抑制基因型1b之C型肝炎病毒NS3絲胺酸蛋白酶活性之化合物鑑定

Abstract

C型肝炎病毒(hepatitis C virus, HCV)為一種屬於黃熱病毒科(Flaviviridae)的單股RNA病毒，其主要構造包含外套膜和內部的遺傳物質正向單股RNA。C型肝炎病毒的基因體約9.6 kb，能合成出結構性蛋白質core、E1、E2、p7以及非結構性蛋白質NS2、NS3、NS4A、NS4B、NS5A、NS5B。C型肝炎病毒主要經由血液與體液感染，全球約有超過1.5億的人口感染C型肝炎病毒，其所造成的慢性感染是目前肝癌形成的主因。然而C型肝炎病毒疫苗尚未發展出來，且當前治療C型肝炎的方法副作用大，亦無法有效治療所有基因型之病毒株所造成的C型肝炎。因此尋找有效並能廣泛治療C型肝炎的藥物為醫學界致力的目標。本研究利用轉染病毒基因體cDNA的細胞培養系統釋放出的基因型2a之C型肝炎病毒直接感染Huh7細胞，並同時施予待篩選化合物系列A、B、C及D。結果顯示A及C系列中具有能有效降低細胞中基因型2a病毒RNA的化合物。進一步以抑制效果較為顯著的藥物施予在基因型1b之C型肝炎病毒非結構性蛋白質次基因體複製子的細胞株時，發現其中數種化合物亦能抑制基因型1b之病毒RNA的複製。本研究同時也建立了人類正常肝細胞HuS-E/2之三維培養系統，此系統能更能切合人體內病毒感染的實際生理條件，當以此三維培養系統進行分析同時進行藥物安全性測試時，也發現部分A及C系列的化合物能降低基因型2a之病毒RNA於細胞中的累積。接著將該數種化合物施予在穩定表現病毒NS3/NS4A蛋白質的細胞株中，發現類似於槲皮素，化合物A6、C35、C38皆能抑制NS3蛋白質的絲胺酸蛋白酶活性，這可能是造成C型肝炎病毒複製受抑制的原因之ㄧ。基因型1b為台灣主要的C型肝炎病毒分離株，而其造成的C型肝炎目前治癒率偏低。本研究所篩選出的化合物能有效的抑制基因型1b之C型肝炎病毒複製，因此具有發展成藥物的潛力。

Hepatitis C virus (HCV) is a member of the Faviviridae family. The genome of HCV is a positive-sense, single-stranded RNA of 9.6 kilobases which contains genes encoding the structural proteins core, E1, E2, p7 and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. HCV is spread by blood and body fluid contact. There are more than 150 million people worldwide infected by HCV. The majority of HCV-infected patients fail to clear the virus and develop chronic liver diseases including cirrhosis and hepatocellular carcinoma. Unfortunately, there is currently no vaccine to prevent hepatitis C infection. Although pegylated interferon-α and ribavirin are effective drugs in the treatment of chronic hepatitis C, there are side effects to patients and the sustained virological response rates vary for patients with different genotypes. In this study, chemical compounds of A~D groups were screened for anti-HCV activity. Genotype 2a HCVcc were used to infect naive Huh7 cells pretreated with the specific chemical compounds. Results showed that several compounds from groups A and C downregulated the expression level of both HCV genomic and anti-genomic RNAs in Huh7 cells. Some of the chemical compounds also show their inhibitory effects on the replication of genotype 1b HCV RNA in a subgenomic replicon system and of genotype 2a HCV RNA in a 3D human hepatocyte-derived HuS-E/2 culture system. To further understand the possible mechanisms involved in the inhibition, effects of the chemical compounds were further examined on Huh7-TR-NS(3-4A) cell line which expresses the genotype 1b HCV NS3/NS4A proteins. Results demonstrated that like quercetin, the chemical compounds A6, C35, and C38 inhibited the serine protease activity of the NS3 protein, implying a potential mechanim of the chemical compounds in inhibiting HCV RNA replication. Taken together, the chemical compounds A6, C35, and C38 were identified in this study as lead compounds for anti-HCV therapeutics.