* authors contributed equally

Abstract 

The NLRP3 inflammasome is a key driver in inflammatory, infectious, metabolic, and neurodegenerative diseases. Although the NLRP3 inhibitor CRID3 (also known as MCC950) exhibits potent activity, it cannot inhibit several hyperactive NLRP3 mutations associated with autoinflammatory syndromes and has not progressed clinically, underscoring the need for the development of new NLRP3 inhibitors. Through a high-throughput screening, we identified LOC14, an isothiazolinone-containing small molecule, as a selective NLRP3 inhibitor. Distinct from CRID3, which targets the NACHT domain, LOC14 binds to or near the LRR domain of NLRP3 and inhibits both CRID3-responsive and CRID3-non-responsive hyperactive or gain-of-function NLRP3 variants. Furthermore, we identified that the carbonyl oxygen of the isothiazol-3(2H)-one moiety is critical for inhibitory activity. In vivo, LOC14 exerted antiinflammatory activity in mouse models of colitis, sepsis, and psoriasis, demonstrating broad physiological and therapeutic relevance. Our findings highlight isothiazolinone-containing compounds as selective NLRP3 inhibitors and provide a promising foundation for developing therapies targeting NLRP3-driven inflammatory diseases.