DNA intercalation by quinacrine and methylene blue: a comparative binding and thermodynamic characterization study

There is compelling evidence that cellular DNA is the target of many anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA-binding agents, quinacrine (QNA) and methylene blue (MB), with calf thymus (CT) DNA. The study revealed noncooperative binding phenomena for both the drugs to DNA with an affinity one order higher for QNA compared to MB as observed from diverse techniques, but both bindings obeyed neighbor exclusion principle. The data of the salt dependence of QNA and MB from the plot of log K versus log [Na+] revealed a slope of 1.06 and 0.93 consistent with the values predicted by theories for the binding of monovalent cations, and have been analyzed for contributions from polyelectrolytic and nonpolyelectrolytic forces. The binding of both drugs was further characterized by strong stabilization of DNA against thermal strand separation in both optical melting and differential scanning calorimetry studies. The binding data analyzed from the thermal denaturation and from isothermal titration calorimetry (ITC) were in close proximity to those obtained from spectral titration data. ITC results revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. The heat capacity changes obtained from temperature dependence of enthalpy indicated -146 and -78 cal/(mol.K), respectively, for the binding of QNA and MB to CT DNA. Circular dichroism study further characterized the structural changes on DNA upon intercalation of these molecules. Molecular aspects of interaction of these molecules to DNA are discussed.     

Elsamicin A binding to DNA. A comparative thermodynamic characterization

The antitumor drug elsamicin A contains a coumarin-related chartarin chromophore that intercalates into DNA. It differs from other related molecules in its disaccharide moiety, which bears an amino sugar. Its binding to DNA was analyzed using isothermal titration calorimetry and UV thermal denaturation, and characterized thermodynamically. For the association of elsamicin A with DNA we found DeltaG degrees = -8.6 kcal mol(-1), DeltaH = -10.4 kcal mol(-1), DeltaS = -6.1 cal mol(-1) K(-1), and Kobs = 2.8(+/- 0.2) x 10(6) M(-1) at 20 degrees C in 18 mM Na+. The contributions to the free energy of binding that lead to the DNA-elsamicin complex are compared with the binding to DNA of chartreusin, another chartarin-containing drug. The results are discussed in terms of the contributions of the disaccharide moieties into the strength of binding.     

Studies on DNA binding of caffeine and derivatives: evidence of intercalation by DNA-unwinding experiments

Caffeine and derivatives are compounds with pleiotropic effects on the genetic material which are supposed to originate from drugs binding to DNA. Here we show, by using two different topological methods, that methylated oxypurines, at biologically relevant concentrations, unwind DNA in a fashion similar to known intercalators. Methylated oxypurines could be ranked by decreasing unwinding potency: 8-methoxycaffeine greater than 8-ethoxycaffeine greater than 8-chlorocaffeine greater than caffeine greater than theophylline. These findings confirm, with a different assay, interaction of caffeine with DNA and add additional support to an intercalative mode of binding of these drugs to DNA.     

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.     

Porphyrin intercalation and non-specific 'edge on' outside binding to natural DNA

A theoretical two-mode binding model for porphyrin binding to natural DNA is presented. One of the binding modes is assumed to be base sequence specific with binding sites n base-pairs long. The other binding mode has binding sites which consist of only one base-pair and can involve cooperativity. The model fits satisfactorily to data for H2TMPyP-4, Cu(II)TMPyP-3 and Cu(II)TMPyP-4 binding to calf thymus DNA in both a high (mu congruent to 1.0 M) and a low (mu congruent to 0.2 M) ionic strength buffer. The results show that the fraction of porphyrin bound in the non-specific mode reaches a maximum at certain input DNA to porphyrin concentrations ratios. The value of this maximum decreased, and its position shifted to higher DNA to porphyrin concentration ratios for binding in the high ionic strength buffer. The value of the cooperativity parameter obtained through the fitting process suggests that the non-specific binding is positively cooperative. The results are compared with the data analysed using other techniques.     

Molecular dynamics of structural transitions and intercalation in DNA

The large-scale flexibility of DNA and the intercalation of actinomycin D have been studied by computer simulation using molecular dynamics. The stretching and unwinding of B and Z forms of DNA and intercalation in B-DNA were examined through molecular dynamics simulations, and the energetics of transitions were calculated by the conformational energy minimization method. The principal results of this research are as follows: (1) A dynamic conformational pathway is presented for longitudinal stretching and unwinding of the double helix to open an intercalation site. (2) Large-scale transitions are possible in both B and Z forms of DNA through a conformationally allowed kinetic pathway. (3) The stretching and untwisting of a 5′(CG)3′ step is energetically more favorable than for a GC step in B-DNA. (4) The formation of an adjacent second cavity in B-DNA requires larger energy than the formation of the first cavity, affirming the neighbor-exclusion principle of intercalation. (5) Docking an intercalated actinomycin D in the stretched structure is shown to be geometrically and energetically feasible.     

Aryl substituted ruthenium bis-terpyridine complexes: intercalation and groove binding with DNA

The non-covalent interaction of five novel ruthenium(II) bis-terpyridine complexes with calf thymus DNA and, where appropriate, with poly[d(G-C)](2) and poly[d(A-T)](2) is described. Each complex is functionalised with aryl tail groups in the 4' position of the terpyridine ligands ((i) 9-anthracenyl, (ii) 4,4'-biphenyl, (iii) beta-naphthyl, (iv) 9-phenanthrenyl, and (v) 1-pyrenyl). Circular dichroism and linear dichroism show that the binding of three of the complexes (phenanthrenyl, anthracenyl and pyrenyl) at low metal complex concentration is dominated by intercalation of the aryl tail groups between the DNA bases. The complex with the biphenyl tail predominantly exhibits groove binding with no significant tail intercalation. The naphthyl derivative binds both by intercalation and a non-intercalative mode even at low metal complex concentrations. At high metal complex concentrations, aggregation of the complexes on the DNA is observed. Resonance light scattering indicates that the aggregates are of low nuclearity along the groove.     

HU binding to DNA: evidence for multiple complex formation and DNA bending

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.     

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding.

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA. At low to moderate [K(+)] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size. In this [K(+)] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K(+)]. Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K(+)]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K(+)]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K(+)] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.     

Molecular recognition and binding of a GC site-avoiding thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: 1H-NMR evidence for thiazole intercalation

The structural and dynamic aspects of the interaction of the thiazole containing lexitropsin (1) with an oligodeoxyribonucleotide were studied by high field 1H-NMR spectroscopy. Complete assignment of the 1H-NMR resonances of lexitropsin 1 was accomplished by 2D-NMR techniques. The complexation-induced chemical shifts and NOE cross peaks in the NOESY map of the 1:1 complex of lexitropsin (1) and d-[CGCAATTGCG]2 reveal that the thiazole ring of the lexitropsin (1) intercalates between dA4.A5 bases and the rest of the ligand resides in the minor groove of the AT rich core of decamer, thus occupying the 5'-AATT sequence on the DNA. Intercalation of the thiazole moiety of the drug has been detected by the presence of intermolecular NOEs both in the major and the minor groove of the decamer helix. The absence of intranucleotide NOEs between base protons and H1'/H2' protons suggested local unwinding of the binding site on the DNA. From COSY and NOESY methods of 2D-NMR, it was established that the N-formyl (amino) terminus of the thiazole lexitropsin (1) is projecting into the major groove towards A5H8 while the amidinium terminus lies in the minor groove towards the T7G8 base pairs of the opposite strand. The expected intranucleotide NOEs confirmed that the decadeoxyribonucleotide in the 1:1 complex exists in a right handed B-conformation. The presence of exchange signals along the binding site 5'-AATT indicated an exchange of the bound drug process wherein the rate of exchange between the two equivalent sites was estimated to be congruent to 130 s-1 at 30 degrees C and with delta G degrees of 62.4 kJ mol-1. Force field and Pi calculations permitted a rationalization of the experimentally observed binding mode in terms of preferred conformation of the ligand and repeat length in lexitropsins compared with the DNA receptor.     

Methylene blue binding to DNA with alternating AT base sequence: minor groove binding is favored over intercalation

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5'-YpR-3' intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute-solvent interactions, which are assumed to be nearly constant for the compared complexes of MB with DNA, seem to be justified by the results.     

Interaction of DNA with a porphyrin ligand: evidence for intercalation.

The porphyrin photosensitizer, meso-Tetra (4-N-methylpyridyl) porphine tetraperchlorate binds to calf thymus DNA by intercalation and by external electrostatic association. This was concluded from the results of measruements involving Scatchard analysis, viscometry, thermal denaturation, and circular dichroism.     

A detailed thermodynamic profile of cyclopentyl and isopropyl derivatives binding to CK2 kinase

The detailed understanding of the molecular features of a ligand binding to a target protein, facilitates the successful design of potent and selective inhibitors. We present a case study of ATP-competitive kinase inhibitors that include a pyradine moiety. These compounds have similar chemical structure, except for distinct terminal hydrophobic cyclopentyl or isopropyl groups, and block kinase activity of casein kinase 2 subunit α (CK2α), which is a target for several diseases, such as cancer and glomerulonephritis. Although these compounds display similar inhibitory potency against CK2α, the crystal structures reveal that the cyclopentyl derivative gains more favorable interactions compared with the isopropyl derivative, because of the additional ethylene moiety. The structural observations and biological data are consistent with the thermodynamic profiles of these inhibitors in binding to CK2α, revealing that the enthalpic advantage of the cyclopentyl derivative is accompanied with a lower entropic loss. Computational analyses indicated that the relative enthalpic gain of the cyclopentyl derivative arises from an enhancement of a wide range of van der Waals interactions from the whole complex. Conversely, the relative entropy loss of the cyclopentyl derivative arises from a decrease in the molecular fluctuation and higher conformational restriction in the active site of CK2α. These structural insights, in combination with thermodynamic and computational observations, should be helpful in developing potent and selective CK2α inhibitors.     

A comparative study of estramustine and pregnenolone binding to prostatic binding protein: evidence for subunit cooperativity

The binding of estramustine, a nitrogen mustard derivative of oestradiol to purified rat prostatic binding protein was studied as a test for a possible identity between this protein and the very similar estramustine-binding protein, described by Forsgren et al. In accordance with this hypothesis estramustine binds to purified prostatic binding protein with a high affinity (2.5 X 10(7)M-1). This affinity markedly exceeds the affinity of pregnenolone for this protein (0.9 X 10(6)M-1) or for a complex of prostatic binding protein, with prostatic proline-rich polypeptide, (4.7 X 10(6)M-1). In competition experiments estramustine completely suppresses the binding of [3H]pregnenolone, whereas the binding of [3H]estramustine is only partially suppressed by pregnenolone, even at high concentrations. Prostatic binding protein was separated in its F- and S-subunit by DEAE-Sepharose chromatography performed in the presence of 8 M urea. Only the S-subunit, most probably in its dimer form, displays marked estramustine and pregnenolone binding, with affinities of respectively 3.7 and 1.2 X 10(6)M-1. Recombination of both subunits results in a strong increase of estramustine binding, but not of pregnenolone binding.     

DNA binding of tilorone: 1H NMR and calorimetric studies of the intercalation

The fluorene derivative tilorone has received great attention as a DNA intercalator and has been widely recognized as an inducer of interferon. The biological activity of tilorone is known to be related to its binding mode with DNA; however, few structural and thermodynamic studies have elaborated on this issue. This paper presents two-dimensional (2-D) NMR and isothermal titration calorimetry (ITC) for the tilorone/DNA complex, coupled with circular dichroism (CD) spectroscopy and viscosity measurements. NMR investigation suggests that tilorone binds to DNA through intercalation, showing greater affinity for insertion between AT base pairs than between CG pairs. CD spectral changes were observed for T/B (tilorone/DNA base pair molar ratio) ratios greater than the stoichiometric ratio generally expected for intercalators (i.e., T/B = 0.5, according to the neighbor-exclusion principle). However, there was a clear plateau in the CD intensity between T/B < 0.35 and T/B > 0.45. From comparison with NMR and other measurements, we postulate that CD changes below the plateau should be related to the intercalation and the latter to electrostatic interactions and nonspecific bindings. ITC data showed that DeltaH < -TDeltaS < 0, which indicated that tilorone/DNA binding is enthalpy controlled. The magnitude of Kb (the binding constant) was of the same order as that of ethidium bromide. The stoichiometric number, obtained from ITC, CD, and UV data, implied a relatively smaller value (0.28-0.35) than that of the neighbor-exclusion principle. This is because side chains located in the groove disrupt further intercalation to the adjacent sites.