Mechanism of Muscimol as a Novel Pyroptosis Inhibitor

项目摘要 细胞凋亡是一种程序性的溶解性、促炎性细胞死亡过程，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转导激活caspase-1 家族蛋白酶，通过切割成孔蛋白gasdermin D来启动焦亡。最近的数据 表明ninjurin-1蛋白在细胞凋亡过程中发生寡聚化， 破裂或细胞裂解，gasdermin D孔形成的下游。热性下垂时释放的细胞因子 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作为治疗靶点，还没有很好的理解。我们最近发现 蝇酚作为一种新型的热解抑制剂，但其作用机制尚不清楚。蝇蕈醇是 作为神经元GABA受体的激动剂得到了很好的研究，但我们的初步数据表明， 热解裂解不是由这些受体介导的。该提案旨在了解蝇蕈醇如何预防 热解和鉴定具有有效和特异活性的蝇蕈醇类似物。 本提案中概述的实验将系统地检查焦亡过程中的步骤， 蝇蕈醇抑制作用。基于初步的数据，我们将集中实验的假设，蝇蕈醇 干扰ninjurin-1寡聚化，同时也测试其他可能性。我们将使用互补 由炎性小体依赖性和非依赖性刺激诱导的焦亡模型，并进一步采用 还原论系统的基础上，我们的发现。我们的初步数据表明，有特定的分子 蝇蕈醇抑制热解裂解的决定因素，因为类似物表现出与母体不同的效力 分子，与GABA受体活性无关。我们将系统地测试一组合理选择的， 已经合成的蝇蕈醇类似物用于抑制热解。我们假设我们的结果 揭示了蝇蕈醇抑制质膜破裂的新的结构-活性关系， 它在神经元受体上的典型活性。此外，这些实验可以产生具有增加的生物活性的类似物。 防止热解溶解的效力和/或特异性活性，对GABA受体没有活性。最后我们将 利用蝇蕈醇独特的化学性质，再加上蛋白质组学的进步和 我们的合作者，以确定新的蝇蕈醇结合蛋白。这些实验的结果将 提供对破坏热解裂解的作用机制的精确分子理解，并提供 为许多涉及焦亡的疾病的新治疗策略奠定了基础。

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.