Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR
Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR
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DOI：
10.1016/j.antiviral.2009.03.008
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发表时间：
2009-08-01
影响因子：
7.6
通讯作者：
Fukazawa, Hidesuke
中科院分区：
文献类型：
--
作者：
Murakami, Yuko;Noguchi, Kohji;Fukazawa, Hidesuke
我们设计了一种利用JFH 1病毒培养系统筛选丙型肝炎病毒（HCV）抑制剂的方法。将培养基中释放的病毒RNA吸附到PCR板上，并通过将一步RT-PCR反应混合物直接加入到威尔斯孔中进行实时RT-PCR。“管捕获-RT-PCR”方法避免了劳动密集型RNA分离的需要，并应允许高通量筛选HCV抑制剂。为了证实药物筛选试验的有效性，对由95种化合物组成的抑制剂文库进行了中试筛选。除了库中包含的已知HCV复制抑制剂外，该测定还鉴定了PKC抑制剂双吲哚基马来酰亚胺I（BIM I）作为HCV复制抑制剂。BIM I也有效降低基因型1b和2a亚基因组复制子细胞中的病毒蛋白水平，表明抑制HCV复制。进一步的分析表明，广泛的双吲哚马来酰亚胺和吲哚并咔唑抑制HCV，但没有发现PKC抑制模式和抗HCV活性之间的相关性。这一系列化合物代表了可能需要进一步开发的新型抑制剂。(C)2009 Elsevier B. V.保留所有权利。
We devised a screening method for hepatitis C virus (HCV) inhibitors by exploiting the JFH1 viral culture system. The viral RNA released in the medium was adsorbed onto PCR plates, and real-time RT-PCR was performed by directly adding the one-step RT-PCR reaction mixture to the wells. The "tube-capture-RT-PCR" method obviates the need for labor-intensive RNA isolation and should allow high-throughput screening of HCV inhibitors. To substantiate the validity of the assay for drug screening, a pilot screen of an inhibitor library composed of 95 compounds was performed. In addition to the known inhibitors of HCV replication included in the library, the assay identified the PKC inhibitor bisindolylmaleimide I (BIM I) as an HCV replication inhibitor. BIM I was also effective in reducing the viral protein level in genotype 1b and 2a subgenomic replicon cells, indicating inhibition of HCV replication. Further assays revealed that a broad range of bisindolylmaleimides and indolocarbazoles inhibit HCV, but no correlation was found between the PKC inhibition pattern and anti-HCV activity. These series of compounds represent new classes of inhibitors that may warrant further development. (C) 2009 Elsevier B.V. All rights reserved.