Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.     

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.     

NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer

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.     

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.     

1H NMR study of the complexation of the quinolone antibiotic norfloxacin with DNA

Complexation of antibiotic norfloxacin (NOR) with DNA fragments 5'-d(TpGpCpA) and 5'-d(CpGpCpG) has been studied in aqueous solution by 1H NMR spectroscopy (500 MHz). Equilibrium parameters of the complexation with single-stranded and duplex forms of DNA oligomer--equilibrium constants, enthalpy and entropy--have been obtained for the first time. Based on the analysis of the complexation parameters as well as induced chemical shifts of the antibiotic protons within different complexes, it was found that NOR binds with the tetramer duplexes mainly by intercalation. The complexation with the single-stranded form may occur either by intercalation and external binding. The site of preferential binding of the antibiotic with DNA duplex is GC site.     

Investigation of the self-association and hetero-association interactions of H-NS and StpA from Enterobacteria

The nucleoid-associated protein H-NS and its paralogue StpA are global regulators of gene expression and form an integral part of the protein scaffold responsible for DNA condensation in Escherichia coli and Salmonella typhimurium . Although protein oligomerization is a requirement for this function, it is not entirely understood how this is accomplished. We address this by reporting on the self-association of H-NS and its hetero-association with StpA. We identify residues 1–77 of H-NS as being necessary and sufficient for high-order association. A multi-technique-based approach was used to measure the effects of salt concentration on the size distribution of H-NS and the thermal stability of H-NS and StpA dimers. The thermal stability of the StpA homodimer is significantly greater than that of H-NS₁₋₇₄. Investigation of the hetero-association of H-NS and StpA proteins suggested that the association of H-NS with StpA is more stable than the self-association of either H-NS or StpA with themselves. This provides a clear understanding of the method of oligomerization of these important proteins in effecting DNA condensation and reveals that the different associative properties of H-NS and StpA allow them to perform distinct, yet complementary roles in the bacterial nucleoid.     

De-intercalation of ethidium bromide and propidium iodine from DNA in the presence of caffeine

Caffeine (CAF) is capable of interacting directly with several genotoxic aromatic ligands by stacking aggregation. Formation of such hetero-complexes may diminish pharmacological activity of these ligands, which is often related to its direct interaction with DNA. To check these interactions we performed three independent series of spectroscopic titrations for each ligand (ethidium bromide, EB, and propidium iodine, PI) according to the following setup: DNA with ligand, ligand with CAF and DNA-ligand mixture with CAF. We analyzed DNA-ligand and ligand-CAF mixtures numerically using well known models: McGhee-von Hippel model for ligand-DNA interactions and thermodynamic-statistical model of mixed association of caffeine with aromatic ligands developed by Zdunek et al. (2000). Based on these models we calculated association constants and concentrations of mixture components using a novel method developed here. Results are in good agreement with parameters calculated in separate experiments and demonstrate de-intercalation of EB and PI molecules from DNA caused by CAF.     

Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and with double stranded DNA

Caffeine (CAF) inhibits the intercalation of acridine orange (AO) into cellular DNA. Optical absorption and fluorescence spectroscopy were employed to determine the molecular interactions of AO with itself, with CAF, and with double stranded herring sperm DNA (dsDNA). AO dimerization was observed at concentrations >2 micromol. The sharp increase in fluorescence (lambda(em)=530 nm) at 5 micromol of AO was attributed to AO multimer formation. From 0.5 to 5.0 micromol, the AO self-association binding constant (K(assoc)) was determined to be 38620 mol(-1), however, the presence of 150 mmol NaCl increased K(assoc) to 118000 mol(-1) attributed to the charge neutralization. The K(assoc) for AO with CAF was confirmed to be 256 mol(-1). K(assoc) for the binding of AO with 20 micromol DNA ranged from, 32000 mol(-1) at 2 micromol AO, to approximately 3700 mol(-1) at 10 micromol AO, in the absence of NaCl. This AO concentration dependency of K(assoc) value with DNA was attributed to AO intercalation into dsDNA at high dsDNA/AO ratios, and electrostatic binding of AO to dsDNA at low AO ratios. The findings provide information used to explain fluorescence intensity values at lambda(em) at 530 nm from studies that combine AO, caffeine, and dsDNA.     

Complexation of the quinolone antibiotic norfloxacin with DNA

¹H-NMR spectroscopy (500 MHz) was used to study the complexation of the antibacterial agent norfloxacin (NOR) with DNA tetramers 5′-d(TpGpCpA) and 5′-d(CpGpCpG) in aqueous solution. For the first time, the equilibrium parameters (equilibrium constants, enthalpy, and entropy) were obtained for NOR binding with single-stranded and duplex DNA tetramers. By analyzing the complexation parameters and the induced proton chemical shifts in NOR in various complexes, the character of NOR binding was identified as intercalation in the case of the duplex tetramers and as intercalation with external binding in the case of single-stranded tetramers. NOR proved to preferentially bind to GC sites in DNA duplexes.     

Modulation of acridine mutagen ICR191 intercalation to DNA by methylxanthines—Analysis with mathematical models

Caffeine (CAF) and other methylxanthines (MTX) may interact directly with several aromatic, intercalating ligands through mixed stacking aggregation. Formation of such stacking hetero-complexes may decrease their free form concentration and, in consequence, diminish their biological activity, which is often related to their direct interaction with DNA. In this paper interactions of acridine mutagen (ICR191) with DNA in the presence of three MTX: caffeine (CAF), pentoxifylline (PTX) and theophylline (TH) are investigated. Several mathematical models are used to calculate all association constant values and every component concentration in each analyzed mixture. Model McGhee–von Hippel is used to analyze ligand–DNA interaction, and model Zdunek et al.—to analyze ligand–MTX interactions. Finally, two distinct mathematical models are employed to analyze three-component mixture containing ligand, MTX and DNA molecules. The first model describes possible interactions of ligand with DNA and MTX, and rejects direct MTX interactions with DNA. The second model describes all interactions mentioned above and, additionally, allows MTX to interact directly with DNA. Results obtained using these models are similar. However, correspondence of theoretical results to experimental data is better for the first model than the second one. In this paper possible interactions of ICR191 with eukaryotic cell chromatin are also analyzed, showing that CAF reduces acridine mutagen potential to interact directly with cell chromatin. Additionally, it is demonstrated that MTX inhibit mutagenic activity of ICR191 in a dose-dependent manner. Furthermore, biological activity of ICR191–MTX mixtures corresponds with concentration of free mutagen form calculated using appropriate mathematical model.     

Complexation of Biologically Active Aromatic Compounds with DNA in the Presence of Theophylline

¹H 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.     

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.     

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.     

Caffeine and other xanthines as cytochemical blockers and removers of heterocyclic DNA intercalators from chromatin

Caffeine (CAF) and other xanthines non-covalently bind with the cationic fluorescent dye acridine orange (AO) and with other heterocyclic mutagens and carcinogens that are known to intercalate into double-stranded DNA (dsDNA). Fluorescence microscopy and spectrofluorometry studies were employed to test the ability of caffeine and certain other methyl substituted xanthines, with different binding affinities for AO, to inhibit and to reverse the intercalation of AO and other heterocyclic agents from intercalation with the DNA of nuclear chromatin of air-dried cells. Results indicated that xanthines with binding affinity for AO greater than 150 m(-1) block the AO molecule in a concentration dependent manner and comply with mass action kinetics. Thus CAF and other xanthines can be used to either inhibit intercalation of AO into nuclear DNA or to remove AO once intercalated into nuclear DNA. The interactions between other planar heterocyclics, xanthines, and nuclear chromatin dsDNA were also found to be non-covalent. Studies are needed to determine the ability of CAF and other xanthines to block and/or remove polyaromatic hydrocarbon (PAH) intercalators from the DNA of living cells.     

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.     

Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein.

The ordered copolymerization of viral proteins to form the herpes simplex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes. In common with capsid assembly in double-stranded DNA bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions. These regions also contain conserved sequences which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.     

E. coli trp repressor forms a domain-swapped array in aqueous alcohol

The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 A X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol.