Novel Amino-Carbonitrile-Pyrazole Identified in a Small Molecule Screen Activates Wild-Type and ∆F508 Cystic Fibrosis Transmembrane Conductance Regulator in the Absence of a cAMP Agonist

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.