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）收缩长期以来一直作为治疗靶点受到关注，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中的气流阻塞。我们的策略是削弱收缩后ASM保持缩短的能力。在稳定负荷下收缩的ASM可以无限期地缩短，而在波动负荷下收缩的ASM先缩短后延长；这种力波动诱导的再强化（FFIR）可以是相当可观的，可以在药理学上进一步增强。在完整的肺中，ASM不断暴露于潮汐呼吸所施加的力波动。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对气道管腔的挤压，从而缓解支气管收缩。我们的目标是鉴定和评估针对这种非常有效但在很大程度上尚未研究的支气管扩张途径的化合物。初步研究表明，破坏肌动蛋白-肌球蛋白-肌动蛋白连接（AMAC）是增强FFIR的一种有吸引力的策略。在孤立的ASM中，减少肌动蛋白或肌球蛋白丝聚合的干预措施既减少了AMAC，又促进了FFIR。在分阶段筛选设计中，我们将使用大规模高通量初级分析来鉴定新的或已经存在于人体中的化合物：1)抑制肌球蛋白聚合成粗肌丝；和/或2)减少培养的人ASM产生的力。一种新的低通量测定方法将用于测试这些化合物中的哪一种可以增强FFIR或抑制精确切割的人体肺薄片内完整气道中的支气管收缩。最后，我们将通过定量快速冷冻对照和药物处理的完整人ASM的肌纤维分子长度来评估药物的作用机制，通过EM断层图的肌纤维分子超微结构的3D重建，并通过评估药物作用的潜在替代机制，包括细胞内钙动员的改变，MLC20磷酸化和/或HSP27磷酸化。我们的研究结果应该能鉴别出增强FFIR或抑制支气管收缩的哮喘新药。相关性（见说明书）：该项目的目的是通过促进支气管收缩的快速逆转，确定可能在哮喘中发挥新型治疗作用的药物。这种方法完全补充了当前旨在抑制气道炎症或放松气道肌肉的治疗方法，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.