The resulting white solid was suspended in CH2Cl2 (1.5 ml) inhibitor Trichostatin A and treated with p-bromoaniline (90 mg, 0.526 mmol) and triethylamine (100 ml, 0.719 mmol). The reaction was stirred at room temperature for 14 h, mixed with silica gel, and concentrated to dryness. Chromatography [silica, CH2Cl2:methanol (98:2 to 1:3)] yielded an off-white solid (48 mg, 53%), decomposition point 203��C. 1H NMR (300 MHz, DMSO-d6) d 7.58 (d, J = 4.9, 2H), 7.79 (d, J = 4.9, 2H), 7.85 (��, J = 8.0, 1H), 8.10�C8.18 (m, 2H), 8.47�C8.51 (m, 1H), 10.22 (s, 1H), and 10.62 (br, 1H, NH). 13C NMR (75 mHz, DMSO-d6) �� 120.18, 122.08, 123.92, 128.50, 130.11, 131.30, and 142.25. ESI-MS is calculated for C14H10BrN5O 344.0141; found 344.0152.
RESULTS Discovery and characterization of TMEM16A activators TMEM16A activators were identified from screening of ~110,000 synthetic drug-like compounds, purified natural products, and approved/investigational drugs. Our cell-based screen utilized FRT cells coexpressing human TMEM16A and the I?-sensing yellow fluorescent protein YFP-H148Q/I152L/F46L. FRT cells were chosen because of their low basal I? and Cl? transport, rapid growth on uncoated plastic, strong stable expression of transfected proteins, and formation of tight junctions for measurements of transepithelial short-circuit current (23). As diagrammed in Fig. 1A, test compounds at 25 ��M final concentration were added 10 min prior to I? addition. The 10-min incubation was chosen to allow for compound transport into cytoplasm and to minimize false positives from compounds that elevate cytoplasmic Ca2+ transiently.
Fluorescence from individual wells of 96-well plates was measured just prior to and for 6 s after I? addition for computation of initial I? influx rate. Figure 1B shows representative fluorescence data from single wells showing positive (ionomycin) and negative (vehicle only) controls, and examples of inactive and active compounds. Figure 1. Identification of small-molecule TMEM16A activators by high-throughput screening. A) Screening protocol. FRT cells stably expressing TMEM16A and the halide-sensitive cytoplasmic fluorescent sensor YFP-H148Q/I152L/F46L were incubated for 10 min with test … Primary screening yielded 40 compounds that increased I? influx by >2 mM/s at 25 ��M (>50% of maximal I? influx produced by 100 ��M ATP) and had EC50 of <10 ��M.
Figure 1C shows structures of active compounds from six chemical classes. Secondary screens were done to identify compounds that increased I? influx by targeting TMEM16A. Measurements on Dacomitinib TMEM16A null cells (expressing YFP alone) showed that none of the active compounds increased I? influx. However, Cact and Dact increased TMEM16A-mediated I? efflux by producing sustained elevation of cytoplasmic Ca2+ (Fig. 2A). Though such Ca2+ agonists are of potential interest, they were not studied further.