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

哮喘和囊性纤维化（CF）的中心问题是气道中大量粘液的积累。CF中，粘液特别粘稠，粘连，并出现脱水，导致气道和肠道粘液阻塞。慢性炎症引起杯状细胞化生和钙激活的Cl-通道TMEM16A上调。然而，目前尚不清楚TMEM16A是否对粘液的产生和/或分泌至关重要。此外，尚不清楚TMEM16A的上调和激活是否有利于粘膜纤毛清除，或者是否有助于气道清除受损。我们对组织特异性TMEM16A和TMEM16F敲除小鼠的已发表和初步（未发表）结果表明，TMEM16A对粘液释放至关重要，而TMEM16F与粘液产生有关。TMEM16A和16F是在小鼠和人类气道和肠道中表达的Cl-通道，是CFTR运行所必需的。我们报道了TMEM16A-/-小鼠基本没有液体分泌，但粘液纤毛运输甚至增强。此外，在哮喘小鼠的初步实验中，抑制TMEM16可改善气道功能。使用TMEM16A和16F敲除小鼠和多能性人气道上皮细胞，我们将澄清：i) TMEM16F是否对粘液产生至关重要。ii) TMEM16A是否对粘液分泌不可或缺，而对粘液产生不重要。iii)哮喘小鼠阻断TMEM16是否会减弱黏液分泌，增强黏毛清除，改善气道功能。iv)激活TMEM16是否对小鼠气道有不良影响。将在小鼠体内、体外、小鼠和猪气道以及小鼠肠道中测量粘液运输和粘纤毛清除。我们将研究fda批准的化合物和TMEM16抑制剂氯胺醇胺是否抑制粘液产生和粘液分泌。我们将测试药物是否减轻哮喘小鼠气道堵塞和CF小鼠肠梗阻。类似的实验将使用AMGEN的一种新型高效抑制剂（ANO1-InhibA）进行。使用TMEM16的激活剂（Eact或C5），我们将证明TMEM16的激活对气道功能有负面影响。这是第一个明确证明TMEM16在炎性气道疾病中气道粘液清除和粘液堵塞中的作用的研究。它将为粘液释放的细胞机制提供新的见解。这项研究是紧迫的，因为它的结果需要指导进一步的基于tmem16的策略，以减少哮喘和CF中的气道粘液堵塞。

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 Our published and preliminary (unpublished) results from tissue specific TMEM16A and TMEM16F knockout mice suggest that TMEM16A is essential for mucus release, while TMEM16F is relevant for mucus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.