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Antimicrobial Agents and Chemotherapy, February 2009, p. 631-638, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00866-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Specific Interactions between the Viral Coreceptor CXCR4 and the Biguanide-Based Compound NB325 Mediate Inhibition of Human Immunodeficiency Virus Type 1 Infection{triangledown}

Nina Thakkar,1 Vanessa Pirrone,1 Shendra Passic,1 Wei Zhu,2 Vladyslav Kholodovych,2 William Welsh,2 Robert F. Rando,3 Mohamed E. Labib,3 Brian Wigdahl,1 and Fred C. Krebs1*

Department of Microbiology and Immunology, and Center for Molecular Therapeutics and Resistance, Center for Sexually Transmitted Disease, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102,1 Robert Wood Johnson Medical School, University of Medicine & Dentistry of New Jersey, Piscataway, New Jersey 08854,2 Novaflux Biosciences, Inc., Princeton, New Jersey 085403

Received 30 June 2008/ Returned for modification 26 August 2008/ Accepted 24 November 2008


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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.


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INTRODUCTION
 
The critical involvement of the cellular chemokine receptors CXCR4 and CCR5 in the process of human immunodeficiency virus type 1 (HIV-1) attachment and entry has made these viral coreceptors attractive targets in the development of effective HIV-1 entry inhibitors (15, 43, 44). A number of CXCR4 antagonists, such as the bicyclam AMD3100 and the polypeptides T22 and ALX40-4C, have been identified as effective inhibitors of viruses with an X4 phenotype (strains that use CXCR4 as the coreceptor). Similarly, antagonists of CCR5, such as TAK-779, SCH-C, and SCH-D (vicriviroc), have been shown to potently inhibit infection by R5 viruses (viruses that use CCR5). As proof of the therapeutic value of coreceptor inhibitors, the CCR5 antagonist maraviroc (developed as UK-427,857) was recently approved for clinical use under the name Selzentry (50).

Our efforts to develop inhibitors of HIV-1 infection have focused on biguanide (BG)-based molecules, including polybiguanides (PBG). BG-based compounds have a long history of safe and effective use. Chlorhexidine digluconate, a bis-BG, has been used as a general vaginal disinfectant for over 30 years with a high level of safety (36, 45, 47). The PBG compound polyhexamethylene biguanide (PHMB) is used as an antibacterial agent in contact lens solutions (25) and in other applications (29, 32, 42), as a treatment for Acanthamoeba keratitis (27), and as an environmental biocide (53). PHMB also has potent antiviral activity against herpes simplex virus type 1 (49). Although PHMB was also shown to inhibit HIV-1 infection, the in vitro cytotoxicity of this molecule precluded its further development as an HIV-1 inhibitor (26).

Recent developmental efforts have focused on the PBG compound polyethylene hexamethylene biguanide (PEHMB; Fig. 1A), which was also shown to have anti-HIV-1 activity (26) and activity against herpes simplex virus type 2 (our unpublished data). This compound, which carries an overall positive charge, is composed of BG subunits flanked by alternating linkers containing two or six methylenes (26).


Figure 1
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  		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.

Recently, refinements in the synthesis of PEHMB resulted in a preparation of the compound designated NB325, which was also shown to be an effective HIV-1 inhibitor with minimal cytotoxicity. The demonstration that NB325 was an effective inhibitor of X4 HIV-1 infection prompted investigations into the mechanisms responsible for its antiviral activities. Previous experiments, which indicated that PEHMB had its greatest activity in the presence of both virus and target cell (26), suggested that mechanism of action experiments should investigate the effects of NB325 on cell surface molecules involved in HIV-1 binding and entry: CD4, CXCR4, and CCR5. The present study provides evidence for a specific interaction between CXCR4 and NB325. Results from flow cytometric analyses, coreceptor function assays, and assessments of antiviral activity point to inhibition of HIV-1 through a direct interaction between NB325 and the second extracellular loop (ECL2) of CXCR4. These results will now be used to further the development of NB325 and similar molecules as coreceptor inhibitors effective against HIV-1.


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MATERIALS AND METHODS
 
Cells and viruses. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood (Biological Specialty Corporation, Colmar, PA) using Ficoll-Paque Plus gradient density centrifugation, as described by the manufacturer (GE HealthCare). Cells were cultured in RPMI supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 U/ml each), and 0.05% sodium bicarbonate (RPMI complete media). PBMC preparations were stimulated with 5 µg/ml phytohemagglutinin (catalog no. L-1668; Sigma-Aldrich, St. Louis, MO) and 20 U/ml interleukin-2 (IL-2; catalog no. 136; NIH AIDS Research & Reference Reagent Program, Germantown, MD) for 48 h, and then IL-2 (20 U/ml) alone for 24 h. Subsequent to stimulation, CD4+ T cells were isolated by magnetic separation using a CD4+ T-cell enrichment protocol and Auto MACS Pro (Miltenyi Biotech, Auburn, CA). HIV-1 strain IIIB was obtained from Advanced Biotechnologies, Inc. (Columbia, MD).

Compounds. NB325 (Fig. 1A) was synthesized using procedures similar to those previously described for PEHMB (26) and PHMB (35). In this case, the reaction time was extended to 24 h and the synthesis was performed in a closed vessel under an ambient atmosphere. NB325 was readily soluble in water and polydisperse, with molecular masses ranging from approximately 900 to 1,900 Da (1,400-Da median molecular mass). NB325 and PEHMB (26) are presumed to be similar in both structure and biological function. Dextran sulfate (DS; Dextralip 50; catalog no. D8787, lot no. 71K1278) was purchased from Sigma-Aldrich (St. Louis, MO). DS was included as a control in the initial assays of antiviral activity and cytotoxicity, since DS combines potent activity against HIV-1 with low cytotoxicity.

Viral infection inhibition assay. Primary CD4+ T lymphocytes, stimulated as described above, were seeded at a density of 1 x 105 cells/well in a 96-well plate. For the preincubation component of the experiment, compounds were incubated with HIV-1 IIIB (0.3 multiplicity of infection) for 10 min at 37°C, followed by a 1:100 dilution in RPMI complete media and addition to cells for 2 h. Cells were otherwise infected with HIV-1 IIIB (0.3 multiplicity of infection) for 2 h in the absence or presence of compound. After 2 h, cells were washed and subsequently cultured for 3 days, at which time the cells were washed and supplied with new media supplemented with IL-2 (20 U/ml). Following an additional 3 days, HIV-1 release into the supernatant was measured using an HIV-1 p24 antigen enzyme-linked immunosorbent assay (catalog no. 0801169; ZeptoMetrix Corporation, Buffalo, NY). The infectivity remaining was expressed relative to mock-treated, HIV-1-infected cells.

Cytotoxicity assay. Stimulated primary CD4+ T lymphocytes, seeded at a density of 1 x 105 cells/well in a 96-well plate, were incubated with compound or mock exposed for 2 h at 37°C. Levels of cell viability were determined immediately following exposure or six days postexposure using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (Cell Titer 96 aqueous nonradioactive cell proliferation assay; Promega, Madison, WI).

Cytotoxicity was also determined by flow cytometry. Briefly, following a 2-h incubation with NB325, stimulated CD4+ T lymphocytes were reacted with fluorochrome-conjugated Annexin V (BD Biosciences, San Jose, CA), which indicates early apoptosis (51), and propidium iodide (BD Biosciences, San Jose, CA), which indicates necrotic cell death (23).

Flow cytometry. Primary CD4+ T lymphocytes, stimulated as described above, were incubated with NB325 or mock exposed for 2 h at 37°C. Following incubation, cells were washed three times with Hanks' balanced salt solution (Cellgro, Herdon, VA) supplemented with 3% fetal bovine serum and 0.02% NaN3 and then reacted with fluorochrome-conjugated antibodies or their corresponding isotype controls for 45 min at 4°C. Assays included the following antibodies: anti-CD4 (clone RPA-T4; BD Biosciences, San Jose, CA), anti-CD3 (clone UCHT1; eBioscience, San Diego, CA), anti-CD45RO (clone UCHL1; BD Biosciences, San Jose, CA), and anti-CXCR4 (clones 12G5 and 1D9 [BD Biosciences, San Jose, CA] and clone 173 [R&D Systems, Minneapolis, MN]). Isotype control antibodies used in these studies included the following: mouse immunoglobulin G1 (IgG1) (eBioscience, San Diego, CA), mouse IgG2a (eBioscience, San Diego, CA), mouse IgG2b (eBioscience, San Diego, CA), and rat IgG2a (BD Bioscience, San Jose, CA). Antibodies (and their corresponding isotype controls) were used in the following quantities per assay: anti-CD4 and anti-CXCR4, 1 µg; anti-CD45RO, 0.06 µg; and Annexin V, 0.01 µg (as described by the manufacturer). Subsequently, cells were washed with supplemented Hanks' balanced salt solution and then fixed with 1% formaldehyde at 4°C.

Samples were collected on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and analyzed using Flow Jo software version 8.5.3 (Tree Star, Ashland, OR). Annexin V analysis was performed on every sample, and Annexin V-positive cells were not included in the evaluation of experimental results. Isotype antibodies were used as controls as appropriate. Briefly, Annexin V-negative cells were selected based on a control sample prepared by mixing viable cells with cells induced to undergo apoptosis. Viable cells were then gated for the selection of memory CD4+ T lymphocytes based on the results obtained with corresponding isotype control antibodies. CD45RO+ CD4+ cells were selected and examined for expression levels of CXCR4.

Chemotaxis assay. Primary CD4+ T lymphocytes were seeded at 1 x 106 cells in 0.1 ml in the upper well of a 24-well transmigration chamber (5-µm-pore-size polycarbonate filter; Transwell, Costar, Cambridge, MA). NB325, CXCL12 (250 ng/ml), or a combination of NB325 and CXCL12 in 0.6 ml of RPMI complete media was added to the lower well. Plates were incubated for 2 h at 37°C. Following the incubation, cells that had migrated to the lower chamber were counted using trypan blue dye exclusion on a hemacytometer. Chemotaxis in the presence of CXCL12 and NB325 is expressed relative to the extent of cell migration induced by CXCL12 alone.

Competitive binding assay. Competitive binding between CXCL12 and NB325 was examined using a Fluorokine-based assay as described by the manufacturer (R&D Systems, Minneapolis, MN). Briefly, primary CD4+ T lymphocytes were incubated with NB325, biotinylated CXCL12, biotinylated negative control reagent, NB325 plus biotinylated CXCL12, or NB325 plus biotinylated negative control reagent or were mock exposed for 1 h at 4°C. Avidin-fluorescein was added to each sample (except the mock-exposed sample) and incubated for 1 h at 4°C. As a control, CXCL12 was preincubated with the supplied blocking antibody for 15 min at room temperature before being added to cells. Subsequently, cells were washed three times with 1x RDF-1 buffer and reacted with fluorochrome-conjugated antibodies specific for CD45RO (clone UCHL1) and CXCR4 (clone 12G5) or their corresponding isotype control antibodies for 45 min at 4°C. Cells were washed again with 1x RDF-1 buffer and fixed with 1% formaldehyde. Samples were collected for flow cytometry and analyzed using Flow Jo. Annexin V antibodies were included in every sample examined, and Annexin V-positive cells were excluded from all analyses.

Peptide competitive binding assay. Interactions between CXCR4-derived peptides and NB325 were investigated using flow cytometry. ECL2 (amino acids 182 to 196; sequence, DRYICDRFYPNDLWV) and N-terminal (amino acids 1 to 14; MEGISIYTSDNTYE) peptides derived from CXCR4 (3, 30) were obtained from Leinco Technologies (St. Louis, MO). Scrambled peptides corresponding to ECL2 (DDCIYRRDLWFVYP) and N-terminal (ISTIGNDYYETMES) peptides were custom produced by Genscript Corporation (Piscataway, NJ). Stimulated primary human CD4+ T lymphocytes were incubated with NB325, peptide, or NB325 plus peptide (at a ratio of 1:1, 3:1, or 10:1) or were mock exposed for 2 h at 37°C. Cells were collected for flow cytometry and analyzed by Flow Jo as described above.


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RESULTS
 
HIV-1 IIIB infection is inhibited by NB325. Previous studies demonstrated that BG-based compounds (including PEHMB) inhibited HIV-1 infection with greater efficacy when incubated with target cells and virus compared to virus alone (26). In similar experiments using primary human CD4+ T lymphocytes as the target cells, NB325 inhibited HIV-1 IIIB infection with 50% inhibitory concentrations (IC50s) of approximately 0.01% (~71 µM) (Fig. 1B). The antiviral activity of NB325 following preincubation with virus was comparable to the antiviral activity achieved when compound and virus were added concurrently to PBMCs. In contrast, preincubation of DS and cell-free virus resulted in an increase in antiviral activity (0.001% to 0.00016% IC50). Compound cytotoxicity did not impact the calculation of the IC50, since NB325 was not cytotoxic at concentrations below 0.0316% and 0.01%, as indicated by Annexin V and propidium iodide, respectively (data not shown). As measured by the MTS assay, NB325 cytotoxicity following a 2-h exposure to NB325 was evident only at concentrations exceeding 0.316% (Fig. 1C). Cell viability after compound exposure and 6-day incubation in the absence of compound was similar to that measured immediately after exposure (Fig. 1C), indicating the absence of any delayed cytotoxicity that may have affected the outcome of the antiviral assay.

NB325 inhibits antibody recognition of CXCR4 but not CD4 or CD3. The results of the previous experiment (Fig. 1) were consistent with the hypothesis that the antiviral activity of NB325 involves (i) interactions with cell surface molecules involved in HIV-1 infection or (ii) inhibition of dynamic events (e.g., gp41-driven fusion) that occur as the virus and cell interact. To examine this hypothesis, the effect of NB325 exposure on the recognition of CD4 and CXCR4 epitopes by flow cytometry was examined. Following a 2-h exposure of stimulated CD4+ T lymphocytes to a range of NB325 concentrations, no change in the recognition of CD4 by the RPA-T4 antibody was observed at any concentration examined (Fig. 2). Similar results (data not shown) were obtained using the OKT4 antibody, which binds to an epitope distinct from that recognized by the RPA-T4 antibody (6, 41). In contrast, the recognition of CXCR4 by the 12G5 antibody was decreased in a concentration-dependent manner by NB325. Recognition of CD3 (a cell surface molecule not involved in HIV-1 attachment and entry) was also unaffected by the presence of NB325, supporting the conclusion that the reduction in CXCR4 epitope recognition was the result of a specific interaction with NB325.


Figure 2
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  		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.

Interactions between NB325 and CXCR4 are domain specific. Introduction of NB325 may have simply blocked the interaction between the CXCR4-specific antibody and its corresponding epitope, or it may have caused CXCR4 internalization, as does CXCL12, which is the natural ligand of CXCR4 (17, 33). To examine these possibilities, the effect of NB325 on the recognition of CXCR4 epitopes was investigated using the 12G5 antibody in conjunction with two additional antibodies specific to different CXCR4 epitopes (Fig. 3). Monoclonal antibody 12G5 recognizes a conformation-dependent epitope involving the first and second extracellular domains (ECL1 and ECL2) of CXCR4 (14). CXCR4-specific antibody 173 (R&D Systems) preferentially recognizes ECL2 (8), while the epitope recognized by antibody 1D9 (BD Biosciences) is contained within the N terminus of CXCR4 (16).


Figure 3
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  		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).

As shown in the previous experiment, exposure of stimulated memory CD4+ T lymphocytes to NB325 resulted in a concentration-dependent decrease in the recognition of CXCR4 ECL1 and ECL2 epitopes by antibody 12G5. An even greater decrease in ECL2 epitope recognition by antibody 173 was observed in the presence of NB325 (Fig. 3). However, there was no change observed in the recognition of the CXCR4 N terminus in the presence of NB325 when using the 1D9 antibody. Antibody-dependent changes in CXCR4 epitope recognition have been shown as scatter plots (Fig. 3A) and summarized in graph form (Fig. 3B). From these results, it has been concluded that (i) NB325 interacts with the ECL2 of CXCR4, thus inhibiting the binding of monoclonal antibodies 12G5 and 173, and (ii) NB325 does not interact with the N terminus of CXCR4, as indicated by the undiminished recognition of CXCR4 in the presence of NB325.

The previous results also suggested that CXCR4 internalization was not induced by NB325. CXCL12, the sole ligand for CXCR4 (4, 5, 34), has been shown to inhibit infection by X4 strains of HIV-1 (19). The effect of CXCL12 on HIV-1 infection is twofold. First, binding sites for CXCL12 and HIV-1 gp120 on CXCR4 are distinct but overlapping (12, 46). Therefore, CXCL12 acts as a physical inhibitor of HIV-1 binding (1). Second, CXCL12 induces CXCR4 recycling as a means to regulate CXCL12-induced signaling. As a result, less CXCR4 is available on the cell surface to support HIV-1 infection (1). To confirm that the differential recognition of CXCR4 following NB325 exposure was not the result of induced receptor recycling, CXCR4 detection by antibody 1D9 following exposure to NB325 was examined in greater detail. The average recognition of surface CXCR4 by 1D9, as indicated by mean fluorescence intensity (MFI), was unchanged after exposure to NB325 (data not shown). In contrast, incubation of T lymphocytes with CXCL12 resulted in a clear reduction in MFI (data not shown). These results indicated that NB325 does not induce CXCR4 internalization.

NB325 affects CXCL12-induced signaling through CXCR4 without affecting CXCL12 binding. One possible outcome of signaling initiated by CXCL12 through CXCR4 is the induction of cell chemotaxis (52). To examine the effect of NB325 on this specific CXCR4 function, chemotaxis assays were performed using a transmigration experimental design (Fig. 4). In these experiments, CXCL12 potently induced the migration of CD4+ T lymphocytes. However, in the presence of NB325, CXCL12-induced chemotaxis was effectively inhibited at concentrations as low as 0.0003%. NB325 alone did not induce the migration of CD4+ T lymphocytes (data not shown). These results demonstrate functional inhibition of CXCR4 by NB325 and provide further support for a specific interaction with CXCR4.


Figure 4
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  		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).

The implication of the preceding result was that NB325 was inhibiting chemotaxis by preventing CXCL12 binding. CXCL12 binds CXCR4 through a two-site binding and activation model. First, CXCL12 binds to the N terminus of CXCR4. This binding induces a conformational change in the receptor, allowing for the interaction between CXCL12 and ECL2, through which signaling is then initiated (12, 18, 19, 48, 54). To determine the effect of NB325 on CXCL12 binding, stimulated CD4+ T lymphocytes were incubated with labeled CXCL12 in the absence or presence of NB325. In the presence of NB325, the binding of CXCL12 was unchanged relative to CXCL12 binding in the absence of NB325 (Fig. 5A). In contrast, preincubation of CXCL12 with a blocking antibody specific to CXCL12 completely abrogated the binding of CXCL12 (Fig. 5A). Because these experiments were performed at 4°C, additional analyses were performed to confirm NB325 binding and its impact on CXCR4 detection at this temperature. As shown in Fig. 5B, the partial reduction in CXCR4 epitope recognition by antibody 12G5 and the greater reduction in CXCR4 epitope recognition by antibody 173 caused by exposure to NB325 were still evident at 4°C and comparable to results obtained at 37°C (Fig. 3).


Figure 5
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  		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).

The effect of CXCL12 occupancy of CXCR4 on antibody-mediated recognition of CXCR4 was also examined (Fig. 5C). The MFI of CXCR4 detection by the N-terminal antibody (1D9) was reduced in the presence of CXCL12 but not NB325 (Fig. 5C). This result was expected, since CXCL12 binds first to the N terminus of CXCR4 in the two-step model of binding (12, 18, 19, 48, 54), and NB325 presumably interacts with ECL2 but not the N terminus. Again, the presence of NB325 had no effect on CXCL12 binding at the N terminus, since the reduced detection of the N terminus caused by CXCL12 was unchanged in the absence or presence of NB325 (Fig. 5C). Correspondingly, the presence of CXCL12 alone did not reduce the detection of either ECL1 or ECL2 (using antibodies 12G5 and 173), suggesting that the conformational change in CXCR4 necessary to facilitate the interaction between CXCL12 and ECL2 does not take place at 4°C. The conclusions from these results were that (i) NB325 was bound to ECL2, while CXCL12 was bound to the N terminus, and (ii) inhibition of CXCL12-induced chemotaxis by NB325 was the result of blocking the interaction of CXCL12 with ECL2 but not blocking CXCL12 binding to the N terminus of CXCR4.

Interactions between NB325 and CXCR4 are specifically abrogated by an ECL2-derived peptide. To further demonstrate the specificity of interactions between NB325 and CXCR4 ECL2, competitive binding analyses were performed using CXCR4-derived peptides to show specific abrogation of the effect of NB325 on CXCR4 epitope recognition. Peptides derived from amino acid sequences in ECL2 and the N terminus of CXCR4 were examined for their effects on differential CXCR4 epitope recognition by antibodies 12G5, 1D9, and 173 in the presence of NB325.

When stimulated CD4+ T lymphocytes were incubated with NB325 and CXCR4 ECL2 peptide at ratios of 1:1 or 3:1 (Fig. 6A), reductions in CXCR4 epitope recognition caused by NB325 were completely abrogated. This effect was concentration dependent, since abrogation of the effect of NB325 was no longer apparent with a greater excess of NB325 (10:1, NB325 to peptide). Consistent with the proposed specificity of the interaction between NB325 and CXCR4, introduction of an N terminus-derived peptide at ratios of 1:1, 3:1, and 10:1 (NB325 to peptide) (Fig. 6B) had no effect on reduced recognition of CXCR4 epitopes by antibodies 12G5 and 173 in the presence of NB325. ECL2 and N-terminal peptides with scrambled sequences also had no effect on the differential recognition of CXCR4 epitopes by NB325 (data not shown), confirming the specificity of the effect of the ECL2 peptide. These results provide further evidence for a specific interaction between NB325 and CXCR4 ECL2 but not with the N terminus of CXCR4.


Figure 6
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  		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).


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DISCUSSION
 
Although the initial working hypothesis was that PBG compounds would disrupt the integrity of the HIV-1 envelope and render the virus noninfectious, our studies involving BG-based compounds with greater antiviral activity and less cytotoxicity relative to PHMB (26) indicated that the mechanism of action was not virucidal in nature but rather related to interactions that take place between the virus and cell surface molecules on the host cell. The present studies provide evidence to support the conclusion that NB325 (also known as PEHMB) functions as a coreceptor inhibitor by interacting specifically with ECL2 and interfering with interactions with this domain of CXCR4 (Fig. 7A). As a result, NB325 inhibits HIV-1 infection by blocking the interaction between the coreceptor binding site on gp120 and ECL2 on CXCR4 (Fig. 7A). Similarly, NB325 inhibits CXCL12-induced chemotaxis by interfering with the initiation of signaling through an interaction between CXCL12 and ECL2 (Fig. 7B).


Figure 7
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  		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.

The experimental observation that NB325, like the natural CXCR4 ligand CXCL12, interacts specifically with CXCR4 and inhibits HIV-1 infection provides further insight into the mechanism of action of NB325. CXCL12 is expressed in a tissue-specific manner as six different splice variants of differing lengths and amino acid contents. All six splice variants contain one or more KHLK (lysine-histidine-leucine-lysine) motifs, which have been identified in numerous proteins (including heparin binding proteins) as BBXB domains (amino acids of "basic-basic-any-basic" charge) (38). Recent studies of these variants demonstrated that the lambda variant of CXCL12 had the greatest antiviral activity against X4 virus (IC50 ca. 25 nM against IIIB). This variant includes the highest number of BBXB motifs (five total) out of all of the CXCL12 splice variants. Furthermore, the order of antiviral activity correlated positively with the number of BBXB motifs found in the six splice variants. These results are consistent with previous experiments (11) that demonstrated the necessity of the C-terminal BBXB domain for the anti-HIV-1 activity of a CXCL12-derived C-terminal peptide. Because each of the BG units in the oligomeric NB325 contributes a positive charge to the molecule, NB325 has a charge structure that may function in a manner similar to the BBXB motifs in CXCL12. These observations suggest that charge and perhaps the relative positioning of the charge play important roles in the mechanism of NB325 antiviral activity.

The demonstrated inhibition of CXCL12-induced chemotaxis by NB325 provides the basis for two conclusions. First, inhibition of one of several functional outcomes of CXCL12-induced signaling provides further support for a specific interaction between NB325 and CXCR4. Second, inhibition of cell recruitment by NB325 suggests that BG-based molecules like NB325 may also have anti-apoptotic and anti-inflammatory potential. Previous studies have shown that gp120 can initiate signaling through CXCR4, resulting in apoptosis of the affected cell (10, 21). In addition, gp120 can also act as a chemotactic factor through CXCR4 (20, 22). As an inhibitor of CXCR4, NB325 may directly inhibit HIV-1 entry and also block CXCR4-mediated events that contribute to viral pathogenesis.

Previous experiments (26) demonstrated that PEHMB also functions as an inhibitor of HIV-1 strain BaL, which uses CCR5 as a coreceptor. Activity against R5 strains of HIV-1, which are thought to play a dominant role in HIV-1 sexual transmission and the initial spread of the virus, suggests the potential for the development of BG-based molecules as microbicidal agents (2, 28). Investigations of the mechanism of action of NB325 (PEHMB) specific to R5 HIV-1 strains are under way. These preclinical studies will advance the development of NB325 and similar compounds as potential anti-HIV-1 agents to be used in a preventative capacity as microbicides or therapeutically as HIV-1 inhibitors.


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ACKNOWLEDGMENTS
 
These studies were supported through a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (8 U19 AI076965), awarded to M.E.L. (Principal Investigator) and B.W. (Co-Principal Investigator), as well as faculty development funds awarded to F.C.K. from the Department of Microbiology and Immunology, Drexel University College of Medicine, and the Institute for Molecular Medicine and Infectious Disease.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Drexel University College of Medicine, Mail stop 1013-A, 245 N. 15th Street, Philadelphia, PA 19102. Phone: (215) 762-7398. Fax: (215) 762-7784. E-mail: fkrebs{at}drexelmed.edu Back

{triangledown} Published ahead of print on 1 December 2008. Back


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Antimicrobial Agents and Chemotherapy, February 2009, p. 631-638, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00866-08
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