Agonists of the RIG-I Innate Immune Pathway

描述（由申请人提供）：对具有新型作用机制且靶向广谱病毒的新型抗病毒产品存在巨大的商业需求。大多数以前和正在进行的药物开发计划涉及筛选基本病毒酶的抑制剂，对调节宿主对感染的免疫反应的药物的投资相对较少。在这项提案中，我们将药物开发工作集中在病毒-宿主相互作用的宿主方面，以发现和开发具有新作用机制的新抗病毒产品，这些新作用机制更有效，毒性更小，对病毒通过突变逃逸不敏感。简而言之，我们建议实施高通量筛选以鉴定细胞RIG-I途径的小分子激动剂。RIG-I是一种双链RNA解旋酶，其作为胞质病原体识别受体发挥功能，该受体对于触发对多种RNA病毒的免疫是必需的。我们的团队开发了一种基于细胞的筛选平台，该平台基于使用在ISG 56基因启动子控制下用荧光素酶报告基因稳定转染的Huh 7细胞（Huh 7-ISG 56-Luc）。ISG 56被RIG-I效应分子IRF-3激活。该平台适用于高通量化合物筛选，其中通过荧光素酶活性的定量鉴定RIG-I信号传导。在具体目标1中，我们将简化实践现有的用于RIG-I激动剂的高通量筛选的细胞系和方法。这将包括按比例缩放至微量滴定板、优化阳性和阴性对照以及定义所有试验参数。在具体目标2中，我们将通过库筛选，计数器筛选和验证分析来确定一组先导化合物。这将包括筛选RIG-I途径激动剂的20，000-化合物最大多样性文库。将针对靶特异性和细胞毒性对化合物命中进行反筛选，并验证Huh 7-ISG 56-Luc表达的剂量依赖性激活和诱导天然Huh 7细胞中内源性ISG 56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作用和机制作用，以确定适合优化和药物开发的经验证化合物列表。这些试验将包括检查化合物对多种病毒的抗病毒作用，包括水泡性口炎病毒（VSV）、新城堡病病毒（NDV）、丙型肝炎病毒、西尼罗河病毒、呼吸道合胞病毒、流感病毒和人免疫缺陷病毒-1。我们还将开始通过检查化合物对IRF-3活化动力学的影响来评估化合物功能的机制方面，包括IRF-3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。这种基于细胞的系统和RIG-I的靶向构成了独特的药物发现策略。由于RIG-I对于触发对各种RNA病毒的免疫是必不可少的，因此这种方法提供了定义广谱抗病毒化合物的希望。 公共卫生相关性：我们将使用一种独特的药物发现策略来识别潜在的新的抗病毒药物，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。我们的目标是确定药物，将工作对各种病毒，包括流感病毒，丙型肝炎病毒（HCV），西尼罗河病毒，和人类免疫缺陷病毒-1（HIV-1）。由于这些药物会激活天然免疫反应，因此它们可能更安全，更有效，并且由于病毒产生耐药性的能力而不太容易失败。

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.