Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications

Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNAI were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNAI was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent Tm of ∼60°C. In contrast, the Apical Stem unfolds independently and with much higher apparent Tm of ∼83°C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNAI Central Domain structure essential for PKR inhibitory activity was identified

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

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

<p>The antiretroviral consequences of hypusine pathway inhibition are conceptualized into two categories: <b>i</b>) inhibition <b>before</b> deoxyhypusyl-eIF5A formation (green symbols), which causes lack of hypusyl-eIF5A due to deoxyhypusyl-eIF5A depletion and results in <u><i>HIV-1 suppression</i></u>; and <b>ii</b>) inhibition <b>after</b> deoxyhypusyl-eIF5A formation (red symbols), which causes lack of hypusyl-eIF5A despite deoxyhypusyl-eIF5A accumulation and results in <u><i>HIV-1 ablation</i>.</u> DOHH blockade in primary cells, <i>if they are HIV-infected</i>, coincides with enhanced deoxyhypusyl-eIF5A accumulation jointly with their preferential apoptotic death (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g002" target="_blank">Fig 2B and 2A</a>, respectively; also [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref028" target="_blank">28</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref043" target="_blank">43</a>]), incurring loss of HIV-1 protein, HIV-1 RNA, and HIV-1 DNA (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g004" target="_blank">4</a>) in an irreversible manner (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g005" target="_blank">Fig 5</a>). <b>A</b>, antiretroviral effect by inhibition at the level of ornithine decarboxylase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref117" target="_blank">117</a>]; <b>B</b>, antiretroviral effect by inhibition at the level of S-adenosyl-L-methionine decarboxylase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref114" target="_blank">114</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref117" target="_blank">117</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref118" target="_blank">118</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref121" target="_blank">121</a>]; <b>C</b>, antiretroviral effect by inhibition at the level of S-adenosyl-L-homocysteine hydrolase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref119" target="_blank">119</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref120" target="_blank">120</a>]; <b>D</b>, antiretroviral effect by inhibition at the level of deoxyhypusyl synthase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref115" target="_blank">115</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref116" target="_blank">116</a>]; <b>E</b>, antiretroviral effect by inhibition at the level of deoxyhypusyl hydroxylase (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.s001" target="_blank">S1 Fig</a>; and refs. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref028" target="_blank">28</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref043" target="_blank">43</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref046" target="_blank">46</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref059" target="_blank">59</a>]). 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.ref187" target="_blank">187</a>]). 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; <i>n</i>.<i>d</i>., not determined.</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

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

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

<p>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 (<i>Y</i>), the hypothesized threshold we defined on the basis of cell culture results detailed in the text (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#pone.0154842.g001" target="_blank">Fig 1</a>). In Group B (bottom left) serum levels remained at <149 μM (<i>X)</i>. 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 (<b>Δ</b>log) 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 c_max ≥275 μM; subsequently Subject 2 discontinued oral intake on Day 5 (after the 13^th dose) and Subject 22 discontinued oral intake on Day 3 (after the 7^th dose), as indicated by their white line segments in S1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154842#sec002" target="_blank">Results</a> 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).</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

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