Studies of viomycin, an anti-tuberculosis antibiotic: copper(ii) coordination, DNA degradation and the impact on delta ribozyme cleavage activity.

Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(ii) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(ii)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(ii)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme.

Amiodarone affects membrane water permeability properties of human erythrocytes and rat mitochondria.

Dose-dependent water exchange times and intracellular water contents were measured by NMR (nuclear magnetic resonance) in erythrocytes and mitochondria interacted with the anti-anginal and anti-arrhytmic agent, amiodarone. Addition of the drug up to 26 microM yielded 80% enhancement of the water exchange rate in erythrocytes at 37 degrees C and 41% enhancement at 22 degrees C with 40% and 9%, respectively, increases in the intracellular water content. Similar enhancements were obtained in mitochondria at 22 degrees C. The data suggests a somewhat higher affinity of amiodarone to mitochondrial than to erythrocyte membranes.

1H NMR studies on the interaction of beta-carboline derivatives with human serum albumin.

1H NMR studies were performed on two beta-carboline derivatives interacting with human serum albumin. The spin-lattice relaxation rates of the two derivatives, having side chains of different length and polarity, were used to demonstrate a diverse motional behavior in solution together with slightly different relaxation pathways. Single- and double-selective excitation made it possible to evaluate dynamics in the free and protein-bound states. Occurrence of a relatively long hydrophilic chain interacting with the proton-acceptor nitrogen of the beta-carboline moiety was shown to yield lower association constants, slower dissociation rates, and diverse interacting modes with the indole hydrophobic site of the protein.

1H NMR relaxation investigation of inhibitors interacting with Torpedo californica acetylcholinesterase.

Two naphthyridines interacting with Torpedo californica acetylcholinesterase (AChE) were investigated. (1)H NMR spectra were recorded and nonselective, selective, and double-selective spin-lattice relaxation rates were measured. The enhancement of selective relaxation rates could be titrated by different ligand concentrations at constant AChE (yielding 0.22 and 1.53 mM for the dissociation constants) and was providing evidence of a diverse mode of interaction. The double-selective relaxation rates were used to evaluate the motional correlation times of bound ligands at 34.9 and 36.5 ns at 300 K. Selective relaxation rates of bound inhibitors could be interpreted also in terms of dipole-dipole interactions with protons in the enzyme active site.

1H-NMR studies on the self-association of chloroquine in aqueous solution.

The concentration dependences of 1H-NMR chemical shifts and spin-lattice relaxation rates were measured for chloroquine in aqueous solution. The weak self-association constant was evaluated according to a dimerization equilibrium with the formation of self-stacked adducts (Kd = 4.52 +/- 0.68 l mol-1). The motional correlation times were evaluated for the monomer and the dimer by measuring intramolecular dipolar cross-relaxation rates of aromatic vicinal protons (tau cm = 0.06 ns and tau cd = 0.26 ns). The geometry of the stacked dimer was elucidated by measuring intermolecular dipolar cross-relaxation rates and interpreted in terms of partial superposition of quinoline moieties.

Selective 1H-NMR relaxation investigations of membrane-bound drugs in vitro. 1. Colchicine.

Binding of colchicine to dipalmitoylphosphatidylcholine bilayer vesicles was detected by measuring the 1H-NMR selective spin-lattice relaxation rates of the low-field protons of colchicine. From the temperature dependence of the selective rates, preferential binding was observed above the temperature of transition. In the same way, binding of colchicine to red blood cells was detected and the equilibrium constant determined. Binding to the lipid matrix of red blood cells accounted only partially for the binding of colchicine to whole cells.

Selective 1H-NMR relaxation investigations of membrane-bound drugs in vitro. 2. Angiotensin II.

Binding interactions between human angiotensin II and dipalmitoylphosphatidylcholine bilayer vesicles have been detected by measuring the selective proton spin-lattice relaxation rates of aromatic protons within the peptide. Involvement of the imidazole moiety of the His-6 residue has been demonstrated by the pH dependence of the NMR observables. A lower limit of the binding constant has been evaluated at 78.12 mol-1 dm3 for the interaction involving nonionic intermolecular forces between aromatic residues and the lipid matrix.

Co-ordination behaviour of nucleic acid bases against the Fe(I)(NO)-2 group in solution. I. Purines: ESR study.

The binding sites of purine bases in the presence of the Fe(I)(NO)-2 group were investigated on the basis of the nuclear hyperfine structure of the electron spin resonance spectra. Selective isotopic substitution with 15-NO was used to clarify co-ordindination. Coupling constants and different types of complexes were determined by means of computer-simulated ESR spectra. Comparison was made with the binding sites of nucleotides and it was concluded that N-7, in the imidazole ring, is the preferred binding site. A structure with two base molecules bonded to the iron atom was proposed. Finally 8-azaguanine, an antitumoral agent, was studied and the special behaviour of this antimetabolite was demonstrated in the biologically-interesting pH values.

Conformational dynamics of cytochalasin B in solution as detected by 13C and 1H NMR relaxation rates.

13C and 1H NMR spin-lattice relaxation rates have been measured for cytochalasin B in [2H]6DMSO. Motional features have been interpreted in terms of nearly isotropic reorientation of the whole molecule with few additional internal motions. The 'isotropic' reorientational correlation time was evaluated at 0.21 ns at room temperature. By using selective and double-selective excitation techniques, relevant cross-relaxation terms have been obtained wherefrom proton-proton distances have been calculated. A Dreiding model of the 'preferred' conformation in solution has been built, yielding evidence of a strong similarity between solution and solid state structures of cytochalasin B.

Effect of morphine analogues on the conformational state of polyriboadenylic acid in solution: an NMR study.

The interaction of morphine analogues with polyadenylic acid in aqueous solution at pH 7 has been investigated. The NMR spectra measured at several temperatures were shown to give evidence of a lowering of the transition temperature from the helix to the random-coil structure. The measured chemical shifts and relaxation times were interpreted in terms of an ionic interaction between the + N - CH3 group of the drug and phosphate residue of the polynucleotide.

Conformational features of cell-bound drugs as detected by selective proton relaxation rate investigations.

The nonselective and selective longitudinal relaxation rates were measured for procaine protons in the presence of model lipid membranes, biological membranes and whole cells. Unlike the nonselective relaxation rates, the selective rate was shown to be particularly sensitive in detecting binding interactions with macromolecular cell constituents. It was shown that the aromatic moiety of procaine is involved in binding the cell plasma membrane.

Conformational and dynamic features of cocaine in DMSO-d6 solution.

13C spin-lattice relaxation rates, 13C {1H} NOESs, 1H spin-spin relaxation rates and 1H two-dimensional magnetization transfer spectroscopy were used for delineating conformational features of cocaine in DMSO-d6 solution. Two main conformations differing in the orientation of the plane made by the benzoxy substituent with respect to the piperidine ring principal axis were observed. Relatively slow interconversions of the piperidine ring were delineated together with the main motional features of the whole molecule.

The water proton spin-lattice relaxation times in virus-infected cells.

The water proton spin-lattice relaxation times in HEp-2 cell cultures were determined immediately after 1 h of polio-virus adsorption. The shortening of the water T1 was closely related to the multiplicity of infection, allowing direct inspections of the virus--cell interaction since the first steps of the infectious cycle. Virus-induced structural and conformational changes of cell constituents were suggested to be detectable by NMR investigation of cell water.

Selective 1H-NMR relaxation investigations of membrane-bound drugs in vitro. 3. Calcium-entry blockers and adenosine.

Selective proton relaxation rates (SPRR) were measured for selected protons of nimodipine or diltiazem in the presence of neutrophils, allowing detection of binding to the cell membrane. Fast exchange exchange of drug molecules between bound and free environments was shown to be the main factor determining the enhancement of SPRR, whereas viscosity effects could be neglected. The SPRR enhancement was almost completely cancelled out by the presence of adenosine as a cosolute in a dose-dependent fashion, leading to the suggestion that the endogenous mediator 'adenosine' affects binding of calcium-entry blockers to the neutrophil surface.

NMR investigations of Mn(II)-leucine-enkephalin complexes in [2H6]-DMSO solution.

Structural and kinetic features of the Mn(II)-Leu-enkephalin binding equilibria were delineated by measuring 13C and 1H NMR spin-lattice relaxation rates. The temperature dependence of such rates showed that some carbons were experiencing slow exchange regimes such that kinetic parameters at room temperature could be calculated (k(off) = 1400 sec-1, delta H* = 12.0 kcal/mol, delta S* = -9.9 e.u.). The paramagnetic rates of fast exchanging carbons were interpreted by the Solomon-Bloembergen-Morgan theory to provide structural parameters. The terminal carboxyl and amino groups were shown to be the binding sites. The motional correlation time (tau c = 0.6 nsec at 298 K) was calculated by measuring selective and double-selective 1H spin-lattice relaxation rates for the free peptide. The number of coordinated ligands was evaluated by considering the distance of the Leu CO in the complex at 2.54 A, as shown by molecular models. Finally, carbon-Mn(II) distances were calculated and the molecular model of the 1:1 complex was built.

The effect of binding divalent metal ions on the conformation of human angiotensin-II in solution as probed by nuclear and electron paramagnetic resonance investigations.

The interaction between human angiotensin-II and Mn(II) ions in water solution has been delineated by nmr and esr experiments. A pH-dependent binding interaction with the imidazole moiety of His 6 has been shown, ratifying the suggestion that His 6 is involved in intramolecular structuring within the peptide molecule.

NMR investigation of the copper(II)-carnosine complex in water solution.

The predominant species in water solution of excess carnosine and Cu2+ ions was characterized by measuring 13C and 1H spin-lattice and spin-spin relaxation rates. Four peptide molecules were calculated to be coordinated to the metal through the imidazole nitrogen. Evaluation of ion-proton distances demonstrated that metal complexation does not involve severe changes in conformation of the peptide.

Structural, motional, and kinetic features of the Cu(II)-(L-His)2 complex in aqueous solution.

A careful analysis by 1H and 13C FT-NMR on the Cu(II) (L-histidine)2 complex was carried out which allows delineation of structure and dynamics in solution. A mixture of complexes was shown such that 24% of the Cu(II) (L-histidine)2 complex contains both histidines bound in the histaminelike way, while the remaining 76% contains one L-His molecule bound in the histaminelike way and the other L-His molecule bound in the glycinelike way. The motional correlation time and relevant features of the exchange process were also delineated.

Nuclear magnetic resonance spectrometric investigations of calcium antagonist drugs. III: Conformational and dynamic features of diltiazem in solution.

Conformational features of diltiazem in D2O have been delineated by measuring 13C and 1H NMR parameters. The data obtained by interpretation of spin-lattice relaxation rates and 13C-[1H] nuclear Overhauser effects were used to build a model of the most probable arrangement in solution. The molecular motion of the two aromatic rings and of the thiazepinic ring is anisotropic, and the assumption that the molecule is an ellipsoid undergoing Brownian motion leads to calculation of the diffusion constants for the rotation around the longer axis and the shorter axis. The dimethylaminoethyl chain seems folded back, and it displays the typical features of segmental motion; in the same way, the methoxy- and the acetyloxymethyls behave like free rotors.

Nuclear magnetic resonance investigations of calcium antagonist drugs. II: Conformational and dynamic features of verapamil in [2H6]DMSO.

Conformational features of verapamil in [2H6]DMSO have been delineated by measuring 13C and 1H NMR parameters. Spin-lattice relaxation rates were interpreted within the frame of an extended alkane chain with segmental motions hampered by the aromatic substituents at both ends. The effective correlation times were all evaluated in the range 0.16-0.22 ns at 293 K. The aromatic rings were found to lie somewhat out-of-plane with the alkane chain. The NMR data were used to construct a Dreiding model of the most probable spatial arrangement and a molecular modeling system was utilized to represent the "preferred" conformation of verapamil in solution.