Cell-based assay for high-throughput quantitative  screening of CFTR chloride transport agonists

Galietta, Luis V. J.; Jayaraman, Sujatha; Verkman, A. S.

doi:10.1152/ajpcell.2001.281.5.c1734

Cited by 200 publications

(182 citation statements)

References 22 publications

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“…FRT cells stably expressing the halide sensor YFP-H148Q/I152L (YFP-FRT cells) 36, 46 were a generous gift of AS Verkman (University of California, San Francisco). YFP-FRT cells were cultured as described previously for FRT cells 38 except that media contained 10% fetal bovine serum, 2 mM glutamine and the selection agent G418 (0.5 mg/mL).…”

Section: Methodsmentioning

confidence: 99%

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

Valkenier

Judd³

et al. 2015

Nature Chem

155

160

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The transport of anions across cell membranes has become an important goal for supramolecular chemistry [1][2][3][4] . It is well-known that cation carriers (cationophores) can serve as antibiotics and toxins 5,6 , and it seems likely that anion carriers might also show useful biological activity. However, suitable anionophores have only recently become available 7-10 , and the nature of their biological effects is still in question. A particular hope is that anionophores might be used to replace the activity of endogenous anion channels which are missing or defective 11,2 . Such deficiencies underlie a number of medical conditions including Best disease, Startle disease, Bartter's syndrome and, most notably, the widespread lifeshortening genetic disease cystic fibrosis (CF) 12,13 .If anionophores are to be used to treat these "channelopathies", it must be shown that they can be delivered to cells in sufficient quantities to produce substantial effects, of the same order of magnitude as endogenous anion channels, and that these quantities are not toxic to the cells. However, while a wide variety of anionophores have been studied in synthetic membranes (principally large unilamellar vesicles, or LUVs), the range of systems subjected to biological investigations is still quite limited. Moreover, from the point of view of CF treatment, the results thus far have been mixed. On the positive side, a few systems have been tested in whole cells, epithelia or (in one case) genetically modified mice 14 , using electrophysiological methods such as patch-clamp and Ussing chamber, and shown to induce anion conduction without obvious toxic effects. These anionophores include synthetic 3 peptides conceptually related to natural anion channels 11,[15][16][17] , as well as the steroid-based system 1 from the authors' laboratory 18 , and other small organic molecules 14,19,20 .Less encouragingly, a number of molecules showing anion transport in LUVs have tested positive for anti-cancer cytotoxicity (potentially valuable, but incompatible with CF treatment) 8,[21][22][23][24][25] . Many of these systems, including the well-studied prodigiosin 2 26 , transport protons as well as anions 8,[21][22][23] . Intracellular acidification can lead to apoptosis 26 , so in these cases proton transport could underlie toxicity. On the other hand, a recent study on calixpyrroles 3 suggested that anion transport as such could be cytotoxic 25 . As the transport activity of 3 was only weak, this raises the concern that powerful anionophores might be highly toxic and thus unsuitable for CF treatment. Thus far the only biological testing of these "1,5-diaxial" systems has been the early study on 1, referred to above 18 . The assay involved the application of 1 to the apical membrane of oriented Madin Darby canine kidney cell (MDCK) epithelia, mounted in an Ussing chamber, and measurement of the resulting electrical current caused by Cl − transport. The methodology is well-established, but not so convenient for screening large numbers of compounds.Mor...

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Section: Methodsmentioning

confidence: 99%

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

Valkenier

Judd³

et al. 2015

Nature Chem

155

160

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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%

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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“…FRT-CFTR cells were a gift from Professor A. S. Verkman, University of California, San Francisco, CA, U.S.A. 15) T84 cells were cultured in a mixture of Dulbecco's Modified eagle's Medium (DMeM) and Ham's F-12 Medium (1 : 1) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. FRT-CFTR cells were cultured in Coon's Modification Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Activation of AMP-Activated Protein Kinase by a Plant-Derived Dihydroisosteviol in Human Intestinal Epithelial Cell

Muanprasat

Sirianant

Sawasvirojwong

et al. 2013

Biological & Pharmaceutical Bulletin

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Our previous study has shown that dihydroisosteviol (DHIS), a derivative of stevioside isolated from Stevia rebaudiana (Bertoni), inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial chloride secretion across monolayers of human intestinal epithelial (T84) cells and prevents cholera toxin-induced intestinal fluid secretion in mouse closed loop models. In this study, we aimed to investigate a mechanism by which DHIS inhibits CFTR activity. Apical chloride current measurements in Fisher rat thyroid cells stably transfected with wild-type human CFTR (FRT-CFTR cells) and T84 cells were used to investigate mechanism of CFTR inhibition by DHIS. In addition, effect of DHIS on AMP-activated protein kinase (AMPK) activation was investigated using Western blot analysis. Surprisingly, it was found that DHIS failed to inhibit CFTR-mediated apical chloride current in FRT-CFTR cells. In contrast, DHIS effectively inhibited CFTR-mediated apical chloride current induced by a cell permeable cAMP analog CPT-cAMP and a direct CFTR activator genistein in T84 cell monolayers. Interestingly, this inhibitory effect of DHIS on CFTR was significantly (p<0.05) reduced by pretreatment with compound C, an AMPK inhibitor. AICAR, a known AMPK activator, was able to inhibit CFTR activity in both FRT-CFTR and T84 cells. Western blot analysis showed that DHIS induced AMPK activation in T84 cells, but not in FRT-CFTR cells. Our results indicate that DHIS inhibits CFTR-mediated chloride secretion in T84 cells, in part, by activation of AMPK activity. DHIS therefore represents a novel candidate of AMPK activators.

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Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists

Cited by 200 publications

References 22 publications

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

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

Activation of AMP-Activated Protein Kinase by a Plant-Derived Dihydroisosteviol in Human Intestinal Epithelial Cell

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