Biochemical Screens for Modulators of Muscle Force

描述（由申请人提供）：我们的长期目标是建立简单的生化分析，这将使我们能够i)评估肌肉力量的广泛生理和疾病相关效应物的机械效应，ii)快速筛选小分子文库，以寻找新的肌肉力量治疗调节剂。我们的一般假设是，等长肌肉力F的大多数变化都有明确的生化基础。特别是等长肌力，F =Funi。N受肌动蛋白与肌动蛋白强结合的肌球蛋白分子的数目N和每肌动蛋白结合的肌球蛋白的平均作用力Funi的影响，最近的研究表明，Funi与肌动蛋白-肌球蛋白的结合能g成比例地变化。突变、辅助蛋白和小分子对等长肌肉力的生化决定因素deltaG和N的影响，可以直接在试管中测量。表明高通量筛选（HTS）肌肉力效应器是可能的。利用生物物理和生化技术，我们开发了几种分析方法，证明了HTS用于肌肉力效应器的可行性。在本提案中，我们将（目标1）建立肌肉力调节剂的HTS，我们将（目标2）验证这些筛选是F变化的准确预测因子，使用体外力测定和肌肉力学实验。该提案依赖于我们实验室在肌肉生理学/生物物理学、最先进的生物物理技术和肌肉机械化学理论模型方面的综合专业知识。本提案中开发的肌肉力效应器的HTS将极大地加速表征广泛的已知和可疑的肌肉力调节剂，并将为发现肌肉力的小分子抑制剂和激活剂提供强大的工具。公共卫生相关性。肌肉力量的精细控制对大多数肌肉骨骼和器官系统的正常功能、适应和发育至关重要，破坏这种机械平衡可导致各种疾病，如高血压导致心力衰竭和与哮喘相关的气道高反应性。开发快速评估新化合物对肌肉力学影响的方法将使我们能够快速发现新的疗法，通过这种疗法，我们可以帮助处于疾病状态的肌肉恢复正常的机械功能。

DESCRIPTION (provided by applicant): Our long term goal is to establish simple biochemical assays that will allow us i) to assess the mechanical effects of a broad range of physiological and disease-related effectors of muscle force, and ii) to rapidly screen small molecule libraries for new therapeutic modulators of muscle force. Our general hypothesis is that most changes in isometric muscle force, F, have a well-defined biochemical basis. Specifically isometric muscle force, F =Funi.N, is influenced by the number, N, of myosin molecules strongly bound to actin and the average force, Funi, per actin bound myosin, where recent studies indicate that Funi varies proportionally with the actin- myosin binding energy, G. The effects of mutations, accessory proteins, and small molecules on the biochemical determinants of isometric muscle force, deltaG and N, can be measured directly in a test tube, suggesting that high throughput screens (HTS) for effectors of muscle force are possible. Using biophysical and biochemical techniques, we have developed several assays that demonstrate the feasibility of a HTS for effectors of muscle force. In this proposal we will (Aim 1) establish a HTS for modulators of muscle force, and we will (Aim 2) verify that these screens are accurate predictors of changes in F, using both in vitro force assays and muscle mechanics experiments. This proposal relies on our laboratory's combined expertise in muscle physiology/biophysics, state-of-the-art biophysical techniques, and theoretical models of muscle mechanochemistry. The HTS for effectors of muscle force developed in this proposal will dramatically accelerate characterization of a wide range of known and suspected modulators of muscle force and will provide a powerful tool for discovering small molecular inhibitors and activators of muscle force. PUBLIC HEALTH RELEVANCE. Fine control of muscle force is central to the normal function, adaptation, and development of most musculoskeletal and organ systems, and disrupting this mechanical balance can lead to a variety of diseases, such as hypertension resulting in cardiac failure and airway hyper-responsiveness associated with asthma. Developing approaches for rapidly assessing the effects of new compounds on muscle mechanics will allow us to rapidly discover new therapies through which we can help muscles in disease states regain their normal mechanical function.