Mapping the covalent cysteine interactome of Ebselen reveals high-sensitivity target engagement and redox proteome remodeling.

Abstract

Ebselen is a covalent organoselenium compound with broad pharmacological activity, yet its cellular cysteine targets and downstream proteomic consequences remain incompletely defined. Here, we integrated competitive gel-based activity-based protein profiling, reactivity-dependent tandem orthogonal proteolysis-activity-based protein profiling, and TMT-based quantitative proteomics to map Ebselen-induced cysteine engagement and proteome remodeling in living cancer cells. Ebselen exhibited dose-dependent cytotoxicity and markedly perturbed intracellular thiol-redox balance, as reflected by glutathione depletion and altered reactive oxygen species-associated fluorescence readouts. Competitive gel-based profiling confirmed concentration-dependent engagement of protein cysteine residues in live cells. Quantitative rdTOP-ABPP further identified hundreds of dose-responsive cysteine sites in HeLa and HepG2 cells and revealed a preference for cysteine microenvironments enriched with basic residues. Cross-cell-line comparison highlighted CDK5 Cys53, SMU1 Cys298, and RPSA2 Cys163 as conserved covalent nodes, among which CDK5 Cys53 showed high sensitivity to Ebselen treatment, a finding validated by competitive labeling and MS-based site assignment. Global TMT proteomics revealed extensive remodeling of redox-related and cell-survival-associated pathways, including compensatory upregulation of selenoproteins such as TXNRD1 and GPX family members. Together, these results define a chemical proteomic atlas of Ebselen-cysteine interactions and provide a framework for understanding and optimizing covalent organoselenium therapeutics.