morrisville, NC) at 450 nm with 690 nm as reference wavelength after 3 h incubation at 37 °C. GraphPad Prism 8 was used in the analysis of GI50values from the data of three independent experiments.TR-FRET Assay for p53Y220C-DBD and TAF1-BD2 Protein-Protein Interaction

[0080] The TR-FRET assay for determining ternary complex formation was performed by Reaction Biolog)’ Corp. (Malvern, PA). Briefly, 10 nM of TAF1-BD2 (GST-tagged) and 10 nM of p53Y220C-DBD (His-tagged) were prepared in 50 mM Tris pH 7.5, 100 mM NaCl, 0.05% CHAPS and 1 mM DTT buffer. GNE-371 or MS 172 at 1% final DMSO concentration wasAttorney Docket No. 27527-0233WO1incubated with the proteins at room temperature for 30 mins. 6.7 nM HTRF MAb Anti-6HIS Tb cryptate Gold and 0.5 nM HTRF MAb Anti GST-d2 were used as the detection antibody.Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR)

[0081] RT-qPCR was performed as previously described.1Briefly, cells were treated with DMSO or indicated compounds at specific time points. Total RNA was extracted using the Monarch Total RNA Miniprep Kit (T2010S, New England Biolabs), and cDNA was generated using the SuperScript IV First-Strand Synthesis System (18091050, Thermo Fisher). qPCR was performed using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, A25742) on Agilent Technologies Stratagene Mx3005p qPCR system. RxnReady™ premixed primer pairs for MT IX, MT IN, PLK1 and DKK1 were purchased from Integrated DNA Technologies, Inc. (Coralville, Iowa). GAPDH forward: 5'-ACAACTTTGGTATCGTGGAAGG-3' (SEQ ID NO: 1), GAPDH reverse: 5'-GCCATCACGCCACAGTTTC-3' (SEQ ID NO: 2) was used as controls. The mRNA expression for each target gene was first normalized to internal GAPDH and then calculated relative to the DMSO control. Experiments were performed in triplicates.RNA-seq study

[0082] The RNA-seq study was conducted as previously described.1BxPC3 cells were treated with DMSO or 5 pM of MS 172 or PK9328 for 4 h in triplicates. Cells were washed three times using ice-cold 1XPBS and subsequently pelleted by centrifuging at 12,000 rpm at 4°C. The pellet was flash frozen and sent to Azenta Life Sciences for further studies. The total RNA in 9 samples were extracted using Qiagen RNeasy Plus Mini Kit according to the protocols in the RNeasy Plus Mini Handbook published by Qiagen. RNA-seq libraries were constructed from the Poly A selected mRNA using the TruSeq RNA sample preparation guide (Illumina) and paired-end 150 base pairs sequencing on a HiSeq2500 system (Illumina) was performed at Azenta Life Sciences. The raw data in FASTQ format was analyzed as previously described.1

[0083] After quality control of FASTQ files using the FASTQC tool (version 0.11.7) (http: / / www. bioinformatics. babraham,ac. uk / proj ects / fastqc / ). we trimmed low-quality bases (Phred < 10) and adapter sequences and then discarded short reads (length < 60 nt) using the bbduk tool (version 37.53) (https: / / jgi.doe.gov / data-and-tools / bbtools / bb-tools-user-guide / ). When either forward or reverse of a paired-end read were discarded, we discarded the complete paired-end read. We quantified the expression of transcripts using cleaned paired-end reads with the Salmon tool (version 0.9.1)4using the human reference transcriptome from TheAttorney Docket No. 27527-0233WO1Cancer Genome Atlas (TCGA; GDC.h38 GENCODE v22).5We performed the differential expression between DMSO. PK9328 or MS 172 treated BxPC3 cells using the R tximport library (version 1.26.1) on R (version 4.2.2).6We pre-filtered genes to keep only genes that had at least 10 reads in total (Ngene = 22,454) and then performed differential gene expression using the R DESeq2 library7(version 1.38.3). We identified as differential expressed between two conditions when the P value adjusted was at 5% and log2(fold change) was more than 1. The heatmap was drawn using the R ComplexHeatmap library (version 2.14.0). We next performed GSEAto capture pathways perturbed towards both directions simultaneously using the 22,454 ranked genes identified in our dataset and annotated in ENSEMBL (version 94) against the 343 p53 target genes listed in previously reported study7using GSEAPreranked (version 4.3.2.; number of permutations = 1000, no collapse) and the KEGG pathways using R GAGE (version 2.48.0).Data Availability

[0084] RNA-seq data have been deposited in the GEO database (GEO accession no. GSE277017). There are no restrictions on data availability.Mouse pharmacokinetic study

[0085] Three male Swiss Albino mice were administered intraperitoneally with solution formulation of MS 172 at a 50 mg / kg dose. The following formulation vehicle was used in the study: 10% v / v NMP, 10% v / v Solutol and 80% v / v normal Saline. Blood samples (approximately 60 µL) were collected from the three test mice at 0.5, 2 and 8 h. Plasma was harvested by centrifugation of blood and stored at −70 ± 10 °C until analysis. Plasma samples were quantified by fit-for-purpose LC-MS / MS method. Compound concentrations in plasma at each time point are average values from 3 test mice. Error bars represent ± SEM. Experiments involving mice were performed according to the Institutional Animal Care and Use Committee (IACUC)-approved protocol.Statistics and Reproducibility

[0086] Experimental data are presented as the mean ± SD or SEM of three independent experiments unless otherwise noted. Statistical analysis was performed using an unpaired two-sided Student’s t test for comparing two sets of data with assumed normal distribution. The results for immunoblotting are representative of at least three biologically independentAttorney Docket No. 27527-0233WO1experiments unless otherwise noted. All statistical analyses and visualizations were performed using GraphPad (Prism v8.4.2) and BioRender.CHEMISTRY EXPERIMENTAL SECTION

[0087] Chemistry General Procedures. All commercially available chemical reagents were used directly in syntheses without further purification. A Teledyne ISCO CombiFlash RT instrument and HP Cl 8 RediSep Rf reverse phase columns equipped with UV detector were used to conduct flash chromatography. All final compounds for biological evaluation were purified with preparative high-performance liquid chromatography (HPLC) on an Agilent Prep 1200 series with the UV detector set to 254 or 220 nm with a flow rate of 40 mL / min at room temperature. Crude samples were injected into a Phenomenex Luna 750 x 30 mm, 5 p C18 column, wi th the gradient program set to 10% of methanol or acetonitrile (B) in H2O containing 0.1% TFA (A) progressing from 10 % to 100% of methanol or acetonitrile (B). The purity of all compounds for biological testing is > 95% assessed by HPLC-HRMS. All HPLC spectra was obtained by using an Agilent 1200 series system with DAD detector and a 2.1 mm × 150 mm Zorbax 300SB-C18 5 µm column for chromatography. Samples (0.5 pL) were injected onto a Cl 8 column at room temperature with the flow rate of 0.4 mL / min. Chromatography was performed with the solvent as follows: water containing 0.1% formic acid was designated as Solvent A while acetonitrile containing 0.1% formic acid was designated as solvent B. The linear gradient was set such that 1% B was used from 0-1 min, 1-99% B from 1-4 min, and 99% B from 4-8 min. High-resolution mass spectra (HRMS) data w as acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. All compounds were also characterized using a Bruker (Billerica, MA) DRX Nuclear Magnetic Resonance (NMR) spectrometer (400 MHz, ¹H NMR, 101 MHz ¹³C NMR). Chemical shifts for all compounds are reported in units of parts per million (ppm, 5) relative to residual solvent peaks. ¹H NMR data are reported in the following format: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant, and integration.Linkers 1–121, intermediate I-22and intermediate I-32were prepared following the reported procedures.Attorney Docket No. 27527-0233WO1Scheme SI. General synthetic route for preparing intermediate 1-5“

[0088] “Reaction and conditions: (a) 4-Bromobut-l-ene, NaH, DMF, 0 °C to rt, 2 h; (b) I-2, Pd(dppf)Cl2, CS2CO3, dioxane / H2O, 100 °C, 3 h; (c) K2CO3, CH3I, DMF, 3 h; (d) NaOH, MeOH / H2O, 80 °C, 2 h; (e) tert-butyl piperazine-1-carboxylate, HATU, DIPEA, DMF, rt; (f) TFA / DCM, rt

[0089] 4-Bromo-6-(but-3-en-l-yl)-l-tosyl-l,6-dihydro-7 / / -pyrrolo[2,3-i|pyridin-7-one (I— 1). To a solution of 4-bromo-1-tosyl-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (950 mg, 2.57 mmol, 1.0 equiv) in DMF (6 rnL) were added sodium hydride (123 mg, 3.08 mmol, 1.2 equiv) under 0 °C ice bath for 15 min, then 4-bromobut-1-ene (555 µL, 5.14 mmol, 2 equiv) and the reaction solution was stirred at rt for 2 h. The resulting system was quenched with H2O and extracted with EA (50 mL × 3). The combined organic layers were concentrated in vacuo and the residue was purified by flash chromatography (hexane / ethyl acetate = 20%) to afford the desired intermediate 1-1 as white solid (400 mg, 37% yield).1H NMR (400 MHz, Methanol-d4) δ 8.04 (d. J = 3.5 Hz, 1H), 7.90 (d, J= 8.0 Hz, 2H), 7.52 (d, J= 3.1 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 6.59 (d, J= 3.5 Hz, 1H), 5.82 - 5.50 (m, 1H), 4.96 - 4.89 (m, 2H), 4.04 - 3.83 (m, 2H), 2.46 - 2.30 (m, 5H).

[0090] 4-(6-(But-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-1-methyl-1H-benzo[d]imidazole-6-carboxylic acid (I-4). To a solution of intermediate 1-1 (400 mg, 0.95 mmol, 1.0 equiv) and intermediate 1-2 (313 mg, 1.425 mmol, 1.5 equiv) in dioxane / H2O (6 mL, 5 / 1) were added CS2CO3 (618 mg, 1.90 mmol, 2.0 equiv) and PdCh(dppf) (69 mg, 0.095 mmol, 0.1 equiv), then the mixture was heated at 100 °C under N2. After 3 h, theAttorney Docket No. 27527-0233WO1reaction mixture was concentrated in vacuo, and the residue was extracted with EA (40 mL × 3) and washed with brine (40 mL × 3). The combined organic layers were concentrated in vacuo and the residue was purified by flash chromatography (DCM / MeOH = 99% to 25%) to afford the desired intermediate 1-3 as black solid (410 mg, 84% yield). HRMS (ESI-TOF) m / z: [M + H]+calcd for C27H25N4O5S, 517.1540; found, 517.1528.

[0091] To a solution of last step desired intermediate 1-3 (275 mg, 0.63 mmol, 1.0 equiv) in DMF (2 mL) were added K2CO3 (146 mg, 1.06 mmol, 2 equiv) and Mel (50 pL, 0.636 mmol, 1.5 equiv). After being stirred at rt for 2 h. the resulting mixture was purified by reverse-ISCO to afford intermediate. To the obtained intermediate in MeOH / H2O (5:1, 3 mL) was added sodium hydroxide (NaOH, 106 mg, 2.65 mmol, 5 equiv). After being stirred at 80 °C for 1 h, the resulting mixture was purified by reverse-ISCO to afford desired intermediate 1-4 as white solid (65 mg. 34% yield for two steps). ¹H NMR (400 MHz, Methanol-d4) δ 9.09 (s, 1H), 8.49 (s, 1H), 8.30 (s, 1H), 7.54 (s, 1H), 7.46–7.34 (m, 1H), 6.29 (s, 1H), 5.96–5.76 (m, 1H), 5.12–4.99 (m, 2H), 4.28–4.03 (m, 5H), 2.69–2.50 (m, 2H).

[0092] 6-(But-3-en-1-yl)-4-(1-methyl-6-(piperazine-1-carbonyl)-1H-benzo[d]imidazol-4-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (I-5). To a solution of 1 - 4 (47 mg, 0.13 mmol, 1.0 equiv) in DMF (1 mL) were added tert-butyl piperazine-1-carboxylate (24.2 mg, 0.13 mmol, 1.0 equiv), O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 59 mg, 0.156 mmol, 1.2 equiv), and diisopropylethylamine (DIPEA, 66 µL, 0.39 mmol, 3 equiv). The resulting mixture was stirred at room temperature for 30 min followed by purified by reverse-ISCO to afford the intermediate. To a solution of the obtained intermediate in DCM (1 mL) was added TFA (1 mL). The resulting mixture was stirred at rt for 30 min. Then all the volatiles were removed, the 1 - 5 was obtained as a yellow solid (43 mg, 50% yield). ¹H NMR (400 MHz, Methanol-d4) δ 9.25 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.50 (s, 1H), 7.38 (s, 1H), 6.26 (s, 1H), 5.95–5.79 (m, 1H), 5.15–4.98 (m, 2H), 4.23–4.13 (m, 5H), 4.06–3.79 (m, 4H), 3.42–3.31 (m, 4H), 2.63–2.49 (m, 2H).Scheme S2. General synthetic route for preparing compound 1-12Attorney Docket No. 27527-0233WO1Linker 1: n » 1PS Linker 2: nI-S Link»r 3: n ~ 3Linker 4: n ®4 Linker 9: m ® 1Linker5: n ®S Linker M: m s 2Linkers: n «S Linker 11; m ® 3Linker 7: n « 7 Linker 12: m = 4Linker 3* n « 8

[0093] " Reaction and conditions: (a) HATU, DIPEA, DMF, rt (b) HC1, MeOH, rt, 30 min.

[0094] 6-(But-3-en-l-yl)-4-(6-(4-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol-3-yl)methyl)glycyl)piperazine- 1-carbonyl)- 1-methyl- 1 / / -benzo [ z / | imidazol-4-yl)- 1,6-dihydro-7E -pyrrolo[2,3-c]pyridin-7-one (1). To a solution of linker 1 (3.7 mg, 0.0075 mmol, 1.0 equiv) in DMF (0.5 mL) were added I - 5 (5 mg, 0.0075 mmol, 1.0 equiv), O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 3.4 mg, 0.009 mmol, 1.2 equiv), and diisopropylethylamine (DIPEA, 4 pL, 0.0225 mmol, 3 equiv). The resulting mixture was stirred at room temperature for 30 min followed by purified by preparative HPLC to afford compound 1 as a white solid (3.9 mg, 51% yield). ¹H NMR (400 MHz, Methanol-t / 4) 6 9.11 (s, 1H), 8.21 (s, 1H). 8.09 (d, J= 8.0 Hz, 1H), 7.89 (s, 1H), 7.76 -7.69 (m, 1H), 7.65 (s, 1H), 7.58 (q, J= 8.6 Hz, 2H), 7.53 - 7.44 (m, 2H), 7.38 (d, J = 2.8 Hz, 1H), 7.32 (s, 1H), 6.95 (s, 1H), 6.27 (d, J= 2.9 Hz, 1H), 5.88 (td, J= 17.0, 7.1 Hz, 1H), 5.05 (dd, J = 24.3, 13.6 Hz, 2H), 4.57 - 4.38 (m, 4H), 4.21 (t, J = 7.4 Hz, 2H), 4.11 (s, 5H), 3.56 (dd, J= 51.4, 32.0 Hz, 8H), 2.58 (q, J = 7.3 Hz, 2H), 2.29 (s, 3H), 1.41 (t, J= 7.1 Hz, 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C46H47N8O3S. 791.3486; found, 791.3475.

[0095] 6-(But-3-en-l-yl)-4-(6-(4-(3-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol- 3-yl)methyl)amino)propanoyl)piperazine- 1-carbonyl)- 1-methyl- l / / -benzo|r / ]imidazol-4-yl)-l,6-dihydro-7 / / -pyrrolo[2,3-c]pyridin-7-one (2). Compound 2 was synthesized following the standard procedure for preparing compound 1 from linker 2 and 1-5. White solid, 2.9 mg. 37% yield. ’H NMR (400 MHz, Methanol-^) 59.45 (s, 1H), 8.24 (s, 1H), 8.13 - 7.98 (m. 2H), 7.80 - 7.68 (m. 2H), 7.62 - 7.56 (m. 2H), 7.53 - 7.46 (m. 2H), 7.40 (s. 1H), 7.34 (s,Attorney Docket No. 27527-0233WO11H), 6.96 (s, 1H), 6.25 (s, 1H), 5.96 - 5.76 (m, 1H), 5.14 – 4.99 (m, 2H), 4.53 - 4.40 (m, 4H), 4.23 - 4.16 (m, 4H). 3.85 - 3.49 (m, 9H), 2.97 - 2.83 (m, 2H), 2.66 - 2.52 (m, 2H), 2.30 (s, 3H), 1.46 - 1.39 (m, 3H), 1.29 - 1.26 (m, 2H).13C NMR (101 MHz, Methanol-^) 5 171.00, 170.63, 156.13, 145.98, 144.72, 142.31, 139.99, 139.84, 135.76, 135.18, 134.61, 134.23, 132.47, 131.04, 130.47, 129.44, 128.44, 127.31, 126.77, 126.59, 124.81, 124.56, 123.18, 123.01. 122.64, 121.85, 121.25, 118.82, 117.99, 112.10, 111.67, 110.52, 106.71. 103.31, 52.87, 44.00, 38.50, 35.10, 34.00, 29.87. 17.54, 15.86, 15.81. 14.07. HRMS (ESI-TOF) m / z: [M + H]+calcd for C47H49N8O3S, 805.3643; found, 805.3630.

[0096] 6-(But-3-en-l-yl)-4-(6-(4-(4-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol- 3-yl)methyl)amino)butanoyl)piperazine-l -carbonyl)-! -methyl-17 / -benzo[r / |imidazol-4-yl)-l,6-dihydro-77 / -pyrrolo[2,3-c]pyridin-7-one (3). To a solution of linker 3 (5.1 mg, 0.01 mmol, 1.0 equiv.) were added 1-5 (6.6 mg. 0.01 mmol, 1.0 equiv) <9-(7-Azabenzotriazol-l-yl)-A'. AA. / V'-tetramethyluronium hexafluorophosphate (HATU, 4.6 mg, 0.012 mmol, 1.2 equiv), and diisopropylethylamine (DIPEA, 5 pL, 0.03 mmol, 3 equiv). The resulting mixture was stirred at room temperature for 30 min followed by purified by preparative HPLC to afford intermediate. The obtained product was dissolved in MeOH (0.5 mL) followed by HC1 (4M in dioxane, 0.5 mL). The resulting mixture was stirred at room temperature for 0.5 h. Then all the volatiles were removed, then 3 was obtained as a white solid (7.8 mg, 87% yield).XH NMR (400 MHz, Methanol-^) 59.59 (s, 1H), 8.24 (s, 1H), 8.18 - 8.02 (m, 2H), 7.87 - 7.70 (m, 2H), 7.63 - 7.34 (m, 6H), 6.94 (s, 1H), 6.26 (s, 1H), 5.99 - 5.86 (m, 1H), 5.14 - 5.04 (m, 2H), 4.55 - 4.39 (m, 4H), 4.33 - 4.21 (m, 4H), 3.75 - 3.55 (m. 9H), 3.22 - 3.08 (m, 2H), 2.77 - 2.56 (m, 4H), 2.29 (s, 3H), 2.12 - 2.00 (m. 2H), 1.41 (s. 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C48H51N8O3S, 819.3799; found, 819.3778.

[0097] 6-(But-3-en-l-yl)-4-(6-(4-(5-(((9-ethyl-7-(4-methylthiophen-2-yl)-9H-carbazol- 3-yl)methyl)amino)pentanoyl)piperazine-l -carbonyl)-! -methyl-! 7 / -benzoh / |imidazol-4-yl)-l,6-dihydro-777-pyrrolo[2,3-c]pyridin-7-one (4). Compound 4 was synthesized following the standard procedure for preparing compound 3 from linker 4 and 1-5. White solid, 9.7 mg, 39% yield. 'H NMR (400 MHz. Methanol-ti4) 59.56 (s, 1H). 8.24 (s. 1H), 8.18 - 7.95 (m, 2H), 7.85 - 7.67 (m, 2H), 7.66 - 7.38 (m, 5H), 7.32 (s, 1H), 6.95 (s, 1H), 6.25 (s, 1H), 5.99 - 5.83 (m, 1H), 5.12 - 5.01 (m, 2H), 4.52 - 4.33 (m, 4H), 4.29 - 4.16 (m, 4H), 3.75 - 3.57 (m, 9H), 3.19 - 2.99 (m, 2H), 2.70 - 2.44 (m, 4H), 2.29 (s, 3H), 1.87 - 1.69 (m, 4H), 1.40 (s. 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C49H53N8O3S. 833.3956; found, 833.3967.Attorney Docket No. 27527-0233WO1

[0098] 6-(But-3-en-1-yl)-4-(6-(4-(6-(((9-ethyl-7-(4-methylthiophen-2-yl)-9H-carbazol- 3-yl)methyl)amino)hexanoyl)piperazine- 1-carbonyl)- 1-methyl- 1 / / -benzo \d\ imidazol-4-yl)-l,6-dihydro-7 / / -pyrrolo[2,3-c]pyridin-7-one (5). Compound 5 was synthesized following the standard procedure for preparing compound 3 from linker 5 and 1-5. White solid, 7.6 mg, 83% yield. 'H NMR (400 MHz, Methanol-^) 59.60 (s, 1H), 8.24 (s, 1H), 8.19 - 7.95 (m, 2H), 7.84 - 7.67 (m, 2H), 7.63 - 7.20 (m, 6H), 6.94 (s, 1H), 6.25 (s, 1H), 6.08 - 5.85 (m, 1H), 5.12 - 5.02 (m, 2H), 4.53 - 4.33 (m, 4H), 4.30 - 4.04 (m, 4H), 3.89 - 3.50 (m, 9H). 3.20 - 3.00 (m, 2H), 2.76 - 2.37 (m, 4H), 2.29 (s, 3H), 1.90 - 1.63 (m, 4H), 1.53 - 1.32 (m, 5H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C50H55N8O3S, 847.4112; found, 847.4141.

[0099] 6-(But-3-en-l-yl)-4-(6-(4-(7-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol- 3-yl)methyl)amino)heptanoyl)piperazine-l -carbonyl)-! -methyl-l / / -benzo[r / |imidazol-4-yl)-l,6-dihydro-77 / -pyrrolo[2,3-c]pyridin-7-one (6) Compound 6 was synthesized following the standard procedure for preparing compound 3 from linker 6 and 1-5. White solid, 6.6 mg, 71% yield. ‘H NMR (400 MHz, Methanol^) 59.58 (s, 1H), 8.34 - 8.03 (m, 3H), 7.85 - 7.70 (m, 2H), 7.60 - 7.33 (m, 6H), 6.94 (s, 1H), 6.26 (s, 1H), 6.08 - 5.81 (m, 1H), 5.12 -5.00 (m, 2H), 4.47 - 4.34 (m, 4H), 4.31 - 4.21 (m, 4H), 3.76 - 3.58 (m, 9H), 3.15 - 2.99 (m, 2H), 2.63 - 2.42 (m, 4H), 2.29 (s, 3H). 1.75 - 1.58 (m, 4H), 1.47 - 1.37 (m. 7H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C5iH57N8O3S, 861.4269; found, 861.4255.

[0100] 6-(But-3-en-l-yl)-4-(6-(4-(8-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol- 3-yl)niethyl)amino)octanoyl)piperazine-l -carbonyl)- l-methyl-l / / -benzo[r / |imidazol-4-yl)-l,6-dihydro-7 / / -pyrrolo[2,3-c]pyridin-7-one (7). Compound 7 was synthesized following the standard procedure for preparing compound 3 from linker 7 and 1-5. White solid, 8.5 mg, 90% yield. ' H NMR (400 MHz. Methanol-^) 59.56 (s, 1H), 8.22 (s. 1H), 8.14 - 7.93 (m, 2H), 7.85 - 7.67 (m, 2H), 7.66 - 7.46 (m, 4H), 7.46 - 7.33 (m, 2H), 6.94 (s, 1H), 6.25 (s, 1H), 5.98 - 5.79 (m, 1H), 5.14 - 5.02 (m, 2H), 4.51 - 4.34 (m, 4H), 4.27 - 4.15 (m, 4H), 3.79 - 3.59 (m, 9H), 3.12 - 2.97 (m, 2H), 2.68 - 2.52 (m, 2H), 2.49 - 2.34 (m, 2H), 2.29 (s, 3H), 1.75 - 1.67 (m, 2H), 1.64 - 1.54 (m, 2H), 1.44 - 1.34 (m, 9H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C52H59N8O3S, 875.4425; found, 875.4428.

[0101] 6-(But-3-en-l-yl)-4-(6-(4-(9-(((9-ethyl-7-(4-methylthiophen-2-yl)-9H-carbazol- 3-yl)methyl)amino)nonanoyl)piperazine- 1-carbonyl)- 1-methyl- 1 / / -benzo \d\ imidazol-4-yl)-l,6-dihydro-7 / / -pyrrolo[2,3-c]pyridin-7-one (8). Compound 8 was synthesized following the standard procedure for preparing compound 3 from linker 8 and 1-5. White solid, 8.0 mg. 83% yield. 'H NMR (400 MHz, Methanol-t / 4) 59.62 (s, 1H), 8.27 - 7.98 (m, 3H), 7.80Attorney Docket No. 27527-0233WO1- 7.66 (m, 2H), 7.59 - 7.32 (m, 6H), 6.94 (s, 1H), 6.27 (s, 1H), 6.08 - 5.74 (m, 1H), 5.22 -5.06 (m, 2H), 4.53 - 4.39 (m, 4H), 4.35 - 4.21 (m, 4H), 3.80 - 3.50 (m, 9H), 3.20 - 3.03 (m, 2H), 2.68 - 2.54 (m, 2H), 2.51 - 2.36 (m, 2H), 2.29 (s, 3H), 1.77 - 1.59 (m, 4H), 1.49 - 1.32 (m, 11H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C53H61N8O3S, 889.4582; found, 889.4576.

[0102] 6-(Bbut-3-en-l-yl)-4-(6-(4-(3-(2-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / f-carbazol-3-yl)methyl)amino)ethoxy)propanoyl)piperazine-l-carbonyl)-l-methyl-l / / -benzo[rf|>n>idazol-4-yl)-l,6-dihydro-77 / -pyrrolo[2,3-c]pyridin-7-one (9). Compound 9 was synthesized following the standard procedure for preparing compound 3 from linker 9 and I-5. White solid, 9.8 mg, 81% yield. 'H NMR (400 MHz, Methanol-^) 5 9.53 (s, 1H), 8.34 -7.97 (m, 3H), 7.83 - 7.54 (m, 5H), 7.43 - 7.25 (m, 3H), 6.93 (s, 1H), 6.20 (s, 1H), 6.00 - 5.74 (m, 1H), 5.12 - 5.01 (m, 2H), 4.49 - 4.35 (m, 4H), 4.30 - 4.16 (m, 4H), 3.79 - 3.62 (m, 15H), 2.82 - 2.68 (m, 2H), 2.64 - 2.51 (m, 2H), 2.28 (s, 3H). 1.43 - 1.30 (m, 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C49H53N8O4S, 849.3905; found, 849.3914.

[0103] 6-(But-3-eii-l-yl)-4-(6-(4-(3-(2-(2-(((9-etliyl-7-(4-metliylthiophen-2-yl)-9 / / -carbazol-3-yl)methyl)amino)ethoxy)ethoxy)propanoyl)piperazine-l-carbonyl)-l-methyl-17 / -benzo[r / |imidazol-4-yl)-l,6-dihydro-7 / / -pyriolo[2,3-c|pyiidin-7-one (10). Compound 10 was synthesized following the standard procedure for preparing compound 3 from linker 10 and 1-5. White solid, 6.8 mg. 59% yield. 'H NMR (400 MHz, Methanol-^) 59.57 (s. 1H), 8.31 - 8.02 (m, 3H), 7.70 - 7.52 (m, 5H), 7.46 - 7.31 (m, 3H), 6.90 (s, 1H), 6.23 (s, 1H), 6.01 - 5.71 (m, 1H), 5.07 - 5.00 (m, 2H), 4.50 - 4.42 (m, 4H), 4.29 - 4.18 (m, 4H), 3.73 - 3.55 (m, 19H), 2.70- 2.55 (m, 4H), 2.26 (s, 3H), 1.45 - 1.39 (m, 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C51H57N8O5S, 893.4167; found, 893.4195.

[0104] 6-(Biit-3-en-l-yl)-4-(6-(4-(l-(9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol-3-yl)-5,8, ll-trioxa-2-azatetradecan- 14-oyl)piperazine- 1-carbonyl)- 1-methyl- 1H-benzo[rf]iniidazoI-4-yl)-l,6-dihydro-7 / 7-pyrrolo[2,3-c]pyridin-7-one (11). Compound 11 was synthesized following the standard procedure for preparing compound 3 from linker 11 and 1-5. White solid, 8.5 mg, 70% yield. 'H NMR (400 MHz, Methanol-c / 4) 59.58 (s, 1H), 8.25 (s, 1H), 8.16 - 7.94 (m, 2H), 7.78 - 7.56 (m, 4H), 7.52 - 7.25 (m, 4H), 6.90 (s, 1H), 6.25 (s, 1H), 5.99 - 5.72 (m, 1H), 5.08 - 5.00 (m, 2H), 4.51 - 4.38 (m, 4H), 4.29 - 4.15 (m, 4H), 3.80 - 3.53 (m, 23H), 2.74 - 2.46 (m, 4H), 2.26 (s, 3H), 1.45 - 1.37 (m, 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for CssHeiNsOeS, 937.4429; found, 937.4445.Attorney Docket No. 27527-0233WO1

[0105] 6-(Biit-3-en-l-yl)-4-(6-(4-(l-(9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -caibazol-3-yl)-5,8,ll,14-tetraoxa-2-azaheptadecan-17-oyl)piperazine-l-carbonyl)-l-methyl-lH-beiizoh / |iinidazol-4-yl)-l.6-diliydr()-7 / / -pynolo|2.3-< |pyridin-7-one (12). Compound 12 was synthesized following the standard procedure for preparing compound 3 from linker 12 and 1-5. White solid, 10.1 mg, 80% yield. 'H NMR (400 MHz, Methanol-c / r) 8 9.55 (s, 1H), 8.24 (s, 1H), 8.16 - 7.98 (m, 2H), 7.78 - 7.67 (m, 2H), 7.61 - 7.40 (m, 5H), 7.32 (s, 1H), 6.92 (s, 1H), 6.24 (s, 1H), 6.06 - 5.70 (m, 1H), 5.21 - 5.02 (m. 2H), 4.53 - 4.39 (m, 4H). 4.26 -4.18 (m, 4H), 3.77 - 3.50 (m, 27H), 2.66 - 2.50 (m, 4H), 2.27 (s, 3H), 1.44 - 1.38 (m, 3H). HRMS (ESI-TOF) m / z: [M + H]+calcd for C55H65N8O7S, 981.4691; found, 981.4687.Attorney Docket No. 27527-0233WO1REFERENCES:(1) Choudhary, C.: Kumar, C.; Gnad, F.; Nielsen. M. L.; Rehman, M.; Walther, T. C.: Olsen, J. V.; Mann, M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325 (5942), 834-840. DOI: 10.1126 / science.1175371 From NLM. (2) Narita, T.; Weinert, B. T.; Choudhary, C. Functions and mechanisms of non-histone protein acetylation. Nat. Rev. Mol. Cell Biol. 2019, 20 (3), 156-174. DOI: 10.1038 / s41580-018-0081-3 From NLM.(3) Shvedunova, M.; Akhtar, A. Modulation of cellular processes by histone and non-histone protein acetylation. Nat. Rev. Mol. Cell Biol. 2022, 23 (5), 329-349. DOI: 10.1038 / s41580-021 -00441-y From NLM.(4) Shang, S.; Liu, J.; Hua, F. Protein acylation: mechanisms, biological functions and therapeutic targets. Signal Transduct. Target Ther. 2022, 7 (1), 396. DOI: 10.1038 / s41392-022-01245-y From NLM.(5) Kabir, M.; Sun, N.: Hu, X.; Martin, T. C.; Yi, J.; Zhong, Y.; Xiong. Y.: Kaniskan, H.; Gu, W Parsons, R.; et al. Acetylation targeting chimera enables acetylation of the tumor suppressor p53. J. Am. Chem. 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TAF1 histone acety ltransferase activity in Spl activation of the cyclin DI promoter. Mol. Cell Biol. 2005, 25 (10), 4321-4332. DOI: 10.1128 / mcb.25.10.4321-4332.2005 From NLM.(10) Bhattacharya, S.; Lou, X.; Hwang, P.; Rajashankar, K. R.; Wang, X.; Gustafsson, J.; Fletterick, R. J.; Jacobson, R. H.; Webb, P. Structural and functional insight into TAF1-TAF7, a subcomplex of transcription factor II D. Proc. Natl. Acad. Sci. U SA 2014, 111 (25), 9103-9108. DOI: 10.1073 / pnas.1408293111 From NLM.(11) Bernardini, A.; Mukherjee, P.; Scheer, E.; Kamenova. I.; Antonova, S.; Mendoza Sanchez, P. K.; Yayli, G; Morlet, B.; Timmers, H. T. M.; Tora, L. Hierarchical TAF1 -dependent co-translational assembly of the basal transcription factor TFIID. Nat. Struct. Mol. Biol. 2023, 30 (8), 1141-1152. DOI: 10.1038 / s41594-023-01026-3 FromNLM.(12) Wei. Y.; Resetca, D.; Li, Z.; Johansson-Akhe, L; Ahlner, A.; Helander, S.; Wallenhammar, A.; Morad, V.; Raught, B.; Wallner, B.; et al. 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Commun. 2019, 10 (1), 1915. DOI: 10.1038 / s41467-019-09672-2 From NLM.(16) Bouché, L.; Christ, C. D.; Siegel, S.; Fernández-Montalván, A. E.; Holton, S. J.; Fedorov, O.; Ter Laak, A.; Sugawara, T.; Stöckigt, D.; Tallant, C.; et al. Benzoisoquinolinediones as potent and selective inhibitors of BRPF2 and TAF1 / TAF1L bromodomains. J. Med. Chem.2017, 60 (9), 4002-4022. DOI: 10.1021 / acs.jmedchem.7b00306 FromNLM.Attorney Docket No. 27527-0233WO1(17) Clegg, M. A.; Theodoulou, N. H.; Bamborough, P.; Chung, C. W.; Craggs, P. D.; Demont, E. H.; Gordon. L. J.; Liwicki, G. M.; Phillipou, A.; Tomkinson, N. C. O.; et al. Optimization of Naphthyridones into selective TATA-binding protein associated factor 1 (TAF1) bromodomain inhibitors. ACS Med. Chem. Lett. 2021, 12 (8), 1308-1317. DOI: 10.1021 / acsmedchemlett.1 c00294 From NLM.(18) Karim, R. M.; Yang, L.; Chen, L.; Bikowitz, M. J.; Lu, J.; Grassie, D.; Shultz, Z. P.; Lopchuk. J. M.; Chen, J.; Schonbrunn. E. Discovery of dual TAF1-ATR inhibitors and ligand-induced structural changes of the TAF1 tandem bromodomain. J. Med. Chem. 2022, 65 (5), 4182-4200. DOI: 10.1021 / acs.jmedchem.lc01999 From NLM.(19) Wang, S.; Tsui, V.; Crawford, T. D.; Audia, J. E.; Burdick, D. J.; Beresini, M. H.; Cote, A.; Cummings, R.; Duplessis, M.; Flynn, E. M.; et al. GNE-371, a potent and selective chemical probe for the second bromodomains of human transcription-initiation-factor TFIID subunit 1 and transcription-initiation-factor TFIID subunit 1 -like. J. Med. Chem. 2018, 61 (20), 9301-9315. DOI: 10.1021 / acs.jmedchem.8b01225 FromNLM.(20) Liu, X.; Wilcken, R.; Joerger, A. C.; Chuckowree, I. S.; Amin, J.; Spencer, J.; Fersht, A. R. Small molecule induced reactivation of mutant p53 in cancer cells. Nucleic Acids Res. 2013, 41 (12), 6034-6044. DOI: 10.1093 / nar / gkt305 FromNLM.(21) Baud, M. G. J.: Bauer. M. R.; Verduci, L.; Dingler, F. A.; Patel, K. J.; Horil Roy, D.; Joerger, A. C.; Fersht, A. R. 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Claims

Attorney Docket No. 27527-0233WO1Claims1. An acetylation targeting chimera (AceTAC), comprising a p53Y220C binder conjugated to TATA-binding protein-associated factor 1 (TAF1) binder by a linker.

2. Compound MS 172, having the following structure:

3. A method of inducing acetylation of p53Y220C in p53Y220C-harboring cells by contacting the cells with MS 172.

4. A method of suppressing tumor cell growth in a subject by administration of MS 172.

5. 6-(But-3-en-l-yl)-4-(6-(4-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- yl)methyl)glycyl)piperazine-l -carbonyl)-! -methyl-lF7-benzo[<7]imidazol-4-yl)-l, 6- dihydro-7 / 7-pyrrolo[2,3-c]pyridin-7-one.

6. 6-(But-3-en-l-yl)-4-(6-(4-(3-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- yl)methyl)amino)propanoy I )piperazine-l -carbonyl)- 1 -methyl- 177-benzo|<: / |imidazol- 4-yl)-l,6-dihydro-7 / / -pyrrolo[2,3-c]pyridin-7-one.

7. 6-(But-3-en-l-yl)-4-(6-(4-(4-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- y l)methy l)an io )butanoyl)piperazme- 1 -carbonyl)- 1 -methyl- 17 / -benzo|r / |imidazol-4- yl)-1.6-dihydro-7F7-pyrrolo[2,3-c]pyridin-7-one.Attorney Docket No. 27527-0233WO18. 6-(But-3-en-l-yl)-4-(6-(4-(5-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / 7-carbazol-3- yl)methyl)amino)pentanoyl)piperazine-l-carbonyl)-l-methyl-l / f-benzo[< / |imidazol-4- yl)-!,6-dihydro-77 / -pyrrolo[2,3-c]pyridin-7-one.

9. 6-(But-3-en-l-yl)-4-(6-(4-(6-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- yl)methy l)amino)hexanoy l)piperazine- 1 -carbonyl)- 1 -methyl - 177-benzo [<7] imidazol-4- yl)-1.6-dihydro-7 / 7-pyrrolo[2,3-c]pyridin-7-one.

10. 6-(But-3-en-l-yl)-4-(6-(4-(7-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- l)methyl)amino)heptano l)piperazine- 1 -carbon l )- 1 -meth l- 17 / -benzo|c / |imidazol-4- yl)-l,6-dihydro-7 / 7-pyrrolo[2,3-c]pyridin-7-one.

11. 6-(But-3-en-l-yl)-4-(6-(4-(8-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- yl)methyl)amino)octanoyl)piperazine-l-carbonyl)-l-methyl-177-benzo[<7]imidazol-4- yl)-l,6-dihydro-727-pyrrolo[2,3-c]pyridin-7-one.

12. 6-(But-3-en-l-yl)-4-(6-(4-(9-(((9-ethyl-7-(4-methylthiophen-2-yl)-9H-carbazol-3- y l)methyl)amino)nonanoyl)piperazine- 1 -carbonyl)- 1 -methyl- 177-benzo [< ] imidazol -4- yl)-l.6-dihydro-7 / / -pyrrolo|2.3-c|pyridm-7-one.

13. 6-(Bbut-3-en-l -yl)-4-(6-(4-(3-(2-(((9-ethyl-7-(4-methylthiophen-2-yl)-977-carbazol-3- yl)methyl)amino)ethoxy)propanoyl)piperazine-l -carbonyl)-! -methyl-177- benzo[6f|imidazol-4-yl)-l,6-dihydro-7 / 7-pyrrolo[2,3-c]pyridin-7-one.

14. 6-(But-3-en-l-yl)-4-(6-(4-(3-(2-(2-(((9-ethyl-7-(4-methylthiophen-2-yl)-9 / / -carbazol- 3-yl)methyl)amino)ethoxy)ethoxy)propanoyl)piperazin e-1 -carbonyl)-! -methyl-177- benzo[t / |imidazol-4-yl)-l,6-dihydro-777-pyrrolo[2,3-c]pyridin-7-one.

15. 6-(But-3 -en- 1 -y l)-4-(6-(4-( 1 -(9-ethyl -7 -(4-methy I th io phen-2-v I )-9H-carbazol-3 -yl)- 5,8, 1 l-trioxa-2-azatetradecan-14-oyl)piperazine-l -carbonyl)-! -methyl-177- benzo[rf|imidazol-4-yl)-l,6-dihydro-777-pyrrolo[2,3-c]pyridin-7-one.Attorney Docket No. 27527-0233WO116. 6-(But-3 -en-1 -yl)-4-(6-(4-(l -(9-ethy 1-7 -(4-methy lthiophen-2-y I )-9 / / -carbazol-3 -yl)- 5.8.11,14-tetr aoxa-2-azaheptadecan-17-oyl)piperazine- 1 -carbonyl)-l-methyl-l77- benzo[<flimidazol-4-yl)-l,6-dihydro-77 / -pyrrolo[2,3-c]pyridin-7-one.

17. A pharmaceutical composition, comprising a compound of any of claims 5-16 and a pharmaceutically acceptable carrier.

18. A compound having the following structure:wherein n=l-8.

19. A compound having the following structure:wherein m=l-4.

20. A pharmaceutically acceptable salt of a compound according to any one of claims 1, 2 and 5-19.

21. Pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric or racemates of a compound according to any one of claims 1, 2, and 5-19.

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