Chiritoiu, G.; Ghenea, S.; Isvoranu, G.; Villeneuve, J.; Munteanu, C.; Pop, C.; Bender, A.; Petrescu, S.; Chiritoiu-Butnaru, M.

Unconventional protein secretion mediated by gasdermin D (GSDMD) pores is essential for the release of pro-inflammatory cytokines such as interleukin-1{beta} (IL-1{beta}), a major driver of many inflammatory pathologies. Despite extensive investigation over the years, discovery of IL-1{beta} secretion modulators has been hindered by the lack of robust, scalable experimental platforms. To date, IL-1{beta} related studies largely rely on primary cells and animal models, suitable for mechanistic studies but not readily scalable for high-throughput applications. Here, we engineered a CRISPR-based reporter cell line that allows quantitative monitoring of endogenous IL-1{beta} secretion while preserving the physiologically relevant inflammasome signaling. This platform faithfully recapitulated the response of primary macrophages to pathogen-associated molecular pattern (PAMPs) stimulation and supported the screening of an FDA-approved drug library comprising 1,398 compounds. Form this screen, we identified tolcapone as a potent inhibitor of IL-1{beta} secretion, reducing cytokine release by more than 80% across the screening pipeline. Mechanistically, tolcapone suppressed caspase-1 activation, thereby limiting GSDMD cleavage, pore formation, and the downstream maturation and secretion of IL-1{beta} and IL-18 in vitro. In vivo, tolcapone administration attenuated the acute inflammatory response in a lipopolysaccharide-induced endotoxemia model. Together, these findings establish our reporter platform as a robust tool for discovery of endogenous IL-1{beta} secretion modulators and identify tolcapone as a promising inhibitor of inflammasome-driven immune pathology.