Potential new anti-human immunodeficiency virus type 1 compounds depress virus replication in cultured human macrophages

Abstract

We report that the amiloride analogues 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride inhibit, at micromolar concentrations, the replication of human immunodeficiency virus type 1 (HIV-1) in cultured human blood monocyte-derived macrophages. These compounds also inhibit the in vitro activities of the HIV-1 Vpu protein and might represent lead compounds for a new class of anti-HIV-1 drugs.

FIG. 1.

Chemical structures of amiloride, HMA, and DMA.

FIG. 2.

Effects of HMA on cell viability and proliferation. (A) HeLa cell monolayers were incubated in the presence of various concentrations of HMA (diamonds), DMA (squares), or amiloride (triangles) for 3 days. The cells were then detached from tissue culture wells by treatment with trypsin and were incubated with trypan blue to stain the dead cells. The proportion of live cells was determined by counting in a hemocytometer. (B and C) Effects of HMA at 0.1 μM (squares), 1.0 μM (triangles), and 10 μM (crosses) on the proliferation of CEMT4 cells (B) and T lymphocytes (C) cultured for 5 days. The ordinate shows the number of live cells after trypan blue staining. The diamonds indicate the proliferation of cells in drug-free control cultures. (D) Five-day-old MDMs (approximately 10⁶ cells) were exposed to various concentrations of HMA for 7 days before they were harvested and stained with PI, followed by flow cytometry and fluorescence-activated cell sorter analysis. HMA was dissolved in dimethyl sulfoxide to give a concentration of 500 mM and was then serially diluted 1:9 in 0.1 N HCl and 1:4 in phosphate-buffered saline prior to final dilution in growth medium (RF10/10 [RPMI containing 10% human type AB serum and 10% fetal calf serum]). Solvent controls were prepared in the same way in which the compound was diluted. Ethanol (100%) was used as a positive control. The results are presented as the proportion of viable cells (PI negative) in HMA-treated cultures relative to the number of viable cells in the medium control.

FIG. 3.

Effects of HMA (A), DMA (B), and amiloride (C) on replication of HIV-1_BaL in MDMs. On day 1, 5-day-old macrophages were infected with HIV-1 at an MOI of 0.02/cell, and samples of the culture supernatant were taken at the indicated number of days postinfection for measurement of HIV-1 p24 antigen levels by ELISA. The groups of four bars for each day of measurement represent the average p24 levels for triplicate samples from cultures incubated in the presence of 1 μM HMA, 10 μM HMA, no-drug control, and 20 μM zidovudine, from left to right, respectively. The inset figures (labeled “preincubation”) show the effects of preincubation of identical cultures in the presence of the same concentrations of drug for 1 h prior to infection with HIV-1 and subsequent culture in drug-free medium. Replication of HIV-1 occurred at levels equivalent to those for the untreated controls, indicating that preexposure to compounds did not irreversibly inhibit an enzyme or damage a cellular structure necessary for HIV-1 replication or cell viability. The lower limit of p24 antigen detection is 7 pg/ml.

FIG. 4.

Effects of HMA on release of p24 antigen into MDM culture supernatants (A) and accumulation of intracellular HIV-1 DNA (B and C). Five-day-old MDM cultures were infected with HIV-1_BaL (MOI, 0.02/cell) and exposed to 1, 4, 7, or 10 μM HMA for 10 days, as described in the text. Uninfected macrophages were used as negative controls, and cells infected with HIV-1_BaL in the absence of HMA were considered positive controls. Either supernatants or cell lysates were collected at days 1, 3, 5, 7, and 10 postinfection. (A) Extracellular supernatants were assayed for p24 antigen by ELISA (Coulter). (B) Cell lysates were assayed for HIV DNA by a semiquantitative PCR with input cellular DNA. Simultaneously, a cellular 110-bp β-globin fragment was amplified by PCR. (C) The 320-bp LTR/gag band density, measured by densitometry with a Fluor-S Multi-Imager (gel documentation system; Bio-Rad), is plotted versus the number of days postinfection. By densitometry (data not shown) the β-globin band intensities are close to the control levels in all samples except those exposed to 10 μM HMA on days 7 and 10, in which a decrease of intensity of about 70% was measured. Potentially, this might indicate an increased rate of cell death for cells from this donor in the 10 μM HMA culture. Note, however, that this effect was not seen with HMA at concentrations of 7 μM or lower.

FIG. 5.

Effects of 10 μM HMA on production of p24 antigen (A), HIV-1 DNA (B), and HIV-1 RNA (C) in macrophage cultures. Macrophage cultures (in triplicate) were infected with HIV-1 and exposed to 10 μM HMA or the no-drug control over 14 days. (A) The levels of p24 antigen in culture supernatants, sampled on the indicated days postinfection, were measured by ELISA. As indicated, the solid bars for each day represent the results for the no-drug control and the shaded bars represent the presence of 10 μM HMA. (B) In the same experiment whose results are shown in panel A, total DNA was isolated from duplicate no-drug control and drug-treated macrophages at days 7 and 14 postinfection. PCRs were performed to simultaneously amplify a 320-bp LTR/gag fragment from HIV-1 DNA and a 110-bp β-globin gene fragment from cellular genomic DNA, and samples from the reaction mixtures were run on agarose gels and stained with ethidium bromide. Note that in contrast to the experiment whose results are shown in Fig. 4, with the cells from the donor whose results are shown here, the β-globin band intensity remains near the control level even after 14 days; this is consistent with observations that 10 μM HMA is well tolerated by MDMs from most donors. (C) Total RNA was isolated from control and drug-treated HIV-1-infected macrophages at day 5 and day 9 postinfection. RT-PCRs were performed to simultaneously amplify the 320-bp LTR/gag fragment from HIV-1 RNA and a 196-bp fragment from mRNA for the cellular housekeeping enzyme GAPDH. The groups of three lanes represent serial dilutions of the samples, used to facilitate quantitation by densitometry.