Identification of Hepatitis C Virus Inhibitors Targeting Different Aspects of Infection Using a Cell-Based Assay

Abstract

With 2 to 3% of the worldwide population chronically infected, hepatitis C virus (HCV) infection continues to be a major health care burden. Unfortunately, current interferon-based treatment options are not effective in all patients and are associated with significant side effects. Consequently, there is an ongoing need to identify and develop new anti-HCV therapies. Toward this goal, we previously developed a cell-based HCV infection assay for antiviral compound screening based on a low-multiplicity-of-infection approach that uniquely allows for the identification of antiviral compounds that target cell culture-derived HCV (HCVcc) at any step of the viral infection cycle. Using this assay, here we report the screening of the NCI Diversity Set II library, containing 1,974 synthesized chemical compounds, and the identification of compounds with specific anti-HCV activity. In combination with toxicity counterscreening, we identified 30 hits from the compound library, 13 of which showed reproducible and dose-dependent inhibition of HCV with mean therapeutic indices (50% cytotoxic concentration [CC 50 ]/50% effective concentration [EC 50 ]) of greater than 6. Using HCV pseudotype and replicon systems of multiple HCV genotypes, as well as infectious HCVcc-based assembly and secretion analysis, we determined that different compounds within this group of candidate inhibitors target different steps of viral infection. The compounds identified not only will serve as biological probes to study and further dissect the biology of viral infection but also should facilitate the development of new anti-HCV therapeutic treatments. Hepatitis C virus (HCV) is a hepatotropic enveloped positivestrand RNA virus (family Flaviviridae) that infects the parenchymal cells of the liver (i.e., hepatocytes) (69, 70). HCV infection begins with interaction of the viral particle with the target cell via a relatively undefined multistep process of binding between the virion and multiple cell surface receptors, which include the cluster of differentiation 81 (CD81) tetraspanin protein (7,34,56,80,84), the scavenger receptor class B member I (SR-BI) (also known as SCARB1) (7,30,38,63,83), and the tight-junction proteins claudin-1 (CLDN1) (20) and occludin (OCLN) (8,47,57). In addition to these four host factors, we more recently showed that the cellular Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor also functions as an HCV entry factor via a virion particle cholesterol-associated mechanism (60), and Lupberger et al. have recently shown that the host cellular epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) function as cofactors during viral cell entry (49).Following entry, the viral genome (ϳ9.6 kb) is translated by an internal ribosome entry site (IRES)-dependent process into a single viral polyprotein that is subsequently co-and posttranslationally cleaved into structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins by host and viral proteases (29,46). The NS prote...

Repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis C virus infection

“…One of the antihistamines, clemizole, was shown previously to have anti-HCV activity by interfering with the nonstructural protein 4B functions (21), but our screen indicated a much less potent activity of clemizole as compared to CCZ. The anti-HCV properties of cyclizines and phenothiazines have been reported previously and are consistent with our results (22)(23)(24)(25)(26). Compared to the phenothiazines in table S1, CCZ analogs were chosen for further studies on the basis of the following aspects: (i) higher potency and selectivity against HCV than phenothiazines, (ii) fewer CNSrelated side effects than phenothiazines, and (iii) potential for further structure modification to reduce side effects and increase anti-HCV activity.…”

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

“…Recently, several groups have reported cell-based HCV infection systems for the screening of HCV inhibitors in various assay formats (12)(13)(14)(15). Traditionally, HTS was performed at a single fixed concentration of each candidate compound, which is prone to false positives and false negatives and requires extensive follow-up studies.…”

“…Further studies demonstrated that reporter genes can be genetically engineered into certain locations of the HCV genome without impairing the viability of the virus (11). Several groups have reported cell-based HCV infection systems for the screening of HCV inhibitors in various assay formats (12)(13)(14)(15). Gastaminza et al developed a colorimetric assay measuring HCV E2 protein produced by HCVinfected cells in a 96-well plate format (12).…”

“…In the system, an HCV inhibitor could rescue the cells from the HCV-induced cytopathic effect. Based on the ability of the HCV NS3 protease to cleave synthetic peptides containing the enzyme's natural viral cleavage sites, Yu et al applied a cell-based hepatitis C virus infection fluorescence resonance energy transfer (FRET) assay for antiviralcompound screening (13). Using a luciferase reporter that is directly inserted into the HCV genome, Wichroski et al adapted the infection system in a 384-well format for the screening of HCV inhibitors (15).…”

Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay

“…Reporter genes inserted into certain locations of the HCV genome could yield viable HCV capable of infection and replication in the cell culture (Koutsoudakis et al, 2006). In recent years, several groups have applied either wild-type HCV or modified HCV with reporter genes to develop cell-based HCV infection systems for the screening of HCV inhibitors (Chockalingam et al, 2010; Gastaminza et al, 2010; Kim et al, 2007; Wichroski et al, 2012; Yu et al, 2012). Cell-based HCV infection systems have the advantage of targeting various stages of HCV life cycle, thus significantly improving the efficiency of the drug screening process.…”