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（申请人提供）：心肌病中肌节基因突变的一个标志是其改变心肌收缩的钙调节的能力。一般而言，扩张型（DCM）心肌病收缩的Ca 2+敏感性降低;而在肥厚型（HCM）和限制型（RCM）心肌病中，敏感性增加。由于存在多种形式的心肌病，因此鉴定使Ca 2+敏感性增敏（+）或脱敏（-）的新药可能潜在地逆转（+或-）这些异常变化。因此，本提案的目标是使用高通量筛选（HTS）来鉴定可以调节心肌收缩的Ca 2+敏感性的小分子。为了实现这一点，我们将使用由心肌调节细丝（RTF）组成的模型系统，RTF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）组合构成收缩器中发现的主要蛋白质。在没有肌球蛋白的情况下，RTF保留了所有对肌肉激活和松弛至关重要的Ca 2+调节功能。拟定试验将使用含有荧光标记肌钙蛋白C（CTn C）（CTn复合物的Ca 2+结合亚基）的心脏Tn（CTn）复合物。这将使我们能够监测Ca 2+结合CTnC调控位点时RTF荧光的变化。因此，在固定的[Ca 2 +]和波长下检测标记的RTF荧光强度响应于化合物或来自HTS筛选的“命中”的增加或减少（+或-）将指示CTnC的表观Ca 2+亲和力发生了变化（+或-）。将使用两个生物二次筛选进一步验证HTS的命中。基于上述，RTF系统可以提供稳健、稳定和生理学的测定，以鉴定特异性改变RTF Ca 2+敏感性而不是通过跨桥-药物相互作用改变力的化合物。为了实现我们的目标，这项建议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理学试剂，用于心肌病，高血压和其他形式的心血管疾病的研究和治疗。 公共卫生关系：在过去的十年中，三种主要类型的遗传性心肌病（包括扩张型、肥厚型和限制型）的遗传基础已被深入研究。本文提出的研究将使用现代高通量筛选技术鉴定新的低分子量化合物，其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，用于治疗这些和潜在的其他形式的心血管疾病。