Conformation of calcium antagonists and their interaction with model membranes

Abstract

Calcium antagonists are a class of drugs with diverse chemical identities and a wide range of pharmacological activities. At least four different chemical classes can be identified within this group of drugs. This heterogeneity of chemical structures and biological activity, together with the variety of therapeutic actions, has stimulated extensive research on the molecular mechanism of action as well as on conformational features of these drugs. It is generally believed that the binding to macromolecular receptors involves a process of conformational selection. Therefore, a study of the conformation of calcium antagonists, their calcium ion complexes, and their interaction with membranes is very important for an understanding of the structure-activity relationships of these drugs.

The thesis is divided into seven chapters. Each chapter can be broadly divided into two parts: conformational studies and interactions with model membranes. Conformational studies were carried out using Nuclear Magnetic Resonance (NMR) techniques and molecular modeling. The studies on the interaction of these drugs with model membranes have been carried out using a variety of techniques such as Differential Scanning Calorimetry (DSC), lipid monolayer techniques, and NMR techniques, especially ^31P NMR.

Chapter 1 gives a brief introduction on the importance of calcium and its regulation in living cells. Brief descriptions of various regulatory processes in controlling calcium concentration in the cell are also provided. A brief introduction to calcium channels is given, followed by a discussion on the need for calcium antagonism and the discovery of this important class of drugs called calcium antagonists. The various studies aimed at characterizing the binding sites of these drugs in the calcium channel are described briefly.

Chapter 2 gives an outline of the various techniques and procedures used for this investigation. Brief descriptions are given of various important experiments involved in conformational analysis by NMR. Two-dimensional NMR methods such as Double Quantum Filtered Correlation Spectroscopy (DQFCOSY), used for assignment of various protons of the drug, Rotating frame Overhauser Spectroscopy (ROESY), used to estimate the spatial proximities of the protons, heteronuclear correlation experiments, and SEFT pulse experiments to assign ^13C resonances have been extensively used in this investigation. Descriptions of the techniques used to understand the interaction of the drugs with membranes, such as DSC, ^31P NMR, and the lipid monolayer techniques, as well as a brief introduction to the optimization algorithms used and the molecular dynamics technique (which was used for refining the modeled drug structures), are also given in this chapter.

Chapter 3 deals with the studies on the drug Lidoflazine. Its conformation in two solvents, chloroform and acetonitrile, has been elucidated using NMR. Two different conformations were observed for the drug in these solvents, which were modeled. An extended conformation was observed in acetonitrile, whereas in chloroform, it assumed a bent conformation. The calcium complex of the drug in acetonitrile was characterized with the help of ^1H and ^13C NMR chemical shift data, the coupling constants, and the ROE data from the two-dimensional NMR experiments. NMR titration experiments suggested a 2:1 drug-to-calcium stoichiometry for the drug-calcium complex. Molecular modeling was carried out using all the NMR parameters, and a 2:1 drug-calcium complex was modeled consistent with the data. The magnesium complex of lidoflazine was also modeled on the same lines as the calcium complex. Significantly, the coordination of the magnesium complex was not very different from what was observed for calcium.

In the second part of this chapter, the membrane-interacting properties of lidoflazine were probed using DSC and ^31P NMR. The analysis of the results showed that the location of lidoflazine was near the acyl chain region, deeper in the hydrocarbon core of the membrane bilayer.

Chapter 4 describes the conformational and membrane-interacting properties of the drug Bepridil. The conformation of this drug was characterized in two solvents, namely water and acetonitrile, by NMR followed by molecular modeling. Dramatic changes were observed in ^1H and ^13C NMR chemical shifts when calcium was added to the drug in acetonitrile. NMR titration experiments showed a 2:1 drug-to-calcium stoichiometry for the calcium complex. Modeling was done on the basis of the observed coupling constants of the complex and the ROE distances estimated from the ROESY experiment. Coordination in the complex involved one nitrogen atom and one oxygen atom from each of the drug molecules. A cup-like structure is formed where the calcium ion sits surrounded by hydrophobic groups.

The second part of this chapter describes the interaction of this drug with the model membrane system by DSC and ^31P NMR. DSC data showed that the drug abolishes the pretransition temperature even at a lipid-to-drug molar ratio of 240:1. Further, the drug in increasing concentrations decreased the gel-liquid phase transition temperature T_m linearly and decreased the enthalpy of transition. The data indicated that the Bepridil molecule is located within the first 7–8 methylene carbons of the acyl chain region of the lipid bilayer. DSC thermograms in the presence of calcium showed further broadening of the main transition peak, showing a further decrease in the cooperativity of the phase transition. The drug-calcium complex possibly resides at the same location as the free drug.

An effort was made to locate the position of the drug in the membrane bilayer by two-dimensional NMR techniques, especially the cross-relaxation experiment. A brief description of the problems encountered in such an experiment is given. ROE buildup and decay rates were analyzed, and the position of the drug was located near the acyl chain region of the bilayer.

Chapter 5 describes the studies aimed at modeling the conformation of the drug Flunarizine and its calcium complex in solution and its membrane location by NMR. The conformation of Flunarizine in water and in chloroform was modeled using NMR data. The calcium complex of the drug was characterized in chloroform. The interesting aspect of the complexation of the drug with calcium was the fact that only nitrogen atoms are available for coordination with calcium. A 2:1 drug-to-calcium complex was modeled based on the NMR data. DSC results showed that Flunarizine in the presence of calcium induces further broadening of the main transition peak, and it was concluded that the drug in the calcium-complexed form is located at the same position as the drug itself, near the acyl chain region of the membrane bilayer. Two-dimensional NMR experiments were carried out to find the location of the drug in the bilayer. ROEs were obtained which suggested a possible aggregation of the drug in the lipid medium. A simple dimer model was constructed to explain the various unusual ROEs observed.

Chapter 6 deals with the conformation and membrane-interacting properties of the important calcium antagonists, Verapamil and Diltiazem. Circular dichroism spectroscopy revealed temperature-dependent and solvent-dependent conformational changes in Diltiazem. The conformation of Diltiazem was investigated in methanol using NMR. The conformation was modeled on the basis of the various NMR data. Further, the magnesium and lithium complexes of Diltiazem were characterized, and the conformations of 2:1 drug-to-metal ion complexes were modeled. Monolayer techniques were used to probe the effect of calcium on the interaction of Diltiazem with membranes. Results showed that the presence of calcium significantly alters the ease with which the drug incorporates into the monolayer. Secondly, it also has a significant effect on the exclusion pressure, the limiting pressure at which the drug no longer can penetrate the membrane.

The conformation of Verapamil in two solvents, water and chloroform, was modeled from the NMR data. Two distinct conformations were observed in these solvents. Previous reports suggested a synergism between lithium and Verapamil when they were administered together to patients with manic disorders. The lithium complex of Verapamil was characterized by NMR, and the drug-lithium complex was modeled. Monolayer studies indicated that Verapamil is more surface-active compared to Diltiazem. Calcium exhibited a significant effect on the interaction of Verapamil with the lipid. It also significantly affects the exclusion pressure parameter of the drug.

The overall summary of the results obtained on the conformation of calcium antagonists, their calcium ion complexes, and their interaction with model membranes is given in Chapter 7. It is interesting to note that the most preferred conformation of the calcium complex of these drugs studied has a 2:1 drug-to-calcium structure with interesting coordination involving nitrogen atoms. These results, along with the observations on their mode of interaction with model membranes, are likely to be significant in terms of the calcium channel-blocking activity of the drugs studied.