Interaction of unfused tricyclic aromatic cations with DNA: a new class of intercalators

Unfused tricyclic aromatic ring systems 1-6 with one or two cationic side chains have been synthesized and their interactions with DNA and synthetic polymers probed with a variety of techniques. Molecular mechanics calculations indicate that the torsional angle between ring planes in the minimum energy conformation of the tricyclic molecules can range from 0 degree to as high as 50 degrees depending on the type of rings and substituents. Viscometric titrations with linear and supercoiled DNA, linear dichroism, and NMR studies indicated that all compounds with torsional angles of approximately 20 degrees or less bind to DNA by intercalation. The more highly twisted intercalators caused significant perturbation of DNA structure. Unfused intercalators with twist angles of approximately 20 degrees have reduced binding constants, suggesting that they could not form an optimum interaction with the DNA base pairs. Unfused intercalators with twist less than 20 degrees formed strong complexes with DNA. The structures of these unfused intercalators are more analogous to typical groove-binding molecules, and an analysis of their interaction with DNA provides a better understanding of the subtle differences between intercalation and groove-binding modes for aromatic cations. The results indicate that intercalation and groove-binding modes should be viewed as two potential wells on a continuous energy surface. The results also suggest design strategies for intercalators that can optimally complement DNA base pair propeller twist or that can induce bends in DNA at the intercalation site.     

Virtual screening,synthesis and biological evaluation of DNA intercalating antiviral agents

This paper describes computer-aided design of new anti-viral agents against Vaccinia virus (VACV) potentially acting as nucleic acid intercalators. Earlier obtained experimental data for DNA intercalation affinities and activities against Vesicular stomatitis virus (VSV) have been used to build, respectively, pharmacophore and QSAR models. These models were used for virtual screening of a database of 245 molecules generated around typical scaffolds of known DNA intercalators. This resulted in 12 hits which then were synthesized and tested for antiviral activity against VaV together with 43 compounds earlier studied against VSV. Two compounds displaying high antiviral activity against VaV and low cytotoxicity were selected for further antiviral activity investigations.     

Analysing DNA complexes by circular and linear dichroism

The application of linear and circular dichroism (LD and CD) in nucleic acid research id illustrated by recent results aimed at answering specific structural problem in the interaction of DNA with molecules of biological importance. We first consider the circumstances under which ligands, such as DAPI (4′, 6-diamidino-2-phenylindole), change their preferred binding mode in the minor groove to major groove binding or intercalation. As an extension of this problem we refer to the switch between groove binding and intercalation of structurally similar ligands such as ellipticines and trigonal ruthenium complexes. We also explore the use of LD and CD in the determination of the structure of the complex formed between the polynucleotide poly(dA) and the novel ‘peptide nucleic acid’, consisting of nucleic acid bases joined by a polyamide homomorphous with the deoxyribose-phosphate backbone of DNA. Finally, the structure and interaction of the recombination enzyme RecA with DNA is discussed, in particular the influence of the presence of the intercalators, groove binders or covalent DNA adducts.     

Anticooperative Binding Governs the Mechanics of Ethidium-Complexed DNA

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.     

The influence of N-dialkyl and other cationic substituents on DNA intercalation and genotoxicity

DNA intercalation by small chemical molecules can result in frameshift mutagenesis and chromosomal breakage. With evidence mounting that broadly diverse structures are capable of intercalating between DNA base pairs, it becomes important to better define those structural features that enhance intercalation strength and those that confer genotoxicity particularly among those intercalators that do not have the classical planar tricyclic fused ring structure. A chemical substituent that is present on many pharmaceutical and other biologically active molecules is the N-dialkyl group. In the present study, we investigate if and how the presence of an aromatic N-dialkyl or other cationic group affects the genotoxicity and DNA intercalation ability of 26 selected acridines, phenothiazines, benzophenones, triphenylethylenes and other classes of molecules. The data were obtained from the literature, from experiments using a cell-based DNA intercalation assay, and from modeling studies using a three-dimensional computational DNA docking program. It is demonstrated that cationic substitution can enhance both genotoxicity and electrostatic interactions within a chemical/DNA intercalation complex.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by the pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in live (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: the anthracycline anticancer drug doxorubicin and the nucleic acid dyes ethidium bromide (E) and 4',6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, a competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in native (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: anthracycline anticancer drug doxorubicin and nucleic acid dyes ethidium bromide and 4′,6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

Chirality as a tool in nucleic acid recognition: principles and relevance in biotechnology and in medicinal chemistry

The understanding of the interaction of chiral species with DNA or RNA is very important for the development of new tools in biology and of new drugs. Several cases in which chirality is a crucial point in determining the DNA binding mode are reviewed and discussed, with the aim of illustrating how chirality can be considered as a tool for improving the understanding of mechanisms and the effectiveness of nucleic acid recognition. The review is divided into two parts: the former describes examples of chiral species interacting with DNA: intercalators, metal complexes, and groove binders; the latter part is dedicated to chirality in DNA analogs, with discussion of phosphate stereochemistry and chirality of ribose substitutes, in particular of peptide nucleic acids (PNAs) for which a number of works have been published recently dealing with the effect of chirality in DNA recognition. The discussion is intended to show how enantiomeric recognition originates at the molecular level, by exploiting the enormous progresses recently achieved in the field of structural characterization of complexes formed by nucleic acid with their ligands by crystallographic and spectroscopic methods. Examples of application of the DNA binding molecules described and the role of chirality in DNA recognition relevant for biotechnology or medicinal chemistry are reported.     

Circular dichroism and the interactions of water soluble porphyrins with DNA

The size, sign, and profile of induced circular dichroism (CD) features in the Soret region are reliable indicators of the binding modes of porphyrins and metalloporphyrins to DNA. Porphyrins shown (using such CD criteria) to be intercalators in monodispersed DNA duplexes prove extremely useful for the detection and characterization of organized, condensed forms of nucleic acids (psi-condensates). In addition, certain select porphyrin derivatives can form extended assemblies on nonaggregated DNA templates. A combination of CD and resonance light scattering (RLS) measurements allows for sensitive detection and characterization of these porphyrin arrays.     

Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.

An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.     

The interaction with DNA of unfused aromatic systems containing terminal piperazino substituents. Intercalation and groove-binding

A number of unfused tricyclic aromatic intercalators have shown excellent activity as amplifiers of the anticancer activity of the bleomycins and the 4',6-diphenylpyrimidines, 2a and 2b, with terminal basic functions (4-methylpiperazino groups) have been synthesized to test the structural requirements for amplifier-DNA interactions. The terminal piperazine rings are bulky, have limited flexibility, and are twisted out of the phenyl ring plane in both 2a and 2b. With 2a the pyrimidine is unsubstituted at position 5 and the conformation predicted by molecular mechanics calculations has a 25-30 degrees twist between the phenyl and pyrimidine ring planes. With 2b the 5-position is substituted with a methyl group and this causes a larger twist angle (50-60 degrees) between the phenyl and pyrimidine planes. These conformational variations lead to markedly different DNA interactions for 2a and 2b. Absorption, CD and NMR spectral, viscometric, flow dichroism and kinetics results indicate that 2a binds strongly to DNA by intercalation while 2b binds more weakly in a groove complex. The general structure and conformation of 2a, a slightly twisted, unfused-aromatic system with terminal piperazino groups is more similar to groove-binding agents such as Hoechst 33258 than to intercalators. The fact that 2a forms a strong intercalation complex with DNA is unusual but in agreement with studies on other amplifiers of anticancer drug action. Molecular modeling studies provide a second unusual feature of the 2a intercalation complex. While most well-characterized intercalators bind with their bulky and/or cationic substitutents in the DNA minor groove, the cationic piperazino groups of 2a are too large to bind in the minor groove in an intercalation complex but can form strong interactions with DNA in the major groove. The tricyclic aromatic ring system of 2a stacks well with adjacent base-pairs in the major-groove complex and the piperazino groups have good electrostatic and van der Waals interactions with the DNA backbone.     

Half‐Intercalation Stabilizes Slipped Mispairing and Explains Genome Vulnerability to Frameshift Mutagenesis by Endogenous “Molecular Bookmarks”

Some 60 years ago chemicals that intercalate between base pairs of duplex DNA were found to amplify frameshift mutagenesis. Surprisingly, the robust induction of frameshifts by intercalators still lacks a mechanistic model, leaving this classic phenomenon annoyingly intractable. A promising idea of asymmetric half‐intercalation‐stabilizing frameshift intermediates during DNA synthesis has never been developed into a model. Instead, researchers of frameshift mutagenesis embraced the powerful slipped‐mispairing concept that unexpectedly struggled with the role of intercalators in frameshifting. It is proposed that the slipped mispairing and the half‐intercalation ideas are two sides of the same coin. Further, existing findings are reviewed to test predictions of the combined “half‐intercalation into the slipped‐mispairing intermediate” model against accumulated knowledge. The existence of potential endogenous intercalators and the phenomenon of “DNA bookmarks” reveal ample possibilities for natural frameshift mutagenisis in the cell. From this alarming perspective, it is discussed how the cell could prevent genome deterioration from frameshift mutagenesis.     

Interactions of molecules with nucleic acids. XI. Generalization of techniques to generate nucleic acid structures with applications to intercalation sites and kinked structures

A generalized procedure to generate nucleic acid structures is presented. In this procedure, the bases of a base pair are oriented first for characterization of particular DNA receptor sites. The resultant sites are then used in the study of specific molecule–DNA interactions. For example, intercalation sites, kinked DNA, and twisted and tilted bases are envisioned. Alterations of structures via anti → syn orientations of bases, as well as crankshaft motion about collinear bonds, provide additional conformations without disrupting the overall backbone structure. These approaches to the generation of nucleic acid structures are envisioned as required in studies of the intercalation phenomenon, minor adjustments of DNA to accommodate denaturation, binding of carcinogens to DNA, complex formation of transition metals with DNA, and antitumor agents as ligands. For these base-pair and base orientations, backbone orientations are calculated by the AGNAS technique to yield physically meaningful conformations, namely, those conformations for which nonbonded contacts are favourable. A procedure is presented to generate dimer duplex units that are physically meaningful and to assemble these units into a polynucleotide duplex. Double helices that begin with B-DNA, undergo a transition to one of the above-mentioned receptor sites, and return to B-DNA can be assembled from a catalog of dimer duplexes. Stereographic projections of the various receptor sites already being used to model binding to DNA are presented.     

The Interaction Between the Novel Intercalator Diethidium Cation and B-Form DNA: a Theoretical Study

Abstract

This research is an effort to further understand the physicochemical interaction between the novel drug molecule diethidium (2,7-diamino 9-[2,7 diamino 10-nN- phenanthridium] 10- nN- phenanthridium) and its biological receptor DNA. The ultimate goal is the elucidation of this novel class of drugs as potential pharmaceutical agents. Understanding the physico- chemical properties of this drug as well as the mechanism by which it interacts with DNA should ultimately allow the rational design of novel anti-cancer or anti-viral drugs.

A novel binding structure for the diethidium cation to B-form DNA is herein described. Molecular modeling on the complex formed between diethidium and a dodecamer of double-stranded B-form DNA, CGCGAATTCGCG, has shown that this complex is indeed fully capable of participating in the formation of a stable intercalation site. It was expected that diethidium would have a mechanism of intercalation significantly different from other classical intercalators because a) Its structure, that of two perpendicular planes, each known to have excellent intercalation properties, is novel b) The linker region length is zero c) The tilt between the two planes of the drug matches the geometry of the space available to this drug in the major groove.

We have studied the complex formed when diethidium enters the central site of the B-DNA dodecamer through the major groove. The complex forms several classes of intercalation structures, which are all stable and vary from “partially intercalate” to “fully intercalated”. Multiple minimizations show the drug to be very mobile within the intercalation site. Further, some structures show organization and concomitant stiffening of the DNA above the intercalation site, with a disorganization and disruption of the regular B-DNA structure immediately below the intercalation site. This particular phenomena may be expected to lead to significantly different physicochemical properties for the diethidium complex with respect to other known intercalators, because this sort of vectorial difference in structure above and below the site of intercalation is unknown in existing intercalators, as far as the authors are aware. In addition, we expect the mechanism of interaction between drug and DNA to be described by “direct ligand transfer”, wherein the drug is transferred from duplex DNA to duplex DNA without re-entering the solvent.¹

This work is the first instance known to the authors of a novel drug entity that was deduced solely by mathematical reasoning ² and described subsequently by computational methods. Evidence that diethidium should interact with its target site DNA differently from other known intercalators is strong.     

Thermodynamics of drug-DNA interactions

Many anticancer, antibiotic, and antiviral drugs exert their primary biological effects by reversibly interacting with nucleic acids. Therefore, these biomolecules represent a major target in drug development strategies designed to produce next generation therapeutics for diseases such as cancer. In order to improve the clinical efficacy of existing drugs and also to design new ones it is necessary to understand the molecular basis of drug-DNA interactions in structural, thermodynamic, and kinetic detail. The past decade has witnessed an increase in the number of rigorous biophysical studies of drug-DNA systems and considerable knowledge has been gained in the energetics of these binding reactions. This is, in part, due to the increased availability of high-sensitivity calorimetric techniques, which have allowed the thermodynamics of drug-DNA interactions to be probed directly and accurately. The focus of this article is to review thermodynamic approaches to examining drug-DNA recognition. Specifically, an overview of a recently developed method of analysis that dissects the binding free energy of these reactions into five component terms is presented. The results of applying this analysis to the DNA binding interactions of both minor groove drugs and intercalators are discussed. The solvent water plays a key role in nucleic acid structure and consequently in the binding of ligands to these biomolecules. Any rational approach to DNA-targeted drug design requires an understanding of how water participates in recognition and binding events. Recent studies examining hydration changes that accompany DNA binding by intercalators will be reviewed. Finally some aspects of cooperativity in drug-DNA interactions are described and the importance of considering cooperative effects when examining these reactions is highlighted.     

Using an aryl phenanthroimidazole moiety as a conjugated flexible intercalator to improve the hybridization efficiency of a triplex-forming oligonucleotide

When inserting 2-phenyl or 2-naphth-1-yl-phenanthroimidazole intercalators (X and Y, respectively) as bulges into triplex-forming oligonucleotides, both intercalators show extraordinary high thermal stability of the corresponding Hoogsteen-type triplexes and Hoogsteen-type parallel duplexes with high discrimination to Hoogsteen mismatches. Molecular modeling shows that the phenyl or the naphthyl ring stacks with the nucleobases in the TFO, while the phenanthroimidazol moiety stacks with the base pairs of the dsDNA. DNA-strands containing the intercalator X show higher thermal triplex stability than DNA-strands containing the intercalator Y. The difference can be explained by a lower degree of planarity of the intercalator in the case of naphthyl. It was also observed that triplex stability was considerably reduced when the intercalators X or Y was replaced by 2-(naphthlen-1-yl)imidazole. This confirms intercalation as the important factor for triplex stabilization and it rules out an alternative complexation of protonated imidazole with two phosphate groups. The intercalating nucleic acid monomers X and Y were obtained via a condensation reaction of 9,10-phenanthrenequinone (4) with (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde (3a) or (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-naphthaldehyde (3b), respectively, in the presence of acetic acid and ammonium acetate. The required monomers for DNA synthesis using amidite chemistry were obtained by standard deprotection of the hydroxy groups followed by 4,4'-dimethoxytritylation and phosphitylation.     

On the fidelity of DNA replication. Mechanisms of misincorporation by intercalating agents

Two distinct mechanisms of action for intercalating agents have been delineated: one leading to the production of frameshift misincorporations and the other leading to the production of single-base substitutions. Addition misincorporations are competitive with respect to DNA template (a measure of classical intercalation) but are not competitive with respect to deoxynucleotide substrates. Single-base substitutions are not competitive with template, polymerase, or deoxynucleotide as tested individually, but are proportional to the absolute drug concentration, indicating a ternary complex involving intercalator, polymerase, and template. Increased frequencies of single-base substitutions have not been considered as a general property of intercalators. Using a mutant phi X174 DNA, we demonstrate that intercalators also induce single-base substitutions with natural DNA templates. Reversion of am3 phi X174 DNA occurs only by single-base substitutions at position 587; this is increased 8-fold when the DNA is copied in vitro in the presence of intercalators.