Bromodomains are ~110 amino acid protein modules that bind acetylated lysine residues on histones and other nuclear proteins to epigenetically regulate gene expression. Bromodomains are promising epigenetic targets in cancer, diabetes, and other inflammatory disorders, and bromodomain inhibitors are in clinical trials for multiple cancers and cardiovascular disease. To aid mechanistic inquiries into the association between aberrant bromodomain binding to chromatin and pathogenesis and further drug development, we are developing small molecule inhibitors that selectively target individual bromodomains. The lack of selectivity of existing bromodomain inhibitors may be responsible for the dose-limiting toxicity reported in recent clinical trials. Using fragment screening by mass spectrometry and protein-detected NMR spectroscopy, we report a novel ligandable site on a bromodomain outside the acetyl-lysine binding site and novel inhibitors that are selective for individual bromodomain-containing proteins. Subsequent structure-activity relationship (SAR) analyses led to inhibitors with nanomolar potency, cellular activity, and a substantial improvement in selectivity over previously reported compounds for bromodomains from BRD4 and PBRM1. Fluorescence polarization (FP), differential scanning fluorimetry (DSF), AlphaScreen, isothermal titration calorimetry (ITC), and bioluminescence resonance energy transfer (BRET) were used to establish the potency and selectivity of these inhibitors <i>in vitro</i> and in cellular assays. The best-identified PBRM1 ligand inhibits the association of full-length PBRM1 to acetylated histone peptides in cell lysates and selectively inhibits the growth of PBRM1-dependent prostate cancer cell lines. These inhibitors will help distinguish the differential activities of bromodomains in disease models and may lead to therapeutics targeting the bromodomain axis in cancer, diabetes, and cardiovascular disease.

Discovery of novel prostate cancer-cell active inhibitors and ligandable sites of bromodomain-containing proteins

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ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.