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. 2004 Dec;78(23):12996-3006.
doi: 10.1128/JVI.78.23.12996-13006.2004.

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Katrien Princen  1 , Sigrid Hatse, Kurt Vermeire, Stefano Aquaro, Erik De Clercq, Lars-Ole Gerlach, Mette Rosenkilde, Thue W Schwartz, Renato Skerlj, Gary Bridger, Dominique Schols
Affiliations

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Katrien Princen et al. J Virol. 2004 Dec.

Abstract

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.

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Figures

FIG. 1.
FIG. 1.
Chemical structure of AMD3451.
FIG. 2.
FIG. 2.
Dose-dependent inhibition of HIV-1 NL4.3 (X4 virus) and HIV-1 ADA (R5 virus) replication in PBMCs by AMD3451. The data shown are the mean IC50s of the results from four experiments.
FIG. 3.
FIG. 3.
PCR-based detection of virus (HIV-1 NL4.3 and HIV-1 ADA) entry into U87.CD4.CXCR4 and U87.CD4.CCR5 cells. Two hours after infection with HIV-1, total DNA was extracted from the cells and analyzed by PCR with HIV-1-specific primers from the LTR R/U5 region. Lane 1, positive control; lane 2, 200 μM AMD3451; lane 3, 1 μM AMD3100; lane 4, 2 μM TAK-779. DNA recovery was controlled by PCR with β-actin-specific primers. The data shown are from one representative experiment out of two.
FIG. 4.
FIG. 4.
Dose-dependent inhibitory effect of AMD3451 on CXCL12-induced Ca2+ flux in SUP-T1 cells (top) and U87.CD4.CXCR4 cells (middle) and on CCL4-induced Ca2+ flux in U87.CD4.CCR5 cells (bottom). After loading with the fluorescent calcium indicator Fluo-3, the cells were preincubated for 10 min with AMD3451 at the indicated concentrations and then stimulated with chemokine (10-ng/ml CXCL12 or 5-ng/ml CCL4). The transient increase in intracellular calcium concentration was recorded by monitoring the change in fluorescence of the cells as a function of time by using the FLIPR. Each data point represents the average fluorescence of four identical microplate wells. The data shown are from one representative experiment out of three.
FIG. 5.
FIG. 5.
Effects of AMD3451 on the Ca2+ flux induced by CXCL12, CCL2, CCL3, CCL4, CCL5, and CCL7 in freshly isolated human monocytes. After loading with the fluorescent calcium indicator Fluo-3, the cells were preincubated for 10 min with (open symbols) or without (closed symbols) compound and then stained with chemokine (50 ng/ml). The transient increase in intracellular calcium concentration was recorded by monitoring the change in fluorescence of the cells as a function of time by using the FLIPR. Each data point represents the average fluorescence of four identical microplate wells. The data shown are from one representative experiment out of three.
FIG. 6.
FIG. 6.
Concentration-dependent inhibitory effect of AMD3451 on CXCL12- and CCL4-induced chemotaxis of CCR5-transfected Jurkat cells. The cells were preincubated for 10 min with AMD3451 at the indicated concentrations. Then 5-μm-pore-size filter inserts were loaded with 106 cells and transferred to a 24-well plate containing 100-ng/ml CXCL12 or 500-ng/ml CCL4 in 600 μl of buffer. Transmigration of the cells from the insert to the lower compartment, containing the chemokine, was monitored microscopically. After 4 h of incubation, the membrane inserts were removed and the cells in the wells were collected and counted by flow cytometry. The percentage of migrated cells matches the ratio of the numbers of migrated cells in the lower wells to the number of migrated cells in the positive control (no pretreatment with AMD3451). The exact number of cells in the lower wells was calculated by linear regression with a standard curve. The data shown are from one representative experiment out of two.
FIG. 7.
FIG. 7.
Inhibitory effect of AMD3451 on CXCL12- and CCL3L1-induced endocytosis of CXCR4 (top) and CCR5 (bottom), respectively. U87.CD4.CXCR4-GFP and CEM.CCR5-GFP cells were preincubated for 15 min in the absence (left and middle panels) or presence (right panels) of 200 μM AMD3451. Then the cells were stimulated, except there was no stimulus under the control conditions (left panels). U87.CD4.CXCR4-GFP cells were stimulated with 1-μg/ml CXCL12 (top), and CEM.CCR5 cells were stimulated with 100-ng/ml CCL3L1 (bottom). After incubation at 37°C, the subcellular localization of the fluorescently labeled CXCR4 and CCR5 proteins was examined in the different cell samples by fluorescence microscopy. The pictures were obtained from one representative experiment which was repeated twice with comparable results.
FIG. 8.
FIG. 8.
(A) Opposite effects of AMD3451 and AMD3100 on the binding of anti-CXCR4 (12G5) MAb to Molt-4 cells. After preincubation for 30 min on ice with or without the compounds at the indicated concentrations, the cells were stained with phycoerythrin-conjugated 12G5 MAb and analyzed by flow cytometry. The upper panel shows the aspecific background fluorescence of Molt-4 cells, as determined by an isotype control MAb. The second panel shows Molt-4 cells stained with the antibody in the absence of any compound. The MFI of the cell population is indicated in each histogram. The data shown are from one representative experiment out of four. (B) Effect of AMD3451 on competition binding of anti-CXCR4 antibody 12G5. The binding assay was performed with COS-7 cells transfected with wild-type (wt) CXCR4 with 125I-12G5 as a radioligand. The data are shown as means ± standard errors of the means (n = 3).
FIG. 9.
FIG. 9.
Effect on AMD3451 competition for CXCL12 binding of the Asp-to-Asn substitution at position 171 in the CXCR4 receptor. The binding assay was performed with COS-7 cells transfected with wild-type (WT) CXCR4 or CXCR4 [D171N] with 125I-Met-CXCL12 as a radioligand. The data are shown as means ± standard errors of the means (n = 3).
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