Specific interactions between the viral coreceptor CXCR4 and the biguanide-based compound NB325 mediate inhibition of human immunodeficiency virus type 1 infection

Abstract

The present studies were conducted to better define the mechanism of action of polyethylene hexamethylene biguanide (PEHMB) (designated herein as NB325), which was shown in previous studies to inhibit infection by the human immunodeficiency virus type 1 (HIV-1). Fluorescence-activated flow cytometric analyses of activated human CD4(+) T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 epitope recognition in the absence of altered recognition of selected CD4 or CD3 epitopes. NB325 also inhibited chemotaxis of CD4(+) T lymphocytes induced by the CXCR4 ligand CXCL12. However, NB325 did not cause CXCR4 internalization (unlike CXCL12) and did not interfere with CXCL12 binding. Additional flow cytometric analyses using antibodies with distinct specificities for extracellular domains of CXCR4 demonstrated that NB325 specifically interfered with antibody binding to extracellular loop 2 (ECL2). This interaction was confirmed using competitive binding analyses, in which a peptide derived from CXCR4 ECL2 competitively inhibited NB325-mediated reductions in CXCR4 epitope recognition. Collectively, these results demonstrate that the biguanide-based compound NB325 inhibits HIV-1 infection by specifically interacting with the HIV-1 coreceptor CXCR4.

FIG. 1.

Inhibition of HIV-1 infection by NB325 occurs in the presence of both virus and target cell. (A) Chemical structure of NB325. (B) Stimulated CD4⁺ T lymphocytes were infected with cell-free HIV-1 IIIB for 2 h in the absence or presence of NB325 or DS. For the preincubation portion of the experiment, NB325 and virus were first incubated for 10 min prior to dilution and addition to target T cells. Inhibition of HIV-1 infection was determined as described in Materials and Methods. Infectivity remaining was expressed relative to mock-treated, HIV-1-infected cells and graphed against the compound concentration achieved during the 2-h incubation. (C) Stimulated CD4⁺ T lymphocytes were incubated in the absence or presence of NB325 for 2 h. NB325 cytotoxicity was assessed following 2-h exposure or 6 days postexposure by MTS assay. Each panel incorporates results from two independent assays, in which each concentration was assessed in triplicate. These (and subsequent) figures depict mean values and standard deviations.

FIG. 2.

NB325 perturbs detection of CXCR4, but not CD4 or CD3, on primary human T lymphocytes. Stimulated CD4⁺ T lymphocytes were incubated in the absence or presence of NB325 for 2 h, reacted with fluorochrome-conjugated antibodies specific for CD4 (clone RPA-T4), CD3 (clone UCHT1), and CXCR4 (clone 12G5), and subsequently analyzed using flow cytometry. Results depicted were derived from two independent assays, in which each concentration was assessed in duplicate.

FIG. 3.

Changes in CXCR4 detection in the presence of NB325 are dependent on antibody epitope specificity. Stimulated CD4⁺ T lymphocytes were incubated in the presence or absence of NB325 for 2 h at 37°C. Cells were reacted with fluorochrome-conjugated antibodies specific for CD4 (clone RPA-T4), CD45RO (clone UCHL1), and CXCR4 (clones 12G5, 1D9, or 173) and analyzed by flow cytometry. (A) Scatter plots show CD4 and CXCR4 detection on CD45RO⁺ CD4⁺ T lymphocytes in the absence or presence of NB325. Ab, antibody. (B) The frequency of CXCR4 on CD45RO⁺ CD4⁺ T lymphocytes, as detected by clones 12G5, 1D9, or 173, is summarized. Results shown were derived from two independent experiments, in which each concentration was tested in duplicate. Statistical significance was calculated using a one-tailed, unpaired Student's t test (**, P ≤ 0.01; ***, P ≤ 0.001).

FIG. 4.

NB325 inhibits CXCL12-mediated chemotaxis. Chemotaxis of stimulated CD4⁺ T lymphocytes was induced by CXCL12 (250 ng/ml) in a transmigration chamber (Costar, Inc., Cambridge, MA) in the absence or presence of NB325. The extent of chemotaxis (%) was calculated with respect to the number of cells that migrated in response to CXCL12 alone. Results displayed are representative of three independent assays, in which each condition was examined in triplicate. Statistical significance with respect to chemotaxis induced in the presence of CXCL12 alone was calculated using a one-tailed, unpaired Student's t test (**, P ≤ 0.01).

FIG. 5.

NB325 does not interfere with CXCL12 binding to CXCR4. Stimulated CD4⁺ T lymphocytes were incubated with biotinylated CXCL12 in the absence or presence of NB325 for 1 h at 4°C (as described in Materials and Methods). (A) Detection of labeled CXCL12 (expressed as MFI). (B and C) Cells incubated with labeled CXCL12 and NB325 were also reacted with fluorochrome-conjugated antibodies specific for CD45RO (antibody UCHL1) and CXCR4 (antibodies 12G5, 1D9, or 173) prior to performing flow cytometry. CXCR4 detection by each CXCR4-specific antibody is expressed as (B) percent cells with detectable CXCR4 and (C) MFI of detectable CXCR4. Each panel incorporates results from three independent experiments, in which each concentration was assayed in duplicate. Statistical significance was determined using a one-tailed, unpaired Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

FIG. 6.

Reduced detection of CXCR4 in the presence of NB325 is specifically abrogated by an ECL2-derived peptide. Stimulated CD4⁺ T lymphocytes were incubated with NB325 alone (0.003%), peptide alone, or NB325 (0.003%) and peptide (1:1, 3:1, and 10:1 ratios) or were mock exposed for 2 h. Cells were subsequently reacted with antibodies specific for CD4 (clone RPA-T4), CD45RO (clone UCHL1), or CXCR4 (clones 12G5, 1D9, or 173) prior to performing flow cytometry. Experiments were conducted using the (A) ECL2 peptide or (B) N-terminal (Nt) peptide. The frequency of CXCR4 detection on CD45RO⁺ CD4⁺ T lymphocytes, as indicated by antibodies 12G5, 1D9, or 173, is shown. Each concentration within each panel was examined in triplicate and is representative of data from two independent assays. Statistical significance was calculated using a one-tailed, unpaired Student's t test (**, P ≤ 0.01; ***, P ≤ 0.001).

FIG. 7.

Models for inhibition of HIV-1 and CXCL12 interactions with CXCR4 by NB325. (A) Experimental results indicate that NB325 interacts specifically with CXCR4 ECL2 but not with the N terminus. No information is available regarding NB325 interactions with ECL3. Binding of NB325 to ECL2 inhibits HIV-1 infection, which normally requires protein-protein interactions between the coreceptor binding sites on gp120 and CXCR4 ECL2 (7-9, 12, 13, 24, 31, 37, 39, 40, 48, 54). (B) In a two-site binding model, CXCL12 binds first to the N terminus of CXCR4, which subsequently facilitates an interaction with ECL2 and the initiation of CXCR4-mediated signal transduction. NB325 interacts with CXCR4 ECL2 but not the CXCR4 N terminus, thereby inhibiting CXCL12-induced signaling (chemotaxis) but not CXCL12 binding to the N terminus.