General analysis of competitive binding in drug-interceptor-DNA systems.

A general model of competitive binding in drug-interceptor-DNA systems has been developed in order to quantify both the interceptor and protector mechanisms. The model involves full parameterization of the basic equations governing the mutual competition between drugs binding to DNA and incorporates as partial cases various similar models existing in the literature. The generality of the model results from strict accounting of the statistical effects of the binding of the drug and interceptor with DNA according to the McGhee-von Hippel formalism, and to the strict treatment of hetero-association between the drug and interceptor, which includes formation of all possible types of self- and hetero-complexes in solution. Indirect experimental evidence is provided for the importance of the protector mechanism in drug-caffeine-DNA systems, which is sometimes ignored in the literature because of the small magnitude of the CAF-DNA binding constant.

Quantification of the interceptor action of caffeine on the in vitro biological effect of the anti-tumour agent topotecan.

Using published in vitro data on the dependence of the percentage of apoptosis induced by the anti-cancer drug topotecan in a leukaemia cell line on the concentration of added caffeine, and a general model of competitive binding in a system containing two aromatic drugs and DNA, it has been shown to be possible to quantify the relative change in the biological effect just using a set of component concentrations and equilibrium constants of the complexation of the drugs. It is also proposed that a general model of competitive binding and parameterization of that model may potentially be applied to any system of DNA-targeting aromatic drugs under in vitro conditions. The main reasons underpinning the proposal are the general feature of the complexation of aromatic drugs with DNA and their interaction in physiological media via hetero-association.

Complexation of biologically active aromatic compounds with DNA in the presence of theophylline.

(1)H NMR measurements (500 MHz) have been used to determine the equilibrium hetero-association constants of theophylline (THP) with various biologically active aromatic compounds (daunomycin, novantrone, ethidium bromide, proflavine, norfloxacin) and the complexation constants of THP with both single- and double-stranded oligonucleotides in solution. The results provide a quantitative estimation of the effect of THP on the binding of aromatic ligands with DNA, and a determination of the fraction of aromatic ligand removed from DNA on addition of THP.

Electrostatic contribution to the energy of binding of aromatic ligands with DNA.

The method of solution of the nonlinear Poisson-Boltzmann equation was used to calculate electrostatic energy of binding of various aromatic ligands with DNA oligomers of different length. Analysis of the electrostatic contribution was made in terms of a two-step DNA binding process: formation of the intercalation cavity and insertion of the ligand. The total electrostatic energy was also partitioned into components: the energy of atom-atom coulombic interactions and the energy of interaction with surrounding water. The results indicate that electrostatic interactions are, as a whole, unfavorable to the intercalation process and that a correct analysis of structure-energy interrelation for Ligand-DNA interactions should only be accomplished at the level of the components rather than at the level of total electrostatic energy.

Quantitation of the molecular mechanisms of biological synergism in a mixture of DNA-acting aromatic drugs.

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.

On the origin of the decrease in stability of the DNA hairpin d(GCGAAGC) on complexation with aromatic drugs.

Molecular dynamics simulations of drug-DNA complexes have been carried out in order to explain the experimentally observed decrease in thermal stability of the DNA hairpin d(GCGAAGC) on binding the aromatic drug molecules, daunomycin, ethidium bromide, novantrone and proflavine. This complexation behavior is in contrast to the stabilizing effect of the same aromatic drug molecules on DNA duplexes. Analysis of the energy parameters and the hydration properties of the complexes shows that the main factor correlating with the decrease in melting temperatures of the drug-hairpin complexes is the number of water bridges, with a reduction of at least 40% on ligand binding.

Structural basis for the binding affinity of a homologous series of synthetic phenoxazone drugs with DNA: NMR and molecular mechanics analysis.

The molecular basis of the marked structure-activity relationship for a homologous series of DNA-binding phenoxazone drugs (ActII-ActIV) has been investigated by NMR spectroscopy and molecular mechanics. The spatial structures of the complexes between the drugs and a model deoxytetranucleotide, 5'-d(TpGpCpA), have been determined by molecular mechanics methods using homonuclear (1)H-(1)H 2D-NOESY and heteronuclear (1)H-(31)P (HMBC) NMR spectroscopic data. Observed intermolecular NOE contacts and equilibrium binding studies confirm that the binding affinity of the synthetic phenoxazone derivatives with d(TGCA) decreases with an increase in the number of CH(2) groups in the dimethylaminoalkyl side chains, i.e., ActII > ActIII > ActIV, in agreement with the observed biological activity of these compounds. Molecular mechanics calculations of the spatial structures of the intercalated complexes of ActII-ActIV with d(TGCA) indicate that the different binding constants of the phenoxazone derivatives with the DNA oligomer are due to the different degrees of intercalation of the chromophore and the different steric arrangements of aminoalkyl side chains in the minor groove of the tetramer duplex; this results in different distances between the negatively-charged phosphates of the DNA duplex and the terminal positively-charged N(CH(3))(2) groups of the side chains.

Complexation of anthracycline drugs with DNA in the presence of caffeine.

The competitive binding of anthracycline antitumour drugs, [daunomycin (DAU), doxorubicin (DOX) or nogalamycin (NOG)], with caffeine (CAF) to a model DNA oligomer has been investigated by 500 MHz 1H NMR spectroscopy under physiological solution conditions. The method depends on the stepwise analysis of one-component (self-association), two-component (hetero-association and DNA complexation) and three-component interactions, in order to de-convolute the overall binding of the anthracycline antibiotic and CAF to DNA into two competing processes, viz. hetero-association of the antibiotic-CAF ('interceptor' action of CAF) and CAF-DNA complexation ('protector' action of CAF). It is found that the complexation of DAU with DNA in the presence of CAF is mainly affected by the CAF-DNA complexation, whereas the binding of either DOX or NOG to DNA is affected approximately equally by both the CAF-DNA complexation and CAF-antibiotic hetero-association. Quantitative evaluation of the three-component mixture of drug-CAF-DNA has enabled the proportion of the antibiotic displaced from DNA on addition of CAF to be calculated over a large range of CAF concentration, which may provide a quantitative basis for the change in anthracycline-related toxicity on addition of CAF.

[Heteroassociation of antibiotic norfloxacin with aromatic vitamins in aqueous solution].

The heteroassociation of the antibacterial antibiotic norfloxacin with aromatic vitamins nicotineamide and flavin mononucleotide in aqueous solution has been studied by 1H NMR spectroscopy (503 MHz). Equilibrium constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH, deltaS) of the heteroassociation of molecules were determined from the concentration and temperature dependences of chemical shifts of protons of interacting aromatic molecules. An analysis of the results indicates the formation of heterocomplexes between the molecules of the vitamins and norfloxacin, which is caused by stacking interactions between aromatic chromophores and an additional intermolecular hydrogen bond in the norfloxacin-nicotinamide system. Based on the analysis of induced chemical shifts of protons of molecules, the most probable spatial structures 1:1 of norfloxacin-flavin mononucleitide and norfloxacin-nicotinamide heterocomolexes were determined by the methods of molecular modeling using the X-PLOR program.

Outcomes of pancreas transplantation in the United States using cardiac-death donors.

Organs donated after cardiac death (DCD) are used to expand the donor pool. We analyzed the outcomes in the United States of pancreatic transplantation of organs from DCD donors performed between 1993 and 2003. We used the OPTN/UNOS Registry to compare outcomes of primary pancreas allografts from DCD donors and donors after brain death (DBD). The primary endpoints were graft failure and patient death. A national survey regarding the use of DCD donors in pancreas transplantation was conducted among the directors of pancreas transplant centers. Data were obtained on 47 simultaneous pancreas-kidney transplants (SPK) and 10 solitary pancreas transplants from DCD donors and on 2431 SPK and 1607 solitary pancreas transplants from DBD donors. Recipients of a SPK transplants from DCD and DBD donors had equivalent patient and graft survival rates at 1, 3 and 5 years. For recipients of SPK transplants, the wait for organs from DCD donors was significantly shorter than that for organs from DBD donors. SPK recipients of organs from DCD donors had longer hospital stays than did recipients of organs from DBD donors. With renal allografts, the incidence of delayed graft function was almost four times higher with organs from DCD donors than with organs from DBD donors. Selective use of organs from DCD donors is safe for pancreas transplantation.

[Interaction between aromatic antibiotics and vitamins: 1H NMR analysis of the heteroassociation of nicotinamide and anthracycline antitumor antibiotics].

The interaction between anthracycline antitumor antibiotics daunomycin and novatrone and the vitamin nicotinamide has been investigated by one- and two-dimensional 1H NMR spectroscopy (500 MHz). Due to significant differences in structures of the chromophores of interacting molecules, a two-site heteroassociation model has been developed, allowing the arrangement of one and two nicotinamide molecules on the chromophore of the antibiotic. The equilibrium association constant, thermodynamical parameters (deltaH, deltaS) of the heteroassociation of nicotinamide with daunomycin and novatrone and the induced proton chemical shifts in the heterocomplexes have been determined from the concentration and temperature dependences of proton chemical shifts of interacting molecules. The most favorable structures of 1:1 nicotinamide--daunomycin and nicotinamide-novatrone heteroassociation complexes have been determined using both the molecular mechanics methods (X-PLOR software) and the calculated values of induced proton chemical shifts. Analysis of the results obtained allows one to conclude that two nicotinamide molecules cannot simultaneously bind on one side of the chromophore of antibiotic. Heterocomplexes of the vitamin with the antibiotics with a stoichiometry 1:1 are mainly stabilized by the stacking of aromatic chromophores.

Effect of a mixture of caffeine and nicotinamide on the solubility of vitamin (B2) in aqueous solution.

The effect of caffeine (CAF) and nicotinamide (NMD) on the solubility of a vitamin B2 derivative (FMN) has been evaluated for mixtures containing either a single hydrotrope (CAF or NMD) or the two hydrotropes simultaneously. A model for analysis of ternary systems, which takes into account all possible complexes between the molecules, has been developed and tested with experimental NMR data on the three-component mixture FMN-CAF-NMD. The results indicate that special attention should be given to the concentration of a hydrotropic agent used to enhance the solubility of a particular drug. A decrease in the efficacy of solubility of the vitamin on addition of large amounts of hydrotropic agent is expected in the two-component systems due to the increased proportion of self-association of the hydrotrope. It is found that a mixture of two hydrotropic agents leads to an increase in the solubility of the vitamin in three-component compared to the two-component system. Rather than using just one hydrotropic agent, it is proposed that a strategy for optimising the solubility of aromatic drugs is to use a mixture of hydrotropic agents.

Complexation of norfloxacin with DNA in the presence of caffeine.

(1)H NMR spectroscopy (500 MHz) has been used to quantify the complexation of the antibacterial antibiotic Norfloxacin (NOR) with DNA in the presence of Caffeine (CAF). Separate studies have been made for the self-association of NOR, its hetero-association with CAF and complexation with a model self-complementary DNA tetramer, 5'-d(TpGpCpA), in order to determine the equilibrium parameters (induced chemical shifts, association constants, enthalpy and entropy) of the two-component mixtures to aid the analysis of the three-component systems. Investigations of the self-association of NOR and its hetero-association with CAF show that the aggregation of NOR molecules and association with CAF in solution are driven by the stacking of aromatic chromophores. The complexation of NOR with d(TGCA) has been analysed in terms of intercalation with the double-stranded form and non-intercalative binding with the single-stranded form of DNA. Investigations of the competitive binding of NOR and CAF with DNA show that at physiological concentrations of NOR (muM) and CAF (mM) the dominant mechanism influencing the affinity of NOR with DNA is the displacement of bound NOR molecules from DNA due to CAF-DNA complexation (i.e. the protector action of Caffeine).

Complexation of daunomycin with a DNA oligomer in the presence of an aromatic vitamin (B2) determined by NMR spectroscopy.

The effect of simultaneous binding of the anthracycline antibiotic Daunomycin (DAU) and the Vitamin B2 derivative, Flavin-mononucleotide (FMN), with the DNA oligomer, d(TGCA)2, in solution has been investigated quantitatively by 1I-NMR spectroscopy (500 MHz). The equilibrium reaction constants and the thermodynamical parameters (DeltaH, DeltaS) of the hetero-association FMN-DAU and complexation of FMN with d(TGCA)2 have been determined by analysis of the concentration and temperature dependences of chemical shifts of the aromatic protons in terms of a competitive binding model. A criterion for discrimination between hetero-association and DNA complexation has been developed and applied to the analysis of the simultaneous binding of the antibiotic and the vitamin with DNA. Under the conditions of the experiment, it is found that both the hetero-association of FMN with DAU and the complexation of FMN with DNA contribute approximately equally to the decrease of DAU binding with DNA oligomer. Such competitive complexation of aromatic vitamin and drug with DNA could affect the biological activity of such drugs.

[A structural and thermodynamic analysis of novatrone and flavin mononucleotide heteroassociation in aqueous solution by 1H NMR spectroscopy].

A heteroassociation of antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied by one- and two-dimentional 1H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and vitamin. The equilibrium reaction constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH and deltaS) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores. An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solutions, which could result in a modulation of their medical and biological action.

Hetero-association of anticancer antibiotics in aqueous solution: NMR and molecular mechanics analysis.

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.

Pulmonary hypertension in advanced sarcoidosis: epidemiology and clinical characteristics.

Pulmonary hypertension (PH) is a predictor of poor outcome in sarcoidosis. Little is known about the epidemiology of PH in sarcoidosis. The current authors reviewed the records of patients with sarcoidosis listed for lung transplantation in the USA between January 1995 and December 2002. PH was defined as a mean pulmonary artery pressure of >25 mmHg and severe PH as a mean pulmonary artery pressure of > or =40 mmHg. The cohort included 363 patients of whom 73.8% had PH. Neither spirometric testing nor the need for corticosteroids was associated with PH. Subjects with PH required more supplemental oxygen (2.7+/-1.8 L.min(-1) versus 1.6+/-1.4 L.min(-1)). The cardiac index was lower in individuals with PH, whereas the pulmonary capillary wedge pressure was higher. In multivariate analysis, supplemental oxygen remained an independent predictor of PH, whereas the relationship between cardiac index and PH was no longer significant. As a screening test, the need for oxygen had a sensitivity and specificity of 91.8% and 32.6%, respectively. Pulmonary hypertension is common in advanced sarcoidosis. The need for oxygen correlates with pulmonary hypertension. Since pulmonary hypertension is associated with poor outcomes and because simple clinical criteria fail to identify patients with sarcoidosis and pulmonary hypertension, more aggressive screening for this should be considered.

[Distinctive features of the self-association of deoxyhexanucleotide 5'-D(GpCpApTpGpC) in aqueous solution: 1H NMR analysis]. / Osobennosti protsessa samoassotsiatsii molecul dezoksigeksanukleotida 5'-D(GpCpApTpGpC) v vodnom rastvore: 1H-IAMR-analiz.

The self-association of self-complementary deoxyhexanucleotide d(GCATGC) was investigated in aqueous salt solution. Homonuclear 1H NMR correlation spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignments of nonexchangeable protons of the hexamer. The equilibrium reaction constants and thermodynamical parameters of duplex d(GCATGC)2 formation were determined from experimental concentration and temperature dependences of proton chemical shifts of the deoxyhexanucleotide. Distinctive features of the concentration dependences in the range of small concentrations at relatively low temperatures of solution enable one to assume that one single-stranded hexamer sequence forms a compact structure (similar to a hairpin) in aqueous solution. A possible spatial hairpin structure of the hexamer was proposed. Comparative analysis of the experimental and theoretical (using the "nearest neighbor" model) thermodynamical parameters of duplex formation was made.

[1H-NMR analysis of the heteroassociation of caffeine with the antibiotic actinocyl-bis(3-dimethylaminopropylamine) in aqueous solution]. / 1H-IaMP-analiz geteroassotsiatsii kofeina s antibiotikom aktinotsil-bis(3-dimetilaminopropilaminom) v vodnom rastvore.

The heteroassociation of caffeine (CAF) and the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) (ACT) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The equilibrium reaction constants, thermodynamic parameters (delta H and delta S) of ACT heteroassociation with CAF, the limiting values of proton chemical shifts of their molecules in the heteroassociation complex, and the spatial structure of the ACT-CAF complex were determined from the experimental dependences of proton chemical shifts of the aromatic molecules on concentration and temperature. The parameters of CAF heteroassociation with the phenoxazone antibiotic actinomycin D and its synthetic analogue ACT were comparatively analyzed and conclusions were made on the crucial role of stacking interactions of the chromophores of CAF and the phenoxazone antibiotics in the formation of the heterocomplexes in aqueous solution.

[1H-NMR analysis of self association of an antibiotic actinocyl-bis(3-dimethylaminopropylamine) in water solutions]. / 1H-IaMR-analiz samoassotsiatsii antibiotika aktinotsil-bis(3-dimetilaminopropilamina) v vodnom rastvore.

The self-association of the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The two-dimensional homonuclear correlation NMR techniques (TOCSY and ROESY) were used to completely assign all the proton signals of the antibiotic and to quantitatively analyze the mutual arrangement of the antibiotic molecules in their aggregates. The concentration and temperature dependences of proton chemical shifts were used to determine the equilibrium constants and the thermodynamic parameters (delta H and delta S) of the self-association, as well as the limiting values of proton chemical shifts in associates. The experimental results were analyzed using both the indefinite noncooperative and cooperative models of the molecular self-association. The calculated value of the cooperativity coefficient (sigma approximately 1.1) for our synthetic antibiotic confirmed a substantially lower anticooperative effect at the aggregation of its molecules in comparison with that of the antitumor antibiotic actinomycin D (sigma approximately 1.5). We calculated the most favorable structure of the dimeric associate of the synthetic antibiotic in aqueous solution and found that, like in the actinomycin D dimer, the antiparallel orientation of the phenoxazone chromophore planes of interacting molecules is characteristic of its dimer. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.