1H and 13C-NMR and Molecular Dynamics Studies of Cyclosporin A Interacting with Magnesium(II) or Cerium(III) in Acetonitrile. Conformational Changes and cis-trans Conversion of Peptide Bonds

Abstract

Cyclosporin A (CsA) is an important drug used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is related to the inhibition of T-cell activation through binding with the proteins Cyclophilin (Cyp) and, subsequently, Calcineurin (CN). In the complex with its target (Cyp), CsA adopts a conformation with all trans peptide bonds and this feature is very important for its pharmacological action. Unfortunately, CsA can cause several side effects, and it can favor the excretion of calcium and magnesium. To evaluate the possible role of conformational effects induced by these two metal ions in the action mechanism of CsA, its complexes with Mg(II) and Ce(III) (the latter as a paramagnetic probe for calcium) have been examined by two-dimensional NMR and relaxation rate analysis. The conformations of the two complexes and of the free form have been determined by restrained molecular dynamics calculations based on the experimentally obtained metal-proton and interproton distances. The findings here ratify the formation of 1:1 complexes of CsA with both Mg(II) and Ce(III), with metal coordination taking place on carbonyl oxygens and substantially altering the peptide structure with respect to the free form, although the residues involved and the resulting conformational changes, including cis-trans conversion of peptide bonds, are different for the two metals.

Differentiation of cyclosporin A from isocyclosporin A by liquid chromatography/electrospray ionization mass spectrometry with post-column addition of divalent metal salt

“…[13] The calculated structure of the CsA-Mg complex in water shows an octahedral coordination of the Mg 2+ ion with the carbonyl oxygens of residues 1, 4, 5, and 6 and water molecules. [14] These facts agree with the proposed mechanism of fragmentation, and they also explain the loss of MeLeu subsequent to the loss of the MeBmt side chain. The loss of MeLeu 6 is reasonable after an initial ring opening between residues 5 and 6 (or 6 and 7), especially in the case of metal binding to carbonyl oxygens 1, 4, 5, and 6 (as was proven in the case of Mg(II)), or at least in the region between amino acids 11 to 5.…”

“…) . The calculated structure of the CsA‐Mg complex in water shows an octahedral coordination of the Mg 2+ ion with the carbonyl oxygens of residues 1, 4, 5, and 6 and water molecules . These facts agree with the proposed mechanism of fragmentation, and they also explain the loss of MeLeu subsequent to the loss of the MeBmt side chain.…”

“…Differentiation of isomers by the use of metal complexation and electrospray was previously achieved, for example in hydroxypyridine N ‐oxides and polyaromatic hydrocarbons . The same strategy was explored for the differentiation of CsA and isoCsA by metal complexation, based on the ability of cyclosporins to form stable adducts with Na + , Mg 2+ , Ca 2+ , Cu 2+ and Zn 2+ in solution . On the other hand, post‐column addition of metal solutions in LC/MS has been successfully employed for the improvement of signal response and in isomer differentiation of flavonoids and carbohydrates .…”

“…[11] The same strategy was explored for the differentiation of CsA and isoCsA by metal complexation, based on the ability of cyclosporins to form stable adducts with Na + , Mg 2+ , Ca 2+ , Cu 2+ and Zn 2+ in solution. [12][13][14][15] On the other hand, post-column addition of metal solutions in LC/MS has been successfully employed for the improvement of signal response [16] and in isomer differentiation of flavonoids [17] and carbohydrates. [18] Since CsA and isoCsA are usually analyzed by LC in complex natural matrices, the differentiation of both metabolites was investigated by the use of post-column addition of metal ion solutions.…”

1H and 19F NMR relaxation studies of fleroxacin with Micrococcus luteus

“…receptors and enzymes) in case of fast exchange between the free and the bound state [13]. Various methods such as chemical shift differences [14][15][16], line width analysis, relaxation measurements, determination of diffusion coefficients [17] and intermolecular magnetization transfer techniques such as tr-NOE [18,19] or WaterLOGSY experiments [20] have been reported. There are a number of studies based on relaxation measurements for determination of ligand-macromolecule binding [21][22][23][24][25][26].…”