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. ? ? ?
