Differential scanning calorimetry of antitumor antibiotics-plasmid DNA interaction

Differential scanning calorimetry (DSC) can detect stepwise melting of plasmid DNA along the molecular chain with high resolution. This method was applied to study interaction of some antitumor antibiotics with the plasmid pJL3-TB5 DNA (5277 base-pairs in length). Analysis of DSC curves of the plasmid DNA in the presence of, for example, adriamycin, an antitumor antibiotics of anthracycline group, together with theoretical analysis of the DNA melting curves obtained by calculation from the entire base sequence, led to the conclusion that adriamycin bound preferentially to the four particular regions with high G + C content. The DSC method would thus be useful for the study of properties of drugs which bind to DNA.     

Modulation of platinum antitumor drug binding to DNA by linked and free intercalators

We report the DNA binding site preferences of the novel molecule AO-Pt, in which the anticancer drug dichloro(ethylenediamine)platinum(II) is linked by a hexamethylene chain to acridine orange. The sequence specificity of platinum binding was mapped by exonuclease III digestion of 165 and 335 base pair restriction fragments from pBR322 DNA. Parallel studies were carried out with the unmodified anticancer drugs cis-diamminedichloroplatinum(II) (cis-DDP) and dichloro(ethylenediamine)platinum(II), [Pt(en)Cl2]. Oligo(dG) sequences are the most prevalent binding sites for AO-Pt, with secondary binding occurring mainly at d(AG) sites. cis-DDP and [Pt(en)Cl2] bind less readily to the secondary sequences, with cis-DDP showing greater binding site selectivity than [Pt(en)Cl2]. The DNA intercalator ethidium bromide promotes binding of [Pt(en)Cl2] and cis-DDP to many sites containing d(CGG) and, to a lesser extent, d(AG) sequences. AO-Pt exhibits enhanced binding to these sequences without the need for an external intercalator. Unlinked acridine orange, however, does not promote binding of [Pt(en)Cl2] and cis-DDP to d(CGG) and d(AG) sequences. These results are discussed in terms of the sequence preferences, stereochemistry, and relative residence times of the intercalators at their DNA binding sites. By modulating local structure in a sequence-dependent manner, both linked and, in the case of ethidium, free intercalators can influence the regioselectivity of covalent modification of DNA by platinum antitumor drugs.     

A scanning calorimetric study of natural DNA and antitumoral anthracycline antibiotic-DNA complexes

Thermal denaturation of natural DNA in the absence and presence of antitumor anthracycline antibiotics has been studied by adiabatic differential scanning calorimetry. The helix-coil transition is operationally irreversible as measured by DSC. Both the melting temperature and the overall molar transition enthalpy of the DNA samples was dependent on the percentage of GC base pairs. Calorimetric traces of anthracycline-DNA complexes have qualitatively similar features and the significance of this characteristic is discussed. The unsaturated drug-DNA complex melts through complex thermal transitions with one broad endotherm in the same temperature region as free DNA and the other at a higher temperature which is rf (mol ligand per mol DNA in base pairs) value dependent. Antibiotic binding at concentrations close to saturating conditions (rf = 0.2) reverts the melting range to a value near to its original one and increases the thermal stability of the duplex structure by around 30 degrees C. In addition, the calorimetric enthalpy is increased by between 64% and 150%, depending on which ligand was used.     

Anthracycline antibiotic, beromycin. The formation of complexes with DNA and the suppresion of nucleic acid biosynthesis]

Beromycin, an antitumor anthracycline antibiotic formed in vitro complexes with native and denaturated DNA and ribosomal RNA. Beromycin had a comparatively low constant of DNA binding and to a less extent increased the melting temperature and viscosity of DNA than the other anthracycline antibiotics. A peculiar property of beromycin was very slow binding with DNA, the complex formation was completed in 60 minutes. Beromycin had a selective inhibitory effect on synthesis of nucleic acids in bacterial and tumor cells. Beromycin inhibited synthesis of RNA in the DNA-dependent RNA-polymerase reaction when both the native and denaturated DNA were used as the template. A lower biological activity of beromycin as compared to the other anthracycline antibiotics, such as rubomycin or carminomycin may be explained by lower affinity of this antibiotic to DNA.     

Relationship between helix-coil transition and gene organization of ColE1 plasmid DNA. Differential scanning calorimetric and theoretical studies

Differential scanning calorimetry (DSC) was carried out to analyze the transition of helix to coil state of DNA, using ColE1 DNA molecules digested with EcoRI. The DSC curves showed multimodal transition, consisting of nine to 11 peaks over a temperature range, depending on the ionic strength of the DNA solution. These DSC curves were essentially in good agreement with the optical melting curves of ColE1 DNA. The theoretical melting profiles of ColE1 DNA were predicted from calculations based on the helix-coil transition theory and the nucleotide sequence of the DNA. These profiles resembled the DSC curves and made it possible to assign the peaks seen in the DSC curves to the helix-coil transition of particular regions of the nucleotide sequence of ColE1. The helix-coil transition of each of the small genes gave rise to a single peak in the DSC curve, while the helix-coil transition of large genes contributed to two or more peaks in the DSC curve. This multimodal transition within a single coding region might correspond to the melting of individual segments encoding the different domains of the proteins. The helix-coil transition at the specific sites including ori, the origin of replication of ColE1, was also found to occur in a particular temperature range. DSC, a simple method, is thus useful for analyzing the multimodal helix-coil transition of DNA, and for providing information on the genetic organization of DNA.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by the pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in live (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: the anthracycline anticancer drug doxorubicin and the nucleic acid dyes ethidium bromide (E) and 4',6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, a competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in native (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: anthracycline anticancer drug doxorubicin and nucleic acid dyes ethidium bromide and 4′,6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

Intercalation and induction of strand breaks by adriamycin and daunomycin: a study with human genomic DNA

The anticancer drugs Adriamycin (ADR) and Daunomycin (DNM) of the anthracycline family are effective in treating a variety of cancers. Although their interactions with other cellular targets may play a role in the selective cytotoxicity of these drugs, it is generally believed that intercalation with DNA is essential for their activity. However, a relationship has not yet been established between intercalation and cellular processes leading to cytotoxicity. The present study was designed to investigate the relationship, if any, between intercalation and DNA strand breaks. ADR and DNM were observed to be strong intercalators of human genomic DNA by absorption and fluorimetric methods that were further substantiated by rise in thermal melting temperature. DNM is the better intercalator of the two, which is also evident from circular dichroic spectral changes. DNA strand breaks, considered to be an index of genotoxicity, was assayed by single cell gel electrophoresis (SCGE; comet assay). ADR and DNM induced equivalent genotoxicity in normal human lymphocytes at a clinically used dose, which was observed to be independent of intercalation efficiency though positively correlated to yield of reactive oxygen species.     

The influence of intercalator binding on DNA triplex stability: correlation with effects on A-tract duplex structure

The triplex form of DNA is of interest because of a possible biological role as well as the potential therapeutic use of this structure. In this paper the stabilizing effects of two intercalating drugs, ethidium and the quinoxaline derivative 9-OH-B220, on DNA triplexes have been studied by thermal denaturation measurements. The corresponding duplex structures of the DNA triplex systems investigated are either A-tract or normal B-DNA. The largest increases in the triplex melting temperatures caused by the intercalators were found for sequences having A-tract duplex structures. Inserting a single base pair with an N2-amino group in the minor groove, e.g. a G-C pair, breaks up the A-tract duplex structure and also reduces the stabilizing effect of the drugs on the triplex melting temperatures. The large drug-induced increase in triplex melting temperature for complexes having an original duplex A-tract structure is correlated with a low initial melting point of the triplex, not with the triplex being unusually stable in the presence of the drug. Hence, we conclude that the large thermal stabilizing effect exhibited by ethidium and 9-OH-B220 on dTn.dAn-dTn triplexes is partly caused by the intercalators breaking up the intrinsic A-tract structure of the underlying duplex.     

The interaction of platinum and anthrapyrazole antitumor drugs with mouse thymocytes studied by terbium fluorescence

The fluorescent lanthanide, terbium has been employed to study the effect of a series of platinum and anthracycline drugs and an anthrapyrazole (oxanthrazole) on terbium binding to mouse thymocytes. It was observed that terbium fluorescence intensity was markedly decreased by two platinum drugs (cis-dichlorodiammine platinum(II) (cis-DDP) and cis-dichloro-trans-dihydroxybis(isopropylammine) platinum(IV) (CHIP)) and an anthrapyrazole (oxanthrazole), but that the lipophylic derivative cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) had a small but significant effect and the anthracyclines (at low concentrations) had no effect. The calcium channel blocker, verapamil also had no effect. The effect of cis-DDP was markedly dependent on ionic strength in contradistinction to CHIP. The decreases in phosphorescence decay produced by cis-DDP also showed a marked dependence on ionic strength. It is proposed that cis-DDP interacts with the membrane primarily by a charge effect, but that CHIP may produce a conformational change in the membrane. These data are interesting, since the lipophylic platinum drugs (CHIP and CBDCA) also increased significantly the amount of bound intracellular calcium, but all the drugs decreased mitogen-stimulated calcium uptake into mouse thymocytes.     

Calmodulin inhibitor trifluoperazine selectively enhances cytotoxic effects of strong vs weak DNA binding antitumor drugs in doxorubicin-resistant P388 mouse leukemia cells

Doxorubicin-resistant P388 mouse leukemia cells are cross-resistant to anthracycline and non-anthracycline DNA intercalators as well as to natural and semisynthetic anthracyclines which bind weakly or not at all to DNA. In the presence of a non-lethal concentration of 5 microM trifluoperazine cytotoxic effects of the strong DNA binding drugs actinomycin-D, mitoxantrone and m-AMSA were enhanced less than 2 fold in doxorubicin-sensitive cells and up to 50 fold in doxorubicin-resistant cells. Additionally, trifluoperazine induced a greater than 2-fold enhancement in the cytotoxic effects (but not accumulation and retention) of the strong DNA binder N,N-dimethyladriamycin-14-valerate only in doxorubicin resistant cells. In contrast, cell kill, drug accumulation and retention in P388/S and P388/DOX cells treated with the weak DNA binders N-benzyl-adriamycin-14-valerate and 7(R)-O-methylnogarol, and DNA-nonbinding N,N-dibenzyldaunorubicin was similar with or without trifluoperazine treatment. The study demonstrates that the calmodulin inhibitor trifluoperazine induces a specific and marked enhancement in the cytotoxic effects of strong vs weak DNA binding antitumor drugs in doxorubicin-resistant cells.     

A simple model for predicting the free energy of binding between anthracycline antibiotics and DNA

A theoretical model for predicting the free energy of binding between anthracycline antibiotics and DNA was developed using the electron density functional (DFT) and molecular mechanics (MM) methods. Partial DFT-ESP charges were used in calculating the MM binding energies for complexes formed between anthracycline antibiotics and oligodeoxynucleotides. These energies were then compared with experimental binding free energies. The good correlation between the experimental and theoretical energies allowed us to propose a model for predicting the binding free energy for derivatives of anthracycline antibiotics and for quickly screening new anthracycline derivatives.     

Energetic and conformational contributions to the stability of Okazaki fragments

A combination of spectroscopic and calorimetric techniques was used to determine complete thermodynamic profiles accompanying the folding of a model Okazaki fragment with sequence 5'-r(gagga)d(ATCTTTG)-3'/5'-d(CAAAGATTCCTC)-3' and control DNA (with and without thymidine substitutions for uridine), RNA, and hybrid duplexes. Circular dichroism spectroscopy indicated that all DNA duplexes are in the B conformation, the RNA and hybrid duplexes are in the A conformation, and the Okazaki fragment exhibits a spectrum between the A and B conformations. Ultraviolet and differential scanning calorimetry melting experiments reveal that all duplexes unfold in two-state transitions with thermal stabilities that follow the order RNA > OKA > DNA (with thymidines) > hybrids > DNA (with uridines). The dependence of the transition temperature on salt concentration yielded counterion releases in the following order: DNA (with thymidines) > RNA > DNA (with uridines) > OKA > hybrids. Thus, Okazaki fragments have a conformation and charge density between those of its components DNA and hybrid segments. However, the presence of the RNA-DNA/DNA junction confers on them higher stabilities than their component hybrid and DNA segments. The binding of intercalators to an Okazaki hairpin of sequence 5'-r(gc)d(GCU5GCGC)-3' and to its control DNA hairpin has also been studied. The results show that the binding of intercalators to Okazaki fragments is accompanied with higher heats and lower binding affinities, compared with DNA duplexes. This suggests that the presence of an RNA/DNA junction yields a larger surface contact to interact with the phenanthroline ring of the intercalators, which may lead to a larger disruption of the flexible flanking bases of the junction. The overall results suggest that the presence of this junction stabilizes Okazaki fragments and provides a structural feature that can be exploited in the design of drugs to specifically target these molecules.     

Interaction between second generation anthracyclines and DNA in the nucleosomal structure.

The interaction between three anthracycline antibiotics of second generation (9-deoxydoxorubicin, 9-DAM, 4-demethoxydaunorubicin, 4-DDM, 4'-deoxydoxorubicin, 4'-DAM) and DNA in the nucleosomal structure was investigated using fluorescence and circular dichroism techniques. The thermodynamic parameters of the binding process were obtained at different ionic strength and temperature conditions, thus allowing the calculation of the electrostatic contribution to the free energy and the enthalpy of the process. The same measurements were performed on linear double stranded DNA for comparison. The parent compounds adriamycin and daunomycin were additionally considered. Although the examined drugs greatly vary in biological activity, their binding parameters are only slightly different. Like the parent compounds, 9-DAM, 4-DDM and 4'-DAM exhibit preference for isolated regions of the polynucleotide rather than for nucleosomes. This fact suggests a non-homogeneous distribution of the antibiotics in vivo. The enthalpy values are remarkably lower than the ones characterizing the interaction of adriamycin and daunomycin to DNA. According to CD spectra, all derivatives, but 4-DDM, intercalate into nucleosomal or free DNA in a manner similar to the first generation compounds, namely with the chromophore perpendicular to the hydrogen bonds between the bases. The demethoxy compound, on the other hand, seems to be able to insert its planar moiety in different orientations, which are related to the structure of the nucleic acid being examined. The lack of the methoxy group in the intercalating part of the molecule appears to be responsible for this behaviour. As far as biological activity is concerned, our findings indicate a qualitative correlation between cell cytotoxicity and ability of interaction with nucleosomes at physiological conditions, rather than with free DNA. The modified binding stereochemistry of 4-DDM could play an additive role in modulating the pharmacological effectiveness of the above compounds.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Ligand self-association at the surface of liposomes: A complication during equilibrium-binding studies

Daunomycin and carminomycin, two anthracycline antibiotics known to bind phospholipid bilayers, appear to self-associate at the surface of liposomes at high bound drug/lipid ratios (r). Fluorescence intensity, lifetime, and anisotropy measurements have been used to monitor the equilibrium binding of these drugs to small unilamellar solid-phase dipalmitoylphosphatidylcholine vesicles. Association of an anthracycline with excess liposome (low r) resulted in an increase in both the observed intensity and the fluorescence lifetime. At low vesicle concentrations (high r), a decrease in the total emission intensity was observed which was not paralleled by the excited-state lifetime. The data from these experiments are consistent with the formation of nonfluorescent anthracycline complexes at the surface of liposomes. Such ligand self-association is a potential complication in any studies on the interaction of amphipathic molecules with liposomes conducted at high r values. Because ligand self-association limits the collection of binding data over certain concentration ranges, this consequently results in greater uncertainty in the determination of the maximum value of r (n) in equilibrium binding studies.     

Analysis of the effects of antibiotics on the structure of nucleosome core particles determined by DNAase I cleavage

Patterns of sensitivity to DNAase I cleavage have been analysed in order to investigate the effects of anti-tumour antibiotics and related drugs on nucleosome core particles containing different DNA restriction fragments. In this article, we review the experimental results which show that after controlled digestion of defined-sequence core particles, new cleavage products appear in the enzyme digestion patterns which lie approximately mid-way between the strong bands characteristic of native nucleosome core particles. The effects of the antibiotics, which include bis-intercalators as well as minor groove-binding ligands (but not monofunctional intercalators), are explained in terms of an induced change in rotational setting (phasing) of the core DNA. The new rotational positioning of DNA induced by antibiotic binding appears to be almost independent of DNA sequence, although some differences can be observed with the various pieces of DNA, most likely reflecting the exact number and disposition of antibiotic binding sites.     

Abrogation of the selectivity of adriamycin for negatively-charged phospholipids by 14-valerate side chain substitution

The impressive affinity of Adriamycin and related anthracycline antibiotics for negatively-charged phospholipids has been implicated in the mechanism of the cardiac toxicity of these drugs. In this report we employ the method of fluorescence anisotropy titration to examine the manner in which 14-valerate side chain substitution modulates anthracycline drug associations with electroneutral vesicles composed of dimyristoyl phosphatidylcholine as well as negatively-charged vesicles composed of dimyristoyl phosphatidylglycerol or a binary mixture of dimyristoyl phosphatidylcholine and cardiolipin. Equilibrium binding data gathered on several anthracycline analogs indicate that incorporation of a hydrophobic valerate side chain abolished the high levels of preferential drug binding to negatively-charged membranes. Thus, we propose that 14-O-acyl substitution may prove to be a useful synthetic modification to prevent the selective accumulation of positively-charged anthracyclines in tissues or membrane domains rich in negatively-charged lipid.     

Interaction of unfused tricyclic aromatic cations with DNA: a new class of intercalators

Unfused tricyclic aromatic ring systems 1-6 with one or two cationic side chains have been synthesized and their interactions with DNA and synthetic polymers probed with a variety of techniques. Molecular mechanics calculations indicate that the torsional angle between ring planes in the minimum energy conformation of the tricyclic molecules can range from 0 degree to as high as 50 degrees depending on the type of rings and substituents. Viscometric titrations with linear and supercoiled DNA, linear dichroism, and NMR studies indicated that all compounds with torsional angles of approximately 20 degrees or less bind to DNA by intercalation. The more highly twisted intercalators caused significant perturbation of DNA structure. Unfused intercalators with twist angles of approximately 20 degrees have reduced binding constants, suggesting that they could not form an optimum interaction with the DNA base pairs. Unfused intercalators with twist less than 20 degrees formed strong complexes with DNA. The structures of these unfused intercalators are more analogous to typical groove-binding molecules, and an analysis of their interaction with DNA provides a better understanding of the subtle differences between intercalation and groove-binding modes for aromatic cations. The results indicate that intercalation and groove-binding modes should be viewed as two potential wells on a continuous energy surface. The results also suggest design strategies for intercalators that can optimally complement DNA base pair propeller twist or that can induce bends in DNA at the intercalation site.     

Unprecedented dual binding behaviour of acridine group of dye: a combined experimental and theoretical investigation for the development of anticancer chemotherapeutic agents

Acridine group of dyes are well known in the field of development of probes for nucleic acid structure and conformational determination because of their relevance in the development of novel chemotherapeutic agents, footprinting agents and for gene manipulation in biotechnology and medicine. Here, we report the interaction of 9-N,N-dimethylaniline decahydroacridinedione (DMAADD), a new class of dye molecule with calf thymus DNA (CT-DNA) which has been studied extensively by means of traditional experimental and theoretical techniques. The changes in the base stacking of CT-DNA upon the binding of DMAADD are reflected in the circular dichroic (CD) spectral studies. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in presence of DNA was quenched by the addition of DMAADD indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (3.3+/-0.2)x10(5) M(-1). This competitive binding study and further fluorescence experiments reveal that DMAADD is a moderate binder of CT-DNA, while viscosity measurements show that the mode of binding is partial intercalation. Generally, one would expect increase in the melting temperature (T(m)) of DNA in presence of intercalators. Interestingly, an unusual decrease in melting temperature (DeltaT(m) of -4+/-0.2 degrees C) of DNA by the addition of DMAADD was observed. From our knowledge such a decreasing trend in melting point was not reported before for all the possible modes of binding. Molecular modeling gave the pictorial view of the binding model which clearly shows that of the various mode of binding, the dye prefers the major groove binding to the sites rich in GC residues and to the sites rich in AT residues it prefers intercalation mode of binding either through major or minor groove with the inclusion of the N,N-dimethylaniline (DMA) group inside the double helix which has been stacked in between the bases, under physiological relevant pH of 7.5.