Interaction of Cardiotonic Drugs with Cardiac Troponin

强心药与心肌肌钙蛋白的相互作用

• 批准号: 7129805
• 负责人: Brian Douglas Sykes
• 金额: $ 13.5万
• 依托单位: UNIVERSITY OF ALBERTA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-04 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7129805
• 关键词: binding sites; calcium binding protein; calcium flux; cardiotonic agents; chemical structure function; computer program /software; conformation; heart contraction; heart pharmacology; intermolecular interaction; mathematical model; microfilaments; myocardium; nuclear magnetic resonance spectroscopy; stoichiometry; structural biology; troponin

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to elucidate the molecular mechanisms involved in the regulation of cardiac muscle contraction by troponin and to determine its role as a target for the development of therapeutic protocols that can be used to attenuate heart disease, the leading cause of morbidity and mortality in the Western society. The hypothesis behind the proposed research is that troponin is a molecular switch, directly regulating the Ca2+-dependent activation of myofilament in cardiac muscle contraction and therefore serves as an attractive and logical target for the design of cardiotonic drugs. These drugs are clinically useful because they can be used to modulate the Ca2+-sensitivity of troponin. This approach is therapeutically desirable because intracellular [Ca2+] is not perturbed, preserving the regulation of other Ca2+-based signaling pathways. Towards defining cardiotonic structure-activity relationships, we have shown that several cardiotonic agents specifically bind troponin C (cTnC) and determined high resolution structures of two cTnC-drug complexes, the C-domain of cTnC in complex with EMD 57033, cCTnC.2Ca2+.EMD57033, and the N-domain of cTnC in complex with bepridil and the switch region of cardiac troponin I (cTnI), cNTnC.Ca2+.cTnl147-163.bepridil. These structures have provided a basis for our ongoing effort in addressing the key issues such as: 1) what is the mechanism underlining the mode of action of cardiotonic drugs on cTnC; 2) what is the common pharmacophore responsible for the action of these drugs; 3) what are the effects of these drugs on the structure of troponin in vitro and in situ. Our major tool is nuclear magnetic resonance (NMR) spectroscopy, especially the use of multi-nuclear and multi- dimensional NMR techniques combined with the computational techniques of energy minimization and molecular dynamics to determine the structure of proteins in solution. We will also develop wide line 19F NMR methodologies that can be utilized to study proteins and drugs embedded in muscle fibers. Our specific aims are to 1) elucidate the nature and significance of the effective binding sites of drugs on cTnC and determine the three dimensional solution structures of cTnC-drug complexes, 2) evaluate the relevance of these structures in the whole troponin complex, 3) test the relevance of these structures in muscle fibers. Knowledge gained from this research will help to understand the molecular mechanism underlining the pharmacological effects of the drugs under investigation and will provide insights into the features that are important for the design of cardiotonic drugs in general.

描述（由申请人提供）：拟议研究的总体目标是阐明肌钙蛋白调节心肌收缩的分子机制，并确定其作为开发可用于减弱心肌收缩的治疗方案的目标的作用。心脏病是西方社会发病和死亡的主要原因。这项研究背后的假设是，肌钙蛋白是一种分子开关，直接调节心肌收缩过程中肌丝的 Ca2+ 依赖性激活，因此作为强心药物设计的一个有吸引力且合乎逻辑的目标。这些药物在临床上很有用，因为它们可用于调节肌钙蛋白的 Ca2+ 敏感性。这种方法在治疗上是理想的，因为细胞内 [Ca2+] 不会受到干扰，从而保留了其他基于 Ca2+ 的信号通路的调节。为了定义强心结构-活性关系，我们已经证明了几种强心剂特异性结合肌钙蛋白 C (cTnC)，并确定了两种 cTnC-药物复合物的高分辨率结构，即 cTnC 与 EMD 57033 复合物的 C 结构域、cCTnC.2Ca2+。 EMD57033，以及与贝普地尔复合的 cTnC N 结构域和心肌肌钙蛋白的开关区域I (cTnI)，cNTnC.Ca2+.cTnl147-163.bepridil。这些结构为我们持续努力解决关键问题提供了基础，例如：1）强心药物对 cTnC 作用模式的机制是什么？ 2）负责这些药物作用的共同药效团是什么； 3）这些药物对体外和原位肌钙蛋白结构有何影响。我们的主要工具是核磁共振（NMR）光谱，特别是使用多核和多维NMR技术结合能量最小化和分子动力学计算技术来确定溶液中蛋白质的结构。我们还将开发宽线 19F NMR 方法，可用于研究嵌入肌纤维中的蛋白质和药物。我们的具体目标是 1) 阐明药物在 cTnC 上有效结合位点的性质和意义，并确定 cTnC-药物复合物的三维溶液结构，2) 评估这些结构在整个肌钙蛋白复合物中的相关性，3)测试这些结构在肌纤维中的相关性。从这项研究中获得的知识将有助于了解所研究药物药理作用的分子机制，并将提供对一般强心药物设计重要特征的见解。