Abstract 2997: Cell-based assay platforms for NLRP3 inflammasome drug discovery

Abstract Inflammasomes have recently emerged as the exciting and promising drug targets for inflammatory disease therapy. The best characterized is the NLRP3 inflammasome. The NLRP3 inflammasome is associated with onset and progression of various diseases, including metabolic disorders, multiple sclerosis, inflammatory bowel disease, cryopyrin-associated periodic fever syndrome, as well as other auto-immune and auto-inflammatory diseases. The NLRP3 inflammasome is a cytosolic multiprotein complex composed of the innate immune receptor protein NLRP3, adapter protein ASC, and inflammatory protease caspase-1 that responds to microbial infection, endogenous danger signals, and environmental stimuli. The assembled NLRP3 inflammasome can activate the protease caspase-1 to induce gasdermin D-dependent pyroptosis and facilitate the release of IL-1β and IL-18, which contribute to innate immune defense and homeostatic maintenance. We have established cell-based Caspase-Glo 1 Inflammasome Assay and NanoBRET target engagement intracellular NLRP3 assay for high throughput screening of inflammasome inhibitors. MCC950 is a cell-permeable, bioavailable, non-toxic sulfonylurea-derived compound. Here, we demonstrate that MCC950 inhibits LPS/Nigericin-induced caspase-1 activation and pyroptotic cell death in dose response mode in PMA primed THP-1 cells in Caspase-Glo 1 inflammasome and CellTiter-Glo viability assays. Our NanoBRET target engagement NLRP3 assay results show that MCC950 binds to cellular NLRP3 within 2 hours of incubation. Interestingly, NanoBRET NLRP3 residence time analysis in living cells indicates that a slow dissociation kinetics (long residence time) is observed for MCC950. Furthermore, Western blot analysis confirms that MCC950 blocks LPS/Nigericin-induced caspase-1 cleavage and activation in PMA primed THP-1 cells. Taken together, our results indicate Caspase-Glo 1 inflammasome and NanoBRET target engagement NLRP3 assays serve as great cell-based assay platforms to facilitate inflammasome drug discovery against inflammatory diseases. Citation Format: Jianghong Wu, Li Liang, Peter Gallagher, Yong Wan, Haiching Ma. Cell-based assay platforms for NLRP3 inflammasome drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2997.