Mechanistic analysis of necrostatins: specific inhibitors of programmed necrosis

描述（由申请人提供）：坏死是由压倒性压力引起的灾难性细胞死亡，是人类疾病的主要原因。然而，由于其不可控制的性质，几乎没有努力开发靶向病理性坏死的疗法。这一概念最近受到了挑战，因为发现坏死可以由内在细胞调节途径的激活引起，这表明坏死可以特异性靶向抑制。作为这种方法的可行性的直接证明，我们已经开发了这种调节性坏死或"坏死性凋亡"的有效和选择性小分子抑制剂。此外，我们已经使用这些分子之一，Necrostatin-1，以证明坏死性凋亡是在缺血性脑和心脏损伤和内毒素性肝破坏的情况下体内急性病理损伤的重要组成部分。necrostatin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+化合物的高通量筛选中鉴定了五种结构不同的necrostatin。令人惊讶的是，所有的necrostatin抑制在RIP激酶复合物的水平上的坏死性凋亡，坏死性凋亡诱导的一个既定的介质，这表明它是一个关键步骤，这一途径容易受到抑制。有趣的是，虽然所有的necrostatin都通过类似的靶点起作用，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。在本研究中，我们将进一步研究necrostatin在体内外的作用机制。在目的1中，我们将使用生物化学、质谱、药物化学和结构生物学方法的组合来研究坏死抑制素与RIP和相关因子的体外相互作用。在目标2中，我们将应用目标1的见解来确定定义对necrostatin的差异细胞敏感性的关键参数，以便开发用于预测在各种病理性坏死范例中抑制坏死性凋亡的最佳策略的特异性标志物。在目标3中，我们建议研究necrostatin的体外活性如何在急性肝损伤的动物模型中转化为治疗益处。总的来说，我们的项目将进一步确定坏死性凋亡作为病理损伤的关键组成部分，并为开发新的坏死性凋亡特异性治疗方法提供机制基础。公共卫生相关性：坏死是从中风、心肌梗塞到感染性休克的许多人类疾病中急性组织损伤的关键组成部分。我们建议的目标是表征在我们的初步研究中鉴定的新型细胞坏死化学抑制剂的作用机制，并确定其作为人类疾病动物模型治疗剂的潜力。这些分子可能为许多破坏性人类疾病的药物开发提供了一个主要的新方向。

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.