Interaction of anthracycline antibiotics with biopolymers: 9. Comparative study of the interaction kinetics of daunomycin,adriamycin and iremycin with DNA

The interaction kinetics of the three anthracycline antibiotics, daunomycin, adriamycin and iremycin, with calf thymus DNA has been investigated using the temperature-jump technique. Experimental data obtained at high binding ratio have been fitted by a kinetic theory which, for the binding of large ligands to a linear polymer chain, takes into account both nearest-neighbour ligand interaction and the overlap of potential binding sites. The kinetics of such cooperative binding according to a single-step mechanism can be described completely by two independent microscopic parameters, namely one rate constant and a kinetic cooperativity parameter. Both these parameters have been determined for the three anthracyclcine antibiotics, making use of the known equilibrium binding parameters. The association rate constant in the singly contiguous case turns out to be almost the same for all three antibiotics ($\text{7 × 10}{}^6\text{to 8 × 10}{}^6\text{1 mol}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$), while the corresponding dissociation rate constant ranges from 3.5 s^?1 for adriamycin to 10 s^?1 for daunomycin and about 35 s^?1 for iremycin. The different equilibrium binding constants thus correspond to different mean attachment times of the antibiotics at the polymer chain, which positively correlate with the inhibitory action of these drugs on in vitro DNA synthesis. Nearest-neighbour interaction in the case of adriamycin-DNA binding kinetics implies that adriamycin molecules dissociate from an isolated binding site nine times more frequently than from a site between two adjacent ligands.     

Effect of the molecular structure of anthracycline compounds on their intercalation into DNA

Kinetics of interaction of rubomycin, carminomycin, and their C9-analogues with deoxyribonucleic acid have been studied by the stop - flow method. Possible reasons of nonexponentiality of the kinetic curves are discussed. The rate constants of direct and reverse intercalation of the drugs between DNA base pairs were determined and relatively small differences found for various drugs. These data and of equilibrium binding studies of interaction of the antibiotics and their analogs with DNA led to conclusion that C9-modification of anthracyclines has little effect on the binding process. That was confirmed by quantum-mechanic calculations of potential energy of interaction between various anthracycline aglicones and DNA base-pairs.     

Intercalation of manganese-mefenamic acid complex into double stranded of calf thymus DNA

Abstract

The interaction of the [Mn(mef)₂(phen)H₂O] complex in which mef is mefenamic acid drug and phen is 1,10 phenanthrolin ligand with calf thymus DNA (ct-DNA) was studied by using different spectroscopic methods, molecular docking and viscometery. The competitive fluorescence and UV–Vis absorption spectroscopy indicated that the complex interacted with ctDNA via intercalating binding mode with the binding constant of 1.16?×?10⁴ Lmol^?1. The thermodynamic studies showed that the reaction between the complex and ctDNA is exothermic. Furthermore, the complex induced changes in DNA viscosity. Circular dichroism spectroscopy (CD) was employed to measure the conformational changes of ctDNA in the presence of the complex and verified intercalation binding mode. The molecular modeling results illustrated that the complex interacted via intercalation by relative binding energy of ?28.45?kJ mol^?1.     

Dynamic states of the DNA repair enzyme AlkB regulate product release

The 2-oxoglutarate (2OG)- and Fe(2+)-dependent dioxygenase AlkB couples the demethylation of modified DNA to the decarboxylation of 2OG. Extensive crystallographic analyses have shown no evidence of significant structural differences between complexes binding either 2OG or succinate. By using nuclear magnetic resonance spectroscopy, we have shown that the AlkB-succinate and AlkB-2OG complexes have significantly different dynamic properties in solution. 2OG makes the necessary contacts between the metal site and the large beta-sheet to maintain a fully folded conformation. Oxidative decarboxylation of 2OG to succinate leads to weakening of a main contact with the large beta-sheet, resulting in an enhanced dynamic state. These conformational fluctuations allow for the replacement of succinate in the central core of the protein and probably contribute to the effective release of unmethylated DNA. We also propose that the inherent dynamics of the co-product complex and the subsequent increased molecular ordering of the co-substrate complex have a role in DNA damage recognition.     

Formation of DNA-adducts and induction of DNA-crosslinks and chromosomal aberrations by the new potent anthracycline antitumor antibiotics: morpholinodaunomycin,cyanomorpholinodaunomycin and cyanomorhpolinoadriamycin

The DNA-interaction of three newly developed semisynthetic anthracyclines with high antitumor potency MoDNM³, CNMoDNM, and CNMoADM, was investigated. When primary rat hepatocytes were incubated with tritium labeled MoDNM and CNMoDNM and their DNA was purified and enzymatically hydrolized, the formation of DNA-adducts could be demonstrated by the HPLC chromatography of the resulting mononucleoside mixtures. The parent compound, daunomycin (DNM), also formed covalent adducts with hepatocyte DNA, but to a lesser extent. These findings correlate well earlier observaitons that MoDNM and CNMoDNM are potent inducers of DNA-repair in primary rat hepatocytes, whereas DNM is only weakly active in this regard. Aklaline elution studies were performed with L 1210 mouse leukemia cells and V79 Chinese hamster fibroblasts. The cyanomorpholinyl derivatives showed dose-dependant DNA crosslinking activities in both cell lines at concentrations 5 nMol/l. The formation of crosslinks began a few minutes after treatment of the cells and reached a maximum after 1 hr. In contrast, MoDNM, at concentrations of up to 10 Mol/l, had only a limited capacity to induce single strand breaks in L 1210 cells but did not induce DNA-crosslinks. In addition, chromosomal aberrations (chromatid breaks and translocations) were induced by the treatment of Friend and L 1210 leukemia cells with CNMoADM at concentrations between 0.07–0.6 n Mol/l. At higher doses, chromosome clumping was observed. These results indicate that the high capacity of MoDNM, CNMoDNM and CNMoADM to induce DNA repair in primary rat hepatocytes is due to the formation of covalent adducts with DNA. The cyanomorpholino compounds have alkylating capacities also in cell lines such as L 1210 and V79, whereas MoDNM requires rat hepatocytes for activation. The ready formation of DNA crosslinks and chromosomal aberrations could be responsible for the high cytotoxicity of these compounds.Abbreviations ADM adriamycin - CNMoADM cyanomorpholinoadriamycin - CNMoDNM cyanomorpholinodaunomycin - DNM daunomycin - FLC Friend leukemia cells - (G³H) generally tritium labeled - HPLC high performance liquid chromatography - MoDNM morpholinodaunomycin - Rf retention factor - (Mo³H) tritium labelled at morpholinyl site - Rad radiation unit - RT retention time - SDS sodium dodecylsulphate - Tris tris (hydroxymethyl)aminomethan     

Exploring the intercalation binding of tiamulin with DNA using multispectroscopy and a molecular modelling approach

The binding of tiamulin with calf thymus DNA was systematically investigated using multispectroscopy and molecular modelling techniques. For DNA, once tiamulin was added, viscosity (η) and melting temperature (T_m) both exhibited an uptrend. The fluorescence performance of the tiamulin–DNA complex did not change with the ionic strength changes. The binding constant (K_a) of tiamulin for single-stranded DNA (ssDNA, 1.48 × 10⁴ M⁻¹) was obviously higher than that for double-stranded DNA (dsDNA, 9.51 × 10³ M⁻¹) at 291 K. The helix structure became looser and the base stack force became stronger for DNA due to the presence of tiamulin as seen from circular dichroic (CD) spectra. The intercalation binding mode of tiamulin with DNA was disclosed. Molecular modelling also revealed tiamulin inserting into the base pairs with the lowest binding free energy of −18.73 kJ mol⁻¹ using van der Waals forces as well as hydrogen bonds.     

Energetics of the lattice: Packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice—these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine · cytosine⁺ (C · C⁺) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other—for example, when folded in more complicated higher order structures, such as mRNA and chromatin. © 1998 John Wiley & Sons, Inc. Biopoly 44: 257–267, 1997     

Interaction of anthracycline antibiotics with biopolymers: 7. Equilibrium binding studies on the interaction of iremycin and DNA

We report spectrophotometric equilibrium studies of both the self-association of the new antibiotic iremycin and of its binding to calf thymus DNA in solution (ionic strength 0.2 M; pH 6.0). Iremycin forms dimers in this solution with a dimerization constant K₄=(1.19 ± 0.10) × 10³ M⁻¹. This equilibrium is taken into account in the evaluation of the interaction of iremycin with DNA. The binding behaviour can be completely described by a single binding mechanism of monomeric iremycin to DNA with allowance both for neighbour exclusion and for cooperativity of interaction. The three intrinsic binding parameters for the homogeneous model were determined simultaneously by a least squares fit of the original titration data: equilibrium constant of cooperative binding K = (2.72 ± 0.66) × 10⁵ M⁻¹ cooperativity parameter σ=0.38±3.27 ± 0.32. The binding parameters of iremycin and adriamycin and their microbial activities are compared.     

New alkaloid antibiotics that target the DNA topoisomerase I of Streptococcus pneumoniae

Streptococcus pneumoniae has two type II DNA-topoisomerases (DNA-gyrase and DNA topoisomerase IV) and a single type I enzyme (DNA-topoisomerase I, TopA), as demonstrated here. Although fluoroquinolones target type II enzymes, antibiotics efficiently targeting TopA have not yet been reported. Eighteen alkaloids (seven aporphine and 11 phenanthrenes) were semisynthesized from boldine and used to test inhibition both of TopA activity and of cell growth. Two phenanthrenes (seconeolitsine and N-methyl-seconeolitsine) effectively inhibited both TopA activity and cell growth at equivalent concentrations (～17 μM). Evidence for in vivo TopA targeting by seconeolitsine was provided by the protection of growth inhibition in a S. pneumoniae culture in which the enzyme was overproduced. Additionally, hypernegative supercoiling was observed in an internal plasmid after drug treatment. Furthermore, a model of pneumococcal TopA was made based on the crystal structure of Escherichia coli TopA. Docking calculations indicated strong interactions of the alkaloids with the nucleotide-binding site in the closed protein conformation, which correlated with their inhibitory effect. Finally, although seconeolitsine and N-methyl-seconeolitsine inhibited TopA and bacterial growth, they did not affect human cell viability. Therefore, these new alkaloids can be envisaged as new therapeutic candidates for the treatment of S. pneumoniae infections resistant to other antibiotics.