A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase
A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase
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DOI：
10.1177/1087057113489883
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发表时间：
2013-10-01
影响因子：
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通讯作者：
White, Peter A.
中科院分区：
文献类型：
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作者：
Eltahla, Auda A.;Lackovic, Kurt;White, Peter A.
丙型肝炎病毒（HCV）RNA依赖性RNA聚合酶（RdRp）在HCV复制中起重要作用，是新型抗病毒治疗的关键靶点。几种RdRp抑制剂正在进行临床试验，当与目前基于干扰素的治疗方法联合用于基因型1（G1）HCV患者时，其应答率增加。然而，这些抑制剂对非G1基因型（包括G3a）的疗效较差，G3a占全球HCV病例的20%。在这里，我们使用市售的荧光染料来表征G3a HCV RdRp在体外。通过从单链RNA poly（C）模板合成双链RNA来评估RdRp活性。将该测定小型化为384孔微孔板格式，并使用随机选择的10，208种铅样化合物进行中试高通量筛选，以鉴定HCV G3a RdRp的抑制剂。在150种表现出最大抑制作用的化合物中，有10种使用荧光和放射性测定法进行了确认。前两种抑制剂（HAC001和HAC002）表现出比活性，IC50分别为12.7 μ M和1.0 μ M。总之，我们描述了简单的，基于荧光的高通量筛选（HTS）的从头RdRp活性的抑制剂的识别，使用HCV G3a RdRp作为目标。HTS系统可用于对抗任何不能培养的正义RNA病毒。
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.