Covalent inhibition by a natural product-inspired latent electrophile

Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-bromo-4,5-dihydroxazole is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here we define the global cysteine reactivity and selectivity of a set of 3-bromo-4,5-dihydroxazole-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that 3-bromo-4,5-dihydroxazoles capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by 3-bromo-4,5-dihydroxazoles is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, 3-bromo-4,5-dihydroxazoles showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We further validate that 3-bromo-4,5-dihydroxazoles form covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anti-cancer targets. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroxazole as a cysteine-reactive electrophile.