Mechanism of Action of Hepatitis C Virus NS5A Inhibitors

 描述(申请人提供)：慢性丙型肝炎病毒(丙型肝炎病毒)感染是美国肝硬变、终末期肝病和肝癌的主要原因。目前还没有疫苗，直到2011年，护理的标准是聚乙二醇化干扰素(PEG化干扰素)和利巴韦林(RBV)的双重抗病毒治疗。聚乙二醇化干扰素/RBV治疗耐受性差，仅在50%感染(GT)1a丙型肝炎的患者中导致持续病毒学应答(SVR)，这是在美国最常见的丙型肝炎。为了解决这一问题，人们进行了密集的努力，以确定针对丙型肝炎病毒的小分子抑制剂。作为这项研究的结果，许多直接作用的抗病毒药物(DAA)已经被确定，其中几种正在临床开发中。在过去的三年里，已经批准了5种新的DAA用于治疗慢性丙型肝炎，其中包括专门针对丙型肝炎病毒蛋白酶和聚合酶设计的小分子。这些药物可提高SVR率，但必须与其他DAA或与聚乙二醇-干扰素/RBV联合使用，以避免出现耐药问题。目前正在临床开发的丙型肝炎病毒蛋白水解酶和聚合酶抑制剂是基于对结构-活性关系的详细了解而设计的。临床开发中的另一类抗病毒药物是NS5A抑制剂。与蛋白酶和聚合酶抑制剂不同，NS5A抑制剂是通过基于细胞的筛选试验来鉴定具有抗丙型肝炎病毒活性的化合物的。根据对抗性变异体的序列分析，NS5A随后被确定为这类化合物的靶标。临床开发中的NS5A抑制剂将成为未来用于慢性丙型肝炎治疗的无干扰素DAA组合的关键组成部分。事实上，第一个无干扰素的、全口服治疗慢性丙型肝炎的药物已于2014年10月获得批准，其中包括一种聚合酶抑制剂和一种NS5A抑制剂的组合。丙型肝炎病毒NS5A蛋白是丙型肝炎病毒及相关病毒所特有的，它没有酶活性，结构信息有限。实验数据表明，它在丙型肝炎病毒生命周期的多个方面发挥作用。然而，NS5A的特性仍然很差，NS5A抑制剂如何影响其正常功能也不是很清楚。在拟议的研究中，我将使用细胞培养的传染性丙型肝炎病毒GT 1a株H77S.3来研究NS5A抑制剂的作用机制。具体地说，我将确定NS5A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，将使用定量蛋白质组学方法来确定NS5A抑制剂如何影响NS5A与细胞和病毒蛋白的相互作用，以阻止病毒组装。我将研究抑制剂如何影响亚细胞定位。 在高空间分辨率下，NS5A对于相互作用伙伴和细胞内膜结构的影响。这些研究将揭示这种有效的新型抗病毒药物作用的机制细节。

 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.