title: Colony-forming assays reveal enhanced suppression of hepatitis C virus replication using combinations of direct-acting antivirals.
abstract: The current standard of care for patients infected with hepatitis C virus (HCV) is not effective universally and is associated with severe side effects. Direct-acting antiviral molecules have potential to transform treatment of HCV-infected individuals but emergence of drug-resistant virus will be problematic. It is anticipated that, to limit the emergence of drug-resistant virus, future HCV therapies must consist of multiple direct-acting antivirals. In the present study, cell culture-based colony-forming assays were used to demonstrate enhanced suppression of HCV RNA replication following simultaneous treatment of HCV replicon-containing cells with two direct-acting antivirals. Specifically, combinations of NS5Ai and Filibuvir (small molecule inhibitors of HCV-encoded NS5A and NS5B proteins respectively) were able to suppress colony formation fully at concentrations that individually they could not. HCV replicon RNA isolated from colonies that emerged following treatment with suboptimal concentrations of NS5Ai were found to encode resistance substitutions in the NS5A gene, which rendered them insensitive to subsequent high doses of NS5Ai. Furthermore, both NS5Ai and Filibuvir were effective at suppressing colony formation in combination with BILN 2061, an inhibitor of HCV-encoded NS3. Collectively, these data underscore the increased inhibitory capacity of direct-acting antivirals to suppress HCV RNA replication when present in combination.
authors.affiliation: Pfizer Global Research & Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK.
authors.forename: Emily J S
authors.initials: EJ
authors.lastname: Graham
authors.forename: Rachael
authors.initials: R
authors.lastname: Hunt
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Shaw
authors.forename: Chris
authors.initials: C
authors.lastname: Pickford
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hammond
authors.forename: Mike
authors.initials: M
authors.lastname: Westby
authors.forename: Paul
authors.initials: P
authors.lastname: Targett-Adams
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003114:Colony-Forming Units Assay; D004357:Drug Synergism; D016174:Hepacivirus / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects / Q000821:virology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D011753:Pyrones / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects*; D011743:Pyrimidines; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D011753:Pyrones; D014230:Triazoles; D017361:Viral Nonstructural Proteins; C550357:filibuvir; D011743:Pyrimidines; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase