Identification of HCV E1 an E2 Inhibitors

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)被认为是一个世界性的健康问题，影响着超过1.7亿人。丙型肝炎病毒导致一系列疾病，从无症状的携带者状态到终末期肝病；其中包括肝硬变和肝细胞癌。目前还没有丙型肝炎病毒疫苗。目前治疗丙型肝炎病毒感染的方法通常不会导致病毒清除，难以管理，并导致严重的毒副作用。开发治疗丙型肝炎病毒感染的新药物是一项重大的公共卫生优先事项。由于在细胞培养中复制效率低，对丙型肝炎病毒的研究具有挑战性。丙型肝炎病毒复制子系统是基于细胞培养中工程微型基因组的自我复制，已被证明对研究丙型肝炎病毒基因组复制、翻译和多蛋白加工具有非常重要的价值。利用复制子系统，丙型肝炎病毒编码的丝氨酸蛋白酶(NS3)和RNA聚合酶(NS5B)已成为最受欢迎的药物靶点。然而，该系统不支持丙型肝炎病毒颗粒的产生，也不允许研究病毒进入，这是丙型肝炎病毒生命周期中的第一个事件。我们的战略是开发抗丙型肝炎病毒的药物，以阻断病毒复制周期的新的非酶阶段，特别是病毒宿主细胞进入机制。病毒与靶细胞的选择性结合是由病毒糖蛋白和特定的细胞表面受体(S)之间的相互作用决定的，在启动感染中是必不可少的。丙型肝炎病毒编码两种包膜糖蛋白，E1和E2，它们聚集在内质网中，内质网是丙型肝炎病毒组装和萌发的建议地点。在缺乏合适的天然丙型肝炎病毒模型的情况下，伪型病毒已发展成为研究病毒附着/进入的替代模型。这些模型系统允许研究病毒进入细胞，但不复制病毒生命周期的其他方面。多年来，在(I)包膜糖蛋白的特性和(Ii)了解E1和E2糖蛋白的胞外区在识别宿主细胞表面分子中的功能作用方面取得了重大进展。此外，丙型肝炎病毒有很高的变异率，出现耐药病毒的可能性很大。因此，我们认为抑制丙型肝炎病毒进入是一种很有前途的方法，它将补充其他机制方法。我们的目标是发现和开发用于治疗丙型肝炎病毒感染的丙型肝炎病毒进入抑制剂。在第一阶段，我们将利用含有荧光素酶报告基因的HIV来源的丙型肝炎病毒假型病毒开发一个原型高通量检测方法来测量病毒感染。本实验将成为筛选结构不同的小分子文库的有价值的工具，以确定有效的丙型肝炎病毒进入抑制物。我们将在继发性丙型肝炎病毒假型感染空斑分析中确认其抗病毒活性。将评估先导化合物在所有基因类型中的活性。在第二阶段，我们将通过合理的药物设计程序开发最活跃的支架，并将在动物模型中测试先导化合物的有效性和毒性。最有希望的化合物将进入两个物种的IND毒理学和药理学研究(第三阶段)。

DESCRIPTION (provided by applicant): Hepatitis C Virus (HCV) is recognized as a worldwide health problem affecting over 170 million people. HCV causes a spectrum of disease ranging from an asymptomatic carrier state to end-stage liver disease; which includes cirrhosis and hepatocellular carcinoma. A vaccine for HCV is not available. The present therapies for HCV infection do not often result in viral clearance, are difficult to administer and result in serious toxic side effects. The development of new therapeutic agents for HCV infection is a major public health priority. Studies on HCV are challenging due to its inefficient replication in cell culture. The HCV replicon system, based on the self-replication of engineered mini- genomes in cell culture, has proven invaluable for studying HCV genomic replication, translation and polyprotein processing. Utilizing the replicon system, the HCV-encoded serine protease (NS3) and RNA polymerase (NS5B) have emerged as favorite pharmaceutical targets. However, this system does not support the production of HCV particles or allow for the study of viral entry, the first event in the HCV lifecycle. Our strategy is to develop anti-HCV agents that will block novel non-enzymatic stages of the viral replicative cycle, specifically the viral host cell entry mechanisms. The selective association of a virus with a target cell is determined by an interaction between viral glycoproteins and specific cell-surface receptor(s) and is essential in the initiation of infection. HCV encodes two envelope glycoproteins, E1 and E2, which accumulate in the endoplasmic reticulum, the proposed site for HCV assembly and budding. In the absence of a suitable native HCV model, pseudotype viruses have been developed as surrogate models to study virus attachment/entry. These model systems allow the study of viral entry into the cell but do not replicate other aspects of the viral life cycle. Significant advancements have been made over the years in (i) the characterization of envelope glycoproteins and (ii) understanding the functional role for the ectodomains of E1 and E2 glycoproteins in the recognition of host cell surface molecules. Furthermore, HCV has a high mutation rate and the emergence of drug- resistant virus is highly likely. Therefore, we believe that inhibition of HCV entry is a promising approach, which will complement other mechanistic approaches. Our objective is to discover and develop HCV entry inhibitors for the treatment of HCV infection. In Phase I we will develop a prototype high-throughput assay utilizing a HIV derived HCV pseudotype virus, containing a luciferase reporter gene, to measure virus infection. This assay will become a valuable tool for screening libraries of structurally diverse small molecules, in order to identify potent inhibitors of HCV entry. We will confirm antiviral activity in secondary HCV pseudotype infection plaque assays. Lead compounds will be evaluated for activity across all genotypes. In Phase II, we will progress the most active scaffolds through a rational drug design program and lead compounds will be tested for efficacy and toxicity in animal models. The most promising compound will advance to IND-enabling toxicology and pharmacology studies in two species (Phase III).