[Antiviral and Antimicrobial Nucleoside Derivatives: Structural Features and Mechanisms of Action].

The emergence of new viruses and resistant strains of pathogenic microorganisms has become a powerful stimulus in the search for new drugs. Nucleosides are a promising class of natural compound, and more than a hundred drugs have already been created based on them, including antiviral, antibacterial and antitumor agents. The review considers the structural and functional features and mechanisms of action of known nucleoside analogs with antiviral, antibacterial, or antiprotozoal activity. Particular attention is paid to the mechanisms that determine the antiviral effect of nucleoside analogs containing hydrophobic fragments. Depending on the structure and position of the hydrophobic substituent, such nucleosides can either block the process of penetration of viruses into cells or inhibit the stage of genome replication. The mechanisms of inhibition of viral enzymes by compounds of nucleoside and non-nucleoside nature have been compared. The stages of creation of antiparasitic drugs, which are based on the peculiarities of metabolic transformations of nucleosides in humans body and parasites, have been considered. A new approach to the creation of drugs is described, based on the use of prodrugs of modified nucleosides, which, as a result of metabolic processes, are converted into an effective drug directly in the target organ or tissue. This strategy makes it possible to reduce the general toxicity of the drug to humans and to increase the effectiveness of its action on cells infected by the virus.

Comparative Analysis of the Biosynthesis of Isoprenoid and Aromatic Cytokinins.

To compare the biosynthesis pathways of aromatic and isoprenoid cytokinins, a series of nucleoside derivatives of natural cytokinins was synthesized and their cytokinin activity was determined in a test system based on the model plant Arabidopsis thaliana. Cytokinin nucleosides are known to lack the hormonal activity until cleaving the ribose moiety at the position 9. Our experiments have shown that both ribo- and 5'-deoxyribo derivatives of N6-isopentenyladenine were able to turn into active cytokinins in planta exhibiting cytokinin activity. By contrast, 5'-deoxy nucleosides of aromatic cytokinins did not show similar activity. Since 5'-deoxy nucleosides cannot phosphorylate in vivo, the direct pathway of active cytokinin formation by cleavage of nucleotides is blocked here. The detected activity in 5'-deoxy nucleosides of isoprenoid cytokinins and the lack of the activity in 5'-deoxy nucleosides of aromatic cytokinins indicates the difference in the biosynthesis of these compounds.

[Biodegradablescaffolds based on chitosan: Preparation, properties, and use for the cultivation of animal cells].

The influence of the conditions of the formation of chitosan hydrogels crosslinked with glutaraldehyde (GA) or genipin (the polysaccharide molecular weight, pH level, and concentration of the chitosan solution) on the gel time and the properties of biopolymer scaffolds for tissue engineering obtained by the freeze-drying of hydrogels was studied. The resulting scaffolds had different structures (morphology, degree of anisotropy, average pore size) and moisture-retaining capacities. The cytotoxicity of biodegradable scaffolds based on chitosan with a low content of genipin and GA was studied for the first time. Using the L929 mouse fibroblasts model line, we demonstrated that scaffolds based on chitosan with a molecular weight of 320 and 190 kDa crosslinked with genipin and GA (0.005 and 0.01 mol/mol of chitosan amino groups) are biocompatible. Using confocal laser microscopy, we demonstrated that the cells are uniformly distributed in all scaffold samples and they successfully grew and proliferated when cultured in vitro for 4 days.

[Replication-competent γ-retrovirus Mo-MuLV expressing green fluorescent protein gene as an efficient tool for screening of inhibitors of retroviruses that use heparan sulfate as the primary cell receptor].

The effect of sulfated polysaccharides on the efficiency of infection of mouse embryonic fibroblast cell lines SC-1 and NIH-3T3 by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the eGFP gene was investigated. It was shown that used polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH 3T3 cells inthe concentrations from 0.01 to 100 µg/ml and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit cell infection by Mo-MuLV, that prevents further expansion of viral infection. It was detected that sulfated polysaccharides are effective inhibitors of other retroviruses, including lentiviruses, that use heparan sulfate as cell receptors for non-specific binding.

Antiviral and Antimicrobial Nucleoside Derivatives: Structural Features and Mechanisms of Action.

The emergence of new viruses and resistant strains of pathogenic microorganisms has become a powerful stimulus in the search for new drugs. Nucleosides are a promising class of natural compounds, and more than a hundred drugs have already been created based on them, including antiviral, antibacterial and antitumor agents. The review considers the structural and functional features and mechanisms of action of known nucleoside analogs with antiviral, antibacterial or antiprotozoal activity. Particular attention is paid to the mechanisms that determine the antiviral effect of nucleoside analogs containing hydrophobic fragments. Depending on the structure and position of the hydrophobic substituent, such nucleosides can either block the process of penetration of viruses into cells or inhibit the stage of genome replication. The mechanisms of inhibition of viral enzymes by compounds of nucleoside and non-nucleoside nature have been compared. The stages of creation of antiparasitic drugs, which are based on the peculiarities of metabolic transformations of nucleosides in humans body and parasites, have been considered. A new approach to the creation of drugs is described, based on the use of prodrugs of modified nucleosides, which, as a result of metabolic processes, are converted into an effective drug directly in the target organ or tissue. This strategy makes it possible to reduce the general toxicity of the drug to humans and to increase the effectiveness of its action on cells infected by the virus.

[Disaccharide nucleosides, an important group of natural compounds].

Main structural features of important group of natural compounds disaccharide nucleosides are reviewed. The preparation and properties of modified oligonucleotides on their basis and incorporation of reactive groups are summarized. Several examples of the use of the obtained compounds for investigation of enzymes of nucleic acids methabolism are given.

[Mechanism of the reaction of glutaraldehyde with chitosan].

The regularities of the reaction of aminopolysaccharide chitosan with glutaraldehyde (GA) have been considered. The equilibrium forms of GA in water have been thoroughly studied by NMR spectroscopy. It has been established that at pH 5.6, the exchange of the protons of O=CHCH2 groups for deuterium occurs, indicating the presence of an anion, a product of the first stage of the aldol reaction; at pH > 7.2, the formation of the products of an aldol reaction and aldol condensation takes place. The kinetics of the reaction between the amino groups of chitosan and GA, the kinetics of gel formation in chitosan solutions in the presence of GA, and the kinetics of changes in the rigidity of gels formed have been studied by UV spectroscopy. IR spectra of cross-linked chitosan have been obtained. It has been shown that chitosan catalyzes the polymerization of GA to form inhomogeneous products; in this process, the length of oligomeric chains in modified or cross-linked chitosan and the concentration of coupled bonds increase with the GA concentration and pH of the reaction medium.

[Topographic anatomy of human thoracic organs in the early fetal period].

The purpose of the present investigation was to evaluate the holotopy, skeletotopy and sintopy of human heart, trachea and esophagus in the early fetal period. The quantitative evaluation of the topography of these organs was given as a result of study of 100 human fetuses aged 16-24 week with the modification of N.I. Pirogov method and using the multiplane histotopographical sections in the multidimensional coordinate system. The horizontal sections were selected, that could be proposed as the standard ones. he results of this investigation may be recommended for the improvement of the ultrasonographic and magnetic resonance tomographic diagnosis and fetal surgery development.

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis.

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2'-, 3'- and 5'-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2'- or 5'-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2'- and 5'-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3'-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2'-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.

Inhibition of Poly(ADP-Ribose) Polymerase by Nucleic Acid Metabolite 7-Methylguanine.

The ability of 7-methylguanine, a nucleic acid metabolite, to inhibit poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) has been identified in silico and studied experimentally. The amino group at position 2 and the methyl group at position 7 were shown to be important substituents for the efficient binding of purine derivatives to PARPs. The activity of both tested enzymes, PARP-1 and PARP-2, was suppressed by 7-methylguanine with IC50 values of 150 and 50 µM, respectively. At the PARP inhibitory concentration, 7-methylguanine itself was not cytotoxic, but it was able to accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin, the widely used DNA-damaging chemotherapeutic agents. 7-Methylguanine possesses attractive predictable pharmacokinetics and an adverse-effect profile and may be considered as a new additive to chemotherapeutic treatment.

Inhibition of growth of estrogen receptor positive and estrogen receptor negative breast cancer cells in culture by AA-etherA, a stable 2-5A derivative.

The design, chemical synthesis and biological activities of a nuclease-resistant, nontoxic bioactive 2-5A derivative, AA-etherA [i.e., adenylyl-(2'-5')-adenylyl-(2'-2")-9-[(2'-hydroxyethoxy)-methyl]adenine], are described as a new approach to the inhibition of breast cancer cell growth. AA-etherA inhibits DNA replication and cell division of both estrogen receptor positive (MCF-7) and estrogen receptor negative (BT-20) breast cancer cells in culture in a dose-dependent manner. Maximal inhibition in MCF-7 and BT-20 cells was obtained with 100 microM AA-etherA after four days of treatment, with an GI50 of 58 and 37 microM, respectively. AA-etherA is stable in the cytoplasm. Treated cells accumulate within the late G1/early S phase of the cell cycle and then progress only very slowly through S phase. AA-etherA does not activate RNase L, as do 2-5A and other 2-5A derivatives, nor does it increase p68 kinase (PKR) content of the cells. High resolution, two-dimensional protein gel electrophoresis reveals twofold or greater inhibition of synthesis of 92 proteins out of 682 proteins that were reproducibly detected as high quality spots with average rates of synthesis of > or = 20 p.p.m. in untreated cells. The specificity of the effects of AA-etherA on select proteins and its failure to activate RNase L indicate that AA-etherA does not act through a general effect on mRNA translation or stability, but rather inhibits cell proliferation through a block to DNA replication, with a concommitant reduction in the synthesis of specific proteins, some of which may be required for cell cycle transit. Two likely targets to account for the AA-etherA inhibition of DNA replication are DNA topoisomerase I, which is inhibited by AA-etherA in other cell lines, and thymidine kinase, which could be inhibited in a manner similar to the effect of acyclovir. These data indicate that 2-5A analogs, particularly bifunctional 2-5A analogs like AA-etherA, will be useful for controlling cancer cell growth. Further development of such 2-5A analogs may provide highly specific compounds for chemotherapy and chemoprevention.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2',5'-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3'-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2'-5')adenylyl(2'-5')adenosine.

Substrate properties of C'-methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation.

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5' position) were shown to be weak substrates of T7 RNAP. 3'Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2'Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the chi angle range of 192-256 degrees of N-type conformation.

Mapping of T7 RNA polymerase active site with novel reagents--oligonucleotides with reactive dialdehyde groups.

Oligonucleotides of a novel type containing 2'-O-beta-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein-nucleic acid interactions.

Synthesis and properties of O-beta-D-ribofuranosyl-(1"-2')-adenosine-5"-O-phosphate and its derivatives.

The synthesis of O-beta-D-ribofuranosyl-(1"-2')-adenosine-5"-O-phosphate and its suitably protected derivative for oligonucleotide synthesis have been developed.

Chemical incorporation of 1-methyladenosine, minor tRNA component, into oligonucleotides.

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.

Probing the MVAI methyltransferase region that interacts with DNA: affinity labeling with the dialdehyde-containing DNA duplexes.

Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2'-O-beta-D-ribofuranosylcytidine or 1-beta-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA.

Fluorescent 2-pyrimidinone nucleoside in parallel-stranded DNA.

Stretches of parallel-stranded (ps) double-helical DNA can arise within antiparallel-stranded (aps) Watson-Crick DNA in looped structures or in the presence of sequence mismatches. Here we studied an effect of a pyrimidinone-G (PG) base pair on the stability and conformation of the ps DNA to explore whether P is useful as a structural probe.

Oligonucleotides containing disaccharide nucleosides: synthesis, physicochemical, and substrate properties.

The efficient synthesis of oligonucleotides containing 2'-O-beta-D-ribofuranosyl (and beta-D-ribopyranosyl)nucleosides, 2'-O-alpha-D-arabinofuranosyl (and alpha-L-arabinofuranosyl)nucleosides. 2'-O-beta-D-erythrofuranosylnucleosides, and 2'-O-(5'-amino-5-deoxy-beta-D-ribofuranosyl)nucleosides have been developed.