Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

<p>Abstract</p> <p>Background</p> <p>Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture.</p> <p>Results</p> <p>Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression.</p> <p>Conclusion</p> <p>At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication <it>ex vivo</it>. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.</p

Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

<div><p>HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.</p></div

DOHH inhibition, apoptosis, and structure-dependent chelation of intracellular iron.

<p><b>A</b>. Covalent structures of the medicinal chelators DFOX and DEF, and of the antifungal agent CPX and its chelation homolog Agent P2. DFOX, CPX, and Agent P2 interact with iron via a hydroxyurea-like hydroxamate moiety that is similar to the chelating domain of DEF (shaded). Arrows indicate this moiety’s uniform bidentate mode of metal binding. DFOX contains three of these moieties and is a hexadentate chelator. <b>B.</b> Effect of drugs and Agent P2 on the expression of iron-dependent (IRE; hatched bars) and retrovirally-encoded (HIV; filled bars) gene expression in transfected 293T cells. Results (mean ± standard deviation) are expressed relative to untreated controls. <b>C.</b> Inhibition of DOHH activity in H9-HIV by CPX (<i>blue</i>), but not by its chelation homolog Agent P2 (<i>cyan</i>). <i>Triangles</i>, peptide-bound hypusine; <i>squares</i>, peptide-bound deoxyhypusine. <b>D.</b> Induction of apoptosis by CPX and by DFOX. H9-HIV were treated for 24 hr and then assayed by flow cytometry using TUNEL. Results are expressed as percentage of cells that are TUNEL-positive (± SEM).</p

Long-term suppression of HIV-1 infection in PBMC cultures by ciclopirox.

<p>Multiple-donor PBMC cultures were infected with isolate #990,010 and replenished with fresh cells and medium as indicated by arrowheads; on each occasion, half of the culture was replaced. After one week (period <b>1</b>) to establish infection ex vivo, the culture was treated with 30 µM CPX for one month (period <b>2</b>), then the drug was withdrawn (asterisk) and the culture was assayed for viral copy number during three post-treatment months (period <b>3</b>) to monitor for re-emerging productive infection. p24 assays: open circle, HIV-exposed untreated cultures; closed circles, HIV-exposed cultures, treated with CPX. HIV-1 RNA assays: open squares, HIV-exposed untreated cultures; closed triangles, HIV-exposed cultures during CPX treatment; open triangles, HIV-exposed cultures after withdrawal of CPX. Arrows <b>a</b> and <b>b</b> denote the detection limits of the p24 and HIV-1 RNA assays, respectively. Due to the continuous replenishment with freshly isolated uninfected PBMCs, the viability of cultured cells was consistently above 90% as assessed by computerized vital dye exclusion.</p

Inhibitory action of ciclopirox in rapid-onset infection of primary cells.

<p><b>A-C.</b> Blockade of acute HIV-1 infection and activation of HIV-enhanced apoptosis. Uninfected PBMCs from a single-donor were cultured without infection (open symbols) or were infected with 58,500 copies/ml of HIV-1 isolate #990,010 (filled symbols). After 12 hr, CPX was added to 30 µM (open squares) or cultures were left untreated (triangles). HIV-1 p24 (<b>A</b>) and RNA (<b>B</b>) were assayed at intervals and apoptotic cells were enumerated by TUNEL (<b>C</b>). Active retroviral gene expression occurs in Phase I, preceding suppression of apoptosis in Phase II (green line segments). In CPX-treated infected cultures, retroviral gene expression is inhibited in Phase I and apoptosis is activated in Phase II (red line segments). <b>D, E.</b> Response of innate cytokines. Cells were treated as above, except that CPX addition was coincident with infection. IFN-γ (<b>D</b>) and IL-10 (<b>E</b>) were analyzed during Phase I in the same samples by flow cytometric bead assay. Values are the mean of two independent experiments (initial levels in pg/10⁶ vital cells for HIV-exposed CPX-treated, HIV-exposed untreated, and uninfected CPX-treated cells: 657, 209, and 634 for IFN-γ; 34, 13, and 40 for IL-10).</p

Treatment of mouse vaginal mucosa with ciclopirox.

<p>A, B: Histology of vaginal mucosa of medroxyprogesterone-synchronized mice, untreated (A) or intravaginally treated (B) for four consecutive days with the antifungal gynecological formulation of CPX (1% Batrafen Vaginalcrème™, equivalent to 28.8 mM CPX). A1 and B1, stained with hematoxylin-eosin; A2 and B2, stained with anti-active caspase-3. Due to the progestin synchronization of all animals, the vaginal mucosa of untreated (A) and treated (B) animals displays a luminal surface of living cuboidal mucinous cells, overlying uncornified strata of living squamous epithelial cells. C, D: Tissue reactivity to anti-active caspase-3 for two organs known to contain cells undergoing apoptosis, human neonatal thymus (C) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074414#pone.0074414-ACTG1" target="_blank">[216]</a> and mouse ovary (D1-D3) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074414#pone.0074414-Chen2" target="_blank">[217]</a>. Active caspase-3 locates to the nuclei of cortical lymphocytes and folliculogenic cells, respectively, consistent with its established nuclear occurrence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074414#pone.0074414-Kamada1" target="_blank">[115]</a>, and generates a characteristic, punctate staining pattern. Batrafen-treated vaginal mucosa does not display this apoptotic pattern (<b>B2</b>), showing instead the faint cytoplasmic reactivity of untreated controls (<b>A2</b>). The images of <b>B2</b>, evidencing absence of apoptotic cells after vaginal Batrafen exposure, and of <b>D1</b>-<b>D3,</b> evidencing presence of physiologically apoptotic cells in the ovary, were taken from the same longitudinal cut that sections an animal’s entire reproductive tract.</p

Model for the antiretroviral action of ciclopirox and deferiprone via TRAP.

<p>The model is based on two drug effects: (<b>1</b>) enhanced pro-apoptotic activity in cells, and (<b>2</b>) decreased viral suppression of infection-activated apoptosis due to increased viral pro-apoptotic factors and decreased viral anti-apoptotic factors. In uninfected cells, the apoptotic threshold is decreased (<b>1</b>), but they largely escape apoptosis in the absence of infection-activated apoptosis that is released from viral control by drug treatment (<b>2</b>). See text for details. Yellow boxes, cellular events; green boxes, viral events. Events measured in this study are specified in bold within bold-lined boxes; predicted consequences are specified in italics within thin-lined boxes.</p

Apoptotic activity of ciclopirox and deferiprone in uninfected and infected H9 cells.

<p><b>A-C.</b> Apoptosis in H9-HIV cells treated with 30 µM CPX (circles) or 200 µM DEF (triangles) and in untreated controls (squares). The annexin V- positive and 7-AAD - negative population was quantified by flow cytometry (<b>A</b>); cell diameter was quantified by image analysis (<b>B</b>); and live cells were quantified by computerized enumeration of trypan blue-stained samples (<b>C</b>). <b>D.</b> Mitochondrial membrane potential (▵Ψ collapse) and apoptotic proteolysis (89-kDa PARP accumulation) in untreated H9-HIV (red) and H9 (blue) cells. Assays were conducted by flow cytometry 24 hr after plating. Data (mean ± SEM) are calculated as percentage of cell population displaying ▵Ψcollapse or 89-kDa PARP, and <i>P</i> values are indicated. <b>E, F.</b> Concentration-dependent degradation of mitochondrial membrane potential (▵Ψcollapse) in H9-HIV (red) and H9 (blue), exposed for 24 hr to 30 µM CPX (<b>E</b>) or 200 µM DEF (<b>F</b>). Results (mean ± SEM) were obtained by flow cytometry using JC-1 and are expressed relative to untreated control cells. <i>P</i> values are indicated.</p

Antiretroviral activity of ciclopirox in slow-onset infection of primary cells.

<p>Uninfected PBMCs from a single-donor were infected with isolate #990,135. Cultures were left untreated (open squares) or either CPX (red triangles) or Agent P2 (green circles) was added at 48 hr after plating/inoculation to 30 µM (small symbols) or 60 µM (large symbols). HIV-1 protein (p24; <b>A</b>) and copy number (HIV-1 RNA; <b>B</b>) were assayed at 24-hr intervals.</p