Cell-based assay directly monitoring viral polymerase activity for drug discovery.

项目摘要/摘要 我们的目标是开发一种新的基于细胞的HTS平台，可以用于改进抗病毒药物的发现。 登革热病毒感染每年导致约1亿人患病，暴发严重和频率高 随着时间的推移而增加。所有的RNA病毒都是如此，包括寨卡病毒、埃博拉病毒、流感、黄热病和 丙型肝炎，仅举几例。除了登革热本身带来的健康负担外，登革热还构成了 巨大的全球财政负担(约390亿美元)，世界上20%的人口生活在高危地区 目前迫切需要登革热疗法。目前还没有批准的通用疫苗或登革热 特定的抗病毒药物。目前市场上的抗病毒药物针对的是病毒酶；病毒酶通常有 哺乳动物细胞中不存在的结构，是它们生命周期所必需的，使它们 很好的目标。登革热药物发现的高通量筛选试验要么在体外进行，要么在 活着。体外检测是靶标特异性的，但在细胞中进行检测时，结果不能很好地转化。体内检测 在临床上更相关，但药物的靶点尚不清楚，因此很难预测毒性和量身定制 提高疗效的药物。尽管人们对登革热抗病毒药物感兴趣，但还没有一种登革热抗病毒药物成功通过 临床测试，建议需要一种更好的抗病毒筛查平台，可以结合 体外和体内检测。我们计划使用我们合适的RNA适配子技术来开发一种针对目标的 针对登革病毒RNA依赖的RNA聚合酶(RdRP)的细胞HTS平台。RDRP是一种 登革热的理想靶标是因为其在不同血清型中的高度同一性，而在RNA病毒中的理想靶标是因为 它在病毒复制中的关键作用，以及它缺乏哺乳动物的对应物。此应用程序的目标是 开发一种能够在稳定表达病毒的细胞系中监测Rdrp转录的荧光传感器 促进登革热抗病毒发现的蛋白质。这个应用程序的第二阶段是开发一个完全 稳定表达病毒蛋白和用于HTS检测的荧光传感器的综合细胞系。 第二阶段还将包括开发针对寨卡病毒和埃博拉病毒等其他病毒的类似检测方法， 提供此阶段第一阶段授予的设计知识。

Project Abstract/Summary Our goal is to develop a new cell-based HTS platform that can be used to improve antiviral drug discovery. Infection by Dengue virus causes ~100 million illnesses annually, with outbreak severity and frequency increasing with time. This is true of all RNA viruses, which includes Zika, Ebola, Influenza, Yellow Fever, and Hepatitis C, to name a few. In addition to the health burden Dengue alone represents, dengue fever constitutes a large global financial burden (~39 billion USD), and with 20% of the world’s population living in at-risk areas there is an urgent need for dengue therapeutics. There is currently no approved universal vaccine or dengue specific antiviral drug. Current antiviral drugs on the market target viral enzymes; viral enzymes often have structures that are not present in mammalian cells and are absolutely required for their life cycle making them good targets. High throughput screening assays for dengue drug discovery are either performed in vitro or in vivo. In vitro assays are target specific, but results do not translate well when performed in cells. In vivo assays are more clinically relevant, but the target of the drug is unknown, making it hard to predict toxicity and tailor the drug to improve efficacy. Despite interest in dengue antivirals, no dengue antiviral has successfully passed clinical testing, suggesting a need for a better antiviral screening platform that can combine the advantages of in vitro and in vivo assays. We plan to use our propriety RNA aptamer technology to develop a target-specific cell-based HTS platform targeting the RNA-dependent RNA polymerase (RdRP) of dengue virus. RdRP is an ideal target in Dengue due to its high identity across serotypes, and an ideal target in RNA viruses because of its critical role in viral replication, and its lack of a mammalian counterpart. The goal of this application is to develop a fluorescent sensor capable of monitoring RdRP transcription in a cell line stably expressing viral proteins to advance dengue antiviral discovery. Phase II of this application is to develop a completely comprehensive cell line stably expressing both viral proteins and a fluorescent sensor for use in HTS assays. Phase II will also include the development of similar assays for other viruses, including Zika and Ebola, based off design knowledge from this Phase I grant.