In this work, complexes of cysteine proteases, namely bromelain, papain and ficin, with a graft copolymer of chitosan and poly(N,N-dimethyl amino ethyl methacrylate) were obtained. It was determined that the enzyme catalytic activity in the complexes is reduced compared to their native forms. The results of molecular docking showed that modified polysaccharide located in the catalytic pocket of cysteine proteases globules. The resulting complexes have increased stability when stored under physiological conditions, which makes them promising candidates for use in the development of treatments for wounds.
The toxicity and aggressive nature of hydrogen fluoride have resulted in development of alternative strong Lewis acid-based reagents for final deprotection and cleavage steps in Boc/Bzl peptide synthesis. The acids employed are high-boiling liquids unlike hydrogen fluoride, however most peptides could be quite easily isolated from the cleavage cocktails due to their favorable physico-chemical properties: they are usually precipitated with ether. We found that this simple procedure is not suitable for the isolation of Deltorphin II peptides and its analogues. Therefore we developed alternative isolation methods and successfully purified these peptides. The procedures developed could be utilized in purification of other hydrophibic peptides.
We report a series of orto-substituted aryliden-imidazolones and their derivatives containing styrene moiety. These compounds can be used like ligands of NanoLuc protein. Together with NanoLuc this fluorogens can be used for genetically encoded labeling in fluorescence microscopy, as demonstrated by staining HEK293 cells.
A new approach to the synthesis of Anti-Reverse Cap Analog (ARCA) of the classical structure – dimethylated guanosine dinucleotide 2mGpppG is proposed. The classical approach to obtaining consists in condensation of activated 5′-diphosphate of 7,3′-O-dimethylguanosine with 5′-guanosine monophosphate. We suggest to use a commercially available 3′-O-methylguanosine for the synthesis of 5′-monophosphate of dimethylated guanosine only, and to use an activated 5′-guanosine diphosphate in the condensation. The conditions of 3′-O-methylguanosine phosphorylation were determined to avoid the formation of a side product 3′-O-methyl-5′-deoxy-5′-chloroguanosine-3′-phosphate. ARCA was synthesized with a high yield (89% at the condensation stage). Our approach to the synthesis is easily scaled and can be used for a preparative synthesis of ARCA (several grams).
We report a series of 3-aminocoumaranones possess significant chemiluminescence under various conditions. The introduce of the electron-donor groups in the coumaranone moiety and increasing of the conjugated π-system of coumaranones allow to shift 30–40 nm of the chemiluminescence maxima to the long-wavelength region. We showed that some 3-aminocoumaranones can be used for detecting of the urease and hydrogen peroxide.
A method for the synthesis of ketosteroid oxime conjugates with a fluorescein (6-FAM) derivative connected via a short bifunctional aminooxypropyl linker is proposed. The conjugates were used as tracers for fluorescence polarization immunoassay (FPIA) with monoclonal antibodies. Stereoisomers by the oxime group were separated by preparative RP TLC and HPLC and tested in the FPIA method. Binding parameters of of tracers with monoclonal antibodies (mAbs) to progesterone were studied. Tracers showed high efficiency in the analysis of steroid hormones, the detection limit of progesterone being lower than that for a previously described analog. The Z-isomer was found to be more sensitive in the FPIA analysis compared to the E-isomer.
We report about a series of arylidene-rhodanines and their acyclic analogues. The fluorescent properties of these substances were studied. We showed that the derivatives containing a 2,5-dimethoxybenzylidene fragment or similar groups are characterized by a noticeable variation in the fluorescence quantum yield depending on the properties of the medium. We discovered that two of the synthesized compounds – dimethoxy-benzylidene-rhodanine and dimethoxy-benzylidene-malononitrile – can be used as selective fluorogenic dyes for lipid droplets (adiposomes) of living cells for labeling under fluorescent microscopy conditions.
Previously, we found that the venom of the pit viper Gloydius saxatilis inhibited the muscle-type nicotinic acetylcholine receptor (nAChR). Using liquid chromatography, a protein glosaxin was isolated from the venom that inhibited the binding of the α-bungarotoxin to the nAChR of muscle type from Torpedo californica. The amino acid sequence of the isolated protein was analyzed by high resolution mass spectrometry. Subsequent bioinformatic analysis showed that it is homologous to the amino acid sequences of disintegrin-like proteins, consisting of non-catalytic domains of type PIII metalloproteinases from the venom of pit vipers of genus Gloydius. A study of the biological activity of the isolated protein showed that it inhibits the binding of α-bungarotoxin to Torpedo californica nAChR with IC50 = 51 μM. The protein also inhibited acetylcholine-induced functional responses of the human neuronal nAChR of α3β2 subtype. This is the first evidence of the ability of proteins consisting of non-catalytic domains of snake venom type PIII metalloproteinase to inhibit the nAChR.
Transmission electron microscopy (TEM) with contrast staining by uranyl acetate solution was used to study morphological differences between soil humic acids (HAs) and their A, B and C + D fractions obtained using coupling preparative low-pressure size exclusion chromatography and analytical polyacrylamide gel electrophoresis. The electrophoretic mobility of fractions varied in order C + D B A. The distribution of various morphological elements between fractions showed that large structures such as vesicle-like formations 70–150 nm long and 30–80 nm wide with clear edges were found exclusively in fraction A and occupied ~55% of the TEM image area. On the other hand, long fibrils with a length of 60–100 nm, a width of 4–6 nm and a thickness of 2–3 nm, as well as their bundles with a length of 150 nm and a diameter of 30–70 nm were identified only in the C + D fraction and occupied ~59 % area of TEM images. Smaller morphological elements such as point particles with a diameter of 2–3 nm, ring particles with a diameter of 4–6 nm, worm-shaped short particles with a length of 20–30 nm, and spheroids with a diameter of 10– 30 nm were observed in all samples, but in varying quantities. Significant morphological differences between the fractions can be explained by their composition, previously established by using a few physico-chemical methods. The ratio Car(165–108 ppm)/Calk(108–0 ppm), or aromaticity index, calculated from 13C-NMR, could be one of the indicators of the various morphological structures formation. The obtained TEM results clearly confirm the supramolecular organization of soil HAs.
Despite a large number of works focused on the search for the mechanisms of formation of IgE-producing B cells, the question of the relative contribution of germinal centers and extrafollicular foci B cells in this process still remains controversial. Of particular interest is the study of the mechanisms of stimulation of the allergic immune response under the influence of air pollutants. The aim of the work was to study the connection between the adjuvant effect of benzo(a)pyrene (BaP) on the production of specific IgE in a novel low-dose allergy model with changes in the subpopulation composition of B-cells in the tissue of the immunization site and secondary lymphoid organs. Antigen without any stimuli was administrated to one group of BALB/c mice for 9 weeks in a low (0.3 μg) dose. BaP was administrated to another group of mice along with antigens at a dose of 4 ng. B-cell subpopulations were analyzed by flow cytometry. BaP significantly stimulated the production of allergen-specific IgG1 at early (3 weeks) time point, and allergen-specific IgE at late (9 weeks) time point. The aeropollutant increased the content of CD19+CD38–CD95+B220+ germinal center B-cells with the phenotype and their precursors (CD19+CD38+CD95+B220+) with the phenotype in the spleen at early and late time points, but not in the lungs or regional lymph nodes. Under its influence, the content of CD19+CD38–CD95+B220– and CD19+CD38+CD95+B220+ extrafollicular plasmablasts in the spleen at an early time point and in lung tissue at a later time point also increases. In the spleen, BaP increased the content of CD138+CD19–B220+ and CD138+CD19–B220– mature plasma cells, and in regional lymph nodes the content of CD138+CD19+B220– immature plasma cells at a later time point. The adjuvant effect of BaP on the production of specific IgE was largely associated with stimulation of the formation of germinal centers in the spleen and with extrafollicular activation of B cells in lung tissue.
New cationic amphiphiles containing lactose or D-mannose residues were synthesized and cationic liposomes with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) were obtained. The cytotoxicity and transfection activity of new carbohydrate-containing amphiphiles and cationic liposomes against HEK 293, BHK and BHK IR-780 cells were studied. It has been shown that cationic amphiphiles effectively deliver only short fluorescein-labeled oligodeoxyribonucleotide into eukaryotic cells, while cationic liposomes formed by lactose containing amphiphile and DOPE effectively mediate the transport of short oligonucleotide and small interfering RNA and were non-toxic to cells. The resulting cationic amphiphiles can be used for intracellular delivering of nucleic acids both individually and part of cationic liposomes.
A number of molecules expressed on mammalian cells are involved in the formation of autotolerance. These primarily include CTLA-4/B7 and PD1-PD-L1 signaling pathways. Blockers of these signaling pathways, called checkpoint inhibitors (CPIs) of immunity, are used in the clinic for the treatment of various forms of cancer. Antibodies to CTLA-4 cause systemic toxicity and are approved only for some tumors. Antibodies against PD1 or PD-L1 have been successfully used for the treatment of various forms of cancer and are characterized by low toxicity. However, the response to therapy using CPIs is not always observed. The development of more effective approaches to cancer therapy based on PD1/PD-L1 inhibitors requires additional research. The aim of this work was to express the extracellular part of the murine PD-L1 protein (exPD-L1) and obtain antibodies to PD-L1. The mouse exPD-L1 protein was obtained and characterized in the bacterial expression system. exPD-L1 protein was used to immunize mice in order to produce anti-PD-L1 antibodies. Using hybridomic technology, 5 clones expressing antibodies to exPD-L1 were obtained. Antibodies of the B12 clone were developed in the ascitic fluid of BALB/c mice and purified by affinity chromatography. The ELISA method for purified antibodies showed specific binding to the exPD-L1 protein and the commercial protein of the extracellular part of murine PD-L1. Experiments using flow cytometry and confocal microscopy have shown that the antibodies obtained bind the intracellular form of the PD-L1 protein, unlike commercial antibodies binding the membrane form.
Many regulatory neuropeptides contain a large amount of proline residues. The unique proline peptide bond conformation protects these peptides from enzymatic degradation; therefore enzymes cleaving the proline peptide bonds in neuropeptides are of particular interest. The abnormal activity of serine peptidases that cleave peptides at the carboxyl group of proline residues prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP-IV) were observed in patients with anxiety disorders. PEP is involved in the maturation and degradation of neuropeptides and peptide hormones, it also is associated with the regulation of blood pressure and various disorders of the central nervous system. DPP-IV is involved in many physiological processes, in particular in glucose homeostasis in type II diabetes and immunity. When studying the metabolism of the N-acyl derivative of the aminoacyl-2-cyanopyrrolidine PEP inhibitor a decreasing in the activity of DPP-IV at the initial time was detected. This was an unexpected effect observed for inhibitors of the general formula X-Y-2-S-cyanopyrrolidine, where X represents the N-protective group and Y represents the amino acid (any besides glycine and proline). Molecular dynamics simulations of inhibitor complexes with proteases revealed the possibility of PEP inhibitors binding in the DPP-IV active site with hydrogen bonds and hydrophobic interactions that allow linkage of the nitrile group with the catalytic serine residue in the DPP-IV active site. The present study opens the prospect of creating new pharmacologically active ligands of PEP and DPP-IV.
The single-nucleotide mutation rs17713054GA in the promoter region of LZTFL1 (leucine zipper transcription factor like 1) gene is a factor in the severe course of coronavirus infection COVID-19. Computer statistical analysis of the gene by principal component analysis (PCA-seq) revealed the presence of a high correlation between the first principal component of the translated amino acid sequence and eleven amino acid indices of the AAindex database, characterizing the physicochemical and biochemical properties of the protein. The indices BEGF750102, CHOP780209, PALJ810110, GEIM800107, QIAN880121, LEVM780102, PRAM900103 are associated with β-folding parameters. The LZTFL1 protein is part of the Bardet-Biedl Syndrome (BBS) protein complexes that regulate intracellular transport in the ciliated epithelium of the lungs. It is assumed that the presence of β-sheet elements in the structure of the LZTFL1 protein plays an important role in ACE2 receptor-mediated endocytosis, stimulating the rate of angiotensin-converting enzyme 2 recycling and accelerating the delivery of adherented coronavirus SARS-CoV-2 virions into the cell during the initiation of severe acute respiratory syndrome COVID-19.
Vitamin B12 is a vital biologically active compound for human and is involved in a wide range of metabolic processes. The widespread vitamin B12 deficiency and vitamin’s low penetrating ability into cells determine the urgency of delivery systems development for the design of formulations with improved biopharmaceutical properties. This work provides a brief discussion of the main chemical and biochemical properties of the vitamin B12, as well as considers oral, injectable and transdermal multicomponent dosage forms of vitamin B12 that are aimed at solving the issue. Moreover, the literature analysis of the prospects of using vitamin B12 as an auxiliary component for both passive and active delivery of other drug molecules, for example, peptide nucleic acids and antitumor drugs, is presented. The review describes in detail the types of proposed delivery systems for biologically active compounds, in which vitamin B12 is one of the components.
Objective: The systematic use of P–NH2 analogs of nucleic acids as objects and/or tools in molecular biology and biomedicine is limited by the complexity of their synthesis. For almost 40 years, researchers have been looking for effective synthetic approaches to P–NH2 oligonucleotides. These analogs, which are isostructurally identical to natural oligonucleotides, have not been further developed even though a lot of publications on their synthesis and characteristics. To develop a more straightforward and cost-effective method than those being practiced today, our group set out to modify the phosphoramidite protocol of oligonucleotide (ON) synthesis for preparing the P–NH2 analogs of oligodeoxyribonucleotides. Methods: For the synthesis of the P–NH2 analogs, a standard, presently widely used protocol of the phosphoramidite synthesis of oligonucleotides was taken as a basis. The P–NH2 modification was introduced at the oxidation step via the Staudinger reaction, using (9-fluorenyl)methoxycarbonyl azide (FmocN3). The subsequent formation of an N-unsubstituted phosphoramidate moiety in the oligonucleotide was accomplished by the removal of the Fmoc group by treatment with a strong base. The thermodynamic properties of the P–NH2 analogs as part of complementary nucleic acid complexes formed in low-ionic-strength solutions were studied by thermal denaturation analysis with optical signal registration. Results and Discussion: It was found that to increase the efficiency of synthesis of electroneutral P–NH2 oligonucleotides additional Fmoc cleavage step should be introduced to the protocol of automated synthesis. This step should be added after each step of oxidation of the growing oligomer chain via the Staudinger reaction. It was shown that the yield of the P–NH2 oligonucleotide was almost entirely independent of the type of the dinucleotide fragment being modified, as well as of the localization of the P–NH2 linkage in the chain. The attenuation of the destabilizing effect of the introduction of a single P–NH2 linkage with decreasing ionic strength of the solution provided additional evidence for the electroneutral state of the inserted phosphoramidate linkage. Conclusions: A new approach to the automated synthesis of partially modified oligonucleotide derivatives bearing uncharged N-unsubstituted phosphoramidate linkages isostructural to native P–O linkages by an optimized solid-phase phosphoramidite protocol using the Staudinger reaction has been proposed.
Objective: Stability and monodispersity are important properties of nanoparticles and nanocomposites, that ensure the reliability of their application in biological systems and the reproducibility of results. The preparation of non-agglomerated oligonucleotide-containing nanocomposites based on anatase titanium dioxide nanoparticles (Ans ODN) is the aim of this study. Methods: The immobilization of oligodeoxynucleotides on TiO2 nanoparticles was studied by dynamic light scattering and transmission electron microscopy. The antiviral activity of the synthesized samples was evaluated against VERO cells infected with herpes simplex virus type 1. Results and Discussion: The effect of NaCl on the agglomeration of the nanoparticles and the nanocomposites in aqueous solutions was studied. The presence of NaCl leads to agglomeration of the nanoparticles and the nanocomposites. It was shown that the nanocomposites are formed in an aqueous solution in the absence of NaCl. A comparison of the biological activities of the nanocomposites prepared in water and in saline solution was carried out on the example of inhibition of replication of the herpes simplex virus type 1 in the cell culture. The studied nanocomposite, regardless of the preparation method (in water or in 0.9
The study of the interaction of nucleic acids with ligands is relevant both in terms of scientific interest and high potential practical significance. Nucleic acid complex formation with ligands affects the biochemical functions of the most important carrier of genetic information, which opens up opportunities for treating genetic diseases and controlling the aging of both cells and the organism as a whole. Among the huge variety of potential ligands, porphyrins and related compounds occupy a special placedue to their ability to generate reactive oxygen species under irradiation with light. The photocatalytic properties of porphyrins can be used in the creation of molecular tools for genetic engineering and the treatment of viral and bacterial infections at the genetic level. Modification of porphyrin compounds allows targeting of the ligand to a specific biological target.The review summarizes the literature data describing the process of nucleic acid complex formation with aromatic ligands, mainly with porphyrins. The influence of the structure of macroheterocycles on the features of interaction with nucleic acids is analyzed. Promising directions for further research are outlined.
This review presents an analysis of literature, including our own work, on various aspects of using red blood cells (RBCs) as an in vitro model in the comprehensive evaluation of antioxidant activity of a wide range of natural and synthetic compounds, their mixtures, and plant extracts. The characteristics of the most commonly used initiators of oxidative stress in such studies, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and H2O2, as well as the mechanisms underlying the development of the hemolytic process are discussed. A critical analysis of methodological approaches to assessing the level of hemolysis is provided. The review further discusses the evaluation of erythrocyte survival under oxidative stress conditions and the ability of the tested compounds to act as membrane protectors. The review considers the criteria for a comprehensive assessment of erythrocytes, facilitating the study of cellular, and molecular mechanisms underlying antioxidant activity of a wide range of substances in a model of oxidative hemolysis of erythrocytes. Traditional methods include assessment of the intensity of membrane lipid peroxidation (LPO) processes through measurement of concentration of products that react with 2-thiobarbituric acid, as well assessment of relative content of oxidized forms of hemoglobin in erythrocytes. The use of modern fluorescent methods is another promising approach. In particular, the fluorescence of heme degradation products, the decrease in intensity of which can indicate antioxidant activity in the investigated compounds, is a sensitive marker of oxidative stress in erythrocytes. Another prominent fluorescent method is the assessment of oxidative stress level by measuring the intracellular concentration of ROS in erythrocytes. Analysis of our own and literature data allows us to recommend to use the method of oxidative hemolysis of erythrocytes to screen newly developed compounds in order to select the most interesting candidates for further in-depth studies. This method is appropriate for establishing the structure-activity relationship and developing a strategy for the targeted synthesis of new biologically active compounds combining high hemocompatibility and antioxidant activity, which are promising for biomedical applications.
An acute toxicity test was conducted on derivatives of bis(3,4-dihydroquinoxalin-2-one) and bis(3,4-dihydro-2H-1,4-benzoxazin-2-one) synthesized based on 3,5-diacetyl-2,6-dimethylpyridine by single intraperitoneal injection in guinea pigs. It was found that according to K.K. Sidorov’s classification, one of the pyridine derivatives, bis(3,4-dihydroquinoxalin-2-one), exhibited low toxicity, as evidenced by the absence of lethal outcomes when administered to animals in the range of 100 to 400 µg/kg. However, it was accompanied by signs of neurological disorders regardless of the compound dose, which disappeared within a day. When guinea pigs were inoculated with another pyridine derivative, bis(3,4-dihydro-2H-1,4-benzoxazin-2-one), more pronounced and prolonged intoxication symptoms were observed, including convulsive movements of the hind limbs, decreased mobility, and delayed reaction to stimuli, leading to the death of 33% of subjects at a dose of 100 mg/kg, 66% at a dose of 200 mg/kg, and 100% of animals at a dose of 400 mg/kg. Analysis of hematological and biochemical studies conducted on the 15th day after the administration of the test compounds showed no significant deviations from normal physiological values, despite some individual parameters showing differences compared to the control group. Thus, the acute toxicity parameters of the studied compounds were not the same and were more pronounced in the pyridine derivative bis(3,4-dihydro-2H-1,4-benzoxazin-2-one); however, both compounds can be recommended for subsequent study of antibacterial and antiviral activity in guinea pigs.
