Biochemical Screens for Modulators of Muscle Force

描述(申请人提供)：我们的长期目标是建立简单的生化分析，使我们能够i)评估一系列与肌肉力量有关的生理和疾病效应器的机械效应，以及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，大多数等长肌力F的变化都有明确的生化基础。具体地说，等长肌力F=Funi.N受与肌动蛋白强烈结合的肌球蛋白分子的数量N和每个肌球蛋白结合的平均作用力Funi的影响，其中最近的研究表明Funi与肌动蛋白-肌球蛋白结合能G成比例变化。突变、辅助蛋白和小分子对等长肌力的生化决定因素deltaG和N的影响可以在试管中直接测量，这表明高通量筛选(HTS)肌肉力量的效应器是可能的。利用生物物理和生化技术，我们已经开发了几种分析方法，证明了高温超导用于肌肉力量效应器的可行性。在这项提案中，我们将(目标1)建立肌肉力量调节器的HTS，我们将(目标2)使用体外力量分析和肌肉力学实验来验证这些筛查是F变化的准确预测因子。这一建议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力化学的理论模型方面的综合专业知识。该提案中开发的肌肉力量效应器的HTS将极大地加速对一系列已知和可疑的肌肉力量调节器的表征，并将为发现肌肉力量的小分子抑制物和激活剂提供一个强大的工具。与公共卫生相关。对肌肉力量的精细控制是大多数肌肉骨骼和器官系统正常功能、适应和发展的核心，破坏这种机械平衡可能会导致各种疾病，如高血压导致心力衰竭和与哮喘相关的呼吸道高反应性。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迅速发现新的治疗方法，通过这些方法，我们可以帮助处于疾病状态的肌肉恢复其正常的机械功能。

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.