Differential Impact of Random GC Tetrad Binding and Chromatin Events on Transcriptional Inhibition by Olivomycin A.

Olivomycin A (OA), an antibiotic of the aureolic acid family, interferes with gene transcription upon forming complexes with GC-rich regions in the DNA minor groove. We demonstrate that the mechanism of transcriptional deregulation is not limited to OA interaction with GC-containing binding sites for transcription factors. Using electrophoretic mobility shift assays and DNAse I footprinting of cytomegalovirus (CMV) promoter fragments carrying OA-preferred GC tetrads (CMVwt), we showed OA binding specifically to GC islands. Replacement of G for A in these tetrads (CMVmut) abrogated OA binding. Furthermore, OA decreased RNA polymerase II (RNAPII) binding to the CMVwt promoter and inhibited the reporter gene expression. In line with the absence of OA binding sites in CMVmut DNA, the expression driven from this promoter was weakly sensitive to OA. In the endogenous genes OA decreased RNAPII on promoters and coding regions. In certain cases this phenomenon was concomitant with the increased histone 3 abundance. However, the sensitivity to OA did not correlate with GC patterns around transcription start sites, suggesting that certain GC stretches play unequal roles in OA-induced transcriptional perturbations. Thus, OA affects transcription via complex mechanisms in which GC tetranucleotide binding causes RNAPII/chromatin alterations differentially manifested in individual gene contexts.

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction.

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug-DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA-DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

The Effect of Antitumor Antibiotic Olivomycin A and Its New Semi-synthetic Derivative Olivamide on the Activity of Murine DNA Methyltransferase Dnmt3a.

Olivomycin A is a highly active antitumor drug that belongs to the family of aureolic acid antibiotics. The antitumor effect of olivomycin A is related to its ability to bind to the DNA minor groove in GC-rich regions as Mg2+-coordinated complexes. Characterization of cellular targets of olivomycin A and its mechanism of action is crucial for the successful application of this antibiotic in clinical practice and development of semi-synthetic derivatives with improved pharmacological properties. Previously, we have shown that minor groove ligands are able to disrupt the key epigenetic process of DNA methylation. In this paper, we have studied the impact of olivomycin A and its improved semi-synthetic analogue N,N-dimethylaminoethylamide of 1'-des-(2,3-dihydroxy-n-butyroyl)-1'-carboxy-olivomycin A (olivamide) on the functioning of de novo DNA methyltransferase Dnmt3a (enzyme that carries out methylation of cytosine residues in the DNA CG-sites in eukaryotic cells) using an in vitro system consisting of the murine Dnmt3a catalytic domain and a 30-mer DNA duplex containing four consecutive GC pairs. We have shown that olivomycin A and olivamide inhibit Dnmt3a with IC50 of 6 ± 1 and 7.1 ± 0.7 µM, respectively. Neither olivomycin A nor olivamide interfered with the formation of the specific enzyme-substrate complex; however, olivomycin A prevented formation of the covalent DNA-Dnmt3a intermediate that is necessary for the methylation reaction to proceed. The inhibitory effects of olivomycin A and olivamide can be explained by the disruption of the enzyme catalytic loop movement through the DNA minor groove (the reaction stage that precedes the covalent bond formation between DNA and the enzyme). The results of this work indicate the epigenetic contribution to the antitumor effect of aureolic acid group antibiotics.

Divalent cations are dispensable for binding to DNA of a novel positively charged olivomycin A derivative.

The current model of binding of the antitumor antibiotic olivomycin A (1) to GC-rich DNA regions presumes that coordination of the magnesium divalent cation with drug dimers is necessary for binding of 1 into the minor groove of the DNA duplex. Previously we have synthesized the derivatives of 1 termed 'short acid' (2) and its N,N-dimethylaminoethylamide (3). The latter compound demonstrated an improved tolerance in vivo compared to 1 and good therapeutic potency in animal models. We herein report that compound 3 is able to form stable complexes with DNA in the absence of Mg2+, in striking contrast to 1 whose binding to the DNA absolutely requires Mg2+. The mode of binding of 3 to DNA is similar in the presence or absence of Mg2+ as determined by circular dichroism. The affinity to DNA of 3 in Mg2+-free solution was similar to that of 1 or 3 in the presence of Mg2+ at low ionic strength. Non-electrostatic contributions to total free energy of binding of 1 and 3 to DNA were comparable for Mg2+-free complexes. Our data strongly suggest that electrostatic interaction of the positively charged 3 can compensate for the absence of divalent ions in complexes with DNA. This new property of the olivomycin A derivative expands the mechanistic knowledge of the modes of interaction with DNA of small molecular weight drug candidates.

Role of the acyl groups in carbohydrate chains in cytotoxic properties of olivomycin A.

A series of olivomycin A derivatives containing different combinations of the acyl residues in the carbohydrate chains was obtained. The formation of complexes of Mg(2+)-coordinated dimers of these compounds with double-stranded DNA was studied using spectral methods such as absorption, fluorescence and circular dichroism (CD) spectral analyses. There was a good correlation of the values of binding constants of complexes (antibiotic)2Mg(2+)-DNA, the quantum yields of fluorescence and changes of the induced CD spectra with topoisomerase I inhibition and cytotoxicity. We demonstrate that the presence of the acyl groups in the saccharide residues of olivomycin A derivatives is absolutely necessary for a high cytotoxic potency of these antibiotics. On the basis of the experimental results and quantum chemical calculations, we presume that the acyl residue in the 4-O-position in the A-sugar residue is involved, to the most part, in the antibiotic-antibiotic interactions in the (olivomycin)2Mg(2+) dimers, whereas the O-acyl group in E-olivomicose residue largely participates in the formation of the (olivomycin)2Mg(2+)-DNA complexes.

Modification of olivomycin A at the side chain of the aglycon yields the derivative with perspective antitumor characteristics.

A novel way of chemical modification of the antibiotic olivomycin A (1) at the side chain of the aglycon moiety was developed. Interaction of olivomycin A with the sodium periodate produced the key acid derivative olivomycin SA (2) in 86% yield. This acid was used in the reactions with different amines in the presence of benzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate (PyBOP) or diphenylphosphoryl azide (DPPA) to give corresponding amides. Whereas olivomycin SA was two orders of magnitude less cytotoxic than the parent antibiotic, the amides of 2 demonstrated a higher cytotoxicity. In particular, N,N-dimethylaminoethylamide of olivomycin SA showed a pronounced antitumor effect against transplanted experimental lymphoma and melanoma and a remarkably high binding constant to double stranded DNA. The therapeutic effects of this derivative were achievable at tolerable concentrations, suggesting that modifications of the aglycon's side chain, namely, its shortening to methoxyacetic residue and blocking of free carboxyl group, are straightforward for the design of therapeutically applicable derivatives of olivomycin A.

Reaction of the antitumor antibiotic olivomycin I with aryl diazonium salts. Synthesis, cytotoxic and antiretroviral potency of 5-aryldiazenyl-6-O-deglycosyl derivatives of olivomycin I.

The azo coupling of the antibiotic olivomycin I (1) with aryl diazonium tetrafluoroborates produced 5-aryldiazenyl-6-O-deglycosyl derivatives of 1. The structures of new compounds were confirmed by (1)H NMR and mass spectrometry analysis. A quantum-chemical study was performed to analyze the possible directions of electrophilic substitution of 1 and the easiness of 6-O-disaccharide hydrolysis in the course of azo coupling. The antiproliferative and anti-retroviral activities of novel derivatives were studied.

2,3-Anhydrosugars in glycoside bond synthesis. Application to 2,6-dideoxypyranosides.

We describe here the first use of 2,3-anhydrosugars as glycosylating agents for the preparation of 2-deoxypyranosides. In particular, the methodology was used to assemble 2,6-dideoxysugar glycosides. Glycosylation of a panel of alcohols with one of two 6-deoxy-2,3-anhydrosugar thioglycosides (8 and 9) in the presence of a Lewis acid afforded 2,6-dideoxy-2-thiotolyl glycoside products in generally excellent yields with an exclusively syn relationship between the aglycon and the C-3 hydroxyl group. Removal of the 2-thiotolyl group can be achieved upon reaction with tri-n-butyltin hydride and AIBN to give the corresponding 2,6-dideoxy pyranosides. Once developed, the method was applied to the synthesis of oligosaccharide moieties in the natural products apoptolidin and olivomycin A.

Modification of the antibiotic olivomycin I at the 2'-keto group of the side chain. Novel derivatives, antitumor and topoisomerase I-poisoning activity.

A novel way of chemical modification of the antibiotic olivomycin I at the 2'-keto group of the side chain of the aglycone moiety was developed. Reaction of olivomycin I with the carboxymethoxylamine hemihydrochloride gave the key intermediate, 2'-carboxymethoxime-olivomycin I, which was further reacted with different amines in the presence of benzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate to give the corresponding amides. The antiproliferative and topoisomerase I (Topo-I)-poisoning activities of the novel derivatives were examined. One of the novel derivatives showed a marked inhibitory activity against Topo-I, a pronounced antitumor activity in in vivo experiments on mice bearing leukemia P-388 and lower toxic side effects compared with the parent olivomycin I.

Olivomycin induces tumor cell apoptosis and suppresses p53-induced transcription.

Olivomycin (DNA-binding antibiotic) in nanomolar concentrations induces apoptosis of human tumor cells and inhibits p53-dependent transcription of the reporter gene (basal and induced by antitumor drugs). Olivomycin aglycon induces no cytotoxicity and does not block transcription.

Characterization of an MDR1 retroviral bicistronic vector for correction of X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) is a hereditary disorder characterized by severe T cell lymphopenia and abnormal B cell function. The disease is caused by mutations in IL2RG, the gene encoding the interleukin-2 receptor common gamma chain (gamma c) shared by several interleukin receptors. A Harvey retroviral bicistronic vector containing an IL2RG cDNA and cDNA encoding the multidrug transporter (MDR1) was constructed to investigate the correction of XSCID. Translation of the MDR1 cDNA is achieved from an internal ribosome entry site (IRES). Mouse fibroblasts transfected or transduced with the vector expressed both membrane proteins as detected with specific monoclonal antibodies by fluorescence activated cell sorting. Two human XSCID B cell lines were transduced using a filter concentration method in combination with phosphate depletion. Significant expression of both proteins was detected by Western blot analysis. This construct might be particularly useful if high expression of gamma c is required, as might be achievable through in vivo selection for drug resistance of recipient lymphocytes.

Genome size and GC-percent in vertebrates as determined by flow cytometry: the triangular relationship.

Genome size and GC-percent were determined by means of a special method of DNA flow cytometry in 154 vertebrate species. For the total dataset, a highly significant positive correlation was found between both parameters. The overall distribution of points is not linear but triangular: a wide range of GC-percent values is observed at the lower end of genome size range, whereas with an increase in genome size the lower limit for GC-percent is elevated, gradually approaching the upper limit (about 46%). In teleost fishes, which occupy the lower part of genome size range, the negative relationship between both parameters was observed. Two positive linear relationships were found between mean genome size and GC-percent of the main vertebrate groups (one includes fishes, amphibians, and mammals, the other consists of reptiles and birds, which show the higher GC-percent for their genome sizes). Distribution of variance between taxonomic levels indicates that GC-percent is more evolutionarily conservative than genome size in anamniotes. Anuran amphibians show the greatest part of genome size variability at the lower taxonomic levels as compared to other vertebrates (with no additional variance already above the genus level). The data obtained with different methods are compared. It is shown that the proposed method can provide useful data for studies on genome evolution and biodiversity.

[The effect of olivomycin on the spontaneous regression of experimental atherosclerosis in rabbits of different ages]. / Vplyv olivomitsynu na spontannu rehresiiu eksperymental'noho aterosklerozu u kroliv riznoho viku.

Development and regression of experimental atherosclerosis in rabbits is an expressed age-related phenomenon, showing the increase in occurrence and pronouncement of atherosclerotic process with age. A new approach to the disease treatment by means of olivomycin is described. It has shown that while influencing considerably the protein biosynthesis of experimental animal, one can accelerate the regression of atherosclerosis by means of olivomycin and reduce protein and lipid metabolism indicators characterizing the disease development.

Cell membrane-dependent chromatin condensation.

It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non-ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNA-specific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed.

Measurement by flow cytometry of genomic AT/GC ratio and genome size.

Flow cytometry with the AT-specific fluorochrome Hoechst 33258 (HO) and the GC-specific fluorochrome olivomycin (OM) was used for measurement of base pair specific DNA content in 20 species of vertebrates. The results were found to be in good correlation with the biochemical literature on base pair frequencies (r = 0.972, P < 1 x 10(-8). This correlation allows one to determine the percent of GC/AT-pairs and genome size from flow cytometric data. The genome sizes obtained were compared with the literature data on flow cytometric genome size values determined with the use of propidium iodide (PI) that is usually believed to be non-base pair specific. The results were found to be in general agreement; however, the previously reported slight GC-preference of PI is confirmed. The optimal conditions for flow cytometry of AT/GC ratio and genome size with the use of OM and HO are discussed. The approach can be useful for research in ecology, fisheries science, species conservation, and other environmental studies as a tool for rapid survey of a vast array of specimens.

[Use of the DNA-specific fluorochrome olivomycin for work with cell cultures]. / Ispol'zovanie DNK-spetsifichnogo fliuorokhroma olivomitsina dlia raboty s kletochnymi kul'turami.

A high-sensitive, easy and rapid proximate method has been developed to reveal mycoplasmas in the monolayer cell cultures using home fluorescent antibiotic olivomycin. This method has been used to screen many cell lines with its high effectiveness being shown. As based on this method the following control methods are worked out: indication of mycoplasmas in animal blood sera, used for the cultivation of cell lines detection of contaminant microorganisms of the cell cultures (fungi and bacteria) and human ureaplasmas.