Structure–Activity Relationship for Inflammasome Inhibition by Thiomuscimol

Abstract

Inflammasomes are central mediators of innate immune defense but can also drive pathological inflammation and pyroptotic cell death in numerous diseases. While several small-molecule inhibitors have been described, many selectively target individual inflammasomes or act through the adaptor protein ASC, leaving ASC-independent pathways unaffected. We previously identified thiomuscimol as a broad-spectrum inflammasome inhibitor that blocks both ASC-dependent and ASC-independent activation, although the structural basis for this activity remains unclear. Here, we examined the structure–activity relationship of thiomuscimol using related compounds and synthetic analogs. In primary macrophages, inflammasome activation and pyroptosis were assessed by live cell imaging of ASC speck formation, gasdermin D-mediated dye uptake, and cellular ATP levels. Structurally related sulfur-containing molecules, including taurine and isothiazole, failed to inhibit inflammasome activation, indicating that neither the sulfur in an electron-rich environment nor the heterocyclic scaffold confer activity. Replacement of the primary amine with a carbonyl group abolished activity, whereas substitution with a secondary amine preserved inhibitory potency comparable to thiomuscimol. Incorporation of the amine into an annulated piperidine ring reduced potency and revealed sensitivity to the precise positioning of the amine within the ring. Together, these findings identify key structural features required for thiomuscimol-mediated inflammasome inhibition and provide a framework for future studies to define its mechanism of action and guide the development of improved inhibitors.