Isoalantolactone targets NLRP3 to disrupt NLRP3-NEK7 interaction and suppress inflammasome activation.

NLRP3 inflammasome plays a critical role in innate immunity and has been implicated in the pathogenesis of multiple inflammatory diseases. However, pharmacological agents that directly target NLRP3 remain limited. In the present study, we investigated the inhibitory effect of isoalantolactone (IAL), a naturally occurring sesquiterpene lactone, on NLRP3 inflammasome activation. IAL markedly suppressed caspase-1 activation and IL-1β/IL-18 maturation induced by canonical NLRP3 stimuli in macrophages, while showing little effect on affecting AIM2 or NLRC4 inflammasomes, indicating selective inhibition of the NLRP3 inflammasome. Mechanistically, IAL inhibited NLRP3 inflammasome assembly, as evidenced by reduced NLRP3 oligomerization, ASC speck formation, and disruption of the NLRP3-NEK7 interaction. Cellular thermal shift assay (CETSA) and microscale thermophoresis (MST) confirmed direct binding between IAL and NLRP3. Molecular docking, CETSA mutational analyses (E695A, H916A, and the double mutant E695A/H916A), and chemical validation using a reduced, inactive IAL derivative (Re-IAL) collectively demonstrated that residues E695 and H916 are critical for IAL binding and its subsequent inhibitory activity. Furthermore, IAL alleviated monosodium urate-induced gouty inflammation and methionine-choline deficient diet-induced metabolic dysfunction-associated steatotic liver disease (MASLD) in wild-type mice, whereas these protective effects were absent in Nlrp3-deficient mice. Together, these findings identify IAL as a small-molecule inhibitor that directly targets NLRP3 at residues E695/H916 to disrupt inflammasome assembly, highlighting its therapeutic potential for NLRP3-driven inflammatory diseases.