In cardiac and skeletal muscle, the troponin complex turns muscle contraction on and off in a calcium-dependent manner. Many small molecules are known to bind to the troponin complex to modulate its calcium binding affinity, and this may be useful in a broad range of conditions in which striated muscle function is compromised, such as congestive heart failure. As a tool for developing drugs specific for the cardiac isoform of troponin, we have designed a chimeric construct (cChimera) consisting of the regulatory N-terminal domain of cardiac troponin C (cNTnC) fused to the switch region of cardiac troponin I (cTnI), mimicking the key binding event that turns on muscle contraction. We demonstrate by solution NMR spectroscopy that cChimera faithfully reproduces the native interface between cTnI and cNTnC. We determined that small molecules based on diphenylamine can bind to cChimera with a KD as low as 10μM. Solution NMR structures show that minimal structural perturbations in cChimera are needed to accommodate 3-methyldiphenylamine (3-mDPA), which is probably why it binds with higher affinity than previously studied compounds like bepridil, despite its significantly smaller size. The unsubstituted aromatic ring of 3-mDPA binds to an inner hydrophobic pocket adjacent to the central beta sheet of cNTnC. However, the methyl-substituted ring is able to bind in two different orientations, either inserting into the cNTnC-cTnI interface or "flipping out" to form contacts primarily with helix C of cNTnC. Our work suggests that preservation of the native interaction between cNTnC and cTnI is key to the development of a high affinity cardiac troponin-specific drug.

One approach to improve contraction in the failing heart is the administration of calcium (Ca(2+)) sensitizers. Although it is known that levosimendan and other sensitizers bind to troponin C (cTnC), their in vivo mechanism is not fully understood. Based on levosimendan, we designed a covalent Ca(2+) sensitizer (i9) that targets C84 of cTnC and exchanged this complex into cardiac muscle. The NMR structure of the covalent complex showed that i9 binds deep in the hydrophobic pocket of cTnC. Despite slightly reducing troponin I affinity, i9 enhanced the Ca(2+) sensitivity of cardiac muscle. We conclude that i9 enhances Ca(2+) sensitivity by stabilizing the open conformation of cTnC. These findings provide new insights into the in vivo mechanism of Ca(2+) sensitization and demonstrate that directly targeting cTnC has significant potential in cardiovascular therapy.