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

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 DF508-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-carbonitrilepyrazole C_act-A1 identified here is unique among prior CFTRactivating 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.