ABCMdb

PMID: 17578899

Routaboul C, Norez C, Melin P, Molina MC, Boucherle B, Bossard F, Noel S, Robert R, Gauthier C, Becq F, Decout JL
Discovery of alpha-aminoazaheterocycle-methylglyoxal adducts as a new class of high-affinity inhibitors of cystic fibrosis transmembrane conductance regulator chloride channels.
J Pharmacol Exp Ther. 2007 Sep;322(3):1023-35. Epub 2007 Jun 19., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Here we report on the synthesis and screening of a small library of nontoxic ␣-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl-channels. Login to comment 4 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/ F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). Login to comment 25 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Here we present the synthesis and identification of ␣-aminoazaheterocycle-methylglyoxal adducts as highly potent inhibitors of wild-type CFTR and CFTR affected by the following CF mutations: F508del, G551D, and G1349D. Login to comment 73 ABCC7 p.Gly551Asp All cell lines were grown under standard culture conditions, as follows. CHO cells stably transfected with pNUT vector alone (mock-CHO) or containing wild-type CFTR (WT-CFTR-CHO) or the mutant G551D-CFTR were provided by J. R. Riordan and X. B. Chang (Scottsdale, AZ) (Tabcharani et al., 1991; Becq et al., 1994, 1999). Login to comment 74 ABCC7 p.Gly551Asp They were maintained at 37°C in 5% CO2 in ␣-minimal essential medium-GlutaMAX containing 7% fetal bovine serum (FBS), 50 IU/ml penicillin and 50 ␮g/ml streptomycin, and methotrexate for cell selection (WT-CFTR-CHO: 100 ␮M, G551D-CHO: 20 ␮M) (Tabcharani et al., 1991; Becq et al., 1994). Login to comment 75 ABCC7 p.Gly1349Asp The G1349D mutation was created by site-directed mutagenesis as described previously (Melin et al., 2004) and transiently expressed in COS-7 cells, 12 to 24 h after seeding, using cationic lipids (jetPEI; Qbiogene Inc., Carlsbad, CA) with 1 ␮g/ml of plasmid. Login to comment 116 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp CFTR-dependent iodide efflux was stimulated either by forskolin (WT-CFTR) or by a cocktail containing forskolin with genistein (F508del- and G551D-CFTR) or with the benzo[c]quinolizinium derivative MPB-91 (G1349D-CFTR). Login to comment 190 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp The present study was undertaken on the following cells: CHO cells overexpressing WT-CFTR or mutated G551D-CFTR and COS-7 cells expressing G1349D-CFTR (both G551D and G1349D are class III CF mutations); two human airway epithelial cells, Calu-3 and 16HBE14o-, expressing endogenous WT-CFTR; and the human airway epithelial cells JME/CF15, endogenously expressing F508del-CFTR (class II CF mutation). Login to comment 209 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Experiments were performed in the presence of 1 ␮M Fsk (WT-CFTR), 10 ␮M Fsk ϩ 30 ␮M genistein (F508del- and G551D-) or 10 ␮M ϩ 250 ␮M MPB-91 (G1349D-CFTR). Login to comment 210 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Compounds (Ratio a,b Determined by NMR Spectrometry) Endogenous CFTR Heterologous Expression of CFTR WT-CFTR F508del-CFTR WT-CFTR G551D-CFTR G1349D-CFTR Glibenclamide 11.7 Ϯ 1.1 ␮M 11.8 Ϯ 1.1 ␮M 14.7 Ϯ 1.3 ␮M 7.9 Ϯ 1.6 ␮M 9.0 Ϯ 1.4 ␮M CFTRinh-172 N.D. N.D. 1.2 Ϯ 0.9 ␮M N.D. N.D. 5a 93.3 Ϯ 1.3 pM 67.3 Ϯ 1.3 pM 71.0 Ϯ 1.2 pM 43.1 Ϯ 1.5 nM 72.3 Ϯ 1.1 pM 8a,b (60:40) 15.7 Ϯ 1.1 nM 8.7 Ϯ 1.2 nM 2.5 Ϯ 1.7 nM 190 Ϯ 2 nM 79.4 Ϯ 2 nM 7a,b (60:40) 2.3 Ϯ 1.5 nM 6.3 Ϯ 2.1 nM 3.4 Ϯ 1.2 nM 154 Ϯ 2 nM 7.0 Ϯ 1.1 nM 3a,b (75:25) 11.2 Ϯ 1.6 ␮M 7.0 Ϯ 1.4 ␮M 4.4 Ϯ 1.3 M 35.5 Ϯ 1.6 ␮M 9.0 Ϯ 1.8 ␮M 4a,b (60:40) 11.9 Ϯ 1.7 ␮M 6.0 Ϯ 3.3 ␮M 5.7 Ϯ 1.3 ␮M 110 Ϯ 2 ␮M 2.9 Ϯ 1.4 ␮M N.D., complete dose-response curve was not determined, but the inhibition for each cell type with 10 ␮M concentrations of inhibitors was confirmed. Login to comment 256 ABCC7 p.Gly1349Asp Responses to various adducts were also studied on CFTR chloride channels affected by class III CF mutations, G551Dand G1349D-CFTR. Login to comment 258 ABCC7 p.Gly551Asp G551D-CFTR activity, expressed in CHO cells, was analyzed in response to ␣-aminoazaheterocycle-methylglyoxal adducts. Login to comment 260 ABCC7 p.Gly551Asp Glibenclamide inhibited G551D-CFTR with an IC50 of 7.9 Ϯ 1.6 ␮M, similar to that for WT-CFTR (Table 1). Login to comment 261 ABCC7 p.Gly551Asp It is interesting to note that to inhibit G551D-CFTR (whereas glibenclamide is equally potent com- Fig. 7. Login to comment 268 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp For G1349D-CFTR, iodide efflux was stimulated by a mixture of 10 ␮M Fsk plus CFTR activator MPB-91 (250 ␮M) because genistein failed to stimulate G1349D-CFTR as reported earlier (Melin et al., 2004). Login to comment 269 ABCC7 p.Gly1349Asp The IC50 for glibenclamide inhibition of G1349D-CFTR (9 Ϯ 1.4 ␮M; Table 1) was in this case similar to that of WT-CFTR. Login to comment 270 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In contrast with G551D, adducts 3a,b, 4a,b, 5a (IC50 of 72.3 Ϯ 1.1 pM), 7a,b, and 8a,b inhibited G1349D-CFTR with affinities similar to that of WT-CFTR (Table 1). Login to comment 271 ABCC7 p.Gly1349Asp Thus, G551Dand G1349D-CFTR channels are both inhibited (although with different potencies) by the ␣-aminoazaheterocycle-methylglyoxal adducts. Login to comment 288 ABCC7 p.Gly1349Asp Discussion In this report, a new chemical class of CFTR channel inhibitors was discovered; among these are new, highly potent, water-soluble, nontoxic molecules suitable for studying CFTR with iodide efflux, microcytofluorimetry, and whole-cell patch-clamp techniques in epithelial and nonepithelial cells and for CFTR-dependent transepithelial current analysis with a Ussing chamber in mouse colon. To our knowledge, the 2Ј-deoxyadenosine derivative 5a represents the most potent inhibitor of CFTR channels with picomolar affinity in WT-, F508del- and G1349D-CFTR-expressing cells and nano- Fig. 8. Login to comment 293 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp B, with CHO cells overexpressing G551D-CFTR and COS-7 cells expressing G1349D-CFTR. Login to comment 296 ABCC7 p.Gly551Asp molar affinity in G551D-CFTR cells. Login to comment