Dual‐Site Covalent Targeting Enables BD2‐Selective BET Inhibition With Potent Antitumor Activity in Mice

ABSTRACT Bromodomain and extra‐terminal (BET) proteins are validated therapeutic targets for cancer, but clinical translation of pan‐BET inhibitors is limited by dose‐limiting toxicities from non‐selective inhibition of BD1/BD2 domains. Herein, we report the first‐ever domain‐selective covalent inhibitor, named i p AE1 , of BET BD2 domains by installing an epoxide warhead onto the scaffold of ABBV‐744. i p AE1 was shown to irreversibly modify Glu438 and His437 (to a lesser extent) located within the BRD4(2) binding pocket via a dual‐covalent mechanism, as evidenced by its chemical probe p AE1 . Furthermore, i p AE1 exhibited exceptional potency against BRD4(2) ( K d = 0.096 nM) with > 1900‐fold selectivity over BRD4(1), leading to potent and sustained antiproliferative activity in MV4‐11 cells (GI 50 = 1.5 nM). Subsequent live‐cell proteome‐wide profiling validated BRD4 as the primary cellular target of i p AE1 . Consistent with cellular activities, i p AE1 possessed a significantly enhanced antitumor efficacy in an MV4‐11 xenografted mouse model compared to ABBV‐744, presumably due to its on‐target covalent engagement in vivo. Our study thus establishes for the first time a novel targeted covalent inhibition (TCI) strategy that engages two weakly nucleophilic residues within a single bromodomain, an approach generalizable to other compounds targeting E/H, providing a highly selective platform for future development of next‐generation BET inhibitors.