Low systemic absorption of topically applied PPAR β/δ antagonists.

<p><b>A</b>. Peak blood concentrations of PPAR β/δ agonist GW501516, and antagonists GSK0660 and compound 3 h, respectively, at 1 h after topical application to skin. Left: Amount of drugs detected in systemic circulation, expressed as fraction of total amount applied, was calculated as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">methods</a>. Right: Drug concentration expressed as molar concentration. <b>B</b>. GSK0660 concentration in blood (left) and total amount of circulating drug as fraction of amount applied (right, calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>) at the indicated time points after drug application. The horizontal dashed line represents the reported IC50 for GSK0660 acting on PPAR β/δ reported previously (300 nmol/L). Data shown represent average ± s.d. of n = 3 mice per data point.</p

Absence of inflammatory changes induced by PPAR β/δ antagonists in skin after topical application.

<p>(a) C57Bl/6j wild type mice were treated with ointments containing GSK0660 or compound 3 h applied twice daily to shaved dorsal skin for one week. Mice were sacrificed 1 h after the last ointment application and skin tissue processed for H&E based histology and mass spectrometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Data shown represent average ± s.d. of n = 3 mice per data point (left) treated with GSK (blue columns) or compound 3 h (red). Representative histology sections of all treated mice are shown on right. The inset in the middle panel shows a section of GSK0660-treated epidermis showing apoptotic looking cells (marked by red arrow head). Horizontal bar represents 5 µm. (b) Representative H&E sections of C57Bl/6j wild type mice treated for one week with either GSK0660 (top) or GSK3787 (bottom). Red arrow-heads denoting apoptotic looking cells.</p

Prevention of epidermal hyperplasia by transdermal application of selective PPAR β/δ antagonists.

<p>Both the PPAR β/δ agonist GW501516 (GW) and the antagonists GSK0660 (GSK) or compound 3 h were applied topically to the skin, as described in the text. Left: representative H&E-stained paraffin-sections of dorsal skin from PPAR β/δ transgenic mice after treatment with ointments containing the indicated drugs for twenty days, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Horizontal bar represents 5 µm. Right: quantification of H&E-based epidermal thickness observed in n = 4 mice per group, performed as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. * p<0.05 in a two-sided independent t-test.</p

Half – life of GSK0660 after topical application to skin.

<p>42 mg of GSK0660 ointment was applied to dorsal skin of C57Bl/6j wild type mice. Mice were sacrificed at the time after drug application indicated in the figure and drug concentration determined by mass spectrometry, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Data shown represent average ± s.d. of n = 3 mice per data point.</p

PPAR β/δ selective antagonists used in this study.

<p>Chemical structures and in vitro pharmacodynamic data shown are taken from the references listed. The structure of the PPAR β/δ selective agonist GW501516 used in this study is given for comparison.</p

Control of PPAR β/δ – mediated skin disease using reduced-frequency application of ointment containing an irreversible PPAR β/δ antagonist.

<p>Skin disease in PPAR β/δ transgenic mice was induced by i.p. injection of the agonist GW501516. Additionally, mice were shaved on their abdomen and were treated with vehicle-ointment or ointment containing either GSK0660 (twice daily) or GSK3787 at the indicated frequencies. Red arrow denotes apoptotic cells noted in the GW-only treatment group. <b>A</b>. Top: Representative H&E stains from 3 different mice in each treatment group. Horizontal bar represents 5 µm. Bottom: Quantification of epidermal thickness (p<0.01 in all treatment groups vs. GW-only). <b>B</b>. Quantification of dermal infiltrate. Data shown represent average ± s.d. of five mice per group.</p

Sensitivity of WT, (<i>R</i>)-PA-824 resistant and transgenic <i>L</i>. <i>donovani</i> promastigotes to nitroheterocyclic compounds.

<p>Sensitivity of WT, (<i>R</i>)-PA-824 resistant and transgenic <i>L</i>. <i>donovani</i> promastigotes to nitroheterocyclic compounds.</p

Characterisation of LdNTR2.

<p>(A) Purification of recombinant LdNTR2 from <i>E</i>. <i>coli</i> BL21(DE3)pLysS [pET15b-LdNTR2]. Lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, pooled fractions from Ni²⁺-affinity chromatography; lane 4, soluble protein following removal of histidine tag; and lane 5, pooled fractions from size-exclusion chromatography. MALDI analysis confirmed that the minor bands represent NTR2 degradation products. (B) Gel filtration profile of the LdNTR2. The inset shows a plot of elution volume against the log molecular mass (MW) of a standard protein mixture (black circles). The red circle represents the elution volume of NTR2. (C) Metabolism of nitroheterocyclic compounds by recombinant NTR2. Initial rates of metabolism were measured in assays containing 100 μM nitro-compound, 100 μM NADPH and 500 nM NTR2. Rates of metabolism with NADPH and NADH alone were 0.0124 ± 0.001 and 0.0084 ± 0.0006 μmol min^-1 mg^-1, respectively. Rates represent the mean ± SD of triplicate measurements. (D) Immunoblots of whole cell extracts (equivalent of 5×10⁶ parasites in each lane) from <i>L</i>. <i>donovani</i> mid-log promastigotes (L), metacyclic promastigotes (M) and axenic amastigotes (A) were probed with LdNTR2-specific polyclonal antiserum. Known amounts of purified recombinant LdNTR2 were loaded as standards for the quantification of the cellular levels of NTR2.</p

Chemical structures of the bicyclic and monocyclic compounds used in this study.

<p>Procedures for the synthesis of DNDI-VL-2098 and CGI-17341 are described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005971#ppat.1005971.s001" target="_blank">S1 Text</a>.</p

The role of NTR2 in resistance to (<i>R</i>)-PA-824 in <i>Leishmania</i>.

<p>(A) Plot of the proteomics data from (<i>R</i>)-PA-824 resistant clone RES III. Each protein identification is represented by a point plotted as the Log₂ of their heavy-to-light isotope ratio (x axis) versus the Log₁₀ value of the intensities of the peptides belonging to each protein (y axis). Proteins plotted in light blue were determined to have significantly different expression levels compared to those in WT parasites, with the most significantly changed protein shown in red (LinJ.12.0730; NADH:flavin oxidoreductase/NADH oxidase, putative). The identity of the proteins found to be consistently over- or under-expressed are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005971#ppat.1005971.s004" target="_blank">S1 Table</a>. (B) Dose-response curve of WT promastigotes (black circles) and promastigotes overexpressing NTR2 (red circles) to (<i>R</i>)-PA-824. EC₅₀ values of 140 ± 4.6 and 3.5 ± 0.2 nM were determined for WT and NTR2-overexpressing parasites, respectively. Overexpression of NTR2 was confirmed by western blotting (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005971#ppat.1005971.s003" target="_blank">S2 Fig</a>). (C) The susceptibility of RES III parasites (red circles) and RES III parasites overexpressing NTR2 to (<i>R</i>)-PA-824 (black circles). RES III parasites were insensitive to (<i>R</i>)-PA-824 at concentrations up to and including 100 μM while these promastigotes overexpressing NTR2 returned an EC₅₀ value of 1 ± 0.02 nM. Data are the mean ± SD of triplicate cultures. (D) Representation of the frame shift and premature termination of NTR2 translation that results from deletion of a cytosine at genomic position 483544 in (<i>R</i>)-PA-824-resistant clones. The frame shift results in a shortened open reading frame and corresponding amino acid sequence (highlighted in bold), with red indicating the frame-shifted part of this sequence.</p