Data and refinement statistics.

<p>Data and refinement statistics.</p

Domain organization and overall structures of <i>Pf</i>CNB-D.

<p>(A) Domain organization of <i>Pf</i>PKG and sequence alignment between <i>Pf</i>CNB-D and <i>Hs</i>CNB-B (Human PKG I). Identical residues are highlighted in yellow and the capping residues in both proteins are highlighted in red. The capping triad residues are also marked with arrows. (B) cGMP and cAMP affinities of <i>Pf</i>CNB domains. Competition FP curves for cGMP are shown on the left and EC₅₀ values on the right. (C) Overall structure of <i>Pf</i>CNB-D without cGMP. The secondary structure elements are labeled. The phosphate binding cassette (PBC) is colored in yellow, the αB and αC helices in light cyan and blue, the N-terminal helices in light green and the β-barrel in gray. The N- and C-termini are labeled with their corresponding residue number seen in the final model. The sulfate ion co-crystallized with the protein is colored with its sulfur in yellow and oxygen in red. (D) Overall structure of the <i>Pf</i>CNB-D:cGMP complex. The structure is shown with the same color scheme as above except for cGMP. The cGMP is colored by atom type (carbon, white; nitrogen, blue; oxygen, red; and phosphorus, orange). All structure images were generated using <i>PyMOL</i> (Delano Scientific).</p

Structural comparison between the apo- and cGMP bound <i>Pf</i>CNB-D.

<p>The apo and <i>Pf</i>CNB-D:cGMP complex structures are aligned at the β-barrel region (not colored). The helical subdomain of the apo structure is colored in light cyan and that of the cGMP complex structure in yellow.</p

Structural comparison between <i>Pf</i>CNB-D and CNB-B and cGMP binding pocket of <i>Pf</i>CNB-D.

<p>(A) The cGMP pockets <i>Pf</i>CNB-D and CNB-B from human PKG Iβ (PDB code: 4KU7) are shown. The cGMP pocket of <i>Pf</i>CNB-D is colored in yellow (left) and the pocket of PKG Iβ CNB-B in gray (right). Key residues that stabilize the C-helix including the capping residues are shown with transparent surface in the following color theme: side chain carbon, black; oxygen, red; nitrogen. A water molecule captured between E483, R484, and Q532 is shown as a blue sphere. The C atoms of glycine residues located between at the αB and αC helices are shown as black spheres. Hydrogen bonds are shown as dotted lines. (B) Detailed interactions between <i>Pf</i>CNB-D and cGMP. Zoomed in views for each cGMP binding site are shown on either side. The backbone amide of A485 is marked with a blue dot. The individual cGMP interacting residues are shown with the following color theme: side chain carbon, black; oxygen, red; nitrogen, blue. The residues binds cGMP with VDW contacts including the capping residues are shown with transparent surface. Hydrogen bonds are shown as dotted lines with their distances in Å units.</p

Long-Acting Beta Agonists Enhance Allergic Airway Disease

<div><p>Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (β₂-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related β₂-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related β₂-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.</p></div

Salmeterol potentially binds aberrantly to the β₂-AR active site and alters signaling function.

<p>(<b>a</b>) Model of the β₂-AR bound to salmeterol. Salmeterol is docked to the β₂-AR bound to hydroxybenzyl isoproterenol (PDB Code: 4LDE) and energy minimized using Coot [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142212#pone.0142212.ref062" target="_blank">62</a>]. The entire model is shown with electrostatic surface potential calculated with APBS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142212#pone.0142212.ref038" target="_blank">38</a>] on the left and a zoom in view of the binding pocket is shown on the right. Salmeterol is colored by atom type, with carbon colored black, nitrogen colored blue, and oxygen colored red. (<b>b</b>) Similar rendering of the β₂-AR binding pocket with adrenaline bound [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142212#pone.0142212.ref048" target="_blank">48</a>], but with the carbon atoms of adrenaline colored yellow. (<b>c-h</b>) A549 human airway epithelial cells were exposed to IL-13 for 5 days and then to DLPC liposome vehicle, albuterol, salmeterol, or nadolol for 5 days after which (<b>c</b>) total and phosphorylated signaling proteins were determined in relation to beta actin as indicated. (<b>d</b>) Induction of <i>Ccl26</i> mRNA as assessed by real time qPCR was determined under similar conditions in A549 cells. *: P < 0.05 determined by ANOVA (n = 3 technical replicates). (<b>e</b>) Total and phosphorylated STAT6 and (<b>f</b>) <i>Ccl26</i> mRNA were quantitated from IL-13-stimulted human bronchial epithelial cells that were exposed to salmeterol or albuterol as indicated. *: P < 0.05 determined by ANOVA (n = 3 technical replicates). The effect of the phosphopeptidomimetic PM-242H on (<b>g</b>) STAT6 phosphorylation and (<b>h</b>) <i>Ccl26</i> mRNA expression were further determined in IL-13-stimulated A549 cells. *: P < 0.05 determined by Mann Whitney test. Data are from one of 3 or more independent and comparable biological experiments.</p

PM-242H inhibits development of allergic lung disease in Balb/c mice.

<p>Mice were treated every other day for two weeks with vehicle (DLPC) or one of two doses of PM-242H (242H; 5 mg or 50 μg i.n.) and challenged with <i>A</i>. <i>niger</i> conidia (AN) or PBS i.n. after which the allergic airway disease phenotype was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142212#pone.0142212.g003" target="_blank">Fig 3</a>. (<b>a</b>) Airway responsiveness (*: P < 0.05 determined by ANOVA), (<b>b</b>) bronchoalveolar lavage fluid inflammatory cells, (<b>c</b>) total lung IL-4-secreting cells, and (<b>d</b>) fungal colony forming units (CFU) recovered from the lungs of infected mice are shown. *: P < 0.05 determined by Kruskal-Wallis test (n = 4 mice/treatment group). Data are from one of 4 independent and comparable biological experiments.</p

Long acting beta agonists promote allergic airway disease.

<p>(<b>a</b>) Wild type mice were challenged every other day intranasally (i.n.) over 10 days with the spores of <i>A</i>. <i>niger</i> (AN) and every day as indicated with the drugs salmeterol (SX), formoterol (FX), carvedilol (CV), albuterol (Alb) or salmeterol or the combination of salmeterol and fluticasone (SX/FT) and compared to mice challenged with vehicles (PBS and dilauroylphosphatidylcholine (DLPC) liposomes) and (<b>b, c</b>) the effect on airway hyperresponsiveness (AHR) was determined (all data from C57BL/6 mice). *: P < 0.05 determined by ANOVA. (<b>d</b>) Genotype matched wild type and β₂-AR- and ßarr2-deficient mice were challenged with salmeterol or DLPC alone i.n. and assessed for airway hyperresponsiveness. *: P < 0.05 determined by ANOVA. The effect of salmeterol on lung (<b>e</b>) IL-4- and (<b>f</b>) IL-17A-secreting cells, (<b>g</b>) total bronchoalveolar lavage fluid (BALF) inflammatory cells (eos: eosinophils; mac: macrophages; lym: lymphocytes; neut: neutrophils) and (<b>h</b>) airway goblet cell metaplasia was determined after <i>A</i>. <i>niger</i> challenge i.n. *: P < 0.05 determined by Mann Whitney test. Data are from one of 3 or more independent and comparable biological experiments with n = 4 mice/treatment group.</p

PM-242H reverses established airway hyperresponsiveness.

<p>(<b>a</b>) Mice were intranasally challenged with <i>A</i>. <i>niger</i> spores daily i.n. for two weeks after which airway responsiveness to acetylcholine was determined. PM-242H was then given i.n. every other day as spore challenges were continued. Airway responsiveness was determined 7 and 14 days after the initial determination and airway inflammation, lung cytokines and pathology were examined at the end of the experiment. (<b>b</b>) Airway responsiveness of vehicle (DLPC) and PM-242H treated animals. *: P < 0.05 determined by ANOVA. (<b>c</b>) Total BALF inflammatory cells. (<b>d, e</b>) Total lung IL-4 and IL-17A-secreting cells. (<b>f</b>) Total recovered lung fungal CFU. (<b>g</b>) Representative lung bronchovascular bundles depicting airway epithelial goblet cell metaplasia (periodic acid-Schiff stain; bar represents 100 μm). *: P < 0.05 determined by Mann-Whitney test (n = 4 or 5 mice/treatment group as indicated). Data are from one of 4 independent and comparable biological experiments.</p

PM-242H reverses salmeterol-dependent exaggerated allergic airway disease.

<p>(<b>a</b>) Mice were treated daily with DLPC, 50 μg of salmeterol (SX), or salmeterol and PM-242H (50 μg) i.n. and challenged intranasally every other day for 10 days with PBS or <i>A</i>. <i>niger</i> (AN) i.n. The effects of combined therapy on (<b>b</b>) airway hyperresponsiveness (*: P < 0.05 determined by ANOVA), (<b>c</b>) total bronchoalveolar lavage fluid (BALF) inflammatory cells (eos: eosinophils; mac: macrophages; lym: lymphocytes; neut: neutrophils), lung (<b>d</b>) IL-4- and (<b>e</b>) IL-17A-secreting cells, and (<b>f</b>) <i>Muc5AC</i> expression was determined. (<b>g</b>) Total fungal colony forming units (CFU) recovered from lungs of challenged mice. *: P < 0.05 determined by Kruskal-Wallis test (n = 4 or 5 mice/treatment group). Data are from one of 4 independent and comparable biological experiments.</p