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.

Competing interests: MS, FT & JC are employees of ApoPharma Inc. UMDNJ (now Rutgers, The State University of New Jersey), Cornell University and NIH have one patent ("Method of inhibiting viral replication in eukaryotic cells and of inducing apoptosis of virally-infected cells," United States patent 5,849,587) and four pending patent applications. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. There are no further patents, patent applications, products in development or marketed products to declare.

Funding: This work was supported by a grant from the Foundation of UMDNJ to MBM and HMHA. The BIRCWH (Building Interdisciplinary Research Careers in Women's Health) Program of the National Institutes of Health (HD-1457), in which HMHA was a Scholar, supported this study. MBM and TP were funded by the National Institutes of Health (AI034552 and AI060403). MHP and ECW were supported by the Intramural Research Program of the National Institutes of Health (NIH-NIDCR); BMC by the State of New Jersey, through its Commission on Cancer Research (05-2405-CCR-EO); PEP by the Pediatric AIDS Clinical Trials Group, the National Institutes of Health, and DAIDS (202PVCL05); and ARH by the Foundation IND Synergen. ADL and TDC were recipients of grants (08/50355-1 and 04/040181-6) from The State of SÃ£o Paulo Research Foundation (FAPESP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Here we address the generality of this antiretroviral pro-apoptotic activity and define its mechanism. Our results show that both CPX and DEF overcome retrovirally-induced resistance to apoptosis and activate apoptosis selectively in a chronically HIV-infected CD4 + T cell line. The apoptotic mechanism is triggered through the intrinsic mitochondrial pathway. Prior to apoptosis, CPX suppresses acute infection of primary human cells (peripheral blood mononuclear cells, PBMCs) exposed to patient-isolated HIV-1. Self-sustaining HIV-1 infection in long-term PBMC cultures was effectively cleared by CPX and productive infection did not return after cessation of treatment. Despite its pro-apoptotic activity at low µM concentrations in culture, preferentially in infected cells, topical CPX at mM concentrations did not cause deleterious effects in a mouse model predictive for damage to human tissues. Parallel experiments with DEF led to a double-blinded proof-of-concept trial, to be reported elsewhere (Saxena et al., unpublished data). We propose a novel antiretroviral target, the inhibition of protein hydroxylation, and suggest that CPX and DEF can serve as pioneer drugs for exploratory trials and as leads for chemical optimizations with the goal to obtain selectively cytocidal antivirals.

CPX and DEF are iron-chelating hydroxypyridinones (HOPOs), classified among the 1,2- and 3,4-HOPOs, respectively [25] . Both drugs were identified as candidate inhibitors of protein hydroxylation by searching drug libraries for structures that fit the stereochemical parameters of the catalytic mechanism proposed for 2-oxoacid-utilizing protein hydroxylases [26] – [33] . CPX, DEF, and mimosine, a veterinarily employed DEF analogue, were confirmed to inhibit protein hydroxylation by these dioxygenases at therapeutically achievable concentrations [34] , [35] . The compounds were predicted [36] and confirmed [22] , [34] , [35] , [37] , [38] to be inhibitors of the hydroxylation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyhypusine hydroxylase (DOHH). DOHH is an apparent monooxygenase whose active site pocket contains a non-heme iron center essential for activity and inhibition [39] , [40] . DOHH forms the distinctive hypusine residue of eIF5A, a cellular protein involved in the control of apoptosis [22] , [41] – [48] and the replication of HIV-1 and FIV-1 [22] , [23] , [49] – [54] . DEF and CPX inhibit HIV-1 gene expression at the level of transcript initiation [23] , potentially disrupting viral control over cellular apoptosis, acute infection, and the function of the immune system. Consistent with this hypothesis, DEF triggers apoptosis in a latently HIV-infected cell line after mitogen stimulation, but not in its uninfected parent [22] , although the underlying pro-apoptotic mechanism was not established.

To test the concept of pro-apoptotic therapy in HIV-1 infection, we examined the activity of two medicines previously shown to inhibit HIV-1 gene expression in cellular models and HIV-1 replication in infected PBMCs cultured ex vivo [22] , [23] . Ciclopirox (CPX; 6-cyclohexyl-1-hydroxy-4-methylpyridin-2[1H]-one: e.g., Batrafen™) is a well-tolerated topical fungicide in gynecological and dermatological preparations. Deferiprone (DEF; 3-hydroxy-1,2-dimethylpyridin-4(1H)-one: e.g., Ferriprox™) is a systemically active medicinal chelator administered orally to transfusionally iron-overloaded thalassemia patients. If successful, our approach would bypass expensive and high-risk preclinical stages of de novo drug development by exploiting the off-target activity of approved, globally available non-HIV medicines to define novel, therapeutically desirable on-target effects directly in humans. Precedents for this drug-based drug discovery approach, which builds on easily overlooked side activities of clinically established medicines, include the development of diuretics and oral antidiabetics from the sulfonamide antibiotics, and of antipsychotics from the antihistamines [24] .

The prominent role of apoptosis in HIV/AIDS was recognized early [16] – [18] , suggesting that inhibitors of apoptosis could be combined with antiretrovirals to preserve immune system function by promoting the survival of infected cells and uninfected ‘bystanders’ [13] , [19] . While this suggestion remains viable, the studies reported here support an alternative approach, namely the use of activators of apoptosis for the ablation of pathogenic HIV-infected cells that destroy the immune system. In oncology, the intentional ablation of pathogenic cells by interventions that activate apoptosis is widely practiced and a leitmotif in anti-cancer drug development [20] . Therapeutic recruitment of the apoptotic mechanism has also been exploited to control graft-versus-host disease in patients [21] , but this strategy has not been well explored in virology.

After HIV-1 entry, apoptosis remains functional for a brief period [6] . Marked resistance to pro-apoptotic stimuli occurs in HIV-infected cell lines and cultured primary cells, but not their uninfected counterparts, mediated by retroviral proteins and miRNAs [7] – [10] . In brain and blood, infected monomyelocytic cells are protected against apoptosis [11] . Their stable anti-apoptotic gene expression secures viability as mobile infective units and long-term reservoirs [12] . Only 0.1% of productively infected cells in lymph nodes become apoptotic [13] . Furthermore, HIV-1 re-programs susceptible cells to kill uninfected ‘bystanders’ [9] , [13] , resulting in extensive apoptosis of HIV-specific cytotoxic lymphocytes [14] . T cell depletion, due to virally promoted apoptotic death of uninfected and eventually of infected cells, is the major cause of immune deficiency [12] – [15] .

Human immunodeficiency virus type 1 (HIV-1) evades the innate and adaptive responses of the immune system, and exploits both to its advantage. In susceptible cells, HIV-1 establishes infection that resists clearance by all current antiretrovirals. Only rarely and under special circumstances may combination antiretroviral therapy (cART) restrain HIV-1 from re-establishing productive infection upon cART cessation, eliciting post-treatment control [1] . The continued presence of HIV-1 DNA in these patients reaffirms the robust resistance of HIV-1 to clearance by pharmacological means. A major feature of this resistance is HIV-1 interference with the primal cellular defense against viral invasion and takeover, programmed cell death (apoptosis) [2] – [5] .

Results

CPX and DEF trigger apoptosis in H9 cells via the intrinsic pathway An earlier study showed that treatment of HIV-infected H9 cells (H9-HIV) with DEF dramatically reduced virion formation and p24 levels [22]. The drug caused nuclear condensation and enhanced DNA fragmentation in latently infected ACH-2 cells induced by phorbol ester, but not in parental CEM cells [22]. These observations, together with accumulating data on the inhibitory action of both CPX and DEF on eIF5A hydroxylation [35], [38], HIV-1 infection in PBMCs and viral gene expression in model systems [23], suggested that the two drugs might have a common mode of action via the induction of apoptosis in HIV-infected cells. We first determined whether CPX and DEF elicit apoptosis in H9-HIV cells. Annexin-V binding, cell diameter and cell survival were assayed in a time-dependent manner. By 24 hr, drug treatment led to a ∼5-fold increase in the percentage of the 7-amino-actinomycin D (7-AAD)–negative cell population capable of binding annexin-V to exposed membrane phospholipid phosphatidylserine, with little further increase at 48 hr (Fig. 1A). Both drugs caused a decrease in mean cell diameter of ∼15% within 24 hr and ∼30% within 48 hr (Fig. 1B), indicating apoptotic volume decrease (AVD) [55], an early and prerequisite event in apoptosis [56] that reflects cell dismantling into apoptotic bodies [57] and contrasts with the cell volume increase typical of necrosis [58]. Concomitantly, cell survival decreased ∼2 fold at 24 hr and ∼5 fold at 48 hr (Fig. 1C). CPX and DEF exerted similar effects with similar kinetics on these apoptotic indicators, suggesting that both drugs trigger apoptosis in this T lymphocytic cell line chronically infected with HIV-1. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. Apoptotic activity of ciclopirox and deferiprone in uninfected and infected H9 cells. A-C. 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 (A); cell diameter was quantified by image analysis (B); and live cells were quantified by computerized enumeration of trypan blue-stained samples (C). D. 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 P values are indicated. E, F. Concentration-dependent degradation of mitochondrial membrane potential (▵Ψcollapse) in H9-HIV (red) and H9 (blue), exposed for 24 hr to 30 µM CPX (E) or 200 µM DEF (F). Results (mean ± SEM) were obtained by flow cytometry using JC-1 and are expressed relative to untreated control cells. P values are indicated. https://doi.org/10.1371/journal.pone.0074414.g001 Collapse of the mitochondrial membrane potential, Δψ, is an early event in apoptotic death triggered via the intrinsic pathway, leading to proteolytic activation of initiator and effector caspases including caspase-3 [59]. One consequence of caspase-3 activation is the cleavage of poly (ADP-ribose) polymerase (PARP), resulting in the accumulation of an 89-kDa PARP fragment indicative of nuclear proteolysis [60]. We therefore monitored the Δψ and PARP status of H9 and H9-HIV cells. Flow cytometric analysis showed that both the collapse of Δψ and the cleavage of PARP were attenuated in HIV-H9 cells relative to H9 cells (Fig. 1D). Specifically, Δψ collapse was about half as frequent in HIV-H9 cells as in uninfected H9 cells, and approximately one-third as many cells were positive for PARP cleavage in HIV-H9 cell cultures as in uninfected H9 cultures. These data indicate that apoptosis in H9 cells is triggered via the intrinsic pathway and is attenuated by HIV-1 infection.

Drug-mediated reversal of resistance to apoptosis in HIV-infected cells We next compared the effect of the drugs on apoptosis in H9 and H9-HIV cells. Both CPX and DEF increased the collapse of Δψ in a manner that was concentration-dependent and accentuated by viral infection (Fig. 1E,F). Relative to uninfected cells, HIV-infected cells displayed significantly increased collapse of Δψ at the standard drug concentrations used in this study (30 µM CPX; 200 µM DEF). Furthermore, the H9-HIV cultures exhibited enhanced Δψ collapse at lower drug concentrations (5 and 15 µM CPX; 50 and 100 µM DEF) than in uninfected H9 cells (30 µM CPX; 200 µM DEF). Thus, exposure to CPX or DEF counteracted the HIV-mediated reduction of Δψ collapse and rendered infected cells more susceptible than uninfected cells to this early step of apoptosis. To determine whether the differential effects of the drugs extend into late apoptosis, we measured PARP fragmentation in H9 and H9-HIV cells. CPX caused an ∼8-fold increase in H9-HIV cells positive for 89-kDa PARP, compared to ∼2 fold in H9 cells (Fig. 2A). By 24 hr, twice as many cells stained positive for 89-kDa PARP in the infected treated cultures as in uninfected treated cultures (27% compared to 14%; Fig. 2B). Furthermore, the fluorescence intensity was approximately one order of magnitude higher in the presence of HIV-1 (Fig. 2B). These differences persisted after 48 hr of CPX treatment (Fig. 2C). DEF gave similar but less pronounced effects (Saxena et al., unpublished data). We conclude that the retroviral suppression of initiation and execution of apoptosis [7]–[13] is reversed by the drugs and transformed into enhanced susceptibility of HIV-infected cells to apoptosis (Fig. 1D vs. Fig. 2A). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 2. Ciclopirox increases apoptosis preferentially in HIV-infected H9 cells. A. Increased formation of the caspase-3–fragmented 89-kDa form of PARP in H9-HIV (red) and uninfected H9 (blue) after 24 hr of treatment with 30 µM CPX. Results (mean ± SEM) are presented as the fold-increase in PARP fragment-positive cells relative to untreated cells. B, C. Cell counts over the fluorescence intensity spectrum for 89-kDa PARP reactivity, quantified by flow cytometric single cell analysis after 24 hr (B) and 48 hr (C) of treatment with 30 µM CPX. Percentages of frag-PARP–positive H9-HIV (red) and uninfected H9 (blue) are calculated. https://doi.org/10.1371/journal.pone.0074414.g002

Cellular and viral protein levels during drug-induced apoptosis The enhancement of Δψ collapse suggested that the drugs might repress anti-apoptotic proteins that stabilize Δψ, in particular Bcl-2 [61]. CD4+ T cells isolated from infected individuals have increased Bcl-2 levels compared to uninfected lymphocytes [62] and several reports implicate HIV-1 Tat in preventing apoptosis in persistently infected cells by inducing Bcl-2 transcription [63]–[65]. Since CPX blocks HIV-1 gene expression [23], we determined Bcl-2 expression in H9 and H9-HIV cells. After treatment with CPX for 24 hr, ∼35% of cells stained positive for Bcl-2, irrespective of infection (Fig. 3A). Although uninfected cells displayed a modest dose-dependent decrease in Bcl-2 content, contrasting with a slight increase in infected cells, neither of these measures achieved statistical significance (Fig. 3B). DEF gave similar results (not shown). These data do not support a role for Bcl-2 suppression in the drug-induced enhancement of apoptosis in H9-HIV cells, in accord with conclusions drawn from a study of other agents that cause apoptosis in HIV-infected cells [8]. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. Effects of ciclopirox on cellular and retroviral proteins in H9 cells. A, B. Bcl-2 reactivity of cells was quantified by flow cytometry after 24 hr of treatment with CPX. H9-HIV are shown in red, H9 in blue. A: Cell counts over the fluorescence intensity spectrum for Bcl-2 reactivity in cells treated with 30 µM CPX. B: CPX concentration dependence of Bcl-2 reactivity expressed as the geometric mean of fluorescence (mean ± SEM). C. Response of proteins in H9-HIV cells to 30 µM CPX after exposure for 24 hr (hatched bars) and 48 hr (filled bars). Retroviral and cellular proteins were labeled immunocytochemically and quantified in the same sample by flow cytometry. Data are presented as the geometric mean of fluorescence, normalized to time-identical infected untreated controls (100% values at 24/48 hr: p24, 36.2/38.2; Tat, 168.1/141.6; Rev, 7.8/6.4; Vpr, 1.7/1.7; activated caspase-3, 1.3/1.3). P values for deviation from respective controls are indicated: * = 0.02; ** ≤ 0.004; *** ≤ 0.0004. https://doi.org/10.1371/journal.pone.0074414.g003 Retroviral proteins can control apoptosis. We therefore measured the response of individual retroviral proteins in H9-HIV cells (Fig. 3C) to the shutdown of the HIV-1 promoter by CPX. This drug reduced intracellular p24 by 30% within 24 hr, and by 70% within 48 hr, consistent with results in 293T cells [23]. Intracellular Tat was similarly reduced. Rev was only marginally affected, however, possibly due to its greater stability (the half-life of p24 and Tat is ∼3 hr [66], [67] while that of Rev is at least 16 hr [68]). Paradoxically, the levels of Vpr increased by ∼15% within 24 hr and ∼70% within 48 hr, suggesting a degree of autonomy from transcription-dependent accumulation consistent with previous reports [69], [70]. DEF elicited a similar response, sparing Rev, decreasing p24 and Tat, and increasing Vpr (not shown). The rise in Vpr paralleled that of active caspase-3 (Fig. 3C). Vpr at increased intracellular levels [71] is proapoptotic [72]–[78], and Vpr-driven cell death is characterized by many of the parameters recorded above, such as increased annexin binding [72], changes in cell size and volume [74], Δψ collapse [75], PARP cleavage [76], and DNA fragmentation [77]. By contrast, intracellular Tat displays antiapoptotic activity [63], [65], conducive to establishment of infection and latency [79]. Although Tat and Vpr can each display either anti- or proapoptotic activities depending on the test system [78], [79], in the context of our study the divergence in their levels (Fig. 3C) suggests a functional imbalance between these viral controllers of apoptosis that contributes to the drug-induced apoptotic death of HIV-infected cells (see Discussion).

Termination of infection in drug-treated PBMCs We next asked whether CPX can control established HIV-1 infection in primary cells. Long-term PBMC cultures were employed as a model for on-going, self-sustaining HIV-1 production. As before, infection was initiated by exposure to patient isolate-infected cells. To emulate the bulk flow of susceptible cells from a generative into an infective compartment that occurs in vivo, we followed a replenishment protocol. Freshly isolated uninfected primary cells were infused into the infected cultures at regular intervals during multi-month monitoring of viral parameters. HIV-1 RNA reached the range of 106 copies/ml within a week of patient isolate inoculation (Fig. 7, period 1), and this robust infection was sustained for 4 months (Fig. 7; open squares). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 7. Long-term suppression of HIV-1 infection in PBMC cultures by ciclopirox. 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 1) to establish infection ex vivo, the culture was treated with 30 µM CPX for one month (period 2), then the drug was withdrawn (asterisk) and the culture was assayed for viral copy number during three post-treatment months (period 3) 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 a and 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. https://doi.org/10.1371/journal.pone.0074414.g007 The introduction of CPX on day 7, adjusted daily to maintain a constant level of 30 µM, reduced the virus to the limits of detectability with a week-to-week median decline of ▵log -1.0 (Fig. 7, period 2); individual decline kinetics varied with the donor-isolate combination. Apoptosis parameters (TUNEL and AVD) did not differ from untreated controls (not shown), attributed to the on-going replenishment of the cultures with freshly isolated PBMCs. In the experiment shown here, HIV-1 RNA levels declined during the four week treatment by four orders of magnitude (Fig. 7; closed triangles), whereas the suppression of p24 occurred within 14 days (Fig. 7; closed circles). We attribute the apparent lag in HIV-1 RNA inhibition to the broad dynamic range of the PCR-based RNA assay (note logarithmic scale) compared to the relatively narrow range of the ELISA-based p24 assay (linear scale), and possibly to the packaging of RNA into apoptotic bodies that protect against degradation by RNases [98], [99]. Mathematical modeling (Fig. S2) indicated that the viral RNA level decreases more slowly than the rate calculated for depletion by medium replenishment, arguing against a protocol-related artifactual decline. Evidently, CPX dramatically suppressed viral production in continuous PBMC culture and did not allow viral breakthrough during prolonged monotherapy (≥30 days, Fig. 7). Similar results were obtained with DEF (not shown). By contrast, breakthrough occurs after as few as 20 days of monotherapy with standard antiretrovirals like lamivudine, emtricitabine, zidovudine, nevirapine, or foscarnet [100]–[103], and is prevented only by combining several of these drugs [104]-[107]. To determine whether any productively infected cells survived the suppressive effect of CPX monotherapy, we examined the possibility of viral resurgence following withdrawal of drug. Cultures were maintained for extended post-treatment observation periods and monitored for the re-emergence of HIV-1 RNA (Fig. 7, period 3). Strikingly, after drug cessation (asterisk in Fig. 7) HIV-1 infection did not recur during post-treatment observation periods extending up to 90 days. Similar results were obtained in repeated experiments (not shown). DEF likewise produced off-drug suppression and consistent with apoptotic ablation of infected cells, reduced HIV-1 DNA to the limit of detection (Saxena et al., unpublished data). By contrast, monotherapy with standard antiretrovirals (including zidovudine, lamivudine, nevirapine, delavirdine, loviride, tenofovir, ritonavir, indinavir, saquinavir, stavudine, festinavir, didanosine, or emitricitabine [108], [109]) uniformly fails to delay resurgence of HIV-1 production for more than 3 days off drug, despite an initial report that HIV-1 “became negative” [110]. Marked reduction of HIV-1 RNA without off-drug resurgence requires combination of several of these antiretrovirals [104]–[106]. We conclude that the apparent functional sterilization of HIV-infected primary cultures treated with CPX or DEF correlates with the preferential apoptotic ablation of HIV-infected cells, and thus the destruction of the proviral reservoir, by each of these drugs.