酸性天然凝胶电泳法研究HIV进入抑制剂ADS-J1的作用机制  

Acid native polyacrylamide gel electrophoresis:a method for studying the mechanism of action of HIV entry inhibitor ADS-J1

作　　者：毛芹超[1] 王洪涛[1] 李旭桂[1] 夏承来[1] 姜世勃[1,2] 刘叔文[1] 

机构地区：[1]南方医科大学药学院,广东广州510515 [2]美国纽约血液中心LFK研究所,纽约ny10085

出　　处：《中国药理学通报》2010年第1期25-28,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30729001;U0832001)

摘　　要：目的ADS-J1是通过虚拟筛选从20000个化合物中获得的靶向HIVgp41的小分子HIV进入抑制剂。该研究探讨ADS-J1与gp41的结合位点和作用机制。方法采用酸性天然聚丙烯酰胺凝胶电泳技术(AN-PAGE),研究ADS-J1与衍生于gp41不同功能区的多肽的结合。结果此前采用的天然聚丙烯酰胺凝胶电泳(N-PAGE)等方法,由于不能显示衍生于gp41的N-端多肽,未能确定ADS-J1的作用位点。该研究建立的AN-PAGE技术,能直接显示N-端多肽的条带,证实ADS-J1能与gp41的N-端螺旋重复序列(NHR)复合螺旋核中的深穴结合,从而抑制gp41六螺旋束结构的形成,而且深穴中第574位的赖氨酸残基(K574)与ADS-J1的抑制作用密切相关。结论ADS-J1通过与gp41 NHR靶穴中的K574结合,抑制gp41六螺旋束结构的形成,从而抑制HIV进入靶细胞。此外,该研究建立的AN-PAGE技术,为研究靶向Ⅰ型包膜病毒的病毒进入抑制剂的作用机制提供了一个简便有效的实验方法。Aim ADS-J1 is a low molecular HIV entry inhibitor targeting HIV transmembrane subunit gp41 through virtual screening from a compound library containing 20 000 molecules. This study is to investigate the binding sites of ADS-J1 on gp41. Methods Acid native polyacrylamide gel electrophoresis （AN-PAGE） assay was applied to test the binding ability of ADS-J1 with the peptides derived from gp41 N-terminal heptad repeat （NHR） region. Results It was reported previously that ADS-J1 could block the gp41 six-helix bundle （6-HB） formation using native polyacrylamide gel electrophoresis （N-PAGE）. However,the binding sites could not be found because positive charged N-peptides derived from gp41 NHR could not show bands on the gel. In the present study,the AN-PAGE assay which could show N-peptides in the gel was established,and it was found that ADS-J1 could inhibit the gp41 6-HB formation. Moreover,ADS-J1 bound directly to the gp41 cavity region of NHR. The positively charged residue （K574） located in this region was critical for the binding of ADS-J1. Conclusions ADS-J1 inhibits HIV entry by targeting the cavity region of gp41 NHR,whereas K574 in the cavity plays a critical role for the binding. Furthermore,the AN-PAGE assay provides a simple method for studying the mechanism of action of virus entry inhibitors targeting the transmembrane protein of type I enveloped virus.

关 键 词：HIV进入抑制剂 GP41 酸性天然聚丙烯酰胺凝胶电泳 ADS-J1 Ⅰ型包膜病毒 六螺旋束结构 

分 类 号：R341.6[医药卫生—基础医学] R373.9