Inhibitory effects of small-molecule CCR5 antagonists on human immunodeficiency virus type 1 envelope-mediated membrane fusion and viral replication

We established a human immunodeficiency virus type I (HIV-1) envelope (Env) -mediated membrane fusion assay and examined the small-molecule CCR5 antago nist TAK-779 and its derivatives for their inhibitory effects on HIV-1 Env- mediated membrane fusion and viral replication. The membrane fusion assay i s based on HIV-1 long terminal repeat-directed beta -D-galactosidase report er gene expression in CD4- and CCR5-expressed HeLa (MAGI-CCR5) cells after cocultivation with effector 293T cells expressing HIV-1 Env. Inhibition of HIV-1 replication was also determined in MAGI-CCR5 cells infected with the corresponding cell-free HIV-1. TAK-779 effectively suppressed R5 HIV-1 (str ain JR-FL) Env-mediated membrane fusion as well as viral replication. Its 5 0% inhibitory concentrations (IC(50)s) for membrane fusion and viral replic ation were 0.87 +/- 0.11 and 1.4 +/- 0.1 nM, respectively. These values cor responded well to the IC50 for I-125-RANTES (regulated on activation, T cel l expressed, and secreted) binding to CCR5 (1.4 nM). The inhibitory effects of 18 TAK-779 derivatives on membrane fusion differed from one compound to another. However, there was a close correlation among their inhibitory eff ects on membrane fusion, viral replication, and RANTES binding. The correla tion coefficient between their IC(50)s for membrane fusion and viral replic ation was 0.881. Furthermore, since this assay depends on Env expressed in the effector cells, it is also applicable to the evaluation of CXCR4 antago nists. These results indicate that the HIV-1 Env-mediated membrane fusion a ssay is a useful tool for the evaluation of entry inhibitors.