Mechanism of Action of Hepatitis C Virus NS5A Inhibitors

丙型肝炎病毒 NS5A 抑制剂的作用机制

• 批准号: 9100620
• 负责人: David R McGivern
• 金额: $ 19万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9100620
• 关键词: Address; Affect; Antiviral Agents; Antiviral Therapy; Biochemical; Biological Assay; Cell Culture Techniques; Cells; Chronic Hepatitis C; Cirrhosis; Clinic; Clinical; Clinical Trials; Complex; Data; Development; Drug Targeting; Drug resistance; Electron Microscopy; Environment; Enzymes; FDA approved; Fluorescence Microscopy; Future; Genetic; Genotype; Goals; Half-Life; Health; Hepatitis C Therapy; Hepatitis C virus; Hepatocyte; Imaging Techniques; Interferons; Intracellular Membranes; Kinetics; Knowledge; Life Cycle Stages; Liver diseases; Malignant neoplasm of liver; Membrane; Molecular; Oral; Orthologous Gene; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacotherapy; Polymerase; Protease Inhibitor; Proteins; Proteomics; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Replicon; Research; Resistance; Resolution; Ribavirin; Sequence Analysis; Staging; Structure; Structure-Activity Relationship; Vaccines; Variant; Viral; Viral Proteins; Virus; Virus Assembly; Virus Inhibitors; Virus Replication; Work; anti-hepatitis C; base; burden of illness; design; improved; inhibitor/antagonist; next generation; novel; novel therapeutics; replicase; response; screening; small molecule; small molecule inhibitor; standard of care; viral RNA

 DESCRIPTION (provided by applicant): Chronic Hepatitis C virus (HCV) infection is a leading cause of cirrhosis, end-stage liver disease and liver cancer in the USA. There is no vaccine and until 2011, the standard of care was dual antiviral therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). PEG-IFN/RBV therapy is poorly tolerated and only leads to sustained virological response (SVR) in 50% of patients infected with genotype (gt) 1a HCV, the most commonly found gt in the USA. To address this problem, intensive efforts were made to identify small molecule inhibitors targeting HCV. As a result of this research, many direct-acting antivirals (DAA) have been identified and several are in clinical development. The past three years have seen the approval of 5 new DAAs for the treatment of chronic hepatitis C including small molecules specifically designed to target the HCV protease and polymerase enzymes. These drugs improve SVR rates but must be administered in combination with other DAAs or with PEG-IFN/RBV to avoid problems of drug resistance emergence. Inhibitors of the HCV protease and polymerase enzymes that are currently in clinical development were designed based on a detailed knowledge of structure-activity relationships. Another class of antivirals in clinical development is the NS5A inhibitors. Unlike protease and polymerase inhibitors, NS5A inhibitors were identified by cell-based screening assays for compounds with anti-HCV activity. NS5A was subsequently identified as the target for this class of compounds based on sequence analysis of resistant variants. NS5A inhibitors in clinical development will form a key component of future interferon-free DAA combinations for chronic hepatitis C therapy. Indeed the first interferon-free, all oral therapy for chronic hepatitis C was approved in October 2014 and comprises a polymerase inhibitor in combination with an NS5A inhibitor. The HCV NS5A protein is unique to HCV and related viruses, it has no enzymatic activity and limited structural information is available. Experimental data suggest it acts in multiple aspects of the HCV lifecycle. However NS5A remains poorly characterized and how its normal functions are affected by NS5A inhibitors is not well understood. In the proposed studies, I will study the mechanism of action of NS5A inhibitors using H77S.3, a cell culture infectious gt 1a strain of HCV. Specifically, I will determine how NS5A inhibitors affect virus RNA synthesis, virus assembly and egress. At the molecular level, quantitative proteomic approaches will be employed to determine how NS5A inhibitors affect NS5A interactions with cellular and viral proteins to block virus assembly. I will examine how inhibitors affect the subcellular localization of NS5A with respect to interaction partners and also intracellular membrane structures at high spatial resolution. These studies will reveal mechanistic details underlying the action of this potent new class of antiviral.

 描述（由申请人提供）：慢性丙型肝炎病毒（HCV）感染是美国肝硬化、终末期肝病和肝癌的主要原因。没有疫苗，直到2011年，护理标准是使用聚乙二醇干扰素（PEG-IFN）和利巴韦林（RBV）的双重抗病毒治疗。PEG-IFN/RBV治疗耐受性差，仅在50%感染基因型（gt）1a HCV（美国最常见的gt）的患者中导致持续病毒学应答（SVR）。为了解决这个问题，人们进行了大量的努力来鉴定靶向HCV的小分子抑制剂。作为这项研究的结果，许多直接作用的抗病毒药物（DAA）已被确定，其中一些正在临床开发中。在过去的三年中，已经批准了5种新的DAA用于治疗慢性丙型肝炎，包括专门针对HCV蛋白酶和聚合酶的小分子。这些药物提高了SVR率，但必须与其他DAA或PEG-IFN/RBV联合给药，以避免出现耐药性问题。目前在临床开发中的HCV蛋白酶和聚合酶的抑制剂是基于结构-活性关系的详细知识设计的。临床开发中的另一类抗病毒药物是NS 5A抑制剂。与蛋白酶和聚合酶抑制剂不同，NS 5A抑制剂是通过基于细胞的筛选测定来鉴定具有抗HCV活性的化合物。NS 5A随后基于抗性变体的序列分析被鉴定为这类化合物的靶标。临床开发中的NS 5A抑制剂将成为未来无干扰素DAA组合治疗慢性丙型肝炎的关键组成部分。事实上，第一种不含干扰素的全口服治疗慢性丙型肝炎的药物于2014年10月获得批准，包括聚合酶抑制剂与NS 5A抑制剂的组合。HCV NS 5A蛋白是HCV和相关病毒所特有的，它没有酶活性，并且可获得的结构信息有限。实验数据表明，它在HCV生命周期的多个方面发挥作用。然而，NS 5A仍然缺乏表征，并且其正常功能如何受到NS 5A抑制剂的影响尚不清楚。在拟议的研究中，我将研究NS 5A抑制剂的作用机制，使用H77S.3，一种细胞培养感染性HCV gt 1a株。具体来说，我将确定NS 5A抑制剂如何影响病毒RNA合成，病毒组装和出口。在分子水平上，将采用定量蛋白质组学方法来确定NS 5A抑制剂如何影响NS 5A与细胞和病毒蛋白的相互作用以阻断病毒组装。我将研究抑制剂如何影响亚细胞定位 的NS 5A相对于相互作用的合作伙伴，也在高空间分辨率的细胞内膜结构。这些研究将揭示这种新型强效抗病毒药物作用的机制细节。