Novel CFTR Chloride Channel Activators Identified by Screening of Combinatorial Libraries Based on Flavone and Benzoquinolizinium Lead Compounds

Galietta, Luis J. V.; Springsteel, Mark F.; Eda, Masahiro; Niedzinski, Edmund J.; By, Kolbot; Haddadin, Makhluf J.; Kurth, Mark J.; Nantz, Michael H.; Verkman, A. S.

doi:10.1074/jbc.m101892200

Cited by 207 publications

(151 citation statements)

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“…Last, using an established fluorescence measurement method (27), we verified that the CFTR chimeras containing single GFP moieties were functional halide transporters. COS7 cells were co-transfected with halide-sensitive fluorescent protein EYFP-H148Q and each of the CFTR constructs.…”

Section: Resultsmentioning

confidence: 99%

“…CFTR Halide Transport Assay-Transport was assayed in COS7 cells co-transfected with a CFTR-GFP construct and the halide-sensitive EYFP-H148Q protein (27). Cells were imaged 1 day after transfection on a Nikon TE2000E microscope using a Nikon ϫ20, 0.75 NA S Fluor objective, 31001 filter set (Chroma), and QuantEM 512SC CCD camera.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were imaged 1 day after transfection on a Nikon TE2000E microscope using a Nikon ϫ20, 0.75 NA S Fluor objective, 31001 filter set (Chroma), and QuantEM 512SC CCD camera. Cells were bathed for 5 min in PBS containing 20 M forskolin prior to solution exchange to give a 100 mM iodide gradient, as described (27). The kinetics of single cell fluorescence was analyzed using NIS Elements AR software.…”

Section: Methodsmentioning

confidence: 99%

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Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

Haggie

Verkman

2008

Journal of Biological Chemistry

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel. There is indirect and conflicting evidence about whether CFTR exists in cell membranes as monomers, dimers, or higher order oligomers. We measured fluorescence intensities and photobleaching dynamics of distinct fluorescent spots in cells expressing functional CFTR-green fluorescent protein (GFP) chimeras. Intensity analysis of GFP-labeled CFTR in live cells showed singlecomponent distributions with mean intensity equal to that of purified monomeric GFP, indicating monomeric CFTR in cell membranes. Fluorescent spots showed single-step photobleaching, independently verifying that CFTR is monomeric. Results did not depend on whether GFP was added to the CFTR N terminus or fourth extracellular loop or on whether CFTR chloride conductance was stimulated by cAMP agonists. Control measurements with a CFTR chimera containing two GFPs showed two-step photobleaching and a single-component intensity distribution with mean intensity twice that of monomeric GFP. These results provide direct evidence for monomeric CFTR in live cells.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is a member of the ATP-binding cassette protein family that forms cAMP-regulated chloride channels (1). CFTR is expressed in epithelial cells in the airways, pancreas, intestine, and other tissues (2). Loss-of-function mutations in CFTR cause the hereditary lethal disease cystic fibrosis, in which chronic lung infection produces morbidity and mortality (1, 2). Excessive CFTR activity in the intestine in response to bacterial enterotoxins produces secretory diarrheas (3, 4). There is considerable interest in CFTR structure and assembly in cell membranes as CFTR is an important drug target for therapy of cystic fibrosis, secretory diarrheas, and polycystic kidney disease (3, 5-7).The assembly state of CFTR has been controversial, with indirect evidence reported for CFTR monomers, dimers, and mixed monomers/dimers. Patch clamp analysis of constructs containing linked wild-type (WT) CFTRs or WT and mutant CFTRs suggested that two CFTR polypeptides form a single chloride conductance pathway (8). Conflicting data from reconstituted membranes containing WT and mutant CFTRs did not reveal intermediary conductance states, consistent with independently functioning CFTR monomers (9). Electron crystallography has indicated that CFTR is a monomer with two conformations, likely the open and closed channel states (10). These data are in accord with high resolution crystal structures of bacterial ATP-binding cassettetype transporters showing unit cells containing two transmembrane-nucleotide binding domains (11). Biochemical approaches including velocity-gradient centrifugation, coimmunoprecipitation, gel filtration, and cross-linking have generated conflicting data suggesting monomeric CFTR (9, 12), dimeric CFTR (13), and mixed monomeric/dimeric CFTR (14, 15). Data supporting dimeric CFTR have also come from patch clamp of CFTR in the presence of...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

Haggie

Verkman

2008

Journal of Biological Chemistry

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“…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%

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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“…There are several chemical classes of CFTR activators that may function by direct interaction with CFTR, including flavones/isoflavones (e.g., genistein and apigenin, (6 -8), benzo[c]quinoliziniums (9), xanthines (10), benzimidazolones (11), and fluorescein derivatives (12)). Using lead-based combinatorial synthesis, we recently identified improved flavone-type CFTR activators; the most potent had a 7,8-benzoflavone core structure (13).…”

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confidence: 99%

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

Ma¹,

Vetrivel²,

Yang³

et al. 2002

Journal of Biological Chemistry

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Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl؊ conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 M together with a low concentration of forskolin (0.5 M) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl ؊ sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl

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Novel CFTR Chloride Channel Activators Identified by Screening of Combinatorial Libraries Based on Flavone and Benzoquinolizinium Lead Compounds

Cited by 207 publications

References 29 publications

Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

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

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

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