Lysine‐Targeting, Covalent Inhibitors of Bromodomain BD1 of BET Proteins in Live Cells and Animals

Abstract The bromodomain extra‐terminal (BET) family of proteins are valuable therapeutic targets for cancer and other diseases. The adverse events of current pan‐BET inhibitors (BETi) make the development of BET BD1‐ or BD2‐selective inhibitors as a fresh avenue to overcome safety challenges. On the basis of various lysine‐reactive covalent warheads herein we report a set of activity‐based probes (ABPs; P3 – P7 ) capable of global profiling of ligandable lysines within bromodomains (BRDs) in live cells and animals. Chemoproteomic experiments with P7, which utilizes 2‐ethynylbenzaldehyde (EBA), identified 16 endogenous BRDs, thus giving a global landscape of ligandable lysines in BRDs. By further introducing EBA and salicylaldehyde into PLX51107 (a noncovalent BETi), we generated lysine‐reactive, irreversible ( BDS1 – 4 ) and reversible ( BDS5 – 6 ) BD1 covalent inhibitors. Mass spectrometry and X‐ray crystallography confirmed the successful covalent engagement between EBA and K91 near the acetylated lysine (Kac)‐binding site of BD1 in BRD4. BDS4 showed 104‐fold selectivity for BD1 over BD2 with prolonged anticancer effects. Importantly, BDS4 retained robust activity against fibrosis in cells and animals when compared to RVX‐208 (a reported BD2‐selective noncovalent inhibitor), which showed only marginal effects. Our work serves as a useful tool to delineate distinct functions of BD1 and BD2 in future studies.