Inhibition of TMEM16 proteins to attenuate mucus secretion and to improve airway clearance

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A central problem in asthma and cystic fibrosis (CF) is the accumulation of large amounts of mucus in the airways. In CF, the mucus is particularly viscous, adhesive, and appears dehydrated, leading to mucus obstructions in airways and intestine. Chronic inflammation causes goblet cell metaplasia and upregulation of the calcium activated Cl- channel TMEM16A. It is, however, unclear if TMEM16A is essential for mucus production and/or secretion. In addition, it is unknown whether upregulation and activation of TMEM16A is beneficial for mucociliary clearance or whether this contributes to impaired airway clearance. Our published and preliminary (unpublished) results from tissue specific TMEM16A and TMEM16F knockout mice suggest that TMEM16A is essential for mucus release, while TMEM16F is relevant for mucus production. TMEM16A and 16F are Cl- channels expressed in mouse and human airways and intestine, and are essential for CFTR to operate. We reported that fluid secretion was essentially absent in TMEM16A-/- mice, yet mucociliary transport was even enhanced. Moreover, inhibition of TMEM16 improved airway function in preliminary experiments with asthmatic mice. Using TMEM16A and 16F knockout mice and pluripotent human airway epithelial cells, we will clarify: i) Whether TMEM16F is essential for mucus production. ii) Whether TMEM16A is indispensable for mucus secretion but unimportant for mucus production. iii) Whether blockade of TMEM16 in asthmatic mice will attenuate mucus secretion, enhance mucociliary clearance, and improve airway function. iv) If activation of TMEM16 has adverse effects in mouse airways. Mucus transport and mucociliary clearance will be measured in mice in vivo and ex vivo in perfused mouse and porcine airways, and in mouse intestine. We will investigate whether the FDA-approved compound and inhibitor of TMEM16, niclosamide ethanolamine, inhibits mucus production and mucus secretion. We will test whether the drug attenuates airway plugging in asthmatic mice and intestinal obstruction in CF mice. Similar experiments will be performed using a novel and highly potent inhibitor from AMGEN (ANO1-InhibA). Using activators of TMEM16 (Eact or C5), we will demonstrate that activation of TMEM16 has a negative impact on airway function. This is the first study that will clearly demonstrate the role of TMEM16 for airway mucus clearance and mucus plugging in inflammatory airway disease. It will provide novel insights into the cellular mechanisms of mucus release. The study is pressing because its results are needed to guide further TMEM16-based strategies to reduce airway mucus plugging in asthma and CF.