Hepatitis C virus (HCV) is a major cause of morbidity and mortality, with an estimated global prevalence of 0.5-1.5%. Approximately 80% of infected persons develop chronic hepatitis; 20% of these go on to develop cirrhosis. Chronic HCV infection can lead to hepatocellular carcinoma and liver failure. While interferon alpha (IFN-alpha) is approved for treatment of hepatitis C, nonresponse rates are high and cure rates are low. There is a clear need for new therapeutic drugs to control hepatitis C. HCV is genetically heterogeneous; 9 distinct genotypes and numerous subtypes exist. Genotypes show differences in protein antigenicity, clinical disease manifestations, and responsiveness to IFN. In order to develop an efficacious HCV antiviral, it will be necessary for the drug to be effective against most (or all) of the major viral genotypes. It is therefore imperative that inhibitors be evaluated against multiple genotypes of HCV early in the drug discovery process. The HCV NS3 RNA helicase enzyme has been selected as a target for antiviral drug discovery. This activity was discovered with the HCV protein derived from a genotype Ia virus. Using this enzyme, inhibitors have been identified, and screening of chemical libraries for additional actives continues. Here, it is proposed that work on the HCV RNA helicase be expanded to include the enzyme from additional genotypes. Specific aims are to: (i) acquire primary materials containing HCV RNA of genotypes 1b, 2a, 2b, and 3a, and confirm their genotypes by sequence analysis; (ii) molecularly clone NS3 genes from each genotype; and (iii) construct recombinant baculoviruses that express the NS3 protein of each genotype. The results of this Phase l work will provide the basis of our SBIR Phase II proposal, which will include: (i) purifying the individual genotype NS3 proteins; (ii) characterizing the RNA helicase and polynucleotide-stimulated ATPase activities of each genotype NS3 protein; (iii) evaluating proprietary HCV RNA helicase inhibitors against the genotype NS3 helicases and cellular helicases; and (iv) synthesizing chemical analog series of promising compounds to identify inhibitors with improved potency and specificity. On completion of our SBIR Phase II work, lead inhibitor compounds with the desired specificity and spectrum of activity will be advanced as preclinical HCV antivirals in our follow-on development work.Proposed commercial application:While annual sales of IFN-alpha for hepatitis C exceed $ 2 billion, this drug alone is not sufficient to deal with this worldwide disease problem. New drugs are desperately needed. It is imperative that new drug discovery take into account the genetic heterogeneity of HCV. New drug candidates must be effective against multiple virus genotypes. It is the objective of this Phase I, and subsequent Phase II, work to evaluate inhibitors against the HCV RNA helicase derived from multiple genotypes. Our Phase I and Phase II work will directly contribute to the identification of new, broad spectrum compounds for the treatment of hepatitis C.National Institute of Allergy and Infectious Diseases (NIAID)
