


Vol 51, No 3 (2025)
ОБЗОРНАЯ СТАТЬЯ
The role of phospholipid derivatives of cyclodextrins in the formation of stable lipid nanoparticles for drug delivery
Abstract
This review article deals with physical methods for investigating the structural characteristics of inclusion complexes of supramers of phospholipid derivatives of cyclodextrins. Phospholipid derivatives of cyclodextrins are formed by attaching a phospholipid moiety to the cyclodextrin molecule. This modification imparts additional structural features to the cyclodextrin, increasing its solubility and stability in aqueous media. These new compounds can self-assemble in aqueous media into different types of supramolecular nanocomplexes. Biomedical applications are envisaged for nanoencapsulation of drug molecules in hydrophobic interchain volumes and nanocavities of amphiphilic cyclodextrins (serving as drug carriers or pharmaceutical excipients), antitumour phototherapy, gene delivery, and protection of unstable active ingredients by complexation of inclusions in nanostructured media. The focus is on the study of nanoparticle morphology, as efficient delivery systems must fulfil certain requirements. Classical physical methods cannot provide detailed information on the properties of potential structures for biomedical applications. For this purpose, the search for new non-invasive approaches is necessary.



Mass spectrometric analysis of Xenopus laevis cytoskeletal protein zyxin post-translational modifications
Abstract
In addition to its involvement in fundamental cellular processes, zyxin, a LIM-domain protein in the cytoskeletal system, is actively studied because it plays an important role in mechanosensory functions, actin polymerization regulation at cell junctions, as well as gene expression regulation. The disruption of zyxin expression and processing has been associated with carcinogenesis and cardiovascular disease. Zyxin plays an important role in the invasion and metastasis of tumors. The post-translational modification of zyxin in mammals regulates its activity and subcellular location. Given that zyxin is an evolutionarily highly conserved protein, we conducted a search for post-translational modifications of the zyxin homolog from Xenopus laevis using chromatographic mass spectrometry. To identify modified peptides, an enrichment method was employed using co-immunoprecipitation of endogenous zyxin from gastrula-stage embryonic cell lysates. As a result, previously unknown modifications of this protein were discovered, specifically N-terminal acetylation at methionine position 1 and phosphorylation at Ser197 and Ser386. To identify zyxin isoforms with different electrophoretic mobilities, separation was performed using polyacrylamide gel electrophoresis. Zyxin was found in bands with electrophoretic mobilities of 70 and 105 kDa. Thus, this study presents entirely new data on the post-translational modifications of zyxin from X. laevis. Since defects in mechanical signal transduction are associated with developmental disorders, oncogenesis, and metastasis, the study of mechanosensitive protein zyxin modifications and processing on the model organism X. laevis opens up opportunities for diagnostic studies at the molecular level, which can be used in the future to determine drugs use prospective in pharmacology.



Combined effects of cytokine TRAIL-based DR5-specific fusion protein with olaparib on tumor cell lines with different BRCA mutation status
Abstract
Tumor cell death induction via activation of TRAIL (tumor necrosis factor-related apoptosis inducing ligand) cytokine signaling pathway is a promising strategy for anticancer therapy. Previously, we developed a fusion protein SRH-DR5-B-iRGD based on the DR5 (death receptor 5)-specific cytokine TRAIL variant DR5-B with antiangiogenic peptides. The SRH peptide specifically binds to the VEGFR2 (vascular endothelial growth factor receptor 2) receptor and blocks its VEGF-mediated activation; the iRGD peptide binds to integrin αvβ3 and the NRP-1 (neuropilin-1) receptor. All of these targets are known to be overexpressed on the surface of tumor cells. In the current study, we investigated the cytotoxic activity of the SRH-DR5-B-iRGD fusion protein in comparison with DR5-B in vitro in ovarian and breast adenocarcinoma cell lines with different BRCA mutation status in combination with a targeted poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Olaparib synergistically enhanced the cytotoxicity of TRAIL-based proteins regardless of the presence of BRCA mutations in the cells, and this effect was more pronounced for SRH-DR5-B-iRGD. Thus, the combination of SRH-DR5-B-iRGD with olaparib can be considered as a new approach to treatment of ovarian and breast adenocarcinomas regardless of the presence of BRCA mutations.



Sequence specificity of dimeric bisbenzimidazoles to AT-sequences of DNA of different nucleotide composition determined by footprinting
Abstract
The study aimed to investigate the site-specificity of binding to DNA of three series of minor groove ligands – dimeric bisbenzimidazoles DB2(n), DB2P(n), and DB2Py(n) – using DNAase I footprinting. The compounds consist of two bisbenzimidazole units linked by oligomethylene linkers of varying lengths (n), with structural modifications to enhance DNA-binding properties. The binding specificity of the compounds was determined using DNAase I footprinting. The DB2(n) and DB2P(n) series are analogs of Hoechst 33342, modified by removing hydrophobic ethoxyphenol cores and introducing hydrophilic aminomethylene groups. The DB2Py(n) series incorporates a pyrrolcarboxamide group, a structural unit of the AT-specific antibiotic netropsin. The interaction of these compounds with DNA sequences was analyzed to identify their binding preferences. All studied compounds demonstrated specificity for AT-rich DNA sequences. The DB2P(n) and DB2(n) series exhibited increased affinity for (AATT)3 and TTTT sequences. The DB2Py(n) series showed high specificity to AT-rich regions, with a preference for the TTTT motif. None of the compounds interacted with sequences containing fewer than four AT base pairs. These findings highlight the influence of structural modifications on DNA-binding specificity and affinity. The study revealed that dimeric bisbenzimidazoles DB2(n), DB2P(n), and DB2Py(n) exhibit distinct binding preferences for AT-rich DNA sequences, with DB2Py(n) showing a pronounced affinity for the TTTT motif. The results demonstrate the potential of these compounds as tools for targeting specific DNA sequences, with implications for molecular biology and drug design.



Synthesis and study of cytotoxicity of 3β-acetoxyurs-12-en-28-oyl-thiourea derivatives
Abstract
The interaction of 3β-acetoxyurs-12-en-28-oyl chloride with potassium rhodanide afforded 3β-acetoxyurs-12-en-28-oyl isothiocyanate. A series of substituted 3β-acetoxy-urs-12-en-28-oyl-thioureas was synthesised in yields of 69–88% by condensation of triterpene acyl isothiocyanate with a series of amino derivatives. The CuAAC cycloaddition reaction of N-(2-azidoethylcarbamothioyl)-3-acetoxyurs-12-en-28-oyl-amide with propargyl alcohol and 3-(prop-2-inyloxy)-4,5-((R,S)-methoxymethylenedioxy)-benzoate led to the formation of hybrid acylthioureas containing a 1,2,3-triazole linker in 72 and 75% yields. In CuAAC reactions of N-(prop-2-ynylcarbamothioyl)-3β-acetoxyurs-12-en-28-oylamide with substituted acylthiourea azides containing 1,2,3-triazole were isolated in moderate yields of 48–62%. The use of one-pot-reactor version of the synthesis with the preparation of substituted (1H-1,2,3-triazol-4-yl)methanamines in the reaction of propargylamine with substituted azides followed by condensation with 3β-acetoxyurs-12-en-28-oyl isothiocyanate increased the yield of 1,2,3-triazole-containing acylthioureas to 65–85%. Polar triterpene acylthioureas containing carboxyl or alcohol groups exhibited high inhibitory activity against HepG2 cells, significantly superior to the parent compound ursolic acid, and were also more selective than the drug doxorubicin. Among the acylthioureas, products of CuAAC cycloaddition, the most active was the polar derivative with (1H-1,2,3-triazol-4-yl)methanol substituent, which was cytotoxic to all cells tested, including the non-tumour control, but superior in selectivity to doxorubicin. Ursane hybrids with acylthiourea derivatives are of interest for further investigation as promising antitumour agents.



Effect of different coatings on immobilization of biomolecules in brush polymer cells
Abstract
Biochips with protein and oligonucleotide probes are used to analyze protein and nucleic acid samples. The key challenges of the technology are the selection of substrate materials and surface functionalization. Polybutylene terephthalate substrates were modified by coating them with photoactive polymers: poly(ethylene-co-propylene-co-5-methylene-2-norbornene), acetylcellulose, polyvinyl acetate and polyvinyl butyral. The coatings were applied by centrifugation and dried. The effect of the coating on the biochip characteristics was investigated. A matrix of hydrophilic cells made of brush polymers with epoxy groups for immobilization of DNA probes and human immunoglobulins was prepared by photoinitiated radical polymerization. The functionality of probes was investigated by hybridization analysis and reaction with specific antibodies. The binding efficiency of probes to molecular targets was evaluated on biochips with different coatings. Cells on substrates coated with polyvinyl butyral and poly(ethylene-co-propylene-co-5-methylene-2-norbornene) showed the best binding efficiency and weak adsorption of targets, providing high contrast fluorescence images after probe binding. Biochips on such substrates are promising for lab-on-a-chip microanalysis technology.



Comparison of DNA analysis on biochips with brush polymer cells and cross-linked polymer cells
Abstract
The regulation of substrate surface properties in biochip technology opens the possibility of optimizing platforms for efficient biomolecule recognition. The research is aimed at exploring the use of brush polymers to improve the sensitivity and speed of DNA analysis on biochips. Brush polymer cells for biochips were prepared by UV-initiated polymerization of monomers from the surface on polyethylene terephthalate substrates. Cross-linked hydrogel polymer cells for biochips were prepared on polybutylene terephthalate substrates by copolymerization of gel components with DNA probes. The probes in brush polymer cells were immobilized through activated carboxyl groups. A single-stranded DNA target with a length of 124 nucleotides corresponding to the 7th exon of the human ABO gene was used for hybridization analysis. Hybridization of the DNA target was studied on biochips with cells made of brush polymers and cross-linked polyacrylamide hydrogels. The results of hybridization analysis on biochips were evaluated by digital fluorescence microscopy. Higher intensity of fluorescence signals and higher ratio of signals of cells with perfect duplexes to those of cells with imperfect duplexes were observed in cells from brush polymers compared to cells from 3D cross-linked polymers. Achievement of hybridization signal up to 90% of saturation occurred in the same time in both cell types. The relevance of this work stems from the need for highly accurate and efficient diagnostic methods to analyze biomolecules with minimal time and reagent consumption. The development of biochips based on brush polymers will increase the accuracy and sensitivity of molecular studies, which is especially important for early diagnosis of diseases.



A method for increasing the efficiency of selection of aptamers to cellular receptors
Abstract
A method has been proposed to increase the efficiency of selection of aptamers to cellular receptors by the cell-Selex method, in particular to the receptor tyrosine kinase c-KIT. The use of Tween 20 in buffer solutions in concentrations not exceeding 0.01%, as well as trypsinolysis of surface proteins at the stage of elution of the combinatorial library of oligonucleotides bound to the cell surface, led to an increase in the specificity of aptamers and a decrease in nonspecific sorption according to the results of fluorescence microscopy, thermofluorimetric analysis and high-precision sequencing.



Combined effects of senexin B and antitumour agents on neuroblastoma and glioblastoma cell lines
Abstract
Neurogenic tumors such as neuroblastoma and glioblastoma are highly heterogeneous and particularly aggressive: they are characterized by rapid growth, metastasis and resistance to treatment. Both tumors exhibit MYCN oncogene copy-number disruption, impaired gene transcription, and overall high transcriptional deregulation. In this study, we evaluated the survival of glioblastoma and neuroblastoma cells and performed real-time PCR analysis to assess the change in expression of MYCN, HAND2, PHOX2A, and PHOX2B oncogenes after exposure to senecin B in combination with temozolomide and the potential therapeutic agent 10058-F4. As a result, a trend of increased PHOX2B gene expression and decreased PHOX2A gene expression after drug exposure was observed in one of the neuroblastoma lines and both glioblastoma lines, an increase in MYCN and HAND2 gene expression was also observed. Viability tests showed that substance 10058-F4 was effective against neuroblastoma line but not glioblastoma lines. However, senexin B enhanced the inhibitory effect of 10058-F4 on glioblastoma cell lines and also enhanced the effect of temozolomide on the T98G cell line.



2-fluorocordycepin: chemoenzymatic synthesis and study of anticancer activities in vitro
Abstract
Two methods for obtaining 2-fluorocordycepin were proposed and implemented: chemical synthesis from 2-fluoroadenosine with a yield of 34% and chemical-enzymatic synthesis with a yield of 66%, including the production of 3-deoxyerythropentofuranose-1-phosphate and subsequent transglycosylation using E. coli purine nucleoside phosphorylase. The cytotoxic activity of 2-fluorocordycepin in vitro was assessed. It was shown that 2-fluorocordycepin exhibits an anti-metabolic effect on a number of tumor cell lines (Jurkat, Raji, MCF-7, THP-1, U937, A549, LS174T), which allows us to consider this compound as a promising candidate for the development of antitumor drugs.



Regulation of pou5f3 family pluripotency gene transcripts stability by Ybx1 ribonucleoprotein complexes in Xenopus laevis early development
Abstract
Here, we studied the regulation of pou5f3 family transcripts stability by association with Ybx1, a protein of ribonucleoprotein complexes. It is known that the clawed frog Xenopus laevis has three genes belonging to the POU5 family: pou5f3.1/oct91, pou5f3.2/oct25, and pou5f3.3/oct60. The Pou5f3 family factors are orthologues of the mammalian embryonic stem cell OCT4 pluripotency factor. However, the expression patterns of these genes differ over time. Pou5f3.3/oct60 transcripts are stored in oocytes, are present in large quantities in fertilized eggs, and then degrade only after fertilization. Pou5f3.2/oct25 transcripts are also present in the zygote, but their numbers increase even more during the development process. Finally, pou5f3.1/oct91 transcription begins only after the activation of the embryo genome at the middle blastula stage. In the present work, we revealed a much higher specificity of the Ybx1 factor to form a complex with the maternal mRNA of the pou5f3.3/oct60 gene compared to zygotic mRNAs of the pou5f3.1/oct91 and pou5f3.2/oct25 genes. Since Ybx1 is a protein that, on the one hand, is involved in interaction with cytoskeletal proteins, and, on the other hand, binds and stabilizes pluripotency genes mRNA, it can play a linking role in between the degradation of these maternal transcripts and cytoskeletal rearrangements during the onset of morphogenetic cell movements in the process of formation of germ layers.



Searching for possible sites of electrophils conjugation with biomolecules using molecular modeling methods
Abstract
The ability to rapidly form adducts with nucleophilic groups of proteins, nucleic acids and lipids largely determines the toxic effects of electrophiles. Considering that the number of toxic electrophiles is practically unlimited, and they can form adducts with many molecular targets, a purely empirical approach to characterizing the adductome is obviously unproductive. The aim of this study is to develop a method for primary in silico assessment of the probability of conjugation of electrophiles with a particular modification site. For the model group of electrophiles, the quantum-chemical indices were calculated using the density functional theory method, and the molecular docking method was used to search for priority sites of covalent binding of the studied compounds. Based on the obtained results, a scale for assessing the hardness of electrophiles was developed and an algorithm for computer selection of possible conjugation sites of electrophiles with biological macromolecules was compiled.



Synthesis, study of antitumor activity, and effect of (4-alkoxyphenyl)(3-aryloxy-2-yl)-methanones on tumor DNA in vitro
Abstract
Condensation of 1-(4-alkoxyphenyl)ethanones with aromatic aldehydes in the presence of sodium hydroxide in an aqueous-ethanol solution yielded (2E)-3-aryl-1-(4-alkoxyphenyl)prop-2-en-1-ones. Subsequent oxidation of the latter with hydrogen peroxide in a water-ethanol system in a basic medium at room temperature yielded the corresponding epoxy derivatives. The effect of the synthesized compounds on tumor DNA methylation processes in vitro and the antitumor activity of [4-(pentyloxy)phenyl)](3-phenyloxiran-2-yl)methanone in vivo were assessed.



ПИСЬМА РЕДАКТОРУ
Intrinsically disordered regions affect the expression of Toll-like receptor TIR domains in soluble form
Abstract
Here we investigate the effect of the C-terminal region on the solubility of TLR4 and TLR10 TIR domains upon the heterologous expression in Escherichia coli. We demonstrated that the absence of this region significantly enhances the yields of globular domains in a soluble form. These findings complement the protocols, previously established to synthesize the TLR TIR domains for the structural studies by various techniques, including by NMR spectroscopy.



TERT promoter mutation analysis in glioma samples by allele-specific biochip hybridization
Abstract
Somatic mutations in the promoter of the telomerase reverse transcriptase gene TERT can cause reactivation of the telomerase enzyme, which stimulates neoplastic processes in the body. C228T and C250T mutations of the TERT gene promoter (TERTp) are most often found in brain gliomas, for which they are important diagnostic and prognostic markers. To detect TERTp mutations, an approach involving amplification of the promoter region and subsequent hybridization with immobilized probes on a biological microarray (biochip) has been developed. Using this approach, the mutational status of TERTp in 94 glioma samples (astrocytoma, oligodendroglioma, glioblastoma) was investigated. To verify the genotyping results, we used data from Illumina platform targeting sequencing and Sanger direct sequencing. In total, TERTp mutations were detected in 62 of 94 samples (66%), most commonly in patients with glioblastoma (71%). The C228T mutation (69%) was significantly more frequent compared to the C250T mutation (31%). The results of biochip validation on a collection of clinical samples show that it can be used as a convenient and reliable diagnostic tool in genetic analysis of CNS tumors.



Fluorescence labeling of GC-rich DNA matrix with different nucleotide derivatives for hybridization analysis on a biological microarray
Abstract
We investigated the efficiency of labeling the GC-rich promoter region of the TERT gene in the human genome with derivatives of 5′-triphosphates of 2′-deoxyuridine (dU) and 2'-deoxycytidine (dC) containing cyanine dyes Cy5 and Cy7 as a fluorescent label. The effect of modified nucleotides on the efficiency of the polymerase chain reaction was evaluated by real-time PCR, and the extent of nucleotide incorporation into the PCR product was also determined. The efficiency of DNA matrix labeling was determined by the intensity of fluorescent signal during allele-specific hybridization on a biological microarray. The highest level of biochip cell fluorescence was observed for dU-Cy7 derivatives compared to dU- and dC-Cy5 derivatives. At the same time, in the case of GC-rich DNA matrix, the use of dC-Cy5 derivatives gave a fundamentally better result compared to dU-Cy5 derivative. Thus, modified Cy7 analogs capable of incorporation into DNA during PCR are less dependent on the GC composition of the DNA matrix and are more universal fluorescent labels for diagnostic purposes. Further application of modified Cy7 analogs in the development of laboratory-on-a-chip test systems seems to be the most promising.


