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.