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. [1 ]
Lackovic, Kurt [2 ,3 ]
Marquis, Christopher [1 ]
Eden, John-Sebastian [1 ,4 ]
White, Peter A. [1 ]
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.
引用
收藏
页码:1027 / 1034
页数:8
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