Drug recognition of DNA. Proposal for GC minor groove specific ligands: vinylexins

In a previous publication in this journal we have proposed an isolexin-like prototype of a GC minor groove specific ligand. The present paper is devoted to refinements of this prototype (increase in specificity and in DNA binding energy). It is shown that only a very limited improvement can be obtained by increasing the proton accepting capabilities of the heteroaromatic ring systems of the prototype, although these rings interact directly with the proton donating NH2 group of guanine. On the other hand a significant increase both in GC specificity and in DNA binding energy is obtained by replacing the NH linkers of the isolexin by C = C double bonds (yielding what we term "vinylexins"). Specificity is still largely conserved and the DNA binding energy is significantly increased in monocationic vinylexins, which should thus be efficient GC minor groove specific ligands. The outstanding importance for the GC specificity of the C = C linkers is evidenced by the disappearance of this specificity when these linkers are replaced by peptide bonds (peptilexins). On the other hand vinylexins with proton donating heteroaromatic rings are, as expected, AT specific. The vinylexin family may thus represent universal minor groove binding agents susceptible to bind to any given base pair sequence of DNA, following the positioning of their proton donor and proton acceptor rings. This study confirms the insufficiency of purely geometrical and/or hydrogen bonding considerations for the correct estimation of GC versus AT specificity of groove binding ligands. These can only be accounted for by taking into consideration the overall electronic properties of the interacting species and explicitly calculating the energies of complex formation including all the relevant contributions.     

Theoretical Study of the Sequence Selectivity of Isolexins,Isohelical DNA Groove Binding Ligands. Proposal for the GC Minor Groove Specific Compounds

Abstract

Atheoretical study is presented of complex formation between DNA fragments of different base sequences and isolexins, “isohelical base reading polymers”, formed of heteroaromatic pentagonal rings joined by appropriate linkers. Extensive computations are performed for the isolexin composed of the furan-pyrrole-furan sequence. They involve charged ligands with propioamidinium groups at both ends as well eis neutral molecules with terminal methyl, carbonyl and amino groups. Two different groups (C=O and NH) are used as linkers between the base reading moieties. The role of these elements on the binding preference of the ligands has been examined. The results show that the mere possibility of formation of hydrogen bonds between a ligand and the nucleic acid bases is not sufficient to ensure its binding specificity which is determined largely by the interplay of electrostatic factors. Thus the dicationic isolexins uniformly prefer AT sequences. For the neutral isolexins the nature of the groups forming the linkers is a major factor in defining the specificity, although these groups do not participate directly in the interaction with DNA The C=O linkers favour binding to AT sequence while the N-H linkers permit preferential binding to the GAG sequence. Finally, for the first time in theoretical computations, a ligand is proposed which should bind preferentially to the minor groove of GC sequences: this ligand is a neutral isolexin composed of three furan rings linked by two N-H groups. This ligand is considered as an improvable prototype. Altogether the results presented open the path for the designing of minor groove ligands specific for any desirable DNA base sequence.     

Design of Sequence-Specific DNA Binding Ligands That Use a Two-Stranded Peptide Motif for DNA Sequence Recognition

Abstract

The design and DNA binding activity of β-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides - S,S'-bis(Lys-Gly-Val-Cys-Val- NH-NH-Dns) - bridged by an S-S bond binds at least 10 times more strongly to poly(dG)?poly(dC) than to poly(dA)?poly(dT). This peptide can also discriminate between 5′-GpG-3′ and 5′-GpC-3′ steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides - Gly-Cys-Gly- or Val-Cys-Val - bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5′-GpG-3′ steps on DNA, whereas the netropsin-like fragments bind preferentially to tuns of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5′-GpG-3′-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.     

Energetics and stereochemistry of DNA complexation with the antitumor AT specific intercalators tilorone and m-AMSA.

Computations by the SIBFA method on the intercalative interaction energies of tilorone and m-AMSA with B-DNA representative oligonucleotides account for the specificity of these antitumor drugs for AT sites and minor groove intercalation. In tilorone this specificity is due to the strong preference of the side chains for the minor groove, which overcomes the preference of the chromophore for a GC intercalation site. In m-AMSA the specificity is due to the combined preference of both the chromophore and the anilino side chain for AT intercalation site and minor groove, respectively. o-AMSA is shown to manifest a similar (although significantly less pronounced specificity) as m-AMSA but a higher affinity for DNA. A comparison of the energetics and stereochemistry of intercalative binding to DNA of m-AMSA (AT minor groove specific) and 9-aminoacridine-4-carboxamide (GC major groove specific), which possess the same chromophore and differ only by the nature and position of the side chains, shows the possibility of important variations in the intercalative behaviour of chromophoric drugs as a function of the substituent groups attached to them.     

Low-temperature crystallographic analyses of the binding of Hoechst 33258 to the double-helical DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G.

The crystal structure of the complex of Hoechst 33258 and the DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G has been solved from X-ray data collected at three different low temperatures (0, -25, and -100 degrees C). Such temperatures have permitted collection of higher resolution data (2.0, 1.9, and 2.0 A, respectively) than with previous X-ray studies of the same complex. In all three cases, the drug is located in the narrow central A-A-T-T region of the minor groove. Data analyses at -25 and -100 degrees C (each with a 1:1 drug/DNA ratio in the crystallizing solution) suggest a unique orientation for the drug. In contrast, two orientations of the drug were found equally possible at 0 degrees C with a 2:1 drug/DNA ratio in solution. Dihedral angles between the rings of Hoechst 33258 appear to change in a temperature-dependent manner. The drug/DNA complex is stabilized by single or bifurcated hydrogen bonds between the two N-H hydrogen-bond donors in the benzimidazole rings of Hoechst and adenine N3 and thymine O2 acceptors in the minor groove. A general preference for AT regions is conferred by electrostatic potential and by narrowing of the walls of the groove. Local point-by-point AT specificity follows from close van der Waals contacts between ring hydrogen atoms in Hoechst 33258 and the C2 hydrogens of adenines. Replacement of one benzimidazole ring by purine in a longer chain analogue of Hoechst 33258 could make that particular site GC tolerant in the manner observed at imidazole substitution for pyrrole in lexitropsins.     

Cationic porphyrins as probes of DNA structure.

The DNA binding specificity of a group of cationic manganese porphyrin complexes has been examined using DNase I footprinting methodology and by observing the sites of porphyrin-induced DNA strand scission in the presence of potassium superoxide. The compounds, which possess systematic changes in total charge, its distribution on the periphery on the macrocycle and ligand shape, bind in the minor groove of AT rich regions of DNA. While changes in total charge and charge arrangement do not significantly influence specificity, a shape change which blocks close ligand contact with the minor groove relaxes the original AT specificity causing the compound to cleave at both AT and GC sites. The observed changes in binding sequence specificity were interpreted in terms of electrostatic and steric factors associated with both the compounds and DNA.     

DNA binding of the nonintercalative ligands SN-6132, SN-6131 and SN-6113: minor variations of the ligand structure may cause changes in the base pair preference.

The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary ammonium heterocycles has been investigated by means of CD spectroscopy and melting measurements. From the spectroscopic results and binding data it is concluded that the agents SN-6132, SN-6131 and SN-6113 have relatively high affinity to AT base pair sequences whereas the binding to GC pairs is very low. The binding selectivity to AT base pair sequences decreases in the order netropsin > SN-6132 > SN-6113 > SN-6131. Poly(dA).poly(dT) has the highest binding preference for SN-6132 relative to that of SN-6131. The different binding behavior of the ligands is related to their distinct changes in the chemical structure and to the DNA minor groove properties which determines the adaptability of the ligands in the groove.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Structural analysis of the binding modes of minor groove ligands comprised of disubstituted benzenes

Two-dimensional homonuclear NMR was used to characterize synthetic DNA minor groove-binding ligands in complexes with oligonucleotides containing three different A-T binding sites. The three ligands studied have a C₂ axis of symmetry and have the same general structural motif of a central para-substituted benzene ring flanked by two meta-substituted rings, giving the molecules a crescent shape. As with other ligands of this shape, specificity seems to arise from a tight fit in the narrow minor groove of the preferred A-T-rich sequences. We found that these ligands slide between binding subsites, behavior attributed to the fact that all of the amide protons in the ligand backbone cannot hydrogen bond to the minor groove simultaneously.     

Sequence-dependent binding of bis-amidine carbazole dications to DNA.

The conventional wisdom argues that DNA intercalators possess a condensed polyaromatic ring whereas DNA minor groove binders generally contain unfused aromatic heterocycles, frequently separated by amide bonds. Recently, this view has been challenged with the discovery of powerful intercalating agents formed by unfused aromatic molecules and groove binders containing a polyaromatic nucleus. Bis-amidinocarbazoles belong to this later category of drugs having a planar chromophore and capable of reading the genetic information accessible within the minor groove of AT-rich sequences [Tanious, F.A., Ding, D., Patrick, D.A., Bailly, C., Tidwell, R.R. & Wilson, W.D. (2000) Biochemistry 39, 12091-12101]. But in addition to the tight binding to AT sites, we show here that bis-amidinocarbazoles can also interact with GC sites. The extent and mode of binding of 2,7 and 3,6 substituted amidinocarbazoles to AT and GC sequences were investigated by complementary biochemical and biophysical methods. Absorption, fluorescence, melting temperature and surface plasmon resonance (SPR) measurements indicate that the position of the two amidine groups on the carbazole ring influences significantly the drug-DNA interaction. SPR and DNase I footprinting data confirm the AT-preference of the compounds and provide useful information on their additional interaction with GC sequences. The 3,6-carbazole binds approximately twice as strongly to the GC-containing hairpin oligomer than the 2,7-regioisomer. The high tendency of the 3,6 compound to intercalate into different types of DNA containing G.C base pairs is shown by electric linear dichroism. This work completes our understanding of the sequence-dependent DNA binding properties of carbazole dications.     

Specificity in the interaction of non intercalative groove binding ligands with nucleic acids

This paper presents results of theoretical computations on the interaction energies and geometries for the binding to nucleic acids of a number of representative groove binding non intercalating drugs: netropsin, distamycin A, SN 18071, etc. The computations account for the specificity of binding in all cases and demonstrate that the formation of hydrogen bonds is not necessary neither for binding nor for the preference for the minor groove of AT sequences of B-DNA. It appears that if a relatively good steric fit can be obtained in the minor groove, the interaction will be preferentially stabilized there by the favorable electrostatic potential generated in this groove by the AT sequences. The computation of the interaction energies in free space does not reproduce, however, the order of affinities of the ligands studied and yields too great values of the binding energies. The introduction of the solvent effect, through the computation of the hydration and cavitation effects, confirms the specificity, improves the ordering and brings the values of the energies close to the experimental ones. The theoretical account of the “surprising” effect of netrospin binding to the major groove of theTψC stem of tRNA^Phe confirms the decisive significance of the distribution of the molecular electrostatic potential for the selection of the binding site. The inclusion in the computations of the flexibility of DNA enables to predict correctly the main features of the macromolecular deformation upon the binding of the ligand.     

A study of the interaction of Cr(III) complexes and their selective binding with B-DNA: a molecular modeling approach

Molecular modeling and energy minimisation calculations have been used to investigate the interaction of chromium(III) complexes in different ligand environments with various sequences of B-DNA. The complexes are [Cr(salen)(H(2)O)(2)](+); salen denotes 1, 2 bis-salicylideneaminoethane, [Cr(salprn)(H(2)O)(2)](+); salprn denotes 1, 3 bis- salicylideneaminopropane, [Cr(phen)(3)](3+); phen denotes 1, 10 phenanthroline and [Cr(en)(3)](3+); en denotes ethylenediamine. All the chromium(III) complexes are interacted with the minor groove and major groove of d(AT)(12), d(CGCGAATTCGCG)(2) and d(GC)(12) sequences of DNA. The binding energy and hydrogen bond parameters of DNA-Cr complex adduct in both the groove have been determined using molecular mechanics approach. The binding energy and formation of hydrogen bonds between chromium(III) complex and DNA has shown that all complexes of chromium(III) prefer minor groove interaction as the favourable binding mode.     

Methylene Blue Binding to DNA with Alternating AT Base Sequence: Minor Groove Binding is Favored over Intercalation

Abstract

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.     

Use of Electric Linear Dichroism and Competition Experiments with Intercalating Drugs to Investigate the Mode of Binding of Hoechst 33258, Berenil and DAPI to GC Sequences

Abstract

The drugs Hoechst 33258, berenil and DAPI bind preferentially to the minor groove of AT sequences in DNA Despite a strong selectivity for AT sites, they can interact with GC sequences by a mechanism which remains so far controversial. The 2-amino group of guanosine represents a steric hindrance to the entry of the drugs in the minor groove of GC sequences. Intercalation and major groove binding to GC sites of GC-rich DNA and polynucleotides have been proposed for these drugs. To investigate further the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences, we studied by electric linear dichroism the mutual interference in the DNA binding reaction between these compounds and a classical intercalator, proflavine, or a DNA-threading intercalating drug, the amsacrine-4-carboxamide derivative SN16713. The results of the competition experiments show that the two acridine intercalators markedly affect the binding of Hoechst 33258, berenil and DAPI to GC polynucleotides but not to DNA containing AT/GC mixed sequences such as calf thymus DNA Proflavine and SN16713 exert dissimilar effects on the binding of Hoechst 33258, berenil and DAPI to GC sites. The structural changes in DNA induced upon intercalation of the acridine drugs into GC sites are not identically perceived by the test compounds. The electric linear dichroism data support the hypothesis that Hoechst 33258, berenil and DAPI interact with GC sites via a non-classical intercalation process.     

FTIR study of specific binding interactions between DNA minor groove binding ligands and polynucleotides.

The use of FTIR spectroscopy is made to study the interactions between polynucleotides and two series of minor groove binding compounds. The latter were developed and described previously as part of an ongoing program of rational design of modified ligands based on naturally occurring pyrrole amidine antibiotic netropsin, and varying the structure of bisbenzimidazole chromosomal stain Hoechst 33258. Characteristic IR absorptions due to the vibrations of thymidine and cytosine keto groups in polynucleotides containing AT and GC base pairs respectively are used to monitor their interaction with the added ligands. Although the two thiazole based lexitropsins based on netropsin structure differ in the relative orientation of nitrogen and sulfur atoms with respect to the concave edge of the molecules, they interact exclusively with the thymidine C2 = O carbonyl groups in the minor groove of the alternating AT polymer as evidenced by specific changes in the IR spectra. In the second series of compounds based on Hoechst 33258, the structure obtained by replacing the two benzimidazoles in the parent compound by a combination of pyridoimidazole and benzoxazole, exhibits changes in the carbonyl frequency region of poly dG.poly dC which is attributed to the ligand interaction at the minor groove of GC base pairs. In contrast, Hoechst 33258 itself interacts only with poly dA.poly dT. Weak or no interaction exists between the ligands and any of the polynucleotides at the levels of the phosphate groups or the deoxyribose units.     

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.     

Theoretical Exploration of Netropsin Binding to tRNAPhe

Abstract

Theoretical exploration of the possible interaction of netropsin with tRNA^Phe indicates that binding should occur preferentially with the major groove of the TψC stem of the macromolecule, specifically with the bases G51, U52, G53 and phosphates 52, 53, 61 and 62. This agrees with the recent crystallographic result of Rubin and Sundaralingam. It is demonstrated that the difference with respect to netropsin binding with B-DNA, where it occurs specifically in the minor groove of AT sequences, is due to the differences in the distribution of the electrostatic molecular potential generated by these different types of DNA: this potential is sequence dependent in B-DNA (located in the minor groove of AT sequences and the major groove of GC sequences), while it is sequence independent and always located in the major groove in A-RNA. The result demonstrates the major role of electrostatics in determining the location of the binding site.     

Comparative thermodynamics for monomer and dimer sequence-dependent binding of a heterocyclic dication in the DNA minor groove

Phenylamidine cationic groups linked by a furan ring (furamidine) and related symmetric diamidine compounds bind as monomers in the minor groove of AT sequences of DNA. DB293, an unsymmetric derivative with one of the phenyl rings of furamidine replaced with a benzimidazole, can bind to AT sequences as a monomer but binds more strongly to GC-containing minor-groove DNA sites as a stacked dimer. The dimer-binding mode has high affinity, is highly cooperative and sequence selective. In order to develop a better understanding of the correlation between structural and thermodynamic aspects of DNA molecular recognition, DB293 was used as a model to compare the binding of minor-groove agents with AT and mixed sequence DNA sites. Isothermal titration calorimetry and surface plasmon resonance results clearly show that the binding of DB293 and other related compounds into the minor groove of AT sequences is largely entropy-driven while the binding of DB293 as a dimer into the minor groove of GC-containing sequences is largely enthalpy-driven. At 25 degrees C, for example, the AT binding has DeltaG degrees, DeltaH degrees and TDeltaS degrees values of -9.6, -3.6 and 6.0 kcal/mol while the values for dimer binding to a GC-containing site are -9.0, -10.9 and -1.9 kcal/mol (per mol of bound compound), respectively. These results show that the thermodynamic components for binding of compounds of this type to DNA are very dependent on the structure, solvation and sequence of the DNA binding site.