Non-A non-B hepatitis (NANBH) is a major cause of morbidity and mortality. The principal etiologic agent of NANBH is hepatitis C virus (HCV). The estimated worldwide prevalence of HCV is 0.5-1-5%. HCV can establish a lifelong asymptomatic carrier state. At least 50% of infected persons will develop chronic hepatitis; 20% of these will go on to develop cirrhosis. Chronic HCV infection can lead to development of hepatocellular carcinoma. Currently, interferon alpha (IFN-alpha) is the only drug approved for treatment of hepatitis due to HCV. While IFN-alpha treatment has been associated with improved serum liver enzyme response in 20-40% of patients, its effectiveness is variable and its cure rate is low. Clearly, additional therapeutic drugs are needed for control of hepatitis C. In this proposal, the RNA helicase activity of the HCV NS3 protein will be the focus of antiviral drug discovery and development. This activity has been identified with a purified recombinant NS3 protein, and a gel migration assay for the activity has been established. These preliminary observations will be extended to the development of a high capacity drug screening assay.There are three specific aims of this Phase I project:i) further characterization of the enzymologic features and the nucleic acid substrate requirements of the NS3 helicase;ii) development of a high capacity assay for the quantitative measurement of NS3 helicase activity; andiii) validation of this assay in a microplate format for screening purposes.Results of this Phase I work will provide the basis for a Phase II project which will involvei) utilization of this microplate assay for screening compound libraries for inhibitors;ii) establishment of an analogous assay with other viral and host cell helicases to allow rapid assessment of inhibitor selectivity and specificity;iii) development of a secondary cell-based confirmatory assay;iv) medicinal chemistry (SAR) studies to develop more potent and selective HCV helicase inhibitors; andv) evaluation of active compounds for cellular toxicity, bioavailability, and metabolic stability.Compounds meeting our criteria of potency, specificity, cellular toxicity, and bioavailability will be considered lead compounds and will be further advanced as HCV antiviral drug candidates in our Phase III follow-on development work.National Cancer Institute (NCI)
