Mechanistic analysis of necrostatins: specific inhibitors of programmed necrosis

描述（由申请人提供）：坏死是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，由于其不可控制的性质，很少有人努力开发针对病理性坏死的治疗方法。这一观点最近受到了挑战，因为发现坏死可由细胞内在调节途径的激活引起，这表明坏死可以被特异性地靶向抑制。为了直接证明这种方法的可行性，我们已经开发出有效的、选择性的小分子抑制剂来抑制这种受调节的坏死或“坏死下垂”。此外，我们已经使用其中一种分子Necrostatin-1来证明，在缺血性脑和心脏损伤以及内毒素肝破坏的情况下，坏死性上垂是体内急性病理性损伤的一个重要组成部分。坏死他汀类药物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100,000多个化合物的基于细胞的高通量筛选中发现了五种结构不同的necro他汀类药物。令人惊讶的是，所有的坏死性他汀类药物都在RIP激酶复合物水平上抑制坏死性死亡，RIP激酶复合物是一种已知的坏死性死亡诱导介质，这表明它是这一途径的关键步骤，容易受到抑制。有趣的是，虽然所有的坏死性他汀都通过相似的靶点起作用，但它们在不同的细胞中表现出不同的活性，这表明有可能开发出细胞类型特异性抑制剂。在本课题中，我们将进一步研究坏死他汀类药物在体外和体内的活性机制。在Aim 1中，我们将结合生化、质谱、药物化学和结构生物学方法，研究坏死他汀类药物与RIP及相关因子的体外相互作用。在Aim 2中，我们将应用Aim 1的见解来确定定义不同细胞对坏死他汀敏感性的关键参数，以便开发特定标记物，用于预测各种病理性坏死范式中抑制坏死性上睑下垂的最佳策略。在Aim 3中，我们建议研究坏死他汀类药物的体外活性如何在急性肝损伤动物模型中转化为治疗效益。总之，我们的项目应该进一步明确坏死性上睑下垂是病理性损伤的关键组成部分，并为开发新的坏死性上睑下垂特异性治疗方法提供机制基础。公共卫生相关性：坏死是许多人类疾病急性组织损伤的关键组成部分，从中风、心肌梗死到感染性休克。我们建议的目标是描述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.