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，全球约有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化干扰素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，开发新型和特异性治疗方法的进展受到很大阻碍。我们开发了基于抑制HCV核心二聚化的高通量筛选方法，HCV核心是病毒衣壳蛋白，对病毒颗粒的组装至关重要。这些检测方法现已被制药行业所采用，使我们能够识别新的HCV生产抑制剂。基于我们的实验验证的筛选结果被用作精细的分子探针来研究core与HCV NS3解旋酶的相互作用。然而，它对与NS5A的相互作用没有影响，我们在实验室也对其进行了生化表征。因此，我们开发了一种新的、基于珠子的能量转移试验来鉴定核心- ns5a相互作用的抑制剂。由此产生的化合物将构成新的分子探针，用于了解丙型肝炎病毒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联合的药物的基础，这应该比目前的单靶点或非特异性药物更广泛地适用。

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.