Publications by authors named "Malcolm Begg"

33 Publications

Dominant effect of gap junction communication in wound-induced calcium-wave, NFAT activation and wound closure in keratinocytes.

J Cell Physiol 2021 Jun 27. Epub 2021 Jun 27.

Institute of Translational and Clinical Medicine, Medical School, Newcastle University, Newcastle upon Tyne, UK.

Wounding induces a calcium wave and disrupts the calcium gradient across the epidermis but mechanisms mediating calcium and downstream signalling, and longer-term wound healing responses are incompletely understood. As expected, live-cell confocal imaging of Fluo-4-loaded normal human keratinocytes showed an immediate increase in [Ca ] at the wound edge that spread as a calcium wave (8.3 µm/s) away from the wound edge with gradually diminishing rate of rise and amplitude. The amplitude and area under the curve of [Ca ] flux was increased in high (1.2 mM) [Ca ] media. 18α-glycyrrhetinic acid (18αGA), a gap-junction inhibitor or hexokinase, an ATP scavenger, blocked the wound-induced calcium wave, dependent in part on [Ca ] . Wounding in a high [Ca ] increased nuclear factor of activated T-cells (NFAT) but not NFkB activation, assessed by dual-luciferase receptor assays compared to unwounded cells. Treatment with 18αGA or the store-operated channel blocker GSK-7975A inhibited wound-induced NFAT activation, whereas treatment with hexokinase did not. Real-time cell migration analysis, measuring wound closure rates over 24 h, revealed that 18αGA essentially blocked wound closure whereas hexokinase and GSK-7975A showed relatively minimal effects. Together these data indicate that while both gap-junction communication and ATP release from damaged cells are important in regulating the wound-induced calcium wave, long-term transcriptional and functional responses are dominantly regulated by gap-junction communication.
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http://dx.doi.org/10.1002/jcp.30488DOI Listing
June 2021

Exploring PI3Kδ Molecular Pathways in Stable COPD and Following an Acute Exacerbation, Two Randomized Controlled Trials.

Int J Chron Obstruct Pulmon Dis 2021 3;16:1621-1636. Epub 2021 Jun 3.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, UK.

Background: Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) exerts corrective effects on the dysregulated migration characteristics of neutrophils isolated from patients with chronic obstructive pulmonary disease (COPD).

Objective: To develop novel, induced sputum endpoints to demonstrate changes in neutrophil phenotype in the lung by administering nemiralisib, a potent and selective inhaled PI3Kδ inhibitor, to patients with stable COPD or patients with acute exacerbation (AE) of COPD.

Methods: In two randomized, double-blind, placebo-controlled clinical trials patients with A) stable COPD (N=28, randomized 3:1) or B) AECOPD (N=44, randomized 1:1) received treatment with inhaled nemiralisib (1mg). Endpoints included induced sputum at various time points before and during treatment for the measurement of transcriptomics (primary endpoint), inflammatory mediators, functional respiratory imaging (FRI), and spirometry.

Results: In stable COPD patients, the use of nemiralisib was associated with alterations in sputum neutrophil transcriptomics suggestive of an improvement in migration phenotype; however, the same nemiralisib-evoked effects were not observed in AECOPD. Inhibition of sputum inflammatory mediators was also observed in stable but not AECOPD patients. In contrast, a placebo-corrected improvement in forced expiratory volume in 1 sec of 136 mL (95% Credible Intervals -46, 315mL) with a probability that the true treatment ratio was >0% (Pr(θ>0)) of 93% was observed in AECOPD. However, FRI endpoints remained unchanged.

Conclusion: We provide evidence for nemiralisib-evoked changes in neutrophil migration phenotype in stable COPD but not AECOPD, despite improving lung function in the latter group. We conclude that induced sputum can be used for measuring evidence of alteration of neutrophil phenotype in stable patients, and our study provides a data set of the sputum transcriptomic changes during recovery from AECOPD.
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http://dx.doi.org/10.2147/COPD.S309303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184158PMC
June 2021

An Inhaled PI3Kδ Inhibitor Improves Recovery in Acutely Exacerbating COPD Patients: A Randomized Trial.

Int J Chron Obstruct Pulmon Dis 2021 3;16:1607-1619. Epub 2021 Jun 3.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, UK.

Purpose: This study evaluated the safety and efficacy of inhaled nemiralisib, a phosphoinositide 3-kinase δ (PI3Kδ) inhibitor, in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD).

Methods: In this double-blind, placebo-controlled study, 126 patients (40-80 years with a post-bronchodilator forced expiratory volume in 1 sec (FEV) ≤80% of predicted (previously documented)) were randomized 1:1 to once daily inhaled nemiralisib (1 mg) or placebo for 84 days, added to standard of care. The primary endpoint was specific imaging airway volume (siVaw) after 28 treatment days and was analyzed using a Bayesian repeated measures model (clintrials.gov: NCT02294734).

Results: A total of 126 patients were randomized to treatment; 55 on active treatment and 49 on placebo completed the study. When comparing nemiralisib and placebo-treated patients, an 18% placebo-corrected increase from baseline in distal siVaw (95% credible intervals (Cr I) (-1%, 42%)) was observed on Day 28. The probability that the true treatment ratio was >0% (Pr(θ>0)) was 96%, suggestive of a real treatment effect. Improvements were observed across all lung lobes. Patients treated with nemiralisib experienced a 107.3 mL improvement in posterior median FEV (change from baseline, 95% Cr I (-2.1, 215.5)) at day 84, compared with placebo. Adverse events were reported by 41 patients on placebo and 49 on nemiralisib, the most common being post-inhalation cough on nemiralisib (35%) vs placebo (3%).

Conclusion: These data show that addition of nemiralisib to usual care delivers more effective recovery from an acute exacerbation and improves lung function parameters including siVaw and FEV. Although post-inhalation cough was identified, nemiralisib was otherwise well tolerated, providing a promising novel therapy for this acutely ill patient group.
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http://dx.doi.org/10.2147/COPD.S309129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184151PMC
June 2021

Bronchial Epithelial Calcium Metabolism Impairment in Smokers and Chronic Obstructive Pulmonary Disease. Decreased ORAI3 Signaling.

Am J Respir Cell Mol Biol 2019 10;61(4):501-511

Department of Respiratory Diseases and Addictology, Hôpital Arnaud de Villeneuve, Centre Hospitalier Universitaire Montpellier, Montpellier, France.

The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca) signaling. Ca is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca stores was decreased in patients with COPD and that the Ca influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca release, significant in smoker subjects, and to a decrease in Ca influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.
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http://dx.doi.org/10.1165/rcmb.2018-0228OCDOI Listing
October 2019

Translation of Inhaled Drug Optimization Strategies into Clinical Pharmacokinetics and Pharmacodynamics Using GSK2292767A, a Novel Inhaled Phosphoinositide 3-Kinase Inhibitor.

J Pharmacol Exp Ther 2019 06 2;369(3):443-453. Epub 2019 Apr 2.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, United Kingdom (M.B., C.D.E., J.N.H., E.M.H.); Clinical Pharmacology & Model Stimulation, (E.P., R.W.), Drug Metabolism & Pharmacokinetics, (G.V., D.M., J.M.), Refractory Respiratory Inflammation DPU, (S.U.), In Vitro In Vivo Translation, (C.F.N.), Discovery Medicine, (A.C) GlaxoSmithKline, Stevenage, United Kingdom; Global Clinical Science & Delivery, GlaxoSmithKline, Stockley Park, Uxbridge, United Kingdom (M.M., J.G.); In Vitro In Vivo Translation, (A.H.), Drug Product Design & Development, (M.I.H.), GlaxoSmithKline, Ware, United Kingdom (M.I.H.); Department of Biological Chemistry, Babraham Institute, Cambridge, United Kingdom (J.C.); and Clinical Unit Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom (F.E., D.F.)

This study describes the pharmacokinetic (PK) and pharmaco-dynamic (PD) profile of -(5-(4-(5-(((2,6)-2,6-dimethylmorpholino)methyl)oxazol-2-yl)-1-indazol-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (GSK2292767A), a novel low-solubility inhaled phosphoinositide 3-kinase delta (PI3K) inhibitor developed as an alternative to 2-(6-(1H-indol-4-yl)-1H-indazol-4-yl)-5-((4-isopropylpiperazin-1-yl)methyl)oxazole (nemiralisib), which is a highly soluble inhaled inhibitor of PI3K with a lung profile consistent with once-daily dosing. GSK2292767A has a similar in vitro cellular profile to nemiralisib and reduces eosinophilia in a murine PD model by 63% ( = 5, < 0.05). To explore whether a low-soluble compound results in effective PI3K inhibition in humans, a first time in human study was conducted with GSK2292767A in healthy volunteers who smoke. GSK2292767A was generally well tolerated, with headache being the most common reported adverse event. PD changes in induced sputum were measured in combination with drug concentrations in plasma from single (0.05-2 mg, = 37), and 14-day repeat (2 mg, = 12) doses of GSK2292767A. Trough bronchoalveolar lavage (BAL) for PK was taken after 14 days of repeat dosing. GSK2292767A displayed a linear increase in plasma exposure with dose, with marginal accumulation after 14 days. Induced sputum showed a 27% (90% confidence interval 15%, 37%) reduction in phosphatidylinositol-trisphosphate (the product of phosphoinositide 3-kinase activation) 3 hours after a single dose. Reduction was not maintained 24 hours after single or repeat dosing. BAL analysis confirmed the presence of GSK2292767A in lung at 24 hours, consistent with the preclinical lung retention profile. Despite good lung retention, target engagement was only present at 3 hours. This exposure-response disconnect is an important observation for future inhaled drug design strategies considering low solubility to drive lung retention.
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http://dx.doi.org/10.1124/jpet.119.257311DOI Listing
June 2019

Use of autologous Technetium-labelled neutrophils to quantify lung neutrophil clearance in COPD.

Thorax 2019 07 23;74(7):659-666. Epub 2019 Jan 23.

Department of Medicine, University of Cambridge, Cambridge, UK.

Rationale: There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung.

Aim: To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT).

Methods: 20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with Technetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis.

Results: There was increased accumulation of Tc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge.

Conclusions: This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.
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http://dx.doi.org/10.1136/thoraxjnl-2018-212509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585304PMC
July 2019

A Multicentre, Randomized, Double-Blind, Placebo-Controlled, Crossover Study To Investigate the Efficacy, Safety, Tolerability, and Pharmacokinetics of Repeat Doses of Inhaled Nemiralisib in Adults with Persistent, Uncontrolled Asthma.

J Pharmacol Exp Ther 2018 12 14;367(3):405-413. Epub 2018 Sep 14.

Respiratory Therapy Area Unit (S.K.), Clinical Pharmacology Science and Study Operations (M.M.), Global Clinical Safety and Pharmacovigilance (Y.C.), and Clinical Pharmacology Modelling and Simulation, Quantitative Sciences, RD Projects Clinical Platforms and Sciences (S.Y.), GSK, Stockley Park, Uxbridge, Middlesex, United Kingdom; Discovery Medicine (A.C.), Refractory Respiratory Inflammation Discovery Performance Unit (M.B., H.W., J.N.H., E.M.H.), Clinical Pharmacology Science and Study Operations (A.H.), and Clinical Statistics (J.R.), GSK, Stevenage, Hertfordshire, United Kingdom; Clinical Operations Department, GSK, Zeist, The Netherlands (C.L.); KLB Gesundheitsforschung Lübeck, Lübeck, Germany (A.L.-S.); and IKF Pneumologie Frankfurt, Clinical Research Centre Respiratory Diseases, Frankfurt, Germany (O.K.)

Phosphoinositide 3-kinase (PI3K) is a lipid kinase involved in leukocyte recruitment and activation. Activation of PI3K has been linked to airway inflammation and asthma pathogenesis. This randomized, double-blind, placebo-controlled, crossover study investigated the efficacy, safety, tolerability, and pharmacokinetics of a PI3K inhibitor, nemiralisib (GSK2269557), in patients with persistent, uncontrolled asthma. Patients ( = 50) received once-daily inhaled nemiralisib (1000 g) or placebo for 28 days, with a crossover to the alternative treatment following a 4-week washout period. Spirometry demonstrated no discernible difference in trough forced expiratory volume in 1 second (FEV) from baseline (adjusted posterior median 7 ml; 95% credible interval -83, 102 ml) between nemiralisib and placebo treatment at day 28 (primary endpoint). These results were supported by most secondary endpoints, including weighted mean FEV (0-4 hours) and change in trough forced vital capacity at day 28. Nemiralisib was generally well-tolerated, with few side effects except for post-inhalation cough (nemiralisib: 35%; placebo: 9%). At day 14, sputum interleukin (IL)-5, IL-13, IL-6, and IL-8 levels were reduced by a median of 17%, 7%, 15%, and 8%, respectively, when comparing nemiralisib with placebo [ = 15 (IL-5, IL-8) or 16 (IL-6, IL-13); posterior probability of a true ratio >0%: 78%, 64%, 76%, and 63%, respectively]. These results suggest that nemiralisib inhibited PI3K locally; however, this did not translate into meaningful clinical improvement. Further studies will investigate the potential efficacy of nemiralisib in patients with asthma with other specific more severe phenotypes, including those who are colonized with bacteria and frequently exacerbate.
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http://dx.doi.org/10.1124/jpet.118.249516DOI Listing
December 2018

Orai and TRPC channel characterization in FcRI-mediated calcium signaling and mediator secretion in human mast cells.

Physiol Rep 2017 Mar;5(5)

Department of Biomedical Science, University of Sheffield Western Bank, Sheffield, UK

Inappropriate activation of mast cells via the FcRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KKEE - TRPC1 gating mutant) failed to alter FcRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcRI-mediated calcium influx through Orai is necessary for histamine and TNF release but is differentially involved in the generation of cytokines and eicosanoids.
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http://dx.doi.org/10.14814/phy2.13166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350174PMC
March 2017

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

Am J Respir Crit Care Med 2016 10;194(8):961-973

1 Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied.

Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition.

Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays.

Measurements And Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures.

Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.
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http://dx.doi.org/10.1164/rccm.201509-1818OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067816PMC
October 2016

2,8-Diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine potent CCR4 antagonists capable of inducing receptor endocytosis.

Eur J Med Chem 2016 Jun 27;115:14-25. Epub 2016 Feb 27.

Department of Respiratory Biology, Respiratory TAU, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom.

A number of potent 2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine CCR4 antagonists binding to the extracellular allosteric site were synthesised. (R)-N-(2,4-Dichlorobenzyl)-2-(2-(pyrrolidin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine (R)-(18a) has high affinity in both the [(125)I]-TARC binding assay with a pKi of 8.8, and the [(35)S]-GTPγS functional assay with a pIC50 of 8.1, and high activity in the human whole blood actin polymerisation assay (pA2 = 6.7). The most potent antagonists were also investigated for their ability to induce endocytosis of CCR4 and were found to internalise about 60% of the cell surface receptors, a property which is not commonly shared by small molecule antagonists of chemokine receptors.
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http://dx.doi.org/10.1016/j.ejmech.2016.02.058DOI Listing
June 2016

Epithelial-mesenchymal transition, IP3 receptors and ER-PM junctions: translocation of Ca2+ signalling complexes and regulation of migration.

Biochem J 2016 Mar 12;473(6):757-67. Epub 2016 Jan 12.

Department of Cellular and Molecular Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.

Disconnection of a cell from its epithelial neighbours and the formation of a mesenchymal phenotype are associated with profound changes in the distribution of cellular components and the formation of new cellular polarity. We observed a dramatic redistribution of inositol trisphosphate receptors (IP3Rs) and stromal interaction molecule 1 (STIM1)-competent endoplasmic reticulum-plasma membrane junctions (ER-PM junctions) when pancreatic ductal adenocarcinoma (PDAC) cells disconnect from their neighbours and undergo individual migration. In cellular monolayers IP3Rs are juxtaposed with tight junctions. When individual cells migrate away from their neighbours IP3Rs preferentially accumulate at the leading edge where they surround focal adhesions. Uncaging of inositol trisphosphate (IP3) resulted in prominent accumulation of paxillin in focal adhesions, highlighting important functional implications of the observed novel structural relationships. ER-PM junctions and STIM1 proteins also migrate to the leading edge and position closely behind the IP3Rs, creating a stratified distribution of Ca(2+) signalling complexes in this region. Importantly, migration of PDAC cells was strongly suppressed by selective inhibition of IP3Rs and store-operated Ca(2+) entry (SOCE), indicating that these mechanisms are functionally required for migration.
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http://dx.doi.org/10.1042/BJ20150364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785603PMC
March 2016

The role of CRAC channel in asthma.

Pulm Pharmacol Ther 2015 Dec 4;35:67-74. Epub 2015 Sep 4.

University of Manchester, Institute of Inflammation and Repair, University Hospital of South Manchester Foundation Trust, Southmoor Road, Manchester, M23 9LT, UK.

Asthma is increasing globally and current treatments only manage a proportion of patients. There is an urgent need to develop new therapies. Lymphocytes are thought to play a central role in the pathophysiology of asthma through the production of inflammatory mediators. This is thought to be via the transcription factor NFAT which in turn can be activated through Ca(2+) release-activated Ca(2+) (CRAC) channels. The aim of this work was to investigate the role of CRAC in clinical and pre-clinical models of allergic asthma. Initial data demonstrated that the NFAT pathway is increased in stimulated lymphocytes from asthmatics. To confirm a role for the channel we showed that a selective inhibitor, Synta 66, blocked mediator production from lymphocytes. Synta 66 inhibited CD2/3/28 induced IL-2, IL-7, IL-13 & IFNΥ in a concentration-dependent manner in healthy and severe asthma donors, with over 60% inhibition observed for all cytokines. NFAT pathway was also increased in a pre-clinical asthma model. In this model we have demonstrated that CRAC played a central role in the airway inflammation and late asthmatic response (LAR). In conclusion, our data provides evidence that suggests targeting CRAC channels could be of therapeutic benefit for asthma sufferers.
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http://dx.doi.org/10.1016/j.pupt.2015.09.002DOI Listing
December 2015

Genome-wide transcription profiling in neutrophils in acute respiratory distress syndrome.

Lancet 2015 Feb;385 Suppl 1:S55

University of Cambridge, Cambridge, UK. Electronic address:

Background: Acute respiratory distress syndrome (ARDS) is characterised by diffuse neutrophil-mediated alveolar inflammation. Recently, we demonstrated that blood polymorphonuclear leucocytes (PMNs) in ARDS are basally activated, and exhibit aberrant oxidative burst and survival responses. The molecular mechanisms governing ARDS PMN function and longevity are incompletely understood. We aimed to use genome-wide transcriptional profiling of ARDS blood PMNs to explore underlying disease mechanisms and identify therapeutic targets aimed at manipulating PMN function and longevity.

Methods: GeneChip Affymetrix oligonucleotide arrays were used to assess global transcriptional profiles in highly pure PMNs from ventilated patients fulfilling the Berlin ARDS definition (n=10), in freshly isolated PMNs from age-matched and sex-matched healthy volunteers (n=10), and in healthy volunteer PMNs exposed in vitro to recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (1 ng/mL for 6 h). Ingenuity Pathway Analysis software was used to map probes identified as important onto specific pathways.

Findings: Transcriptomic analysis showed that 1319 genes were altered in ARDS PMNs relative to healthy volunteer PMNs. Compared with well established reference databases, the gene expression profile in ARDS PMNs showed near-complete correlation to datasets derived from patients with sepsis and burns. Transcripts enriched in ARDS PMNs were differentially expressed in known functional network pathways associated with cancer, cellular compromise, apoptotic mechanisms, and chemotaxis. Of the observed gene changes, only 292 (22%) were seen in healthy volunteer PMNs after exposure to rhGM-CSF, of which 216 showed the same directional change as ARDS PMNs.

Interpretation: Existing genome-wide studies in ARDS use total blood leucocytes; our study is the first, to our knowledge, to use unbiased global genomic profiling of highly pure ARDS blood PMNs in parallel with age-matched and gender-matched healthy volunteer PMNs treated with rhGM-CSF. Collectively our results show that ARDS PMNs display important de-novo transcriptional activity. The global transcriptomic changes were consistent with the observed aberrant ARDS PMN survival and functional phenotype that we have previously reported, and show near-complete correlation to existing sepsis and burns datasets, but only limited transcriptomic overlap with healthy volunteer PMNs treated with rhGM-CSF.

Funding: National Institute for Health Research, GlaxoSmithKline.
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http://dx.doi.org/10.1016/S0140-6736(15)60370-1DOI Listing
February 2015

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

Gastroenterology 2015 Aug 25;149(2):481-92.e7. Epub 2015 Apr 25.

Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom. Electronic address:

Background & Aims: Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.

Methods: Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.

Results: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.

Conclusions: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.
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http://dx.doi.org/10.1053/j.gastro.2015.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556985PMC
August 2015

Store-Operated Ca2+ Channels in Mesangial Cells Inhibit Matrix Protein Expression.

J Am Soc Nephrol 2015 Nov 18;26(11):2691-702. Epub 2015 Mar 18.

Department of Integrative Physiology and Anatomy and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas;

Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca(2+) signals mediated by store-operated Ca(2+) channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store-operated Ca(2+) channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store-operated Ca(2+) channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release-activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II-induced fibronectin protein expression, whereas thapsigargin abrogated high glucose- and TGF-β1-stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno-associated virus-encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store-operated Ca(2+) channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes.
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http://dx.doi.org/10.1681/ASN.2014090853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625675PMC
November 2015

The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells.

Biochem J 2015 Feb;465(3):405-12

*Department of Cellular and Molecular Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.

The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca(2+) concentration ([Ca(2+)]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as 'initiating' organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca(2+) influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca(2+) influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca(2+) entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca(2+) elevation and endocytic vacuole formation.
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http://dx.doi.org/10.1042/BJ20140398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303308PMC
February 2015

"Chemokine receptors as therapeutic targets: Why aren't there more drugs?".

Eur J Pharmacol 2015 Jan 10;746:363-7. Epub 2014 Jul 10.

Refractory Respiratory Inflammation DPU, Respiratory Therapy Area Unit, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.

Chemokines are a family of around 40 small proteins, which are secreted by a variety of cells, including structural cell types and leukocytes of the immune system. Chemokines bind to their specific 7-transmembrane G protein-coupled receptors (GPCRs) and induce a variety of downstream signals which notably modulate polymerization of the actin cytoskeleton and thus drive cellular motility. Excessive or inappropriate release of chemokines is observed in many inflammatory diseases and so there has been a great effort in industry to target chemokine receptors. The large family of GPCRs regulate many physiological cellular processes and they have proved to be highly amenable to pharmacological intervention with small chemicals. Consequently GPCRs make attractive targets for drug discovery and indeed a large number of successful current therapeutics are either agonists or antagonists of GPCRs. The apparent lack of success with chemokine receptors has been frustrating and in this paper we discuss potential reasons for previous failures and also why there is considerable cause for optimism.
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http://dx.doi.org/10.1016/j.ejphar.2014.06.060DOI Listing
January 2015

Lead identification of benzimidazolone and azabenzimidazolone arylsulfonamides as CC-chemokine receptor 4 (CCR4) antagonists.

Bioorg Med Chem 2014 Aug 21;22(15):4298-311. Epub 2014 May 21.

Department of Respiratory Biology, Respiratory TAU, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom.

A knowledge-based library of 2,3-dichlorophenylsulfonyl derivatives of commercially available aryl amines was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. Hits were required to be more potent than an existing indazole series, have better physicochemical properties (clogP <3.5, chrom logD₇.₄ <5.3 and CLND solubility >116 μg/mL), and be stable to acid and light. The benzimidazol-2-one core was identified as a hit suitable for further investigation. Substitution at N1 with small alkyl groups was tolerated; however, these analogues were inactive in the whole blood assay (pA₂ <5). Azabenzimidazolone analogues were all found to be active, with compound 38 exhibiting whole blood activity of 6.1, low molecular weight (389) and chrom logD₇.₄ (2.4), high LE (0.43), and solubility (152 μg/mL). In addition, 38 had human serum albumin binding of around 93% and met all the criteria for progression to lead optimisation.
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http://dx.doi.org/10.1016/j.bmc.2014.05.021DOI Listing
August 2014

High glucose and diabetes enhanced store-operated Ca(2+) entry and increased expression of its signaling proteins in mesangial cells.

Am J Physiol Renal Physiol 2014 May 12;306(9):F1069-80. Epub 2014 Mar 12.

3500 Camp Bowie Blvd., Dept. of Integrative Physiology, Univ. of North Texas Health Science Center, Fort Worth, TX 76107.

The present study was conducted to determine whether and how store-operated Ca(2+) entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7-day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca(2+) channels. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca(2+) currents were significantly enhanced in the MCs treated with HG for 7 days, and the enhanced response was abolished by both GSK-7975A and La(3+). In contrast, receptor-operated Ca(2+) entry in MCs was significantly reduced by HG treatment. Western blotting showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real-time RT-PCR revealed that a 6-day HG treatment significantly increased the mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription but increases Orai1 protein through a posttranscriptional mechanism.
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http://dx.doi.org/10.1152/ajprenal.00463.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010683PMC
May 2014

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists.

Eur J Pharmacol 2014 Apr 15;729:75-85. Epub 2014 Feb 15.

Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK; Airway Disease Infection Section, National Heart and Lung Institute, Imperial College, Norfolk Place, London W2 1PG, UK. Electronic address:

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.
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http://dx.doi.org/10.1016/j.ejphar.2014.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989064PMC
April 2014

CRAC channel inhibition produces greater anti-inflammatory effects than glucocorticoids in CD8 cells from COPD patients.

Clin Sci (Lond) 2014 Feb;126(3):223-32

*University of Manchester NIHR Translational Research Facility, Manchester Academic Health Science Centre, University Hospital of South Manchester Foundation Trust, Manchester M23 9LT, U.K.

There are increased numbers of pulmonary CD8 lymphocytes in COPD (chronic obstructive pulmonary disease). CRAC (calcium release-activation calcium) channels play a central role in lymphocyte activation though the regulation of the transcription factor NFAT (nuclear factor of activated T-cells). We studied the expression of NFAT in lungs from COPD patients compared with controls, and evaluated the effects of CRAC channel inhibition compared with corticosteroids on NFAT activation and cytokine production in CD8 cells from COPD patients. The effects of the corticosteroid dexamethasone, the calcineurin inhibitor cyclosporin and the CRAC channel inhibitor Synta 66 were studied on cytokine production and NFAT activation using peripheral blood and isolated pulmonary CD8 cells. NFAT1 and CD8 co-expression in the lungs was compared in COPD patients and controls using combined immunohistochemistry and immunofluorescence. NFAT inhibition with either cyclosporin or Synta 66 resulted in significantly greater maximal inhibition of cytokines than dexamethasone in both peripheral blood and pulmonary CD8 cells [e.g. >95% inhibition of IFNγ (interferon γ) production from pulmonary CD8 cells using cyclosporin and Synta 66 compared with <50% using dexamethasone]. The absolute number of pulmonary CD8 cells co-expressing NFAT1 was significantly raised in lungs from COPD patients compared with controls, but the percentage of CD8 cells co-expressing NFAT1 was similar between COPD patients and controls (80.7% compared with 78.5% respectively, P=0.3). Inhibition of NFAT using the CRAC channel Synta 66 produces greater anti-inflammatory effects on CD8 cells from COPD patients than corticosteroids. NFAT is expressed at a high level in pulmonary CD8 cells in COPD.
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http://dx.doi.org/10.1042/CS20130152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401013PMC
February 2014

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy.

Proc Natl Acad Sci U S A 2013 Aug 22;110(32):13186-91. Epub 2013 Jul 22.

Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3AX Wales, United Kingdom.

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca(2+) concentration inside pancreatic acinar cells ([Ca(2+)]i), due to excessive release of Ca(2+) stored inside the cells followed by Ca(2+) entry from the interstitial fluid. The sustained [Ca(2+)]i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca(2+) release-activated Ca(2+) channels (CRAC) would prevent sustained elevation of [Ca(2+)]i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca(2+) ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca(2+). Ca(2+) entry then occurred upon admission of Ca(2+) to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca(2+) entry in a concentration-dependent manner within the range of 1 to 50 μM (IC50 = 3.4 μM), but had little or no effect on the physiological Ca(2+) spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca(2+)]i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca(2+)]i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.
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http://dx.doi.org/10.1073/pnas.1300910110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740877PMC
August 2013

Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations.

Eur J Pharmacol 2013 Mar 27;704(1-3):49-57. Epub 2013 Feb 27.

Respiratory Therapy Area Unit, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK.

Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders.
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http://dx.doi.org/10.1016/j.ejphar.2013.02.022DOI Listing
March 2013

Synthesis and structure-activity relationships of indazole arylsulfonamides as allosteric CC-chemokine receptor 4 (CCR4) antagonists.

J Med Chem 2013 Mar 27;56(5):1946-60. Epub 2013 Feb 27.

Department of Medicinal Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom.

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.
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http://dx.doi.org/10.1021/jm301572hDOI Listing
March 2013

The action of selective CRAC channel blockers is affected by the Orai pore geometry.

Cell Calcium 2013 Feb 5;53(2):139-51. Epub 2012 Dec 5.

Institute of Biophysics, University of Linz, 4040 Linz, Austria.

As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC(50) of approximately 4μM and exhibited a substantially slower rate of onset than the typical pore blocker La(3+), together with almost no current recovery upon wash-out over 4min. For the less Ca(2+)-selective Orai1 E106D pore mutant, I(CRAC) inhibition was significantly reduced. FRET experiments indicated that neither STIM1-STIM1 oligomerization nor STIM1-Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.
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http://dx.doi.org/10.1016/j.ceca.2012.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580291PMC
February 2013

CRACM/Orai ion channel expression and function in human lung mast cells.

J Allergy Clin Immunol 2012 Jun 10;129(6):1628-35.e2. Epub 2012 Mar 10.

Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Leicester, United Kingdom.

Background: Influx of extracellular Ca(2+) into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca(2+) influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca(2+) release-activated Ca(2+) current are candidates.

Objectives: To investigate the expression and function of CRACM channels in HLMCs.

Methods: CRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus.

Results: CRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca(2+)-selective current obtained under both conditions was blocked by 10 μM La(3+) and Gd(3+), known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers-GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca(2+) influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C(4), and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue.

Conclusions: The presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca(2+) influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases.
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http://dx.doi.org/10.1016/j.jaci.2012.01.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526795PMC
June 2012

In vitro characterisation of the duration of action of the histamine-1 receptor antagonist azelastine.

Eur J Pharmacol 2011 Nov 21;670(2-3):586-92. Epub 2011 Sep 21.

Respiratory CEDD Biology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, UK.

Azelastine is a selective antagonist at the human histamine-1 receptor and is used clinically in the treatment of allergic rhinitis. In this study we have investigated its duration of action in vitro in an effort to characterise the receptor and tissue components involved. Chinese hamster ovary cell membrane fragments were used to determine the kinetics of azelastine at the H₁ receptor in a radioligand binding assay. Further duration of action studies were completed in tissue preparations using guinea-pig trachea and human bronchus. In radioligand binding studies, azelastine reached steady state at the H₁ receptor after approximately 41 min and exhibited a significantly slower dissociation rate constant from the receptor than the first generation antihistamine, diphenhydramine. In washout studies completed in guinea-pig and human airway in vitro tissue preparations, azelastine continued to antagonise the effects of histamine at the H₁ receptor for at least 18 h post-washout of the antagonist. This outcome was reversed following removal of the epithelium from guinea-pig isolated tracheal strips. These studies indicate there is a tissue component contributing to azelastine's duration of action, in addition to its direct H₁ receptor binding, with evidence suggesting a role for the epithelial layer.
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http://dx.doi.org/10.1016/j.ejphar.2011.09.017DOI Listing
November 2011

Arthroscopic assessment and classification of Kienbock's disease.

Tech Hand Up Extrem Surg 2006 Mar;10(1):8-13

Royal Adelaide Hospital, Modbury Public Hospital, University of Adelaide.

The authors have utilised arthroscopy to assess and classify Kienbock's avascular necrosis of the lunate. The classification is based on the number of articular surfaces of the lunate and adjacent articulation, which are non-functional. Kienbock's disease usually affects the proximal surface of the lunate first with subsequent secondary changes to the lunate facet of the radius. Advanced cases and those with a coronal fracture of the lunate will cause involvement of the mid carpal joint. Surgery is aimed to debride the joint, classify the level of disease and direct the definitive procedure to be performed. If the articular surfaces are intact, a synovectomy or radial shortening would be indicated. If there is involvement of the lunate but an intact lunate facet a proximal row carpectomy would be indicated. If there is involvement of the proximal lunate and lunate facet then a radio-scapholunate fusion could be utilised. More extensive involvement of the joint would require a wrist fusion. Arthroscopy provides a valuable assessment and subsequent classification of Kienbock's disease.
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http://dx.doi.org/10.1097/00130911-200603000-00003DOI Listing
March 2006

Evidence for novel cannabinoid receptors.

Pharmacol Ther 2005 May 11;106(2):133-45. Epub 2005 Jan 11.

National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, MSC-9413 Bethesda, MD 8092-9413, United States.

Cannabinoids, including the bioactive constituents of the marijuana plant, their synthetic analogs, and endogenous lipids with cannabinoid-like activity, produce their biological effects by interacting with specific receptors. To date, two G protein-coupled cannabinoid receptors have been identified by molecular cloning, CB1 receptors mainly expressed in the brain and mediating most of the neurobehavioral effects of cannabinoids and CB2 receptors expressed by immune and hematopoietic tissues. Recent findings indicate that some cannabinoid effects are not mediated by either CB1 or CB2 receptors, and in some cases there is compelling evidence to implicate additional receptors in these actions. These include transient receptor potential vanilloid 1 (TRPV1) receptors and as-yet-unidentified receptors implicated in the endothelium-dependent vasodilator effect of certain cannabinoids and in the presynaptic inhibition of glutamatergic neurotransmission in the hippocampus. The case for these additional receptors is being reviewed here.
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http://dx.doi.org/10.1016/j.pharmthera.2004.11.005DOI Listing
May 2005
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