Received for publication March 23, 2007.
Revised June 18, 2007.
Accepted for publication June 19, 2007.

Discovery of -aminoazaheterocycle-methylglyoxal adducts as a new class of high affinity inhibitors of CFTR chloride channels

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl^- channel in apical membrane of epithelial cells for cAMP-dependent Cl^- secretion. Here we report on the synthesis and screening of a small library of non-toxic -aminoazaheterocycle-methylglyoxal adducts inhibitors of wild-type CFTR, G551D-, G1349D- and F508del-CFTR Cl^- channels. In whole-cell patch clamp experiments of CHO cells expressing wild-type (wt-) CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC₅₀ = 14.7 µM), CFTR_inh-172 (IC₅₀ = 1.2 µM) and -aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC₅₀ = 71 pM), 8a,b (IC₅₀ = 2.5 nM) and 7a,b (IC₅₀ = 3.4 nM) as the most potent inhibitors of wt-CFTR channel. Similar range of inhibition were also found with these compounds 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). No effect of compound 5a was detected on the volume-regulated or calcium-regulated-iodide efflux. Picomolar inhibition of wt-CFTR with adduct 5a was also found using SPQ fluorescent probe applied to the human tracheobronchial epithelial cell line 16HBE14o-. Finally, we found comparable inhibition by 5a or by CFTR_inh-172 of forskolin-dependent short-circuit currents on mice colon. To the best of our knowledge, these new non-toxic -aminoazaheterocycle-methylglyoxal adducts represent the most potent compounds reported to inhibit CFTR chloride channels.

Key words: CFTR chloride channels, cystic fibrosis, drug discovery, inhibitor, methylglyoxal, pharmacology

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