Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) “killer domains” of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs. [Display omitted] •A disulfide tethering screen identifies small molecules that target BFL-1 C55•Structural analyses reveal the conformational consequences of disulfide formation•Lead molecule 4E14 effectively competes with BH3-binding at the BFL-1 groove•Covalent 4E14 targeting of C55 inhibits BFL-1 suppression of mitochondrial apoptosis Harvey et al. perform a disulfide tethering screen of BFL-1, a BCL-2 protein implicated in cancer, in order to identify molecules capable of covalently targeting a unique C55 residue of the anti-apoptotic groove. 4E14 derivatization (304 Da moiety) effectively blocks BFL-1, informing the design of cysteine-reactive drugs to selectively target BFL-1.