TMEM16A activators are useful research tools for pharmacological dissection of TMEM16A function, and potential drug candidates for treatment of salivary gland dysfunction, as in Sjogren’s syndrome and following radiation injury, as well as for cystic fibrosis, dry eye syndrome, selleck 17-AAG intestinal hypomotility, and other disorders associated with Cl? channel dysfunction (2, 19). In cystic fibrosis, the rationale for CaCC activator therapy is the activation of alternative, non-CFTR chloride channels in airway epithelium where CFTR is dysfunctional. Two CaCC activator therapies for cystic fibrosis have been in clinical trials, including a P2Y2 receptor antagonist (denufosol) (20), which acts through Ca2+ elevation, and a bacterial polycyclic peptide (duramycin) (21).

A P2Y2 receptor agonist is also in clinical trials for dry eye disease (22). CaCC activators that target CaCCs directly without cytoplasmic Ca2+ elevation might offer more targeted therapy than general agonists of Ca2+ signaling and, unlike receptor agonist therapy, could produce more sustained CaCC activation and hence, offer greater efficacy. Here, we report the identification and activation mechanism of TMEM16A-targeted activators and evidence for their potential therapeutic utility in cystic fibrosis, dry mouth, and intestinal hypomotility. MATERIALS AND METHODS Chemicals and solutions Amiloride, ATP, UTP, and other chemicals, unless otherwise indicated, were purchased from Sigma (St. Louis, MO, USA). 1-(2-Methoxyethyl)-2-thiourea was purchased from Oakwood Products (West Columbia, SC, USA).

T16Ainh-A01 and CFTRinh-172 were synthesized as described previously (23). The compound collections used for screening included ~100,000 synthetic small molecules from ChemDiv (San Diego, CA, USA) and Asinex (Winston Salem, NC, USA), and ~7500 purified natural products from Analyticon (Potsdam, Germany), Timtek (Newark, NJ, USA), and Biomol (Plymouth Meeting, PA, USA). Compounds were maintained as DMSO stock solutions. Structure-activity analysis was done on analogs purchased from ChemDiv and Asinex. The HCO3?-buffered solution contained (in mM): 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 d-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4). In the half-Cl? solution, 65 mM NaCl in the HCO3?-buffered solution was replaced by Na gluconate. Cell culture Fisher rat thyroid (FRT) AV-951 cells were stably transfected with human TMEM16A [TMEM16A(abc), cDNA provided by Dr. Luis Galietta, Gaslini Institute, Genoa, Italy] and the halide sensor YFP-H148Q/I152L/F46L. Cells were plated in 96-well black-walled microplates (Corning, Corning, NY, USA) at a density of 20,000 cells/well in Coon’s modified F12 medium supplemented with 5% FCS, 2 mM l-glutamine, 100 U/ml penicillin and 100 ��g/ml streptomycin.