A novel class of selective and potent WRN helicase antagonists identified via a DNA-encoded library screen was rigorously validated by various biophysical assays including ASMS, TSA, and SPR. Preliminary structure-activity-relationship studies identified the key pharmacophores and advanced the biochemical potency to single digit nanomolar. Potent analogs demonstrated anti-proliferative activities specifically in cell lines with a WRN genetic dependency. A crystal structure of a ligand-WRN complex revealed an unexpected large shift of the helicase domain compared to the apo WRN structure with ADP. These structural insights led to the successful design of covalent inhibitors and the identification of compound resistant WRN mutants that confirmed on-mechanism cellular activity. The covalent interaction between the ligand and at WRN Cys727 was confirmed by intact mass spectrometry and a bound crystal structure. The discovery of these unique WRN inhibitors provides more insight into the field and offers an opportunity to further optimize such molecules.