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. 2001 Dec;45(12):3538-43.
doi: 10.1128/AAC.45.12.3538-3543.2001.

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

K Takashima  1 , H Miyake, R A Furuta, J I Fujisawa, Y Iizawa, N Kanzaki, M Shiraishi, K Okonogi, M Baba
Affiliations

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

K Takashima et al. Antimicrob Agents Chemother. 2001 Dec.

Abstract

We established a human immunodeficiency virus type 1 (HIV-1) envelope (Env)-mediated membrane fusion assay and examined the small-molecule CCR5 antagonist TAK-779 and its derivatives for their inhibitory effects on HIV-1 Env-mediated membrane fusion and viral replication. The membrane fusion assay is based on HIV-1 long terminal repeat-directed beta-D-galactosidase reporter 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 (strain JR-FL) Env-mediated membrane fusion as well as viral replication. Its 50% inhibitory concentrations (IC(50)s) for membrane fusion and viral replication were 0.87 +/- 0.11 and 1.4 +/- 0.1 nM, respectively. These values corresponded well to the IC(50) for (125)I-RANTES (regulated on activation, T cell 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 effects on membrane fusion, viral replication, and RANTES binding. The correlation coefficient between their IC(50)s for membrane fusion and viral replication was 0.881. Furthermore, since this assay depends on Env expressed in the effector cells, it is also applicable to the evaluation of CXCR4 antagonists. These results indicate that the HIV-1 Env-mediated membrane fusion assay is a useful tool for the evaluation of entry inhibitors.

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Figures

FIG. 1
FIG. 1
Chemical structures of TAK-779 derivatives.
FIG. 2
FIG. 2
Schematic presentation of the HIV-1 Env-mediated membrane fusion assay. 293T cells transiently expressing HIV-1 Tat and Env were used as the effector cells, and MAGI-CCR5 cells were used as the target cells. When cell-to-cell membrane fusion occurs between the effector and target cells, the reporter gene is activated by Tat under the control of HIV-1 LTR. Reporter activity was detected by chemiluminescence.
FIG. 3
FIG. 3
Inhibitory effects of TAK-779 on HIV-1 Env-mediated membrane fusion (A) and on virus replication (B). Assay procedures are described in Materials and Methods. All data represent means ± standard errors of the means obtained in three separate experiments.
FIG. 4
FIG. 4
Correlation between the inhibitory effects of TAK-779 derivatives on HIV-1 Env-mediated membrane fusion and viral replication. Each point represents the IC50s for membrane fusion and viral replication (r = 0.881).
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