Laboratory of Bioassay
Head of Laboratory
Dmitry Aminin, D.Sc.
Lab staff
Likhatskaya G.N. (senior researcher, docent, PhD), Agafonova I.G. (senior researcher, PhD), Yurchenko E.A. (researcher, PhD), Pislyagin E.A. (senior researcher, PhD), Chingizova (Martiyas) E.A. (senior researcher, PhD), Chaikina E.L. (researcher), Menchinskaya E.S. (senior researcher, PhD).
Dmitry Aminin, D.Sc.
Lab staff
Likhatskaya G.N. (senior researcher, docent, PhD), Agafonova I.G. (senior researcher, PhD), Yurchenko E.A. (researcher, PhD), Pislyagin E.A. (senior researcher, PhD), Chingizova (Martiyas) E.A. (senior researcher, PhD), Chaikina E.L. (researcher), Menchinskaya E.S. (senior researcher, PhD).
The main goals
Study of the biological activity of natural and synthetic compounds. Study of antimicrobial growth-stimulatory activities and phyto-regulatory activity in the models of agricultural plants. Screening of compounds for the detection of cytotoxic, hemolytic, embryotoxic, antibacterial, antifungal and antitumor activity using different cell line cultures. Search for compounds with hepatoprotective and immunomodulatory properties. MRI study of agents having protective properties in experimental models of ischemia, stroke, cerebral arterial hypertension of animals. Reconstruction of biologically active compounds in the bilayer lipid membranes (BLMs) and the investigation of their pore-forming properties. Establishing a relationship between the structure of the substance and its biological activity. Computer simulation of the spatial structure of biological molecules. Modeling the interaction of biologically active compounds with intracellular targets.
Highlights and perspectives
The in vitro interactions between triterpene glycoside, cucumarioside A2-2, isolated from the Far-Eastern holothurian Cucumaria japonica, and mouse splenocyte and peritoneal macrophage biomembranes were studied. Cucumarioside A2-2 exhibited strong cytotoxic effect in the micromolar range of concentrations and showed pronounced immunomodulatory activity in the nanomolar concentration range. Cucumarioside A2-2 effectively interacted with immune cells and increased the cellular biomembrane microviscosity. This interaction led to a dose-dependent reversible shift in cellular membrane potential and temporary biomembrane depolarization; and an increase in [Ca2+]i in the cytoplasm. There are at least two binding sites for [3H]-cucumarioside A2-2 on cellular membranes corresponding to different biomembrane components: a low affinity site match to membrane cholesterol that is responsible for the cytotoxic properties, and a high affinity site corresponding to a hypothetical receptor that is responsible for immunostimulation. The most likely membrane targets of this glycoside can be purinergic receptors of P2X family (predominantly P2X1 and P2X4). Cucumarioside A2-2 acts as an allosteric regulator which able to withdraw purine receptor inactivation by extracellular ATP and provide a recovery of Ca2+ conductivity of macrophage membrane. The pharmacokinetic behavior parameters of cucumarioside A2-2 in mouse spleen was determined after i.p. drug administration. Using differential image analysis of protein 2D gels followed by MALDI-ToF MS identification the number of differentially expressed target proteins were determined after mouse splenocyte incubation with cucumarioside A2-2 and frondoside A. These proteins participate in phagocytosis, cell proliferation, adhesion and motility, and enhance the natural cellular defense barrier that is necessary to fight pathogens.
Cucumarioside A2-2 influences tumor cell viability at micromolar concentrations. The EC50 for glycoside estimated by non specific esterase assay and MTT assay was 2.1 and 2.7 mM, respectively. Cucumarioside A2-2 at sub-cytotoxic range of concentrations exhibits a cytostatic effect by blocking cell proliferation and DNA biosynthesis in the S phase. It may induce apoptosis in tumor cells through caspase-dependent way bypassing activation of p53-dependent segment. The anticancer and pro-apoptotic properties of cucumarioside A2-2 may be due to direct interaction of the glycoside with tumor cells. Cucumarioside A2-2 and ñumaside exhibits antitumor activity against various forms of experimental mouse Ehrlich carcinoma in vivo, both alone and in the combination with cytostatics. Frondoside À, cucumarioside À2-2, and their complexes with cholesterol in non-cytotoxic concentrations block the activity of membrane transport protein, P-glycoprotein in cells of mouse Echrlich carcinoma cells preventing an efflux of fluorescent probe Calcein from the cell. These results in decrease of multidrug resistance (MDR) of cells by glycosides and their complexes with cholesterol may have a perspective in use as blockers of MDR during anticancer therapy.
The anticancer effect of thiacarpine, a synthetic analogue of the known cytotoxic alkaloid polycarpine isolated from the Pacific ascidian Polycarpa aurata, was investigated in vivo experiments using mouse solid Ehrlich carcinoma tumor as the target. A high-resolution magnetic resonance imaging (MRI) technique using a MR tomograph "PharmaScan" US70/16 (Bruker, Germany) was used for visualization and quantification of tumor size. Fluorescence microscopy and image analysis were applied to determine Ehrlich carcinoma cell chromatin condensing (apoptosis) and necrosis in Ehrlich carcinoma cells at the action of thiacarpine in in vitro experiments. The scan and size calculations of the tumor and some mouse organs were carried out during the experiments. Thiacarpine in a total dose of 100 mg/kg was found to exhibit the delay in growth of the mouse tumor. The antineoplastic effect of this compound was accompanied by an increase in the life time of experimental mice in comparison with the control group of animals. The ability of thiacarpine to induce apoptosis in carcinoma cells may contribute to thiacarpine anticancer effects against mice solid Ehrlich carcinoma in vivo detected by MRI.
The influence of histochrome® and mexidol on morphofunctional characteristics of the brain and the behavior of premature aging OXYS rats and Vistar rats were studied. Using the method of MRI it was found that the symptoms of neurodegenerative changes are present in OXYS rats aged 3 months, but in 12 months rats are significantly marked. Histochrome® (at dosage of 1 mg/kg for 5 days) reduced the anxiety and increased search and exploratory activity in one year old OXYS rats that is greater than that of mexidol (4 mg/kg for 7 days). Both drugs had a positive impact upon the morphological and functional characteristics of the brain. Their effects associated with the correction of diffuse white matter changes and reduction of edema were comparable, but only histochrome® reduced the severity of demyelination processes.
Using computer modeling and structural bioinformatics methods high precision full atomic models of 3D-structures were generated for proteins:
porins, immunoglobulin binding protein Skp and phospholipase A of Gram-negative bacteria genus Yersinia; it was found the structural differences OmpC and OmpF porins, the models of Yersinia OmpF porin mutants, a model of complex Skp trimer with human immunoglobulin IgG1;
actinoporins, neurotoxins and Kunitz-type inhibitors from the tropical sea anemone Heteractis crispa (formerly Radianthus macrodactylus), and actinoporins from the sea anemone Oulactis orientalis of the Japan Sea; detected differences actinoporins tropical and northern seas, and it is shown that sphingomyelin-dependent actinoporin-like type of action is due to structural conservative part of the molecules; built models of the anemone peptides complexes with target proteins (G-actin, integrin, trypsin, chymotrypsin and ion channels);
mannan-binding lectin from the coelomic fluid of the sea urchin Strongylocentrotus nudus and show that features substrate specificity of the lectin due to differences in the structure of the loops involved in the formation of the carbohydrate binding site
All-atomic models of the spatial structure of hydrolytic enzymes from marine organisms and bacteria were generated and established the structures of their active sites: α-N-acetylgalactosaminidase of marine bacterium Arenibacter latericius KMM 426T, modifying the blood group A antigen; α-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM, modifying the blood group B antigen; S1-type nuclease of marine fungus Penicillium melinii; endo-1,3-β-D-glucanases from the shellfishes and their complexes with molecules of the substrates, an inhibitor and an acceptor.
All-atomic models of the spatial structure of chitosan and its derivatives, LPS and chitooligosaccharide complexes with LPS, myeloid differentiation factor-2 and Toll-like receptor-4 were generated. LPS complexes with the lipid bilayer modified acyl derivatives of chitosan were generated and potential binding sites and the energy of interaction of LPS with modified bilayer were identified
A theoretical models of the 3D-structure of chitosan, kappa-carrageenan and their complexes in solution were generated. It was found that chitosan can have multiple binding sites with carrageenan double helix
The pore-forming activity and thermal stability of porins were studied and the 3D-structure models were generated (OmpF , OmpC and OmpY porins Yersinia pseudotuberculosis; OmpC and OmpY porins from Y. ruckeri, OmpC and OmpY porins Y. enterocolitica, mutant OmpF-porins Y. pseudotuberculosis with deletion outer loops L1, L6 and L8, porin from the marine bacterium P. haloplanktis).
Phyto-regulatory activity was studied for series of natural and synthetic compounds: virescenosides A, B, C, F, G, M, N, P, Q and V, cyclopentane β, β'- triketones, coruscanone B and its methyl enol ether, coruscanone A and its 14 synthetic analogues, various classes of lipids, sterols, pigments, phenolic compounds, polysaccharides and ethanol extract derived from a medicinal plant Kalanchoe daigremontiana, and extracts of brown algae polysaccharides extracted from seaweed: laminaran and fucoidan from L. cichorioides, polymannuronic acid and fucoidan from F. evanescens, Antivir and β-D- glucooligosaccharides - products of enzymatic transformation of laminarans. Plant cultures which are sensitive to phyto-regulatory action and parameters of this regulatory action were established. Cyclopentane β, β'-triketones were found to be a new class of plant growth regulators.
The effect of decumbenones A, B and C from the marine-derived strain of the fungus Aspergillus sulphureus on the growth of seedling roots of buckwheat, wheat, barley and corn was studied. It was shown that decumbenone B had a stimulatory effect on the growth of seedling roots of buckwheat, decumbenone A - on the growth of seedling roots of spring soft wheat, decumbenone C - on the growth of seedling roots of spring barley, decumbenone A, B and C - on the growth of seedling roots of corn. The stimulatory effect for some substances was shown at ultra-low concentrations of 10−12 - 10−18 M. It is shown that biologically active substances from marine organisms have not only a stimulatory effect on the growth of buckwheat seedling roots, but also stimulate productivity and rutin content there.
Study of the biological activity of natural and synthetic compounds. Study of antimicrobial growth-stimulatory activities and phyto-regulatory activity in the models of agricultural plants. Screening of compounds for the detection of cytotoxic, hemolytic, embryotoxic, antibacterial, antifungal and antitumor activity using different cell line cultures. Search for compounds with hepatoprotective and immunomodulatory properties. MRI study of agents having protective properties in experimental models of ischemia, stroke, cerebral arterial hypertension of animals. Reconstruction of biologically active compounds in the bilayer lipid membranes (BLMs) and the investigation of their pore-forming properties. Establishing a relationship between the structure of the substance and its biological activity. Computer simulation of the spatial structure of biological molecules. Modeling the interaction of biologically active compounds with intracellular targets.
Highlights and perspectives
The in vitro interactions between triterpene glycoside, cucumarioside A2-2, isolated from the Far-Eastern holothurian Cucumaria japonica, and mouse splenocyte and peritoneal macrophage biomembranes were studied. Cucumarioside A2-2 exhibited strong cytotoxic effect in the micromolar range of concentrations and showed pronounced immunomodulatory activity in the nanomolar concentration range. Cucumarioside A2-2 effectively interacted with immune cells and increased the cellular biomembrane microviscosity. This interaction led to a dose-dependent reversible shift in cellular membrane potential and temporary biomembrane depolarization; and an increase in [Ca2+]i in the cytoplasm. There are at least two binding sites for [3H]-cucumarioside A2-2 on cellular membranes corresponding to different biomembrane components: a low affinity site match to membrane cholesterol that is responsible for the cytotoxic properties, and a high affinity site corresponding to a hypothetical receptor that is responsible for immunostimulation. The most likely membrane targets of this glycoside can be purinergic receptors of P2X family (predominantly P2X1 and P2X4). Cucumarioside A2-2 acts as an allosteric regulator which able to withdraw purine receptor inactivation by extracellular ATP and provide a recovery of Ca2+ conductivity of macrophage membrane. The pharmacokinetic behavior parameters of cucumarioside A2-2 in mouse spleen was determined after i.p. drug administration. Using differential image analysis of protein 2D gels followed by MALDI-ToF MS identification the number of differentially expressed target proteins were determined after mouse splenocyte incubation with cucumarioside A2-2 and frondoside A. These proteins participate in phagocytosis, cell proliferation, adhesion and motility, and enhance the natural cellular defense barrier that is necessary to fight pathogens.
Cucumarioside A2-2 influences tumor cell viability at micromolar concentrations. The EC50 for glycoside estimated by non specific esterase assay and MTT assay was 2.1 and 2.7 mM, respectively. Cucumarioside A2-2 at sub-cytotoxic range of concentrations exhibits a cytostatic effect by blocking cell proliferation and DNA biosynthesis in the S phase. It may induce apoptosis in tumor cells through caspase-dependent way bypassing activation of p53-dependent segment. The anticancer and pro-apoptotic properties of cucumarioside A2-2 may be due to direct interaction of the glycoside with tumor cells. Cucumarioside A2-2 and ñumaside exhibits antitumor activity against various forms of experimental mouse Ehrlich carcinoma in vivo, both alone and in the combination with cytostatics. Frondoside À, cucumarioside À2-2, and their complexes with cholesterol in non-cytotoxic concentrations block the activity of membrane transport protein, P-glycoprotein in cells of mouse Echrlich carcinoma cells preventing an efflux of fluorescent probe Calcein from the cell. These results in decrease of multidrug resistance (MDR) of cells by glycosides and their complexes with cholesterol may have a perspective in use as blockers of MDR during anticancer therapy.
The anticancer effect of thiacarpine, a synthetic analogue of the known cytotoxic alkaloid polycarpine isolated from the Pacific ascidian Polycarpa aurata, was investigated in vivo experiments using mouse solid Ehrlich carcinoma tumor as the target. A high-resolution magnetic resonance imaging (MRI) technique using a MR tomograph "PharmaScan" US70/16 (Bruker, Germany) was used for visualization and quantification of tumor size. Fluorescence microscopy and image analysis were applied to determine Ehrlich carcinoma cell chromatin condensing (apoptosis) and necrosis in Ehrlich carcinoma cells at the action of thiacarpine in in vitro experiments. The scan and size calculations of the tumor and some mouse organs were carried out during the experiments. Thiacarpine in a total dose of 100 mg/kg was found to exhibit the delay in growth of the mouse tumor. The antineoplastic effect of this compound was accompanied by an increase in the life time of experimental mice in comparison with the control group of animals. The ability of thiacarpine to induce apoptosis in carcinoma cells may contribute to thiacarpine anticancer effects against mice solid Ehrlich carcinoma in vivo detected by MRI.
The influence of histochrome® and mexidol on morphofunctional characteristics of the brain and the behavior of premature aging OXYS rats and Vistar rats were studied. Using the method of MRI it was found that the symptoms of neurodegenerative changes are present in OXYS rats aged 3 months, but in 12 months rats are significantly marked. Histochrome® (at dosage of 1 mg/kg for 5 days) reduced the anxiety and increased search and exploratory activity in one year old OXYS rats that is greater than that of mexidol (4 mg/kg for 7 days). Both drugs had a positive impact upon the morphological and functional characteristics of the brain. Their effects associated with the correction of diffuse white matter changes and reduction of edema were comparable, but only histochrome® reduced the severity of demyelination processes.
Using computer modeling and structural bioinformatics methods high precision full atomic models of 3D-structures were generated for proteins:
porins, immunoglobulin binding protein Skp and phospholipase A of Gram-negative bacteria genus Yersinia; it was found the structural differences OmpC and OmpF porins, the models of Yersinia OmpF porin mutants, a model of complex Skp trimer with human immunoglobulin IgG1;
actinoporins, neurotoxins and Kunitz-type inhibitors from the tropical sea anemone Heteractis crispa (formerly Radianthus macrodactylus), and actinoporins from the sea anemone Oulactis orientalis of the Japan Sea; detected differences actinoporins tropical and northern seas, and it is shown that sphingomyelin-dependent actinoporin-like type of action is due to structural conservative part of the molecules; built models of the anemone peptides complexes with target proteins (G-actin, integrin, trypsin, chymotrypsin and ion channels);
mannan-binding lectin from the coelomic fluid of the sea urchin Strongylocentrotus nudus and show that features substrate specificity of the lectin due to differences in the structure of the loops involved in the formation of the carbohydrate binding site
All-atomic models of the spatial structure of hydrolytic enzymes from marine organisms and bacteria were generated and established the structures of their active sites: α-N-acetylgalactosaminidase of marine bacterium Arenibacter latericius KMM 426T, modifying the blood group A antigen; α-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM, modifying the blood group B antigen; S1-type nuclease of marine fungus Penicillium melinii; endo-1,3-β-D-glucanases from the shellfishes and their complexes with molecules of the substrates, an inhibitor and an acceptor.
All-atomic models of the spatial structure of chitosan and its derivatives, LPS and chitooligosaccharide complexes with LPS, myeloid differentiation factor-2 and Toll-like receptor-4 were generated. LPS complexes with the lipid bilayer modified acyl derivatives of chitosan were generated and potential binding sites and the energy of interaction of LPS with modified bilayer were identified
A theoretical models of the 3D-structure of chitosan, kappa-carrageenan and their complexes in solution were generated. It was found that chitosan can have multiple binding sites with carrageenan double helix
The pore-forming activity and thermal stability of porins were studied and the 3D-structure models were generated (OmpF , OmpC and OmpY porins Yersinia pseudotuberculosis; OmpC and OmpY porins from Y. ruckeri, OmpC and OmpY porins Y. enterocolitica, mutant OmpF-porins Y. pseudotuberculosis with deletion outer loops L1, L6 and L8, porin from the marine bacterium P. haloplanktis).
Phyto-regulatory activity was studied for series of natural and synthetic compounds: virescenosides A, B, C, F, G, M, N, P, Q and V, cyclopentane β, β'- triketones, coruscanone B and its methyl enol ether, coruscanone A and its 14 synthetic analogues, various classes of lipids, sterols, pigments, phenolic compounds, polysaccharides and ethanol extract derived from a medicinal plant Kalanchoe daigremontiana, and extracts of brown algae polysaccharides extracted from seaweed: laminaran and fucoidan from L. cichorioides, polymannuronic acid and fucoidan from F. evanescens, Antivir and β-D- glucooligosaccharides - products of enzymatic transformation of laminarans. Plant cultures which are sensitive to phyto-regulatory action and parameters of this regulatory action were established. Cyclopentane β, β'-triketones were found to be a new class of plant growth regulators.
The effect of decumbenones A, B and C from the marine-derived strain of the fungus Aspergillus sulphureus on the growth of seedling roots of buckwheat, wheat, barley and corn was studied. It was shown that decumbenone B had a stimulatory effect on the growth of seedling roots of buckwheat, decumbenone A - on the growth of seedling roots of spring soft wheat, decumbenone C - on the growth of seedling roots of spring barley, decumbenone A, B and C - on the growth of seedling roots of corn. The stimulatory effect for some substances was shown at ultra-low concentrations of 10−12 - 10−18 M. It is shown that biologically active substances from marine organisms have not only a stimulatory effect on the growth of buckwheat seedling roots, but also stimulate productivity and rutin content there.