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Vaccines and Antiviral Agents

Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites

Lieven J. Stuyver, Tamara R. McBrayer, Phillip M. Tharnish, Abdalla E. A. Hassan, Chung K. Chu, Krzysztof W. Pankiewicz, Kyochi A. Watanabe, Raymond F. Schinazi, Michael J. Otto
Lieven J. Stuyver
1Pharmasset, Inc., Tucker, Georgia 30084
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  • For correspondence: lstuyver@pharmasset.com
Tamara R. McBrayer
1Pharmasset, Inc., Tucker, Georgia 30084
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Phillip M. Tharnish
1Pharmasset, Inc., Tucker, Georgia 30084
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Abdalla E. A. Hassan
1Pharmasset, Inc., Tucker, Georgia 30084
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Chung K. Chu
2College of Pharmacy, University of Georgia, Athens, Georgia 30602
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Krzysztof W. Pankiewicz
1Pharmasset, Inc., Tucker, Georgia 30084
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Kyochi A. Watanabe
1Pharmasset, Inc., Tucker, Georgia 30084
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Raymond F. Schinazi
3Veterans Affairs Medical Center and Department of Pediatrics, Emory University School of Medicine, Decatur, Georgia 30033
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Michael J. Otto
1Pharmasset, Inc., Tucker, Georgia 30084
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DOI: 10.1128/JVI.77.19.10689-10694.2003
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    FIG. 1.

    Chemical structures of various nucleosides and antimetabolites.

  • FIG. 2.
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    FIG. 2.

    Dynamics of cell growth and HCV RNA levels after exposure to control anti-HCV compounds. HCV replicon cells were seeded at approximately 104 cells per well in a 24-well plate. Over a 7-day period, cells were counted daily, and rRNA and HCV RNA were quantified by quantitative reverse transcription-PCR. (A) IFN-α-2a at 100 IU/ml. (B) Ribavirin at 100 μM. (C) 2′-C-CH3-C at 100 μM. (D) 2′-C-CH3-A at 20 μM. •, cell proliferation in the absence of the compound; ○, cell proliferation in the presence of the compound; ▾, HCV RNA levels in untreated cells; ▿, HCV RNA levels in the presence of the compound. The points shown are averages ± standard deviations (S.D.) (error bars) for three independent experiments.

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    FIG. 3.

    Dynamics of the cell growth and HCV RNA levels after exposure to selected antimetabolites. The experimental method used is described in the legend to Fig. 2. (A) PALA at 10 μM. (B) PZF at 5 μM. (C) CP-C at 25 μM. (D) CPE-C at 2.5 μM. •, cell proliferation in the absence of the compound; ○, cell proliferation in the presence of the compound; ▾, HCV RNA levels in untreated cells; ▿, HCV RNA levels in the presence of the compound. The points are averages ± standard deviations (S.D.) (error bars) for three independent experiments.

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    FIG. 4.

    Replicon RNA changes per cell. The plots were obtained from collected data as shown in Fig. 2 and 3, in which log10 changes for cell count and replicon RNA levels were subtracted from each other. (A) Control antiviral compounds. •, no drug control; ▾, IFN-α-2a at 100 IU/ml; ▪, ribavirin at 100 μM; ⧫, 2′-C-CH3-C at 100 μM; ▴, 2′-C-CH3-A at 20 μM. (B) Selection of the most important antimetabolites. •, no drug control; ▾, dFdC at 50 nM; ▪, CP-C at 25 μM; ⧫, CPE-C at 2.5 μM; ▴, PALA at 10 μM; ⬢, PZF at 5 μM.

Tables

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  • TABLE 1.

    Antiviral and cytotoxicity effects of antimetabolites on HCV replicon-transfected Huh-7 cells

    Compoundlog10 RNA reductiona at 100 μM for:Corrected HCV RNA
    HCVrRNAlog10 reductiona at 100 μMlog10 reduction at 10 μMEC90, μM
    IMPDH inhibitors (EC 1.1.1.205)
        Mizoribine0.29 ± 0.740.21 ± 0.500.08 ± 0.82−0.14 ± 0.12>100
        Tiazofurin0.86 ± 0.270.99 ± 0.35−0.13 ± 0.370.04 ± 0.10>100
        Mycophenolic acid1.15 ± 0.431.09 ± 0.280.07 ± 0.470.22 ± 0.01>100
        C2-MAD1.09 ± 0.211.00 ± 0.150.08 ± 0.240.36 ± 0.21>100
    Ribonucleotide reductase inhibitors (EC 1.17.4.1; EC 1.17.4.2)
        Guanazole0.25 ± 0.110.07 ± 0.030.32 ± 0.080.05 ± 0.08>100
        Hydroxyurea0.17 ± 0.080.25 ± 0.20−0.08 ± 0.160.06 ± 0.04>100
        Tezacytabine1.59 ± 0.081.78 ± 0.69−0.19 ± 0.490.63 ± 0.07>100
        Deferoxamine1.00 ± 0.060.92 ± 0.080.08 ± 0.030.17 ± 0.11>100
    CTP synthase inhibitors (E.C. 6.3.4.2.)
        CP-C1.97 ± 0.380.91 ± 0.131.06 ± 0.260.64 ± 0.1025
        CPE-C2.47 ± 0.331.21 ± 0.161.26 ± 0.511.43 ± 0.012.5
        3-DU1.41 ± 0.090.48 ± 0.110.94 ± 0.200.13 ± 0.10∼100
        dFdC1.87 ± 0.160.59 ± 0.051.29 ± 0.111.32 ± 0.08Too toxic
    OMPDC inhibitors (EC 4.1.1.23)
        6-Azauridine0.25 ± 0.090.61 ± 0.18−0.36 ± 0.160.12 ± 0.05>100
        2-Thio-6-azauridine0.16 ± 0.04−0.02 ± 0.120.19 ± 0.090.12 ± 0.10>100
        PZF1.88 ± 0.050.42 ± 0.031.46 ± 0.081.16 ± 0.213.80
    ATC inhibitors (EC 2.1.3.2)
        PALA1.77 ± 0.020.48 ± 0.021.30 ± 0.051.18 ± 0.117.60
    Dihydroorotate dehydrogenase inhibitors (E.C. 1.3.3.1)
        Brequinar (NSC-368390)−0.05 ± 0.050.29 ± 0.01−0.34 ± 0.04−0.17 ± 0.09>100
        Dichloroallyl lawsone (NSC-126771)1.27 ± 0.022.13 ± 0.15−0.86 ± 0.17−0.52 ± 0.01>100
    Thymidylate synthase inhibitors (E.C. 2.1.1.45)
        2′-Deoxy-5-fluorouridine0.76 ± 0.060.73 ± 0.350.04 ± 0.250.23 ± 0.05>100
        Methotrexate0.18 ± 0.010.07 ± 0.100.11 ± 0.090.15 ± 0.01>100
    Controls
        IFN-α-2a1.57 ± 0.260.21 ± 0.211.36 ± 0.37NAb4.5 IU/ml
        Ribavirin1.96 ± 0.280.91 ± 0.121.05 ± 0.290.16 ± 0.10∼100
        2′-C-CH3-A2.32 ± 0.112.96 ± 0.08−0.64 ± 0.182.051.4
        2′-C-CH3-C2.20 ± 0.52−0.02 ± 0.052.21 ± 0.471.010.4
    • ↵ a IFN tested at 100 IU/ml; dFdC tested initially at 50 μM; a meaningful EC90 value could not be attributed because of extreme toxicity.

    • ↵ b NA, not applicable.

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Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites
Lieven J. Stuyver, Tamara R. McBrayer, Phillip M. Tharnish, Abdalla E. A. Hassan, Chung K. Chu, Krzysztof W. Pankiewicz, Kyochi A. Watanabe, Raymond F. Schinazi, Michael J. Otto
Journal of Virology Sep 2003, 77 (19) 10689-10694; DOI: 10.1128/JVI.77.19.10689-10694.2003

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Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites
Lieven J. Stuyver, Tamara R. McBrayer, Phillip M. Tharnish, Abdalla E. A. Hassan, Chung K. Chu, Krzysztof W. Pankiewicz, Kyochi A. Watanabe, Raymond F. Schinazi, Michael J. Otto
Journal of Virology Sep 2003, 77 (19) 10689-10694; DOI: 10.1128/JVI.77.19.10689-10694.2003
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  • Top
  • Article
    • ABSTRACT
    • Specificity of the antiviral effect in the HCV replicon system.
    • Antiviral effect of antimetabolites.
    • Dynamics of the antiviral effect of inhibitors of the de novo synthesis of ribopyrimidines.
    • Reduction of replicon RNA copy number per cell.
    • Prevention studies.
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
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KEYWORDS

Antimetabolites
antiviral agents
hepacivirus
Nucleosides
RNA, Viral
replicon

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