Mechanism of Muscimol as a Novel Pyroptosis Inhibitor

项目摘要 上睑下垂是一种程序性的溶解、促炎症细胞死亡的过程，参与了 导致全球死亡的主要原因。炎症体介导的先天免疫信号激活caspase-1 Gasdermin D.最近的数据显示，家族蛋白酶通过裂解造孔蛋白来启动下垂。 证明忍者蛋白-1在下垂过程中寡聚，并且是质膜所必需的。 破裂，或细胞裂解，在Gasdermin D孔形成的下游。焦油中毒过程中释放的细胞因子 溶解引起局部和全身炎症和病理，但调节质膜的过程 破裂以及这些是否可以在治疗上有针对性，还没有很好的理解。我们最近确认了 蝇草酚是一种新型的焦链裂解抑制剂，但其作用机制尚不清楚。麝香酚是 被认为是神经元GABA受体的激动剂，但我们的初步数据表明，抑制 焦磷脂的溶解不是由这些受体介导的。这项提案旨在了解蝇草酚如何预防 焦链裂解，并鉴定具有强大和特定活性的蝇草酚类似物。 这项提案中概述的实验将系统地检查下垂过程中的步骤，以 被麝香酚抑制。在初步数据的基础上，我们将重点进行实验，假设 干扰忍者蛋白-1齐聚，同时也测试其他可能性。我们将使用互补性 炎性刺激和非依赖刺激诱导的下睑下垂模型及其进一步应用 以我们的发现为基础的简化论系统。我们的初步数据表明，有特定的分子 由于类似物显示出不同于亲本的效力，因此蝇草酚抑制焦磷脂溶解的决定因素 分子，与GABA受体活性无关。我们将系统地测试一组经过合理选择的， 已经合成了抑制焦磷脂溶解的蝇草酚类似物。我们假设我们的结果将 揭示了一种新的蝇硫醇抑制质膜破裂的构效关系 它在神经元感受器上的典型活动。此外，这些实验可能会产生类似的增加 防止嗜热性溶解的效力和/或比活性，不作用于GABA受体。最后，我们会 利用麝香酚的独特化学性质，再加上蛋白质组学的进展和 我们的合作者，以确定新的蝇草酚结合蛋白。总而言之，这些实验的结果将 告知准确的分子理解破坏焦链裂解的作用机制，并提供 为许多涉及上睑下垂的疾病的新的治疗策略奠定基础。

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.