Abstract
<jats:p>Unconventional protein secretion mediated by gasdermin D (GSDMD) pores is essential for the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β), a major driver of many inflammatory pathologies. Despite extensive investigation over the years, discovery of IL-1β secretion modulators has been hindered by the lack of robust, scalable experimental platforms. To date, IL-1β 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-1β 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-1β 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-1β 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-1β secretion modulators and identify tolcapone as a promising inhibitor of inflammasome-driven immune pathology.</jats:p>