Tunable Cysteine-Targeting Electrophilic Hetero-Aromatic Warheads Induce Ferroptosis

Covalent inhibitors have historically been considered potential liabilities in medicinal chemistry. The modern era witnesses a renaissance of interest in irreversible binders. The available toolbox of electrophilic warheads is limited with constraints on tuning reactivity and selectivity. Following our initial work on a new class of ferroptotic agents termed CETZOLEs, we discovered new tunable heterocyclic electrophiles, which are capable of inducing ferroptosis. Extensive biological evaluation demonstrated that thiazoles with an alkyne electrophile at the 2-position selectively induce ferroptosis with high potency. Density functional theory (DFT) calculations and NMR kinetic studies demonstrated the ability of our heterocycles to undergo thiol addition, which appears to be a prerequisite for cytotoxicity. Chemoproteomic analysis by in-gel fluorescence and pulldown experiments indicated several potential targets, the most prominent among them being GPX4 protein. The results from the proteomic data were further validated by western blot analysis and CETSA. Incorporation of our heterocycles into appropriate pharmacophores generates highly cytotoxic agents such as the analog BCP-T. A, which demonstrated low nM IC50 values in a series of ferroptosis-sensitive cell lines.