基于假病毒技术探讨黄芩苷抗HIV-1活性研究

Study on anti-HIV-1 activity of baicalin based on pseudovirus technology

目的:基于假病毒技术探究黄芩苷抗HIV-1活性作用。方法:利用HIV-1骨架质粒NL4-3 mCherry Luciferase、HIV-1包膜蛋白质粒pCMV-VSV-G和人胚肾细胞HEK-293T构建HIV-1假病毒药物筛选体系,并对该体系进行优化和安全性验证。测定黄芩苷对293T细胞活性、HIV-1假病毒活性、HIV-1逆转录酶(RT)活性、RNA依赖的DNA聚合酶(RDDP)活性以及核糖核酸酶H(RNase H)活性的影响。将黄芩苷分别与HIV-1 RT的晶体结构(2ZD1)、RNase H的晶体结构(3QIN)进行分子对接,探讨黄芩苷与HIV-1 RT、RNase H的相互作用及结合性能。结果:成功构建出了具有双报告检测基因的HIV-1假病毒药物筛选体系,该体系只有单轮感染活性;黄芩苷对293T细胞活性的抑制作用随浓度的增大而增加,黄芩苷在100μmol·L^(-1)浓度以下对293T细胞的活力影响较小(P>0.05);黄芩苷对HIV-1假病毒具有一定的抑制作用,给药浓度越高,其相应的萤光素酶基因和红色荧光基因表达则越低,在100μmol·L^(-1)高浓度下其抑制率可达60%左右;黄芩苷对HIV-1 RT活性和RNase H表现出明显的抑制作用(P<0.05),且呈现浓度依赖性,其半数抑制浓度(IC;)分别约为58.4μmol·L^(-1)、78.4μmol·L^(-1);但黄芩苷对HIV-1 RDDP活性的抑制作用较弱,其IC;约为1.686 mmol·L^(-1);黄芩苷与HIV-1 RT、RNase H蛋白的结合区域均为对应的酶活区域,其分子对接打分值分别为|-10.991|、|-8.485|;黄芩苷能够与HIV-1 RT酶蛋白的LYS101、ILE180氨基酸残基形成氢键作用,与TRP229氨基酸残基形成π-π作用,与VAL106、TYR181、TYR188、VAL108等氨基酸残基形成多个疏水作用;黄芩苷结构母核上的苯环可以与RNase H酶蛋白中的一个Mn;形成一个阳离子-π作用,黄芩苷还能与GLY444、ASP498、GLN500、TYR501氨基酸残基形成氢键作用。结论:黄芩苷在体外具有较好的抗HIV-1活性,其主要的作用靶点为HIV-1 RT和HIV-1 RNase H。

OBJECTIVE To investigate the anti-HIV-1 activity of baicalin based on pseudovirus technology.METHODS The drug screening system of HIV-1 pseudovirus was constructed by using the HIV-1 skeleton plasmid NL4-3 mCherry Luciferase, HIV-1 envelope protein plasmid pCMV-VSV-G and HEK-293 T cells.And the system was optimized and its safety was verified.The effects of baicalin on the activity of 293 T cells, HIV-1 pseudovirus, HIV-1 reverse transcriptase(RT),RNA-dependent DNA polymerase(RDDP)and ribonuclease H(RNase H)were determined.The molecular docking of baicalin with the crystal structure of HIV-1 RT(2 ZD1)and RNase H(3 QIN)was conducted to investigate the interaction and binding properties of baicalin with HIV-1 RT and RNase H.RESULTS The drug screening system of HIV-1 pseudovirus with dual reporter assay gene was successfully constructed, which only had the activity of single round infection.The inhibitory effect of baicalin on the viability of 293 T cells increased with the increase of the concentration.The effect of baicalin on the viability of 293 T cells was small when the concentration of baicalin was below 100 μmol·L^(-1)(P>0.05).Baicalin had a certain inhibitory effect on HIV-1 pseudovirus.The higher the concentration of baicalin, the lower the expression of luciferase gene and red fluorescent gene.The inhibition rate of baicalin on HIV-1 pseudovirus was about 60% at the high concentration of 100 μmol·L^(-1).Baicalin significantly inhibited the activity of HIV-1 RT and RNase H in a concentration-dependent manner(P<0.05),and their half inhibitory concentrations(IC;)were about 58.4 μmol·L^(-1)and 78.4 μmol·L^(-1).However, the inhibitory effect of baicalin on the activity of HIV-1 RDDP was weak, and its IC;was approximately 1.686 mmol·L^(-1).The binding regions of baicalin to HIV-1 RT and RNase H proteins were the corresponding enzyme-active regions, and their docking scores were |-10.991| and |-8.485|.Baicalin could form hydrogen bond with LYS101 and ILE180 amino acid residues of HIV-1 RT protein, and it could form π-π interaction with TRP229 amino acid residues, and it could form multiple hydrophobic interactions with amino acid residues such as VAL106,TYR181,TYR188 and VAL108.The benzene ring on the structural parent core of baicalin could form a cation-π interaction with one Mn;in RNase H protein, and baicalin could also form hydrogen bonds with GLY444,ASP498,GLN500,and TYR501 amino acid residues.CONCLUSION Baicalin shows good anti-HIV-1 activity in vitro,and its main targets are HIV-1 reverse transcriptase and HIV-1 ribonuclease H.