Ethidium Analogs Research Articles

Influence of the Amino Substituents in the Interaction of Ethidium Bromide with DNA

A key step in the rational design of new DNA binding agents is to obtain a complete thermodynamic characterization of small molecule-DNA interactions. Ethidium bromide has served as a classic DNA intercalator for more than four decades. This work focuses on delineating the influence(s) of the 3- and 8-amino substituents of ethidium on the energetic contributions and concomitant fluorescent properties upon DNA complex formation. Binding affinities decrease by an order of magnitude upon the removal of either the 3- or 8-amino substituent, with a further order-of-magnitude decrease in the absence of both amino groups. The thermodynamic binding mechanism changes from enthalpy-driven for the parent ethidium to entropy-driven when both amino groups are removed. Upon DNA binding, fluorescence enhancement is observed in the presence of either or both of the amino groups, likely because of more efficient fluorescence quenching through solvent interactions of free amino groups than when buried within the intercalation site. The des-amino ethidium analog exhibits fluorescence quenching upon binding, consistent with less efficient quenching of the chromophore through interactions with solvent than within the intercalation site. Determination of the quantum efficiencies suggests distinct differences in the environments of the 3- and 8-amino substituents within the DNA binding site.

Covalent attachment of ethidium to DNA results in enhanced topoisomerase II-mediated DNA cleavage.

The classic DNA intercalator, ethidium, was used to probe the effects of (i) intercalation and (ii) covalent modification of the DNA on the catalytic activity of topoisomerase II. Ethidium bromide, which binds reversibly to DNA via intercalation, does not stimulate topoisomerase II-mediated DNA cleavage at concentrations up to 100 microM, indicating that the intercalative binding of this molecule to DNA is not sufficient to alter the activity of the enzyme. In contrast, covalent attachment of the photoreactive ethidium analog to DNA resulted in marked enhancement of topoisomerase II-mediated single- and double-stranded DNA cleavage. This increase in DNA cleavage was observed at very low drug binding densities (<1 drug per 10-80 base pairs) which correspond to nanomolar concentrations, as compared with other topoisomerase II poisons such as etoposide or m-AMSA which require micromolar concentrations to elicit comparable DNA cleavage levels. Over the past decade, topoisomerase II has been an important target for a variety of clinically relevant anticancer agents due to the abilities of these agents to convert this enzyme to a cellular toxin resulting in an increase in the levels of enzyme-mediated DNA breaks. Modification of DNA by covalently attaching a DNA-targeting intercalating agent (i.e., ethidium bromide) resulted in a marked shift of the cleavage/religation equilibrium of the enzyme toward the cleaved state "poison" topoisomerase II as observed by the enhancement in single- and double-stranded cleavage; thus, key insight was gained into the mechanism(s) through which DNA binding agents may influence the catalytic properties of topoisomerase II. These data demonstrate that conversion of a reversible ethidium-DNA complex to an irreversible adduct results in the transformation of an ineffective intercalating drug into a potent topoisomerase II-targeted agent. Finally, they provide support for the recently proposed "positional poisoning model" for the actions of DNA lesions and anticancer drugs on the type II enzyme.

An ethidium analogue that binds with high specificity to a base-bulged duplex from the TAR RNA region of the HIV-I genome.

An ethidium analogue that binds with high specificity to a base-bulged duplex from the TAR RNA region of the HIV-I genome.

Subcellular Localization of Photoreactive Ethidium Analogs in Trypanosoma brucei by Fluorescence Microscopy

To identify the in vivo targets of the trypanocide, ethidium bromide, the fluorescent staining of T. brucei was examined for a series of ethidium analogs using fluorescence microscopy. Determination of the biological targets for most drugs is limited by the reversible nature of their interactions. To overcome this limitation, photoaffinity (azido) analogs of ethidium, which are capable of covalent attachment with photoactivation, were used to identify the ethidium binding sites within the parasites. Two of these compounds, when covalently attached, demonstrated an enhancement of fluorescent staining and were selective for the kinetoplast at low drug concentrations. These compounds were also those found previously to have the highest trypanocidal activity. Propidium, a phenanthridinium analog identical to ethidium except for a larger, more ionic substitution at R5, showed more nonspecific binding as determined by its general staining of the cytoplasm.

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling.

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.

Petite induction in Saccharomyces cerevisiae by ethidium analogs action on mitochondrial genome

Petite induction of ethidium analogs was examined in both resting and growing yeast cells. All of the analogs used in these experiments were active in dividing cells of Saccharomyces cerevisiae; only the parent ethidium bromide was mutagenic under resting conditions. Incorporation of adenine into mitochondrial DNA appeared to be prevented completely by ethidium and partially inhibited by other analogs. Treatment of growing cells with analogs affected fragmentation of pre-existing DNA as seen by the loss of a mitochondrial antibiotic resistance marker. The rates of elimination of the marker were different; ethidium generated greater loss than the monoamino analogs (3-amino and 8-amino-); and the deaminated analog was least effective. However, in resting yeast the marker was partially eliminated only with treatment of the parent ethidium. The degradation of the mitochondrial DNA by exposure to ethidium compounds was confirmed by agarose gel electrophoresis. Electrophoretic patterns of the mitochondrial DNA treated with each of the analogs under growing conditions and only with ethidium under resting conditions showed degradation of the mitochondrial DNA.

Synthesis and characterization of ethidium analogs: Emphasis on amino and azido substituents

AbstractA series of eight ethidium derivatives has been synthesized in which the substituents at R3 and R8 have been varied with hydrogen, azido and amino functions. Three of these compounds are new and their synthesis and characterization by uv‐visible, ir and 1H nmr spectroscopy are presented. The synthesis and characterization of the other compounds are also given for comparison, because the compounds served as precursors, or the synthetic route undertaken for these compounds differed from that reported previously.

Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with an ethidium analog.

We have used photoaffinity labeling of pBR322 DNA with 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride to demonstrate high affinity ethidium-binding sites. Plasmid equilibrated with as little as 1 drug/DNA molecule was photoactivated, freed of uncomplexed drug by ethanol precipitation, and subjected to restriction analysis. There was highly specific, rather than random, blockage of HhaI sites (d(GCGC)) at low drug concentrations. Furthermore, the same 7 new digestion fragments were generated at drug to nucleotide ratios ranging from 1:100 to 1:8000. All the new DNA fragments had chain lengths greater than the largest HhaI fragment (393 base pairs). At higher ligand concentrations closely approximating those needed for equilibrium binding studies, detection of the high affinity sites was greatly masked. Drug binding to HhaI restriction fragments which had been prepared prior to the action of drug did not induce new bands. Furthermore, the larger DNA fragments from drug-labeled plasmid were resistant to HhaI digestion over a wide range of enzyme concentrations. These findings suggest that ligand binding can be highly selective even between sites which have the same tetranucleotide sequence. Therefore, selective drug binding must be dictated not only by local base sequence preference, but also by other long range parameters.

Antitrypanosomal Action Enhanced by Photoaffinity Labeling with Ethidium Analogs

The trypanocidal activity of photoreactive azido analogs of ethidium was tested to determine the suitability of using such compounds as in vivo probes to study the mechanism of the antitrypanosomal activity of ethidium. Eight ethidium analogs, including three nonphotoreactive compounds, were tested for their ability to kill T. brucei both with and without photolytic activation. Two analogs tested, the monoamino-monoazido isomers, showed greater that 100-fold enhancement of trypanocidal activity following photolytic activation in situ. Without photolytic activation, only the nonphotoreactive monoamino precursor analogs showed activity greater than the parent ethidium compound. The availability of suitable ethidium analogs which can be covalently attached by in situ photoactivation provides a new approach for studying the mechanism by which ethidium exerts its trypanocidal activity.

Structure-function characterization for ethidium photoaffinity labels as mutagens in salmonella

The development of photoaffinity probes to characterize the binding process and subsequent biological activity of a drug has recently been emphasized by the synthesis of two ethidium azide analogs. The initial findings showed that one of the azido analogs, the 8-azido-3-amino derivative, was at least 40-fold more mutagenic and toxic in Salmonella tester strain TA1538 than the other analog, the 3,8-diazido derivative. These observations suggested the need to examine the structural requirements of ethidium photoaffinity labels for frameshift mutagenic activity in Salmonella. Thus, the isomer of the monoazide, the 3-azido-8-amino derivative, and two deaminated monoazide derivatives were synthesized and all of the ethidium analogs were screened in two Salmonella frameshift tester strains, TA1537 and TA1538, and in their excision-repair positive isogenic strains. The results presented in this paper demonstrate that two substituents are needed to produce significant mutagenicity and toxicity by the compound. One substituent, usually the amino group, is required for mutagenic activity, perhaps by orienting the phenanthridinium ring into its mutagenic configuration. The other substituent, the azido group, is required for covalent attachment, a requisite for mutagenic activity. Thus, photoaffinity labeling has provided a means of comparing structure with mutagenic activity for ethidium compounds.

Petite induction in Saccharomyces cerevisiae by ethidium analogs : Distinction between resting and growing cells

The importance of specific substituents, especially amino and azide groups, for ethidium induction of petites was evaluated in resting and dividing cells of Saccharomyces cerevisiae through the study of a series of ethidium analogs. The structural requirements in resting and growing cells were found to be different, suggesting that at least two mechanisms are responsible for induction. The significance of particular substituents in the induction processes were recognized by: (1) a dependence upon the ethyl substituent at the ring nitrogen in both actively growing and in resting cells; and (2) the implication that amino substituents are important for the effect in dividing cells and especially in resting cells. Photolytic enhancement of petite induction (via a nitrene which forms a covalent linkage to a biological site) was observed for 3 of the azide analogs, which emphasizes the likelihood that metabolic activation of ethidium to a covalent complex is responsible for its effectiveness. Furthermore, these studies indicate that these monoazide analogs should be ideal probes for examining the mitochondrial mutagenic processes.

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.

Production of frameshift mutations in salmonella by a light sensitive azide analog of ethidium

Frameshift mutations have been produced in specific repair-negative Salmonella tester strains by photoaffinity labeling technique using ethidium azide. Reversions requiring a +1 addition or a −2 deletion were especially sensitive. Mutagenesis was reduced by the simultaneous addition of non-mutagenic ethidium bromide, and was prevented by photolysis of the azide prior to culture addition. Identical tester strains active in DNA excision repair were not mutagenized by the azide. These results are consistent with the interpretation that photolysis of the bound ethidium analog converts the drug from its noncovalent mode of binding (presumably intercalation) to a covalent complex with consequent production of frameshift mutations. Such photoaffinity labeling by drugs which bind to DNA not only confirms the importance of covalent drug attachment for frameshift mutagenesis, but also provides powerful techniques for studying the molecular details of a variety of genetic mechanisms.

Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling

A photosensitive azide derivative of ethidium bromide, 3(8)-amino-8(3)-azido-5-ethyl-6-phenyl phenanthridinium chloride, was prepared which binds covalently to DNA via a light-generated nitrene intermediate. When exposed to light in aqueous solvent the azide loses free nitrogen to yield a reactive nitrene, which reacts with water to form the hydroxylamine derivative. When the drug is photolyzed while tightly bound to DNA, rather than free in solution, a covalent complex is generated in situ due to the attack of the nitrene to form a covalent bond with the nucleic acid. A selectivity for cytoplasmic DNA reported previously for ethidium bromide was also shown for the azido analog by enhanced production of petite mutants in Saccharomyces following the photolysis of the azide in vivo. Isolated mitochondrial DNA following binding of the ethidium analog in vivo also appeared to retain a greater number of drug molecules per nucleotide than nuclear DNA. In vitro binding experiments did not suggest the same extent of preferential binding. This technique of photoaffinity labeling provides a means of studying the precise model of binding nucleic acid active drugs in relation to their biological effects.