RESUMO
Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of cystic fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-cell and single patch-clamp recordings showed that RP193 induced a linear, time- and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GP(inh)5a). Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.
RESUMO
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 (EC(50) = 89 nM) and RP108 (EC(50) = 103 microM) 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.
