KSEF R&D Excellence: Devlopment of High-Throughput Screens for Discovery of Specific Inhibitors against Hepatitis C Virus

Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B (NANB) viral hepatitis, infecting approximately 4 million people in the U.S. and 170 million people worldwide. The majority (75-85%) of individuals with HCV infection develop chronic hepatitis, which leads to cirrhosis (10-20%) and hepatocellular carcinoma (1-5%). HCV associated endstage liver disease is the leading indication for liver transplantation in the U.S. Each year, HCV infection results in 8,000-10,000 deaths in the US alone, which are expected to triple within the next 10-20 years. Currently, there is no specific and effective therapy to treat HCV infection, nor is an HCV vaccine conceivable in the foreseeable future. Recently, we have expressed and purified the HCV protease/helicase (NS3) and RNA dependent RNA polymerase (NS5B). We have also developed a novel system to study HCV RNA replication in the cell. The overall goal of this research proposal is to develop highthroughput screens for discovery of novel anti-HCV agents. Our specific aims are: 1) to develop high-throughput screens for inhibitors of the viral RNA polymerase and helicase in vitro using purified recombinant NS3 and NS5B proteins coupled with scintillation proximity assay (SPA) technology, and 2) to genetically engineer novel subgenomic HCV replicons containing biological reporters and to develop high-throughput screens for identification of specific and potent compounds inhibiting HCV RNA replication in the cell. These high-throughput screens will be used to identify compounds active against HCV replication. Knowledge obtained and technologies developed in these studies will facilitate anti-HCV drug discovery and lead to identification of specific anti-HCV agents. In addition, advancement in knowledge and technologies from these studies will place us in a strong position to compete for additional federal and private-sector funding. Moreover, this application will likely result in intellectual property that will contribute to the economic development of the Commonwealth.