Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction

Abstract

TMEM16A (ANO1) is a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. Cell-based functional screening of ∼110,000 compounds revealed compounds that activated TMEM16A CaCC conductance without increasing cytoplasmic Ca(2+). By patch-clamp, N-aroylaminothiazole "activators" (E(act)) strongly increased Cl(-) current at 0 Ca(2+), whereas tetrazolylbenzamide "potentiators" (F(act)) were not active at 0 Ca(2+) but reduced the EC(50) for Ca(2+)-dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (E(act)) and potentiator (F(act)) produced large and more sustained CaCC Cl(-) currents than general agonists of Ca(2+) signaling, with EC(50) 3-6 μM and Cl(-) conductance comparable to that induced transiently by Ca(2+)-elevating purinergic agonists. Analogs of activators were identified that fully inhibited TMEM16A Cl(-) conductance, providing further evidence for direct TMEM16A binding. The TMEM16A activators increased CaCC conductance in human salivary and airway submucosal gland epithelial cells, and IL-4 treated bronchial cells, and stimulated submucosal gland secretion in human bronchi and smooth muscle contraction in mouse intestine. Small-molecule, TMEM16A-targeted activators may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for pharmacological dissection of TMEM16A function.

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 compound. Fluorescence was monitored in response to addition of iodide. B) Fluorescence measured in single wells of 96-well plates, showing vehicle and positive (ionomycin) controls and examples of inactive and active compounds. C) Structures of TMEM16A activators of six different chemical classes.

Figure 2.

Characterization of TMEM16A activators. A) Cytoplasmic calcium measured by Fluo-4 fluorescence. Arrow indicates addition of 100 μM ATP (gray line) or 10 μM of indicated TMEM16A activators. B) Apical membrane current measured in TMEM16A-expressing FRT cells in the presence of a transepithelial chloride gradient and after basolateral membrane permeabilization. Left and center panels: representative current traces showing ATP (100 μM), E_act- or F_act-stimulated TMEM16A Cl⁻ current. T16A_inh-A01 (10 μM) was added where indicated. Inset shows long-time E_act effect. Right panel: concentration-activation data summary (mean±se, n=4–6). C) Synergistic effect of E_act and F_act. Left and center panels: representative current traces showing synergy. Right panel: data summary of low doses of TMEM16A activation (mean±se, n=5). *P < 0.05. D) Apical membrane current measured in FRT cells transfected with mouse TMEM16A or TMEM16B. E) Effect of E_act and F_act on CFTR and ENaC. Left panel: FRT cells expressing wild-type CFTR and YFP indicator were pretreated for 5 min with 10 μM E_act and F_act. Forskolin (10 μM) was added as indicated. Right panel: HBE cells were pretreated for 5 min with 10 μM E_act and F_act, with amiloride (10 μM) added as indicated.

Figure 3.

Structure-activity analysis and synthesis of TMEM16A activators. A) Structural similarities between TMEM16A inhibitors and activators (common scaffolds shown in red). Apical membrane current measurements show activation of TMEM16A by B_act (top panels), inhibition of ionomycin (1 μM)-induced TMEM16A currents by pretreatment B and E class analogs (each 10 μM; bottom panels). B) Summary of structural determinants for TMEM16A activation. Left panel: E_act class. Right panel: F_act class. C) Synthesis of E_act and F_act analogs (see Materials and Methods).

Figure 4.

Structure-activity analysis of E_act and F_act analogs. EC₅₀ values were determined from fluorescence plate reader assay.

Figure 5.

Patch-clamp analysis of Ca²⁺ requirements for TMEM16A activation by E_act and F_act. A) Apical membrane current measured in TMEM16A-expressing FRT cells. ER calcium stores were depleted by CPA (50 μM, 30 min) and 0 CaCl₂ in bath. ATP (100 μM), F_act (10 μM), E_act (10 μM), and T16A_inh-A01 (10 μM) were added as indicated. B, C) Whole-cell TMEM16A currents were recorded at a holding potential at 0 mV, and pulsing to voltages between ± 80 mV (in steps of 20 mV) in the absence and presence of 3 μM E_act (B) or 10 μM F_act (C). Left panels: free calcium concentration of pipette solutions were clamped at 0 μM (black), 0.07 μM (green), 0.15 μM (red), and 1 μM (blue). E_act (3 μM) or F_act (10 μM) was added as indicated. Right panels: current-voltage (I/V) plots of mean currents at the middle of each voltage pulse. D) TMEM16A inhibited by 10 μM T16A_inh-A01 after stimulation by E_act or F_act.

Figure 6.

Airway epithelial chloride secretion. A) Short-circuit current in CF HBE cells. Left and center panels: E_act and UTP (100 μM) were added in control (left) and IL-4 (10 ng/ml, 24 h, center) treated CF HBE cells. Right panels: summary of E_act-induced, T16A_inh-A01-sensitive peak current (mean±se, n=6–8). *P < 0.05. ENaC was inhibited by 10 μM amiloride. B) E_act (10 μM) and UTP (100 μM) induced CaCC Cl⁻ current measured in primary cultures of non-CF human tracheal gland (HTG) serous cells. Left and center panels: TMEM16A, CFTR, and ENaC were inhibited by pretreatment with T16A_inh-A01, CFTR_inh-172, and amiloride, respectively. Inset: TMEM16A immunoblot in whole cell homogenates of CF HBE and HTG cells. Right panel: summary of UTP and E_act-induced peak current in the presence and absence of T16A_inh-A01 (mean±se, n=3). *P < 0.05.

Figure 7.

Airway submucosal gland fluid secretion in human bronchi. A) TMEM16A immunohistochemistry in CF (left panel) and non-CF (right panel) human bronchi showing apical membrane expression in serous gland epithelial cells (arrows). Scale bar = 20 μm. B) Mucus (fluid) secretion in human bronchi. Top panels: images of mucus bubbles formed under oil in response to basolateral application of 300 nM carbachol (CCh) and 20 μM E_act. TMEM16A was inhibited by 30 μM T16A_inh-A01. Individual fluid bubbles marked with arrowheads. Scale bar = 0.5 mm. Bottom panels: CCh and E_act-induced secretion rates. Where indicated, tissues were pretreated with T16A_inh-A01 (30 μM). Each point is the average of measurements made from 20 glands (mean±se). C) Summary of human gland fluid secretion rates measured at 20 min after addition of 20 μM E_act, and 30 min after application of 300 nM carbachol (CCh) and 10 μM forskolin (20–66 glands from 3 tracheas and 4 bronchi). In CF-bronchi, 6 glands from one donor were stimulated by E_act. *P < 0.05.

Figure 8.

Salivary gland epithelial cell Cl⁻ secretion. A) Expression of TMEM16A in human salivary gland. Left panel: TMEM16A immunostaining in human parotid gland. Scale bar = 20 μm. Right panel: immunoblot of TMEM16A in FRT-TMEM16A and A253 cells. B) Whole-cell patch-clamp recordings in A253 cells. Left panel: CaCC and TMEM16A chloride current induced by 100 μM ATP and 10 μM E_act, respectively. Right panel: current/voltage (I/V) plot of mean currents at the middle of each voltage pulse (voltages between −80 and +80 mV in steps of 20 mV).

Figure 9.

Intestinal smooth muscle contraction. A) Representative traces from mouse ileal segments showing effects of T16A_inh-A01 (10 μM), carbachol (CCh, 1 μM) and E_act (10 μM). B) Effect of CCh (1 μM) and E_act (10 μM) following atropine (1 μM). C) Summary of contraction frequency (left panel) and resting and maximum tone (right panel; mean±se, n=4–7). *P < 0.05.