ABCMdb

PMID: 11262417

Galietta LJ, Springsteel MF, Eda M, Niedzinski EJ, By K, Haddadin MJ, Kurth MJ, Nantz MH, Verkman AS
Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds.
J Biol Chem. 2001 Jun 8;276(23):19723-8. Epub 2001 Mar 21., 2001-06-08 [PubMed]

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No. Mutations Sentence Comment

2 ABCC7 p.Gly551Asp The compounds were screened for their ability to activate CFTR at 50 ␮M concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Login to comment 9 ABCC7 p.Gly551Asp Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. Login to comment 36 ABCC7 p.Gly551Asp 23, Issue of June 8, pp. 19723-19728, 2001 (c) 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 19723 atUniversityofNorthCarolinaatChapelHill,onAugust8,2011www.jbc.orgDownloadedfrom http://www.jbc.org/content/suppl/2001/06/07/276.23.19723.DC1.html Supplemental Material can be found at: compounds for CFTR-activating potency by a cell-based halide transport assay that utilized fluorescent epithelial cells stably expressing wild-type or G551D CFTR together with the green fluorescent protein halide indicator YFP-H148Q. Login to comment 56 ABCC7 p.Gly551Asp Cell Culture and Transfection-Fischer rat thyroid (FRT) cells expressing the human wild-type CFTR or CFTR-G551D were transfected with the plasmid pcDNA3.1 (Invitrogen) containing the cDNA encoding YFP-H148Q and selected in G418 (0.75 mg/ml). Login to comment 84 ABCC7 p.Gly551Asp Short-circuit Current Measurements-FRT cells stably expressing human wild-type or G551D CFTR were cultured on Snapwell inserts (Costar) at a density of 500,000 cells/insert. Login to comment 105 ABCC7 p.Gly551Asp FRT cells coexpressing human wild-type CFTR or G551D CFTR (which causes CF) and YFP-H148Q were cultured on 96-well plates for the monitoring of YFP fluorescence in a plate reader. Login to comment 110 ABCC7 p.Gly551Asp Neither activation of G551D CFTR nor of null cells that do not express CFTR was observed under these conditions (data not shown). Login to comment 113 ABCC7 p.Gly551Asp Screening for activators of wild-type and G551D CFTR. Login to comment 116 ABCC7 p.Gly551Asp Right panel, for G551D CFTR, the addition of a test compound can have no effect (heavy line) or can activate forskolin-independent or -dependent I- transport. Login to comment 120 ABCC7 p.Gly551Asp C, a screening of FRT cells expressing G551D CFTR showing I- transport after the addition of 5 ␮M forskolin. Login to comment 132 ABCC7 p.Gly551Asp A full screen was also carried out in FRT cells expressing G551D CFTR. Login to comment 133 ABCC7 p.Gly551Asp Halide transport by G551D CFTR is not increased by cAMP elevation alone. Login to comment 137 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Many of the same compounds that activated wild-type CFTR also activated G551D CFTR, although relative activating potencies differed, and a few compounds activated preferentially wild-type CFTR (e.g. UCCF-180) or G551D CFTR (e.g. UCCF-023 and UCCF-030). Login to comment 145 ABCC7 p.Gly551Asp Fig. 3B shows a similar comparison for the activation of G551D CFTR. Login to comment 147 ABCC7 p.Gly551Asp Short-circuit current was measured in polarized monolayers of FRT cells expressing wild-type or G551D CFTR. Login to comment 159 ABCC7 p.Gly551Asp Representative fluorescence curves are shown for FRT cells expressing wild-type CFTR (A) and G551D CFTR (B). Login to comment 164 ABCC7 p.Gly551Asp Fig. 4B shows short-circuit current analysis of G551D CFTR activation by genistein versus UCCF-030. Login to comment 166 ABCC7 p.Gly551Asp Interestingly, the activator of wild-type CFTR, UCCF-180, was ineffective in activating G551D CFTR. Login to comment 167 ABCC7 p.Gly551Asp The compounds UCCF-023, UCCF-027, and UCCF-028 were also effective in inducing short-circuit current in cells expressing G551D CFTR (data not shown). Login to comment 170 ABCC7 p.Gly551Asp All compounds with activating potency on wild-type and/or G551D CFTR were nontoxic (cell growth Ͼ90% of control) except for UCCF-027, which was similar to genistein. Login to comment 185 ABCC7 p.Gly551Asp Short-circuit current (Isc) measurements FRT cells expressing wild-type CFTR (A) and G551D CFTR (B). Login to comment 196 ABCC7 p.Gly551Asp The CFTR activators did not induce Cl- currents in null cells, and short-circuit current analysis showed that they had activating potencies for wild-type and G551D CFTR that were substantially better than the existing lead compounds. Login to comment 199 ABCC7 p.Gly551Asp Interestingly, the order of activating potencies of the CFTR activators on wild-type versus G551D CFTR was different. Login to comment 200 ABCC7 p.Gly551Asp For example, UCCF-029 was the most potent compound in activating wild-type CFTR but had less effect on G551D CFTR. Login to comment 201 ABCC7 p.Gly551Asp In contrast, UCCF-023, UCCF-028, and UCCF-030, which were substantially less potent than UCCF-029 in activating wild-type CFTR, were very potent in activating G551D CFTR in synergy with forskolin. Login to comment