Novel inhibitory effects of gamma-glutamylcysteine ethyl ester against human immunodeficiency virus type 1 production and propagation

Abstract

The anti-human immunodeficiency virus type I (anti-HIV-1) effects of gamma-glutamylcysteine ethyl ester (gamma-GCE; TEI-2306) were examined in vitro. In initial studies using a vigorously HIV-1-producing human T-lymphocytic cell line, gamma-GCE displayed a novel biphasic repressive effect on chronic HIV-1 infection that was unlike that of other glutathione prodrugs or other reported antioxidants. In high doses, up to a concentration of 2.5 mM, at which neither glutathione (GSH) nor another GSH precursor has shown inhibitory effects, gamma-GCE potently inhibited the production of HIV-1 by a selective cytopathic effect against infected cells, while the viability and growth of uninfected cells were unaffected at the same gamma-GCE concentrations. At lower concentrations (200 to 400 microM), gamma-GCE significantly repressed the virus production from chronically HIV-1-expressing cells without affecting their viability. The discrepancy of the thresholds of the toxic doses between infected and uninfected cells was found to be more than 10-fold. Relatively high doses of gamma-GCE, utilized in acute HIV-1 infection of T-lymphocytic cells, entirely blocked the propagation of HIV-1 and rescued the cells from HIV-1-induced cell death. Furthermore, gamma-GCE at such concentrations was found to directly inhibit the infectivity of HIV-1 within 4 h. Repressive effects of gamma-GCE on acute HIV-1 infection in human primary human peripheral blood mononuclear cells were also demonstrated. Here, the anti-HIV-1 strategy utilizing gamma-GCE is removal of both HIV-1-producing cells and free infectious HIV-1 in vitro, in place of specific immunoclearance in vivo, which might lead to an arrest or slowing of viral propagation in HIV-1-infected individuals.

FIG. 1

Structural formula of γ-GCE and its property as a GSH prodrug. A double line represents the cell membrane. The figure illustrates that γ-GCE permeates the membrane into the cell (closed arrow) and is metabolized to GSH intracellularly (open arrow) through γ-glutamyl cysteine (γ-GC).

FIG. 2

(A) Effects of 2.5 mM γ-GCE and other GSH prodrugs on the growth of uninfected H-9 cells. Symbols: closed circles, γ-GCE; triangles, GSH; squares, NAC; open circles, control. Error bars may not be visible in cases in which variations among experiments were very small. These results are the mean values of two independent experiments. (B) Effects of 800 μM γ-GCE on growth of H-9/IIIB cells. Growth of H-9/IIIB cells in 800 μM γ-GCE (closed circles) and control experiments without γ-GCE (open circles) are shown. Error bars are indicated. The data are the mean values of two independent experiments. Total cell numbers (in millions) were deduced by adjusting raw values with accumulated dilution factors along with the passage of cell culture.

FIG. 3

(A) Effects of 5 mM γ-GCE on growth and viability of uninfected H-9 cells; (B) effects of 400 μM γ-GCE on growth and viability of H-9/IIIB cells. Columns: 1, without γ-GCE; 2, with γ-GCE. After 6 days of treatment, the relative cell number and viability were monitored for each case. Relative cell numbers were calculated, with the mean number of the control cells at the same time point defined as 100%. These data are the mean values of two independent experiments.

FIG. 4

Inhibition of HIV-1 production from H-9/IIIB cells by high concentrations of γ-GCE. γ-GCE, GSH, and NAC were used at concentrations of 1.25 (A) and 2.5 (B) mM. The mean values of two independent experiments are illustrated. Symbols: closed circles, γ-GCE; triangles, GSH; squares, NAC; open circles, control.

FIG. 5

(A) Inhibitory activity of low concentrations of γ-GCE against HIV-1 production from H-9/IIIB cells. The effects of 400 μM (closed circles) and 200 μM (squares) on viral production are shown with control experimental data (open circles). The data are the mean values of two independent experiments. Error bars may not be visible in cases in which variations were very small. (B) Dose-dependent inhibition of HIV-1 production from H-9/IIIB cells by γ-GCE. The data from panel A on day 6 are comparatively summarized with the results of parallel experiments with 800 μM γ-GCE, at which concentration the cytotoxic effects become evident (see Fig. 2B). Relative values were calculated, with the mean value without γ-GCE defined as 100%.

FIG. 6

Inhibition of acute HIV-1 infection by γ-GCE. Cell growth (A), cell viability (B), and release of HIV-1 p24 antigen (C) were monitored along the course of acute HIV-1 infection of H-9 cells in the presence or absence of γ-GCE. The data shown are from a representative of two independent series of experiments. Symbols: closed circles, 1.6 mM γ-GCE; closed squares, 800 μM γ-GCE; closed triangles, 400 μM γ-GCE; open circles, control without γ-GCE. Cell numbers (A) were deduced by multiplying raw values by dilution factors, which had been accumulating during maintenance of growing cells, as described in Materials and Methods.

FIG. 7

Inhibitory effects of γ-GCE on acute HIV-1 infection in human primary PBMCs at higher viral input. HIV-1 p24 antigen was monitored, along the time course of HIV-1 infection, with 170-fold more virions per cell than that used in the H9 experiments. Symbols: closed circles, 2.5 mM γ-GCE; open circles, control without γ-GCE.

FIG. 8

Effect of preincubation of HIV-1 (strain-NL_4-3) with γ-GCE on viral infectivity. Acute-infection experiments using H-9 cells were performed, and viral production was monitored, after preincubation of the virus in the presence of the indicated concentrations of γ-GCE for 4 h. Symbols: closed circles (all on x axis), 2.5 mM; triangles (all on x axis), 1.25 mM; squares, 625 μM; open circles, control without γ-GCE. These results are representative of two independent sets of experiments.