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ABSTRACT: Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that functions as a calcium activated chloride channel (CaCC). CaCCs are major regulators of various physiological processes, such as sensory transduction, epithelial secretion, smooth muscle contraction and oocyte fertilization. Thirty novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids (B01-B30) were synthesized and evaluated for their TMEM16A inhibitory activity by using short circuit current measurements in Fischer rat thyroid (FRT) cells expressing human TMEM16A. IC(50) values were calculated using YFP fluorescence plate reader assay. Final compounds, having free carboxylic group displayed significant inhibition. Eight of the novel compounds B02, B13, B21, B23, B25, B27, B28, B29 exhibit excellent CaCCs inhibition with IC(50) value <6 μM, with compound B25 exhibiting the lowest IC(50) value of 2.8 ± 1.3 μM. None of the tested ester analogs of final benzofuran derivatives displayed TMEM16A/CaCCs inhibition.
Bioorganic & medicinal chemistry 06/2012; 20(14):4237-44. · 2.82 Impact Factor
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ABSTRACT: The Ca(2+)-activated Cl(-) channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl(-) conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl(-) conductance with single-site IC(50)~150 µM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl(-) channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities.
PLoS ONE 01/2012; 7(5):e38030. · 4.09 Impact Factor
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ABSTRACT: TMEM16A (ANO1) is a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. Cell-based functional screening of ∼110,000 compounds revealed compounds that activated TMEM16A CaCC conductance without increasing cytoplasmic Ca(2+). By patch-clamp, N-aroylaminothiazole "activators" (E(act)) strongly increased Cl(-) current at 0 Ca(2+), whereas tetrazolylbenzamide "potentiators" (F(act)) were not active at 0 Ca(2+) but reduced the EC(50) for Ca(2+)-dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (E(act)) and potentiator (F(act)) produced large and more sustained CaCC Cl(-) currents than general agonists of Ca(2+) signaling, with EC(50) 3-6 μM and Cl(-) conductance comparable to that induced transiently by Ca(2+)-elevating purinergic agonists. Analogs of activators were identified that fully inhibited TMEM16A Cl(-) conductance, providing further evidence for direct TMEM16A binding. The TMEM16A activators increased CaCC conductance in human salivary and airway submucosal gland epithelial cells, and IL-4 treated bronchial cells, and stimulated submucosal gland secretion in human bronchi and smooth muscle contraction in mouse intestine. Small-molecule, TMEM16A-targeted activators may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for pharmacological dissection of TMEM16A function.
The FASEB Journal 08/2011; 25(11):4048-62. · 5.71 Impact Factor
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ABSTRACT: TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate
the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ~110,000 compounds
revealed four novel chemical classes of small-molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with
IC50 < 10 microM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor,
an aminophenylthiazole (T16Ainh-A01), had IC50 of ~1 microM. Two distinct types of inhibitors were identified. Some compounds,
such as tannic acid and the arylaminothiophene CaCCinh-A01, fully inhibited CaCC current in human bronchial and intestinal
cells. Other compounds, including T16Ainh-A01 and digallic acid, poorly inhibited total CaCC current in these cells, but
blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the
transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current
in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a
minor contributor to total CaCC current in airway and intestinal epithelia. The small-molecule inhibitors identified here
permit pharmacological dissection of TMEM16A / CaCC function, and are potential development candidates for drug therapy of
hypertension, pain, diarrhea and excessive mucus production.
Journal of Biological Chemistry 11/2010; · 4.77 Impact Factor
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ABSTRACT: TMEM16A was found recently to be a calcium-activated Cl(-) channel (CaCC). CaCCs perform important functions in cell physiology, including regulation of epithelial secretion, cardiac and neuronal excitability, and smooth muscle contraction. CaCC modulators are of potential utility for treatment of hypertension, diarrhea, and cystic fibrosis. Screening of drug and natural product collections identified tannic acid as an inhibitor of TMEM16A, with IC(50) ∼ 6 μM and ∼100% inhibition at higher concentrations. Tannic acid inhibited CaCCs in multiple cell types but did not affect CFTR Cl(-) channels. Structure-activity analysis indicated the requirement of gallic or digallic acid substituents on a macromolecular scaffold (gallotannins), as are present in green tea and red wine. Other polyphenolic components of teas and wines, including epicatechin, catechin, and malvidin-3-glucoside, poorly inhibited CaCCs. Remarkably, a 1000-fold dilution of red wine and 100-fold dilution of green tea inhibited CaCCs by >50%. Tannic acid, red wine, and green tea inhibited arterial smooth muscle contraction and intestinal Cl(-) secretion. Gallotannins are thus potent CaCC inhibitors whose biological activity provides a potential molecular basis for the cardioprotective and antisecretory benefits of red wine and green tea.
The FASEB Journal 11/2010; 24(11):4178-86. · 5.71 Impact Factor
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ABSTRACT: TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 μM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ∼1 μM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.
Journal of Biological Chemistry 11/2010; 286(3):2365-74. · 4.77 Impact Factor
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ABSTRACT: Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca(2+)-activated chloride channel (CaCC) is activated by Ca(2+) agonists like UTP. We found that most chloride current elicited by Ca(2+) agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca(2+) activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca(2+) agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca(2+) agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca(2+)/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1-CFTR association is responsible for Ca(2+)/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca(2+) agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.
Molecular biology of the cell 08/2010; 21(15):2639-48. · 5.98 Impact Factor
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ABSTRACT: The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to approximately 1-microm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 +/- 0.5 microm (SE 10 cultures) under basal conditions, 8.4 +/- 0.4 microm following ENaC inhibition by amiloride, and 14.5 +/- 1.2 microm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 +/- 0.7 microm under basal conditions, 11.0 +/- 1.7 microm following amiloride, 17.0 +/- 3.4 microm following cAMP agonists, and 7.1 +/- 0.5 microm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth.
AJP Lung Cellular and Molecular Physiology 10/2009; 297(6):L1131-40. · 3.66 Impact Factor
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ABSTRACT: Crofelemer, a purified proanthocyanidin oligomer extracted from the bark latex of Croton lechleri, is in clinical trials for secretory diarrheas of various etiologies. We investigated the antisecretory mechanism of crofelemer by determining its effect on the major apical membrane transport and signaling processes involved in intestinal fluid transport. Using cell lines and measurement procedures to isolate the effects on individual membrane transport proteins, crofelemer at 50 microM had little or no effect on the activity of epithelial Na(+) or K(+) channels or on cAMP or calcium signaling. Crofelemer inhibited the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channel with maximum inhibition of approximately 60% and an IC(50) approximately 7 microM. Crofelemer action at an extracellular site on CFTR produced voltage-independent block with stabilization of the channel closed state. Crofelemer did not affect the potency of glycine hydrazide or thiazolidinone CFTR inhibitors. Crofelemer action resisted washout, with <50% reversal of CFTR inhibition after 4 h. Crofelemer was also found to strongly inhibit the intestinal calcium-activated Cl(-) channel TMEM16A by a voltage-independent inhibition mechanism with maximum inhibition >90% and IC(50) approximately 6.5 microM. The dual inhibitory action of crofelemer on two structurally unrelated prosecretory intestinal Cl(-) channels may account for its intestinal antisecretory activity.
Molecular pharmacology 10/2009; 77(1):69-78. · 4.53 Impact Factor
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ABSTRACT: Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. Screening of approximately 110000 small synthetic and natural compounds for inhibition of halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure-activity relationships. The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4',5'-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chloride current with IC(50) approximately 90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date.
Journal of Medicinal Chemistry 09/2009; 52(20):6447-55. · 4.80 Impact Factor
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ABSTRACT: The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K(+)], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K(+) ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K(+)-selective, increasing >4-fold with increasing [K(+)] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K(+)] was 20.8 +/- 0.3 mm and decreased by inhibition of the Na(+)/K(+) pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTR(inh)-172), or K(+) channels (TEA or XE991). ASL [K(+)] was increased by forskolin but not affected by Na(+)/K(+)/2Cl(-) cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K(+)] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K(+)]. [K(+)] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K(+)] is not a factor in CF lung disease. In intact airways, ASL [K(+)] was also well above extracellular [K(+)]: 22 +/- 1 mm in pig trachea ex vivo and 16 +/- 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K(+)] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K(+)].
Journal of Biological Chemistry 04/2009; 284(23):15916-26. · 4.77 Impact Factor
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ABSTRACT: K+ channels and K+-coupled membrane transporters are important targets for drug discovery. We previously developed a triazacryptand (TAC)-based K+ sensor, TAC-Red, and demonstrated its utility to image K+ waves in mouse brain in vivo (Padmawar et al. Nat. Methods. 2005, 2, 825-827). Here, we synthesized a green-fluorescing dextran conjugate of TAC-bodipy ("TAC-Limedex") for use as an extracellular K+ sensor and demonstrated its utility in measuring K+ transport across cell membranes. TAC-Limedex fluorescence increased by 50% with increasing [K+] from 0 to 2 mM and was insensitive to [Na+], [Cl-], or pH. K+ efflux from cells was quantified from increasing extracellular TAC-Limedex fluorescence following cell immersion in K+-free buffer. In HT-29 cells, K+ efflux was 2.0 +/- 0.1 micromol/cm2/s, increasing 8-fold following K+ channel activation by ATP; the increase in K+ efflux was inhibited by a K+ channel blocker or by preventing cytoplasmic calcium elevation. Electroneutral K+/Cl- cotransport was demonstrated in SiHa cells, in which K+ efflux was increased 3-fold by hypotonic challenge; the increase in K+ efflux was fully inhibited by a K+/Cl- transport blocker. K+ efflux measurements were adapted to a commercial fluorescence platereader for automated screening. The fluorescence-based K+ transport assay largely replaces assays requiring radioactive rubidium and is suitable for high-throughput identification of K+ transport modulators.
Journal of the American Chemical Society 07/2008; 130(25):7794-5. · 9.91 Impact Factor
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ABSTRACT: Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. In a recent study, several highly polar alpha-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (J Pharmacol Exp Ther 322:1023-1035, 2007), more than 10,000-fold better than that of thiazolidinone and glycine hydrazide CFTR inhibitors previously identified by high-throughput screening. In this study, we resynthesized and evaluated the alpha-aminoazaheterocyclic-methylglyoxal adducts reported to have high CFTR inhibition potency (compounds 5, 7, and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 microM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp experiments, and yellow fluorescence protein-fluorescence quenching. As positive controls, approximately 100% of CFTR inhibition was found by thiazolidinone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by alpha-aminoazaheterocyclic-methylglyoxal adducts.
Journal of Pharmacology and Experimental Therapeutics 06/2008; 325(2):529-35. · 3.83 Impact Factor
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ABSTRACT: Calcium-activated chloride channels (CaCCs) are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. Potent, selective CaCC inhibitors have not been available. We established a high-throughput screen for identification of inhibitors of a human intestinal CaCC based on inhibition of ATP/carbachol-stimulated iodide influx in HT-29 cells after lentiviral infection with the yellow fluorescent halide-sensing protein YFP-H148Q/I152L. Screening of 50,000 diverse, drug-like compounds yielded six classes of putative CaCC inhibitors, two of which, 3-acyl-2-aminothiophenes and 5-aryl-2-aminothiazoles, inhibited by >95% iodide influx in HT-29 cells in response to multiple calcium-elevating agonists, including thapsigargin, without inhibition of calcium elevation, calcium-calmodulin kinase II activation, or cystic fibrosis transmembrane conductance regulator chloride channels. These compounds also inhibited calcium-dependent chloride secretion in T84 human intestinal epithelial cells. Patch-clamp analysis indicated inhibition of CaCC gating, which, together with the calcium-calmodulin data, suggests that the inhibitors target the CaCC directly. Structure-activity relationships were established from analysis of more than 1800 analogs, with IC(50) values of the best analogs down to approximately 1 muM. Small-molecule CaCC inhibitors may be useful in pharmacological dissection of CaCC functions and in reducing intestinal fluid losses in CaCC-mediated secretory diarrheas.
Molecular pharmacology 03/2008; 73(3):758-68. · 4.53 Impact Factor
