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

 

A systematic comparison of the profiles of inhaled ENaC blocker candidates on mucociliary clearance: are we under-dosing in clinical studies?

Henry Danahay¹, Clive McCarthy¹ and Martin Gosling¹

¹Enterprise Therapeutics, UK

Objectives: Recent clinical studies with ENaC modulator compounds have challenged the validity of the target for treating CF lung disease. Our aim was to establish retrospective human dose predictions for several of these compounds to understand whether this might be a factor in the failure of the clinical studies to show benefit.

Methods: ENaC modulators used in recent CF clinical studies were assessed in a sheep model of mucociliary clearance (MCC) and were compared with ETD001, a novel, non-amiloride based inhaled ENaC blocker (Enterprise Therapeutics), entering clinical development in 2019. Test compounds were administered directly into the sheep airways by aerosolization and MCC was measured by gamma-scintigraphy between 4-6h after the completion of dosing. Using the dose required to achieve a maximal increase in MCC in the sheep, an estimated ‘minimal effective clinical dose’ was calculated for each ENaC modulator.

Results: Each of the compounds studied induced a dose-dependent increase in MCC, typically reaching a maximum of between 20-25% clearance of the ^99mTc-sulfur colloid tracer. Combining the predicted clinical dose (based on the sheep MCC model) with the actual dose/nebuliser used in the respective clinical study (www.clintrials.gov), suggests that all the ENaC modulators tested in the clinic to date have potentially been under-dosed by as much as 90%. This analysis further assumes that the healthy sheep airways in the MCC model predict for a clinical dose in a CF lung on a 1:1 basis. If a CF lung requires a higher dose than a sheep airway, the potential for under-dosing increases.

Conclusions: The pre-clinical safety profile of ETD001 currently enables doses of between 30-40x above the minimum predicted clinically efficacious dose to be tested in clinical studies. This profile provides a good opportunity to test the ENaC therapeutic hypothesis and ultimately provide a new therapy for CF patients.

The pharmacology of novel TMEM16A potentiator compounds

Henry Danahay1,2, Sarah Lilley1, Holly Charlton1, Roy Fox1, Brian Button3, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK; 3University of North Carolina, Chapel Hill, 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.

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.
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.

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

Identifying pathways & compounds regulating goblet cell metaplasia in vitro

Henry Danahay1, Roy Fox2 and Martin Gosling1
1Enterprise Therapeutics, UK; 2University of Sussex, UK

It is widely accepted that the composition of mucus in the CF airway, and the hydration status, significantly affects its clearance and thereby the potential for form plugs, restrict airflow and create a nidus for chronic microbial colonisation. A variety of largely ion channel-based strategies are being employed to promote mucosal hydration e.g. CFTR repair, ENaC blockers and TMEM16A potentiators. An alternative, but complimentary approach would be to reduce the excessive production and secretion of the mucin proteins that contribute to the solids component of the mucus gel. One approach to reduce excessive mucus production in the diseased lung is to reduce the number of mucus producing goblet cells in the airways.

To identify pathways that could regulate goblet cell formation in primary human bronchial epithelial (HBE) cells, we used ‘bronchospheres’ (Danahay et al., Cell Rep. [2015] 10(2):239) to run a screen of approximately 2,000 compounds. IL-13 treatment of the organoids increased the expression of goblet cell markers, including MUC5AC, and compounds that prevented this increase were identified as hits. This list was then refined, removing compounds that also decreased the expression of the ciliated cell marker FOXJ1, as these likely represented a non-specific, negative effect on epithelial differentiation. The remaining compounds were then validated for effects in air-liquid interface cultures of primary HBE, of which ˜40% significantly reduced an IL-13 induced increase in MUC5AC+ goblet cell numbers using quantitative immunohistochemistry.

Several of these validated hits were identified as not only preventing the IL-13 induced increase in MUC5AC+ goblet cells but also showed the potential to increase ciliated cell numbers. These compounds were prioritized and further tested in primary HBE to understand whether: 1) their pharmacology was specific to an IL-13 driven goblet cell phenotype, and 2) whether they could reverse an established goblet metaplasia.

Examples were demonstrated to attenuate the increase in MUC5AC+ goblet cells that was driven by the TLR5 agonist, flagellin as well as the type III interferon, IL-28. Furthermore, IL-28 also induced a profound increase in the number of MUC5B+ cells that could also be attenuated by some of the compounds.
A small number of the most promising compounds were then introduced to HBE that had an established goblet cell phenotype. Under conditions where HBE had been treated with IL-13 for 14 days to elevate goblet cell numbers, the introduction of test compounds between days 15-28, in the continued presence of IL-13, was able to reverse the increase in goblet cells and to also recover ciliated cell numbers.

Based on the output of this screen, we have identified compounds that can both prevent and reverse a goblet cell phenotype in primary HBE. Next steps will be to understand whether these effects can translate into in vivo models of a goblet cell metaplasia.