Vilazodone inhibits NLRP3 inflammasome assembly by targeting the NACHT domain and alleviates inflammatory disease.

The NLRP3 inflammasome plays a central role in innate immunity and inflammatory diseases, yet effective inhibitors remain limited. Here, we identified vilazodone, an FDA-approved antidepressant, as a potent inhibitor of NLRP3 inflammasome activation by small-molecule screening. Vilazodone significantly suppressed IL-1β secretion in multiple macrophage models in response to diverse NLRP3 stimuli. Mechanistically, vilazodone did not affect the priming step but selectively inhibited inflammasome activation. It disrupted NLRP3 interactions with NEK7 and ASC, thereby blocking inflammasome assembly, caspase-1 activation, and downstream pyroptosis, as evidenced by reduced gasdermin D cleavage. DARTS and CETSA assays indicated that vilazodone directly binds to NLRP3, with CETSA showing increased thermal stability and DARTS showing increased resistance to proteolysis. Domain-mapping and molecular simulations further indicated that vilazodone targets the NACHT domain and forms a stable complex primarily driven by van der Waals interactions. Vilazodone also inhibited NLRC4 inflammasome activation but showed minimal effects on AIM2 inflammasome activation. Importantly, vilazodone exerted anti-inflammatory effects in vivo, reducing cytokine production in mouse models of MSU-induced peritonitis and LPS-induced systemic inflammation. Collectively, these findings identify vilazodone as a direct inhibitor of NLRP3 inflammasome assembly and highlight its potential for repurposing in inflammasome-driven diseases.