[Novel hybrid inhibitors of the phage T7 RNA polymerase: synthesis, docking and screening in vitro].

A number of new hybrid heteroaromatic compounds, consisting of tricyclic fragments (acridone, thioxanthone and phenazine) and bicyclic fragments (benzimidazole, benzothiazole and benzoxazole) were synthesized using the method, developed by the authors. As a result of screening against the transcription model system of the phage T7 DNA-dependent RNA polymerase three effective inhibitors of the RNA syntheses with the IC50 value of 8.9, 5.7 and 19.8 microM were detected. To cast light on the mode of interaction between the synthesized compounds and the target, the molecular docking was applied to the model pocket of the phage T7 RNA polymerase transcription complex. It was established that these ligands form networks of H-bonds with residues of the pocket conservative amino acids and pi-interaction with the Mg2+ ion. A planar geometry of the hybrid molecules, realized due to the intramolecular H-bonds, proved to be an important structural feature, which correlates with an efficacious inhibitory activity.

[Design of transcription inhibitors on the basis of n-arylamides of 9-methyl- and 9-methoxyphenazine-1-carboxylic acids].

A convenient method of synthesis was developed and two series of N-arylamides of 9-methyl- and 9-methoxyphenazine-1-carboxylic acids were obtained. By the molecular docking method the mode of the synthesized compounds interaction with catalytic pocket of the RNA polymerase T7 transcription complex was simulated. Key ligand-receptor intermolecular contacts were identified. They are realized by various types of non-covalent interactions with line of conservative amino acid residues involved in recognition of incoming nucleotide, catalytic act of RNA synthesis as well as in stabilizing the RNA-DNA hybrid at early steps of transcription. In silico data indicate sufficient affinity of ligands for the receptor and allow to predict their ability to inhibit the functioning of RNA polymerase T7 transcription complex that is consistent with preliminary experimental results. Initial testing in a model RNA polymerase T7 transcription system demonstrates significant inhibition of in vitro RNA synthesis by investigated compounds at a concentration of 25 microg/ml (approximately 80 microM).

[New amides of phenazine-1-carboxylic acid: antimicrobial activity and structure-activity relationship].

Complex investigation of new phenazine-1-carboxylic acid (PCA-1) phenylamides allowed to reveal their ability for substantial growth retardation of three gram-positive bacterial strains--Micrococcus sp., Erysipelothrix rhusiopathiae and Staphylococcus aureus. The strong inhibitory activity of PCA-1 derivatives towards the RNA synthesis in in vitro T7-RNA-polymerase transcription system was also shown, and this property depended on concentration and structure of the tested compounds. The methods of computer modeling outlined the possible mechanism of RNA synthesis inhibition by PCA-1 amides: this process is arisen due to formation of stable complex of substances with enzyme at the position of substrate (rNTP) binding site. The revealed accordance of suppressor PCA-1 amides action in the enzymatic transcription system with antibacterial activity of these agents allows assuming that DNA-dependent RNA polymerase might be one of the cellular targets for tested bacteria. Such an approach permits to propose the use of such in vitro transcription model system to reveal biologically active substances among newly synthesized compounds, having close action mechanism.

[Isoforms of Bacillus subtilis IMV B-7014 lectin].

The subunit organization and regulatory features of extracellular sialic acid specific lectin produced by saprophytic strains Bacillus subtilis IMV B-7014 have been investigated. Autofocusing method was used to identify three lectin isoforms that were distinguished by physicochemical and biological properties. Using the transcription system in vitro it was established that one of the targets of bacterial lectin action was DNA-dependent RNA-synthesis. Lectin isoforms affect differently the synthesis: from its full inhibition to the absence of the effect on the yield of RNA-transcription product.

[Regulatory properties of Bacillus subtilis isolectins].

The ability of natural and mutant Bacillus subtilis cultures with imperfect reparation/recombination system to synthesis of extracellular and surface lectins was investigated, and dependence of lectin production process on cultures' genotype was proved. Mutant B. subtilis recP has practically lost its ability to produce the extracellular lectins as a result of mutation of a gene of the reparation/recombination system. The application of the method "autofocusing" allowed to investigate all the spectrum oflectin molecular forms of natural B. subtilis culture and to reveal isoforms distinguished by physico-chemical and hemagglutination properties. It was shown that lectin cathode forms inhibit the transcription process from plasmid promnoter completely, and anodic forms activate the transcript formation slightly in the transcription in vitro with T7 bacteriophage DNA-dependent RNA-polymerase.

[Lectins from Sambucus nigra L inflorescences: isolation and investigation of biological activity using procaryotic test-systems].

Isolation of lectins from extracts of the Sambucus nigra inflorescences and of pollen material have been performed using isoelectric focusing without carrier ampholytes (autofocusing). Fractions active in agglutination tests with different carbohydrate specificity were subjected to SDS-PAGE. The major lectin found in whole inflores-cences was GalNAc specific and is proposed to be a heterotetramer with subunits of about 30 and 33 kDa. It was called SNAflu-I. At least two other lectins were present in the pollen material and supposed to consist of identical subunits. Major positively charged lectin was Glc/Man specific with subunit of 26 kDa and called SNApol-I. Other pollen component (SNApol-II) was Gal specific with subunit of about 20 kDa. In order to elucidate cell targets sensitive for the S. nigra lectin's activity the combined effects of the lectins and transcriptional of phenazine origin on B. subtilis cells growth have been studied. Only SNApol-I demonstrated the antagonistic activity against these inhibitors in vivo. This lectin but not the SNAflu-I can also inhibit transcription in vitro. It is supposed that lectins from the same source may act in different directions on cell metabolism. Particularly one of the common targets may be the DNA-dependent synthesis of RNA.

[Lectins modulate the cytostatic action of the composite bioregulators-conjugates of azaanalogues of pyrimidine and phenazine-1-carboxilic acid in Bacillus subtilis test-system].

The idea of the work was to study a combined effect of some lectins (Con A, PHA, STA, WGA, SNA, VAA) and new composite bioregulators (PhCA-1 and azapyrimidine derivatives) on growth of Bacillus subtilis cells in order to elucidate cell targets sensitive to lectin's activity. Study of combined effects of high- and low-molecular bioregulators may also be the subject of practical interest with a prospect of obtaining new bacterio-and carcinostatic drugs. Using B. subtilis mutants it was shown that lectins can modulate the cytostatic effect of initial substanses and their pyrimidine derivatives in the range: phenazine < PhCA-1 < 6-azacytosine derivative of PhCA-1 < 6-azauracyl derivative of PhCA-1. This modulating effect was absent in recP mutant, which has lost the ability to produce its own bacterial lectin, demonstrating its possible role as a mediator. The antagonistic effect of all plant lectins under study on cytostatic action of <6-azauracyl derivative of PhCA-1 in rec+ culture was observed as the result of possible competition for some common target. As both the B. subtilis lectin and the low-molecular bioregulator can inhibit transcription in vitro, it is supposed that their common target may be the DNA-dependent synthesis of RNA.