Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection

Abstract

Over 170 million people are chronically infected by the hepatitis C virus (HCV) and at risk for dying from liver cirrhosis and hepatocellular carcinoma. Current therapy is expensive, associated with significant side effects, and often ineffective. Discovery of antiviral compounds against HCV traditionally involves a priori target identification followed by biochemical screening and confirmation in cell-based replicon assays. Typically, this results in the discovery of compounds that address a few predetermined targets and are prone to select for escape variants. To attempt to identify antiviral compounds with broad target specificity, we developed an unbiased cell-based screening system involving multiple rounds of infection in a 96-well format. Analysis of a publicly available library of 446 clinically approved drugs identified 33 compounds that targeted both known and previously unexplored aspects of HCV infection, including entry, replication, and assembly. Discovery of novel viral and cellular targets in this manner will broaden the therapeutic armamentarium against this virus, allowing for the development of drug mixtures that should reduce the likelihood of mutational escape.cell-based assay | antivirals | entry | replication | assembly H epatitis C virus (HCV) (Flaviviridae) is an enveloped, positivestranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (1). HCV establishes persistent infection, and more than 170 million people are chronically infected worldwide (2). Chronic infection is associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma (3). Although the mechanisms by which HCV causes liver disease are not entirely understood, immunologically mediated events play an important role in HCV clearance and pathogenesis (4).The HCV plus-stranded RNA genome (9.6 kb) encodes a single polyprotein that is cleaved into structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (5). Infection is initiated by virus-particle binding to cellular receptors, internalization through receptor-mediated endocytosis, and delivery of the viral genome to the cytosol after endosomal acidification (6). Delivery and/or translation of incoming viral genomes depends on cellular autophagy-related factors (7), enabling viral gene expression, replication (8), and production of progeny virus, which depends on VLDL biosynthesis (9-11).Currently, there is no vaccine against HCV, and the standard therapy (pegylated IFN-alfa plus ribavirin) is associated with significant side effects and is only effective in a fraction of the patients (12). Establishment of HCV replicons (13, 14) greatly contributed to the discovery of antiviral compounds that target the viral NS3-4A serine protease and NS5B RNA polymerase (15). Although they are extremely potent, these agents select for resistant variants because of the error-prone RNA polymerase activity of HCV (16).The development of HCV infection models (17-19) that reproduce the entire life cycle of HCV in...

Hepatitis C virus selectively perturbs the distal cholesterol synthesis pathway in a genotype-specific manner

“…17 Pharmacological in vitro research suggests the distal sterol pathway is important for the HCV lifecycle and raises the potential for more selective inhibition of HCV replication by targeting this with small molecules and sterol antagonists. 36,37 Our data would support this. Further targeted, in vitro studies are indicated to better identify the specific point(s) and nature of HCV interference in late cholesterol biosynthesis.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Reporter viruses make such assays adaptable to high-throughput screening, with obvious advantages for antiviral research [76,126,[129][130][131]. Figure 4 illustrates a possible assay setup based on a dual reporter system, typically the Gaussia/Renilla and firefly luciferases.…”

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

“…Moreover, chlorcyclizine inhibits HCVcc but not HCVpp (6). In contrast, some phenothiazines, like fluphenazine, trifluoperazine, and prochlorperazine, inhibit both particle types (4,5,21). These observations show that there may be subtle differences in the ways that these structurally related drugs inhibit HCV.…”

“…Since HCV infects cells by using the endocytic pathway (10), it is reasonable to assume that chlorpromazine and possibly also related compounds inhibit HCV and other viruses by disrupting this process (4,10). While this may be the case for chlorpromazine and its antiviral activity toward HCV and other viruses, this is, however, unlikely to be the primary antiviral mechanism for related phenothiazines, like fluphenazine and trifluoperazine, and for flunarizine, a structurally similar diphenylpiperazine (7).…”

“…They influence the function of muscarinic, ␣-adrenergic, and serotonergic receptors and can modulate cardiac channels, resulting in a relatively broad spectrum of side effects, including sedation, hyperactivity, insomnia, and convulsions (24). Among antihistamines with anti-HCV activity are both first-generation (chlorcyclizine, cyclizine, hydroxyzine, cyproheptadine, ketotifen, mequitazine, and trimeprazine) and second-generation (loratadine and desloratadine) antihistamines (4,6,24), which indicates that both drug classes include molecules that can target HCV.…”