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

Abstract

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, Alzheimer’s 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-κB inhibitor ¹³, potently inhibit GSDMD pore formation in liposomes and inflammasome-mediated pyroptosis and IL-1β secretion in human and mouse cells. Moreover, disulfiram, administered at a clinically well-tolerated dose, inhibits LPS-induced septic death and IL-1β 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-κB, 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.