【作者】 小溪； 刘晨； 郑智慧； 路新华； 张华； 贺建功；

【Author】 Xiaoxi Shui1, Chen Liu1, Zhihui Zheng2* Xinhua Lu2, Hua Zhang 2, Jiangong He2 1 College of Life Science, Hebei Normal University, Shijiazhuang, 050016, China 2 Drug Research &Development Center of North China Pharmaceutical Group Corporation, National Microbial Medicine Engineering &Research Center, Shijiazhuang, 050015, China

【机构】 河北师范大学生命科学学院； 华北制药集团新药研发中心天然药物室；

【摘要】 gp41是HIV-1表面的包膜糖蛋白(Env)成分,介导了病毒膜和宿主细胞膜间的融合,在HIV-1的侵染过程中起关键作用。gp41中的N末端重复序列(NHR)和C末端重复序列(CHR)通过相互作用形成关键的核心三聚体发夹状结构,从而介导HIV膜和宿主细胞膜的融合过程。抑制gp41中的NHR和CHR的核心三聚体形成已经成为抗HIV药物的新靶点。本研究利用哺乳动物双杂交技术检测蛋白之间相互作用的原理,建立新的靶向HIV-1gp41抗HIV药物高通量筛选模型。将PCR扩增的gp41NHR和CHR基因片段分别连接至哺乳动物双杂交的pACT和pBIND载体上构建表达载体pACT-NHR和pBIND-CHR,用脂质体与已经构建好的报告质粒pGl3-GAL4进行共转染动物细胞。通过检测荧光素酶基因的表达水平定量评价化合物对NHR和CHR的相互作用的拮抗活性。经过多种条件的优化,使得筛选模型的信号本底比达10,Z’因子大于0.5。阳性对照化合物XTT在本模型中可以浓度依赖地降低荧光素酶的表达,其IC50为13.54μg/mL(20.1μM),证明该模型具有很好的灵敏度和稳定性,可以用于大量药物筛选。本研究建立的全新靶向gp41的抗HIV小分子药物筛选模型,为发现和研究新一代的抗HIV药物研究打下坚实的基础。更多还原

【Abstract】 gp41 is a major component of the envelope glycoprotein of the HIV-1 membrane, which is responsible for the membrane fusion between the virus and cells. The interaction of its N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) play a key role in the membrane fusion process by forming a trimer-of-hairpins fusion-active gp41 core structure. So the inhibition of formation of trimer-of-hairpins has become a new target of HIV enter inhibitors drugs. In this study, a mammalian two-hybrid system based novel high-throughput screening (HTS) assay targeting gp41 was developed. The fragments of NHR and CHR of gp41 were obtained by PCR and were separately inserted into pACT and pBIND to construct the mammalian two-hybrid expressing vectors pACT-NHR and pBIND-CHR. After optimization on the cotransfection conditions such as host cell line, incubation time, the ratios of plasmids DNA and numbers of cells, the assay produced a signal-to-noise (S/N) ratio of over 10 and Z′ value of over 0.5. The positive compound, XTT, could reduce the luciferase activity in a dose-dependent manner with IC50 value of 13.54μg/mL (20.1μM). These data suggest we provide a novel assay for discovery of specific, small molecules inhibitors of HIV membrane fusion targeting gp41, which is sensitive and robust for HTS.更多还原