Judith Markovits

Ethidium dimer: a new reagent for the fluorimetric determination of nucleic acids.

Abstract A dimeric derivative of ethidium is used for fluorimetric assay of nucleic acids. Due to the very high binding affinity of this derivative for DNA and RNA, a significant increase of sensitivity of the ethidium fluorimetric technique for nucleic acids determination is obtained. Using a commercially available instrument, DNA concentrations in the nanogram per milliliter range are determined. In addition, we have found that an acridine ethidium dimer can also be used for a sensitive fluorimetric assay of DNA concentration, while simultaneously providing a determination of the DNA base composition.

Inhibition of DNA topoisomerases I and II and induction of apoptosis by erbstatin and tyrphostin derivatives

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

DNA Mono and Bisintercalators as Models for the Study of Protein Nucleic Acid Interactions: Origin of the High Affinity and Selectivity

In order to analyse the role of symmetry and dimerization on protein DNA interaction, DNA mono and bisintercalating molecules have been prepared and studied as model compounds. Bisintercalating molecules were obtained which have DNA binding affinity one hundred thousand times higher than the corresponding monomer. This increase corresponds almost to the maximum theoretical one. These data show that the mere symmetrical dimerization of a ligand can lead to a very large increase of its binding affinity for a receptor. On the other hand, monointercalating molecules bearing a side chain terminated by a carboxamide group have been synthetized. These compounds can bind to DNA by intercalating their aromatic ring, while the side chain lies in the small groove. The terminal carboxamide group then forms a pair of hydrogen bonds with guanine. This interaction promotes a specificity for GC rich DNA and is responsible for an additional interaction of -1 Kcal/mole.The contribution of similar hydrogen bonds to specificity in protein DNA complexes is discussed.

Inhibition of tRNA aminoacylation by ethidium dimer and several other bifunctional intercalators with or without antitumor activity.

Abstract The effect of various bifunctional intercalators with or without antitumor activity on the tRNA aminoacylation step was examined. The ethidium dimer appears as the strongest inhibitor of aminoacylation of E. coli methionine, phenylalanine and leucine tRNA and of yeast phenylalanine tRNA. Ethidium dimer binds to tRNA through two classes of sites, I and II ( K I > 10 9 M −1 , K II − 10 6 M −1 ) (C.R. Reinhardt, Biophys. J. 25 , 44242 (1979)]. Surprisingly, binding of the drug to the high affinity class of sites causes only partial inhibition of tRNA aminoacylation, while full inhibition requires saturation of the second class of sites. This inhibition is reversed by displacement of the drug by an excess of DNA. In the studied systems, the 7H pyridocarbazole dimers 1 and 2 which have antitumor activity [B.P. Roques, D. Pelaprat, J. Le Guen, G. Porcher, C. Gosse and J. B. Le Pecq, Biochem. Pharmac . 28 , 1811 (1979); D. Pelaprat, A. Delbarre, J. Le Guen, J. B. Le Pecq and B. P. Roques, J. med. Chem . 23 , 1336 (1980)] inhibit aminoacylation at much higher concentrations than that of ethidium dimer. This behavior is discussed in relation to the particular structures of these drugs.

DNA binding, cytotoxicity and inhibitory effect on RNA synthesis of two new 1-nitro-9-aminoacridine dimers.

Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different.

1H NMR Study of the Nature of Bonding Interactions Involved in Complexes Between Nucleic Acids and Intercalating Compounds

Simple monointercalating compounds (acridine, ethidium, ellipticine) self-associate in aqueous solution with association constant in the range of 100 M-1 to 5000 M-1 depending on structure (nature of the ring and substituent) and environment (pH and salt concentration). Bis-intercalators folding and aggregation were studied and the importance of the rigidity of the linking chain to prevent the autostacking was demonstrated. The sequence specificity of intercalating agents was studied at the level of the minihelix formed by autocomplementary ribo-dinucleotides. Chloro-2 methoxy-6 amino-9 acridine exhibits a strong pyrimidine 3x′-5x′ purine preference and the geometry of its intercalated complex with CpG was determined using isoshielding curves. The addition to this acridine ring of a 9 substituted pentylcarboxamido chain confers a specificity of this compound for guanine. The involvement of hydrogen-bonds between the carboxamide group born by the acridine lateral chain and the guanine was clearly demonstrated from temperature depen-cies of NH protons and from the variation of the linewidths of the pentylcarboxamido chain during the fusion of the intercalated complex.

The Dynamic Structural Fluctuations of Nucleic Acid-Drug Complexes

Different modes of structural fluctuations of the DNA double helix have been characterized. Several of them will be reviewed in this course. Measurements of hydrogen exchange kinetics have been extremely useful to demonstrate and study the structural fluctuations associated with the local melting of hydrogen bonds between base pairs, often referred as the breathing reaction. The studies of DNA breathing have recently been reviewed in detail by Englander and Kallenbach (1983). Most of the data have been obtained by tritium exchange. More recently hydrogen exchange in nucleic acids have been studied by a new approach taking advantage of the modification of UV absorption of the bases upon deuteration (Mandai et al., 1979). Schreir and Baldwin (1976) were the first to show the potential usefulness of hydrogen exchange measurements for the study of ligand macromolecule interactions. They were able to deduce in such measurements very small dissociation rate constants. Mandai et al. (1980) measured by absorption spectroscopy the hydrogen deuterium exchange of ethidium bromide in interaction with DNA.