Virtual Screening And Evaluation In Vitro Of CCR5 Receptor Inhibitors Based On Structure-Activity Relationship

ObjectiveOur study aims at investigating the structure-activity relationship of CCR5 receptor inhibitors.On this basis,computer-virtual screening was performed and the activity of the screened compounds was verified in vitro in order to obtain new anti-HIV lead compounds.Methods1.Construction of 3D-QSAR modelForty-eight CCR5 receptor inhibitors were selected from the previous literature.CoMFA and CoMSIA models were constructed based on common skeleton overlap.The predictive and fitting abilities of the models were evaluated and three-dimensional isopotential maps were analyzed.2.Construction of pharmacophore modelThe HipHop and HypoGen pharmacophore models were constructed.The optimal pharmacophore model was selected according to the statistical index and screening ability of the pharmacophore for virtual screening.3.Construction of molecular docking modelSurflex-Dock and CDOCKEER molecular docking models were reformed to analyze the key interactions and amino acid residues between CCR5 inhibitors and CCR5 receptors,which laid the foundation for virtual screening.4.Virtual screeningThe strategy of multi-stage screening was adopted,and the five principles of drug-like,pharmacophore model and molecular docking model were used for screening step by step.5.Evaluation of in vitro activity of compoundsThe anti-HIV-1 activities of the compounds was determined by detecting the expression of luciferase reporter gene in TZM-bl cells infected with HIV-1 clone virus.MTT assay was used to examines the cytotoxicity of the compounds.Flow cytometry was exerted to analyze the effects of compounds on the internalization of CCR5 receptor.Results1.3D-QSAR modelThe cross-validation coefficient(q2)of CoMFA model was 0.77,the optimal number of components(NOC)was 5,and the non-cross-validation coefficient(r2)was 0.95.The optimal CoMSIA(SAH)model considers the stereo field,hydrogen bond acceptor field and hydrophobic field.In this case,the NOC is 5,q2 = 0.8,and r2 = 0.96.2.Pharmacophore modelThe optimal HipHop pharmacophore had one positive ionizable characteristic,two hydrophobic characteristics and three hydrogen bond acceptor characteristics.Rank value was 105.9.and the area under ROC curve was 0.82.The optimal HypoGen pharmacophore model had two hydrogen bond acceptor characteristics,one positive ionizable characteristic and two hydrophobic characteristics.The value of Total Cost was 150.46,A Cost was 218.66,predictive correlation coefficient(r)was 0.95,root mean square deviation(RMSD)was 1.26,and the area under ROC curve was 0.7.The model has been verified by Fisher at 95%confidence level.3.Molecular docking model Key interactions between CCR5 receptor inhibitors and receptor were hydrogen bond and hydrophobic interaction.The key amino acid residues of CCR5 receptor inhibitors were Glu283,Tyr251,Phe112,Tyr108 and Trp86.4.Virtual screening 6 compounds(B01-B06)were obtained by multi-stage virtual screening.5.Evaluation of in vitro activityThe cloned virus of HIV-1 showed strongest infection ability when diluted four times.Compounds B04 and B06 showed anti-HIV-1 activity.The median inhibition concentration(IC50)of B04 was 29.52± 3.73μmol/L,the cytotoxicity concentration(CC50)was 181.40± 14.22(μmol/L;IC50 of B06 was 42.13± 5.06μmol/L,CC50 was 354.90 ± 28.82 μmol/L.The results of flow cytometry showed that the average fluorescence intensity on the surface of cells treated with B04 and B06 decreased significantly,which proved that B04 and B06 could induce the internalization of CCR5 receptor.Conclusion1.3D-QSAR,pharmacophore and molecular docking models were successfully constructed.The structure-activity relationship of CCR5 receptor inhibitors was deeply discussed.The pharmacophore and molecular docking models were suitable for screen the ZINC database.2.Compounds B04 and B06 had anti-HIV-1 activity in vitro determined,our findings suggested that both compounds had certain anti-HIV-1 activity,which could induce internalization of CCR5 receptor,and could be applied as lead compounds for structural modification in order to obtain better anti-HIV-1 drug.