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.

Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymerization with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small molecules with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degradation by covalent binding to Cys-239 of β-tubulin. The small molecules highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 derivative), T138067, N,N′-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quantitative proteomic analysis revealed that T007-1 promotes tubulin degradation with high selectivity. Mass spectrometry findings showed covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those containing Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small molecules (T138067, EBI, and AITC) also reported to bind covalently to Cys-239 of β-tubulin similarly induced tubulin degradation. Our results strongly suggest that covalent modification of Cys-239 of β-tubulin by small molecules could serve as a novel strategy to promote tubulin heterodimer degradation. We propose that these small molecules represent a third novel class of tubulin inhibitor agents that exert their effects through degradation activity.