中草藥萃取物抗C型肝炎病毒暨病毒聚合酶(RNA dependent RNA polymerase)篩藥平台開發之研究

Herbal component extracted suppresses Hepatitis C Virus replication and a cell-based reporter assay for inhibitor screening of hepatitis C virus RNA dependant RNA Polymerase

曾敬凱(Chin-Kai Tseng)

指導教授 ： 李景欽

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2010年)

摘要

本研究以中草藥萃取物為主要抗C型肝炎病毒藥物之篩選，在多數中草藥中我們發現三黃瀉心湯萃取物AN26對於HCV 複製有良好的抑制效果EC50，約7.5μg/ml，且不具有明顯的細胞毒性，CC50大於 150μg/ml，其SI值大於10 。將AN26與不同的C型肝炎病毒蛋白抑制劑，進行加成作用的實驗中發現，AN26能夠與IFN-α、NS3/NS4A病毒蛋白酶抑制劑Telaprevir (VX-950)以及NS5B病毒聚合酶抑制劑2′-C-Methylcytidine (2′-C-MeC)，產生良好的加成作用。針對AN26作用機制的探討中，發現了AN26能夠有效的抑制NF-κB-COX2細胞傳訊因子路徑，前人研究指出COX2對於HCV的複製有著明顯的調控效果。我們也對於含有HCV複製子的人類肝癌細胞株 Ava5進行COX2 基因過度表現的實驗。AN26抑制HCV複製子的能力在過度表現COX2的細胞株中明顯的降低，這也證明了AN26抑制C型肝炎病毒複製活性與細胞傳訊因子COX2的調控機制有關。除了藥物篩選以外，本研究也針對C型肝炎病毒NS5B gene(RNA dependent RNA polymerase)設計活性分析平台，HCV的NS5B gene 是一個重要的HCV藥物篩選目標，現有RdRp藥物篩選系統最大的缺點為皆是胞外的篩選系統無法有效的反應出藥物對於細胞的毒性以及細胞的吸收。我們建立了一株BHK-NS5B-FRLuc 報導基因細胞株，其內含了持續過量表現的NS5B gene以及Bicistronic的報導基因(+)FLuc-(-)UTR-RLuc，這樣的系統一方面可以篩選抗NS5B病毒蛋白活性之藥物，也可以了解藥物造成的細胞毒性。而報導基因(+)FLuc-(-)UTR-RLuc的構築包含了，螢火蟲冷光蛋白酶基因(firefly luciferase (FLuc))以及被反股病毒非轉譯區域5'UTR、3'UTR所夾住之Renilla luciferase (RLuc) 基因，而FLuc和RLuc的活性分別由細胞的聚合酶以及病毒的聚合酶(RdRp)所調控。我們也利用了具有RdRp專一性的抑制劑針對我們的報導基因系統進行測試，在NM107的測試中其抑制效果與前人研究中的抑制效果有相關性的結果。我們將此系統運用於96wells的細胞培養盤中進行測試其Z'-factor的值為0.75也符合了高速篩選系統的標準。透過電腦模擬試算抗HCV RdRp的藥物，並且利用此系統進行篩選得到了一個imidazole derivative的化合物對於 HCV RdRp病毒蛋白有良好的抑制效果。

關鍵字

c型肝炎病毒 ； 中草藥 ； 聚合酶

並列摘要

Here we report the fractionated extract, referred as AN26, obtained from San-Huang-Xie-Xin-Tang exhibited the inhibitory activity on HCV RNA replication in the HCV replicon assay system, with EC50 and selective index (SI) of 7.5 μg/ml and 150 μg/ml, respectively. By use of different actions of antiviral drugs, AN26 shows antiviral synergy in combination with IFN-α, Telaprevir (VX-950), and 2′-C-Methylcytidine (2′-C-MeC). Furthermore, we demonstrate that the AN26 extract significantly suppresses COX-2 expression in HCV replicon cells. Reversely, overexpression of the COX-2 in HCV replicon cells by a transient transfection results in decreasing AN26’s inhibitory effects on HCV replication in a concentration-dependent fashion, suggesting that the anti-HCV activity of AN26 was associated with down-regulation of COX-2, which was dependent of NF-κB inactivation by AN26. The hepatitis C virus (HCV) NS5B, a RNA-dependant RNA polymerase (RdRp) is an attractive target for anti-HCV agents. The major disadvantages of the commonly used polymerase inhibitor screening involving the assessment of in vitro RNA synthesis is incapable of demonstrating the cellular permeability and the cytotoxicity of compounds. To overcome these limitations, we created the BHK-NS5B-FRLuc reporter cell line that carries stably transfected NS5B and a bicistronic reporter gene, (+)FLuc-(–)UTR-RLuc, which can be used to simultaneously measure cellular toxicity and intracellular RdRp activity. The (+)FLuc-(–)UTR-RLuc construct comprises the firefly luciferase (FLuc) gene and the Renilla luciferase (RLuc) gene in reverse orientation flanked by both the negative strands of the HCV 5′ and 3′ untranslated regions (UTRs), in which FLuc and RLuc reporter proteins are regulated by host polymerase and functional NS5B polymerase, respectively. The reporter system was validated with specific agents against NS5B polymerization. Additionally, this assay was placed in 96-well plates and had a Z′-factor value of approximately 0.75, which is amenable to facilitate high-throughput screening operations. Notably, in combination with the structured-based virtual screening, an imidazole derivative compound was evaluated as a candidate HCV RdRp inhibitor.

並列關鍵字

hepatitis C virus ； Herbal component extracted ； RNA dependant RNA Polymerase

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