Therapeutic Potentiation of Bronchial Dilatation

描述（由申请人提供）：鉴于其在哮喘气流阻塞中的核心作用，气道平滑肌（ASM）收缩长期以来一直受到关注，作为治疗靶点，其策略旨在放松ASM或完全消融ASM。在这里，我们提出了一种全新的方法来缓解哮喘气流阻塞。我们的策略是削弱收缩的ASM在收缩发生后保持缩短的能力。当ASM在稳定载荷下收缩时，可以保持无限缩短，而ASM在波动载荷下收缩时，首先缩短，然后再延长;这种力波动引起的再延长（FFIR）可能相当大，并且可以进一步增强。在完整的肺中，ASM不断暴露于由潮式呼吸施加的力波动。该应用的关键前提是，专门针对扩大呼吸诱导的FFIR的药物应释放ASM对气道腔的挤压，从而缓解支气管收缩。我们的目标是鉴定和评价靶向这种极其有效但大部分未研究的支气管扩张途径的化合物。初步研究揭示了肌动蛋白-肌球蛋白-肌动蛋白连接（AMAC）的中断作为一个有吸引力的策略，以提高FFIR。在孤立的ASM中，减少肌动蛋白或肌球蛋白细丝聚合的干预措施既减少AMAC又促进FFIR。在分阶段筛选设计中，我们将使用大规模高通量初步测定来鉴定新的或已经在人体内的化合物：1）抑制肌球蛋白聚合成厚肌丝;和/或2）减少培养的人ASM的力产生。然后将使用一种新的低通量测定来测试这些化合物中的哪一种在人肺的精确切割薄片内的完整气道中增强FFIR或抑制支气管收缩。最后，我们将通过使用来自EM断层图像的肌丝超微结构的3D重建来量化快速冷冻对照和药物处理的完整人ASM中的肌丝长度，并通过评估潜在的替代药物作用机制（包括细胞内钙动员、MLC 20磷酸化和/或HSP 27磷酸化的改变）来评估药物作用机制。我们的研究结果将确定新的哮喘药物，加强FFIR或抑制支气管收缩。相关性（参见说明）：该项目的目的是确定药物，可以发挥一种新的治疗哮喘的效果-通过促进支气管收缩的快速逆转。这种方法与目前旨在抑制气道炎症或放松气道肌肉的治疗方法完全互补，因此，如果开发成功，应该为现有的哮喘治疗增加一种全新的治疗策略。

DESCRIPTION (provided by applicant): Given its central role in asthmatic airflow obstruction, airway smooth muscle (ASM) contraction has long received attention as a therapeutic target, with strategies directed at relaxing ASM or ablating ASM altogether. Here, we propose an entirely novel approach to relieving airflow obstruction in asthma. Our strategy is to impair the ability of contracted ASM to remain shortened after contraction has occurred. While ASM that has contracted against a steady load can remain shortened indefinitely, ASM contracting against a fluctuating load first shortens then relengthens; such force fluctuation-induced relengthening (FFIR) can be quite substantial and can be further enhanced pharmacologically. In the intact lung, ASM is constantly exposed to force fluctuations imposed by tidal breathing. The key premise of this application is that drugs targeted specifically to exaggerate breathing-induced FFIR should release ASM's squeeze on the airway lumen and thus relieve bronchoconstriction. Our objective is to identify and evaluate compounds that target this extremely potent but largely unstudied bronchodilatory pathway. Preliminary studies disclose disruption of actin-myosin-actin connectivity (AMAC) as an attractive strategy for enhancing FFIR. In isolated ASM, interventions that reduce the polymerization of actin or myosin filaments both reduce AMAC and promote FFIR. In a staged screening design, we will use large scale high throughput primary assays to identify novel or already-in-human compounds that: 1) inhibit polymerization of myosin into thick myofilaments; and/or 2) reduce force generation by cultured human ASM. A novel low throughput assay will then be used to test which of these compounds potentiates FFIR or inhibits bronchoconstriction in intact airways within precision- cut thin slices of human lungs. Finally, we will evaluate mechanisms of drug action by quantifying myofila}} ment lengths in flash frozen control and drug-treated intact human ASM using 3D reconstructions of myofila}} ment ultrastructure from EM tomograms, and by assessing potential alternative mechanisms of drug action, including alteration of intracellular calcium mobilization, MLC20 phosphorylation, and/or HSP27 phosphoryl}} ation. Our results should identify novel drugs for asthma that potentiate FFIR or inhibit bronchoconstriction. RELEVANCE (See instructions): The objective of this project is to identify drugs that could exert a novel therapeutic effect in asthma - by promoting the rapid reversal of bronchoconstriction. This approach is fully complementary to current therapeutic approaches aimed at suppressing airway inflammation or relaxing airway muscle and so, if developed successfully, should add an entirely new therapeutic strategy to existing asthma therapies.