Enrichment-Free, Targeted Covalent Drug Discovery in Live Cells

Live-cell activity-based protein profiling (ABPP) with mass spectrometry enables the proteome-wide quantification of compound reactivity, yet resulting datasets often suffer from low data completeness for high-priority targets and do not give users the option to measure compound-induced protein changes within the same screening assay. To address these limitations, we developed CysDig, an enrichment-free chemoproteomics platform for the targeted covalent drug discovery in live cells. Using the CysDig platform, we screened 288 cysteine-reactive electrophiles against 300 functionally annotated cysteine sites. From this screen, we identified covalent binders that liganded dozens of sites and identified multiple instances of acute compound-induced protein degradation of ACAT1. We validated a molecule that engaged with the active site of HECT E3 ligase HUWE1 and showed that chemical inhibition stabilized known substrates. Together, these findings establish CysDig as a powerful, targeted platform for live-cell covalent drug screening, expanding the current repertoire of available approaches for ligand discovery in live cells.