Persistent interactions between biguanide-based compound NB325 and CXCR4 result in prolonged inhibition of human immunodeficiency virus type 1 infection

Abstract

We previously demonstrated that the biguanide-based compound NB325 inhibits human immunodeficiency virus type 1 (HIV-1) infection by interacting with the CXCR4 viral coreceptor. This interaction also appeared to be persistent, since HIV-1 infection was inhibited even when the virus was introduced subsequent to the removal of NB325 from the cell culture medium. The present studies were conducted to determine the extent and mechanism of this prolonged antiviral activity. Persistent inhibition of HIV-1 infection by NB325 was concentration dependent and was apparent up to 8 h after removal of the compound. Flow cytometric analyses of stimulated CD4(+) T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 extracellular loop 2 epitope recognition that were maintained up to 24 h after removal of the compound. CXCL12-induced chemotaxis was also persistently inhibited following pre-exposure to NB325. These results demonstrate that persistent inhibition of X4 HIV-1 infection by NB325 involves extended perturbation of the viral coreceptor CXCR4.

FIG. 1.

Polyethylene hexamethylene biguanide (PEHMB) structure. The structural formula and space-filling model of PEHMB (also known as NB325) are shown. PEHMB consists of alternating ethylene and hexamethylene linkers connecting biguanide subunits. The compound is readily soluble in water and polydisperse, with molecular weights ranging from approximately 900 to 1,900 (median molecular weight, 1,400).

FIG. 2.

NB325 pre-exposure results in persistent protection from infection by HIV-1 strain IIIB. (A) Stimulated CD4⁺ T lymphocytes were infected with HIV-1 strain IIIB in the absence or presence of NB325 or dextran sulfate (DS) for 1 h at 37°C. These results were used to establish the NB325 IC₅₀ (0.018%) and IC₉₀ (0.05%) for subsequent experiments. The IC₅₀ and IC₉₀ for DS were established similarly. Stimulated CD4⁺ T lymphocytes were incubated with the IC₅₀ (B) or IC₉₀ (C) of NB325 or DS for 1 h at 37°C. Cells were then challenged with HIV-1 IIIB immediately after compound washout (0 h) or at up to 24 h after exposure and washout. The remaining infectivity is expressed as the percentage relative to the results for infected, mock-exposed cells. Each graph is representative of data from two independent experiments in which each data point was examined in triplicate. Statistical significance was calculated in comparison to results for mock-exposed cells (unless otherwise indicated) using the two-tailed, unpaired Student's t test (*, P ≤ 0.05, and ***, P ≤ 0.001).

FIG. 3.

IC₅₀ and IC₉₀ of NB325 have no effect on cell viability. Stimulated CD4⁺ T lymphocytes were incubated with compound for 1 h at 37°C and then washed thoroughly. (A) Cytotoxicity at the indicated concentrations was assessed (as described in Materials and Methods) immediately following incubation or at 24 h after exposure. Error bars show standard deviations. (B) NB325 cytotoxicity at the calculated IC₅₀ (0.018%) and IC₉₀ (0.05%) was assessed at the indicated times after exposure. Viability (%) is expressed with respect to that of mock-exposed cells. Each graph is representative of data from two independent experiments in which each data point was examined in triplicate.

FIG. 4.

Epitope-specific disruption of CXCR4 detection caused by pre-exposure to NB325 is apparent up to 24 h after exposure. Stimulated CD4⁺ T lymphocytes were incubated in the absence or presence of NB325 for 1 h at 37°C. Cells were incubated immediately or up to 24 h after NB325 exposure and washout with fluorochrome-conjugated antibodies specific for CD4 (clone RPA-T4), CD45RO (clone UCHL1), and CXCR4 (clones 1D9, 12G5, and 173). The graphs and selected scatter plots (from assays at 0 h and 8 h after exposure to the IC₅₀ and IC₉₀ of NB325) display the frequencies and numbers (respectively) of CD45RO⁺ CD4⁺ T lymphocytes expressing CXCR4 as detected by clones 12G5 (A, B), 173 (C, D), or 1D9 (E), as well as the frequency of CD4 detection (F). Each graph is representative of data from two independent assays in which each concentration was examined in duplicate. Scatter plots depict results from a single, representative experiment. The number in each scatter plot quadrant indicates the percentage of cells (with respect to the total number of sampled cells) found in that quadrant.

FIG. 5.

Pre-exposure to NB325 inhibits CXCL12-induced chemotaxis through CXCR4. (A) Chemotaxis of stimulated CD4⁺ T lymphocytes was induced for 2 h in the presence of CXCL12 alone or CXCL12 and NB325. In a variation of the experiment (*), cells were pre-exposed to NB325 for 1 h, washed, and then immediately exposed to CXCL12 in a transmigration chamber. In this experiment, 100% chemotaxis equated to migration of 470,000 cells. (B) Stimulated CD4⁺ T lymphocytes were incubated with NB325 or mock exposed for 1 h. Following exposure, chemotaxis of pretreated or mock-exposed cells was induced by CXCL12 in a transmigration chamber in the absence of NB325. Induction by CXCL12 was immediate or at the indicated time points after washout. The extent of chemotaxis (%) was calculated with respect to the number of cells that migrated in response to CXCL12 alone (213,000 cells). Results displayed are representative of two independent assays, in which each condition was examined in triplicate. Error bars show standard deviations.

FIG. 6.

NB325 binds persistently to CD4⁺ T lymphocytes in association with CXCR4. Stimulated CD4⁺ T lymphocytes were incubated with fluorochrome-conjugated NB325 for 1 h at 37°C. Following exposure, cells were washed, incubated with fluorochrome-conjugated monoclonal antibodies specific for CD4, CD45RO, and CXCR4, and collected for flow cytometry. (A and B) The graph (A) and selected scatter plots (from assays at 0 h and 8 h after exposure to the IC₅₀ and IC₉₀ of NB325) (B) display the frequencies and numbers (respectively) of memory CD4⁺ T lymphocytes retaining NB325. (C) Graph depicts the MFI of NB325 on the surface of memory CD4⁺ T lymphocytes. The graphed results are representative of three independent assays in which each condition was examined in triplicate. Scatter plots depict results from a single representative experiment. The number in each scatter plot quadrant indicates the percentage of cells (with respect to the total number of sampled cells) found in that quadrant.