Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

Abstract

bTherapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC 50 s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC 50 s of <10 M were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC 50 s (compound concentrations at 50% cytotoxicity) of <30 M from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.

Selective Inhibition of Hepatitis C Virus Infection by Hydroxyzine and Benztropine

“…We confirmed that this class of inhibitors interferes with viral entry of different HCV genotypes (Fig. 5) using as a representative member of the group perphenazine, which has also been recently identified as an HCV inhibitor by others (34). We have also reported previously that dibenzazepines inhibit HCV entry (12).…”

Clinically Approved Ion Channel Inhibitors Close Gates for Hepatitis C Virus and Open Doors for Drug Repurposing in Infectious Viral Diseases

“…Alternatively, there may be subtle strain-specific differences regarding the membrane requirements to trigger fusion, and the resistance mutations observed may change these requirements. Either way, it is enigmatic that HCV pseudoparticles (HCVpp), i.e., retroviral particles carrying the HCV envelope proteins E1 and E2 at their surfaces (50,51), in contrast to authentic cell culture-derived HCV particles (HCVcc) with exactly the same glycoproteins, are not inhibited by flunarizine (5,7,52). Moreover, chlorcyclizine inhibits HCVcc but not HCVpp (6).…”

Border Control in Hepatitis C Virus Infection: Inhibiting Viral Entry

“…31 This technological advance led to the large-scale screening of compound libraries and the identification of potential therapeutics. Our group and others recently published reports identifying antihistamine chlorcyclizine HCl (CCZ) as a potent inhibitor of HCV replication.…”