Novel Assay for Small Molecule Inhibitors of core-NS5A Interaction

描述（由申请人提供）：丙型肝炎病毒（HCV）是肝脏疾病的主要原因，全世界约有1.3亿人感染。到目前为止还没有开发出疫苗，目前唯一可用的治疗方法是聚乙二醇干扰素α和利巴韦林的组合。由于缺乏新的靶蛋白和合适的感染性HCV培养系统，开发新的特异性治疗方法的进展受到很大阻碍。我们已经开发了基于抑制HCV核心（病毒衣壳蛋白，其对于病毒颗粒的组装是必不可少的）的二聚化的高通量筛选测定。这些检测方法现在被制药业采用，使我们能够识别HCV生产的新型抑制剂。基于我们的检测的筛选的验证命中被用作精细的分子探针来研究核心与HCV的NS3解旋酶的相互作用。然而，它对与NS5A的相互作用没有影响，我们也在实验室中对其进行了生物化学表征。因此，我们开发了一种新的，基于珠的能量转移试验，以确定核心NS5A相互作用的抑制剂。所得化合物将构成新的分子探针，用于了解HCV非结构蛋白如何参与病毒颗粒的组装。这些抑制剂也可以作为与抗病毒治疗相结合的药物的基础，这应该比目前的单一靶点或非特异性药理学药物更广泛地适用。 公共卫生相关性：丙型肝炎影响全球1.3亿人，其中300万人生活在美国，唯一的治疗方法是干扰素和利巴韦林的组合，对不到一半的患者有效。我们将开发一种基于能量转移的特异性检测方法，用于筛选病毒的有效分子探针，该方法基于抑制核心（病毒颗粒组装所必需的衣壳蛋白）和NS5A（病毒复制所必需的HCV蛋白）之间的相互作用。这些化合物可能导致开发用于治疗丙型肝炎的口服药物。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a major cause of liver disease, with about 130 million people infected worldwide. No vaccine has been developed so far, and the only treatment available today is a combination of pegylated interferon alpha and ribavirin. Progress in developing novel and specific treatments has been greatly hampered by the dearth of novel target proteins and suitable infectious HCV culture systems. We have developed high-throughput screening assays based on the inhibition of dimerization of HCV core, the viral capsid protein, which is essential for the assembly of the viral particle. These assays, now adopted by the pharmaceutical industry, have allowed us to identify novel inhibitors of HCV production. A validated hit of a screen based on our assays was used as an exquisite molecular probe to study the interaction of core with the NS3 helicase of HCV. It had however no effect on the interaction with NS5A, which we also characterized biochemically in our laboratory. We have therefore developed a new, bead-based transfer of energy assay to identify inhibitors of the core-NS5A interaction. The resulting compounds will constitute novel molecular probes for understanding how HCV non structural proteins participate in the assembly of the viral particle. These inhibitors may also serve as the basis of drugs to be combined with anti-viral treatments that should be more widely applicable than current single target or non-specific pharmacologic agents. PUBLIC HEALTH RELEVANCE: Hepatitis C affects 130 million individuals worldwide, 3 million of which live in the US, and the only treatment, a combination of interferon and ribavirin, is effective for less than half of the patients. We will develop a specific transfer-of energy-based assay screening assay for potent molecular probes of the virus, based on inhibition of interaction between core, the capsid protein essential for assembly of the viral particle, and NS5A, a HCV protein essential for viral replication. These compounds may lead to the development of orally available drugs for treatment of Hepatitis C.