title: Isolation and characterization of hepatitis C virus resistant to a novel phenanthridinone derivative.
abstract: BACKGROUND
  The novel phenanthridinone derivative HA-719 has recently been identified as a highly potent and selective inhibitor of hepatitis C virus replication. To elucidate its mechanism of inhibition, we have isolated and analyzed a clone of hepatitis C virus replicon cells resistant to HA-719.
  
  
  METHODS
  To isolate HA-719-resistant replicon cells, Huh-7 cells containing subgenomic hepatitis C virus replicons (genotype 1b) with a luciferase reporter (LucNeo#2) were cultured in the presence of G418 and escalating concentrations of HA-719. After several passages, total RNA was extracted from the growing cells, and Huh-7 cells were transfected with the extracted RNA. Limiting dilution of the transfected cells was performed to obtain an HA-719-resistant clone.
  
  
  RESULTS
  The 50% effective concentration (EC50) of HA-719 for hepatitis C virus replication was 0.058 ± 0.012 µM in LucNeo#2 cells. The replicon cells capable of growing in the presence of G418 and 3 µM HA-719 were obtained after 18 passages (72 days). The HA-719-resistant clone LucNeo719R showed 98.3-fold resistant to the compound (EC50= 5.66 ± 0.92 µM), but the clone had no cross-resistance to telaprevir (NS3 inhibitor), daclatasvir (NS5A inhibitor), and VX-222 (NS5B inhibitor). The sequence analysis for the wild-type and LucNeo719R identified 3, 2 and 7 mutations in NS3/4 A, NS4B, and NS5A, respectively, but no mutations in NS5B.
  
  
  CONCLUSIONS
  None of the amino acid mutations in the resistant clone corresponds to those reported to confer drug-resistance to current anti-hepatitis C virus agents, suggesting that the target of HA-719 for hepatitis C virus inhibition differs from those of the existing agents.
authors: 1 Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors: Wataru
authors: W
authors: Ito
authors: 1 Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors: Masaaki
authors: M
authors: Toyama
authors: 1 Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors: Mika
authors: M
authors: Okamoto
authors: 2 Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors: Masanori
authors: M
authors: Ikeda
authors: 3 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Koichi
authors: K
authors: Watashi
authors: 3 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Takaji
authors: T
authors: Wakita
authors: 4 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.
authors: Yuichi
authors: Y
authors: Hashimoto
authors: 1 Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors: Masanori
authors: M
authors: Baba
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral / Q000187:drug effects*; D016174:Hepacivirus / Q000187:drug effects* / Q000302:isolation & purification*; D006801:Humans; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D010616:Phenanthrenes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D010616:Phenanthrenes; C040601:phenanthridone
keywords: Drug resistance; hepatitis C virus; inhibitors