Received for publication March 15, 2006.
Revised July 6, 2006.
Accepted for publication July 6, 2006.

Discovery of pyrrolo[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild-type, G551D and F508del CFTR chloride channels

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. 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 RP-107 as a submicromolar activator of wt-CFTR (human airway epithelial Calu-3 and wtCFTR-CHO cells), G551D-CFTR (G551D-CFTR-CHO cells) and F508del-CFTR (in temperature corrected human airway epithelial F508del/F508del CF15 cells). The structural analogue RP-108 (4-chlorophenyl), contrary to RP-107 (4-hydroxyphenyl), was less potent being activator only at micromolar concentrations. RP-107 and RP-108 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 but not by calixarene or DIDS. Finally, we found significant stimulation of short circuit current (Isc) by RP-107 (EC₅₀ = 89 nM) and RP-108 (EC₅₀ = 103 µM) on colon of Cftr^+/+ but not of Cftr^-/- mice mounted in Ussing chamber. Stimulation of Isc was inhibited by glibenclamide but not affected by DIDS. These results show that RP-107 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.

Key words: CFTR, F508del, G551D, chloride secretion, pharmacology, pyrrolopyrazines

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