Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on-and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Automated summary

Covalent drug discovery has experienced a resurgence and reactive cysteine profiling has emerged as a platform for ligand discovery. However, the scope of this approach at the whole-proteome level has not been fully explored. By combining AlphaFold2-predicted side-chain accessibilities and a meta-analysis of cysteine profiling datasets, accessibility biases in cysteines were revealed. Analysis of cysteine-fragment interactions showed increased bias against buried cysteine residues. Future directions for increasing coverage were proposed.