基于CCR5受体的中药化合物抑制剂的虚拟筛选及活性评价

Virtual screening and evaluation of inhibitor activity of TCM compounds basedupon CCR5 receptor

目的:以C-C趋化因子受体5(C-C chemokine receptor type 5,CCR5)为靶点,利用计算机辅助药物设计技术中的虚拟筛选技术、体外抗病毒活性实验、细胞毒性测试以及分子动力学实验筛选出低毒性、高抗艾滋病病毒(HIV)活性的中药化合物抑制剂,并探究其内在结合机制。方法:接受者操作特征曲线(receiver operating characteristic curve,ROC)评估Ledock、AutoDock Vina对接软件对于CCR5结构灵敏性,选择适合CCR5抑制剂筛选的对接软件,运用多级筛选的策略对中药系统药理数据库和分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)中的中药化合物进行筛选。利用荧光素酶报告基因的表达检测中药化合物的抗HIV活性,细胞毒性实验测试中药化合物对MAGI-CCR5、L02细胞毒性,分子动力学实验验证中药化合物与CCR5的结合作用。结果:ROC曲线确定了分子对接软件(Ledock,AUC=0.899),虚拟筛选得到8种潜在拮抗CCR5的中药天然小分子;抗HIV活性实验及细胞毒性测试获得2个低毒性、高抗HIV活性中药化合物MT[IC50,(21.79±4.12)μmol·L^(-1);CC50MAGI-CCR5,(170.20±7.75)μmol·L^(-1);CC50L02,(108.04±11.64)μmol·L^(-1)]及FY[IC50,(6.69±1.40)μmol·L^(-1);CC50MAGI-CCR5,(97.82±10.57)μmol·L^(-1);CC50L02,(114.70±10.40)μmol·L^(-1)]。分子动力学实验表明,MT-CCR5、FY-CCR5体系均方根偏差(root mean square deviation,RMSD)值均较小,平均分别为2.77?、2.18?,表明两化合物可与CCR5紧密结合,两者与CCR5结合的主要作用力包括氢键(结合氢键平均个数分别为5.19和4.65个)、范德华力(MTΔEvdw=-232.04±3.45 kJ·mol^(-1),FYΔEvdw=-193.66±0.56 kJ·mol^(-1))及非极性相互作用(MTΔEvdw±ΔGnonpolar=-255.73±4.03 kJ·mol^(-1),FYΔEvdw±ΔGnonpolar=-222.39±0.60 kJ·mol^(-1))。结论:中药化合物MT和FY可与CCR5结合从而具有一定的抗HIV活性,可作为先导抗病毒中药化合物进行下一步结构改造。

OBJECTIVE By using the virtual screening technology of computer-aided drug design,antiviral activity test in vitro,cytotoxicity test and molecular dynamic experiment,the inhibitors of TCM compounds with low toxicity and high anti-human immunodeficiency virus(HIV)activity were screened with C-C chemokine receptor type 5(CCR5)as the target and explore their internal conjugating mechanism.METHODS The sensitivity of Ledock and AutoDockVina docking software to the structure of CCR5 was evaluated by receiver operating characteristic curve(ROC).The docking software suitable for screening CCR5 inhibitors was selected and multi-stage screening strategy utilized for screening traditional Chinese medicine compounds in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The anti-HIV activity of TCM compounds was detected by luciferase reporter gene expression,the cytotoxicity of TCM compounds to MAGI-CCR5 and L02 cells examined by cytotoxicity test and the binding effect of TCM compounds with CCR5 verified by molecular dynamic experiments.Finally,molecular dynamic simulation experiment was employed for verifying the binding effect of two TCM compounds with CCR5.RESULTS The molecular docking software(Ledock,AUC=0.899)was determined by ROC curve and eight natural small molecules of TCM potentially antagonizing CCR5 were obtained by virtual screening.Two low-toxicity and highly anti-HIV active TCM compounds MT and FY were obtained by activity detection and cytotoxicity test.MT(IC50,(21.79±4.12)μmol·L^(-1);CC50 MAGI-CCR5,(170.20±7.75)μmol·L^(-1);CC50 L02,(108.04±11.64)μmol·L^(-1)),FY(IC50,(6.69±1.40)μmol·L^(-1);CC50 MAGI-CCR5,(97.82±10.57)μmol·L^(-1);CC50 L02,(114.70±10.40)μmol·L^(-1)).Molecular dynamic experiments showed that the RMSD values of MT-CCR5 and FY-CCR5 systems were small,with an average of 2.77?and 2.18?respectively,indicating that the two compounds could bind to CCR5 closely.The main binding forces of these two compounds with CCR5 included hydrogen bonds(average number of binding hydrogen bonds 5.19 and 4.65 respectively),Vander Waals forces(MTΔEvdw=-232.04±3.45 kJ·mol^(-1),FYΔEvdw=-193.66±0.56 kJ·mol^(-1))and nonpolar interactions(MTΔEvdw±ΔGnonpolar=-255.73±4.03 kJ·mol^(-1),FYΔEvdw±ΔGnonpolar=-222.39±0.60 kJ·mol^(-1)).CONCLUSION MT and FY have certain anti-HIV activity of acting as potential inhibitors for further structural modifications.