Proteome-wide structure-based accessibility analysis of ligandable and detectable cysteines in chemoproteomic datasets

Covalent drug discovery, in particular targeting reactive cysteines, has undergone a resurgence over the past two decades, demonstrated by recent clinical successes of covalent inhibitors for high-priority cancer targets. Reactive cysteine profiling, first pioneered by the Cravatt lab, has emerged in parallel as a powerful approach for proteome-wide on- and off-target profiling. Thus far however, structural analysis of liganded cysteines has been restricted to experimentally determined protein structures. We combined AlphaFold-predicted amino acid side chain accessibilities for >95% of the human proteome with a meta-analysis of thirteen public cysteine profiling datasets, totalling 40,070 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further suggests that exposed cysteine residues are preferentially targeted by elaborated fragments and drug-like compounds. We finally propose a framework for benchmarking coverage of ligandable cysteines in future cysteine profiling approaches, considering both selectivity for high-priority residues and quantitative depth. All analysis and produced resources (freely available at www.github.com/TateLab) are readily extendable to reactive amino acids beyond cysteine, and related questions in chemical biology. ### Competing Interest Statement EWT is or has been employed as a consultant or scientific advisory board member for Myricx Pharma, Samsara Therapeutics, Roche, Novartis and Fastbase. Research in his group has been funded by Pfizer Ltd, Kura Oncology, Daiichi Sankyo, Oxstem, Exscientia, Myricx Pharma, AstraZeneca, Vertex Pharmaceuticals, GSK and ADC Technologies. EWT owns equity in Myricx Pharma, Exactmer and Samsara Therapeutics, and is a named inventor on patents filed by Myricx Pharma, Exactmer, Imperial College London and the Francis Crick Institute. None of these interests conflict with the work described here. JG has acted as a consultant for Unity Biotechnology, Geras Bio, Myricx Pharma and Merck KGaA. Pfizer and Unity Biotechnology have funded research in JGs lab (unrelated to the work discussed here). JG owns equity in Geras Bio. JG is a named inventor in MRC and Imperial College patents, both related to senolytic therapies (the patents are not related to the work discussed here).