DEVELOPMENT OF A SCREENING PROGRAM TO EVALUATE THE EFFICACY, RANGE, AND MECHANISM OF ACTION OF POTENTIAL ANTI-HCV COMPOUNDS

Abstract

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and liver cancer. In this project a screening program has been developed to evaluate potential anti-HCV compounds in vitro, while meeting the Food and Drug Administration's (FDA) recommendations for in vitro pre-clinical drug evaluations. In this project an algorithm was created to test a panel of compounds for their efficacy against the HCV surrogate model bovine viral diarrhea virus (BVDV), and to further attempt to determine the mechanism of action of those showing antiviral activity by utilizing both cell based and molecular target assays. The algorithm consists of cell-based assays to evaluate antiviral efficacy and cytotoxicity, combination drug studies, multiplicity of infection (MOI) effects and time of removal plaque reduction and RNA accumulation assays. A novel molecular target assay has been developed to evaluate compounds for their inhibition of the HCV NS2/3 protease and an assay designed to evaluate compounds for their activity against the HCV internal ribosomal entry site (IRES) mechanism has been optimized and validated for use in this screening program. Three compounds: ribavirin, interferon alpha (1NF-α) and a proprietary compound (Compound "A") have been evaluated in the system. All three compounds have shown antiviral activity in the efficacy assays. The combinations of ribavirin with interferon alpha and Compound "A" with interferon alpha were shown to have synergistic antiviral activity. The combination of ribavirin and compound "A" has shown a reduction in the cytoxicity associated with ribavirin alone. None of the compounds appeared to be entry inhibitors, and none of them showed inhibition of NS2/3 protease. Compound "A" was shown to inhibit HCV IRES driven luciferase expression, showing it is potentially inhibiting IRES function. Neither ribavirin or interferon reduced IRES driven luciferase expression in the same assay. Both the RNA accumulation assay and the time of removal plaque reduction assays have suggested that the three compounds target later phases in the viral replication cycle, however, results have suggested that compound "A" inhibits at an earlier phase than ribavirin and INF-α. Upon completion of the assays in this screening program, results have suggested that the mechanism of action of the three compounds is a target occurring in the later stages of virus replication. The assays and methodologies described in this thesis have yielded results suggesting that the screening algorithm described has the potential to be very useful in evaluating potential anti-HCV compounds in vitro.