Defining the Binding Site of Levosimendan and Its Analogues in a Regulatory Cardiac Troponin C−Troponin I Complex

The interaction of Cardiac Troponin C (cTnC) and Cardiac Troponin I (cTnI) plays a critical role in transmitting the Ca2+ signal to the other myofilament proteins in the activation of cardiac muscle contraction. As such, the cTnC−cTnI interface is a logical target for cardiotonic agents such as levosimendan that can modulate the Ca2+ sensitivity of the myofilaments. Evidence indicates that drug candidates may exert their effects by targeting a site formed by binding of the switch region of cTnI to the regulatory N domain of cTnC (cNTnC). In this study, we utilized two-dimensional 1H−15N HSQC NMR spectroscopy to monitor the binding of levosimendan and its analogues, CMDP, AMDP, CI-930, imazodan, and MPDP, to cNTnC·Ca2+ in complex with two versions of the switch region of cTnI (cTnI147−163 and cTnI144−163). Levosimendan, CMDP, AMDP, and CI-930 were found to bind to both cNTnC·Ca2+·cTnI147−163 and cNTnC·Ca2+·cTnI144−163 complexes. These compounds contain a methyl group that is absent in MPDP or imazodan. Thus, the methyl group is one of the pharmacophores responsible for the action of these pyridazinone drugs on cTnC. Furthermore, the results showed that the cNTnC·Ca2+·cTnI144−163 complex presents a higher-affinity binding site for these compounds than the cNTnC·Ca2+·cTnI147−163 complex. This is consistent with our observation that the affinity of cTnI144−163 for cNTnC·Ca2+ is ∼10-fold stronger than that of cTnI147−163, likely a result of electrostatic forces between the N-terminal RRV extension in cTnI144−163 and the acidic residues in the C and D helices of cNTnC. These results will help in the delineation of the mode of action of levosimendan on the important functional unit of cardiac troponin that constitutes the regulatory domain of cTnC and the switch region of cTnI.