| Objective:In order to develop CCR5 antagonists with novel structure,strong activity and independent intellectual property rights in China,this study conducted a preliminary screening of 27 compounds from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences for anti-HIV-1 activity in vitro,and two compounds DC521042 and DC521043 with the best activity were found.Then,several HIV-1 virus strains and cells were used for their anti-HIV activity pharmacodynamics,and DC521042 was selected for evaluation of combined anti-HIV activity with other types of marketed antiretroviral drugs.Methods:Firstly,the cytotoxicity of DC521042 and DC521043 on different cell lines was detected by MTT assay.The inhibitory effect of DC521042 and DC521043 on the replication of different lab-adapted HIV-1 strains in TZM-bl cells was detected by luciferase activity assay.The inhibitory effect of DC521042 and DC521043 on the replication of three HIV-1 clinical isolates in PBMC and the inhibition of two different cells infected with HIV-lBa-L were tested by ELISA.The luciferase activity assay and ELISA method were used to detect DC521042 in combination with AZT,FTC,TDF,3TC,ETR,NVP,EFV,DTG,RAL,IDV,T-20 and MVC on intracellular replication.Maraviroc(MVC)was used as a positive control drug for all experiments.Results:(1)The cytotoxicity of DC521042 and DC521043 to TZM-bl,PM1,HOS-CD4-CCR5 cells both were low,and the CC50 were more than 200?M.However,there was a certain cytotoxicity to PBMC,with CC50 of 102.51±1.19?M and 81.70±3.63 ?M,respectively.(2)DC521042 and DC521043 could effectively inhibit the replication of three HIV-1 lab-adapted HIV-1 strains in TZM-bl cells.The EC50 of DC521042 against HIV-1Ba-L,HIV-1YU-2,HIV-1SF-162 in TZM-bl cells for replication was 9.96±4.33 nM and 16.93±2.90 nM,2.89±0.55 nM and 8.73±0.34 nM.The EC50 of DC521043 against HIV-1Ba-L,HIV-1YU-2,HIV-1SF-162 in TZM-bl cells for replication was 7.64±0.55 nM and 15.99±3.87 nM,respectively.(3)DC521042 and DC521043 also potently inhibited the replication of HIV-1Ba-L in PM1 and HOS-CD4-CCR5 cells:The EC50 of DC521042 and DC521043 were 0.29±0.12 nM and 0.50±0.36 nM,0.70±0.27 mM and 2.37±1.80 nM,respectively.(4)DC521042 and DC521043 could effectively inhibit the replication of IN resistant strain HIV-1YU-2(G140S/Q148H)in TZM-bl cells,the average EC50 of which were 2.71±0.34 nM and 4.34±1.61 nM,respectively.(5)DC521042 and DC521043 had good inhibitory effects on HIV-1KM018,HIV-1KIZ001 and HIV-1KIZ006.The EC50 of DC521042 and DC521043 for HIV-1KM018,HIV-1KIZ001 and HIV-1KIZ006 were 23.31±13.61 nM and 32.04±11.24 nM,5.26±1.74 nM and 7.82±0.61 nM,respectively.(6)However,X4-or R5/X4-tropism strains were not sensitive to DC521042 and DC521043,with EC50 greater than 1000 nM and 500 nM,respectively,the same as MVC.(7)The combined index(CI)of DC521042 combined with NRTIs(AZT,3TC,TDF,FTC,NVP)to against HIV-1YU-2 was 0.60±0.02,0.69±0.01,0.47±0.25,0.44±0.16,and 0.41±0.03,respectively.The CI of DC521042 and NNRTIs(EFV and ETR)was 0.85±0.08,0.75±0.38,respectively.The CI of DC521042 combined with Pls(IDV)was 0.95±0.04.The CI of DC521042 combined with INIs(RAL and DTG)was 0.46±0.03 and 0.50±0.14,respectively.The CI of DC521042 combined with FIs(T-20)was 0.33±0.05.Finally,the CI of DC521042 in combination with MVC was 1.33±0.08,showing antagonism.Conclusion:DC521042 and DC521043 showed different cytotoxicity to four host cells.They were less toxicity to TZM-b1,PM1,HOS-CD4-CCR5 cells,and had certain cytotoxicity to PBMC,while MVC was low toxicity to the four cells.DC521042 and DC521043 showed excellent antiviral activity against R5-tropism lab-adapted and clinical isolated HIV-1 strains in different cells.However,X4-or R5/X4-tropism strains are not sensitive to them,as well as MVC,indicating that DC521042 and DC521043 are CCR5-specific antagonists.The anti-HIV-1KM018 activity of DC521042 and DC521043 was inferior to MVC,but anti-HIV-1KIZ001 and HIV-1 KIZ006 were better than MVC;the same is true for INs resistant strains.DC521042 showed synergism or addition when combined with NRTIs,NNRTIs,PIs,INIs and FIs,but showed antagonism with CCR5 inhibitors,indicating that DC521042 act on the same target compare with MVC.From the above results indicate that DC521042 has a good inhibitory activity against the R5-tropic HIV-1 strains.The anti HIV activity of DC521042 is superior or equivalent to MVC.The DC521042 can be used as a promising low-toxic CCR5 inhibitors to continue to develop,which is expected to be the candicate compound,and also provides a reference for the design of new CCR5 antagonists. |
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