A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity

Abstract

The beta-chemokine receptor CCR5 is considered to be an attractive target for inhibition of macrophage-tropic (CCR5-using or R5) HIV-1 replication because individuals having a nonfunctional receptor (a homozygous 32-bp deletion in the CCR5 coding region) are apparently normal but resistant to infection with R5 HIV-1. In this study, we found that TAK-779, a nonpeptide compound with a small molecular weight (Mr 531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+ signaling at nanomolar concentrations. The inhibition of beta-chemokine receptors by TAK-779 appeared to be specific to CCR5 because the compound antagonized CCR2b to a lesser extent but did not affect CCR1, CCR3, or CCR4. Consequently, TAK-779 displayed highly potent and selective inhibition of R5 HIV-1 replication without showing any cytotoxicity to the host cells. The compound inhibited the replication of R5 HIV-1 clinical isolates as well as a laboratory strain at a concentration of 1.6-3.7 nM in peripheral blood mononuclear cells, though it was totally inactive against T-cell line-tropic (CXCR4-using or X4) HIV-1.

Figure 1

Chemical structure of TAK-779, N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride.

Figure 2

Characterization of CHO/CCR5 cells and inhibitory effect of TAK-779 on [¹²⁵I]-RANTES binding to the cells. (A) Equilibrium binding of [¹²⁵I]-RANTES to CHO/CCR5 cells. The cells were incubated in the presence of various concentrations of 100 pM [¹²⁵I]-RANTES, as described in Materials and Methods. Specific binding was estimated by subtracting the binding of [¹²⁵I]-RANTES to CHO-K1 cells (nonspecific binding) from the total binding to CHO/CCR5 cells. Data represent means obtained from measurement in triplicate wells. (B) Scatchard analysis for the binding of [¹²⁵I]-RANTES to CHO/CCR5 cells. The K_d value was calculated by the prism program (Takara, Osaka). (C) Inhibitory effect of TAK-779 on the binding of [¹²⁵I]-RANTES to CHO/CCR5 cells. The cells were incubated with [¹²⁵I]-RANTES in the presence of various concentrations of the compound. The percent binding was calculated by an equation of 100 × [(binding with inhibitor − nonspecific binding)/(binding without inhibitor − nonspecific binding)]. The K_i value was calculated by the Cheng-Prusoff derivation (35). Data represent means ± SEM obtained from measurement in six wells from two separate experiments.

Figure 3

Inhibitory effects of TAK-779 on chemokine binding to CCR1-, CCR2b-, CCR3-, or CCR4-expressing CHO cells. CHO/CCR1, CHO/CCR2b, CHO/CCR3, and CHO/CCR4 cells were incubated with [¹²⁵I]-RANTES, [¹²⁵I]-monocyte chemotactic protein 1, [¹²⁵I]-eotaxin, and [¹²⁵I]-thymus- and activation-regulated chemokine, respectively, in the presence of various concentrations of the compound. The percent binding was calculated by the same equation described in the legend of Fig. 2. Data represent means ± SEM obtained from measurements in triplicate wells.

Figure 4

Inhibitory effects of TAK-779 on RANTES-induced Ca²⁺ mobilization in CHO/CCR5 and CHO/CCR1 cells. (A) FuraPE3-AM-loaded CHO/CCR5 cells were incubated in the absence or presence (10, 1, and 0.1 nM) of TAK-779. Changes in the intracellular Ca²⁺ level in response to 20 nM RANTES were determined by a fluorescence spectrometer. (B) CHO/CCR1 cells were treated with 100 nM TAK-779 and then were stimulated with 20 nM RANTES.

Figure 5

Inhibitory effects of TAK-779 and AMD3100 on the replication of R5 (Ba-L) and X4 (III_B) HIV-1 in MAGI-CCR5 cells. The assay methods are described in Materials and Methods. The nuclei of infected cells were stained blue. The concentrations of the compounds were 0.032 μM TAK-779 and 100 μM AMD3100 for Ba-L strain and 20 μM TAK-779 and 0.032 μM AMD3100 for III_B strain. (×100.)