TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis

Abstract

Rationale: Enhancing non-CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca²⁺-activated Cl^- channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.

Keywords: anoctamin-1; calcium-activated chloride channel; mucus clearance; mucus hydration.

Figure 1.

ETX001 is a potent and efficacious potentiator of human recombinant TMEM16A (anoctamin-1). (A and C) Whole-cell patch-clamp electrophysiological recordings from Fischer rat thyroid cells stably expressing hTMEM16A in the absence (black lines) and presence (red lines) of primary screening hit (A) and the optimized compound ETX001 (C) at indicated concentrations. Dashed lines indicate the zero-current level. (B and D) Current–voltage relationships for vehicle and indicated concentrations of compounds are shown. (E) Concentration–response curves for the primary screening hit and ETX001. Mean data (±SEM) (screening hit n = 5; ETX001 n = 14) are shown. EC₅₀ = half-maximal effective concentration.

Figure 2.

ETX001 potentiates the calcium-activated anion secretory current in primary cystic fibrosis (CF)–human bronchial epithelial (HBE) cells. (A) Sample raw data trace illustrating the experimental protocol for assessing the effects of TMEM16A (anoctamin-1) potentiator compounds. Increasing concentrations of ETX001 were added to CF-HBE cells following stimulation of the short-circuit current (ISC) with a submaximal (EC₂₀) concentration of cyclopiazonic acid (CPA). Ani9 (10 μM; downward arrow) was added at the completion of each experiment to confirm the sensitivity of the ISC response to the TMEM16A blocker. (B) Sample concentration–response data derived from the raw data in A. (C) Correlation of data from the CPA ISC assay to the QPatch whole-cell patch-clamp assay. ETX001 is shown as a solid red circle. (D) Sample raw data trace illustrating the effects of ETX001 (1 μM) on the UTP-stimulated anion current response. After inhibition of the ENaC current with amiloride (10 μM), inserts were treated with either DMSO (gray dashed trace) or 1 μM ETX001 (red line) before the cumulative addition of increasing concentrations of UTP. (E) Mean data illustrating the potentiation of the UTP concentration response by ETX001. Data are expressed as the mean of 6 to 8 inserts of CF-HBE cells (donor: KK036H). See online supplement for additional donor-derived CF-HBE cell data. All CF-HBE cells in A to E were IL-13 treated (10 ng/ml) for 48 hours before ion transport assay to optimize the TMEM16A assay window. (F and G) CF-HBE cells that had not been treated with IL-13 also showed a UTP-stimulated (10 μM) anion secretory response that was potentiated by ETX001. *P < 0.005 and **P < 0.001 using a two-way ANOVA with Siadak’s multiple comparison test; ^#P < 0.05 using two-tailed Student’s t test. AUC = area under the curve; EC₅₀ = half-maximal effective concentration; UTP = uridine-5′-triphosphate.

Figure 3.

ETX001 does not affect calcium handling in differentiated cystic fibrosis–human bronchial epithelial (HBE) cells. (A) A cartoon illustrating the experimental protocol for assessing effects of compounds on [Ca²⁺]_i in air–liquid interface cultures of HBE cells. (B and C) ETX001 (1 μM) had no direct effect on [Ca²⁺]_i levels (B) and also had no effect on the UTP-stimulated increase in [Ca²⁺]_i (C). Mean data ± SD (n = 6 inserts per group) are shown from a single donor. addⁿ/incubⁿ = addition/incubation; ALI = air–liquid interface; AM = acetoxymethyl ester; RFU = relative fluorescence units; UTP = uridine-5′-triphosphate.

Figure 4.

ETX001 enhances fluid secretion in cystic fibrosis–human bronchial epithelial (HBE) cells under static and shear stress–stimulated conditions. (A) A cartoon illustrating the physiological regulation of anion secretion in the human airway epithelium. (B) Sample XZ confocal images of airway surface liquid (ASL) visualized using Texas red staining in the absence and presence of ETX001 (1 μM). (C) The change in ASL height from baseline was measured following a 1-hour treatment with ETX001 or vehicle under static conditions (i.e., no shear stress) and after 3 hours of continual shear stress. Data are expressed as the mean change in ASL height (±SEM) for seven to eight inserts from a F508del/F508del HBE cells donor and is representative of three independent experiments. Cystic fibrosis–HBE cells had not been pretreated with IL-13. *P < 0.001 and **P < 0.00001 for ETX001-treated versus time-matched vehicle control using two-tailed Student’s t test. TMEM16A = anoctamin-1.

Figure 5.

Inhaled ETX001 accelerates mucociliary clearance in conscious sheep models. (A) Dosing of conscious sheep with CFTR_Inh172 slowed the rate of tracheal mucus velocity (TMV). At 4 hours after dosing with CFTR_inh172, ETX001 or vehicle was administered as an inhaled, nebulized, DMSO-based solution. TMV values are expressed as the mean (±SEM) % of the initial (pre-CFTR_Inh172) rate (n = 3 sheep per group). (B) To assess whole-lung clearance, ETX001 or vehicle were dosed as an inhaled, nebulized suspension 4 hours before the inhaled administration of technetium-labeled sulfur colloid (^99mTc-SC). Mean (±SEM) clearance of ^99mTc-SC in sheep is expressed as the percentage of the initial deposited lung dose (n = 3–4 sheep per group). Symbols denote a significant difference from the time-matched vehicle control animals using two-way ANOVA with a post hoc Dunnett’s test. ^#P < 0.01, ^##P < 0.001, and *P < 0.0001.