Abstract
Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) Cl− channel. We developed a phenotype-based high-throughput screen to identify small-molecule activators of human airway epithelial Ca2+-activated Cl− channels (CaCCs) for CF therapy. Unexpectedly, screening of ∼110,000 synthetic small molecules revealed an amino-carbonitrile-pyrazole, Cact-A1, that activated CFTR but not CaCC Cl− conductance. Cact-A1 produced large and sustained CFTR Cl− currents in CFTR-expressing Fisher rat thyroid (FRT) cells and in primary cultures of human bronchial epithelial (HBE) cells, without increasing intracellular cAMP and in the absence of a cAMP agonist. Cact-A1 produced linear whole-cell currents. Cact-A1 also activated ΔF508-CFTR Cl− currents in low temperature-rescued ∆F508-CFTR-expressing FRT cells and CF-HBE cells (from homozygous ∆F508 patients) in the absence of a cAMP agonist, and showed additive effects with forskolin. In contrast, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770) and genistein produced little or no ∆F508-CFTR Cl− current in the absence of a cAMP agonist. In FRT cells expressing G551D-CFTR and in CF nasal polyp epithelial cells (from a heterozygous G551D/Y1092X-CFTR patient), Cact-A1 produced little Cl− current by itself but showed synergy with forskolin. The amino-carbonitrile-pyrazole Cact-A1 identified here is unique among prior CFTR-activating compounds, as it strongly activated wild-type and ∆F508-CFTR in the absence of a cAMP agonist. Increasing ∆F508-CFTR Cl− conductance by an “activator,” as defined by activation in the absence of cAMP stimulation, provides a novel strategy for CF therapy that is different from that of a “potentiator,” which requires cAMP elevation.
Footnotes
- Received March 16, 2013.
- Accepted June 19, 2013.
This work was supported by the Yonsei University Research Fund of 2012 and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology [NRF-2012R1A1A1040142]; and by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK72517].
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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