Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial

Abstract

Antiretrovirals suppress HIV-1 production yet spare the sites of HIV-1 production, the HIV-1 DNA-harboring cells that evade immune detection and enable viral resistance on-drug and viral rebound off-drug. Therapeutic ablation of pathogenic cells markedly improves the outcome of many diseases. We extend this strategy to HIV-1 infection. Using drug-based lead discovery, we report the concentration threshold-dependent antiretroviral action of the medicinal chelator deferiprone and validate preclinical findings by a proof-of-concept double-blind trial. In isolate-infected primary cultures, supra-threshold concentrations during deferiprone monotherapy caused decline of HIV-1 RNA and HIV-1 DNA; did not allow viral breakthrough for up to 35 days on-drug, indicating resiliency against viral resistance; and prevented, for at least 87 days off-drug, viral rebound. Displaying a steep dose-effect curve, deferiprone produced infection-independent deficiency of hydroxylated hypusyl-eIF5A. However, unhydroxylated deoxyhypusyl-eIF5A accumulated particularly in HIV-infected cells; they preferentially underwent apoptotic DNA fragmentation. Since the threshold, ascertained at about 150 μM, is achievable in deferiprone-treated patients, we proceeded from cell culture directly to an exploratory trial. HIV-1 RNA was measured after 7 days on-drug and after 28 and 56 days off-drug. Subjects who attained supra-threshold concentrations in serum and completed the protocol of 17 oral doses, experienced a zidovudine-like decline of HIV-1 RNA on-drug that was maintained off-drug without statistically significant rebound for 8 weeks, over 670 times the drug's half-life and thus clearance from circulation. The uniform deferiprone threshold is in agreement with mapping of, and crystallographic 3D-data on, the active site of deoxyhypusyl hydroxylase (DOHH), the eIF5A-hydroxylating enzyme. We propose that deficiency of hypusine-containing eIF5A impedes the translation of mRNAs encoding proline cluster ('polyproline')-containing proteins, exemplified by Gag/p24, and facilitated by the excess of deoxyhypusine-containing eIF5A, releases the innate apoptotic defense of HIV-infected cells from viral blockade, thus depleting the cellular reservoir of HIV-1 DNA that drives breakthrough and rebound.

Trial registration: ClinicalTrial.gov NCT02191657.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy. The authors of this manuscript have the following competing interests: Cornell University, as former employer of HMHA, owns two patents that are cited in the Discussion under reference [24] and [105] and that directly relate to material pertinent to this article. Under applicable US law, the term for either patent is expiring or has expired. Rutgers University, as employer of HMHA, MBM, TP, BH, DS, BMC, former employer of PEP, and representative of ARH, owns patents and has filed patents and patent applications to protect aspects of the intellectual property that is disclosed in this manuscript. Rutgers University has granted option rights for its intellectual property to HMHA and MBM, which relates to material one might perceive as pertinent to this article: U.S. Patents 7,244,814 and 7,141,589; U.S. Patent applications 12/840,270; 13/271,190; 10/581,658; 10/527,453; 13/272,190; US Provisional applications 61/805,056 and 62/073,980. No financial interactions (employment, consultancy, compensations, grants, royalties, personal fees, fund raising, etc.) have occurred as a result of this option agreement. The contributions of ARH to this work predate the current employment by Eli Lilly and Company, Indianapolis, USA; the latter did not play a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript; did not provide financial support; and has been listed solely to identify “Current Affiliation” as stated. ApoPharma Inc., Toronto, Canada, the employer of MS, FT, and JC, holds FDA and EMA approvals for commercialization of deferiprone as medicinal chelator to treat iron overload in thalassemic patients, and also holds patents for deferiprone use and use of certain analogs for indications outside of those disclosed for HIV-AIDS in this manuscript. The clinical trial was funded by ApoPharma Inc., Toronto, Canada, whose employees MS, FT, and JC received funding in the form of salary. MS, FT, and JC also collaborated with university-employed colleagues (PEP and HMHA) on the design of that trial and independently collected the trial data. The university-employed scientists HMHA, BH, PEP, ARH, and BMC lead the statistical analysis of these data, unconditionally provided by ApoPharma, and independently decided on their publication, without involvement of any financial instruments or support by ApoPharma in the form of authors' salaries, consultation fees, non-financial assistance, or research materials other than medicine-grade deferiprone (see Materials and Methods). The contributions of MS, FT, and JC to this work occurred as part of their employment by ApoPharma Inc., Toronto, Canada, and by way of their employment may relate to that company’s patents, products in development, or marketed products. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Effect of deferiprone on HIV-1 in the isolate-infected, long-term replenished primary cell model: Dose dependency.

Cultures were infected with clinical isolate of HIV-1 on Day 0 as described [43]. Once self-sustaining infection was established by Day 12, cultures were treated with 100 μM or 200 μM deferiprone for the indicated duration, with a post-treatment observation period of 11 days. Controls were identically maintained without drug. Each p24 value in Panel A is expressed relative to the respective p24 control on the day of each measurement. Upon complete inhibition of p24 (Panel A), HIV-1 RNA measurements commenced (Panel B). Smaller triangles connected by thin line, 100 μM deferiprone; larger triangles connected by thicker line, 200 μM deferiprone; closed symbols, treatment period; open symbols, pre- and post-treatment periods; black asterisks, cessation of medication; bright green line segments, rebound of HIV-1 protein (as p24) and HIV-1 RNA (as copy number) during the post-treatment period at 100 μM deferiprone; red line segment, HIV-1 RNA decline off-drug at the on-drug rate achieved by 200 μM deferiprone; arrowheads, half of culture replenished with fresh medium, drug, and primary cells; blue, control parameters.

Fig 2. Effect of deferiprone in HIV-infected primary cells: Apoptotic DNA fragmentation, deoxyhypusyl-eIF5A accumulation, and DOHH activity.

Apoptotic DNA fragmentation (Panel A) was detected by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) of single-donor peripheral blood mononuclear cells, maintained in short-term non-replenished cultures as described [43]. Cells were left uninfected and/or untreated; or infected immediately after plating with clinical HIV-1 isolate [43] and 12 hours after infection, treated with 200 μM deferiprone. In these single-donor cultures, metabolic labeling with (1,8-3H)spermidine was performed for quantification of the 3H-labelled deoxyhypusyl substrate and of the 3H-labelled hypusine product of cellular DOHH (Panel B), followed by acid hydrolysis, ion-exchange-based separation of the 3H-labeled amino acids, and fractionation as described [28,43,44,124]; the x-axis of the graph shows 8 of the 16 fractions from a representative experiment. The dose-effect curve for inhibition of cellular DOHH by increasing deferiprone concentrations in short-term non-replenished single-donor cultures (Panel C) was calculated from the accumulation of its 3H-labeled substrate and the decrease of its 3H-labeled product; error bars are at the size of the graph symbols and not shown. Black, HIV-infected untreated cells; green, uninfected untreated cells; blue, uninfected deferiprone-treated cells; red, HIV-infected deferiprone-treated cells; large triangles, HIV-infected cells (red) or uninfected cells (blue) treated with 200 μM deferiprone.

Fig 3. Effect of deferiprone in transwell-cultured confluent ECC-1 cells: Epithelial monolayer integrity.

Cultures at maximal luminal barrier function (TER ≥ 1000 ohms / cm2) were left untreated, or were treated with deferiprone at the indicated concentrations, every day via the apical chamber and every other day via the basolateral chamber. To document the spontaneous TER fluctuation in the untreated cultures, and any drug-induced deviation from those fluctuations reflective of epithelial monolayer disruption [66], TER measurements of untreated and treated wells were made on consecutive days for a week. P values for untreated vs. treated cultures are shown at 96, 120, and 144 hours after start of deferiprone. In this system, chemicals that cause TER collapse are evident within the first 24 hours of exposure, as shown earlier (e.g. [68]). Def, deferiprone; closed small black squares, 100 μM deferiprone; closed large black squares, 200 μM deferiprone; open cyan circles, untreated controls.

Fig 4. Post-treatment effect of deferiprone in infected primary cells: Protein, RNA, and DNA of HIV-1.

Cultures were infected with clinical isolate of HIV-1 on Day 0 as described [43]. Once self-sustaining infection was established by Day 7, cultures were treated with 200 μM deferiprone for the indicated duration. Controls were identically maintained without drug. Observation of virological parameters was extended to 10 days after cessation of medication. Viral load in untreated controls, under culture conditions consistently stabilizing within a narrow range at 106 copies/ml throughout month-long experiments (see Figs 1 and 5), are shown for the transit from on-drug to off-drug. Each p24 value is expressed relative to the respective p24 control on the day of each measurement. Results of the HIV-1 DNA determination are expressed according to the A450-based gradation of the assay, which defines A450 readings of <0.350 as ‘0 copies’, emphasized by the green arrow at the HIV-1 DNA axis, and increases stepwise to ‘20+ copies’ at A450 readings above 3.000 (Roche Amplicor HIV-1 DNA Test; see Materials and Methods). Triangles, viral p24; circles, viral RNA; squares, viral DNA; closed symbols, treatment period; open symbols, pre- and post-treatment periods; black asterisk, cessation of medication; red line segment, HIV-1 RNA decline off-drug; arrowheads, half of each culture replenished with fresh medium, drug, and primary cells; blue, control parameters.

Fig 5. Lasting off-drug antiretroviral activity by deferiprone in isolate-infected, long-term replenished primary cell cultures.

Long-term replenished primary cell cultures were infected with isolate #990,010 on Day 0 as described [43] and replenished as indicated by arrowheads; on each occasion, half of the culture was replaced. After one week to establish infection ex vivo (period 1), cultures were treated with 200 μM deferiprone for one month (period 2), then the drug was withdrawn (asterisk) and the cultures were assayed for viral copy number during three post-treatment months to monitor for re-emerging productive infection (period 3). Each p24 value is expressed relative to the respective p24 control on the day of each measurement. Due to the continuous replenishment with freshly isolated uninfected primary cells, the viability was consistently above 95% as assessed by computerized trypan blue exclusion. The detection limits of the HIV-1 RNA assays are indicated. p24 assays: Open circle, HIV-exposed untreated cultures; closed circles, HIV-exposed cultures, treated with deferiprone. HIV-1 RNA assays: Open squares, HIV-exposed untreated cultures; closed triangles, HIV-exposed cultures during deferiprone treatment; open triangles, HIV-exposed cultures after withdrawal of deferiprone; blue, control parameters.

Fig 6. The double blind, placebo-controlled, dose-escalating, multiple-dose study: Arms, subject enrollment, disposition, and analysis.

Treatment groups are shown in blue-graded boxes according to oral regimen, analysis groups in red-graded boxes according to pharmacokinetic (cmax [per threshold]) or viral (HIV-1 RNA [per DTD]) response. Subjects are indicated by number and were dichotomized after one week on-drug (S1) into those who did achieve the threshold of ≥150 μM in serum (Group A) or who did not (Group B [≤149 μM] and Group C [‘below the threshold, unaffected by medication’]) (see Fig 7); and after 8 weeks off-drug (S2) into those who had or who had not shown a S1 viral response (see Fig 8). Note that as defined, Group C consists of Group B plus three specified subjects; for further details, see Results. Yellow highlight, treatment-naïve HIV-infected subject who achieved the threshold of ≥150μM in serum; gray highlight, treatment-naïve HIV-infected subject who achieved ≤149 μM in serum; red asterisk, treatment-naïve HIV-infected subject with decrease of HIV-1 RNA (‘acute responder’); black asterisk, treatment-naïve HIV-infected subject without decrease of HIV-1 RNA (‘non-responder’); white-in-black, subject removed; AE, adverse events; S1, first stage of protocol (one-week treatment); S2, second stage of protocol (eight-week observation).

Fig 7. Threshold concentration-dependent, acute HIV-1 suppression by deferiprone in treatment-naive HIV-infected subjects.

Color-coded curves on left: Deferiprone levels in serum after the first oral dose, shown for each subject. In Group A (top left), serum levels rose to ≥150 μM (Y), the hypothesized threshold we defined on the basis of cell culture results detailed in the text (see also Fig 1). In Group B (bottom left) serum levels remained at <149 μM (X). Color-coded straight lines on right, within S1: HIV-1 RNA levels in plasma, shown for each subject in Group A (top) and in Group B and Group C (bottom). Group C extends the definition of ‘below the threshold’ to include the subjects on placebo. For each subject, viral load is shown immediately before the first dose of deferiprone on Day 1 (A and C) and after the last dose of deferiprone on Day 7 (B and D). The one-week treatment period is designated as AB for Group A, and as CD for Groups B and C. The intra-cohort response (ICR) to treatment is analyzed as the group’s 95% confidence interval (CI)-limited average of its subjects’ log10-transformed viral load on Day 7 (B and D) relative to Day 1 (A and C); this averaged log10 difference (Δlog) is assessed as decrement (-) or increment (+) of each cohort. Extent and significance of the AB vs. CD inter-cohort log10 differences (ICDs) are indicated. Note that in Group A, after the first dose only Subject 2 and Subject 22 achieved cmax ≥275 μM; subsequently Subject 2 discontinued oral intake on Day 5 (after the 13th dose) and Subject 22 discontinued oral intake on Day 3 (after the 7th dose), as indicated by their white line segments in S1 (see Results for clinical details). S1, first stage (‘treat’) of protocol; closed colored symbols and thin lines, intake of 33 mg/kg orally (Subjects 1, 2, 5, 4, 6, 8, 41 [99 mg/kg total daily dose]); closed colored symbols and bold lines, intake of 50 mg/kg (Subjects 19, 20, 22, 24 [150 mg/kg total daily dose]); open symbols and black lines, placebo (Subjects 7, 18, 23).

Fig 8. Persistent HIV-1 suppression after deferiprone cessation in treatment-naive HIV-infected subjects.

Discontinuation trial design (DTD) is used to analyze long-term rebound following HIV-1 RNA—based segregation into a ‘Decrease’ and a ‘No decrease’ cohort, defined by viral load post-drug on Day 7 relative to viral load pre-drug on Day 1. S1, first stage of protocol (one-week treatment); S2, second stage of protocol (eight-week observation). Left: HIV-1 RNA levels in each trial subject immediately before and after the one-week treatment period (S1). Subject 22 discontinued oral intake on Day 3 (after the 7th dose) and Subject 2 discontinued oral intake on Day 5 (after the 13th dose), as indicated by the white line segments (for clinical details, see Results). Right: Presence or absence of a decrease in HIV-1 RNA after the one-week treatment period (S1) segregates subject into the two subsets for the eight-week observation period (S2). Horizontal arrows (at C in S2 of upper panel) delineate the pre-drug viral load on Day 1 (at A in S1 of upper panel), color-coded to an individual’s post-drug viral load on Day 35 and Day 63 (28 and 56 days after drug cessation). Subject 2 did not enter S2 analysis. Subject 22 did enter S2 analysis and reacquired the pre-medication viral load at 4 weeks post-treatment, verified at 8 weeks post treatment (open circles, S2 of upper panel). A and D, HIV-1 RNA copies immediately before the intake of the first dose of deferiprone on Day 1; B and E, HIV-1 RNA copies immediately after the last dose of deferiprone on Day 7; C and F, HIV-1 RNA copies on Day 63 of protocol, i.e. day 56 off-drug. Two-letter combinations indicate the period of the intra-cohort response (ICR). Extent and significance of the inter-cohort differences (ICDs) are indicated for the identified periods.

Fig 9. The cellular hypusine pathway and proline cluster-containing proteins (PccPs) of HIV-1: Spectrum of antiretroviral activity.

The antiretroviral consequences of hypusine pathway inhibition are conceptualized into two categories: i) inhibition before deoxyhypusyl-eIF5A formation (green symbols), which causes lack of hypusyl-eIF5A due to deoxyhypusyl-eIF5A depletion and results in HIV-1 suppression; and ii) inhibition after deoxyhypusyl-eIF5A formation (red symbols), which causes lack of hypusyl-eIF5A despite deoxyhypusyl-eIF5A accumulation and results in HIV-1 ablation. DOHH blockade in primary cells, if they are HIV-infected, coincides with enhanced deoxyhypusyl-eIF5A accumulation jointly with their preferential apoptotic death (Fig 2B and 2A, respectively; also [28,43]), incurring loss of HIV-1 protein, HIV-1 RNA, and HIV-1 DNA (Figs 1 and 4) in an irreversible manner (Fig 5). A, antiretroviral effect by inhibition at the level of ornithine decarboxylase [117]; B, antiretroviral effect by inhibition at the level of S-adenosyl-L-methionine decarboxylase [114,117,118,121]; C, antiretroviral effect by inhibition at the level of S-adenosyl-L-homocysteine hydrolase [119,120]; D, antiretroviral effect by inhibition at the level of deoxyhypusyl synthase [115,116]; E, antiretroviral effect by inhibition at the level of deoxyhypusyl hydroxylase (Figs 1–5 and S1 Fig; and refs. [,–46,59]). Proline (P) clusters are defined as (P)n type, with n≥2; or as (PxP)n type, with x≤2 and n≥1; or any combination of these two types, e.g. PxPxxPP. The numbering of residues in the HIV-1 PccPs follows the reference genome for HIV-1 (HXB2, accession K03455 [187]). AdoHcy, S-adenosyl-L-homocysteine (SAH); SAM, S-adenosyl-L-methionine (AdoMet); dcSAM, S-3-aminopropyl-5'-methyl-thioadenosine; Ado, adenosine; Hcy, L-homocysteine. P, genetically encoded peptidyl proline within the specified viral PccPs; x, a genetically encoded amino acid residue other than peptidyl proline; n.d., not determined.