ABCMdb

PMID: 16829626

Noel S, Faveau C, Norez C, Rogier C, Mettey Y, Becq F
Discovery of pyrrolo[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild type, G551D, and F508del cystic fibrosis transmembrane conductance regulator chloride channels.
J Pharmacol Exp Ther. 2006 Oct;319(1):349-59. Epub 2006 Jul 7., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp Discovery of Pyrrolo[2,3-b]pyrazines Derivatives as Submicromolar Affinity Activators of Wild Type, G551D, and F508del Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels Sabrina Noel, Christelle Faveau, Caroline Norez, Christian Rogier, Yvette Mettey, and Fre´ de´ ric Becq Institut de Physiologie et Biologie Cellulaires Centre National de la Recherche Scientifique (CNRS) Unite´ Mixte de Recherche 6187, Universite´ de Poitiers, Poitiers, France (S.N., C.F., C.N., C.R., Y.M., F.B.); and Faculte´ de Me´ decine et Pharmacie, Poitiers cedex, France (C.F., Y.M.) Received March 15, 2006; accepted July 6, 2006 ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl-channel in the apical membrane of epithelial cells for cAMP-dependent Cl-secretion. Login to comment 1 ABCC7 p.Gly551Asp Here we report on the synthesis and screening of a small library of 6-phenylpyr- rolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl-channels. Login to comment 2 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Iodide efflux and whole-cell patch-clamp recordings analysis identified RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]- pyrrolo[2,3-b]pyrazine] as a submicromolar activator of wild-type (WT)-CFTR [human airway epithelial Calu-3 and WT-CFTR-Chinese hamster ovary (CHO) cells], G551D-CFTR (G551D-CFTR-CHO cells), and F508del-CFTR (in temperature-corrected human airway epithelial F508del/F508del CF15 cells). Login to comment 36 ABCC7 p.Gly551Asp Moreover, RP107 activates two CF-associated CFTR mutants (G551D-CFTR and F508del-CFTR mutants). Login to comment 61 ABCC7 p.Gly551Asp CHO cells stably transfected with pNUT vector alone (mock-CHO) or containing wild-type CFTR (WT-CFTR-CHO) and the mutant G551D-CFTR were provided by J. R. Riordan and X. B. Chang (Mayo Clinic of Scottsdale, Scottsdale, AZ) (Tabcharani et al., 1991; Becq et al., 1994, 1999). Login to comment 66 ABCC7 p.Gly551Asp maintained in ␣-minimal essential medium-GlutaMAX containing 7% FBS, 50 IU/ml penicillin and 50 ␮g/ml streptomycin, and methotrexate for cell selection (WT-CFTR-CHO, 100 ␮M; G551D-CHO, 20 ␮M; pNUT-CHO, 20 ␮M) (Tabcharani et al., 1991; Becq et al., 1994). Login to comment 83 ABCC7 p.Gly551Asp Four different cell types, WT-CFTR-CHO, G551D-CFTR CHO, Calu-3, and CF-15, were incubated in Multiwell plates at 37°C in Krebs` solution containing 1 ␮M KI and 1 ␮Ci/ml Na125 I (NEN, Boston, MA) for 30 min (CHO cells) or 1 h (Calu-3 and CF15 cells) to permit the 125 I to reach equilibrium. Login to comment 196 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp To determine whether RP107 could activate CFTR mutants, we performed iodide efflux experiments with cells expressing two of the most common CF mutations, i.e., the class III mutation G551D-CFTR studied with CHO cells stably expressing G551D-CFTR and the class II deletion F508del-CFTR studied in human airway epithelial CF15 cells endogenously expressing F508del-CFTR protein. Login to comment 197 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The mutation G551D-CFTR disrupts activation of CFTR, and phosphorylation alone is not able to achieve sufficient stimulation of G551D-dependent Cl-transport (Welsh and Smith, 1993; Becq et al., 1994; Illek et al., 1999; Marivingt-Mounir et al., 2004). Login to comment 198 ABCC7 p.Gly551Asp Iodide efflux experiments on G551D-CFTR CHO cells have been performed in the presence of 10 ␮M fsk. Login to comment 200 ABCC7 p.Gly551Asp However, RP107 stimulates an efflux in G551D-CFTR expressing cells with an EC50 of 1.5 Ϯ 1.1 nM in the presence of 10 ␮M fsk (n ϭ 4; Fig. 6A). Login to comment 202 ABCC7 p.Gly551Asp This delay in response may be related to the gating defect of the G551D-CFTR channel (Welsh and Smith, 1993) and to its inability to response to high concentration of cAMP agonists (Becq et al., 1994; Illek et al., 1999; Marivingt-Mounir et al., 2004). Login to comment 216 ABCC7 p.Gly551Asp A, effect of RP107 in G551D-CFTR-CHO cells. Login to comment 224 ABCC7 p.Gly551Asp this case was not delayed as it was for G551D-CFTR but was similar to that of wild-type CFTR (t3 for WT-CFTR and F508del-CFTR). Login to comment 279 ABCC7 p.Gly551Asp Discussion In the present study, we report on the discovery of 6-phe- nylpyrrolo[2,3-b]pyrazines, a novel family of wild-type CFTR, G551D, and F508del-CFTR activators. Login to comment 282 ABCC7 p.Gly551Asp RP107 is the most potent with an affinity of 140 to 152 nM on wild-type CFTR (Calu-3 and WT-CFTR-CHO cells), 1.5 nM on G551D-CFTR, and 111 nM on temperature-corrected F508del-CFTR. Login to comment 324 ABCC7 p.Gly551Asp It is noteworthy that trifluoromethylphe- nylbenzamine activated G551D-CFTR channels with a Kd Ͼ 10 ␮M (Ma et al., 2002). Login to comment 327 ABCC7 p.Gly551Asp In conclusion, we have identified 6-phenylpyrrolo[2,3b]pyrazines as a new scaffold structure for the selective activation of CFTR, with submicromolar affinity on wild-type CFTR, G551D-, and F508del-CFTR, two of the most common CF mutations. Login to comment