Discovery of a Covalent Inhibitor of Pro-Caspase-1 Zymogen Blocking NLRP3 Inflammasome Activation and Pyroptosis

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (<b>CIB-1476</b>) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, <b>CIB-1476</b> directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1β production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of <b>CIB-1476</b> with caspase-1. <b>CIB-1476</b> showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in <i>Casp1</i>^-/- mice. These results warrant further development of <b>CIB-1476</b> along with its analogues as a novel strategy for caspase-1 inhibitors.