Mechanistic analysis of necrostatins: specific inhibitors of programmed necrosis

描述（由申请人提供）：坏死是一种由巨大压力引起的灾难性细胞死亡，是人类疾病的主要原因。然而，由于病理性坏死具有不可控制的性质，因此很少有人致力于开发针对病理性坏死的疗法。最近发现坏死可能是由内在细胞调节途径的激活引起的，这一观点受到了挑战，这表明可以专门针对坏死进行抑制。作为这种方法可行性的直接证明，我们开发了这种调节性坏死或“坏死性凋亡”的有效且选择性的小分子抑制剂。此外，我们还使用其中一种分子 Necrostatin-1 来证明，在缺血性脑和心脏损伤以及内毒素性肝脏破坏的情况下，坏死性凋亡是体内急性病理损伤的重要组成部分。坏死他汀的发现为开发针对人类疾病的新型坏死特异性疗法提供了前所未有的机会。在初步研究中，我们在基于细胞的 100,000 种化合物的高通量筛选中鉴定出了五种结构不同的坏死抑素。令人惊讶的是，所有坏死他汀类药物都在 RIP 激酶复合物水平上抑制坏死性凋亡，RIP 激酶复合物是坏死性凋亡诱导的既定介质，表明它是该途径中容易受到抑制的关键步骤。有趣的是，虽然所有坏死抑素都通过相似的靶点发挥作用，但它们在不同的细胞中表现出不同的活性，这表明开发细胞类型特异性抑制剂的可能性。在本提案中，我们将进一步研究necrostatins的体外和体内活性机制。在目标 1 中，我们将结合生化、质谱、药物化学和结构生物学方法研究 necrostatins 与 RIP 及相关因素的体外相互作用。在目标 2 中，我们将应用目标 1 的见解来确定定义细胞对坏死他汀的差异敏感性的关键参数，以便开发特定标记来预测各种病理性坏死范例中坏死性凋亡抑制的最佳策略。在目标 3 中，我们建议研究 necrostatins 的体外活性如何转化为急性肝损伤动物模型的治疗效果。总的来说，我们的项目应进一步将坏死性凋亡定义为病理损伤的关键组成部分，并为开发新型坏死性凋亡特异性疗法提供机制基础。公共卫生相关性：坏死是许多人类疾病（从中风、心肌梗塞到感染性休克）中急性组织损伤的关键组成部分。我们提案的目标是表征在我们的初步研究中确定的新型细胞坏死化学抑制剂的作用机制，并确定它们作为人类疾病动物模型中的治疗剂的潜力。这些分子可能为治疗许多破坏性人类疾病的药物开发提供一个主要的新方向。

DESCRIPTION (provided by applicant): Necrosis, a catastrophic cell death caused by overwhelming stress, is a major contributor to human disease. However, very little effort has been made to develop therapies targeting pathologic necrosis due to its perceived uncontrollable nature. This notion has been recently challenged through the discovery that necrosis can result from the activation of intrinsic cellular regulatory pathways, suggesting that necrosis can be specifically targeted for inhibition. As a direct demonstration of the feasibility of this approach, we have developed potent and selective small molecule inhibitors of such regulated necrosis or "necroptosis". Furthermore, we have used one of these molecules, Necrostatin-1, to demonstrate that necroptosis is an important component of acute pathologic injury in vivo in the case of ischemic brain and heart damage and endotoxic liver destruction. Discovery of necrostatins provides an unprecedented opportunity to develop novel necrosis-specific therapies for human disease. In Preliminary studies, we identified five structurally distinct necrostatins in a cell based high-throughput screen of 100,000+ compounds. Surprisingly, all necrostatins inhibit necroptosis at the level of RIP kinase complex, an established mediator of necroptosis induction, suggesting that it is a key step of this pathway susceptible to inhibition. Interestingly, while all necrostatins act through a similar target, they display distinct activities in different cells, suggesting the possibility of developing cell type-specific inhibitors. In this proposal, we will further investigate the mechanism of necrostatins' activity in vitro and in vivo. In Aim 1, we will investigate in vitro interactions of necrostatins with RIP and associated factors using combination of biochemical, mass spectrometry, medicinal chemistry and structural biology methods. In Aim 2, we will apply the insights from Aim 1 to identify key parameters defining differential cellular sensitivity to necrostatins in order to develop specific markers for predicting optimal strategy for necroptosis suppression in various paradigms of pathologic necrosis. In Aim 3, we propose to study how in vitro activities of necrostatins translate into therapeutic benefit in the animal model of acute liver injury. Overall, our project should further define necroptosis as the key component of pathologic injury and provide mechanistic basis for development of novel necroptosis-specific therapies. PUBLIC HEALTH RELEVANCE: Necrosis is a key component of acute tissue injury in many human diseases from stroke and myocardial infarction to septic shock. The goal of our proposal is to characterize the mechanism of action of novel chemical inhibitors of cellular necrosis, identified in our preliminary studies, and establish their potential as therapeutic agents in animal models of human disease. These molecules may present a principally novel direction for drug development for many devastating human disorders.