Comparative reactivity profiling of cysteine-specific probes by chemoproteomics

Cysteines, as one of the most intrinsically nucleophilic amino acids, play important roles in proteins involved in diverse biological processes. They are also targets of covalent drugs for treating cancers and other diseases. Understanding the cysteine reactivity towards different types of electrophilic reactive groups will contribute to design of cysteine-reactive probes and facilitate the development of cysteine-based covalent drugs. In this study, we systematically evaluated the cysteinome reactivity toward two common electrophilic probes that are based on nucleophilic substitution and Michael addition, respectively, using chemical proteomic strategies. Our profiling results showed that each probe had its own preferential reactivity towards different cysteines and the engagement of the ligand fragments could only be distinguished by the probe with the matching reactive group. Our study highlighted the importance of choosing proper cysteine-reactive probes for screening covalent ligands and provided informative guidance for covalent drug development.