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

基于细胞的检测直接监测病毒聚合酶活性以进行药物发现。

• 批准号: 9907408
• 负责人: Ryan O'Hanlon
• 金额: $ 22.43万
• 依托单位: LUCERNA, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-05 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907408
• 关键词: 3' Untranslated Regions; Address; Antiviral Agents; Antiviral Therapy; Aptamer Technology; Area; Binding; Biological Assay; Broccoli - dietary; Cell Line; Cell physiology; Cells; Cellular Assay; Cessation of life; DNA cassette; Dengue; Dengue Fever; Dengue Infection; Dengue Virus; Development; Disease Outbreaks; Drug Design; Drug Targeting; Ebola; Ensure; Enzymes; Extravasation; Family; Financial Hardship; Flaviviridae; Fluorescence; Frequencies; Genetic Transcription; Goals; Grant; Health; Hepatitis C; Human; In Vitro; Industry Standard; Influenza; Internal Ribosome Entry Site; Knowledge; Lead; Life Cycle Stages; Luciferases; Mammalian Cell; Measures; Monitor; Names; Nonstructural Protein; Open Reading Frames; Pathway interactions; Permeability; Pharmaceutical Preparations; Phase; Polymerase; Population; Protein Biosynthesis; Publishing; RNA; RNA Polymerase Inhibitor; RNA Viruses; RNA-Directed RNA Polymerase; Reporter; Resources; Risk; Role; Serotyping; Severities; Signal Transduction; Specificity; Spinach - dietary; Structure; Structure-Activity Relationship; Symptoms; System; Testing; Therapeutic; Time; Toxic effect; Transfection; Translating; Translations; Vaccines; Viral; Viral Genome; Viral Proteins; Virus; Virus Replication; Yellow Fever; ZIKA; aptamer; assay development; base; clinically relevant; combat; design; drug development; drug discovery; high throughput screening; improved; in vitro Assay; in vivo; inhibitor/antagonist; interest; research clinical testing; screening; sensor; small molecule; stable cell line; universal vaccine; viral RNA

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.

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，可用于改善抗病毒药物的发现。 登革热病毒感染每年导致约1亿人患病，其爆发严重性和频率 随着时间而增加。所有RNA病毒都是如此，包括寨卡病毒、埃博拉病毒、流感病毒、黄热病病毒和 丙型肝炎，仅举几例。除了登革热本身所代表的健康负担外，登革热还构成了 巨大的全球财政负担（约390亿美元），世界20%的人口生活在风险地区 迫切需要登革热治疗剂。目前没有批准的通用疫苗或登革热 特异性抗病毒药物目前市场上的抗病毒药物靶向病毒酶;病毒酶通常具有 这些结构在哺乳动物细胞中不存在，并且是它们的生命周期所必需的， 好目标。用于登革药物发现的高通量筛选测定在体外或体外进行。 vivo.体外测定是靶特异性的，但在细胞中进行时结果不能很好地转化。体内测定 更具有临床相关性，但药物的靶点尚不清楚，因此很难预测毒性并定制药物。 药物，以提高疗效。尽管对登革热抗病毒药物感兴趣，但没有登革热抗病毒药物成功通过 临床试验，这表明需要一个更好的抗病毒筛选平台，可以结合联合收割机的优点， 体外和体内测定。我们计划使用我们的专有RNA适体技术来开发一种靶向特异性的 基于细胞的HTS平台靶向登革病毒的RNA依赖性RNA聚合酶（RdRP）。RdRP是一个 由于其在血清型之间的高度同一性，是登革热的理想靶标，并且由于其在RNA病毒中的 它在病毒复制中的关键作用，以及它在哺乳动物中缺乏对应物。此应用程序的目标是 开发能够监测稳定表达病毒的细胞系中RdRP转录的荧光传感器， 蛋白质来推进登革热抗病毒药物的发现。此应用程序的第二阶段是开发一个完整的 稳定表达病毒蛋白和用于HTS测定的荧光传感器的综合细胞系。 第二阶段还将包括开发针对其他病毒的类似检测方法，包括寨卡病毒和埃博拉病毒， 从第一阶段的资助中获得的设计知识。