National Science Centre

The aim of the project will be the synthesis and determination of biological activity of new pyrido [1,2-c] pyrimidine derivatives, compounds with double bond to 5-HT1AR and SERT. The results of in vitro and in vivo biological tests will allow the assessment of the impact of the modifications of the structure of compounds on their biological activity. The premise of the research is to obtain a new third-generation antidepressant from the SSRI + or MTA (Multi Target Agents) group. The research on both groups (SSRI +, MTA) is one of the leading directions of medical chemistry research on a new antidepressant, and in the case of research in the MTA group - pioneering research. In order to achieve the aim of the research, the knowledge of the team implementing the project will be used in the field of medical chemistry, physicochemistry, pharmacokinetics, pharmacology and in silico research. The implementation of the project's research objective will be based on the following methodology: - designing ligand structures based on structural analogies with known ligands with binding to 5-HT1AR or SERT, modifications of the leading structures and preliminary results of molecular modeling final relationships; synthesis of final compounds planned in the project in five independent series (synthesis of over 60 new compounds is planned); - preparation of analytical samples for physicochemical and biological tests and evaluation of their purity (HPLC); - confirmation of the chemical structure (by IR, NMR spectroscopy methods) and composition (elemental analysis and HRMS spectrometry), carried out for all final compounds; - in vitro affinity test for 5-HT1AR and SERT using appropriate radioligands (for all final compounds); - in vitro radioreceptor studies in an extended receptor profile (5-HT2A, 5-HT2c, 5-HT6, 5-HT7, α and D2) for ligands showing the highest affinity for one of the 5-HT1AR or SERT molecular targets, - in vivo study functional activity at pre- and postsynaptic 5-HT1A receptors (mousse induced hypothermia test and rat LLR test); - Phase I transition stability study for selected ligands from both series (ADME study); - study of antidepressant activity for compounds showing the highest binding to 5-HT1AR and SERT and having the highest stability in phase I pass (Porsolt test and mobility test); - in silico testing for ligands of both series (molecular docking, SAR); - SAR analysis for all tested compounds; structure optimization based on SAR, ADME and in silico research, which may significantly affect the effective search for the leading structure in research on new SSRI and or MTA antidepressants. The synthesis carried out under the project will contribute to the knowledge of the medical chemistry of heterocyclic compounds showing biological activity. The promising results of previous studies on pyrido [1,2-c] pyrimidine derivatives indicate a high probability of obtaining ligands with high affinity for 5-HT1AR and SERT, which may lead to an effective new third-generation SSRI antidepressant. On the other hand, the planned research on MTA ligands may lead to an antidepressant with a new mechanism of action, a shorter latency period and greater effectiveness in comparison with the second-generation SSRI drugs most frequently used in the pharmacotherapy of depression. The results of biological research envisaged in the project will allow to determine the influence of the ligand structure modifications on binding to 5-HT | AR and SERT, optimization of the leading structure leading to obtaining new compounds with antidepressant activity in the SSRH or MTA group.

The aim of the presented project is to obtain by biosynthesis a series of new, hitherto undescribed macromolecular compounds - exopolysaccharides of fungal origin (β-glucans) containing selenium atoms in their structure, and then to study the influence of selenium incorporation into the polysaccharide chain on their structural features and immunomodulatory activity. The presented project is a continuation of the research carried out in 2010-2011 as part of the own research project MEiN N N405 613238. As a result of the research carried out in this period, the conditions for the cultivation of mycelium L. edodes on substrates enriched with selenium compounds were optimized, and speciation studies of organic selenium compounds biosynthesized were carried out by mycelial cultures, and one selenium-containing polysaccharide fraction was isolated. Preliminary studies of the effect of the isolated selenium polysaccharide fraction on the proliferation of human blood lymphocytes indicated its strong and selective immunosuppressive effect with very low toxicity. The research results were so promising that the possible use of biosynthesized selenium polysaccharides as immunosuppressants was covered by a patent application in 2012 (P. 402082). Both the mechanism of selenium incorporation into fungal exopolysaccharide molecules, as well as their structure and the mechanism of the immunosuppressive activity detected by us in preliminary studies are so far unknown. Therefore, in the presented project, research is planned to clarify this interesting problem. They also concern the hitherto unexplored issues of biosynthesis, structure and mechanism of action of selenopolysaccharides. We intend to use the methods in three areas of the research: In the biotechnological part of the research: - We will conduct the biosynthesis of selenium-enriched exoplisaccharides by deep mycelial culturing of the medicinal mushroom Lentinula edodes, belonging to the Basidiomycetes class, using culture media enriched with various selenium compounds. - We will use (we will develop) a method of fractionating these compounds that will enable the isolation of polysaccharides with different molar mass, monosaccharide composition and selenium content by using eluents of different polarity and chemical nature for the extraction. In the analytical part of the research: - We will apply the previously unknown method in which selenium is bound to the structure of polysaccharides by the method of X-ray absorption spectroscopy (XANES, XAFS) and nuclear magnetic resonance (selenium NMR spectra) - We will conduct a thorough analysis of the structure of selenopolysaccharides by using the following methods: to determine the monosaccharide composition, after complete hydrolysis, the method of high-performance liquid chromatography, • to determine the molar mass of isolated polysaccharides, we will use the gel chromatography technique (GPLC), • to determine the primary structure, we will use degradation methods, using enzymatic hydrolysis and a modified Hakomori method - by preliminary methylation and then hydrolysis of the analyzed fractions, • to determine the type of glycosidic bonds, we will use the IR and NMR spectral methods, • to determine the spatial structure, we will use (2D) NMR methods. e-polysaccharides with different molecular weight, selenium content and structure, and on the proliferation of human blood T and B lymphocytes, by conducting OKT3, PHA and SAC tests - We will investigate whether Se-polysaccharides have the ability to activate macrophages, NK cells, and whether they affect the dependent response from T lymphocytes. We assume that the results of the planned studies will allow us to formulate a hypothesis regarding the causes of atypical for polysaccharides of fungal origin, found in preliminary studies on the immunosuppressive activity of seleniated polysaccharide fractions. We also assume that we will be able to investigate to what extent selenium incorporation into the exopolysaccharide molecule changes their spatial structure, which in turn probably affects the binding of these compounds with CR-1 and CR-3 receptors. The research results should also broaden the scope of knowledge on the metabolism of selenium compounds by fungi and the possibility of their biosynthesis of selenopolysaccharides. The research should result in publications in journals from the JCR list and congress reports. We assume that the research will contribute to the development of young academic staff, constituting the subject of thesis / doctoral dissertations. It is also probable that their results will be used to design and produce an innovative drug with immunosuppressive activity, belonging to a new chemical group.

The aim of the planned research is to detect the presence of 13 two-component regulatory systems (TCS) identified in the genome of S. mutans UA159 (ATCC 700610) and to investigate the role of four of them, by constructing deletion mutants of S. mutans clinical isolates, in the formation of dental plaque in children and adults, taking into account two types of teeth (permanent and milk). The obtained strains with inactivated genes encoding sensor domains will be analyzed in terms of the produced biomass and the structure of the created biofilm. As part of this project, it is also planned to assess the drug susceptibility of clinical strains of S. mutans and the obtained mutants to selected antibiotics and chemotherapeutic agents. Swabs from the tooth surfaces of children and adults will be taken and inoculated onto Mitis Salivarius Agar supplemented with sucrose, bacitracin and potassium tellurite, and then incubated for 48 h at 37 ° C in 5% CO2 concentration. The identification will involve two steps: identification based on biochemical features and genetic identification using the nested-PCR method. The project will then rely on basic molecular biology techniques (ie isolation of genetic material, PCR, Multiplex PCR, restriction analysis, etc.). Multiplex PCR and / or classical PCR will be used to identify 13 TCS. In turn, the method of ligating mutagenesis will be used to inactivate genes encoding two-component systems. The ability and intensity of in vitro biofilm formation on polystyrene titration plates, both by clinical isolates and mutant strains, will be assessed by crystal violet staining and by MTT staining using a spectrophotometer. The biofilm culture will be established on BHI medium and incubated at 37 ° C in 5% CO concentration for 24 hours to 48 hours. In turn, the structure of the biofilm will be examined using scanning electron microscopy. The assessment of drug susceptibility of clinical strains of S. mutans and mutants will be carried out with the use of the Vitek-2 Compact system. S. mutans is one of the most important species of microorganisms among the microflora inhabiting the oral cavity of adults and children, directly related to the formation of caries in permanent and deciduous teeth. Caries is a very serious and common infectious disease, and despite the advances in dentistry and hygiene, it remains a significant problem in our society. One of the important factors in the virulence of carcinogenic bacteria, including S. mutans, is the ability to create plaque, the so-called natural biofilm. In a diverse environment, which is the oral cavity, bacteria living in the structure of the biofilm are exposed to various adverse environmental factors, such as changes in temperature, nutritional conditions and oxygen intensity. Their ability to adapt, survive and cause such a serious disease as caries indicates the need for an in-depth analysis of the molecular mechanisms of the response of bacteria living in the biofilm to the stress conditions encountered. As demonstrated in the case of S. mutans, the response to environmental changes is mediated by two-component regulatory systems. Therefore, an in-depth understanding of the structure and functioning of TCS seems to be necessary for the complete elimination or modification of the characteristics of this microorganism, the more so as recent years' research has shown that two-component regulatory systems may constitute new potential sites of drug action.