TMEM16A Channel Function Does Not Influence Goblet Cell Numbers or Mucin Secretion In The Human Airway Epithelium – NACFC – Nashville – October 2019

Henry Danahay^1,2, Alexis Flen, Camille Ehre³, Martin Gosling^1,2

¹University of Sussex, Brighton, UK; ²Enterprise Therapeutics, UK; ³University of North Carolina, Chapel Hill, USA.

A role for the calcium activated chloride channel, TMEM16A, in the regulation of airway goblet cell formation and function has been recently reported (Benedetto et al., 2019; Kondo et al., 2017; Lin et al., 2015; Qin et al., 2016). Much of the published data to support a role for TMEM16A in these processes has been based on either gene silencing or the use of non-specific TMEM16A blockers (e.g. niflumic acid). It is therefore unclear whether these proposed functions are dependent on the ion channel activity of TMEM16A, through an alternative aspect of the proteins function or even a TMEM16A-independent activity of a non-selective pharmacological tool.

Using a potent TMEM16A channel blocker (Ani9) and recently identified TMEM16A potentiator compounds (Enterprise Therapeutics; proprietary), we have evaluated whether channel function can regulate goblet cell number and/or mucin secretion in primary cultures of human bronchial epithelial (HBE) cells.

Primary HBE (3 donor codes) were cultured for 2 weeks at air-liquid interface (ALI) on permeable supports and formed a well-differentiated mucociliary epithelium. On ALI day 15, cells were treated with either: 1) vehicle, 2) IL-13 (10 ng/mL) or 3) the TMEM16A potentiator, ETX001 (1 µM) with each group in either the absence or presence of the TMEM16A blocker, Ani9 (10 µM). ETX001 is a TMEM16A potentiator with EC₅₀ values of 114 and 170 nM for the potentiation of chloride secretion in patch clamp (FRT-TMEM16A) and ion transport (CF-HBE) respectively. Ani9 fully blocks TMEM16A function in both patch clamp and HBE ion transport studies. Cells were cultured under these conditions for 96h before fixation and staining with antibodies directed against MUC5AC (goblet cells) and acetylated α-tubulin (ciliated cells). Goblet and ciliated cell numbers were quantified using an automated image acquisition (Zeiss Axiovert) and analysis system (Image J).

IL-13 induced a significant increase in the density of goblet cells based on the increased staining for MUC5AC, that was unaffected by co-administration of Ani9. Neither ETX001 or Ani9 (alone or in combination) had any effect on goblet cell numbers. Finally, the co-administration of ETX001 with IL-13 also failed to modify goblet cell numbers.

In a separate series of experiments, CF-HBE (±IL-13 pre-treatment; 2 donor codes) were treated with TMEM16A potentiator compounds for 24 hours and mucin secretion was quantified. TMEM16A potentiator compounds were without effect on the secretion of either MUC5B or MUC5B

Together, these data do not support a role for the ion channel function of TMEM16A in either the regulation of goblet cell numbers or the secretion of mucins in primary HBE.

 

Benedetto et al. (2019) FASEB J. 33(3):4502-4512

Kondo M et al. (2017) Clin Exp Allergy. 47(6):795-804.

Lin J et al. (2015) Exp Cell Res. 334(2):260-9.

Qin et al. (2016) Int Immunopharmacol. 40:106-114

The In Vitro & In Vivo Pharmacology of Novel Tmem16a Potentiator Compounds – NACFC – Nashville – October 2019

Henry Danahay^1,2, Sarah Lilley¹, Holly Charlton¹, Roy Fox¹, Brian Button³, Juan Sabater⁴, Martin Gosling^1,2

¹University of Sussex, Brighton, UK; ²Enterprise Therapeutics, UK; ³University of North Carolina, Chapel Hill, USA; Mount Sinai Medical Center, Miami, USA

TMEM16A was recently identified as a calcium-activated chloride conductance and a key orchestrator of anion secretion in the human airway epithelium (Caputo et al 2008; Schroeder et al 2008; Yang et al 2008). It is now clinically established that promoting anion secretion in the airway leads to enhanced mucus clearance and reduced exacerbation frequency in CF patients and as such TMEM16A represents an important target for the next generation of mucokinetics. Importantly, positive regulators of TMEM16A function will be expected to be of benefit in all CF patients, irrespective of their CFTR mutational status.

Using 4 parallel screening approaches, we identified several chemically diverse, low molecular weight compounds that potentiated TMEM16A function. These hit compounds were validated for TMEM16A function using a patch-clamp assay under conditions where [Ca²⁺]_i was tightly buffered at an EC₂₀ for TMEM16A channel activity. This enabled hits that activated TMEM16A by non-specifically elevating [Ca²⁺]_i to be rapidly filtered out from the hit list.

The efficacy of bona fide TMEM16A potentiators translated through to function in ion transport studies in CF-HBE. Pre-treatment of CF-HBE with TMEM16A potentiators for between 5 min to 96h resulted in an enhancement of Ca²⁺-mediated anion-secretory responses that were sensitive to the TMEM16A blocker, Ani9. Measurements of [Ca²⁺]_i confirmed that TMEM16A potentiators had no effect on calcium mobilization, consistent with a direct effect on the channel. TMEM16A potentiators also enhanced fluid secretion in CF-HBE, measured as an increase in the height of airway surface liquid (ASL). Inhaled dosing of TMEM16A potentiators induced a dose-dependent increase mucus clearance in vivo, using a sheep model of tracheal mucus velocity.

Together, these data support the concept that potentiators of the alternative airway chloride conductance, TMEM16A, can restore both anion and fluid secretion in primary CF cells and also enhance mucociliary clearance in vivo. Enterprise Therapeutics are advancing TMEM16A potentiators into clinical development.

Caputo et al (2008) Science 322(5901):590-594

Schroeder et al (2008) Cell 134(6):1019-1029

Yang et al (2008) Nature 455(7217):1210-1215

 

ECFS Clinical Meeting Liverpool TMEM16A potentiator

Objectives: TMEM16A (ANO1) is a Ca²⁺-activated, alternative chloride channel expressed in the human airway epithelium. A potentiator of TMEM16A is predicted to enhance the secretion of chloride and thereby increase mucus hydration and clearance in the CF lung. Our aim is to discover and test the efficacy of novel TMEM16A potentiator compounds as a new therapeutic option for the treatment of CF lung disease.

Methods: Four, parallel high-throughput screening approaches were used to identify novel TMEM16A potentiator compounds. Compounds were then optimised and tested in primary CF bronchial epithelial cells for effects on both ion transport and airway surface liquid secretion.

Results: Several classes of TMEM16A potentiators were identified from the high-throughput screens. Only compound series that potentiated TMEM16A activity in a patch-clamp electrophysiology assay and which had no effect on intra-cellular Ca²⁺, were progressed for lead optimization. Pre-treatment of CF-HBE with TMEM16A potentiators for between 5 min to 96h resulted in an enhancement of Ca2+-mediated anion-secretory responses that were sensitive to the TMEM16A blocker, Ani9. Measurements of [Ca2+]i confirmed that TMEM16A potentiators had no effect on calcium mobilization, consistent with a direct effect on the channel. A Series 1 TMEM16A potentiator, ETX001, increased the secretion of airway surface liquid (ASL) in CF-HBE. The ETX001-driven increase in ASL height was further enhanced in cells that had been pre-treated with IL-13 to boost TMEM16A expression. A close structural analogue of ETX001, ETX002, that is inactive on TMEM16A, did not increase ASL height.

Conclusions: Together, these data support the concept that potentiators of the alternative airway chloride conductance, TMEM16A, can restore anion conductance and fluid secretion in both primary CF cells. Enterprise Therapeutics are advancing TMEM16A potentiators into clinical development.

Something old and something new: ENaC & TMEM16A as targets to hydrate mucus in the CF airway

Henry Danahay, Enterprise Therapeutics, Brighton, UK

In the absence of functional CFTR in the apical membrane, the epithelial sodium channel (ENaC) and TMEM16A, a calcium activated chloride channel, are key regulators of airway mucosal hydration. As drug targets, both ENaC blockers and TMEM16A potentiators would provide an opportunity to treat CF patients who are unsuitable for CFTR repair therapies.

Inhaled ENaC blockers are predicted to attenuate fluid absorption out of the airway mucosa and improve mucus clearance. Clinical studies using the ENaC blocker amiloride, were first reported >30 years ago although since then novel inhibitors have failed to deliver efficacy in Phase 2. In contrast, positive modulators of TMEM16A as an alternative anion conductance to compensate for the loss of CFTR function in the airway epithelium are only now becoming available to begin to ask whether this mechanism could provide adequate anion and fluid secretion to hydrate airway mucus.

Why have novel, inhaled ENaC blockers failed to deliver any clinical benefit in Phase 2 studies? Is ENaC simply just not a good target for restoring mucus hydration or have we not developed the right drug candidates yet? Human genetics would support the negative regulation of ENaC as being a core mechanism by which to enhance mucus clearance, in which case the drug candidates are presumably the issue. To this end, we have evaluated the in vivo potency and efficacy of several ENaC blockers that have entered clinical development. A common finding is that doses used for the clinical studies are low when compared to the predicted ‘minimally efficacious dose’ derived from the pre-clinical dose-response relationships. This may be because of safety concerns commonly associated with ENaC blockers, specifically the potential for inducing hyperkalaemia as a side effect of ENaC block in the kidney. Moving forward, perhaps we should consider advancing only candidates with a broader safety window, offering the potential to dose significantly higher than a predicted minimally efficacious dose? Furthermore, clinical studies to confirm target engagement and duration of action would help to understand whether ENaC does represent a meaningful target to inhibit and thereby restore mucus hydration.

TMEM16A-based drug discovery is at a much earlier stage. High-throughput screening has identified novel potentiators of the channel. These compounds do not directly open the channel but rather ‘sensitize’ it to elevations in [Ca²⁺]_i under the control of normal physiology. We can demonstrate that TMEM16A potentiators will enhance airway epithelial anion and fluid secretion and improve mucus clearance with the opportunity to treat patients currently unsuitable for CFTR repair drugs.

E_ACT increases intracellular calcium levels by a TMEM16A-independent mechanism

Sarah Lilley1, Henry Danahay1,2, Holly Charlton1, Kathryn Adley1, Roy Fox1, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK

The N-aroylaminothiazole EACT was first described by Namkung et al. (2011) as an activator of the calcium-activated chloride channel, TMEM16A. Subsequently, EACT has been used as a tool compound by investigators to describe a wide variety of putative physiological functions of TMEM16A. The aim of the present study was to compare the pharmacology of EACT with alternative potentiators of TMEM16A that have been recently discovered by high-throughput screening.

Consistent with the original reported pharmacology, EACT increased anion secretory responses in models of epithelial ion transport that could be attenuated with the TMEM16A blocker, Ani9. Similarly, novel TMEM16A potentiators from 3 structurally distinct chemical series also increased the Ani9-sensitive anion secretion in these ion transport models.

To understand the mechanism of activation of these anion secretory currents, the effects of EACT and the novel TMEM16A potentiators on levels of intracellular calcium ([Ca2+]i) were evaluated. The acute addition of EACT to primary CF-HBE induced a concentration-dependent increase in [Ca2+]i. Pre-treatment of cells with Ani9 had no effect on the EACT-induced rise in [Ca2+]i. In contrast, the novel TMEM16A potentiators had no effect on [Ca2+]i.

The observation that EACT could increase [Ca2+]i questioned the reported pharmacological mechanism of TMEM16A activation by this molecule ie. via a direct interaction with the channel. To address whether EACT could directly activate TMEM16A in the absence of an elevation of [Ca2+]i, patch-clamp studies were performed under conditions of buffered [Ca2+]i. Under these conditions, with [Ca2+]i tightly clamped, EACT showed no evidence of any activity on TMEM16A. In contrast, the novel TMEM16A potentiators all showed a potent increase in channel function.

Together, these data do not support the description of EACT as a direct TMEM16A modulator but are consistent with its activation of TMEM16A being indirect, the result of an as yet undefined mechanism leading to an elevation of [Ca2+]i. Furthermore the recent proposal that TMEM16A positively regulates [Ca2+]i (Cabrita et al., 2017) is not consistent with the lack of effect of either the TMEM16A blocker Ani9 or the novel potent and selective TMEM16A potentiators on [Ca2+]i. Our data suggest that literature reports of TMEM16A function that have relied on the use of EACT as a pharmacological tool should be interpreted with caution.

Namkung et al (2011) FASEB J 25(11):4048-4062