Structure-Based\nOptimization of a Series of Covalent,\nCell Active Bfl‑1 Inhibitors

Bfl-1, a member of the Bcl-2 family of proteins, plays\na crucial\nrole in apoptosis regulation and has been implicated in cancer cell\nsurvival and resistance to venetoclax therapy. Due to the unique cysteine\nresidue in the BH3 binding site, the development of covalent inhibitors\ntargeting Bfl-1 represents a promising strategy for cancer treatment.\nHerein, the optimization of a covalent cellular tool from a lead-like\nhit using structure based design is described. Informed by a reversible\nX-ray fragment screen, the strategy to establish interactions with\na key glutamic acid residue (Glu78) and optimize binding in a cryptic\npocket led to a 1000-fold improvement in biochemical potency without\nincreasing reactivity of the warhead. Compound <b>(<i>R,R,S</i>)-26</b> has a <i>k</i><sub><i>inact</i></sub><i>/K</i><sub><i>I</i></sub> of 4600 M<sup>–1</sup> s<sup>–1</sup>, shows <1 μM caspase activation in\na cellular assay and cellular target engagement, and has good physicochemical\nproperties and a promising <i>in vivo</i> profile.