title: Novel amino-carbonitrile-pyrazole identified in a small molecule screen activates wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator in the absence of a cAMP agonist.
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, C(act)-A1, that activated CFTR but not CaCC Cl⁻ conductance. C(act)-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. C(act)-A1 produced linear whole-cell currents. C(act)-A1 also activated ΔF508-CFTR Cl⁻ currents in low temperature-rescued ΔF508-CFTR-expressing FRT cells and CF-HBE cells (from homozygous ΔF508 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 ΔF508-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), C(act)-A1 produced little Cl⁻ current by itself but showed synergy with forskolin. The amino-carbonitrile-pyrazole C(act)-A1 identified here is unique among prior CFTR-activating compounds, as it strongly activated wild-type and ΔF508-CFTR in the absence of a cAMP agonist. Increasing ΔF508-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.
authors.affiliation: College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon 406-840, Korea. wnamkung@yonsei.ac.kr
authors.forename: Wan
authors.initials: W
authors.lastname: Namkung
authors.forename: Jinhong
authors.initials: J
authors.lastname: Park
authors.forename: Yohan
authors.initials: Y
authors.lastname: Seo
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D000242:Cyclic AMP / Q000819:agonists / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D009154:Mutation; D009570:Nitriles / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D061251:Primary Cell Culture; D011720:Pyrazoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: C492109:CLCA1 protein, human; D018118:Chloride Channels; D009570:Nitriles; D011720:Pyrazoles; D054852:Small Molecule Libraries; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP