Einav Shirit, Gerber Doron, Bryson Paul D, Sklan Ella H, Elazar Menashe, Maerkl Sebastian J, Glenn Jeffrey S, Quake Stephen R
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
Nat Biotechnol. 2008 Sep;26(9):1019-27. doi: 10.1038/nbt.1490.
More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.
迫切需要更有效的疗法来对抗丙型肝炎病毒（HCV），它是病毒性肝炎的主要病因。我们利用体外蛋白质表达和微流控亲和分析来研究HCV跨膜蛋白NS4B与RNA的结合，NS4B在HCV RNA复制中起着至关重要的作用。我们发现HCV NS4B能结合RNA，且这种结合对病毒基因组负链的3'末端具有特异性，解离常数（Kd）约为3.4 nM。对一个化合物库进行的高通量微流控筛选鉴定出18种能显著抑制NS4B与RNA结合的化合物。其中一种化合物盐酸氯咪唑，被发现能通过抑制NS4B的RNA结合来抑制细胞培养中的HCV RNA复制，对宿主细胞几乎没有毒性。这些结果为HCV生命周期提供了新的见解，并为药物开发提供了一种候选化合物。
