Targeting airway smooth muscle chloride fluxes for bronchorelaxation

 描述(申请人提供)：全球有超过3亿人患有哮喘。美国是发病率最高的国家之一(占人口的10.9%)，每年的成本为560亿美元。尽管有这些惊人的统计数据，但几十年来一直没有针对哮喘患者的气道平滑肌(ASM)介导的支气管收缩的新药物。此外，长效激动剂是目前直接针对ASM收缩的主要治疗方法，与哮喘死亡率的增加有关，目前正在接受FDA授权的5项临床研究的安全性审查。我们的初步研究支持这一假设，即针对ASM中控制氯离子流量的通道和转运体的药物可能是松弛ASM的新疗法。我们建议进行功能和机制研究，以证明单独或与钠-钾-氯共转运体联合靶向钙激活的氯通道作为新的哮喘治疗方法的有效性。我们建议吸入性治疗将避免与全身毒性相关的担忧，并将证实现有文献，即阻断这些通道也会减少呼吸道粘液的产生。因此，我们提出了以下三个具体目标：目标1a：通过靶向氯离子通量通路，实现体外人和豚鼠ASM的功能松弛。我们将演示单独或与氯转运体联合阻断氯离子通道在防止收缩或诱导松弛方面的有效性。我们还将确定是否有能力 氯通道/转运体阻断增强人类ASM中2-激动剂的松弛，为临床相关药物的发现提供初步的翻译数据。目的2：在体肺阻力的功能性降低。我们将证明氯离子通道+/-氯离子转运体阻滞剂的急性气雾剂释放在3个小鼠模型中防止体内支气管收缩：(1)幼稚高反应性A/J菌株(2)家尘螨致敏C57小鼠，(3)选择性基因缺失TMEM16A氯离子通道在平滑肌中的小鼠。将在这些模型中评估对上皮粘液产生的影响。目的3：氯通道调节肌动蛋白张力的细胞机制(S)。我们将展示氯离子对质膜(PM)电位的控制与细胞内与收缩/松弛相关的信号通路之间的联系，包括存储操作的钙离子进入，GQ偶联到肌醇磷酸生成，以及收缩调节蛋白(RhoA，MYPT1，MLC)的磷酸化。我们将使用基于FRET的SR特异性钙指示器(D1ER)来区分氯离子对跨PM和SR的钙离子流动的控制。

 DESCRIPTION (provided by applicant): Over 300 million people in the world suffer from asthma. The US is among the countries with the highest incidence (10.9% of the population) with a yearly cost of $56 billion. Despite these staggering statistics, there have been no new classes of medications targeting airway smooth muscle (ASM)-mediated bronchoconstriction in asthma for many decades. Moreover, long-acting -agonists, the current leading therapy that directly targets ASM constriction, have been associated with an increased mortality from asthma and are currently under FDA-mandated safety review in 5 clinical studies. Our preliminary studies support the hypothesis that drugs targeting the channels and transporters that control chloride flux in ASM may be novel therapies for relaxing ASM. We propose functional and mechanistic studies to document the efficacy of targeting calcium-activated chloride channels alone or in combination with sodium-potassium-chloride co-transporters as novel asthma therapies. We propose that inhaled therapy will circumvent concerns related to systemic toxicity and will confirm existing literature that blockade of these channels also reduces airway mucus production. Thus, we propose the following 3 specific aims: Aim 1a: functional relaxation of ex vivo human and guinea pig ASM by targeting chloride flux pathways. We will demonstrate the effectiveness of blockade of chloride channels alone or in combination with chloride transporters in the prevention of contraction or the induction of relaxation. We will also determine the ability of chloride channel/transporter blockade to potentiate relaxation of 2- agonists in human ASM providing initial translational data for clinically relevant drug discovery. Aim 2: functional reduction of in vivo lung resistance. We will demonstrate that acute aerosol delivery of blockers of chloride channels +/- chloride transporters prevents bronchoconstriction in vivo in 3 mouse models: (1) naively hyperresponsive A/J strain (2) house dust mite sensitized C57 mice, and (3) a mouse with a selective genetic deletion of the TMEM16A chloride channel in smooth muscle. The effect on epithelial mucous production will be assessed in these models. Aim 3: the cellular mechanism(s) by which chloride channels regulate ASM tone. We will demonstrate the link between chloride control of plasma membrane (PM) potential and intracellular signaling pathways linked to contraction/relaxation including stored operated Ca2+ entry, Gq-coupling to inositol phosphate generation, and phosphorylation of contractile-regulatory proteins (RhoA, MYPT1, MLC). We will distinguish between chloride control of Ca2+ flux across the PM vs SR using a FRET-based SR-specific Ca2+ indicator (D1ER).