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A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase
被引:31
作者:

Eltahla, Auda A.
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机构:
Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia

Lackovic, Kurt
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机构:
Walter & Eliza Hall Inst Med Res, High Throughput Chem Screening Facil, Melbourne, Vic 3050, Australia
Univ Melbourne, Dept Med Biol, Melbourne, Vic, Australia Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia

Marquis, Christopher
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机构:
Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia

Eden, John-Sebastian
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机构:
Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia
Univ Sydney, Fac Sci, Sch Biol Sci, Sydney, NSW 2006, Australia Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia

White, Peter A.
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Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia
机构:
[1] Univ New S Wales, Sch Biotechnol & Biomol Sci, Fac Sci, Sydney, NSW 2052, Australia
[2] Walter & Eliza Hall Inst Med Res, High Throughput Chem Screening Facil, Melbourne, Vic 3050, Australia
[3] Univ Melbourne, Dept Med Biol, Melbourne, Vic, Australia
[4] Univ Sydney, Fac Sci, Sch Biol Sci, Sydney, NSW 2006, Australia
关键词:
hepatitis C virus;
inhibitors;
RNA-dependent RNA polymerase;
NS5B;
high throughput;
fluorescence;
NS5B POLYMERASE;
ASSAY;
IDENTIFICATION;
RIBAVIRIN;
PEGINTERFERON;
POPULATION;
D O I:
10.1177/1087057113489883
中图分类号:
Q5 [生物化学];
学科分类号:
071010 ;
081704 ;
摘要:
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents similar to 20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 lead-like compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC50 of 12.7 mu M and 1.0 mu M, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.
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页码:1027 / 1034
页数:8
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Tibotec BVBA, Beerse, Belgium Queen Marys Univ London, Biizard Inst Cellular & Mol Sci, Barts & London Sch Med & Dent, London E1 2AT, England

van Baelen, Ben
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Picchio, Gaston
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Beumont, Maria
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[10]
Differential Efficacy of Protease Inhibitors Against HCV Genotypes 2a, 3a, 5a, and 6a NS3/4A Protease Recombinant Viruses
[J].
Gottwein, Judith M.
;
Scheel, Troels K. H.
;
Jensen, Tanja B.
;
Ghanem, Lubna
;
Bukh, Jens
.
GASTROENTEROLOGY,
2011, 141 (03)
:1067-1079

Gottwein, Judith M.
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机构: Copenhagen Univ Hosp, Copenhagen Hepatitis Program CO HEP C, Dept Infect Dis, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark

Scheel, Troels K. H.
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机构: Copenhagen Univ Hosp, Copenhagen Hepatitis Program CO HEP C, Dept Infect Dis, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark

Jensen, Tanja B.
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机构: Copenhagen Univ Hosp, Copenhagen Hepatitis Program CO HEP C, Dept Infect Dis, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark

Ghanem, Lubna
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机构: Copenhagen Univ Hosp, Copenhagen Hepatitis Program CO HEP C, Dept Infect Dis, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark

Bukh, Jens
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机构:
Copenhagen Univ Hosp, Copenhagen Hepatitis Program CO HEP C, Dept Infect Dis, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark
Copenhagen Univ Hosp, Clin Res Ctr, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark Copenhagen Univ Hosp, Copenhagen Hepatitis Program CO HEP C, Dept Infect Dis, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark