High-affinity Activators of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Conductance Identified by High-throughput Screening

Ma, Tonghui; Vetrivel, L.; Yang, Hong; Pedemonte, Nicoletta; Zegarra‐Moran, Olga; Galietta, Luis J. V.; Verkman, A. S.

doi:10.1074/jbc.m205932200

Cited by 175 publications

(151 citation statements)

References 15 publications

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“…Forskolin (1 M; Sigma, St. Louis, MO) was added to the bath solution in all experiments. Where indicated, measurements of acid-equivalent influx were made in the presence of 200 M of the anion exchange inhibitor dihydro-4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid (H 2DIDS; Calbiochem, San Diego, CA) and/or 10 M of the CFTR inhibitor inh-172 (CFTRinh-172; Sigma) (27). All solutions were adjusted to pH 7.4 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

Stewart

Yamamoto²,

Nakakuki³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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Ϫ -rich fluid in response to stimulation by the adenylyl cyclase-coupled hormone secretin. The Cl Ϫ -rich secretion of pancreatic acinar cells is modified as it flows along the pancreatic ductal system, ultimately producing pancreatic juice with final concentrations of ϳ140 mM HCO 3 Ϫ and ϳ20 mM Cl Ϫ (31). In the guinea pig pancreatic duct, the uptake of bicarbonate across the basolateral membrane is mediated by Na ϩ -HCO 3 Ϫ cotransport (NBC1) and by Na ϩ /H ϩ exchange (19). HCO 3 Ϫ efflux across the apical membrane has been proposed to be mediated by CFTR, in concert with apical Cl Ϫ /HCO 3 Ϫ exchanger activity (9). Proximal pancreatic duct HCO 3 Ϫ secretion by apical Cl Ϫ / HCO 3 Ϫ exchange is favored by the high luminal Cl Ϫ concentrations resulting from pancreatic acinar Cl Ϫ secretion. However, since this proximal fluid flows distally toward the ampullary terminus of the duct, its Cl Ϫ concentration progressively decreases in parallel with a gradual increase in its HCO 3 Ϫ concentration. These inverse changes in Cl Ϫ and HCO 3 Ϫ concentrations are predicted to alter relative contributions of apical membrane Cl Ϫ /HCO 3 Ϫ exchange and CFTR-mediated HCO 3 Ϫ conductance and have been suggested to reflect axial variation in expression and regulation of these transport activities (18). The Cl Ϫ /HCO 3 Ϫ exchangers Slc26a3 and Slc26a6 have been detected in mouse pancreatic duct and in the human pancreatic cell line CFPAC-1 (10, 22), leading to the proposal that Cl Ϫ -dependent HCO 3 Ϫ secretion by pancreatic proximal ducts represents combined activities of Slc26a6 and Slc26a3 anion exchangers, positively regulated by activated CFTR, with reciprocal positive regulation of CFTR by the anion exchangers (4, 5, 22, 23). However, a study in native mouse pancreatic duct suggests negative regulation of CFTR by Slc26a6 (43).Secretin-stimulated HCO 3 Ϫ secretion in guinea pig pancreatic duct is not dependent on elevated luminal [Cl Ϫ ] (21) (brackets denote concentration) and occurs even in the presence of luminal fluid containing 125 mM HCO 3 Ϫ and 23 mM Cl Ϫ (17). As luminal [Cl Ϫ ] falls to or below this level (with corresponding elevation of [HCO 3 Ϫ ]), CFTR Cl Ϫ permeability may fall while that for HCO 3 Ϫ may rise (34). Measurements of membrane potential (V m ) and intracellular pH (pH i ) in luminally perfused interlobular ducts from guinea pig pancreas suggest that CFTR HCO 3 Ϫ conductance can account for observed levels of stimulated bicarbonate secretion (15,18,20). A computational model of pancreatic duct HCO 3 Ϫ secretion predicted for similar conditions that ϳ94% of HCO 3 Ϫ efflux across the apical membrane was mediated by HCO 3 Ϫ conductance (36), although the model did not consider contributions from electrogenic Cl Ϫ /HCO 3 Ϫ exchange. Thus the HCO 3 Ϫ conductance of CFTR could provide the main route for apical HCO 3 Ϫ secretion in distal pancreatic ducts from species in which, like human and guinea pig, pancreatic juice contains high concentrations of HCO 3 Ϫ (ϳ140 mM). Studies of the role of SLC26-media...

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Section: Methodsmentioning

confidence: 99%

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

Stewart

Yamamoto²,

Nakakuki³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…The screening was performed in FRT epithelial cells coexpressing human WT CFTR and a cytoplasmic yellow fluorescent protein halide sensor in a 96-well format (27,32,33). Additional details of the primary screening will be reported separately.…”

Section: Characterization Of Small-molecule Cftr Activatorsmentioning

confidence: 99%

“…Both solutions were bubbled with air and maintained at 37°C. The basolateral membrane was permeabilized with 250 mg/ml amphotericin B (27,28). Hemichambers were connected to a DVC-1000 voltage clamp via Ag/AgCl electrodes and 3 M KCl agar bridges for I sc recording.…”

Section: Short-circuit Currentmentioning

confidence: 99%

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Small‐molecule CFTR activators increase tear secretion and prevent experimental dry eye disease

et al. 2016

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Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl 2 channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by highthroughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl 2 -driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTR act -K089, fully activated CFTR in cell cultures with EC 50 ∼250 nM and produced an ∼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTR act -K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTR act -K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTR act -K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

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“…40 Primary screening was done using a collection of 100,000 diverse, drug-like compounds from commercial sources (ChemDiv, San Diego, CA; Asinex, Winston-Salem, NC). For assay of UT-B inhibition, whole blood was diluted to a hematocrit of approximately 1% in PBS containing 1.25 M acetamide and 5 mM glucose.…”

Section: Ut-b Inhibitor Identification By High-throughput Screeningmentioning

confidence: 99%

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

Yao

Anderson

Zhang

et al. 2012

Journal of the American Society of Nephrology

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Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB inh -14, fully and reversibly inhibited urea transport with IC 50 values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB inh -14 competed with urea binding at an intracellular site on the UT-B protein. UTB inh -14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB inh -14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H 2 O lower in UTB inh -14-treated mice than vehicle-treated mice. UTB inh -14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB inh -14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

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High-affinity Activators of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Conductance Identified by High-throughput Screening

Cited by 175 publications

References 15 publications

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

Small‐molecule CFTR activators increase tear secretion and prevent experimental dry eye disease

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

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High-affinity Activators of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Conductance Identified by High-throughput Screening

Cited by 175 publications

References 15 publications

Functional coupling of apical Cl−/HCO3− exchange with CFTR in stimulated HCO3− secretion by guinea pig interlobular pancreatic duct

Functional coupling of apical Cl−/HCO3− exchange with CFTR in stimulated HCO3− secretion by guinea pig interlobular pancreatic duct

Small‐molecule CFTR activators increase tear secretion and prevent experimental dry eye disease

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

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Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct

Functional coupling of apical Cl⁻/HCO₃⁻ exchange with CFTR in stimulated HCO₃⁻ secretion by guinea pig interlobular pancreatic duct