INTERACTION OF CALCIUM-CHANNEL ANTAGONISTS WITH CALCIUM - STRUCTURAL STUDIES ON VERAPAMIL AND ITS CA2+ COMPLEX

The conformation of the calcium channel antagonist verapamil has been determined in acetonitrile, in the absence and presence of Ca2+, using two-dimensional H-1-NMR and molecular modeling techniques. Interproto n connectivities in the drug molecule were identified from the observe d NOESY cross peaks and interproton distances were estimated from the magnitudes of the volume integrals of the cross peaks. The molecular m odeling program utilized the Monte Carlo simulation to generate a rand om ensemble of conformers complying with the NOESY-derived distance co nstraints. The energies of these conformers were subsequently computed . The minimum-energy structure of the free drug obtained in this manne r exhibited some significant differences from the structure of verapam il determined by X-ray crystallography. In particular, the torsional a ngles in the middle region of the molecule containing the aliphatic '' backbone' were such that the two aromatic rings at either end of the d rug molecules were moved farther apart from each other in solution tha n in the crystal structure. The nearly perpendicular orientation of th e aromatic rings seen in the crystal was, however, maintained in the s olution structure as well. The addition of Ca2+ to a solution of verap amil in acetonitrile caused marked changes in the difference absorbanc e of the drug in the 200-300-nm region and in many of its H-1-NMR reso nances. The changes were most significant up to a mole ratio of about 0.5 Ca2+:drug. Analysis of the binding data at 25-degrees-C showed the presence of both 2:1 and 1:1 drug:Ca2+ complexes in equilibrium, the former ''sandwich'' complex being dominant at the lower cation concent rations with an estimated dissociation constant of about 300 muM. All of the NOESY cross peaks of the free drug remained on addition of 0.5 mol ratio of Ca2+ to verapamil in deuterated acetonitrile and only two new connectivities were observed. Using the interproton distances cal culated from these NOESY data, molecular modeling of the 2:1 drug:Ca2 complex was carried out to yield the minimum-energy conformer. In thi s conformer, Ca2+ Was coordinated to two methoxy oxygens from each of the two drug molecules. The implications of the verapamil-Ca2+ interac tion are discussed in terms of available experimental data on the bind ing of verapamil to the dihydropyridine-sensitive channel and in terms of a hypothesis on the formation of a drug-Ca2+-receptor complex in t he lipid bilayer environment.