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

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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.