ABCMdb

PMID: 22927224

Giampieri M, Vanthuyne N, Nieddu E, Mazzei MT, Anzaldi M, Pedemonte N, Galietta LJ, Roussel C, Mazzei M
Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
ChemMedChem. 2012 Oct;7(10):1799-807. doi: 10.1002/cmdc.201200311. Epub 2012 Aug 27., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp [4-6] Compounds able to repair class II CFTR mutations (among which the most relevant is DF508) are defined as "correctors", whereas compounds able to ameliorate the gating defect in class III mutations (which include G551D and G1349D) are defined as "potentiators". Login to comment 10 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp [14] These defects can be corrected by the same potentiators that are effective on G551D and G1349D mutations. Login to comment 16 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp A small set of asymmetric 4-aryl-DHPs was synthesized, and each racemic couple was tested in a functional assay carried out on cells expressing the G1349D, DF508, and G551D mutants. Login to comment 18 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Although three enantiomers demonstrated high potency (Kd values less than 0.09, 0.1, and 0.5 mm in G1349D, DF508, and G551D, respectively), in general, the screening of pure enantiomers did not produce a great diversity in potency values. Login to comment 49 ABCC7 p.Gly1349Asp Results Biological results of racemates Compounds 4a-l were first tested on Fischer rat thyroid (FRT) cells expressing the G1349D mutation to evaluate, by means of the iodide influx assay, whether the synthesized DHPs could ameliorate the gating defect of mutant CFTR. Login to comment 52 ABCC7 p.Gly551Asp Five asymmetric DHPs, namely 4a, 4c, 4d, 4e, and 4g, the Kd values of which are all <0.4 mm (Table 1), were also tested on DF508 and G551D mutations (Table 2), and interesting results relative to felodipine were observed, especially in terms of potency (Figure 2A). Login to comment 65 ABCC7 p.Gly1349Asp Evaluation of G1349D CFTR gating effect by compounds 4a-l tested as racemates. Login to comment 68 ABCC7 p.Gly551Asp Evaluation of rescued DF508 and G551D CFTR gating effects by selected compounds tested as racemates. Login to comment 69 ABCC7 p.Gly551Asp [a] Rescued DF508 G551D Compd Kd [mm] Emax [msÀ1 ] Kd [mm] Emax [msÀ1 ] 4a 0.67Æ0.18 25Æ1.4 1.10Æ0.20 19Æ1.3 4c 0.067Æ0.025 24Æ1.7 0.60Æ0.19 18Æ1.4 4d 0.17Æ0.09 23Æ1.5 0.62Æ0.15 22Æ1.9 4e 0.27Æ0.11 22Æ2.0 0.19Æ0.05 16Æ1.2 4g 0.51Æ0.11 26Æ2.2 1.80Æ0.30 21Æ2.0 felodipine 0.88Æ0.16 29Æ1.9 22.60Æ2.20 21Æ1.7 [a] Kd and Emax values are the mean ÆSEM of n=5-10 experiments. Login to comment 78 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp mers; compounds (À)-4a, (À)-4c, (+)-4d, (+)-4e, and (À)-4g generally show a slightly higher activity than their enantiomeric counterparts in all cell lines. In particular, the Kd ratios of the above values range from 1.7 to 4.7 for G1349D, from 2.0 to 3.1 for rescued DF508, and from 1.5 to 5.5 for G551D. Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Evaluation of G1349D, rescued DF508, and G551D CFTR gating effects by selected DHPs tested as pure enantiomers. Login to comment 96 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp [a] G1349D Rescued DF508 G551D Compd Kd [mm] Emax [msÀ1 ] Kd [mm] Emax [msÀ1 ] Kd [mm] Emax [msÀ1 ] (+)-4a 0.362Æ0.125 126Æ9.8 1.0Æ0.3 29Æ2.2 1.8Æ0.40 18Æ1.6 (À)-4a 0.210Æ0.085 121Æ8.0 0.5Æ0.2 28Æ1.9 0.6Æ0.20 18Æ1.5 (+)-4c 0.088Æ0.055 123Æ8.3 0.16Æ0.02 28Æ2.3 1.1Æ0.30 18Æ1.1 (À)-4c 0.036Æ0.025 120Æ7.8 0.082Æ0.011 27Æ2.0 0.20Æ0.04 18Æ1.0 (+)-4d 0.046Æ0.021 97Æ6.7 0.061Æ0.022 28Æ2.4 0.46Æ0.10 23Æ1.9 (À)-4d 0.216Æ0.031 109Æ8.1 0.187Æ0.025 25Æ2.1 0.77Æ0.12 19Æ1.6 (+)-4e 0.080Æ0.055 130Æ10.2 0.09Æ0.2 30Æ2.5 0.14Æ0.04 17Æ1.6 (À)-4e 0.158Æ0.045 125Æ11.3 0.25Æ0.03 29Æ2.3 0.24Æ0.08 16Æ1.1 (+)-4g 0.559Æ0.155 121Æ9.4 1.4Æ0.3 28Æ2.0 2.3Æ0.5 19Æ1.2 (À)-4g 0.313Æ0.075 122Æ7.2 0.48Æ0.17 30Æ2.5 1.5Æ0.4 21Æ1.8 [a] Kd and Emax values are the mean ÆSEM of n=5-10 experiments. Login to comment 98 ABCC7 p.Gly1349Asp [22-24] In particular, compounds 4c, 4d, and 4e show activity at concentrations <0.1 mm in the G1349D cell line, giving evidence, in this small series, for the importance of a branched substituent at 4`. Login to comment 100 ABCC7 p.Gly1349Asp On the other hand, 4-naphthyl derivatives 4 f-i showed moderate activity (only 4g has a Kd value lower than 0.4 mm in the G1349D cell line). Login to comment 102 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Compounds that showed the best values in the G1349D cell line (Kd <0.4 mm) were also tested against rescued DF508 and G551D mutations, demonstrating high potency (Table 2). Login to comment 103 ABCC7 p.Gly551Asp For example, 4c, 4d, and 4e have Kd values less than 0.3 and 0.7 mm in rescued DF508 and G551D cell lines, respectively. Login to comment 105 ABCC7 p.Gly1349Asp Interestingly, we found that the compounds appear to be more active toward G1349D than toward the DF508 mutant. Login to comment 106 ABCC7 p.Gly1349Asp This is not surprising, as these two mutations affect different NBDs (NBD1 for DF508 and NBD2 for G1349D); therefore, they may affect the potentiator binding site(s) differently (possibly localized at the NBD1/NBD2 interface). Login to comment 107 ABCC7 p.Gly551Asp Moreover, emphasis must be placed on the potency values of 4c, 4d, and 4e with respect to felodipine in the G551D cell line: in particular, 4e shows a gain in potency of about two orders of magnitude (Table 2). Login to comment 112 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The biological tests on FRT cells were repeated for each separated enantiomer, and the results listed in Table 4 indicate a clear, although not dramatic, increase in activity for compounds (À)-4a, (À)-4c, (+)-4d, (+)-4e, and (À)-4g with respect to the enantiomeric counterparts in all cell lines. In particular, compounds (À)-4c and (+)-4d showed Kd values lower than 0.050 and 0.09 mm in G1349D and rescued DF508, respectively, whereas compound (+)-4e had Kd <0.15 mm in G551D. Login to comment 165 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Biology CFTR assays Cell culture: Fischer rat thyroid (FRT) cells, stably transfected with G1349D, G551D, or DF508 CFTR and the halide-sensitive yellow fluorescent protein YFP-H148Q/I152L[32] were cultured in Coon`s modified Ham`s F-12 medium supplemented with 10% fetal calf serum, l-glutamine (2 mm), penicillin (100 UmLÀ1 ), and streptomycin (100 mgmLÀ1 ). Login to comment 170 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Fluorescence assay for CFTR activity: Measurements of CFTR activity were carried out on FRT cells expressing G1349D, G551D, or DF508 CFTR and the halide-sensitive YFP 48 h after plating on microplates. Login to comment