应用假病毒技术研究HIV-1复制抑制剂

建立以HIV-1复制为靶点的细胞水平假病毒药理筛选模型，并运用该模型筛选化合物库中随机选取的化合物。细胞水平的HIV-1假病毒药理筛选模型是应用水泡性口膜炎病毒的外壳糖蛋白(vesicular stomatitis virus glycoprotein，VSV-G)包装HIV-1核心建立的，简称VSVG/HIV模型。细胞被VSVG/HIV感染后，病毒携带的报告基因的表达水平(荧光素酶活性或GFP阳性细胞比例)反映了HIV-1的复制水平。对HIV-1复制有抑制作用的化合物应用VSVG/MLV模型对其特异性地进一步检测。被感染细胞中报告基因的表达水平与病毒稀释度呈显著的剂量依赖效应。阳性药物齐多夫定(zidovudine，AZT)、拉米夫定(lamivudine，3TC)、去羟基苷(didanosine，DDI)可剂量依赖性地抑制HIV-1的复制，其IC₅₀分别为48.5 nmol·L^-1、 0.13 μmol·L^-1和1.73 μmol·L^-1，与文献报道一致。在随机筛选的500个化合物中有3个化合物可以抑制HIV-1的复制，IC₅₀分别为1.92、 5.38和3.39 μmol·L^-1，而对MLV的复制无影响。VSVG/HIV假病毒系统是一种安全、有效的针对HIV-1复制的药理筛选模型。当将此模型与VSVG/MLV模型联合使用时，可判断化合物作用的特异性。实验表明：3个化合物，2-甲硫基-5-(4-甲基苯并)酰胺基-1,3,4-噻二唑(化合物A)、N-(3-羟基苯基)-2-(4-异丁基苯基)丙酰胺(化合物B)和N-(4-甲基吡啶基)-4-甲基苯磺酰胺(化合物C)可以特异性抑制HIV-1的复制。

Screening of HIV-1 replication inhibitors by using pseudotyped virus system

This study is to establish a cell-based pharmacological model targeting HIV-1 replication for compounds screening and to screen compounds randomly selected from compounds library by using this pseudotyped viral system. The cell-based HIV-1 replication pharmacological model was set up by HIV-1 core packed with vesicular stomatitis virus glycoprotein. The level of HIV-1 replication was presented by reporter genes expression (luciferase activity or percentage of GFP positive cells). When a compound has inhibitory effect on VSVG/HIV model, VSVG/MLV model would be used to test for specificity. Vesicular stomatitis virus glycoprotein can efficiently mediate HIV core into a wide range of host cells. Expression level of reporter genes showed dose-dependent manner with virion dilution. Among 500 compounds, three compounds dose-dependently inhibit HIV-1 replication, but not MLV replication. VSVG/HIV pseudotyped viral system can be used as a pharmacological model for HIV-1 replication inhibitor screening. Compounds 2-methylthio-5-(4-methylbenzo)amido-l,3,4-thiadiazole, N-(3-hydroxyphenyl)-2-(4-isobutylphenyl) propionamide, and N-(4-picolyl)-4-methylbenzenesulfonamide can specifically inhibit HIV-1 replication with IC50 of 1.92, 5.38, and 3.39 micromol L(-1) respectively.