Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.     

Binding of nonintercalative antitumor drugs to DNA-polymers: structural effects of bisquaternary ammonium heterocycles

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA.dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA.dT preference in their binding affinity to DNA. Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC).poly(dG-dC), poly(rA).poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA).poly(dT) and poly(dI).poly(dC) indicating a slow kinetics. The preferred binding to dA.dT base pairs in DNA decreases in the order from SN-61367 greater than SN-13232 greater than SN-6324,SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA.dT).poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAse I cleavage of poly(dA-dT).poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNA binding and dA.dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Binding of Nonintercalative Antitumor Drugs to DNA-Polymers: Structural Effects of Bisquaternary Ammonium Heterocycles

Abstract

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA · dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA · dT preference in their binding affinity to DNA.

Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC) · poly(dG-dC), poly(rA) · poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA) · poly(dT) and poly(dI) · poly(dC) indicating a slow kinetics.

The preferred binding to dA · dT base pairs in DNA decreases in the order from SN-61367 > SN-13232 > SN-6324, SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA-dT) · poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAase I cleavage of poly(dA-dT) · poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNAbinding and dA · dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA).poly(dT) and poly(dG).poly(dC), and with triple helical poly(dA).[poly(dT)](2) and poly(dC).poly(dG).poly(dC)(+) were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA).poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG).poly(dC) and -poly(dC).poly(dG).poly(dC)(+) complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Structural motives controlling the binding affinity of 9,10-bis(methylpyridinium)anthracenes towards DNA

In the search of new DNA groove binding agents a series of substituted 9,10-methylpyridiniumanthracenes have been synthesized and their interactions with DNA have been studied by UV/vis absorption, CD and fluorescence spectroscopy. A minor groove binding mode is confirmed by DNA melting studies, strong CD effects, the dependence of the binding affinity on ionic strength, and the differentiation between AT and GC base pairs. No binding occurs to GC sequences. Binding constants to calf thymus DNA (ct-DNA) and poly(dA:dT) in the range between 1 × 10⁴ and 3 × 10⁵ M⁻¹ have been determined. The binding strength decreases with the size of substituents attached at the anthracene site. Variation of the substitution pattern of the charged groups shows that methyl groups in meta position cause slightly stronger binding than methyl groups in para position. In contrast, with these groups in ortho position, no binding interaction has been observed. The strongest binding is achieved with an expansion of the peripheral heterocycle from pyridine to quinoline. Molecular modeling reveals the pivotal role of the substitution pattern: Anthracenes with para and meta pyridines align along the minor grooves. On the other hand, the ortho derivative adopts no groove-alignment.     

Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) footprinting and DNA affinity cleaving

Using two direct methods we have studied the binding locations and site sizes of distamycin and penta-N-methylpyrrolecarboxamide on three DNA restriction fragments from pBR322 plasmid. We find that methidiumpropyl-EDTA.Fe(II) footprinting and DNA affinity cleaving methods report common binding locations and site sizes for the tri- and pentapeptides bound to heterogeneous DNA. The tripeptide distamycin binds 5-base-pair sites with a preference for poly(dA).poly(dT) regions. The pentapeptide binds 6-7-base-pair sites with a preference for poly(dA).poly(dT) regions. These results are consistent with distamycin binding as an isogeometric helix to the minor groove of DNA with the four carboxamide N-H's hydrogen bonding five A + T base pairs. The data supports a model where each of the carboxamide N-H's can hydrogen bond to two bases, either O(2) of thymine or N(3) of adenine, located on adjacent base pairs on opposite strands of the helix. In most (but not all) cases the tri- and pentapeptide can adopt two orientations at each A + T rich binding site.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]₂ and poly(dC)·poly(dG)·poly(dC)⁺ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)⁺ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Variations in duplex DNA conformation detected by the binding of monoclonal autoimmune antibodies.

Four monoclonal antibodies (Jel 229, 239, 241, 242) which bound to duplex DNA were prepared from two autoimmune female NZB/NZW mice. Their binding to various nucleic acids was investigated by a competitive solid phase radioimmune assay which allows the estimation of relative binding constants. None of the antibodies showed any consistent variation of binding constant with base composition and thus they must recognize features of the DNA backbone. Jel 241 binds across the major groove but the interaction with poly(pyrimidine) X poly(purine) DNAs was barely detectable. This antibody appears to recognize the "alternating-B" conformation which is promoted by methylation of pyrimidines in alternating sequences. The other three antibodies bind in the minor groove. In particular, for Jel 229 the preferred antigen was poly(dG) X poly(dC) with only weak binding to poly(dA) X poly(dT). This suggests a requirement for a wide minor groove. Thus autoimmune antibodies provide examples of "analogue" recognition and can be used to detect structural variations in the grooves of duplex DNA.     

Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.     

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

Interaction of a cysteine-containing peptide with DNA

Cystine peptide dimer (Lys-Gly-Val-Cys-Val-N2H2Dns)2 with S-S bridge was synthesized and its interactions with DNA and synthetic polynucleotides have been studied by optical spectroscopy methods. By recording fluorescent titration curves we have shown that the affinity of the peptide to different synthetic polynucleotides decreases in the order: poly(dG).poly(dC) greater than poly(dA).poly(dT) greater than poly(dGC).poly(dGC). The stability of complexes to increasing concentrations of NaCl diminishes in the same order. The association constant is about 20-fold greater for peptide binding to poly(dG).poly(dC) than to poly(dA).poly(dT). By using circular dichroism and fluorescence measurements we have shown that the peptide competes for the binding sites on DNA with two minor-groove binding antibiotics--distamycin A and sybiromycin. These results have suggested that the peptide also binds in the DNA minor groove. Investigation of the interactions between such peptides and DNA may be useful for constructing ligands with combined specificity to DNA.     

Synthesis of a dA-dT base pair analogue and its effects on DNA-ligand binding

Two nucleoside derivatives containing the base analogues 3-deazaadenine and 3-methyl-2-pyridone have been prepared as analogues of dA and dT, respectively. After conversion into the appropriately protected phosphoramidites, DNA sequences were prepared with site-specifically placed analogues. When present in a duplex DNA sequence, the analogues result in the deletion of one or both of the hydrogen bonding functional groups (the N3-nitrogen of dA and the O2-carbonyl of dT) present in the minor groove. Binding by two ligands, 4',6-diamidine-2-phenyl indole (DAPI) and Hoechst 33258 in the minor groove has been probed using a variety of DNA sequences. These sequences contain a d(GAATTC)2 core with analogue nucleosides substituted for one or more of the dA and dT residues. DAPI bound strongly to any sequence that contained both O2-carbonyls of the central two dT residues. The presence of a dc3A residue did in some cases enhance binding. With one of the central O2-carbonyls deleted, the binding was noticeably reduced, and with both absent, no significant binding could be detected. Similar although less dramatic results were observed with Hoechst 33258 binding to analogue sequences.     

A premelting conformational transition in poly(dA)-Poly(dT) coupled to daunomycin binding

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.     

Probing the hydration of the minor groove of A.T synthetic DNA polymers by volume and heat changes

The minor-groove ligand netropsin provides a sensitive probe of the hydration difference between poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)]. We have measured the volume change delta V accompanying binding of netropsin to these polymers, using an improved magnetic suspension densimeter. For poly(dA).poly(dT) we find delta V = +97 mL/mol of bound netropsin at pH 7.0 and 10 mM sodium phosphate buffer. For poly[d(AT)].poly[d(AT)] we find delta V = -16 mL/mol of bound netropsin. This striking differential effect suggests that the poly(dA).poly(dT) duplex compresses more water (or is more extensively hydrated). From our enthalpy and entropy results we estimate the approximately 10 water molecules, immobilized in the minor groove of this system, are displaced by each netropsin bound. The volume increase, however, is substantially larger than can be explained by a simple melting of these immobilized water molecules in the minor groove. A decompression of at least 40 water molecules must attend the complexation to the poly(dA).poly(dT) duplex. This suggests that the conformation change attending the binding of the drug to this polymer duplex causes a further dehydration, whereas no such change in dehydration and configuration for the heteropolymer system is indicated.     

Structure of poly(dA).poly(dT) is not identical to the AT rich regions of the single crystal structure of CGCGAATTBrCGCG. The consequence of this to netropsin binding to poly(dA).poly(dT)

The basic assumption of Dickerson and Kopka (J. Biomole. Str. Dyns. 2, 423, 1985) that the conformation of poly(dA).poly(dT) in solution is identical to the AT rich region of the single crystal structure of the Dickerson dodecamer is not supported by any experimental data. In poly(dA).poly(dT), NOE and Raman studies indicate that the dA and dT units are conformationally equivalent and display the (anti-S-type sugar)-conformation; incorporation of this nucleotide geometry into a double helix leads to a conventional regular B-helix in which the width of the minor groove is 8A. The derived structure is consistent with all available experimental data on poly(dA).poly(dT) obtained under solution conditions. In the crystal structure of the dodecamer, the dA and dT units have distinctly different conformations-dA residues adopt (anti, S-type sugar pucker), while dT residues belong to (low anti, N-type sugar pucker). These different conformations of the dA and dT units along with the large propeller twist can be accommodated in a double helix in which the minor groove is shrunk from 8A to less than 4A. In the conventional right handed B-form of poly(dA).poly(dT) with the 8A wide minor groove, netropsin has to bind asymmetrically along the dA strand to account for the NOE and chemical shift data and to generate a stereochemically sound structure (Sarma et al, J. Biomole. Str. Dyns. 2, 1085, 1985).