Block of CFTR-dependent chloride currents by inhibitors of multidrug resistance-associated proteins

Diena, Tullia; Melani, Raffaella; Caci, Emanuela; Pedemonte, Nicoletta; Sondo, Elvira; Zegarra‐Moran, Olga; Galietta, Luis J. V.

doi:10.1016/j.ejphar.2007.01.051

Cited by 14 publications

(12 citation statements)

References 23 publications

(19 reference statements)

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“…One possible explanation is that TMEM16A is not the intestinal CaCC. Benzbromarone was previously identified as a CFTR blocker in Fischer rat thyroid cells (56). Here, in NHBE cells, we found that benzbromarone does not significantly affect CFTR current.…”

Section: Discussionsupporting

confidence: 40%

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Huang

Zhang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

303

342

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Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calciumactivated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.A sthma is a significant cause of morbidity and mortality worldwide, and the prevalence of this disease is increasing among all age, sex, and racial groups. Characteristic features of asthma include inflammation, subepithelial fibrosis, hyperplasia of mucus-producing cells, accumulation of mucus within airway lumens, hyperplasia of airway smooth muscle (ASM), and ASM hyperresponsiveness. Together, these symptoms impair lung function by limiting the flow of gases to and from the alveoli in the distal lung.The current standard of care for asthma involves inhaled corticosteroids for the management of inflammation combined with long-acting agonists of β2-adrenergic receptors. Despite this treatment, lung function is not improved in 30-45% of asthmatic patients, and exacerbations continue to be a major problem (reviewed in ref. 1). Asthma can be divided into at least two distinct molecular phenotypes defined by the degree of Th2 inflammation (2, 3). Cytokines, including IL-4 and IL-13, promote airway epithelial mucous cell metaplasia, subepithelial fibrosis, and hyperplasia of smooth muscle in Th2-high asthmatics, and these patients generally show improved lung function with inhaled corticosteroid therapy. A greater understanding of this heterogeneity and the molecular and physiological events that lead to airway remodeling might lead to improved diagnosis and treatment.Calcium-activated chloride channels (CaCCs) have been ascribed numerous cellular functions (reviewed in refs. 4 and 5), among these are epithelial fluid secretion and smooth muscle contraction, both of which contribute to the progression and severity of asthma. Moreover, calcium-activated chloride currents in the airway epithelium are enhanced by the Th2 cytokines IL-4 and IL-13, as well as IFN-γ (6). For these reasons, CaCC is an attractive potential therapeutic target for asthma (7). However, the study of CaCC was impeded by lack of information about the gene(s) encoding this channel. It was only relatively recently that TMEM16A (transmembrane p...

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Section: Discussionsupporting

confidence: 40%

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Huang

Zhang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

303

342

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“…We first tested the system by using a compound that binds within the CFTR channel pore (at the interface between the two TMDs) and to see whether it could block cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Benzbromarone is a CFTR inhibitor that blocks channel activity by occupying the pore (18). Accordingly, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were preincubated with various concentrations of benzbromarone and then treated with M11M cross-linker.…”

Section: Resultsmentioning

confidence: 99%

Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein

Wang

Loo

Bartlett

et al. 2007

Journal of Biological Chemistry

123

129

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The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe 508 (⌬F508). The ⌬F508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37°C, we found that maturation could be restored if Val 510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of ⌬F508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

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“…In vivo studies in mice showed that INH 1 (3 mg/kg) reduced 40% of cholera toxin-induced intestinal fluid secretion, whereas INH 2 had no effect (Muanprasat et al, 2007). Finally, inhibitors of multidrug resistanceassociated proteins, among them sulfinpyrazone, probenecid, and benzbromarone, inhibit CFTR activity with low affinity, i.e., K i values of 191, 1244, and 11.5 M, respectively (Diena et al, 2007). This latter observation is nevertheless interesting because it confirms pharmacological similarities within members of the family of ABC transporters.…”

Section: Discussionmentioning

confidence: 99%

Discovery of α-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Routaboul

Norez²,

Melin³

et al. 2007

J Pharmacol Exp Ther

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The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl Ϫ channel in the apical membrane of epithelial cells for cAMP-dependent Cl Ϫ secretion. Here we report on the synthesis and screening of a small library of nontoxic ␣-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl Ϫ channels. In whole-cell patch-clamp experiments of Chinese hamster ovary (CHO) cells expressing WT-CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM concentrations. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC 50 ϭ 14.7 M), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone (CFTR inh -172) (IC 50 ϭ 1.2 M), and ␣-aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC 50 ϭ 71 pM), 8a,b (IC 50 ϭ

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Block of CFTR-dependent chloride currents by inhibitors of multidrug resistance-associated proteins

Cited by 14 publications

References 23 publications

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein

Discovery of α-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

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