NU6300 covalently reacts with cysteine-191 of gasdermin D to block its cleavage and palmitoylation.

Gasdermin D (GSDMD) serves as a vital mediator of inflammasome-driven pyroptosis. In our study, we have identified NU6300 as a specific GSDMD inhibitor that covalently interacts with cysteine-191 of GSDMD, effectively blocking its cleavage while not affecting earlier steps such as ASC oligomerization and caspase-1 processing in AIM2- and NLRC4-mediated inflammation. On the contrary, NU6300 robustly inhibits these earlier steps in NLRP3 inflammasome, confirming a unique feedback inhibition effect in the NLRP3-GSDMD pathway upon GSDMD targeting. Our study reveals a previously undefined mechanism of GSDMD inhibitors: NU6300 impairs the palmitoylation of both full-length and N-terminal GSDMD, impeding the membrane localization and oligomerization of N-terminal GSDMD. In vivo studies further demonstrate the efficacy of NU6300 in ameliorating dextran sodium sulfate-induced colitis and improving survival in lipopolysaccharide-induced sepsis. Overall, these findings highlight the potential of NU6300 as a promising lead compound for the treatment of inflammatory diseases.