Abstract The inflammasomes are crucial innate immune signaling platforms implicated in immune defense against infections and autoimmune/autoinflammatory disorders such as multiple sclerosis, diabetes, and Alzheimer’s disease. Pyroptosis is triggered by inflammatory caspases-1, 4, 5, or 11 upon stimulation of the canonical and noncanonical inflammasomes. Despite the recent progress on the identification and characterization of GSDMD as an effector of pyroptosis, the molecular events underlying the transformation of GSDMD from an autoinhibited resting state to the recognition and cleavage by caspases has remained poorly defined. The lack of mechanistic understanding of GSDMD-mediated pyroptosis has hampered progress on the development of novel therapeutics against infections and autoimmune/autoinflammatory disorders. This proposal targets critical gaps in our understanding of GSDMD/inflammatory caspase-mediated pyroptosis using complementary structure- function approaches. We hypothesize that GSDMD is maintained in an autoinhibited conformation through intramolecular domain interactions between its N- (GSDMD-N) and C-terminal (GSDMD-C) domains. Such conformation is recognized by inflammatory caspases that cleave at the linker region to release the autoinhibition and facilitate membrane pore formation. We propose the following specific aims to test the above hypothesis. Aim 1. Define the mechanism through which GSDMD maintains its autoinhibited state, and how such autoinhibition may be regulated by small molecule tool compounds. We will characterize the structure of the GSDMD domains and the full-length protein in its autoinhibited state using primarily structural approaches, in concert with analysis of the GSDMD-N and GSDMD-C interactions through biochemical techniques. The role of the intramolecular domain interface during Salmonella infection-induced pyroptosis will be probed using GSDMD-deficient cell lines reconstituted with mutant GSDMD. A unique feature of our approach is the incorporation of GSDMD-binding compounds identified from chemical libraries screening into the above efforts to investigate the regulation of GSDMD and pyroptosis by small molecule tool compounds. Aim 2. Define the mechanisms of gasdermin recognition and cleavage by inflammatory caspases. We will elucidate the molecular basis of the conversion of caspases-4 and 11 from zymogens to active conformation, which has been poorly characterized. We will investigate the mechanism of GSDMD recognition by these caspases, which may involve both the linker peptides and tertiary structure of GSDMD. In summary, this project will delineate the molecular mechanisms underlying the autoinhibition and activation of GSDMD during pyroptosis, identify small molecule tool compounds and characterize their mode of acti...