Discovery of Potent, Allosteric GSTO1 Covalent Inhibitors with a New Binding Mode

GSTO1 activates the NLRP3 inflammasome by deglutathionylating NEK7 at Cys253, driving proinflammatory responses. Using PRM-based targeted mass spectrometry, we identified compound A1 as a covalent GSTO1 inhibitor modifying Cys32. Ligand-based optimization generated analogues with diverse activities; among them, A13 exhibited moderate target engagement (kinact/KI = 226 M–1·s–1), excellent cysteine selectivity confirmed by desthiobiotin iodoacetamide (DBIA)- and alkyne-based ABPP, and superior metabolic stability in human liver microsomes. High-resolution crystal structures revealed an unexpected A13 binding mode occupying a new hydrophobic pocket distinct from known GSTO1 inhibitors. Functionally, covalent targeting of GSTO1-C32 by A13 markedly reduced LPS-induced IL-1β and IL-18 secretion in human monocyte-derived macrophages. Collectively, these results identify A13 as a potent, selective, and metabolically stable lead compound for developing next-generation GSTO1 inhibitors targeting inflammatory diseases.