Russian Journal of Bioorganic Chemistry最新文献

Objective: Lactoferrin is a multifunctional glycoprotein with beneficial properties, including immunomodulatory, anti-inflammatory, antibacterial, antiviral and antioxidant properties. hLF contained in breast milk provides natural protection of newborns from infections. Testing the quality of breast milk and colostrum is an important task that will enable the adjustment of the baby’s nutrition. A large number of methods for determining hLF are known, most often instrumental and immune tests are used. However, simple and rapid analysis methods are needed. Methods: 32 colostrum samples and 10 milk were obtained from healthy women. Lactoferrin content was tested by fluorescence polarization immunoassay (FPIA) and ELISA using monoclonal and polyclonal antibodies. Results and Discussion: Previously, we demonstrated the possibility of determining hLF by the FPIA using fluorescently labeled recombinant camel nanobodies, which took no more than 10 min. In this work, we demonstrate the possibility of determining hLF in colostrum samples, which differs in composition from milk. A one-step enzyme immunoassay using polyclonal and monoclonal antibodies has been developed, which allows for faster and minimal-stage determination of hLF in milk and colostrum. The ELISA detection limit was 1.25 ng/mL. 32 colostrum samples and 10 milk samples were tested using FPIA and ELISA. Conclusions: It has been shown that both methods correctly determine hLF in milk and colostrum. It is important to note that the developed ELISA can be extended to other human physiological fluids, such as tear fluid or urine.

Objective: Single embryo transfer is a strategy used in assisted reproductive technologies that can reduce the incidence of multiple pregnancies. In this approach, the importance of methods for assessing embryo quality increases substantially, as it is necessary to select the embryo with the highest implantation potential from among several embryos. One way to improve embryo quality assessment is through non-invasive methods of diagnosing oocyte competence. The aim of the study was to evaluate the potential use of the expression levels of long non-coding RNAs (lncRNAs) NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, VIM-AS1, HSAT2/3, LTR12c-375, HERVS71-int9, MER52c-175 as predictors of oocyte quality. Methods: Nine donors and 19 patients with various IVF outcomes participated in the study. The groups of donors and patients did not differ significantly in age, body mass index, antimüllerian hormone level, duration of stimulation, or daily dose of hormones. RNA was isolated from the cumulus cells and used for cDNA synthesis and subsequent amplification by qPCR. The data were analyzed using statistical methods to determine the correlation between the level of the lncRNAs expression and IVF procedure outcome. Results and discussion: The expression levels of the lncRNAs were analyzed in relation to the IVF outcome and the embryologic status of patients and donors (total number, number of mature oocytes, and number of day 5 blastocysts). Decreased expression of NEAT1 and increased expression of VIM-AS1 were the predictors most strongly associated with successful IVF outcomes, i.e., birth (correlation coefficient r (–0.7) and 0.6 respectively). Logistic regression parameters were determined for NEAT1 to describe the relationship between IVF outcomes and non-coding RNA transcription levels (b0 = 3.852; b1 = –1.418). There was no correlation between the NEAT1 transcription level and the number of oocytes or blastocysts. This suggests that NEAT1-dependent factors that reduce oocyte competence play a role in post-implantation stages. VIM-AS1 expression showed a weak correlation with the total number of oocytes (r = 0.3). Transcript 2 isoform of the VIM-AS1 correlated also with the number of mature oocytes and day 5 blastocysts. Conclusions: NEAT1 and VIM-AS1 non-coding RNAs of cumulus cells are involved in follicle maturation processes and can be considered as potential biomarkers for assessing oocyte quality and predicting the outcome of IVF procedure.

Objective: A complex approach using test organisms from different taxonomic groups (the unicellular green alga Chlorella vulgaris and the onion Allium cepa) was employed to assess the toxicity, cytotoxicity, and genotoxicity of polycarboxylic acids containing cycloaliphatic fragments. Methods: Onion (Allium cepa L.) of the Stuttgarter Riesen variety was used as a test object. Samples were analyzed using a Mikromed microscope (Russia) at 400× magnification. Differences were considered statistically significant at p < 0.05. The unicellular green alga Chlorella vulgaris (Beijer), strain LARG-1, obtained from the Vavilov Institute of General Genetics of the Russian Academy of Sciences, was used to investigate genotoxic effects. The culture was incubated in a Flora-1 photobioreactor (Russia) for 10 days at 25°C. Visible mutations were determined using an Altami stereomicroscope (Russia) at magnifications ranging from 15× to 40×. Results and Discussion: Polycarboxylic acids I–IV were evaluated at concentrations of 1.0%, 0.1%, 0.01%, and 0.001%. Compounds I and II at a concentration of 1.0% significantly inhibited the cell division rate, almost completely stopping it. Compounds I–IV at a concentration of 0.1% inhibited the cell division rate in the onion root meristem, reducing the mitotic index to 8.33 ± 1.12, 8.11 ± 0.90, 5.33 ± 0.70, and 8.33 ± 0.28%, respectively, compared to 11.05 ± 0.73% in the control. In contrast, compounds I–IV at concentrations of 0.01 and 0.001% did not cause a significant decrease in the mitotic index or observable damage to the root meristem. Analysis of the mitotic phase indices showed that compounds I–IV at a concentration of 1.0% exhibited antiproliferative activity. The tested polycarboxylic acids demonstrated mutagenic effects in the bioassay with Chlorella vulgaris. Conclusions: The analysis of the genotoxic activity polycarboxylic acids I–IV in various concentrations carried out using a system of toxicogenetic methods, showed that at high concentrations these compounds can reduce the mitotic index and phase indexes. Although no chromosomal aberrations were observed, the compounds demonstrated significant cytotoxicity and mutagenicity, as evidenced by the inhibition of cell division in Allium cepa and the increased mutation frequency in Chlorella vulgaris. The polycarboxylic acids I–IV with antiproliferative activity can be used as promising scaffolds. These drugs can be recommended for further research. The approach described here in may be applied for obtaining rapid, cost-efficient and useful supplementary data on different types of toxicity in vitro for promising scaffolds as well as for drugs under development.

Objective: Quantitative, rapid and high-throughput analysis of IgG and IgA immunoglobulins is necessary to determine the content of these proteins and their associated molecules in the patient’s physiological fluids. The analysis of these proteins is necessary in the diagnosis of specific antibody deficiency as an auxiliary test for the detection of general variable immunodeficiency, as well as for risk stratification of patients with low IgA levels. IgG content determination can help in prescribing revaccination to patients and supporting their treatment strategy, can be used to monitor the patient's humoral immune system, as well as in the development and subsequent production of most therapeutic antibodies in biopharmaceuticals. Methods: Miniature recombinant single-domain antibodies (nanobodies) have a number of advantages over classical antibodies, such as their relative simplicity of operation, high stability over a wide range of temperature and pH values, the ability to recognize highly specific conformational epitopes of the target protein, as well as the possibility of using them as probes for detecting larger target antigen proteins in the fluorescence polarization method. Results and Discussion: Fluorescently labeled FITC-anti-IgG and FITC-anti-IgA nanobodies to human IgG and IgA were obtained and characterized. The K_D values of the FITC-anti-IgG*IgG and FITC-anti-IgA*IgA complexes were determined; they confirmed the high affinity of the immunoreagents. The possibility of specifically determining IgG and IgA levels in human serum in the range of 35– 120 μg/mL (for IgA) and 75–260 μg/mL (for IgG) was demonstrated. Eighteen human sera were tested for IgG and IgA levels, and the content of antibodies in the samples was confirmed using commercial enzyme immunoassay kits. FITC-anti-IgG and FITC-anti-IgA did not interact with other human proteins: albumin, plasminogen, fibrinogen, lactoferrin, and transferrin. Testing of human and animal sera by FITC-anti-IgG and FITC-anti-IgA demonstrated specific binding to human and monkey antisera, but not to animal sera: bovine, canine, feline, rabbit, and sheep. Conclusions: Thus, the FPIA method can be used for the rapid and specific determination of human IgG and IgA.

Objective: This article focuses on the design, synthesis, and characterization of C-19 acylamino-functionalized isosteviol derivatives, and their evaluation for LSD1 inhibitory activity as well as molecular docking studies. Methods: The novel C-19 acylamino-functionalized isosteviol derivatives were designed based on the principle of drug combination, and were subsequently synthesized via acetylation with acyl chlorides and condensation with amines. The LSD1 inhibitory activity of the synthesized compounds was evaluated using the LSD1 small molecule inhibitor screening platform. Molecular docking studies were performed using MOE (Version 2019). Results and Discussion: The synthesized C-19 acylamino-functionalized isosteviol derivatives were characterized by IR, NMR, and HR-MS spectra. The results of the anti-LSD1 activity assays showed that compounds with cyclic substituents generally exhibited excellent inhibitory activity. In particular, compound IIIl exhibited the best LSD1 inhibitory effect, with an IC₅₀ value of 8.523 ± 0.882 μM. Further molecular docking studies revealed that the 16-carbonyl group of compound IIIl formed a hydrogen bond with the Ser289 residue, and its aromatic ring formed a π-H interaction with the Met332 residue. Additionally, the top-ranked docking pose of compound IIIl showed a strong binding affinity to the LSD1 protein, with a docking score of –5.273.Conclusions: This study lays the groundwork for the development and structural modification of new isosteviol-based drugs.

Objective: Benzothiazole derivatives linked to coumarin exhibit a broad spectrum of biological activities. Based on this rationale, we synthesized several compounds containing triheterocyclic structures, which are connected via a methine (–CH) group and also feature a biologically relevant thioether bond (–C–S). Methods: We used L-proline as a catalyst in a multicomponent one-pot synthesis. The structures of the synthesized compounds were confirmed by various instrumental techniques, including IR, ¹H, ¹³C NMR, and mass spectrometry. Results and Discussion: The obtained benzothiazole thioether derivatives were screened for antibacterial activity against Eю coli and Sю aureus using the zone of inhibition method. Compounds IVa and IVc exhibited promising antibacterial activity. Conclusions: The synthesized benzothiazole thioether derivatives show potential antibacterial activity, particularly compounds IVa and IVc.

Objective: The tetrapeptide Ac–Trp–Pro–Arg–Gly–NH₂, a C-terminal fragment of arginine vasopressin (AVP), is of high interest as a potential pharmaceutical agent for developing new antidepressant and anxiolytic drugs. In this work we aimed to improve synthetic approach for obtaining the compound and perform its toxicological evaluation. Methods: Ac–Trp–Pro–Arg–Gly–NH₂ was synthesized by classical methods of peptide chemistry using a convergent approach to chain assembly and activated ester and carbodiimide methods as condensation methods. The cytotoxicity of the synthesized peptide against human fibroblast cells was assessed by the MTT assay. Acute toxicity was studied on ICR mice (n = 16 in experimental group; n = 16 in control group) with intranasal administration of the tetrapeptide. The toxicity of Ac–Trp–Pro–Arg–Gly–NH₂ was assessed by monitoring changes in the body weight of rodents and visual changed in their internal organs. Results and Discussion: This study shows that the new “block” scheme allows to produce the tetrapeptide in higher yields (2–3 times) than was shown previously. Ac–Trp–Pro–Arg–Gly–NH₂ demonstrated no cytotoxicity on fibroblasts in the range of expected therapeutic doses (IC₅₀ >1000 μM) and did not exhibit any clearly expressed pathological effect on the internal organs of experimental mice under conditions of the experiment. Conclusions: The low toxic effects of tryptophan-containing AVP analog demonstrated in this work makes the tetrapeptide a promising substance for further investigations of biological activity, toxicity and mechanisms of action with the aim of creating new, safer and more effective anxiolytics and antidepressants based on the compound.

Objective: Synthesis of a new series of fluorescent dyes based on Kaede chromophore moiety for selective mitochondria and nucleus staining. Methods: [3+2]-cycloaddition of carboxyimidate to aromatic imines. Reaction of arylidene imidazolone with BBr₃ followed by treatment with aqueous HF was used in synthesis of difluoroboranyl compound. Condensation of corresponding aromatic aldehydes with active methyl group of arylidene-imidazolone was used for Kaede chromophore analogues synthesis. UV-VIS spectroscopy and fluorimetry was used to study optical properties of new chromophores. The widefield fluorescence microscopy was used for imaging of cellular structures. Results and Discussion: We discovered that 5-((Z)-4-(dimethylamino)-3-hydroxybenzylidene)-3-methyl-2-((E)-2-(pyridin-4-yl)vinyl)-3,5-dihydro-4H-imidazol-4-one can act as a fluorogen for mitochondria and nucleus. Conclusions: We created a series of three new arylidene imidazolone fluorogens and showed that compound can be used as a dye for mitochondria and nucleus in widefield fluorescence microscopy.

Objective: One of the most promising approaches to addressing the problem of bacterial resistance is the development of antibacterial drugs with a novel mechanism of action. Oxazolidinones are a class of synthetic antibacterial drugs with high activity against a wide range of Gram-positive bacteria, including resistant strains. A library of 11 pyridoxine derivatives containing 1,3-oxazolidin-2-one fragments at the second, fifth, and sixth positions was synthesized. Methods: The structures of the synthesized compounds were confirmed by ¹H, ¹³C NMR spectroscopy and mass spectrometry. The target oxazolidinone derivatives were obtained using organic synthesis methods. Numerous biological experiments were performed to evaluate the antibacterial activity and toxicity of the synthesized compounds. Results and Discussion: The antimicrobial activity testing on 6 reference strains and 6 clinical isolates of Gram-positive bacteria, as well as in vitro toxicity against a panel of normal human cells (HSF, MSC, and HEK-293), revealed a highly active and low-toxicity lead compound containing a 1,3-oxazolidin-2-one fragment at the fifth position of pyridoxine. Subsequent studies on bacterial biofilms of S. aureus and E. faecium demonstrated comparable and, in some cases, superior efficacy to linezolid. The lead compound, unlike linezolid, did not exhibit a mutagenic effect in the Ames test and also showed high safety upon intragastric administration to mice (LD₅₀ >2000 mg/kg). Conclusions: The results indicate that pyridoxine derivatives containing 1,3-oxazolidin-2-one fragments are of interest for antibacterial drug development.

Objectives: Isatin and 2-oxoindole are valuable synthetic building blocks with diverse biological properties, including antifungal, antibacterial, antitumor, and anticancer activities. Due to the growing threat of pathogen resistance to current antimicrobial, nematicidal, and antienzymatic agents, our research group focuses on discovering new compounds with promising biological potential. Methods: N-1 alkylated isatin was synthesized in the presence of K₂CO₃. The N-1 alkylated isatin was then reacted with 2-oxoindole in the presence of the acid-base catalyst ZrCl₄ to afford N-1 alkylated isoindigo derivatives, which were characterized using ¹H, ¹³C NMR, and FT-IR spectroscopy. The synthesized derivatives were evaluated for their antimicrobial activity against Dickeya sp., Streptomyces sp., Fusarium oxysporum, and Rhizoctonia solani, their nematicidal efficacy against the plant pathogen Meloidogyne incognita, and their inhibitory potential against acetylcholinesterase. Results and Discussion: Among all synthesized derivatives, the isoindigo compound VIj (3-(1-decyl-2-oxoindolin-3-ylidene)indolin-2-one) exhibited the highest biological activity. It demonstrated significant antibacterial activity, with MIC values of 39 and 36 μg/mL against Dickeya sp. and Streptomyces sp., respectively. Similarly, the compound showed remarkable antifungal activity, with MFC values of 278 and 292 μg/mL against Fusarium oxysporum and Rhizoctonia solani, respectively. Regarding its nematicidal activity, VIj displayed potent inhibition against Meloidogyne incognita. The LC₅₀ and LC₉₅ values for egg hatch inhibition were determined as 0.193 and 1.462 mg/mL, respectively, while for juvenile mortality, the LC₅₀ and LC₉₅ values were 0.152 and 0.995 mg/mL, respectively. Additionally, the inhibitory activity of VIj against the acetylcholinesterase enzyme of M. incognita was evaluated, revealing an IC₅₀ value of 210.4 μg/mL. These findings highlight VIj as a novel scaffold with significant antimicrobial, antifungal, and nematicidal properties. Conclusions: Its promising biological activities make it a valuable candidate for the agrochemical sector, where such compounds are in high demand for potential agricultural applications.