Identification of pyroptosis inhibitors that target a reactive cysteine in gasdermin D

Inflammasomes are multi-protein signalling scaffolds that assemble in response to invasive pathogens and sterile danger signals to activate inflammatory caspases (1/4/5/11), which trigger inflammatory death (pyroptosis) and processing and release of pro-inflammatory cytokines (1,2). Inflammasome activation contributes to many human diseases, including inflammatory bowel disease, gout, type II diabetes, cardiovascular disease, Alzheimer9s disease, and sepsis, the often fatal response to systemic infection (3-6). The recent identification of the pore-forming protein gasdermin D (GSDMD) as the final pyroptosis executioner downstream of inflammasome activation presents an attractive drug target for these diseases (7-11). Here we show that disulfiram, a drug used to treat alcohol addiction (12), and Bay 11-7082, a previously identified NF-kappaB inhibitor (13), potently inhibit GSDMD pore formation in liposomes and inflammasome-mediated pyroptosis and IL-1beta secretion in human and mouse cells. Moreover, disulfiram, administered at a clinically well-tolerated dose, inhibits LPS-induced septic death and IL-1beta secretion in mice. Both compounds covalently modify a conserved Cys (Cys191 in human and Cys192 in mouse GSDMD) that is critical for pore formation (8,14). Inflammatory caspases employ Cys active sites, and many previously identified inhibitors of inflammatory mediators, including those against NLRP3 and NF-kappaB, covalently modify reactive cysteine residues (15). Since NLRP3 and noncanonical inflammasome activation are amplified by cellular oxidative stress (16-22), these redox-sensitive reactive cysteine residues may regulate inflammation endogenously, and compounds that covalently modify reactive cysteines may inhibit inflammation by acting at multiple steps. Indeed, both disulfiram and Bay 11-7082 also directly inhibit inflammatory caspases and pleiotropically suppress multiple processes in inflammation triggered by both canonical and noncanonical inflammasomes, including priming, puncta formation and caspase activation. Hence, cysteine-reactive compounds, despite their lack of specificity, may be attractive agents for reducing inflammation.