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)的收缩状态，以避免或 逆转支气管收缩。是否通过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，所有哮喘患者中有55%的人控制不佳。目前所有的支气管扩张剂药物都有局限性 在有效性和安全性方面，最常用的一类支气管扩张剂仍然存在 药物-长效β激动剂(LABA)。我们认为支气管扩张剂药物的局限性是可以克服的 通过一种针对ASM收缩状态的两个最强大调节器的方法：药物机械学 偶联和肌动蛋白细胞骨架。我们假设β-激动剂(主要针对的是 药物-机械耦合)和专门针对肌动蛋白细胞骨架的药物可以非常有效 具有功能性协同作用的支气管扩张剂，药物剂量更低，因此安全性更好 侧写。为了检验这一假设，本文提出了三个目标。在目标1中，我们将利用细胞、组织和体内建立 ASM收缩模型、药物-机械偶联联合靶向的协同性质 肌动蛋白聚合，并确定β-激动剂和细胞骨架靶向药物的最佳组合 放松ASM。在目标2中，我们将通过以下特征来确定这种功能协作性的机制基础 这些药物对控制跨桥循环(肌球蛋白光)的信号中间产物和结果的影响 链激酶和磷酸酶磷酸化)或肌动蛋白聚合态(F/G肌动蛋白比率)，以及在 调节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效果。 β激动剂与肌动蛋白细胞骨架靶向药物联合应用抑制肌间质膜收缩 通过使用来自人类细胞/组织的细胞和组织模型系统来研究呼吸道阻力 哮喘患者，或在体外/体外(细胞、组织)或体内施加哮喘病理生物学(2 不同的活体小鼠模型)。由3个已建立的具有互补性的PI执行的拟议研究 专业知识代表了建立哮喘管理策略的创新方法，该策略克服了 目前疗效和安全性的局限性。此外，拟议的机械论研究将提供新的见解 研究如何以最佳方式破坏跨桥自行车和细胞骨架之间的合作 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.