ABCMdb

PMID: 12161441

Ma T, Vetrivel L, Yang H, Pedemonte N, Zegarra-Moran O, Galietta LJ, Verkman AS
High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening.
J Biol Chem. 2002 Oct 4;277(40):37235-41. Epub 2002 Aug 2., 2002-10-04 [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC7 p.Gly551Asp Several compounds, the most potent being a trifluoromethyl- phenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (Kd > 10 ␮M). Login to comment 23 ABCC7 p.Gly551Asp Such activators could provide information about CFTR activating mechanisms and might activate some CF-causing CFTR mutants, such as G551D-CFTR, that are processed normally but have reduced intrinsic Cl- permeability. Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Novel activators were identified that strongly activated CFTR Cl-conductance at concentrations well under 1 ␮M and without elevation of cAMP, as well as compounds that activated the CF-causing mutants G551D-CFTR and ⌬Phe508 -CFTR. Login to comment 41 ABCC7 p.Gly551Asp MATERIALS AND METHODS Cell Culture-Fischer rat thyroid (FRT) cells stably co-expressing human CFTR (wild-type, or mutants G551D or ⌬Phe508 ) and the yellow fluorescent protein YFP-H148Q were cultured on plastic in Coon`s modified F12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin as described previously (13). Login to comment 157 ABCC7 p.Gly551Asp The activators of wild-type CFTR were screened for their ability to activate two CF-causing CFTR mutants: G551D, which is transported to the plasma membrane but is not active after cAMP elevation, and ⌬Phe508 , which is retained at the endoplasmic reticulum and has impaired function even after transport to the cell surface (by low temperature incubation or chemical chaperones). Login to comment 158 ABCC7 p.Gly551Asp Fig. 5B, top, shows representative fluorescence assays of I- influx in FRT cells coexpressing G551D-CFTR and YFP-H148Q. Login to comment 159 ABCC7 p.Gly551Asp Measurements were performed using 20 ␮M forskolin, which itself does not activate G551D-CFTR, and 50 ␮M of each test compound. Login to comment 160 ABCC7 p.Gly551Asp Of the 57 activators of wild-type CFTR identified in the fluorescence assay, only three compounds activated G551D-CFTR; the most potent compound (structure in Fig. 5B, middle) was not one of the strongest activators of wild-type CFTR. Login to comment 161 ABCC7 p.Gly551Asp The other G551D-CFTR activators (N-[6-methyl-2-(4-methylphenyl)-2H-benzotriazol-5-yl]-4- nitrobenzamide and ␣-methyl-4-oxo-5-(phenylmethylene)-2- thioxo-3-thiazolidineacetic acid) were also not strong activators of wild-type CFTR. Login to comment 162 ABCC7 p.Gly551Asp The potencies of these compounds for activation of G551D-CFTR were relatively poor. Login to comment 204 ABCC7 p.Gly551Asp B, activation of G551D-CFTR. Login to comment 205 ABCC7 p.Gly551Asp Fluorescence assays of I- influx on cells co-expressing G551D-CFTR and YFP-H148Q. Login to comment 217 ABCC7 p.Gly551Asp The compounds that strongly activated wild-type CFTR were screened for their efficacy in activating G551D-CFTR and ⌬Phe508 -CFTR, two mutant CFTRs that cause human CF. Login to comment 218 ABCC7 p.Gly551Asp Of the 57 strong activators of wild-type CFTR, only three compounds activated G551D-CFTR significantly, indicating that CFTR activation is mutation-specific. Login to comment 219 ABCC7 p.Gly551Asp Unfortunately, the compounds that activated G551D-CFTR did so weakly (Kd 10 ␮M or higher), no better than existing flavones. Login to comment