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聚。在CF中，粘液特别粘稠，具有粘性，并且似乎脱水，导致气道和肠道中的粘液阻塞。慢性炎症引起杯状细胞化生和钙激活的Cl-通道TMEM 16 A的上调。然而，尚不清楚TMEM 16 A是否对粘液产生和/或分泌是必需的。此外，尚不清楚TMEM 16 A的上调和激活是否有利于粘膜纤毛清除，或者这是否有助于气道清除受损。我们从组织特异性TMEM 16 A和TMEM 16 F敲除小鼠获得的已发表和初步（未发表）结果表明，TMEM 16 A对粘液释放至关重要，而TMEM 16 F与粘液产生相关。TMEM 16 A和16 F是在小鼠和人类气道和肠中表达的Cl-通道，并且对于CFTR的操作是必需的。我们报道了在TMEM 16 A-/-小鼠中基本上不存在液体分泌，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TMEM 16改善了气道功能。使用TMEM 16 A和16 F敲除小鼠和多能人类气道上皮细胞，我们将阐明：i）TMEM 16 F是否是粘液产生所必需的。ii）TMEM 16 A是否对于粘液分泌是必不可少的，但对于粘液产生是不重要的。iii）哮喘小鼠中TMEM 16的阻断是否将减弱粘液分泌、增强粘液纤毛清除和改善气道功能。iv）TMEM 16的激活是否在小鼠气道中具有不利影响。将在小鼠体内和离体灌注的小鼠和猪气道以及小鼠肠道中测量粘液转运和粘膜纤毛清除。我们将研究FDA批准的化合物和TMEM 16的抑制剂氯硝柳胺乙醇胺是否抑制粘液产生和粘液分泌。我们将测试该药物是否减轻哮喘小鼠的气道堵塞和CF小鼠的肠梗阻。将使用来自AMGEN的新型高效抑制剂（ANO 1-InhibA）进行类似实验。使用TMEM 16的激活剂（Eact或C5），我们将证明TMEM 16的激活对气道功能具有负面影响。这是第一项明确证明TMEM 16在炎症性气道疾病中对气道粘液清除和粘液堵塞的作用的研究。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，因为需要其结果来指导进一步的基于TMEM 16的策略，以减少哮喘和CF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 Our published and preliminary (unpublished) results from tissue specific TMEM16A and TMEM16F knockout mice suggest that TMEM16A is essential for mucus release, while TMEM16F is relevant for mucus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.