Abstract

Covalent drug discovery has experienced a revival since the 2013 approval of the first cysteine-targeting kinase inhibitors. Many drugs that were discovered by serendipity also possess the ability to react with cysteine residues, leading to interactions with multiple proteins. This widespread interaction, known as promiscuity, necessitates a comprehensive study of how these drugs engage with cysteines throughout the proteome. Here we report a large-scale analysis to meet this need by defining proteome-wide cysteine ligandability maps of 70 drugs in native biological systems. We examined over 24,000 cysteines in the human proteome, pinpointing 279 proteins as potential drug targets across diverse functional categories. We further validated several cysteine engagement events, uncovering previously unknown cysteine sites on both established drug targets and proteins that are generally difficult to address with small molecules. Additionally, our findings highlighted an opportunity to harness a drug-cysteine interaction for targeted protein degradation. Together, our analysis provides an invaluable resource for advancing the development of covalent drugs.