Proteome-Wide Ligand and Target Discovery by Using Covalent Probes and Natural Products

Due to the lack of effective therapeutic targets for drug development, many diseases remain difficult to treat. To address this issue, phenotypic screening integrated with chemical proteomics has emerged as an efficient strategy to expand the scope of druggable targets. In this study, we constructed a covalent probe library based on diverse covalent kinase inhibitors and natural products containing an α, β-unsaturated ketone electrophilic warhead. Antiproliferation screening revealed that these probes exhibit potent anticancer activity against triple-negative breast cancer (TNBC) and human colon cancer. Subsequent proteomic studies identified a series of novel covalently ligandable targets, including ASNS, AKR1C1, DDX39B, and PRMT5. Functional validation demonstrated that DDX39B may represent a new therapeutic target for TNBC. Moreover, we identified a series of highly selective covalent probes targeting ARK1C1, PDIA1, and ALDH1A1, which could serve as valuable tools for detecting the expression and activity of these critical proteins.