Targeting airway smooth muscle chloride fluxes for bronchorelaxation

 描述（申请人提供）：世界上有超过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%），每年花费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中气道平滑肌（ASM）介导的支气管收缩的新类别药物。此外，长效激动剂是目前直接靶向ASM收缩的主要疗法，与哮喘死亡率增加相关，目前正在5项临床研究中接受FDA授权的安全性审查。我们的初步研究支持这一假设，即针对控制ASM中氯离子通量的通道和转运蛋白的药物可能是放松ASM的新疗法。我们提出功能和机制的研究，以记录单独或与钠-钾-氯化物共转运蛋白作为新的哮喘治疗靶向钙激活氯通道的疗效。我们建议吸入疗法将避免与全身毒性相关的问题，并将证实现有文献，即阻断这些通道也会减少气道粘液的产生。因此，我们提出了以下3个具体目标：目标1a：通过靶向氯离子通量途径，对离体人和豚鼠ASM进行功能性松弛。我们将证明单独阻断氯离子通道或与氯离子转运蛋白联合阻断氯离子通道在预防收缩或诱导舒张中的有效性。我们还将确定 的氯离子通道/转运蛋白阻断，以加强2-受体激动剂在人类ASM中的松弛，为临床相关药物发现提供初步的翻译数据。目的2：功能性降低体内肺阻力。我们将在3种小鼠模型中证明，氯离子通道+/-氯离子转运蛋白阻断剂的急性气雾剂递送可预防体内支气管收缩：（1）天然高反应性A/J品系，（2）屋尘螨致敏的C57小鼠，和（3）平滑肌中TMEM 16 A氯离子通道选择性基因缺失的小鼠。将在这些模型中评估对上皮粘液产生的影响。目的3：氯通道调节平滑肌张力的细胞机制。我们将证明氯离子控制质膜（PM）电位和细胞内信号通路之间的联系，包括存储操作的Ca 2+进入，Gq耦合磷酸肌醇的产生，和收缩调节蛋白（RhoA，MYPT 1，MLC）的磷酸化收缩/舒张。我们将使用基于FRET的SR特异性Ca 2+指示剂（D1 ER）区分PM与SR中Ca 2+通量的氯化物控制。

 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).