Vol 14, No 2 (2022)


The Evolution of Targeted Radionuclide Diagnosis of HER2-Positive Breast Cancer

Bragina O.D., Deyev S.M., Chernov V.I., Tolmachev V.M.


This review examines the evolution of the radionuclide diagnosis of HER2-positive breast cancer using various compounds as a targeting module in clinical practice: from full-length antibodies to a new group of small synthetic proteins called alternative scaffold proteins. This topic is of especial relevance today in view of the problems attendant to the detection of breast cancer with HER2/neu overexpression, which, in most cases, introduce errors in the treatment of patients. The results of clinical studies of radiopharmaceuticals based on affibody molecules, ADAPTs, and DARPins for SPECT and PET have demonstrated good tolerability of the compounds, their rapid excretion from the body, and the possibility to differentiate tumor sites depending on the HER2/neu status. This indicates that targeted radionuclide diagnosis holds promise and the need to continue research in this direction.

Acta Naturae. 2022;14(2):4-15
pages 4-15 views

Favipiravir and Its Structural Analogs: Antiviral Activity and Synthesis Methods

Konstantinova I.D., Andronova V.L., Fateev I.V., Esipov R.S.


1,4-Pyrazine-3-carboxamide-based antiviral compounds have been under intensive study for the last 20 years. One of these compounds, favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide, T-705), is approved for use against the influenza infection in a number of countries. Now, favipiravir is being actively used against COVID-19. This review describes the in vivo metabolism of favipiravir, the mechanism of its antiviral activity, clinical findings, toxic properties, and the chemical synthesis routes for its production. We provide data on the synthesis and antiviral activity of structural analogs of favipiravir, including nucleosides and nucleotides based on them.

Acta Naturae. 2022;14(2):16-38
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Eukaryotic Ribosome Biogenesis: The 60S Subunit

Moraleva A.A., Deryabin A.S., Rubtsov Y.P., Rubtsova M.P., Dontsova O.A.


Ribosome biogenesis is consecutive coordinated maturation of ribosomal precursors in the nucleolus, nucleoplasm, and cytoplasm. The formation of mature ribosomal subunits involves hundreds of ribosomal biogenesis factors that ensure ribosomal RNA processing, tertiary structure, and interaction with ribosomal proteins. Although the main features and stages of ribosome biogenesis are conservative among different groups of eukaryotes, this process in human cells has become more complicated due to the larger size of the ribosomes and pre-ribosomes and intricate regulatory pathways affecting their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. A previous part of this review summarized recent data on the processing of the primary rRNA transcript and compared the maturation of the small 40S subunit in yeast and human cells. This part of the review focuses on the biogenesis of the large 60S subunit of eukaryotic ribosomes.

Acta Naturae. 2022;14(2):39-49
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Research Articles

Citrobacter freundii Methionine γ-Lyase: The Role of Serine 339 in the Catalysis of γ- and β-Elimination Reactions

Anufrieva N.V., Morozova E.A., Revtovich S.V., Bazhulina N.P., Timofeev V.P., Tkachev Y.V., Faleev N.G., Nikulin A.D., Demidkina T.V.


Serine 339 of the active site of Citrobacter freundii methionine γ-lyase (MGL) is a conserved amino acid in most pyridoxal 5’-phosphate-dependent enzymes of the cystathionine β-lyase subclass, to which MGL belongs. The reaction mechanism of the MGL-catalyzed γ-elimination reaction is poorly explored. We replaced serine 339 with alanine using site-directed mutagenesis. The replacement of serine 339 with alanine led to a significant (by two orders of magnitude) decrease in efficiency in the catalysis of the γ- and β-elimination reactions by the mutant form of the enzyme. The exchange rates of the C-α- and C-β-protons in the amino acids in complexes consisting of the enzyme and competitive inhibitors decreased by one-two orders of magnitude. The spectral characteristics of the mutant form indicated that the replacement did not lead to significant changes in the conformation and tautomerism of MGL internal aldimine. We crystallized the holoenzyme and determined its spatial structure at 1.7 Å resolution. The replacement of serine 339 with alanine did not affect the overall course of the polypeptide chain of the MGL subunit and the tetrameric enzyme structure. An analysis of the obtained kinetic and spectral data, as well as the known spatial structures of C. freundii MGL, indicates that serine 339 is necessary for efficient catalysis of γ- and β-elimination reactions at the stage of C-α-proton abstraction from the external aldimine, the γ-elimination reaction at the stages of coenzyme C4’-atom protonation, and C-β-proton abstraction from a ketimine intermediate.

Acta Naturae. 2022;14(2):50-61
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The Signaling Pathways Controlling the Efficacy of Glioblastoma Therapy

Vasileva N.S., Ageenko A.B., Richter V.A., Kuligina E.V.


The resistance of glioblastoma to existing therapies puts limits on quality-of-life improvements and patient survival with a glioblastoma diagnosis. The development of new effective glioblastoma therapies is based on knowledge about the mechanisms governing tumor resistance to therapeutic agents. Virotherapy is one of the most actively developing approaches to the treatment of malignant neoplasms: glioblastoma in particular. Previously, we demonstrated that the recombinant vaccinia virus VV-GMCSF-Lact exhibits in vitro cytotoxic activity and in vivo antitumor efficacy against human glioblastoma. However, the studied glioblastoma cell cultures had different sensitivities to the oncotoxic effect of the virus. In this study, we investigated cancer stem cell (CSC) surface markers in glioblastoma cells with different sensitivities to VV-GMCSF-Lact using flow cytometry and we assessed the levels of proteins affecting viral entry into cells and virus infection efficiency by western blotting. We showed that cell cultures more sensitive to VV-GMCSF-Lact are characterized by a greater number of cells with CSC markers and a lower level of activated Akt kinase. Akt probably inhibits lactaptin-induced apoptosis in virus-resistant cells. Hence, we suggest that the sensitivity of glioblastoma cells to the oncotoxic effect of VV-GMCSF-Lact is determined by the nature and extent of the disturbances in cell death regulation in various cultures. Further investigation of the factors affecting glioblastoma resistance to virotherapy will test this hypothesis and identify targets for antitumor therapy, combined with VV-GMCSF-Lact.

Acta Naturae. 2022;14(2):62-70
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Imidazole Derivative As a Novel Translation Inhibitor

Lukianov D.A., Buev V.S., Ivanenkov Y.A., Kartsev V.G., Skvortsov D.A., Osterman I.A., Sergiev P.V.


Searching for novel compounds with antibiotic activity and understanding their mechanism of action is extremely important. The ribosome is one of the main targets for antibiotics in bacterial cells. Even if the molecule does not suit the clinical application for whatever reasons, an investigation of its mechanism of action can deepen our understanding of the ribosome function. Such data can inform us on how the already used translational inhibitors can be modified. In this study, we demonstrate that 1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-ium chloride inhibits protein synthesis both in vivo and in vitro.

Acta Naturae. 2022;14(2):71-77
pages 71-77 views

Ras Participates in the Regulation of the Stability of Adenoviral Protein E1A via MAP-kinase ERK

Morshneva A.V., Gnedina O.O., Kindt D.N., Igotti M.V.


The E1A adenoviral protein required for the initiation of the viral life cycle is being actively studied as a sensitizing agent in the combination therapy of cancer, and tumors with activated Ras in particular. We investigated the role played by the Ras signaling pathway in the regulation of E1A protein stability and showed that overexpression of activated Ras increases the basal level of E1A, but enhances the degradation of the E1A protein under treatment with histone deacetylase inhibitors (HDIs). It has been found that the MAP kinase ERK is the key factor in E1A stabilization, and ERK inactivation upon HDI treatment reduces the E1A protein level. Our results indicate that the combination treatment of tumors with activated Ras using adenoviral E1A and HDI has limitations attributed to intense HDI-dependent degradation of E1A. Nevertheless, the established contribution of ERK kinase to the regulation of E1A stability can be used to search for new effective drug combinations based on the adenoviral E1A protein.

Acta Naturae. 2022;14(2):78-84
pages 78-84 views

Antibodies Against Unusual Forms of Sialylated Glycans

Obukhova P.S., Ziganshina M.M., Shilova N.V., Chinarev A.A., Pazynina G.V., Nokel A.Y., Terenteva A.V., Khasbiullina N.R., Sukhikh G.T., Ragimov A.A., Salimov E.L., Butvilovskaya V.I., Polyakova S.M., Saha J., Bovin N.V.


Previous studies have shown that in the blood of healthy donors (1) there are no natural antibodies against sialylated glycoproteins, which contain Neu5Acα (N-acetylneuraminic acid) as the most widespread form of human sialic acid, and (2) there is a moderate level of antibodies capable of binding unnatural oligosaccharides, where Neu5Ac is beta-linked to a typical mammalian glycan core. In the present study, we investigated antibodies against βNeu5Ac in more detail and verified the presence of Kdn (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid) as a possible cause behind their appearance in humans, taking into account the expected cross-reactivity to Kdn glycans, which are found in bacterial glycoconjugates in both the α- and β-forms. We observed the binding of peripheral blood immunoglobulins to sialyllactosamines (where “sialyl” is Kdn or neuraminic acid) in only a very limited number of donors, while the binding to monosaccharide Kdn occurred in all samples, regardless of the configuration of the glycosidic bond of the Kdn moiety. In some individuals, the binding level of some of the immunoglobulins was high. This means that bacterial Kdn glycoconjugates are very unlikely to induce antibodies to βNeu5Ac glycans in humans. To determine the reason for the presence of these antibodies, we focused on noninfectious pathologies, as well as on a normal state in which a significant change in the immune system occurs: namely, pregnancy. As a result, we found that 2/3 of pregnant women have IgM in the blood against Neu5Acβ2-3Galβ1-4GlcNAcβ. Moreover, IgG class antibodies against Neu5Acβ2-3Galβ1-4GlcNAcβ and Neu5Acβ2-6Galβ1-4GlcNAcβ were also detected in eluates from the placenta. Presumably, these antibodies block fetal antigens.

Acta Naturae. 2022;14(2):85-92
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Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis

Shcherbakova T.A., Baldin S.M., Shumkov M.S., Gushchina I.V., Nilov D.K., Švedas V.K.


Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

Acta Naturae. 2022;14(2):93-97
pages 93-97 views

Short communications

High-Affinity Single-Domain Antibodies for Analyzing Human Apo- and Holo-Transferrin

Tillib S.V., Goryainova O.S., Sachko A.M., Ivanova T.I.


A highly efficient technology for generating new monoclonal single-domain recombinant antibodies (nanobodies) was used to obtain a panel of nanobodies recognizing human apo- and/or holo-transferrin. This article is devoted to the primary analysis of the properties of two different variants of the new nanobodies obtained by us, as well as to the demonstration of the unique potential of their application for diagnostic studies. The simultaneous use of immunosorbents based on these nanobodies apparently makes it possible to detect changes in the relative abundance of apo- and holo-transferrin in human biological fluids. Such changes could potentially be indicative of an increased risk or degree of development of pathological processes, such as malignant neoplasms in humans.

Acta Naturae. 2022;14(2):98-102
pages 98-102 views

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