MCC950/CRID3 potently targets the NACHT domain of wildtype NLRP3 but not disease-associated mutants for inflammasome inhibition

The NLRP3 inflammasome drives pathological inflammation in a suite of diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as CAPS. There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome, for the treatment of CAPS and other diseases. However, these efforts have been constrained by the lack of insight in the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome. Here, we show that the NACHT domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. We find photoaffinity labelling of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. Concordantly, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished interleukin (IL)-1β and IL-18 levels in LPS-challenged wildtype mice but not in Nlrp3(L351P) knock-in mice. This work suggests that MCC950/CRID3-based therapies may effectively treat inflammation by wildtype NLRP3, but not CAPS-associated mutants.