ABCMdb

PMID: 17452495

Pedemonte N, Boido D, Moran O, Giampieri M, Mazzei M, Ravazzolo R, Galietta LJ
Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel.
Mol Pharmacol. 2007 Jul;72(1):197-207. Epub 2007 Apr 23., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. Login to comment 7 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca2ϩ channels and strong effect as potentiators on the ⌬Phe508, G551D, and G1349D CFTR mutants. Login to comment 27 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The most common class III mutation is G551D (glycine-to-aspartic acid change at position 551), with a worldwide frequency of 3.1% of CF alleles (Hamosh et al., 1992). Login to comment 28 ABCC7 p.Gly1349Asp Other class III mutations, such as G1349D, are much rarer. Login to comment 31 ABCC7 p.Gly551Asp For example, high concentrations of flavonoids such as genistein can improve ⌬Phe508- and G551D-CFTR channel gating (Hwang et al., 1997; Illek et al., 1999; Zegarra-Moran et al., 2002). Login to comment 34 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Conversely, phenylglycines, another class of compounds identified by high-throughput screening, are effective also on G551D and G1349D channels. Login to comment 36 ABCC7 p.Gly551Asp Among them, felodipine was the most potent compound, having activity on ⌬Phe508- and G551D-CFTR. Login to comment 45 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp To this purpose, we screened a set of 333 felodipine analogs using cell-based assays to determine their activity on three CFTR mutants (⌬Phe508, G551D, and G1349D) and on DHP-sensitive Ca2ϩ channels. Login to comment 48 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Fischer rat thyroid (FRT) cells were stably transfected with ⌬Phe508, G551D, or G1349D-CFTR, and the halide-sensitive yellow fluorescent protein mutant YFP-H148Q/I152L (Galietta et al., 2001a). Login to comment 66 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Measurements of CFTR activity were carried out on FRT cells expressing mutant CFTR and the halide-sensitive YFP 24 h (G551D and G1349D) or 48 h (⌬Phe508) after plating on microplates. Login to comment 124 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The panel of compounds was tested on FRT cells coexpressing the halide-sensitive mutant H148Q/I152L of the fluorescent protein YFP and the CFTR mutants ⌬Phe508, G551D, and G1349D. Login to comment 130 ABCC7 p.Gly1349Asp A, chemical structure of tested compounds. B, representative fluorescence traces showing the response to I- addition in FRT cells expressing G1349D-CFTR under resting conditions (saline) or upon stimulation with forskolin alone (20 ␮M) or in the presence of test compounds. Login to comment 186 ABCC7 p.Gly551Asp We found consistently that G551D was the most refractory to potentiation; the concentrations needed to stimulate this mutant were always 10 to 20 times higher than those active on ⌬Phe508 (Fig. 8A, see also Supplemental Data). Login to comment 187 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Conversely, the G1349D mutant displayed a sensitivity between that of ⌬Phe508 and G551D (Fig. 8, B and C). Login to comment 194 ABCC7 p.Gly1349Asp On the other hand, the same DHPs maintained a good activity on CFTR channels with an apparent Ka for the ⌬Phe508 and G1349D mutants in the range 100 to 700 nM (Fig. 9, C and E). Login to comment 195 ABCC7 p.Gly551Asp In agreement with all previous observations, activity on G551D required higher concentrations, with Ka values of 1 to 6 ␮M (Fig. 9D). Login to comment 199 ABCC7 p.Gly551Asp DHP-194 was the most potent compound, with apparent Ka values of approximately 0.11 and 1.2 ␮M for ⌬Phe508-CFTR and G551D-CFTR, respectively, in agreement with the values derived from the fluorescence assay (0.11 and 2.0 ␮M). Login to comment 201 ABCC7 p.Gly551Asp For example, DHP-229 displayed Ka values of 0.32 and 2.6 ␮M for ⌬Phe508 and G551D channels, respectively, in fluorescence experiments and of 0.31 and 4.0 ␮M in short-circuit current recordings. Login to comment 202 ABCC7 p.Gly551Asp In contrast, genistein had a reduced potency for ⌬Phe508 (Ka of 18 ␮M in fluorescence assays and 14 ␮M in short-circuit current experiments) and for G551D (Ka of 94 ␮M in fluorescence assays and 124 ␮M in short-circuit current experiments). Login to comment 203 ABCC7 p.Gly551Asp The maximal response to the compounds in ⌬Phe508-CFTR cells was comparable with that to genistein, whereas in G551D-CFTR cells, the activity elicited by DHPs was at least 2-fold larger than that evoked by genistein, as already described for antihypertensive DHP drugs (Pedemonte et al., 2005b). Login to comment 206 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In particular, CFTR potentiators are needed to restore activity in those CFTR mutants affected by impaired channel gating (class III mutants), like G551D and G1349D. Login to comment 209 ABCC7 p.Gly551Asp In a previous study, we described the effect of antihypertensive 1,4-dihydropyridines as potentiators of ⌬Phe508 and G551D-CFTR (Pedemonte et al., 2005b). Login to comment 214 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Note that the concentrations needed to stimulate the G551D mutant are always 10 to 20 times higher than those effective on ⌬Phe508 or G1349D. Login to comment 223 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In parallel, we have used the functional assay based on the halide-sensitive YFPs to measure activity of compounds on ⌬Phe508, G551D, and G1349D CFTR mutants. Login to comment 225 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Our first result is that DHP-based structures activate also G1349D-CFTR, besides ⌬Phe508 and G551D mutants. Login to comment 228 ABCC7 p.Gly1349Asp ⌬Phe508 and G1349D were activated at nanomolar concentrations by the most potent compounds. Login to comment 229 ABCC7 p.Gly551Asp In contrast, G551D required consistently higher concentrations, as already found for other CFTR activators (Zegarra-Moran et al., 2002, Pedemonte et al., 2005a; Zegarra-Moran et al., 2007). Login to comment 237 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp B-E, dose-response relationships obtained for indicated compounds on VDCC (B), ⌬Phe508-CFTR (C), G551D-CFTR (D), and G1349D-CFTR (E). Login to comment