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HCV感染中p7蛋白结构及与药物作用机理解析

2019-08-18 09:23
国际权威学术期刊《自然》在线发表了美国哈佛大学医学院、中科院上海生化与细胞所/国家蛋白质科学中心•上海(筹)周界文研究员所带领课题组的最新 成果,首次解析了丙型肝炎病毒(hepatitis C virus)感染宿主过程中重要离子通道蛋白p7的精细空间结构以及p7与抑制剂金刚烷胺类药物相互作用的分子机理。
  丙型肝炎病毒(HCV)与艾滋病病毒(HIV)、流感病毒(influenza virus)一样,属于危害性强的RNA病毒。丙型肝炎病毒所引起的病毒性肝炎,是慢性肝炎的主要病原之一,严重时会导致肝硬化和肝癌。目前估计全世界有 1亿7千万人感染HCV,我国的HCV携带者和患者总数均居世界首位,属于丙肝高发区,迄今为止还没有研发出有效控制HCV的预防或治疗性疫苗。
 
  长期以来,对病人使用的标准治疗方法是持续混合使用长效干扰素(peginterferon-)和利巴韦林(ribavirin)。但这种治 疗方法疗效有限、周期长、费用昂贵,并常伴有毒副作用。最近以标准疗法联用新上市的NS3/4A抑制剂Telaprevir和Boceprevir等,虽 然可以提高治疗效果,但抗药性突变病毒的产生一直是一个潜在的问题。目前国际上抗RNA病毒治疗的发展方向是建立一种基于多种药物靶点的联合疗法,因此迫 切需要开发多个作用靶点,寻找更有效的治疗方法和干预手段。
 
  p7是HCV基因表达的唯一的离子通道蛋白,对病毒颗粒的组装和成熟、病毒颗粒的释放必不可少,突变和完全删除p7会导致HCV病毒不能产生感 染性。离子通道蛋白是一类在许多病毒中广泛存在的蛋白质,对于病毒的生活周期产生重要影响,被广泛作为潜在药物靶点加以研究,例如流感病毒的M2蛋白和艾 滋病病毒的Vpu蛋白。但是以p7为靶点的抗HCV药物研究却进展缓慢,主要原因是由于p7是一个跨膜蛋白,形成多聚体阳离子通道后,结构复杂、构象灵 活,给结构研究、尤其是蛋白质结晶带来极大困难,因此,长期以来缺乏p7离子通道的三维结构及其与小分子化合物结合的作用机理。
 
  在无法得到蛋白质晶体的情况下,周界文研究员和欧阳波博士(文章第一作者)建立了一种基于核磁共振的方法,最终解析了此病毒通道的结构。此通道 结构非常特异,形成花瓣形的六聚体结构,是目前使用核磁共振技术解析出的最大的离子通道结构。由结构带来的启发,周界文课题组与上海巴斯德所、中科院上海 生化细胞所孙兵课题组合作,首次鉴定了金刚烷胺类化合物对p7的离子通道活性发挥抑制作用的结合位点,并通过一系列的功能测试,揭示了p7通道离子转运和 药物抑制的机理。
 
  通过对这些病毒离子通道结构和机制方面的理解,科学家期望在不久的将来可以研制出新一代抗丙型肝炎病毒的治疗手段。

The international authoritative academic journal "Nature" published online the latest results of the research team led by the Shanghai Institute of Biochemistry and Cytology / National Center for Protein Sciences of the Harvard University Medical School and the Chinese Academy of Sciences. For the first time, the fine spatial structure of important Ionic channel protein P7 and the molecular mechanism of interaction between P7 and the inhibitor Jingangwane were analyzed during the infection of the hepatitis C virus(hepatitis C virus).
Hepatitis C virus(HCV), like HIV and influenza virus, is a highly harmful RNA virus. Viral hepatitis caused by the hepatitis C virus is one of the main pathogens of chronic hepatitis and can cause cirrhosis and liver cancer in severe cases. At present, it is estimated that 170 million people in the world are infected with HCV. The total number of HCV carriers and patients in China ranks first in the world and belongs to the high incidence area of HCV. To date, no preventive or therapeutic vaccine has been developed to effectively control HCV.
For a long time, the standard treatment for patients has been the continued use of a combination of long-acting interferon(peginterferon-<UNK>) and ribavirin. However, this therapeutic method has limited efficacy, long cycle, high cost, and often toxic side effects. Recently, the newly listed NS3/4A inhibitors Telaprevir and Boceprevir, etc., have been used in combination with standard therapies. Although treatment effectiveness can be improved, the generation of drug-resistant mutants has been a potential problem. At present, the development direction of anti-RNA virus treatment in the world is to establish a combination therapy based on multiple drug targets. Therefore, it is imperative to develop multiple target targets and find more effective treatment methods and interventions.
P7 is the only ion channel protein expressed by HCV gene. It is indispensable for the assembly and maturity of virus particles and the release of virus particles. Mutations and the complete deletion of P7 will cause HCV viruses to fail to produce susceptibility. Ion channel protein is a type of protein that is widely present in many viruses and has an important impact on the life cycle of the virus. It has been widely studied as a potential drug target, such as M2 protein of influenza virus and Vpu protein of AIDS virus. However, the research on anti-HCV drugs targeting P7 has progressed slowly. The main reason is that P7 is a transmembrane protein. After forming a polymer cation channel, the structure is complex and the structure is active, which brings great difficulties to structural research, especially protein crystallization. Therefore, the three-dimensional structure of the P7 ion channel and its mechanism of binding to small molecular compounds have long been lacking.
In the absence of protein crystals, Zhou Jiwen's researcher and Dr. Ouyangbo(the first author of the article) established a method based on nuclear magnetic resonance that eventually resolved the structure of the virus channel. This channel structure is very specific, forming a petal-shaped hexamer structure, which is currently the largest ion channel structure resolved using nuclear magnetic resonance technology. Inspired by the structure, the Zhoujiewen Group cooperated with the Shanghai Pasteur Institute and the Shanghai Biochemical Cell Institute of the Chinese Academy of Sciences, Sunbing Jingangwane, to identify for the first time the binding site for the inhibition of the ion channel activity of the P7 by the Jingangwane compound. And through a series of functional tests, The mechanism of ion transport and drug inhibition in P7 channel was revealed.
Through the understanding of the structure and mechanism of these virus ion channels, scientists expect to develop a new generation of treatments for hepatitis C virus in the near future.