Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo.

Exosomes support cell-to-cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF-7 breast carcinoma cells, this compound is non-toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin-exosomal pathway, of potential interest for exosome research and oncology.

Structure and gene cluster of a tyvelose-containing O-polysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T related to Yersinia pseudotuberculosis.

An O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T by mild acid hydrolysis and studied by 2D NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: where Tyv indicates 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. entomophaga are related to those of some Y. pseudotuberculosis serotypes.

[DETERMINATION OF PHYLOGENETIC RELATIONSHIP OF YERSINIA PESTIS STRAINS FROM NATURAL PLAGUE FOCI OF THE CAUCASUS BY MULTI-LO- CUS VNTR-ANALYSIS].

AIM: Determination of the degree of phylogenetic relationship of Yersinia pestis strains iso- lated from the territories of natural foci of plague from the Caucasus using VNTR-typing by 25 loci (MLVA25). MATERIALS AND METHODS: 26 strains of Y pestis from Russian natural foci of the Caucasus were used in the study. 25 loci of tandem repeats in Y pestis genome by Le Fleche scheme were used for execution of multi-locus VNTR-analysis. Deciphering of nucleotide sequences was carried out in automatic sequencer ABI 3130 Genetic Analyser. Analysis of confinement of clusters to certain territories, objects and time of isolation of strains was carried out. using Arc GIS 10.1 program. RESULTS: Groups of MLVA25-types of various levels of discrimination were formed: clusters, groups and subgroups. Clusters were formed by strains ofvarious taxonomic membership: main and subspecies of Y pestis. Subgroups reflect membership of strains in certain foci, and MLVA25-types - the degree of genetic relationship. CONCLUSION: Genetic <> of plague causative agents obtained using MLVA25-types circulating in various natural-focal territories allow to solve problems ofboth theoretical and practical character: from interpretation of microevolution processes to the search of the source of infection and ways of its spread during possible epidemic complications.

[COMPARATIVE ANALYSIS OF THE MLVA25- AND MLVA7-TYPING ACCORDING TO THEIR ABILITY TO ASCERTAIN FOCAL AFFILIATION OF YERSINIA PESTIS STRAINS BY THE EXAMPLE OF ISOLATES FROM THE CENTRAL-CAUCASIAN HIGHLAND NATURAL PLAGUE FOCUS].

Comparative analysis of the MLVA25- and MLVA7-typing ability to evaluate focal belonging of Y. pestis strains by the example of bv. medievalis isolates from the Central-Caucasian highland natural plague focus was carried out. The MLVA25-types of-82 isolates from this area were determined and included into the database containing information on 949 Y. pestis strains from other natural foci of Russia and other countries. Categorical-UPGMA dendrograms were created on the bases of the data concerning all 25 VNTR loci or only seven of them, which were recommended by the experts of the Russian Research Anti-Plague Institute "Microbe" for differentiation of the Y. pestis strains according to their affiliation to specific foci. The obtained data indicated greater possibility of diagnostic mistakes in the case of the MLVA7-typing and supported expediency of division of the Central-Caucasian highland natural plague focus into two sub-foci.

[ON THE ORIGIN OF HYPERVIRULENCE OF THE CAUSATIVE AGENT OF PLAGUE].

The attempt to combine Yersinia pseudotuberculosis and Yersinia pestis into one species has been unsupported by microbiologists due to the specific features of the epidemiology and clinical presentations of their induced diseases and to basic differences in their virulence. Pseudotuberculosis is predominantly a relatively mild human intestinal infection transmitted through contaminated food and plague is an acute generalized disease with high mortality, which is most frequently transmitted by the bites of infected fleas. Y. pestis hypervirulence, the ability of single bacteria to ensure the development of predagonal bacteriemia in rodents, which is sufficient to contaminate the fleas, is one of the main events during pathogen adaptation to a new ecological niche. By analyzing the data of molecular typing of the representative kits of naturally occurring Y. pestis isolates, the authois consider the issues of formation of intraspecies groups with universal hypervirulence, as well as biovars that are highly virulent only to their major host. A strategy for searching for selective virulence factors, the potential molecular targets for vaccination and etiotropic treatment of plague, is discussed.

[Application of the methods of molecular modeling to the search for new biologically active substances].

The searching for new chemical compounds possessing specific biological activity is a complex problem that needs the usage of modern methods of molecular modeling. In particular, for the prupose of searching for potentially active compounds for whole class of SH2 domains, a comparison of all available structures, their cluster analysis, molecu- lar docking, selection of all possible pharmacophore models and GTM prediction were done. Obtained results testify to the considerable variability of binding of SH2 domains.

Selective Protective Potency of Yersinia pestis ΔnlpD Mutants.

It has recently been shown that the NlpD lipoprotein is essential to Yersinia pestis virulence and that subcutaneous administration of the nlpD mutant could protect mice against bubonic and pneumonic plague better than the EV vaccine strain [PLoS One 2009. V. 4. № 9. e7023]. In this study, similar ΔnlpD mutants were generated on the basis of other Y. pestis parent strains, including strains from the subspecies microtus, which is avirulent to guinea pigs and humans. Comparative testing confirmed that immunization of mice with ΔnlpD mutants induces immunity 105 times more potent than the one induced by the administration of the EV vaccine strain. At the same time, NlpD- bacteria failed to protect guinea pigs in the case of a subcutaneous challenge with Y. pestis, inducing a 106 times less potent protection compared with that conferred by immunization with the EV vaccine strain. The possible causes of the observed phenomena are discussed.

NEW CONFORMATIONAL PROPERTIES OF SH2 DOMAIN BINDING POCKET.

The conformational changes of proteins play an important role in biological functioning such as ligand-protein and protein-protein interactions. The aim of the work was to investigate the conformational movement of most represented SH2 domains. It was found that SH2 domain binding pocket includes both flexible and not flexible regions: the central area of the binding pocket is the most unflexible, whereas the pTyr-binding and hydrophobic zones are the most flexible. Results of the computer analysis revealed new conformational properties of SH2 domain, which are important for drug design.

[Molecular typing of Yersinia pestis].

Techniques for differentiating single bacterial isolates into intraspecies clusters corresponding to subspecies, biovars, and natural foci are reviewed. The techniques under consideration are reproducible under different laboratory settings. A version of the intraspecies classification of Y. pestis that is in harmony with the International Code of Nomencláture of Bacteria is suggested.

Identification of Novel IGF1R Kinase Inhibitors by Molecular Modeling and High-Throughput Screening.

The aim of this study was to identify small molecule compounds that inhibit the kinase activity of the IGF1 receptor and represent novel chemical scaffolds, which can be potentially exploited to develop drug candidates that are superior to the existing experimental anti-IGF1R therapeuticals. To this end, targeted compound libraries were produced by virtual screening using molecular modeling and docking strategies, as well as the ligand-based pharmacophore model. High-throughput screening of the resulting compound sets in a biochemical kinase inhibition assay allowed us to identify several novel chemotypes that represent attractive starting points for the development of advanced IGF1R inhibitory compounds.

[Molecular characterization of the multidrug-resistant Acinetobacter baumannii strains and assessment of their sensitivity to the phage AP22].

The molecular analysis of 130 multidrug-resistant nosocomial Acinetobacter baumannii strains was performed. The strains were obtained from patients admitted to different Russian hospitals (Chelyabinsk, Moscow, Nizhni Novgorod, and St. Petersburg) in 2005-2010. Species identification was performed using the amplified 16S rRNA gene restriction analysis and by determining intrinsic for A. baumannii blaQXA-51-like genes using PCR. The genetic typing of the strains was performed by RAPD-PCR. All strains fell into two clusters: A and B with dominant RAPD-groups A1 and B1, respectively, including 82% (107 of 130) of all studied strains. The susceptibility to the bacteriophage AP22 of the strains was determined. The phage was found to infect specifically and to constitute 69% of 130 strains and 82% (88 of 107) of the A. baumannii strains from the dominant RAPD groups. The ability of the bacteriophage AP22 to constitute a broad range of the clinically relevant A. baumannii strains makes it an attractive candidate for designing the phage cocktails intended to control the A. baumannii-associated nosocomial infections. Moreover, the phage can be used for the identification of A. baumannii in bacteriological analysis of clinical materials.

[The phylogeography of the Yersinia pestis vole strains isolated from the natural foci of caucasian region].

57 Y pestis bv. caucasica strains were assayed using molecular typing. The results of these assays indicated the presence within this biovar of the three separate clonal clusters and necessity of detachment of the Leninakan mountain mesofocus (subfocus) from the structure of Transcaucasian-highland focus into self-supporting one, as well as inclusion of a part of the Pre-Araks low-mountain natural plague focus in the capacity of the subfocus along with Pre-Sevan mountain and Zanzegur-Karabakh mountain subfoci into the structure of Transcaucasian-highland focus. It was shown that the strains circulating in the East-Caucasian highland plague focus were the most ancient branch of bv. caucasica or even of the entire Y pestis phylogenetic tree.

[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).

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

[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.

Cloning and expression of protective antigens of Mycobacterium tuberculosis Ag85B and ESAT-6 in Francisella tularensis 15/10.

The possibility of expression of genes encoding mycobacterial antigens in Francisella tularensis 15/10 vaccine strain cells has been shown for the first time. To obtain stable and effective expression of mycobacterial antigens in the F. tularensis cells, the plasmid vector pPMC1 and hybrid genes consisting of the leader part FL of the F. tularensis membrane protein FopA and structural moieties of the mature protein Ag85B or the fused protein Ag85B-ESAT-6 were constructed. Recombinant strains F. tularensis RVp17 and RVp18 expressing protective mycobacterial antigens in the fused proteins FL-Ag85B and FL-Ag85B-ESAT-6, respectively, were obtained. Expression of the protective mycobacterial antigens in F. tularensis was analyzed using specific antisera to the recombinant proteins Ag85-(His)6 and ESAT-6-(His)6 isolated from Escherichia coli producer strains created on the basis of the pET23b(+) and pET24b(+) vectors. The expression of heterologous protective antigens in F. tularensis 15/10 is promising for creation of live recombinant anti-tuberculosis vaccines on the basis of the tularemia vaccine strain.

Novel 5,7-disubstituted 6-amino-5H-pyrrolo[3,2-b]pyrazine-2,3-dicarbonitriles, the promising protein kinase inhibitors with antiproliferative activity.

New derivatives of pyrrolo[2,3-b]pyrazine were synthesized and tested on a panel of cultured human tumor cell lines. It was found that 6-amino-5-(3-chlorophenylamino)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[3,2-b]pyrazine-2,3-dicarbonitrile (4j) exhibited a significant antiproliferative activity: GI50 for cell lines RXF 393 (renal cancer) and BT-549 (breast cancer) were 14 and 82 nM, respectively. To identify possible molecular targets, docking of the most active compounds into the active sites of cyclin-dependent kinases was performed. Molecular modeling of the inhibitor-enzyme complexes showed the differences in the binding poses of new pyrrolo[2,3-b]pyrazine derivatives in the kinase ATP-binding site compared with known pyrrolo[2,3-b]pyrazine inhibitors called aloisines. The patterns of drug kinase interactions correlated well with antiproliferative activities of novel derivatives. Key interactions and binding mode of docked compounds are discussed.

[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.

To what extent can methyl derivatives be regarded as stabilized tautomers of xanthine?

Taking xanthine (Xan) as an example, validity of an approach to experimental investigations of nucleotide bases' tautomeric equilibrium, based on the use of methyl derivatives corresponding to their prototropic tautomers, was studied by (1)H NMR in dimethylsulfoxide (DMSO) and by quantum chemical calculations at the B3LYP/6-311++G(d,p) level of theory. From (1)H NMR spectra of m(7)Xan, m(9)Xan, Xan and m(3)Xan conclusion was made that the N7H tautomeric forms of the last two compounds dominate in solution, which was supported by quantum chemical data. Calculated relative energies of the N9H tautomers of Xan and m(3)Xan (8.74 and 9.57 kcal/mol, accordingly) are rather close to the m(9)Xan one (9.11 kcal/mol). Nonspecific influence of DMSO modelled by the COSMO algorithm therewith reduces these values by approximately 2-3 kcal/mol. The data obtained imply that methyl derivatives are rather good models of high-energy tautomers of nucleotide bases, if their relative energies are not less than a few kcal/mol.