Airway Smooth Muscle Relaxation Research Articles

Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAAR is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAARs, affording a seemingly logical site of action, the mechanism(s) by which GABAAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction invivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAAR-dependent, as it is not inhibited by the GABAAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3Rs (IP3 receptors).

Tas2R activation relaxes airway smooth muscle by release of Gαt targeting on AChR signaling

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.

Survey of immunopharmacological effects of botulinum toxin in cell signaling of bronchial smooth muscle cells in allergic asthma.

Asthma is a lung disease that has influenced more than 350 million people worldwide. Airway smooth muscle (ASM) spasm leads to airway hyperresponsiveness (AHR) and bronchial obstruction, which are clinical manifestations of an asthma attack. Botulinum toxin (BTX) is a bacteria toxin that acts as muscle relaxant and may have therapeutic effects on AHR and asthma. In this study, the effect of BTX on AHR and related gene expressions was evaluated. An asthma mice model was developed which was treated with BTX in two ways: intranasally (IN) and via nebulization (N) (0.01, 0.1, and 1 U/mL and 10 U/mL, respectively) on days 25, 27 and 29. AHR was evaluated on days 24, 26, 28, and 30, and gene expressions were evaluated for TrkA, TrkB, M1-M5, α7nAChR, TNF-α, and extracellular signal-regulated kinase 2 (ERK2) proteins. For histopathology of the lungs, perivascular and peribronchial inflammation, production of mucus, and goblet cell hyperplasia were studied. On day 24, treatment with BTX (for all doses) had no significant effect on AHR, but on days 26 and 28, AHR was decreased and this continued up to day 30 for all treated groups. Treatment with BTX had no significant effect on the gene expressions of TrkA, TrkB, M1-M5, α7nAChR, TNF-α, and ERK2 proteins, perivascular inflammation, peribronchial inflammation, hyperplasia of the goblet cell and production of mucus. Besides, mice administered with 10 mg/mL BTX perished. The BTX therapy controlled asthma attacks by decreasing AHR and relaxation of ASMs. However, BTX had no significant effect on airway inflammation and production of mucus. While using BTX, it is necessary to prescribe safe doses in order to prevent adverse reactions.

SPLUNC1 is a negative regulator of the Orai1 Ca2+ channel.

Orai1 is a ubiquitously‐expressed plasma membrane Ca2+ channel that is involved in store‐operated Ca2+ entry (SOCE): a fundamental biological process that regulates gene expression, the onset of inflammation, secretion, and the contraction of airway smooth muscle (ASM). During SOCE, Ca2+ leaves the endoplasmic reticulum, which then stimulates a second, amplifying wave of Ca2+ influx through Orai1 into the cytoplasm. Short Palate LUng and Nasal epithelial Clone 1 (SPLUNC1; gene name BPIFA1) is a multi‐functional, innate defense protein that is highly abundant in the lung. We have previously reported that SPLUNC1 was secreted from epithelia, where it bound to and inhibited Orai1, leading to reduced SOCE and ASM relaxation. However, the underlying mechanism of action is unknown. Here, we probed the SPLUNC1‐Orai1 interactions in ASM and HEK293T cells using biochemical and imaging techniques. We observed that SPLUNC1 caused a conformational change in Orai1, as measured using Forster resonance energy transfer (FRET). SPLUNC1 binding also led to Nedd4‐2 dependent ubiquitination of Orai1. Moreover, SPLUNC1 internalized Orai1 to lysosomes, leading to Orai1 degradation. Thus, we conclude that SPLUNC1 is an allosteric regulator of Orai1. Our data indicate that SPLUNC1‐mediated Orai1 inhibition could be utilized as a therapeutic strategy to reduce SOCE.

Elucidating the Bitter Taste Signaling Paradox in Airway Smooth Muscle Relaxation

RATIONALEAirway smooth muscle (ASM) hypersensitivity and hypercontractility play a pivotal role in the pathophysiology of obstructive airway diseases such as asthma. Furthermore, over half of affected individuals are unable to reliably control these disorders using the current standard of care, β‐(adrenergic) agonists. Previously, our group discovered the expression of a novel group of G protein‐coupled receptors (GPCRs), type 2 taste receptors (TAS2Rs), in the human airway which evoke ASM relaxation. Previous work in our lab suggests that TAS2R agonists evoke relaxation in a G protein beta‐gamma subunit‐, phospholipase C beta‐ and calcium‐dependent manner. We hypothesized that the downstream molecular targets of contractile‐related signaling components are differentially regulated by TAS2Rs compared to other GPCRs. The goal of this study was to establish the molecular mechanism by which TAS2Rs induce ASM relaxation.METHODSWe employed a phosphoproteomic approach, involving dual‐mass spectrometry, to determine differences in the phosphorylation status of contractile‐related proteins in ASM following the stimulation of TAS2Rs and other GPCRs. Healthy human ASM cells were treated with the TAS2R agonist chloroquine or β‐agonist isoproterenol alone or in combination with the bronchoconstrictive agonist histamine to study the ability of each relaxant compound to attenuate the histamine‐mediated phosphorylation profile of contractile‐related proteins. These phosphoproteomic profiles were compared and a select set of targets were validated using western blot analysis.RESULTSOur phosphoproteomic data demonstrated that histamine stimulation results in increased phosphorylation of Ras homolog family member A (RhoA) guanine nucleotide exchange factor (ARHGEF) 12 and A kinase anchoring proteins (AKAP) 2 and 12, as well as decreased phosphorylation of RhoA GTPase‐activating protein (ARHGAP) 6. Furthermore, chloroquine treatment attenuated histamine‐induced phosphorylation of ARHGEF12 but not AKAP2 or AKAP12. Interestingly, isoproterenol attenuated histamine‐induced phosphorylation of AKAP2 and AKAP12 but not ARHGEF12. Validation studies using western blot analysis revealed chloroquine mediated inhibition of histamine‐induced RhoA activation and myosin phosphatase target subunit 1 (MYPT1) phosphorylation at the T853 residue, which was not observed with isoproterenol.CONCLUSIONOur findings were able to show that contractile‐related proteins are phosphorylated differentially by chloroquine and isoproterenol to elicit ASM relaxation. Furthermore, our studies strongly suggest the importance of RhoA and the T853 residue on MYPT1 as points of mechanistic divergence between TAS2R and other GPCR pathways. These findings provide further insight into the mechanism by which TAS2R signaling reduces airway contractility and its potential as a novel therapy for obstructive airway disease.

In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β2AR‐Gs Signaling and Function

Objective & HypothesisActivation of β2‐adrenergic receptors (β2ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β2AR desensitization and activation of non‐canonical β2AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β2AR and potential ligands, and ascertain their effects in‐vitro. We hypothesize that activation of specific allosteric site on β2AR could selectively modulate the activity of β‐agonists to overcome these limitations.MethodsWe employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico‐generated β2AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β2AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β2AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways.ResultsDatabase screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β2AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β2AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM.ConclusionThese findings identify a novel allosteric binding site on the β2AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.

Beta‐agonist Profiling Reveals Novel Biased Signaling Phenotypes for the Beta 2‐adrenergic Receptor with Implications in the Treatment of Asthma

Asthma is characterized by chronic airway inflammation and airway smooth muscle (ASM) cell contraction. Current treatments for asthma include beta‐agonists which activate the beta 2‐adrenergic receptor, a G protein‐coupled receptor (GPCR) that promotes Gs‐dependent production of cAMP leading to ASM relaxation. Unfortunately, beta‐agonists can also promote severe side effects including an increased risk of having a severe asthmatic attack that can result in death. Such side effects have been correlated with the action of beta‐arrestins, scaffolding proteins that mediate GPCR desensitization and internalization as well as G protein independent signaling. In this scenario, biased ligands that promote selective receptor interaction with Gs should be beneficial in promoting airway relaxation while attenuating beta‐arrestin‐induced side effects. To test this, we have used high‐throughput screening to identify Gs‐biased beta‐agonists. The initial lead candidates were further analyzed for their ability to modulate receptor induced Gs and Gi interaction, cAMP production, and beta‐arrestin interaction using bioluminescence resonance energy transfer (BRET) analysis. We identified three compounds with Gs‐biased properties which showed minimal beta‐arrestin recruitment. These compounds were also able to decrease beta‐arrestin mediated outputs including receptor internalization and ERK activation. These compounds also showed reduced GRK‐mediated phosphorylation of the receptor as well as decreased agonist‐promoted desensitization in a physiological model of asthma (human ASM cells). In conclusion, these Gs‐biased beta‐agonists may pave the way for the development of more effective drugs for the treatment of asthma and may help to clarify the structural determinants of receptor mediated biased signaling.

Inhibitory Effect of S100A11 on Airway Smooth Muscle Contraction and Airway Hyperresponsiveness.

S100A11 is a member of the S100 calcium-binding protein family and has intracellular and extracellular regulatory activities. We previously reported that S100A11 was differentially expressed in the respiratory tracts of asthmatic rats as compared with normal controls. Here, we aimed to analyze the potential of S100A11 to regulate both allergen-induced airway hyperresponsiveness (AHR) as well as acetylcholine (ACh)-induced hypercontractility of airway smooth muscle (ASM) and contraction of ASM cells (ASMCs). Purified recombinant rat S100A11 protein (rS100A11) was administered to OVA-sensitized and challenged rats and then the AHR of animals was measured. The relaxation effects of rS100A11 on ASM were detected using isolated tracheal rings and primary ASMCs. The expression levels of un-phosphorylated myosin light chain (MLC) and phosphorylated MLC in ASMCs were analyzed using Western blotting. Treatment with rS100A11 attenuated AHR in the rats. ASM contraction assays showed that rS100A11 reduced the contractile responses of isolated tracheal rings and primary ASMCs treated with ACh. In addition, rS100A11 markedly decreased the ACh-induced phosphorylation of the myosin light chain in ASMCs. Moreover, rS100A11 also suppressed the contractile response of tracheal rings in calcium-free buffer medium. These results indicate that S100A11 protein can relieve AHR by relaxing ASM independently of extracellular calcium. Our data support the idea that S100A11 is a potential therapeutic target for reducing airway resistance in asthma patients.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as β2-adrenergic receptor (β2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABAA R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S.

Biased β-Agonists Favoring Gs over β-Arrestin for Individualized Treatment of Obstructive Lung Disease.

Signals from G-protein-coupled receptors (GPCRs) are the most frequently targeted pathways of currently prescribed therapeutics. Rather than being a simple switch, it is now evident that a given receptor can directly initiate multiple signals, and biasing to achieve signal selectivity based on agonist structure is possible. Biased agonists could direct therapeutically favorable pathways while avoiding counterproductive or adverse reaction pathways. For obstructive lung diseases, β2-adrenergic receptor (β2AR) agonists act at these receptors on airway smooth muscle (ASM) cells to open the airways by relaxing ASM, improving airflow and morbidity. However, these receptors signal to the G protein Gs (increasing cAMP and promoting relaxation), but also to β-arrestin (promoting desensitization and a loss of effectiveness). Indeed, β-agonist use is associated with adverse events in asthma pathogenesis and clinical outcomes which are related to desensitization. β-agonists favoring Gs coupling over β-arrestin binding would provide a means of tailoring bronchodilator therapy. In this review, we show how combinatorial methods with a 40 million compound agnostic library led to a new class of biased β-agonists that do not desensitize, providing an opportunity to personalize therapy in patients who experience poor efficacy or adverse effects from traditional balanced agonists.

Development of Inhaled GABAA Receptor Modulators to Improve Airway Function in Bronchoconstrictive Disorders.

We report the modification of MIDD0301, an imidazodiazepine GABAA receptor (GABAAR) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting β2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABAAR binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1β3γ2 GABAAR structural data, suggests binding of the diazepine core between the α1+/γ2- interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 μM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 μM, with rapid onset (15 min) at 25 μM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally.

Relaxant effect of bioactive component compatibility of San-ao decoction on In vitro guinea pig airway smooth muscle: A dose-response relationship study

Background: Component compatibility is important to the modernization of traditional Chinese medicine. Studies have shown that San-ao decoction (SAD) can treat respiratory diseases by relaxing airway smooth muscle (ASM) and reducing airway hyper-responsiveness. However, whether its bioactive components and compatibility also present with similar relaxant effects remains unknown. This study aims to explore the potential relaxant property, dose-response relationship, and underlying mechanisms of the bioactive component compatibility in SAD. Methods: Network pharmacology was primarily used to identify the bioactive components of SAD and uncover its underlying mechanisms. ASM tension force measuring technique was utilized to verify the relaxant and dose-response effects on in vitro guinea pig ASM. Results: We postulated pseudoephedrine hydrochloride (PH), amygdalin (AM), and diammonium glycyrrhizate (DG) to be the bioactive components of SAD, which could effectively relax ASM in a dose-dependent manner on both acetylcholine-induced and spontaneous contraction. Both PH and AM could lead to DG dose–response curve shift. The regression equation of these three bioactive components was Y = −2.048 × X1 + 0.411 × X2 + 14.052 × X3 (X1, X2, X3 representing PH, AM, and DG, respectively). The underlying mechanisms of these components might be associated with the regulation of smooth muscle contraction. Conclusions: PH, AM, and DG are the bioactive components of SAD, which can relax ASM in a dose–response manner and exert a synergistic effect. Clinically, compatibility of these three bioactive components may serve as a new complementary and alternative treatment for respiratory diseases.

Novel Voltage-Gated Potassium Channel Signaling Pathway and Asthma utilizing Bronchoscopy Data

Asthma is a disease characterized by an excess of free-radical nitric oxide, NO, which leads to airway inflammation and obstruction. S-nitrosothiols (SNO’s) are an important regulator in NO signaling, helping to relieve oxidative stress as well as bronchodilate and relax airway smooth muscle cells. SNO’s exert most of their effects through the NO-cysteine interactions found particularly in S-nitrosocysteine (CSNO). The particular S-nitrosocysteine of interest in this paper is L-CSNO due to its ability to inhibit potassium channels associated with poor lung function, thereby improving ventilation in asthmatic patients. Potassium channels, amongst other ion channels as well, are a new target for therapeutic asthma treatment due to their ability to regulate vascular smooth muscle cells. KCN genes from bronchoscopy data from asthma and control patients were sequenced and analyzed against several measures for asthma and general lung function: asthma severity, FeNO, FEV1%, and androgen receptor gene expression. The goal of this study was to determine which KCN genes, and subsequent Kv channels, are associated with better lung function and which are associated with worse lung function/more severe asthma. The most beneficial KCN genes were found to be KCNA1 and KCNA4, whereas the KCN gene associated with the worst lung function was the KCNK6 gene family. Thus, a potential novel signaling pathway for asthma regulation may involve the binding of L-CSNO to the Kv channels encoded by the KCNK6 gene family in order to inactivate them and improve ventilation and overall lung function.

Ginger metabolites and metabolite-inspired synthetic products modulate intracellular calcium and relax airway smooth muscle.

Asthma affects millions of people worldwide and its prevalence is increasing. It is characterized by chronic airway inflammation, airway remodeling, and pathologic bronchoconstriction, and it poses a continuous treatment challenge with very few new therapeutics available. Thus, many asthmatics turn to plant-based complementary products, including ginger, for better symptom control, indicating an unmet need for novel therapies. Previously, we demonstrated that 6-shogaol (6S), the primary bioactive component of ginger, relaxes human airway smooth muscle (hASM) likely by inhibition of phosphodiesterases (PDEs) in the β-adrenergic (cyclic nucleotide PDEs), and muscarinic (phospholipase C, PLC) receptor pathways. However, oral 6S is extensively metabolized and it is unknown if the resulting metabolites remain bioactive. Here, we screened all the known human metabolites of 6S and several metabolite-based synthetic derivatives to better understand their mechanism of action and structure-function relationships. We demonstrate that several metabolites and metabolite-based synthetic derivatives are able to prevent Gq-coupled stimulation of intracellular calcium [Ca2+]i and inositol trisphosphate (IP3) synthesis by inhibiting PLC, similar to the parent compound 6S. We also show that these compounds prevent recontraction of ASM after β-agonist relaxation likely by inhibiting PDEs. Furthermore, they potentiate isoproterenol-induced relaxation. Importantly, moving beyond cell-based assays, metabolites also retain the functional ability to relax Gq-coupled-contractions in upper (human) and lower (murine) airways. The current study indicates that, although oral ginger may be metabolized rapidly, it retains physiological activity through its metabolites. Moreover, we are able to use naturally occurring metabolites as inspiration to develop novel therapeutics for brochoconstrictive diseases.

VIP/PACAP signaling as an alternative target during hyperoxic exposure in preterm newborns.

The use of oxygen therapy (high doses of oxygen - hyperoxia) in the treatment of premature infants results in their survival. However, it also results in a high incidence of chronic lung disease known as bronchopulmonary dysplasia, a disease in which airway hyper-responsiveness and pulmonary hypertension are well known as consequences. In our previous studies, we have shown that hyperoxia causes airway hyper-reactivity, characterized by an increased constrictive and impaired airway smooth muscle relaxation due to a reduced release of relaxant molecules such as nitric oxide, measured under in vivo and in vitro conditions (extra- and intrapulmonary) airways. In addition, the relaxation pathway of the vasoactive intestinal peptide (VIP) and/or pituitary adenylate cyclase activating peptide (PACAP) is another part of this system that plays an important role in the airway caliber. Peptide, which activates VIP cyclase and pituitary adenylate cyclase, has prolonged airway smooth muscle activity. It has long been known that VIP inhibits airway smooth muscle cell proliferation in a mouse model of asthma, but there is no data about its role in the regulation of airway and tracheal smooth muscle contractility during hyperoxic exposure of preterm newborns.

Mechanism deconvolution of Qing Fei Pai Du decoction for treatment of Coronavirus Disease 2019 (COVID-19) by label-free integrative pharmacology assays

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) has a long history in the prevention and treatment of pandemics. The TCM formula Lung Cleansing and Detoxifying Decoction (LCDD), also known as Qing Fei Pai Du Decoction, has been demonstrated effective against Coronavirus Disease 2019 (COVID-19). Aim of the studyThis work aimed to elucidate the active ingredients, targets and pathway mechanism of LCDD related to suppression of inflammatory, immunity regulation and relaxation of airway smooth muscle for the treatment of COVID-19. Materials and methodsMining chemical ingredients reported in LCDD, 144 compounds covering all herbs were selected and screened against inflammatory-, immunity- and respiratory-related GPCRs including GPR35, H1, CB2, B2, M3 and β2-adrenoceptor receptor using a label-free integrative pharmacology method. Further, all active compounds were detected using liquid chromatography-tandem mass spectrometry, and an herb-compound-target network based on potency and content of compounds was constructed to elucidate the multi-target and synergistic effect. ResultsThirteen compounds were identified as GPR35 agonists, including licochalcone B, isoliquiritigenin, etc. Licochalcone B, isoliquiritigenin and alisol A exhibited bradykinin receptor B2 antagonism activities. Atractyline and shogaol showed as a cannabinoid receptor CB2 agonist and a histamine receptor H1 antagonist, respectively. Tectorigenin and aristofone acted as muscarinic receptor M3 antagonists, while synephrine, ephedrine and pseudoephedrine were β2-adrenoceptor agonists. Pathway deconvolution assays suggested activation of GPR35 triggered PI3K, MEK, JNK pathways and EGFR transactivation, and the activation of β2-adrenoceptor mediated MEK and Ca2+. The herb-compound-target network analysis found that some compounds such as licochalcone B acted on multiple targets, and multiple components interacted with the same target such as GPR35, reflecting the synergistic mechanism of Chinese medicine. At the same time, some low-abundance compounds displayed high target activity, meaning its important role in LCDD for anti-COVID-19. ConclusionsThis study elucidates the active ingredients, targets and pathways of LCDD. This is useful for elucidating multitarget synergistic action for its clinical therapeutic efficacy.

Cooperativity between β-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Anti-Asthmatic Effects of Saffron Extract and Salbutamol in an Ovalbumin-Induced Airway Model of Allergic Asthma

Introduction: Asthma is a chronic inflammatory disorder of the airways often characterized by airway remodeling and influx of inflammatory cells into the airways. Saffron (C. sativus) has been reported to possess anti-inflammatory, anti-allergic and immunomodulatory properties. Salbutamol is known to relax airway smooth muscles. Objective: To investigate the combined anti-asthmatic effect of C. sativus extract (CSE) and salbutamol in an ovalbumin (OVA)-induced asthma in rats. Materials and methods: Airway hyperresponsiveness (AHR) was induced in male Sprague-Dawley rats by OVA challenge and treated with CSE (30 mg/kg and 60 mg/kg i.p.) and salbutamol (0.5 mg/kg p.o) for 28 days. After the induction period, various hematological, biochemical, molecular (ELISA) and histological analyses were performed. Results: OVA-induced alterations observed in hematological parameters (total and differential cell counts observed in Bronchoalveolar Lavage Fluid (BALF) were significantly attenuated (p < 0.01) by CSE (30 mg/kg and 60 mg/kg) and salbutamol (0.5 mg/kg). The treatment combination also significantly decreased (p < 0.01) the levels of total protein and albumin in serum, BALF and lung tissues. Treatment with CSE and salbutamol significantly attenuated (p < 0.01) increase in OVA induced Th2 cytokine levels (TNF-α, IL-1β, IL-4, IL-13). Histopathological analysis of lung tissue showed that combined effect of CSE and salbutamol treatment ameliorated OVA-induced inflammatory influx and ultrastructural aberrations. Conclusion: The results obtained from this study show that the combined effect of CSE and salbutamol exhibited anti-asthmatic properties via its anti-inflammatory effect and by alleviating Th2 mediated immune response. Thus, this treatment combination could be considered as a new therapeutic strategy for management of asthma.

Can GPCRs Be Targeted to Control Inflammation in Asthma?

Historically, the drugs used to manage obstructive lung diseases (OLDs), asthma, and chronic obstructive pulmonary disease (COPD) either (1) directly regulate airway contraction by blocking or relaxing airway smooth muscle (ASM) contraction or (2) indirectly regulate ASM contraction by inhibiting the principal cause of ASM contraction/bronchoconstriction and airway inflammation. To date, these tasks have been respectively assigned to two diverse drug types: agonists/antagonists of G protein-coupled receptors (GPCRs) and inhaled or systemic steroids. These two types of drugs "stay in their lane" with respect to their actions and consequently require the addition of the other drug to effectively manage both inflammation and bronchoconstriction in OLDs. Indeed, it has been speculated that safety issues historically associated with beta-agonist use (beta-agonists activate the beta-2-adrenoceptor (β2AR) on airway smooth muscle (ASM) to provide bronchoprotection/bronchorelaxation) are a function of pro-inflammatory actions of β2AR agonism. Recently, however, previously unappreciated roles of various GPCRs on ASM contractility and on airway inflammation have been elucidated, raising the possibility that novel GPCR ligands targeting these GPCRs can be developed as anti-inflammatory therapeutics. Moreover, we now know that many GPCRs can be "tuned" and not just turned "off" or "on" to specifically activate the beneficial therapeutic signaling a receptor can transduce while avoiding detrimental signaling. Thus, the fledging field of biased agonism pharmacology has the potential to turn the β2AR into an anti-inflammatory facilitator in asthma, possibly reducing or eliminating the need for steroids.