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

Glucocorticoid Resistance Caused by Reduced Expression of the Glucocorticoid Receptor in Cells From Human Vascular Lesions

Mechanisms that control the balance between cell proliferation and death are important in the development of vascular lesions. Rat primary smooth muscle cells were 80% inhibited by low microgram doses of hydrocortisone (HC) and 50% inhibited by nanogram concentrations of transforming growth factor- β1 (TGF-β1), although some lines acquired resistance in late passage. However, comparable doses of HC, or TGF-β1, failed to inhibit most human lesion-derived cell (LDC) lines. In sensitive LDC, HC (10 μg/mL) inhibited proliferation by up to 50%, with obvious apoptosis in some lines, and TGF- β1 inhibited proliferation by more than 90%. Collagen production, as measured by [3H]proline incorporation or RIA for type III pro-collagen, was either unaffected or increased in the LDCs by HC. These divergent responses between LDC lines were partially explained by the absence of the glucocorticoid receptor (GR) and heat shock protein 90 mRNA in 10 of 12 LDC lines, but the presence of the mineralocorticoid receptor and 11β- hydroxysteroid dehydrogenase type II. Western blot analysis confirmed the absence of the GR protein in cells lacking GR mRNA. Immunohistochemistry of human carotid lesions showed high levels of GR in the tunica media, but large areas lacking GR in the fibrous lesion. Considering the absence of the GR in most lines, the effects of HC may be elicited through the mineralocorticoid receptor. Functional resistance to the antiproliferative and antifibrotic effects of HC may contribute to excessive wound repair in atherosclerosis and restenosis

Glucocorticoid resistance caused by reduced expression of the glucocorticoid receptor in cells from human vascular lesions

Mechanisms that control the balance between cell proliferation and death are important in the development of vascular lesions. Rat primary smooth muscle cells were 80% inhibited by low microgram doses of hydrocortisone (HC) and 50% inhibited by nanogram concentrations of transforming growth factor- β1 (TGF-β1), although some lines acquired resistance in late passage. However, comparable doses of HC, or TGF-β1, failed to inhibit most human lesion-derived cell (LDC) lines. In sensitive LDC, HC (10 μg/mL) inhibited proliferation by up to 50%, with obvious apoptosis in some lines, and TGF- β1 inhibited proliferation by more than 90%. Collagen production, as measured by [3H]proline incorporation or RIA for type III pro-collagen, was either unaffected or increased in the LDCs by HC. These divergent responses between LDC lines were partially explained by the absence of the glucocorticoid receptor (GR) and heat shock protein 90 mRNA in 10 of 12 LDC lines, but the presence of the mineralocorticoid receptor and 11β- hydroxysteroid dehydrogenase type II. Western blot analysis confirmed the absence of the GR protein in cells lacking GR mRNA. Immunohistochemistry of human carotid lesions showed high levels of GR in the tunica media, but large areas lacking GR in the fibrous lesion. Considering the absence of the GR in most lines, the effects of HC may be elicited through the mineralocorticoid receptor. Functional resistance to the antiproliferative and antifibrotic effects of HC may contribute to excessive wound repair in atherosclerosis and restenosis

Specific inhibition of eIF-5A and collagen hydroxylation by a single agent. Antiproliferative and fibrosuppressive effects on smooth muscle cells from human coronary arteries.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases

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

<div><p>Antiretrovirals suppress HIV-1 production yet spare the <i>sites</i> 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.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrial.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02191657?term=NCT02191657&rank=1" target="_blank">NCT02191657</a></p></div

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

<p>Cultures at maximal luminal barrier function (TER ≥ 1000 ohms / cm²) were left untreated, or were treated with deferiprone at the indicated concentrations, every day <i>via</i> the apical chamber and every other day <i>via</i> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref066" target="_blank">66</a>], TER measurements of untreated and treated wells were made on consecutive days for a week. <i>P</i> values for untreated <i>vs</i>. 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. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref068" target="_blank">68</a>]). Def, deferiprone; closed small black squares, 100 μM deferiprone; closed large black squares, 200 μM deferiprone; open cyan circles, untreated controls.</p

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

<p>Cultures were infected with clinical isolate of HIV-1 on Day 0 as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref043" target="_blank">43</a>]. 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.</p

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

<p>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 7^th dose) and Subject 2 discontinued oral intake on Day 5 (after the 13^th dose), as indicated by the white line segments (for clinical details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#sec002" target="_blank">Results</a>). 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). <b>A</b> and <b>D</b>, HIV-1 RNA copies immediately before the intake of the first dose of deferiprone on Day 1; <b>B</b> and <b>E</b>, HIV-1 RNA copies immediately after the last dose of deferiprone on Day 7; <b>C</b> and <b>F</b>, 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.</p

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

<p>Treatment groups are shown in blue-graded boxes according to oral regimen, analysis groups in red-graded boxes according to pharmacokinetic (c_max [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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g007" target="_blank">Fig 7</a>); and after 8 weeks off-drug (S2) into those who had or who had not shown a S1 viral response (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g008" target="_blank">Fig 8</a>). Note that as defined, Group C consists of Group B <i>plus</i> three specified subjects; for further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#sec002" target="_blank">Results</a>. 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).</p

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

<p>Long-term replenished primary cell cultures were infected with isolate #990,010 on Day 0 as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref043" target="_blank">43</a>] and replenished as indicated by arrowheads; on each occasion, half of the culture was replaced. After one week to establish infection <i>ex vivo</i> (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.</p