Agonists of the RIG-I Innate Immune Pathway

RIG-I 先天免疫途径激动剂

• 批准号: 7683312
• 负责人: Shawn Patrick Iadonato
• 金额: $ 28.54万
• 依托单位: KINETA, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-05 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7683312
• 关键词: Agonist; Albumins; Antiviral Agents; Antiviral Response; Biological Assay; Cell Density; Cell Line; Cells; Dengue Virus; Development; Dimerization; Dose; Double-Stranded RNA; Drug resistance; Enzymes; Failure; Gene Expression; Gene Targeting; Genes; Goals; HIV-1; Hepatitis C virus; Immune; Immune response; Immunity; Infection; Interferons; Investments; Kinetics; Lead; Libraries; Luciferases; Measures; Methods; Mutation; Nuclear; Nuclear Translocation; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phosphorylation; Play; Production; Program Development; Proliferating; RNA Helicase; RNA Virus Infections; RNA Viruses; Reading; Reagent; Renilla; Reovirus; Reporter Genes; Respiratory syncytial virus; Role; Screening procedure; Serine Protease; Side; Signal Transduction; Specificity; Structure; System; Time; Validation; Vesicular stomatitis Indiana virus; Virus; Virus Diseases; West Nile virus; Work; base; cytotoxic; cytotoxicity; drug development; drug discovery; fighting; high throughput screening; influenzavirus; inhibitor/antagonist; novel; pathogen; promoter; public health relevance; receptor; replicase; respiratory; small molecule; virus host interaction

DESCRIPTION (provided by applicant): There is a tremendous commercial demand for new antiviral products with novel mechanisms of action and which target a broad spectrum of viruses. Most previous and ongoing pharmaceutical development programs involve screening for inhibitors of essential virus enzymes with comparatively little investment in drugs that modulate the host immune response to infection. In this proposal, we focus drug development efforts on the host side of the virus-host interaction in order to discover and develop new antiviral products with novel mechanisms of action that are more effective, less toxic, and less sensitive to virus escape through mutation. Briefly, we propose to implement a high-throughput screen to identify small-molecule agonists of the cellular RIG-I pathway. RIG-I is a double-stranded RNA helicase that functions as a cytosolic pathogen recognition receptor that is essential for triggering immunity to a wide range of RNA viruses. Our group has developed a cell-based screening platform that is based on the use of Huh7 cells that are stably transfected with a luciferase reporter gene under the control of the ISG56 gene promoter (Huh7-ISG56-Luc). ISG56 is activated by IRF-3, a RIG-I effector molecule. This platform is amenable to high-throughput compound screening in which RIG-I signaling is identified through quantification of luciferase activity. In Specific Aim 1, we will reduce to practice the existing cell line and methods for high-throughput screening of RIG-I agonists. This will include scaling to microtiter plates, optimizing positive and negative controls, and defining all assay parameters. In Specific Aim 2, we will identify a set of lead compounds through library screening, counter screens, and validation assays. This will include screening a 20,000-compound maximally diverse library for agonists of the RIG-I pathway. Compound hits will be counter screened for target specificity and cytotoxicity and validated for dose-dependent activation of Huh7-ISG56-Luc expression and the ability to induce endogenous ISG56 gene expression in native Huh7 cells. In Specific Aim 3, we define the antiviral and mechanistic actions of lead compounds to identify a list of validated compounds suitable for optimization and pharmaceutical development. These assays will include examining the antiviral effects of compounds on a variety of viruses, including vesicular stomatitis virus (VSV), New Castle disease virus (NDV), hepatitis C virus, West Nile virus, respiratory syncitial virus, influenza virus, and human immunodeficiency virus-1. We will also begin to evaluate mechanistic aspects of compound function by examining the effects of compounds on IRF-3 activation kinetics, including IRF-3 phosphorylation, dimerization, and nuclear localization, and their action on RIG-I target gene and interferon-stimulated gene expression. This cell-based system and the targeting of RIG-I constitutes a unique drug discovery strategy. Since RIG-I is essential for triggering immunity to a wide range of RNA viruses, this approach offers the promise of defining broad-spectrum antiviral compounds. PUBLIC HEALTH RELEVANCE: We will use a unique drug discovery strategy to identify potential new antiviral drugs that work by activating a faster, more potent immune response to fight off virus infection. Our goal is to identify drugs that will work on a variety of viruses, including influenza virus, hepatitis C virus (HCV), West Nile virus, and human immunodeficiency virus-1 (HIV-1). Because these drugs will activate the natural immune response, they are likely to be safer, more effective, and less prone to failure due to the ability of viruses to develop drug resistance.

描述(由申请人提供)：具有新的作用机制并针对广泛的病毒的新的抗病毒产品有巨大的商业需求。大多数以前和正在进行的药物开发计划都涉及筛选基本病毒酶的抑制剂，而对调节宿主对感染的免疫反应的药物的投资相对较少。在这个建议中，我们将药物开发的努力集中在病毒-宿主相互作用的宿主端，以发现和开发具有新的作用机制的新的抗病毒产品，这些产品更有效，毒性更低，对通过突变逃逸的病毒不那么敏感。简而言之，我们建议实施高通量筛选来识别细胞RIG-I途径的小分子激动剂。RIG-I是一种双链RNA解旋酶，其功能是胞质病原体识别受体，对于触发对多种RNA病毒的免疫是必不可少的。本课题组开发了一种基于细胞的筛选平台，该平台基于ISG56基因启动子(Huh7-ISG56-Luc)控制下稳定转染荧光素酶报告基因的Huh7细胞。ISG56被RIG-I效应分子IRF-3激活。该平台适合高通量化合物筛选，其中RIG-I信号是通过定量荧光素酶活性来鉴定的。在具体目标1中，我们将减少现有的细胞系和高通量筛选RIG-I激动剂的方法。这将包括调整到微量滴度板，优化阳性和阴性对照，并定义所有的检测参数。在具体目标2中，我们将通过文库筛选、计数器筛选和验证分析来鉴定一组先导化合物。这将包括筛选RIG-I途径激动剂的20,000种化合物的最大多样性文库。将对复合HITS进行靶向特异性和细胞毒性的反筛选，并验证其对Huh7-ISG56-Luc表达的剂量依赖性激活以及在天然Huh7细胞中诱导内源性ISG56基因表达的能力。在具体目标3中，我们定义了先导化合物的抗病毒和机制作用，以确定适合优化和药物开发的有效化合物清单。这些检测将包括检测化合物对各种病毒的抗病毒作用，包括水疱性口炎病毒(VSV)、新城疫病毒(NDV)、丙型肝炎病毒、西尼罗河病毒、呼吸道合胞病毒、流感病毒和人类免疫缺陷病毒-1。我们还将通过检测化合物对IRF-3激活动力学的影响，包括IRF-3的磷酸化、二聚化和核定位，以及它们对RIG-I靶基因和干扰素刺激的基因表达的作用，来评估化合物功能的机制方面。这种基于细胞的系统和RIG-I的靶向构成了一种独特的药物发现策略。由于RIG-I对于触发对广泛的RNA病毒的免疫至关重要，这种方法提供了定义广谱抗病毒化合物的前景。 与公共卫生相关：我们将使用一种独特的药物发现策略来确定潜在的抗病毒药物，这些药物通过激活更快、更有效的免疫反应来对抗病毒感染。我们的目标是确定对多种病毒有效的药物，包括流感病毒、丙型肝炎病毒(丙型肝炎病毒)、西尼罗河病毒和人类免疫缺陷病毒-1(HIV-1)。由于这些药物将激活自然免疫反应，它们可能更安全、更有效，而且由于病毒产生抗药性的能力而不易失败。