EMD 57033 interacts directly and stereo-selectively with recombinant human cardiac troponin C in a calcium-dependent manner, with a binding site likely residing in the carboxyl domain.
EMD 57033 binds directly to the carboxyl domain of human cardiac troponin C in a calcium-dependent manner, elucidating its mechanism of action on cardiac myofilaments.
The cardiotonic effects of thiadiazinone derivative EMD 57033 are mediated by direct actions on myofilaments (Lues, I., Beier, N., Jonas, R., Klockow, M., and Haeusler, G. J. (1993) Cardiovasc. Pharmacol. 21, 883-892). Cardiac troponin C has been postulated to be a potential target of the drug (White, J., Lee, J. A., Shah, N., and Orchard, C. H. (1993) Circ. Res. 73, 61-70). This study tested whether EMD 57033 interacts directly with recombinant human cardiac TnC (hcTnC). EMD 57033 caused concentration-dependent quenching of tyrosine (Tyr) fluorescence of hcTnC in the presence of Ca2+ (100 microM) and little change of the fluorescence in the presence of Mg2+ (2 mM). Kd for the drug-hcTnC interaction in the presence of Ca2+, determined by Tyr fluorescence titrations, was approximately 40 microM. The binding of EMD 57033 was stereo-selective: the optical isomer of EMD 57033 bound hcTnC much more weakly. The Ca2+ dependence and stereo-selectivity of EMD 57033 binding were substantiated by a dialysis-based direct binding assay. EMD 57033 was found to interfere with Ca(2+)-dependent binding of hydrophobic probe 1,1'-bi-(4-anili-no)naphthalene-5,5'-disulfonate (bis-ANS) to hcTnC. The relationships between Ca2+ and Tyr fluorescence of hcTnC and between Ca2+ and bis-ANS fluorescence in the presence of hcTnC were substantially altered by EMD 57033 in the range of Ca2+ where Ca2+/Mg2+ sites of hcTnC were titrated by Ca2+. EMD 57033 was found to bind as tightly to 2 Ca2+.hcTnC as to 3 Ca(2+).hcTnC. These observations were interpreted as indicating that a EMD 57033-binding site is induced by Ca2+ binding, but not Mg2+ binding, to the Ca2+/Mg2+ sites of hcTnC. The drug-binding site most likely resides in the carboxyl domain of hcTnC.
Pan et al. (Mon,) reported a other. EMD 57033 was evaluated on Interaction with recombinant human cardiac TnC (hcTnC). EMD 57033 interacts directly and stereo-selectively with recombinant human cardiac troponin C in a calcium-dependent manner, with a binding site likely residing in the carboxyl domain.