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

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 ipAE1, of BET BD2 domains by installing an epoxide warhead onto the scaffold of ABBV-744. ipAE1 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 pAE1. Furthermore, ipAE1 exhibited exceptional potency against BRD4(2) (Kd = 0.096 nM) with > 1900-fold selectivity over BRD4(1), leading to potent and sustained antiproliferative activity in MV4-11 cells (GI50 = 1.5 nM). Subsequent live-cell proteome-wide profiling validated BRD4 as the primary cellular target of ipAE1. Consistent with cellular activities, ipAE1 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.