500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.
"DNA components, then the 'interceptor' and 'protector' mechanisms co-exist in the mixed solution. It was thought that the simplest way to distinguish between these two processes would be to calculate the fraction of X in the hetero-complexes (f h ) and the complexes of X with DNA (f C2 )  . However, both f h and f C2 result from solution of the system of Eq. (2) and therefore they both depend on the effectiveness of Y–DNA complexation, K YN , and the X–Y hetero-association, K h , respectively. "
[Show abstract][Hide abstract] ABSTRACT: It is suggested that the widely reported biological synergism of a mixture of DNA-targeting aromatic drug molecules both in vivo and in vitro can be explained, in part, at the molecular level by competition between two basic mechanisms: the 'interceptor' (hetero-association between Drug1 and Drug2) and 'protector' mechanisms (complexation of Drug1 and Drug2 on DNA-binding sites). In the present work a complete analytical methodology has been developed to quantify these processes, providing an estimate of the relative importance of the interceptor/protector mechanisms using just a set of equilibrium association constants. The general methodology may be applied to other molecules with receptors for aromatic drugs.
"In some cases the heteroassociation of aromatic molecules may lead to formation of stable hetero-complexes in solution as they are energetically more favorable than self-association of the constituent drugs. Our previous investigations have shown that stacked complexes between the antibiotic DAU and the aromatic dyes, proflavine and ethidium bromide, are additionally stabilized by intermolecular hydrogen bonds in aqueous salt solution  . Hence it is likely that physico-chemical investigations of the distinctive features of the interactions between aromatic antibiotics under physiological conditions are very important for the development of chemotherapeutic regimes involving combinations of these drugs. "
[Show abstract][Hide abstract] ABSTRACT: In order to investigate the effect on combinations of aromatic antibiotics used in chemotherapy, the hetero-association of the antitumour antibiotics actinomycin D (AMD) with daunomycin (DAU) or novatrone (NOV) has been studied by the methods of 1D- and 2D 500 MHz 1H-NMR spectroscopy and molecular mechanics calculations. The experimental concentration and temperature dependences of the proton chemical shifts of mixtures of the aromatic drugs have been analyzed in terms of a modified statistical-thermodynamical model of hetero-association to give the equilibrium reaction constants, the thermodynamical parameters (deltaH, deltaS) of hetero-association of AMD with DAU or NOV and the limiting values of proton chemical shifts of the molecules in the hetero-complexes. The most favorable averaged structures of the 1:1 DAU-AMD and NOV-AMD hetero-association complexes have been determined using both the limiting values of proton chemical shifts of the molecules and molecular mechanics methods (X-PLOR software). The results show that intermolecular complexes between DAU-AMD and NOV-AMD are mainly stabilized by stacking interactions of the aromatic chromophores, although the DAU-AMD hetero-complex has additional stabilization, which may be explained by an intermolecular hydrogen bond between a carbonyl group of ring C of DAU and the NH group of D-Val of the pentapeptide side chain ring of AMD. The relative content of each type of molecular complex in the mixed solution has been calculated at different values of the ratio (r) of the initial concentrations of DAU and AMD. It is found that the contributions of hetero-complexes to the general equilibrium in solution are predominant at quite different values of r, viz. at r>12 for AMD with NOV and at r>2 for AMD with DAU, compared to r>0.3 for the DAU-NOV system observed previously. It is concluded that anticancer drugs have quite different affinities for formation of hetero-complexes with other aromatic antibiotics in aqueous solution, which may need to be taken into consideration for their use in combination chemotherapy.
[Show abstract][Hide abstract] ABSTRACT: The structure–activity relations of a series of synthetic phenoxazone drugs with aminoalkyl side chains of variable length and different terminal groups were investigated by examining their biological activity and DNA complexation affinity. Biological activity was determined from their ability to induce apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line. The thermodynamic parameters of drug–DNA complexation were determined by differential scanning calorimetry. By comparing the activities of compounds with different terminal groups (amino, dimethylamino and diethylamino), we found that the existence of a terminal dimethylamino group in the alkylamino side chain is an important factor for anti-tumour activity. Minor modifications in the dimethylaminoalkyl side chain (e.g. elongation by one methylene group) led to notable changes in both the anti-tumour activity and DNA-binding properties of the drug, providing unambiguous evidence of a marked structure–activity relation.
European Journal of Biochemistry 10/2003; 270(20). DOI:10.1046/j.1432-1033.2003.03817.x · 3.58 Impact Factor
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