Antiviral potential of helicase inhibitors

描述(由申请人提供)：为了回应资助机会公告“高通量药物筛选的分析方法的开发”，我们建议在这里开发分析方法来识别和分析RNA病毒复制所需的解旋酶抑制物。所有病毒都需要解旋酶来分离双链DNA或RNA，然而解旋酶仍然是尚未开发的抗病毒药物靶点。尽管一些针对DNA病毒的解旋酶抑制剂正在进行临床试验，但还没有针对RNA病毒的抗病毒药物进入临床舞台。因此，我们将针对RNA病毒，重点关注全球疾病负担异常高的病原体：丙型肝炎病毒(丙型肝炎病毒)、登革热病毒(DENV)、西尼罗河病毒(WNV)、日本脑炎病毒(JEV)、黄热病病毒(YFV)和人类免疫缺陷病毒(HIV)。作为正在进行的假设驱动的合作研究项目的一部分，开发丙型肝炎病毒NS3解旋酶抑制剂作为化学探针，以促进基础科学和药物开发，需要开发这些分析方法。在这一努力中，我们在MLPCN启动了uHTS，使用我们最近开发的基于分子信标的解旋酶分析(MBHA)作为主要筛选。为了评估丙型肝炎病毒解旋酶抑制物的特异性，将利用丙型肝炎病毒相关病毒(DNV、WNV、YFV和JEV)编码的解旋酶和人细胞解旋酶建立MBHAs。待分析的人类蛋白都与艾滋病毒复制有关，包括死盒蛋白DDX1、DDX3、DDX24、MDA-5、RNA解旋酶A(RHA)和Werner综合征蛋白(WRN)。在目标2中，将开发分析细胞抗病毒效果的方法，以及丙型肝炎病毒解旋酶抑制剂的作用机制。机理分析包括ATPase分析、配基结合分析、蛋白质齐聚分析和同时进行的蛋白酶/解旋酶分析。这些机械分析中的大多数可以与丙型肝炎病毒解旋酶或其他十种解旋酶中的一种一起使用，以筛选其他类别的解旋酶抑制剂。 与公共健康相关：与美国国立卫生研究院的高通量筛查中心合作，我的实验室正在搜索数十万个小分子的集合，以寻找可能抑制丙型肝炎病毒编码的解旋酶的化合物。在这个项目中，我们将开发测试，以快速搜索这些丙型肝炎病毒解旋酶抑制剂，寻找可用于治疗丙型肝炎的化合物。我们还将开发类似的测试，以确定可能抑制导致艾滋病、黄热病、登革热、西尼罗河和日本脑炎的病毒的化合物。

DESCRIPTION (provided by applicant): In response to the Funding Opportunity Announcement "Development of Assays for High-Throughput Drug Screening," we propose here to develop assays to identify and analyze inhibitors of helicases needed for RNA virus replication. All viruses require helicases to separate duplex DNA or RNA, yet helicases remain undeveloped antiviral drug targets. Although some helicase inhibitors that target DNA viruses are progressing in clinical trials, no antiviral agents targeting RNA viruses have entered the clinical arena. We therefore will target RNA viruses, focusing on pathogens with abnormally high global burdens of disease: hepatitis C virus (HCV), Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and human immunodeficiency virus (HIV). The assays to be developed are needed as part of an ongoing collaborative hypothesis-driven research project to develop HCV NS3 helicase inhibitors as chemical probes to facilitate basic science and for drug development. In that endeavor, we have initiated an uHTS at the MLPCN that uses our recently developed molecular beacon based helicase assay (MBHA) as a primary screen. To evaluate HCV helicase inhibitor specificity, MBHAs will be developed using helicases encoded by viruses related to HCV (DNV, WNV, YFV, and JEV) and human cellular helicases. The human proteins to be analyzed have all been linked to HIV replication and include the DEAD-box proteins DDX1, DDX3, DDX24, MDA-5, RNA helicase A (RHA), and Werner syndrome protein (WRN). In aim 2, assays will be developed to analyze the cellular antiviral efficacy, and mechanism of action of HCV helicase inhibitors. Mechanistic assays include ATPase assays, ligand binding assays, protein oligomerization assays, and simultaneous protease/helicase assays. Most of these mechanistic assays could be used with either HCV helicase or one of the ten other helicases targeted here to screen for additional classes of helicase inhibitors. PUBLIC HEALTH RELEVANCE: In collaboration with a NIH high throughput-screening center, my lab is searching a collection of hundreds of thousands of small molecules for compounds that might inhibit the helicase encoded by the hepatitis C virus (HCV). In this project, we will develop tests to rapidly search these HCV helicase inhibitors for compounds that could be used to treat hepatitis C. We will also develop similar assays to identify compounds that might inhibit viruses that cause AIDS, yellow fever, Dengue fever, West Nile and Japanese encephalitis.