Antiviral potential of helicase inhibitors

描述（由申请人提供）：为响应资助机会公告“开发高通量药物筛选检测方法”，我们在此提议开发检测方法，以识别和分析RNA病毒复制所需的解旋酶抑制剂。所有病毒都需要解旋酶来分离双链DNA或RNA，但解旋酶仍然是尚未开发的抗病毒药物靶点。虽然一些针对DNA病毒的解旋酶抑制剂正在临床试验中取得进展，但针对RNA病毒的抗病毒药物尚未进入临床领域。因此，我们将以RNA病毒为目标，重点关注全球疾病负担异常高的病原体：丙型肝炎病毒（HCV）、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，需要开发这些检测方法，以开发HCV 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。为此，我们在MLPCN启动了一项uHTS，使用我们最近开发的基于分子信标的解旋酶测定（MBHA）作为主要筛选。为了评估HCV解旋酶抑制剂的特异性，MBHAs将利用与HCV相关的病毒（DNV、WNV、YFV和JEV）和人类细胞解旋酶编码的解旋酶来开发。待分析的人类蛋白都与HIV复制有关，包括DEAD-box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，将开发检测方法来分析HCV解旋酶抑制剂的细胞抗病毒功效和作用机制。机制分析包括atp酶分析、配体结合分析、蛋白质寡聚化分析和蛋白酶/解旋酶同步分析。大多数这些机制试验可用于HCV解旋酶或本文所针对的十种其他解旋酶中的一种，以筛选其他类型的解旋酶抑制剂。

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.