Establishment Of HIV-1env/HIV-1?env Pseudovirus Screening System And Evaluation Of The Anti-HIV-1 Activity Of WYM-17 And WYM-25

Objective:To establish and optimize the HIV=lenv/HIV-l?env pseudovirus system for anti-HIV-1 drugs screening,and evaluation of the anti-HIV-1 activity of the active compounds in vitro.Methods:HIV-1Luc pseudovirus is obtained by co-transfecting 293T cells with the HIV-1 envelope plasmid pCNE49 and the HIV-1 backbone plasmid pNL4-3 Luc R-E-and the luciferase activity analysis system(Promega)is used to evaluate the infectivity of HIV-ILuc in TZM-bl cells in the presence of DEAE-dextran.The antiviral activity of different HIV-1 positive drugs is identified and compared with the HIV-l?B screening system in order to verify the feasibility and effectiveness of the pseudovirus system,and the pseudovirus system will be utilized to screen the anti-HIV-1 drugs.In addition,the cytotoxic effects of the active compounds will examined by MTT assay in different cells.Using the p24 assay and MTT assay as well as luciferase assay to estimate the inhibit activity of the compounds against different virus strains in different cells.Results:The infectivity of HIV-lLuc in TZM-bl cells increased significantly with the increasing concentration of DEAE-dextran,and the expression level of the reporter gene was dose-dependent with the amount of virus.The EC so values of different positive drugs inhibit HIV-lLuc and HIV-I?B were close,which showed that HIV-1Luc,can be used for drug screening.And the 2-thiomethylpyrazole-6-cyclohexyl pyrimidinones displayed significant activity against HIV-1Luc,as well as the activity of WYM-17 and WYM-25 were the most prominent.Further studies about the anti-HIV-1 activity of WYM-17 and WYM-25 in vitro found that:WYM-25 has low cytotoxicity in C8166,MT-4,TZM-bl and PBMC cells,and the CC50 was>200 ?M;WYM-17 and WYM-25 both have broad-spectrum anti-HIV-1 activity against experimental strains(HIV-1?B,HIV-1 Ba-L),clinically isolated strains(HIV-ITc-1,HIV-lwan)and drug-resistant strains(NRTI-resistant HIV-174v,PI-resistant strain HIV-12802)with EC50 at the nanomolar level,whereas the NNRTI-resistant strain HIV-lA17 behaved different degrees of resistance to these compounds.Similarly,WYM-17 and WYM-25 both have a certain inhibitory effect on HIV-1 recombinant reverse transcriptase;and the molecular docking experiments indicated that the compounds have a strong interaction with reverse transcriptase.Conclusion:In this study,we successfully established an anti-HIV-1 drug screening system based on the HIV-lenv/HTV-1?env pseudovirus.This system can be used for the screening of NNRTIs,NRTIs,INIs and FIs.The studies on the anti-HIV-1 activity of the compounds WYM-17 and WYM-25 showed that two compounds both have strong anti-HIV-1 activity against different virus strains in vitro.Molecular docking studies demonstrated that there is potential for further optimization on the basis of the two compounds to develop new types of HIV-1 inhibitors with high activity.