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

• 批准号: 7893300
• 负责人: ALEXEI DEGTEREV
• 金额: $ 7.16万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893300
• 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; 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; 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; public health relevance; receptor; research study; small molecule; structural biology; therapeutic target; therapy development

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(申请人提供)：坏死，一种由压倒性压力引起的灾难性细胞死亡，是人类疾病的主要诱因。然而，由于其被认为是不可控制的性质，很少有人努力开发针对病理性坏死的治疗方法。最近，这一概念受到了挑战，因为发现坏死可能是激活固有的细胞调控通路的结果，这表明可以专门针对坏死进行抑制。作为这种方法的可行性的直接证明，我们已经开发了有效的和选择性的小分子抑制剂来治疗这种调节的坏死或“坏死性下垂”。此外，我们已经使用其中的一个分子，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，坏死性下垂是体内急性病理损伤的重要组成部分。坏死素的发现为开发针对人类疾病的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+化合物的高通量筛选中确定了五种结构不同的坏死素。令人惊讶的是，所有的坏死素都是在RIP激酶复合体的水平上抑制坏死性下垂的，RIP激酶复合体是坏死性下垂诱导的一个公认的中介，这表明它是这一途径的关键步骤，容易受到抑制。有趣的是，虽然所有的坏死素都通过一个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，这表明有可能开发细胞类型特定的抑制剂。在这项提案中，我们将进一步研究死亡素在体外和体内的活性机制。在目标1中，我们将结合生化、质谱学、药物化学和结构生物学的方法，研究核黄素与RIP及其相关因子的体外相互作用。在目标2中，我们将应用目标1的见解来确定定义细胞对坏死素的不同敏感性的关键参数，以便开发特定的标记物来预测在各种病理性坏死范例中抑制坏死性下垂的最佳策略。在目标3中，我们建议研究在急性肝损伤的动物模型中，坏死素的体外活性如何转化为治疗益处。总体而言，我们的项目应该进一步将坏死性下垂定义为病理损伤的关键组成部分，并为开发新的针对坏死性下垂的治疗方法提供机制基础。公共卫生相关性：在许多人类疾病中，从中风、心肌梗死到感染性休克，坏死是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确定的新型细胞坏死化学抑制剂的作用机制，并确定它们在人类疾病动物模型中作为治疗剂的潜力。这些分子可能会为许多毁灭性的人类疾病的药物开发提供一个主要的新方向。