SBBZ Kentucky Cancer Program Demonstration Project

Hepatitis C virus (HCV) infection remains a major area of unmet medical needs. HCV chronically infects approximately 170 million people worldwide, including 3-4 millions of Americans. HCV infection results in 10,000-12,000 annual deaths in the United States alone and the HCV-associated end-stage liver disease is the leading indicator of liver transplantation. The current standard antiviral therapy with pegylated interferon-a and ribavirin is suboptimal for the dominant genotype 1 HCV with less than 50% antiviral efficacy and causes numerous severe side effects. The HCV protease- and polymerase-specific inhibitors currently in clinical trials are showing promising results but are undermined by rapid emergence of drug-resistant mutations due to the error-prone replication of HCV. Future antiviral therapy for hepatitis C likely requires a combination of three or more antiviral drugs targeting different steps of the HCV life cycle. Therefore, there is an urgent need to discover and develop novel drugs inhibiting additional viral targets in order to effectively treat hepatitis C. This R21 application is in response to the program announcement PAR-08-024, Assay Development for High Throughput Molecular Screening. We have successfully developed a groundbreaking cell culture system for robust production of infectious HCV. We have also isolated an infectious HCV variant, which grows to high titer (>107 foci-forming units per milliliter) and causes cytopathic effect (CPE) in a human hepatoma cell line, Huh-7.5. In Specific Aim 1, we will develop a cell protection assay for high-throughput screening of HCV inhibitors. Cells protected by test compounds from HCV infection and replication will be quantified by measuring cell viability. This cell protection assay can serve as a high-throughput screen (96-well format) for both antiviral activity and cytotoxicity of test compounds. Additionally, we have genetically engineered an infectious HCV that expresses a renilla luciferase. In Specific Aim 2, we will use the luciferaseexpressing HCV to develop a high-throughput screen for the identification of specific HCV inhibitors targeting any step of the HCV life cycle. The luciferase activity will be determined in a multiplexed format of 384-well together with the measurement of cytotoxicity by a cell viability assay. In Specific Aim 3, we will develop counter-screens for the determination of efficacy, specificity, cytotoxicity, and molecular targets of compound hits that are identified by high-throughput screening. These assays will facilitate the discovery of novel antiviral drugs against additional viral targets such as HCV infection, replication, assembly, and release as well as cellular factors essential for the completion of the HCV infectious cycle. Project Description