The In Vitro & In Vivo Pharmacology of Novel Tmem16a Potentiator Compounds – NACFC – Nashville – October 2019
Henry Danahay1,2, Sarah Lilley1, Holly Charlton1, Roy Fox1, Brian Button3, Juan Sabater4, Martin Gosling1,2
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