Molecular structure, stereochemistry and interactions of steffimycin B, and DNA binding anthracycline antibiotic

The crystal and molecular structure of anthracycline antibiotic steffimycin B(C29H32O13) has been determined by X-ray diffraction and the stereochemistry revealed. The orthorhombic crystals belong to space group P2(1)2(1)2(1), with the dimensions; a = 8.253 (2), b = 8.198 (2), c = 40.850 (8) A and Z = 4. Intensity data were collected for 2518 independent reflections. The structure was solved by direct methods and refined to an R value of 0.066 for 1410 reflections. The configuration in ring A is 7R,8S,9S. Ring A adopts half chair conformation, while the sugar ring has the regular chair conformation. The molecule most probably binds to double helical DNA through intercalation and hydrogen bonding.     

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

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

Effects of the antitumor antibiotic mithramycin on the structure of repetitive DNA regions adjacent to its GC-rich binding site.

Regions of An.Tn, (GA)n.(TC)n, and (GT)n.(AC)n have been cloned into the SmaI (CCC/GGG) site of plasmid pUC19. HindIII-EcoRI restriction fragments containing these inserts have been used as substrates for footprinting experiments using DNase I, DNase II, and micrococcal nuclease as probes. These present good mithramycin binding sites (GGG) flanking repetitive regions to which the drug does not bind. In each case, mithramycin footprints are observed at the CCC/GGG sites, which are not affected by the nature of the surrounding sequences. Some weaker binding is detected at TCGA and ACCA sites and at regions of alternating GA. No binding is found to regions of alternating GT. An.Tn inserts (n = 23 or 69) are normally resistant to cleavage by all these probes; in the presence of mithramycin, a dramatic increase in DNase I cleavage is observed throughout the entire insert and is indicative of an alteration in DNA structure. Similar changes are seen with DNase II and micrococcal nuclease. These changes cannot be explained by invoking changes in the ratio of free substrate to cleavage agent. In contrast, cleavage of (GA)n.(CT)n and (GT)n.(AC)n inserts is not affected by drug binding. The results are consistent with a model in which mithramycin causes dramatic changes in the width of the DNA minor groove, generating a structure which has some properties of A-DNA, and suggest that this can be propagated into surrounding DNA regions in a sequence-dependent manner. The structural alterations with An.Tn are highly cooperative and can be transmitted over at least three turns of the DNA helix.     

Binding of the antitumor drug nogalamycin and its derivatives to DNA: structural comparison

The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m5CGT(pS)Am5CG] have been determined at 1.7- and 1.8-A resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P6(1)) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences.     

Peroxidase-catalyzed covalent binding of the antitumor drug N2-methyl-9-hydroxyellipticinium to DNA in vitro

In the presence of DNA, the antitumor drug N2-methyl-9-hydroxyellipticinium (elliptinium; NMHE) [Le Pecq, J. B., Gosse, C., Dat-Xuong, N., & Paoletti, C. (1975) C. R. Seances Acad. Sci., Ser. D 281, 1365-1367] is oxidized by the horseradish peroxidase-hydrogen peroxide (HRP-H2O2) system to the quinone imine derivative N2-methyl-9-oxoellipticinium (NMOE) [Auclair, C., & Paoletti, C. (1981) J. Med. Chem. 24, 289-295], which interacts with DNA according to the intercalation mode. When excess H2O2 was used, the major part of the quinone imine was further oxidized to the o-quinone N2-methyl-9,10-dioxoellipticinium [Bernadou, J., Meunier, G., Paoletti, C., & Meunier, B. (1983) J. Med. Chem. 26, 574-579]. In the presence of stoichiometric amounts of H2O2 (H2O2/NMHE = 1), NMOE reacts with DNA, yielding a fluorescent compound irreversibly linked to the nucleic acid, which is related to the covalent binding of the ellipticinium chromophore. Under optimal reaction conditions, NMHE binding occurs according to a first-order process (k = 4.3 X 10(-3) min-1) with a linear increase with respect to drug to nucleotide ratio up to a maximum binding of 1 NMHE per 20 base pairs (r = 0.05). The fluorescence spectra (ex, 330 nm; em, 548 nm) of NMHE bound to DNA, the occurrence of energy transfer from the DNA to the drug, and the DNA length increase of the DNA-NMHE adduct suggest that the binding occurs at the intercalating site with limited denaturation of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site specificity of binding of antitumor antibiotics to DNA

The site specificities for intercalation of steffimycin B, adriamycin, echinomycin, and ethidium bromide with DNA have been determined by CD first-neighbor analysis. The first three, which are antineoplastic antibiotics, all exhibit preferential binding to sites comprised of guanine and cytosine (GpC, CpG, and CpC or its complement GpG). Ethidium bromide displays nonspecific intercalation. The results are compared with findings from “footprinting” studies.     

NMR studies on the binding of antitumor drug nogalamycin to DNA hexamer d(CGTACG).

The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2:1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5' & H5") of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1:1 complex and 2:1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1:1 complex are completely destabilized such that at 65 degrees C only free single-stranded DNA and the 2:1 complex co-exist. At 35 degrees C the equilibrium between free DNA and the 1:1 complex is relatively fast, while that between the 1:1 complex and the 2:1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2:1 complex at low pH showed that the terminal GC base pair is destabilized.     

A theoretical investigation of the sequence specificity in the binding of the antitumor drug anthramycin to DNA

A theoretical study is presented concerning DNA-anthramycin adducts. By explicit energy minimisations using a semi-empirical energy formula and an advanced algorithm the structural properties and the energetics of this system are analysed. The results obtained demonstrate that the formation of a covalently bound adduct in which anthramycin is attached to the N2 site of a guanine within a DNA fragment is accompanied by a considerable change in the nucleic acid conformation as confirmed by recent experimental evidence. With the use of the "SIR" methodology for treating DNA flexibility the general features of this change are characterised. The sequence specificity of anthramycin binding is investigated and the important role of sequence dependent nucleic acid flexibility brought to light. This theoretical treatment thus provides new elements for the interpretation of the origins of ligand binding specificities.     

Detection of drug binding to DNA by hydroxyl radical footprinting. Relationship of distamycin binding sites to DNA structure and positioned nucleosomes on 5S RNA genes of Xenopus.

We report the use of hydroxyl radical footprinting to analyze the interaction of distamycin and actinomycin with the 5S ribosomal RNA genes of Xenopus. There is a qualitative difference in the hydroxyl radical footprints of the two drugs. Distamycin gives a conventional (albeit high-resolution) footprint, while actinomycin does not protect DNA from hydroxyl radical attack, but instead induces discrete sites of hyperreactivity. We find concentration-dependent changes in the locations of distamycin binding sites on the somatic 5S gene of Xenopus borealis. A high-affinity site, containing a G.C base pair, is replaced at higher levels of bound drug by a periodic array of different lower affinity sites that coincide with the places where the minor groove of the DNA would face in toward a nucleosome core that is known to bind to the same sequence. These results suggest that distamycin recognizes potential binding sites more by the shape of the DNA than by the specific sequence that is contained in the site and that structures of many sequences are deformable to a shape that allows drug binding. We discuss the utility of hydroxyl radical footprinting of distamycin for investigating the underlying structure of DNA.     

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.     

Theoretical study of the sequence specificity in the covalent binding of the antitumor drug CC-1065 to DNA

A theoretical modelling is presented of the covalent adducts of the antitumor agent CC-1065 with B-DNA. The optimal complexes are obtained by energy minimisation, taking into account full structure flexibility, including the flexible rings of the ligand and DNA. The binding preference of CC-1065 with respect to base sequence is studied. The results obtained elucidate the origin of the preference for two AT base pairs on the 5'side of the modified adenine. The modifications of the DNA structure upon ligand covalent binding are discussed.     

The binding affinity of HMG1 protein to DNA modified by cis-platin and its analogs correlates with their antitumor activity.

The antitumor activity of cis-platin is believed to result from its interaction with cellular DNA and subsequent processing of DNA adducts by damage recognition proteins. Among them are the high mobility group (HMG) proteins 1 and 2, which have been hypothesized to mediate the effect of cis-platin. One possibility suggests that the tight binding of HMG1 to DNA adducts blocks the repair of damaged DNA. In order to further evaluate such a mechanism, several cis-platinum complexes with known antitumor activity have been used to treat DNA and the affinity of HMG1 to the DNA adduct induced by each drug was determined. The dissociation constants for the complexes of HMG1 with the platinated probe were obtained by gel mobility shift assays. The antitumor activity of the tested platinum compounds was found to correlate with the binding affinity of HMG1 to the respective drug-DNA adduct. These findings support the view that HMG1 contributes to cytotoxicity of cis-platin by shielding damaged DNA from repair. In addition, they offer a fast test for screening new platinum compounds for antitumor activity.     

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone.     

Specific binding of chartreusin, an antitumor antibiotic, to DNA

Chartreusin, an antitumor and antibacterial antibiotic, was found to inhibit negatively superhelical DNA-relaxation catalyzed by prokaryotic topoisomerase I and conversion of the superhelical DNA into unit length linear form catalyzed by single-strand-specific S1 nuclease. The inhibitory effect of the agent was due to the binding to DNA causing the alteration of tertiary structure. To characterize the binding specificity, we investigated the protection of DNA against cleavages by various restriction endonucleases. It was evidenced that the binding of the agent is not at random and correlates to the sequence 5'CGC 3' 3'GCG 5' on DNA stretch.     

One-step synthesis of methylene-bridged bis-carbazole and evaluation of its antitumor activity and G-quadruplex DNA binding property

Most reported carbazolyl G-quadruplex DNA (G4-DNA) ligands possess a rigid structure rather than a flexible one. The conformationally flexible ligands are paid much less attention. In this study, we report a novel class of non-rigid methylene-bridged biscarbazolyl ligand and their G4-DNA binding properties. Moreover, the antitumor activities of all these oligomers have been evaluated. The results show that this family of oligomers could be facilely synthesized via solely one step. Among them, compound 2, the bis-carbazole derivative, displays the best antitumor activity and IC₅₀ values against HT-29, HepG2, A375 and MCF-7 cells are 0.69, 5.09, 3.15 and 3.8 μ mol/L, respectively. Although conformationally flexible, 2 is still capable of binding to as well as stabilizing G4-DNA via π-π stacking interaction. Moreover, 2 selectively binds to G4-DNA over duplex DNA. The current study enriches the category of carbazolyl G4-DNA ligands and paves the way for the search of more efficient G4-DNA ligands and antitumor leads.     

Molecular structure, conformation and interactions of antitumor antibiotic cyanonaphthridinomycin, a covalent binder of DNA

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C22H26N4O5), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P21 with the cell dimensions of a = 5.934(1)b = 20.684(4), c = 16.866(3)A, gamma = 90.9 degrees and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.