title: Development of hepatitis C virus chimeric replicons for identifying broad spectrum NS3 protease inhibitors.
abstract: Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy.
authors.affiliation: Pfizer Worldwide Research and Development, San Diego, CA 92121, USA. joe.binder@pfizer.com
authors.forename: Joseph
authors.initials: J
authors.lastname: Binder
authors.forename: Selwyna
authors.initials: S
authors.lastname: Tetangco
authors.forename: Megan
authors.initials: M
authors.lastname: Weinshank
authors.forename: Karen
authors.initials: K
authors.lastname: Maegley
authors.forename: Laura
authors.initials: L
authors.lastname: Lingardo
authors.forename: Wade
authors.initials: W
authors.lastname: Diehl
authors.forename: Robert
authors.initials: R
authors.lastname: Love
authors.forename: Amy K
authors.initials: AK
authors.lastname: Patick
authors.forename: George J
authors.initials: GJ
authors.lastname: Smith
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002219:Carbamates / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D014644:Genetic Variation; D005822:Genetic Vectors / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D006801:Humans; D047028:Macrocyclic Compounds / Q000737:chemistry / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010802:Phylogeny; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology; D012093:Replicon; D020539:Sequence Analysis, Protein / Q000379:methods; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D047028:Macrocyclic Compounds; D009842:Oligopeptides; D011480:Protease Inhibitors; D011804:Quinolines; D013844:Thiazoles; D017361:Viral Nonstructural Proteins; C479555:BILN 2061; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C486464:telaprevir