Optimizing the affinity selection mass spectrometry workflow for efficient identification and ranking of potent USP1 inhibitors

An optimized Affinity Selection-Mass Spectrometry (AS-MS) workflow has been developed for the efficient identification of potent USP1 inhibitors. USP1 was immobilized on agarose beads, ensuring low small molecule retention, efficient protein capture, and protein stability. The binding affinity of 49 compounds to USP1 was evaluated using the optimized AS-MS method, calculating binding index (BI) values for each compound. Biochemical inhibition assays validated the AS-MS results, revealing a potential correlation between higher BI values and lower IC₅₀ values. This optimized workflow enables rapid identification of high-quality USP1 inhibitor hits, facilitating structure-activity relationship studies and accelerating the discovery of potential cancer therapeutics.