The pharmacology of novel TMEM16A potentiator compounds

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

TMEM16A channel function does not influence goblet cell numbers in the human airway epithelium

TMEM16A channel function does not influence goblet cell numbers in the human airway epithelium

Henry Danahay1,2, Martin Gosling1,2
1University of Sussex, Brighton, UK; 2Enterprise Therapeutics, UK.

A role for the calcium activated chloride channel, TMEM16A, in the regulation of airway goblet cell populations has been recently reported (Kondo et al., 2017; Lin et al., 2015; Qin et al., 2016). The published data to support a role for TMEM16A in driving the formation of goblet cells, are largely based on gene silencing and the use of non-specific TMEM16A blockers (e.g. niflumic acid). It is unclear whether this proposed function is therefore dependent on the ion channel activity of TMEM16A or through an alternative aspect of the proteins function. Using a potent TMEM16A channel blocker (Ani9) and a recently identified TMEM16A potentiator compound (Enterprise Therapeutics; proprietary), we have evaluated whether channel function can regulate goblet cell numbers in primary cultures of human bronchial epithelial (HBE) cells.

Primary HBE (3 donor codes; non-CF) 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 ET003861 (1 µM) each group in either the absence or presence of the TMEM16A blocker, Ani9 (10 µM). ET003861 is a TMEM16A potentiator with an EC50 of 150-200 nM for the potentiation of chloride secretion in patch clamp (FRT-TMEM16A) and ion transport (HBE). 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 (7-25-fold depending on donor code) based on the increased staining for MUC5AC, that was unaffected by co-administration of Ani9. ET003861 also failed to influence goblet cell numbers and Ani9 was likewise without effect. Finally, the co-administration of ET003861 with IL-13 also failed to modify goblet cell numbers.

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

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

EACT increases intracellular calcium levels by a TMEM16A-independent mechanism

EACT 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