Discovery and Characterization of Substituted Diphenyl Heterocyclic Compounds as Potent and Selective Inhibitors of Hepatitis C Virus Replication

TLDR: It is found that R803 was more effective than alpha interferon (IFN-α) at blocking HCV RNA replication in the replicon model, and could potentially be developed as a treatment for HCV infection.

Abstract: Hepatitis C virus (HCV) infection is one of the major causes of viral hepatitis, with a great propensity to induce chronicity (21). Liver inflammation can persist for decades in chronic HCV infection and eventually leads to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. HCV infection is a significant health care problem: it is estimated that approximately 170 million individuals are chronically infected with HCV worldwide, with ∼30,000 cases of new infection each year in the United States alone (1, 2, 46). No vaccine is currently available to prevent HCV infection. The standard treatment for HCV infection, a combination of pegylated alpha interferon (IFN-α) and ribavirin (RBV), is limited by its suboptimal response rate in a significant patient population, side effects, and affordability (11). Thus, it is critical to discover highly effective, safer therapies to improve the clinical management of HCV infection. HCV is an enveloped RNA virus belonging to the family Flaviviridae (9). HCV clinical isolates display high heterogeneity in their genomic RNA and amino acid sequences, and they are classified into six genotypes and numerous subtypes (49). It is documented that infections by different genotypes may produce different clinical outcomes and may respond differently to IFN-α-based antiviral treatment (for a review, see reference 11). Significantly, patients infected with genotype 1 viruses, which account for approximately 70% of HCV infections in the United States, exhibit poor rates of response to the IFN-α-based treatment. An ideal antiviral should, therefore, be effective against the majority, if not all, of the HCV genotypes. Upon entering the host cell, HCV releases its 9.6-kb genomic RNA into the cytoplasm, where it directs the translation of a single polyprotein of about 3,000 amino acids. The giant polyprotein is cotranslationally processed by host and viral proteases into structural proteins (core, E1, and E2) and nonstructural proteins (P7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b). The mature nonstructural proteins (except P7 and NS2) and host factors assemble into membrane-associated RNA replication complexes, where a vast quantity of progeny viral RNA molecules are amplified from the incoming HCV genomic RNA (14, 18, 35). Although all the steps in the HCV life cycle can be targeted for drug discovery against HCV, the viral nonstructural proteins, specifically NS3 and NS5b, which encode well-defined enzymatic activities crucial for viral replication, are the major targets for antiviral discovery (10, 53). However, the replication of HCV viral RNA by the viral replication complex is quickly becoming another focus for drug discovery with the development of the HCV replicon system. Until the establishment of HCV replicons, the analysis of HCV replication was hampered due to the lack of a robust HCV cell culture system (5, 38). The first-generation HCV replicons are human hepatoma Huh-7 cell lines carrying engineered genotype 1b subgenomic RNA with the following genome organization: HCV 5′ nontranslated region (5′ NTR)-neomycin phosphotransferase (NPT) gene (also referred to as the neomycin resistance [Neor] gene)-encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-HCV NS3-4a-4b-5a-5b-HCV 3′ NTR. Subsequent studies have shown that the efficiency of replicon establishment can be enhanced substantially by incorporating cell culture-adaptive mutations, especially those in NS3 and NS5a (5, 26, 37, 38). The HCV replicon system has been an effective tool for studying viral RNA replication and virus-host interactions. It also serves as an important cell-based system with which to evaluate antiviral drugs and to reveal drug resistance mechanisms (for a review, see reference 4). Moreover, the HCV replicon presents a unique drug-screening system, allowing for the screening of compounds inhibiting the viral enzymes as well as other targets of the HCV RNA replication process in a cellular environment. Such screens would perhaps facilitate the discovery of inhibitors that block the functions of NS4b and NS5a or interrupt virus-host interactions, discoveries that cannot be readily achieved with biochemical screens. Several efforts have already been made to screen small-molecule compound libraries against different versions of the HCV replicon system (17, 47, 50, 55). Here we describe the development of an HCV replicon assay for high-throughput screening and the characterization of one of the heterocyclic hits from screens of a 230,000-member chemical library. We show that the lead compound of this hit scaffold is effective at inhibiting HCV replicons of different genotypes and can enhance the inhibitory activity of IFN-α in the replicon model.