Project Summary Pyroptosis is a programmed process of lytic, pro-inflammatory cell death that is involved in the pathogenesis of leading global causes of mortality. Inflammasome-mediated innate immune signaling activates caspase-1 family proteases to initiate pyroptosis by cleaving the pore-forming protein, gasdermin D. Recent data demonstrate that the protein ninjurin-1 oligomerizes during pyroptosis and is required for plasma membrane rupture, or cell lysis, downstream of gasdermin D pore formation. Cellular factors released during pyroptotic lysis cause local and systemic inflammation and pathology, but processes that regulate plasma membrane rupture and whether these can be therapeutically targeted, are not well-understood. We recently identified muscimol as a novel inhibitor of pyroptotic lysis, but its mechanism of action is not yet known. Muscimol is well-studied as an agonist of neuronal GABA receptors, but our preliminary data suggest that inhibition of pyroptotic lysis is not mediated by these receptors. This proposal aims to understand how muscimol prevents pyroptotic lysis and identify muscimol analogs with potent and specific activity. The experiments outlined in this proposal will systematically examine steps in the process of pyroptosis for inhibition by muscimol. Based on preliminary data, we will focus experiments on the hypothesis that muscimol interferes with ninjurin-1 oligomerization, while also testing other possibilities. We will use complementary models of pyroptosis induced by inflammasome-dependent and -independent stimuli, and further employ reductionist systems based on our findings. Our preliminary data suggest that there are specific molecular determinants for muscimol inhibition of pyroptotic lysis, as analogs demonstrate varied potency from the parent molecule, not correlating with GABA receptor activity. We will systematically test a panel of rationally-selected, already synthesized, muscimol analogs for inhibition of pyroptotic lysis. We hypothesize that our results will reveal a novel structure-activity relationship for muscimol inhibition of plasma membrane rupture compared to its canonical activity at neuronal receptors. In addition, these experiments may yield analogs with increased potency and / or specific activity to prevent pyroptotic lysis, without activity at GABA receptors. Finally, we will utilize the unique chemical properties of muscimol, coupled with advances in proteomics and the expertise of our collaborators, to identify novel muscimol-binding proteins. Together, the results of these experiments will inform a precise molecular understanding of the mechanism of action to disrupt pyroptotic lysis and provide the foundation for a novel therapeutic strategy for the many diseases in which pyroptosis has been implicated.