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CELLULAR AND MOLECULAR

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

Institut de Physiologie et Biologie Cellulaires Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 6187, Université de Poitiers, Poitiers, France (S.N., C.F., C.N., C.R., Y.M., F.B.); and Faculté de Médecine et Pharmacie, Poitiers cedex, France (C.F., Y.M.)

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl^- channel in the apical membrane of epithelial cells for cAMP-dependent Cl^- secretion. Here we report on the synthesis and screening of a small library of 6-phenylpyrrolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl^- channels. 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). The structural analog RP108 [7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine], contrary to RP107, was a less potent activator only at micromolar concentrations. RP107 and RP108 did not have any effect on the cellular cAMP level. Activation was potentiated by low concentration of forskolin and inhibited by glibenclamide and CFTR_inh-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone]but not by calixarene or DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). Finally, we found significant stimulation of short circuit current (I_sc) by RP107 (EC₅₀ = 89 nM) and RP108 (EC₅₀ = 103 µM) on colon of Cftr^+/+ but not of Cftr^-/- mice mounted in Ussing chamber. Stimulation of I_sc was inhibited by glibenclamide but not affected by DIDS. These results show that RP107 stimulates wild-type CFTR and mutated CFTR, with submicromolar affinity by a cAMP-independent mechanism. Our preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of CFTR. The potency of these agents indicates that compounds in this class may be of therapeutic benefit in CFTR-related diseases, including cystic fibrosis.

Address correspondence to: Pr. Frédéric Becq, IPBC CNRS UMR 6187, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France. E-mail: frederic.becq{at}univ-poitiers.fr

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