Chemoproteomic discovery of allosteric covalent GSTO1 inhibitors reveals a previously unrecognized binding mode with anti-inflammatory activity

Glutathione S-transferase omega 1 (GSTO1) promotes activation of the NLRP3 inflammasome through deglutathionylation of NEK7, contributing to pro-inflammatory signaling relevant to cancer and inflammatory disease, yet selective and cell-active GSTO1 inhibitors remain limited. Using a live-cell PRM-ABPP platform (ChomiXScreenTM), we identified a covalent small-molecule inhibitor (A1) that engages GSTO1 at Cys32 in intact cells, followed by ligand-based optimization to generate a focused analogue series. Among these, A13 exhibited sustained GSTO1 engagement (kinact/KI = 226 M⁻¹·s⁻¹), high proteome-wide cysteine selectivity as assessed by competitive Ucov-ABPP, and improved metabolic stability in human liver microsomes. High-resolution crystal structures revealed that A13 adopts an unexpected allosteric covalent binding mode distinct from those reported for existing GSTO1 inhibitors. Functionally, covalent targeting of GSTO1 Cys32 significantly suppressed LPS-induced IL-1beta and IL-18 secretion in primary human monocyte-derived macrophages. Together, these findings identify a previously unrecognized allosteric covalent binding mode for GSTO1 inhibition and demonstrate the utility of live-cell chemoproteomics for discovering selective, functional covalent inhibitors to modulate inflammation relevant to cancer.