HTS for Regulated Muscle Thin Filament Function.

HTS 用于调节肌肉细丝功能。

• 批准号: 8038570
• 负责人: JAMES Douglas POTTER
• 金额: $ 3.83万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2011-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038570
• 关键词: ATP phosphohydrolase; Affinity; Binding; Biological; Biological Assay; Biological Models; Calcium; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Cardiovascular Diseases; Chemicals; Complex; Data; Dependence; Detection; Development; Disease; Dose; Drug Interactions; Evaluation; F-Actin; Fiber; Fluorescence; Gene Mutation; Genetic; Goals; Guidelines; Hypertension; Inherited; Investigation; Ionic Strengths; Knowledge; Label; Laboratories; Ligands; Measurement; Measures; Miniaturization; Molecular Weight; Monitor; Muscle; Muscle Contraction; Muscle Fibers; Myocardium; Myosin ATPase; Noise; Pharmaceutical Preparations; Phenotype; Physiological; Probability; Process; Production; Proteins; Quality Control; Reaction; Regulation; Relaxation; Reproducibility; Screening procedure; Signal Transduction; Site; Staging; System; Technology; Temperature; Thick Filament; Thin Filament; Time; Tropomyosin; Troponin; Troponin C; base; comparison group; cost; high throughput screening; inorganic phosphate; instrument; miniaturize; novel therapeutic intervention; public health relevance; research study; response; scale up; small molecule

DESCRIPTION (provided by applicant): A hallmark of sarcomeric gene mutations in cardiomyopathies is their ability to alter the calcium regulation of cardiac muscle contraction. In general, the Ca2+ sensitivity of contraction decreases in dilated (DCM) cardiomyopathy; whereas, in hypertrophic (HCM) and restrictive (RCM) cardiomyopathies, the sensitivity increases. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the Ca2+ sensitivity could potentially reverse (+ or -) these aberrant changes. Therefore, the goal of this proposal is to use high throughput screening (HTS) to identify small molecules that can modulate the Ca2+ sensitivity of cardiac muscle contraction. To achieve this, we will use a model system composed of cardiac muscle regulated thin filaments (RTF) which are comprised of F-actin, tropomyosin and troponin (Tn). The RTF in combination with myosin (thick filament) make up the major proteins found in the contractile apparatus. In the absence of myosin, the RTF retains all of the Ca2+ regulated functions critical for muscle activation and relaxation. The proposed assay will use cardiac Tn (CTn) complexes that contain fluorescently labeled troponin C (CTnC), the Ca2+ binding subunit of the CTn complex. This will allow us to monitor changes in RTF fluorescence that occurs when Ca2+ binds to the CTnC regulatory site. Therefore, detecting an increase or decrease (+ or -) in the labeled RTF fluorescence intensity at a fixed [Ca2+] and wavelength in response to a compound or "hit" from the HTS screen will indicate that a change (+ or -) in the apparent Ca2+ affinity of CTnC has occurred. Hits from the HTS will be further validated using two biological secondary screens. Based on the above, the RTF system can provide a robust, stable and physiological assay to identify compounds that specifically alter the RTF Ca2+ sensitivity and not the force via cross bridge-drug interactions. To achieve our goals, this proposal will pursue two Specific Aims. Knowledge gained from these studies can uncover potentially new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases. PUBLIC HEALTH RELEVANCE: The genetic basis for three major types of inherited cardiomyopathies including dilated, hypertrophic and restrictive have been studied intently over the last decade. The studies proposed here will identify new low molecular weight compounds, using modern high throughput screening technology, which can modulate a key phenotype that has been observed in model systems of these diseases. Results from these studies will ultimately be beneficial in developing new therapeutic approaches for the treatment of these and potentially other forms of cardiovascular disease.

描述（由申请人提供）：心肌病中肌节基因突变的一个标志是它们改变心肌收缩的钙调节的能力。一般来说，扩张型 (DCM) 心肌病的 Ca2+ 收缩敏感性降低；而在肥厚型 (HCM) 和限制型 (RCM) 心肌病中，敏感性会增加。由于存在多种形式的心肌病，因此鉴定出使 Ca2+ 敏感性敏感（+）或脱敏（-）的新药可能会逆转（+或-）这些异常变化。因此，本提案的目标是利用高通量筛选（HTS）来识别可以调节心肌收缩的 Ca2+ 敏感性的小分子。为了实现这一目标，我们将使用由心肌调节细丝 (RTF) 组成的模型系统，该细丝由 F-肌动蛋白、原肌球蛋白和肌钙蛋白 (Tn) 组成。 RTF 与肌球蛋白（粗丝）结合构成了收缩装置中的主要蛋白质。在没有肌球蛋白的情况下，RTF 保留了所有对肌肉激活和放松至关重要的 Ca2+ 调节功能。拟议的检测将使用含有荧光标记肌钙蛋白 C (CTnC)（CTn 复合物的 Ca2+ 结合亚基）的心脏 Tn (CTn) 复合物。这将使我们能够监测 Ca2+ 与 CTnC 调节位点结合时发生的 RTF 荧光变化。因此，检测固定 [Ca2+] 和波长响应 HTS 屏幕中的化合物或“命中”的标记 RTF 荧光强度的增加或减少（+ 或 -）将表明CTnC 的表观 Ca2+ 亲和力已发生。 HTS 的命中将使用两个生物二级筛选进一步验证。基于上述，RTF 系统可以提供稳健、稳定的生理测定，以识别特异性改变 RTF Ca2+ 敏感性的化合物，而不是通过跨桥药物相互作用改变力。为了实现我们的目标，该提案将追求两个具体目标。从这些研究中获得的知识可以发现潜在的新药物，用于研究和治疗心肌病、高血压和其他形式的心血管疾病。 公众健康相关性：在过去十年中，人们对三种主要类型的遗传性心肌病（包括扩张型心肌病、肥厚型心肌病和限制性心肌病）的遗传基础进行了深入研究。这里提出的研究将利用现代高通量筛选技术来鉴定新的低分子量化合物，该技术可以调节在这些疾病的模型系统中观察到的关键表型。这些研究的结果最终将有利于开发新的治疗方法来治疗这些疾病和潜在的其他形式的心血管疾病。