Cooperative targeting of pharmacomechanical coupling and the actin cytoskeleton to regulate ASM contraction

项目摘要 有效的哮喘管理需要调节气道平滑肌（ASM）收缩状态，以避免或 逆转支气管收缩是否尝试使用直接支气管扩张剂（例如，β-激动剂），通过 抗炎剂（例如，皮质类固醇），或两者的某种组合，往往缺乏管理， 据估计，55%的哮喘患者控制不佳。目前所有的支气管扩张药物都有局限性 最常用的一类支气管扩张剂仍然存在疗效和安全性问题， 长效β受体激动剂（LABA）。我们认为支气管扩张药物的局限性是可以克服的 通过一种针对ASM收缩状态的两种最强大的调节剂的方法：药物力学 偶联和肌动蛋白细胞骨架。我们假设β-受体激动剂（主要靶向 药物机械偶联）和特异性靶向肌动蛋白细胞骨架的药物可以非常有效 支气管扩张剂，具有协同作用，能够降低药物剂量，因此安全性更好 profile.提出了三个目标来检验这一假设。在目标1中，我们将建立，使用细胞，组织，和在体内 ASM收缩的模型，药物力学偶联的组合靶向的合作性质， 肌动蛋白聚合，并确定β-激动剂和细胞因子靶向药物的最佳组合， 放松ASM。在目标2中，我们将通过表征 这些药物对控制跨桥周期（肌球蛋白轻）的信号传导中间体和结果的影响 链激酶和磷酸酶磷酸化）或肌动蛋白聚合状态（F/G肌动蛋白比），以及 调控这些结果的上游信号。最后，在目标3中，我们将评估哮喘病理生物学的影响 联合β-激动剂和肌动蛋白细胞因子靶向药物抑制ASM收缩的疗效， 气道阻力，通过使用来源于人的细胞/组织的细胞和组织模型系统， 哮喘，或其中哮喘病理生物学在体外/离体（对细胞、组织）或体内（2）被施加 不同的体内鼠模型）。拟定研究由3名已确立的PI进行， 专业知识代表了一种创新的方法，以建立一个哮喘管理策略，克服了 有效性和安全性的当前限制。此外，拟议的机制研究将提供新的见解 如何最佳地破坏跨桥循环和细胞骨架硬化之间的合作， 在ASM细胞中产生张力。

Project Summary Effective asthma management requires regulating airway smooth muscle (ASM) contractile state to avoid or reverse bronchoconstriction. Whether this is attempted by use of direct bronchodilators (e.g., β-agonists), by anti-inflammatory agents (e.g., corticosteroids), or some combination of both, too often management is lacking, as an estimated 55% of all asthmatics have suboptimal control. All current bronchodilator drugs have limitations which respect to efficacy, and safety issues still persist with the most frequently used class of bronchodilator drugs- long-acting β-agonists (LABAs). We submit that the limitations of bronchodilator drugs can be overcome by an approach that targets the 2 most powerful regulators of ASM contractile state: pharmacomechanical coupling and the actin cytoskeleton. We hypothesize that combinations of beta-agonists (that primarily target pharmacomechanical coupling) and drugs that specifically target the actin cytoskeleton can be highly efficacious bronchodilators, with a functional cooperatively that enables lower drug doses and therefore a better safety profile. Three aims are proposed to test this hypothesis. In Aim 1, we will establish, using cell, tissue, and in vivo models of ASM contraction, the cooperative nature of combined targeting of pharmacomechanical coupling and actin polymerization, and identify optimal combinations of beta-agonists and cytoskeleton-targeting drugs that relax ASM. In Aim 2, we will determine the mechanistic basis for this functional cooperativity by characterizing the effects of these drugs on signaling intermediates and outcomes that control cross bridge cycle (myosin light chain kinase and phosphatase phosphorylation) or actin polymerization state (F/G actin ratio), and on the upstream signals that regulate these outcomes. Lastly, in Aim 3 we will assess the effect of asthma pathobiology on the efficacy of combining β-agonists and actin cytoskeleton-targeting drugs in inhibiting ASM contraction and airway resistance, by employing cell and tissue model systems derived from cells/tissues from human asthmatics, or in which asthma pathobiology is imposed either in vitro/ex vivo (to cells, tissue), or in vivo (2 differrent in vivo murine models). The proposed studies performed by 3 established PIs with complementary expertise represent an innovative approach to establish an asthma management strategy that overcomes the current limitations of efficacy and safety. Moreover, the proposed mechanistic studies will provide new insight into how to optimally disrupt the cooperation between cross bridge cycling and cytoskeleton stiffening that generates tension in the ASM cell.