Mechanistic analysis of necrostatins: specific inhibitors of programmed necrosis

坏死他汀的机制分析：程序性坏死的特异性抑制剂

基本信息

批准号：
8025946
负责人：
ALEXEI DEGTEREV
金额：
$ 41.26万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8025946
关键词：
Acute Animal Model Apoptosis Apoptotic Binding Sites Biochemical Brain Caspase Cell Culture Techniques Cell Death Cells Cessation of life Characteristics Chemicals Chemistry Complex Cultured Cells Cytoprotection Data Death Domain Development Disease Dissection Endotoxins Goals Health Heart Heart Injuries Human In Vitro Injury Ischemia Ligands Liver Mass Spectrum Analysis Mediator of activation protein Methods Molecular Morphology Myocardial Infarction Nature Necrosis Pathologic Pathology Pathway interactions Pharmaceutical Chemistry Phosphotransferases Post-Translational Protein Processing Process Property Regulation Regulatory Pathway Reperfusion Therapy Resistance Resolution Role Septic Shock Site Specificity Stimulus Stress Stroke TNF gene TNFSF10 gene Therapeutic Therapeutic Agents Tissues Translating Traumatic Brain Injury Treatment Efficacy Tumor Necrosis Factor Ligand Superfamily Member 6 Work base cell growth regulation cell type drug development high throughput screening human RIPK1 protein human disease in vitro activity in vivo in vivo Model inhibitor/antagonist insight novel prevent programs protective effect receptor research study small molecule structural biology therapeutic target therapy development

项目摘要

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.

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，是人类疾病的主要因素。但是，由于其可感知的无法控制的性质，几乎没有努力开发针对病理坏死的疗法。最近，通过发现坏死可能是由于内在细胞调节途径的激活而引起的，这表明坏死可以专门针对抑制作用。为了直接证明这种方法的可行性，我们开发了这种调节坏死或“坏死性”的有效和选择性的小分子抑制剂。此外，我们已经使用了这些分子之一Necrostatin-1来证明坏死性是在缺血性脑和心脏损伤和内毒性肝破坏的情况下，是体内急性病理损伤的重要组成部分。坏死蛋白的发现为开发新的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多种化合物的高通量筛选中鉴定了五个结构上不同的坏死蛋白。令人惊讶的是，所有坏死蛋白在RIP激酶复合物（已建立的坏死诱导介质）的水平上抑制坏死性，这表明这是该途径易于抑制的关键步骤。有趣的是，虽然所有死灵蛋白通过相似的靶标作用，但它们在不同细胞中显示出不同的活性，这表明可能产生细胞类型特异性抑制剂。在此提案中，我们将进一步研究核酸固醇活性在体外和体内的机制。在AIM 1中，我们将使用生化，质谱法，药物化学和结构生物学方法的组合研究坏死蛋白与RIP和相关因素的体外相互作用。在AIM 2中，我们将应用来自AIM 1的见解来确定定义差异细胞敏感性的关键参数，以开发特定的标记，以预测在病理坏死的各种范式中抑制坏死性抑制的最佳策略。在AIM 3中，我们建议研究急性肝损伤动物模型中坏死蛋白的体外活性如何转化为治疗益处。总体而言，我们的项目应进一步将坏死作用定义为病理损伤的关键组成部分，并为开发新的坏死性特异性疗法提供机械基础。公共卫生相关性：坏死是从中风和心肌梗死到败血性休克的许多人类疾病中急性组织损伤的关键组成部分。我们建议的目的是表征新型细胞坏死化学抑制剂的作用机理，在我们的初步研究中鉴定出来，并在人类疾病动物模型中确定其作为治疗剂的潜力。这些分子可能为许多毁灭性人类疾病提供了主要的药物发育方向。