400 results on '"Varizhuk A"'
Search Results
202. Triazole‐Linked Oligonucleotides with Mixed‐Base Sequences: Synthesis and Hybridization Properties
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Varizhuk, Anna, primary, Chizhov, Alexandr, additional, Smirnov, Igor, additional, Kaluzhny, Dmitry, additional, and Florentiev, Vladimir, additional
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- 2012
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203. Amino-Functionalized Oligonucleotides with Peptide Internucleotide Linkages
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M. Varizhuk, Anna, primary, V. Kochetkova, Svetlana, additional, U. Fedotov, Alexander, additional, A. Kolganova, Natalia, additional, N. Timofeev, Edward, additional, and L. Florentiev, Vladimir, additional
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- 2012
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204. Synthesis and DNA binding affinity of irregular sequence oligonucleotides with triazole internucleotide linkages.
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Florentiev, Vladimir, primary, Varizhuk, Anna, primary, Kaluzhny, Dmitry, additional, and Smirnov, Igor, additional
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- 2011
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205. Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents
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Varizhuk, Anna, primary, Chizhov, Alexandr, additional, and Florentiev, Vladimir, additional
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- 2011
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- View/download PDF
206. Oligonucleotide Analogs with Peptide Internucleotide Linkages
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Varizhuk, Anna, primary, Kochetkova, Svetlana, additional, Kolganova, Natalia, additional, Timofeev, Edward, additional, and Florentiev, Vladimir, additional
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- 2011
- Full Text
- View/download PDF
207. Stereospecific synthesis of 2'-O-α-D-ribofuranosylnucleosides
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Kulikova, Irina V., primary, Muradova, Daria A., additional, Varizhuk, Ivan S., additional, and Mikhailov, Sergey N., additional
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- 2008
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208. Effective anomerisation of 2'-deoxyadenosine and thymidine derivatives
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Kulikova, Irina V., primary, Varizhuk, Ivan S., additional, Bobkov, Georgii V., additional, Efimtseva, Ekaterina V., additional, and Mikhailov, Sergey N., additional
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- 2005
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209. Oligonucleotide analogues containing a C3′-NH-C(O)-CH2-C5′ amide internucleotide bond.
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Varizhuk, A. M., Kochetkova, S. V., Kolganova, N. A., Timofeev, E. N., and Florent'v, V. L.
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OLIGONUCLEOTIDES , *AMIDES , *THERMODYNAMICS , *SILYLATION , *HYDROXYL group - Abstract
A dinucleotide containing a C3′-NH-C(O)-CH2-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′- O-( tert-butyldimethylsilyl)thymidine with 3′- O-benzyl-2′- O-tert-butyldimethylsilyl-5′-deoxy-5′-carboxymethylribosylthymine, which was obtained from 2′- O-acetyl-3′- O-benzyl-5′-deoxy-5′-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation of liberated hydroxyl and ester saponification. After standard manipulation with protecting groups, the dinucleotide was converted into 3′- O(2-cyanoethyl- N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones. [ABSTRACT FROM AUTHOR]
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- 2010
- Full Text
- View/download PDF
210. Synthesis of 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides, the precursors of oligonucleotides with the amide internucleoside bond C3′-NH-C(O)-CH2-C5′.
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A. Varizhuk, S. Kochetkova, N. Kolganova, E. Timofeev, and V. Florent’ev
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NUCLEOSIDES , *HETEROCYCLIC compounds , *OLIGONUCLEOTIDES , *AMIDES , *CHEMICAL bonds , *CHEMICAL reactions , *SODIUM hydride , *HYDRATION , *NUCLEIC acids - Abstract
Abstract A method for the synthesis of 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides has been developed. 3-O-benzyloxymethyl-1,2-O-isopropylidene-α-D-allofuranose was oxidized by sodium periodate to form a 5′-aldo derivative, which was converted by the reaction with triethylphosphonoacetate in the presence of sodium hydride into a 5-deoxy-5-ethoxycarbonylmethylene derivative. The hydration of the unsaturated compound gave 5-deoxy-5-ethoxycarbonylmethyl-1,2-O-isopropylidene-α-D-ribofuranose. After the benzylation of 3-hydroxyl, the removal of the isopropylidene group by heating with acetic acid, and the subsequent acetylation, 1,2-di-O-acetyl-3-O-benzyl-5-deoxy-5-ethoxycarbonylmethyl-D-ribofuranose was obtained, which reacted with persilylated nucleic acid bases to form 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides. [ABSTRACT FROM AUTHOR]
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- 2009
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211. Oligonucleotide analogues containing internucleotide C3′-CH2-C(O)-NH-C5′ bonds.
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S. Kochetkova, A. Varizhuk, N. Kolganova, E. Timofeev, and V. Florent’ev
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THERAPEUTIC use of oligonucleotides , *NUCLEOTIDES , *PYRIMIDINE nucleotides , *PROTECTIVE groups (Chemistry) - Abstract
Abstract A dinucleoside bearing an amide internucleotide C3′-CH2-C(O)-NH-C5′ bond was synthesized by the interaction of 3′-deoxy-3′-carboxylmethylribothymidine-2′,3′-lactone obtained by hydrolysis of 2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-ethoxycarboxylmethylribothymidine with 5′-deoxy-5′-amino-3′-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3′-O-(2-cyanoethyl-N,N′-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones. [ABSTRACT FROM AUTHOR]
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- 2009
- Full Text
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212. Synthesis of 3′-deoxy-3′-carboxymethylnucleosides, precursors of oligonucleotides with an amide internucleoside bond.
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S. Kochetkova, A. Varizhuk, N. Kolganova, E. Timofeev, and V. Florent’ev
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OLIGONUCLEOTIDES , *SODIUM hydride , *ACETOLYSIS - Abstract
Abstract An improved method for the synthesis of 3-deoxy-3-carboxymethyl nucleosides was suggested. Oxidation of 5-O-benzoyl-1,2-O-isopropylidene-α-D-xylofuranose resulted in the 3-keto derivative, which was treated with triethylphosphonoacetate in the presence of sodium hydride to obtain the 3-deoxy-3-ethoxycarbonylmethylene derivative. Hydrogenation of the unsaturated compound proceeded strictly stereospecifically and gave the product with the ribo-configuration. Acetolysis of the resulting compound with AcOH-Ac2O-CH3SO3H led to 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-ethoxycarbonylmethyl-D-ribofuranose, whose interaction with persilylated nucleic bases gave 3-deoxy-3-ethoxycarbonylmethylnucleosides in a total yield of 42–49% from the starting compound. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
213. Additional file 2: of Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics
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D. Doronin, N. Barykina, O. Subach, V. Sotskov, V. Plusnin, O. Ivleva, E. Isaakova, A. Varizhuk, G. Pozmogova, A. Malyshev, I. Smirnov, K. Piatkevich, K. Anokhin, G. Enikolopov, and F. Subach
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3. Good health - Abstract
Supplementary Methods. (PDF 194Â kb)
214. Oligonucleotide analogues containing internucleotide C3'-CH2-C(O)-NH-C5' bonds
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A. M. Varizhuk, V. L. Florent’ev, E. N. Timofeev, S. V. Kochetkova, and N. A. Kolganova
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Oligonucleotide ,Stereochemistry ,Organic Chemistry ,Oligonucleotides ,Biochemistry ,chemistry.chemical_compound ,Hydrolysis ,chemistry ,Duplex (building) ,Amide ,Polymer chemistry ,Nucleic Acid Conformation ,Thermodynamics ,Bioorganic chemistry ,Thermal stability ,Thymidine - Abstract
A dinucleoside bearing an amide internucleotide C3'-CH2-C(O)-NH-C5' bond was synthesized by the interaction of 3'-deoxy-3'-carboxylmethylribothymidine-2',3'-lactone obtained by hydrolysis of 2'-O-acetyl-5'-O-benzoyl-3'-deoxy-3'-ethoxycarboxylmethylribothymidine with 5'-deoxy-5'-amino-3'-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3'-O-(2-cyanoethyl-N,N'-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones.
215. Additional file 2: of Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics
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D. Doronin, N. Barykina, O. Subach, V. Sotskov, V. Plusnin, O. Ivleva, E. Isaakova, A. Varizhuk, G. Pozmogova, A. Malyshev, I. Smirnov, K. Piatkevich, K. Anokhin, G. Enikolopov, and F. Subach
- Subjects
3. Good health - Abstract
Supplementary Methods. (PDF 194Â kb)
216. Additional file 7: of Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics
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D. Doronin, N. Barykina, O. Subach, V. Sotskov, V. Plusnin, O. Ivleva, E. Isaakova, A. Varizhuk, G. Pozmogova, A. Malyshev, I. Smirnov, K. Piatkevich, K. Anokhin, G. Enikolopov, and F. Subach
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3. Good health - Abstract
Supplementary Results. (PDF 131Â kb)
217. Additional file 4: of Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics
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D. Doronin, N. Barykina, O. Subach, V. Sotskov, V. Plusnin, O. Ivleva, E. Isaakova, A. Varizhuk, G. Pozmogova, A. Malyshev, I. Smirnov, K. Piatkevich, K. Anokhin, G. Enikolopov, and F. Subach
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3. Good health - Abstract
Table S1. In vitro properties of purified iYTnC compared to NTnC. (PDF 115Â kb)
218. Additional file 4: of Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics
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D. Doronin, N. Barykina, O. Subach, V. Sotskov, V. Plusnin, O. Ivleva, E. Isaakova, A. Varizhuk, G. Pozmogova, A. Malyshev, I. Smirnov, K. Piatkevich, K. Anokhin, G. Enikolopov, and F. Subach
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3. Good health - Abstract
Table S1. In vitro properties of purified iYTnC compared to NTnC. (PDF 115Â kb)
219. Oligonucleotides with peptide internucleotide linkage. A novel class of modified oligonucleotides
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Anna Varizhuk, Kochetkova, S. V., Kolganova, N. A., Timofeev, E. N., and Florent Ev, V. L.
220. Oligonucleotide analogues containing a C3'-NH-C(O)-CH(2)-C5' amide internucleotide bond
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N. A. Kolganova, V. L. Florent’ev, E. N. Timofeev, A. M. Varizhuk, and S. V. Kochetkova
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Magnetic Resonance Spectroscopy ,Silylation ,Oligonucleotide ,Stereochemistry ,Organic Chemistry ,Nucleosides ,Biochemistry ,chemistry.chemical_compound ,chemistry ,Oligodeoxyribonucleotides ,Duplex (building) ,Amide ,Polymer chemistry ,Bioorganic chemistry ,Thermodynamics ,Transition Temperature ,Thermal stability ,Thymidine ,Saponification - Abstract
A dinucleotide containing a C3′-NH-C(O)-CH2-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′-O-(tert-butyldimethylsilyl)thymidine with 3′-O-benzyl-2′-O-tert-butyldimethylsilyl-5′-deoxy-5′-carboxymethylribosylthymine, which was obtained from 2′-O-acetyl-3′-O-benzyl-5′-deoxy-5′-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation of liberated hydroxyl and ester saponification. After standard manipulation with protecting groups, the dinucleotide was converted into 3′-O(2-cyanoethyl-N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones.
221. Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.
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Berdyshev, Igor M., Svetlova, Anastasia O., Chukhontseva, Ksenia N., Karaseva, Maria A., Varizhuk, Anna M., Filatov, Vasily V., Kleymenov, Sergey Y., Kostrov, Sergey V., and Demidyuk, Ilya V.
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PROTEASE inhibitors , *PROTEOLYTIC enzyme regulation , *PEPTIDASE , *ENZYME regulation , *GEL permeation chromatography , *RECOMBINANT proteins - Abstract
Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts. [ABSTRACT FROM AUTHOR]
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- 2023
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222. Alpha-Deoxyguanosine to Reshape the Alpha-Thrombin Binding Aptamer.
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Kolganova, Natalia A., Tsvetkov, Vladimir B., Stomakhin, Andrey A., Surzhikov, Sergei A., Timofeev, Edward N., and Varizhuk, Irina V.
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APTAMERS , *QUADRUPLEX nucleic acids , *THERMAL stability , *THROMBIN - Abstract
Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences. [ABSTRACT FROM AUTHOR]
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- 2023
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223. G-Quadruplexes in Nuclear Biomolecular Condensates.
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Pavlova, Iuliia, Iudin, Mikhail, Surdina, Anastasiya, Severov, Vjacheslav, and Varizhuk, Anna
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SMALL molecules , *GENE expression , *TELOMERES , *PHASE transitions , *PROTEIN fractionation , *NUCLEOLUS - Abstract
G-quadruplexes (G4s) have long been implicated in the regulation of chromatin packaging and gene expression. These processes require or are accelerated by the separation of related proteins into liquid condensates on DNA/RNA matrices. While cytoplasmic G4s are acknowledged scaffolds of potentially pathogenic condensates, the possible contribution of G4s to phase transitions in the nucleus has only recently come to light. In this review, we summarize the growing evidence for the G4-dependent assembly of biomolecular condensates at telomeres and transcription initiation sites, as well as nucleoli, speckles, and paraspeckles. The limitations of the underlying assays and the remaining open questions are outlined. We also discuss the molecular basis for the apparent permissive role of G4s in the in vitro condensate assembly based on the interactome data. To highlight the prospects and risks of G4-targeting therapies with respect to the phase transitions, we also touch upon the reported effects of G4-stabilizing small molecules on nuclear biomolecular condensates. [ABSTRACT FROM AUTHOR]
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- 2023
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224. NMR structure of emfourin, a novel protein metalloprotease inhibitor: Insights into the mechanism of action.
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Bozin, Timur N., Berdyshev, Igor M., Chukhontseva, Ksenia N., Karaseva, Maria A., Konarev, Petr V., Varizhuk, Anna M., Lesovoy, Dmitry M., Arseniev, Alexander S., Kostrov, Sergey V., Bocharov, Eduard V., and Demidyuk, Ilya V.
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PROTEASE inhibitors , *SMALL-angle X-ray scattering , *SITE-specific mutagenesis , *NUCLEAR magnetic resonance spectroscopy , *PROTEINS , *PROTEIN structure - Abstract
Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family. [ABSTRACT FROM AUTHOR]
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- 2023
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225. Modeling G4s in chromatin context confirms partial nucleosome exclusion and reveals nucleosome-disrupting effects of the least selective G4 ligands.
- Author
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Pavlova, Iuliia, Barinov, Nikolay, Novikov, Roman, Severov, Vjacheslav, Iudin, Mikhail, Vedekhina, Tatiana, Larin, Andrey, Babenko, Vladislav, Aralov, Andrey, Gnuchikh, Evgeny, Sardushkin, Makar, Klinov, Dmitry, Tsvetkov, Vladimir, and Varizhuk, Anna
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CHROMATIN , *LIGANDS (Biochemistry) , *ATOMIC force microscopy , *QUADRUPLEX nucleic acids , *HISTONES , *NUCLEAR magnetic resonance spectroscopy - Abstract
G-quadruplexes (G4s) are gaining increasing attention as possible regulators of chromatin packaging, and robust approaches to their studies in pseudo-native context are much needed. Here, we designed a simple in vitro model of G4-prone genomic DNA and employed it to elucidate the impact of G4s and G4-stabilizing ligands on nucleosome occupancy. We obtained two 226-bp dsDNA constructs composed of the strong nucleosome positioning sequence and an internucleosomal DNA-imitating tail. The tail was G4-free in the control construct and harbored a "strong" (stable) G4 motif in the construct of interest. An additional "weak" (semi-stable) G4 motif was found within the canonical nucleosome positioning sequence. Both G4s were confirmed by optical methods and 1H NMR spectroscopy. Electrophoretic mobility assays showed that the weak G4 motif did not obstruct nucleosome assembly, while the strong G4 motif in the tail sequence diminished nucleosome yield. Atomic force microscopy data and molecular modeling confirmed that the strong G4 was maintained in the tail of the correctly assembled nucleosome structure. Using both in vitro and in silico models, we probed three known G4 ligands and detected nucleosome-disrupting effects of the least selective ligand. Our results are in line with the negative correlation between stable G4s and nucleosome density, support G4 tolerance between regularly positioned nucleosomes, and highlight the importance of considering chromatin context when targeting genomic G4s. • A minimal model of a G4 structure in native chromatin was designed. • The model was applied to verify mutual effects of G4s, histones, and ligands. • A stable G4 diminished nucleosome assembly at the adjacent 147-bp sequence. • A selective G4 ligand showed no effects on the nucleosome. • Ligands of limited selectivity induced nucleosome disassembly. [ABSTRACT FROM AUTHOR]
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- 2023
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226. Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro.
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Svetlova, Julia, Knizhnik, Ekaterina, Manuvera, Valentin, Severov, Vyacheslav, Shirokov, Dmitriy, Grafskaia, Ekaterina, Bobrovsky, Pavel, Matyugina, Elena, Khandazhinskaya, Anastasia, Kozlovskaya, Liubov, Miropolskaya, Nataliya, Aralov, Andrey, Khodarovich, Yuri, Tsvetkov, Vladimir, Kochetkov, Sergey, Lazarev, Vassili, and Varizhuk, Anna
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NUCLEOSIDE derivatives , *SARS-CoV-2 , *PHASE separation , *PERYLENE - Abstract
The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid–liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi–pi/cation–pi interactions. The set of antivirals included fleximers, 5′-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5′-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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227. cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals.
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Subach, Oksana M., Vlaskina, Anna V., Agapova, Yuliya K., Korzhenevskiy, Dmitriy A., Nikolaeva, Alena Y., Varizhuk, Anna M., Subach, Maksim F., Patrushev, Maxim V., Piatkevich, Kiryl D., Boyko, Konstantin M., and Subach, Fedor V.
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GREEN fluorescent protein , *TROPONIN , *CHEETAH , *HUMMINGBIRDS , *CALCIUM ions , *CALCIUM , *INTRACELLULAR calcium - Abstract
NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively. [ABSTRACT FROM AUTHOR]
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- 2022
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228. Microarray Profiling of Vaccination-Induced Antibody Responses to SARS-CoV-2 Variants of Interest and Concern.
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Svetlova, Julia, Gustin, Dmitry, Manuvera, Valentin, Shirokov, Dmitriy, Shokina, Varvara, Prusakov, Kirill, Aldarov, Konstantin, Kharlampieva, Daria, Matyushkina, Daria, Bespyatykh, Julia, Varizhuk, Anna, Lazarev, Vassili, and Vedekhina, Tatiana
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SARS-CoV-2 , *IMMUNOGLOBULINS , *FIBRINOGEN , *ANTIBODY formation , *VACCINATION complications , *COVID-19 vaccines - Abstract
Mutations in surface proteins enable emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to escape a substantial fraction of neutralizing antibodies and may thus weaken vaccine-driven immunity. To compare available vaccines and justify revaccination, rapid evaluation of antibody (Ab) responses to currently circulating SARS-CoV-2 variants of interest (VOI) and concern (VOC) is needed. Here, we developed a multiplex protein microarray-based system for rapid profiling of anti-SARS-CoV-2 Ab levels in human sera. The microarray system was validated using sera samples from SARS-CoV-2-free donors and those diagnosed with COVID-19 based on PCR and enzyme immunoassays. Microarray-based profiling of vaccinated donors revealed a substantial difference in anti-VOC Ab levels elicited by the replication-deficient adenovirus vector-base (Sputnik V) and whole-virion (CoviVac Russia COVID-19) vaccines. Whole-virion vaccine-induced Abs showed minor but statistically significant cross-reactivity with the human blood coagulation factor 1 (fibrinogen) and thrombin. However, their effects on blood clotting were negligible, according to thrombin time tests, providing evidence against the concept of pronounced cross-reactivity-related side effects of the vaccine. Importantly, all samples were collected in the pre-Omicron period but showed noticeable responses to the receptor-binding domain (RBD) of the Omicron spike protein. Thus, using the new express Ab-profiling system, we confirmed the inter-variant cross-reactivity of the anti-SARS-CoV-2 Abs and demonstrated the relative potency of the vaccines against new VOCs. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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- View/download PDF
229. Anticancer activity of G4-targeting phenoxazine derivatives in vitro.
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Lizunova, Sofia A., Tsvetkov, Vladimir B., Skvortsov, Dmitry A., Kamzeeva, Polina N., Ivanova, Olga M., Vasilyeva, Lilja A., Chistov, Alexey A., Belyaev, Evgeny S., Khrulev, Alexei A., Vedekhina, Tatiana S., Bogomazova, Alexandra N., Lagarkova, Maria A., Varizhuk, Anna M., and Aralov, Andrey V.
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ANTINEOPLASTIC agents , *CELL nuclei , *NUCLEOSIDE derivatives , *LIVER cells , *PROMOTERS (Genetics) , *POISONS , *CANCER cells - Abstract
G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC 50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC 50 in the micromolar range). Importantly, the CC 50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action. • Phenoxazine-based derivatives were probed as G4 ligands. • Some derivatives exhibited remarkably high G4-stabilizing effects and selectivity. • All phenoxazine-based ligands inhibited cellular metabolic activity. • The ligands were particularly toxic for lung adenocarcinoma cells. • Localization in the cell nuclei corroborated G4-mediated mechanisms of action. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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230. Diversifying i-motif-based pH sensors: Labeling patterns tune the intracellular localization.
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Shtork, Alina, Tsvetkov, Vladimir, Slushko, Georgy, Lushpa, Vladislav, Severov, Vjacheslav, Kamzeeva, Polina, Varizhuk, Anna, and Aralov, Andrey
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DETECTORS , *FLUORESCENCE quenching , *OPTICAL sensors , *CYTOSINE , *FLUOROPHORES - Abstract
Intercalated DNA motifs (iMs) provide a convenient scaffold for the design of biocompatible pH sensors. Among unimolecular iM-based sensors, only those labeled with conventional fluorophores or fluorophore/quencher pairs at the 3′ and 5′ termini for FRET/quenching upon pH-dependent iM folding have been tested in cells and tended to accumulate in the nuclei. Here, we used cytosine mimics as internal iM labels and synthesized a new phenoxazine-based non-fluorescent nucleoside analog, tC° Azo , which quenches the fluorescence of a known cytosine mimic, tC°. Incorporation of the tC°/tC° Azo pair into a genomic iM-forming sequence C5T resulted in a high-contrast pH sensor with an increased pH transition point and a working range compatible with physiological conditions. Importantly, unlike the known nuclei-specific C5T-based sensors with conventional labels that provide a fluorescent signal in the green/red channels, the new sensor localized mainly in the cytoplasm and allowed pH monitoring based on the tC° signal in the blue channel. As the labeling scheme was the only unique feature of the new sensor, it must account for the unique distribution pattern, i.e., the accumulation of the sensor in the cytoplasm. These findings highlight the importance of the labeling scheme of unimolecular iM-based pH sensors and open the way for multiplexed pH monitoring in different cellular compartments. [Display omitted] • An optical pH sensor was developed using an i-motif ODN with a new labeling system. • It exploits the quenching of the fluorophore tC° by the new nucleoside analog tC azo • Unlike most i-motif-based tools, the new sensor allows pH monitoring in the cytoplasm. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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- View/download PDF
231. Cytotoxicity reduction by O-nicotinoylation of antiviral 6-benzylaminopurine ribonucleosides.
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Zenchenko, Anastasia A., Oslovsky, Vladimir E., Varizhuk, Irina V., Karpova, Evgenia V., Osolodkin, Dmitry I., Kozlovskaya, Liubov I., Ishmukhametov, Aydar A., and Drenichev, Mikhail S.
- Abstract
One of the promising approaches in the development of nucleoside prodrugs is to use the nucleoside analogs containing lipophilic biodegradable residues, which are cleaved to biologically active forms after metabolic transformations in the cell. The introduction of such fragments makes it possible to reduce the general toxicity of the drug candidate and increase its stability in the cell. In order to study the influence of biodegradable lipophilic groups on antiviral activity and cytotoxicity, in this work we synthesized N 6-benzyl-2′,3′,5′-tri- O -nicotinoyl adenosine and N 6-(3-fluorobenzyl)-2′,3′,5′-tri- O -nicotinoyl adenosine, derivatives of N 6-benzyladenosine (BAR) and N 6-(3-fluorobenzyl)adenosine (FBAR), which had previously shown prominent antiviral activity against human enterovirus EV-A71 but appeared to be cytotoxic. The obtained fully- O -nicotinoylated BAR and FBAR inhibited reproduction of EV-A71 strains BrCr and 46973 and manifested significantly lower cytotoxicity compared to non-protected compounds. In addition, we performed enzymatic hydrolysis of the fully- O -nicotinoylated FBAR in the presence of esterases (CalB and PLE) to investigate metabolic degradation of O -nicotinoylated compounds in cells. Both enzymes hydrolyzed the tested substrate to form the corresponding O -deprotected nucleoside that may suggest the role of hydrolase-type enzymes as general participants of metabolic activation of O -nicotinoylated prodrugs in different cells. [Display omitted] • 6-Benzylaminopurine ribonucleosides with biodegradable O- nicotinoyl ester moieties. • Fully- O -nicotinoylated nucleosides exhibit decrease in cytotoxicity in RD cells. • Fully- O -nicotinoylated nucleosides manifest antiviral activity against EV-A71. • Gradual hydrolysis of fully- O -nicotinoylated nucleosides in the presence of esterases. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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232. Autoimmune Effect of Antibodies against the SARS-CoV-2 Nucleoprotein.
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Matyushkina, Daria, Shokina, Varvara, Tikhonova, Polina, Manuvera, Valentin, Shirokov, Dmitry, Kharlampieva, Daria, Lazarev, Vasily, Varizhuk, Anna, Vedekhina, Tatiana, Pavlenko, Alexander, Penkin, Leonid, Arapidi, Georgij, Pavlov, Konstantin, Pushkar, Dmitry, Kolontarev, Konstantin, Rumyantsev, Alexander, Rumyantsev, Sergey, Rychkova, Lyubov, and Govorun, Vadim
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POST-acute COVID-19 syndrome , *SARS-CoV-2 , *VIRAL proteins , *COVID-19 , *AUTOIMMUNE diseases , *NUCLEOPROTEINS , *AUTOANTIBODIES , *IMMUNOGLOBULINS - Abstract
COVID-19 caused by SARS-CoV-2 is continuing to spread around the world and drastically affect our daily life. New strains appear, and the severity of the course of the disease itself seems to be decreasing, but even people who have been ill on an outpatient basis suffer post-COVID consequences. Partly, it is associated with the autoimmune reactions, so debates about the development of new vaccines and the need for vaccination/revaccination continue. In this study we performed an analysis of the antibody response of patients with COVID-19 to linear and conformational epitopes of viral proteins using ELISA, chip array and western blot with analysis of correlations between antibody titer, disease severity, and complications. We have shown that the presence of IgG antibodies to the nucleoprotein can deteriorate the course of the disease, induce multiple direct COVID-19 symptoms, and contribute to long-term post-covid symptoms. We analyzed the cross reactivity of antibodies to SARS-CoV-2 with own human proteins and showed that antibodies to the nucleocapsid protein can bind to human proteins. In accordance with the possibility of HLA presentation, the main possible targets of the autoantibodies were identified. People with HLA alleles A01:01; A26:01; B39:01; B15:01 are most susceptible to the development of autoimmune processes after COVID-19. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
233. DNA Intercalated Motifs with Non-Nucleoside Inserts.
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Petrunina, N. A., Lebedev, V. V., Kirillova, Yu. G., Aralov, A. V., Varizhuk, A. M., and Sardushkin, M. V.
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GENETIC regulation , *DNA , *DNA repair , *CYTOSINE , *HUMAN genome , *OLIGONUCLEOTIDES - Abstract
A cytosine-rich sequence from the human genome that is presumably involved in the regulation of SHANK1 gene expression through the formation of an intercalated motif (iM) has been investigated. A modification that imitates base excision in the iM loops was shown to significantly accelerate the conformational transitions induced by small pH changes. These results supplement the understanding of the relation between DNA repair and the formation of noncanonical secondary structures. Moreover, they open up new possibilities in the development of oligonucleotide pH sensors with a fast response. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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234. Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring.
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Tsvetkov, Vladimir B., Turaev, Anton V., Petrunina, Nataliia A., Melnik, Denis M., Khodarovich, Yuriy M., Pozmogova, Galina E., Zatsepin, Timofei S., Varizhuk, Anna M., and Aralov, Andrey V.
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SMALL molecules , *HIGH throughput screening (Drug development) , *FLUORESCENCE , *DNA , *DRUG development - Abstract
The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM–BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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- View/download PDF
235. Thiadiazole-, selenadiazole- and triazole-fused anthraquinones as G-quadruplex targeting anticancer compounds.
- Author
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Andreeva, Daria V., Vedekhina, Tatiana S., Gostev, Alexander S., Dezhenkova, Lyubov G., Volodina, Yulia L., Markova, Alina A., Nguyen, Minh Tuan, Ivanova, Olga M., Dolgusheva, Vladislava А., Varizhuk, Anna M., Tikhomirov, Alexander S., and Shchekotikhin, Andrey E.
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THIADIAZOLES , *ANTHRAQUINONES , *ANTHRAQUINONE derivatives , *CELL growth , *INHIBITION of cellular proliferation , *LIGANDS (Biochemistry) , *CELL cycle , *ONCOGENES - Abstract
G-quadruplex (G4) ligands attract considerable attention as potential anticancer therapeutics. In this study we proposed an original scheme for synthesis of azole-fused anthraquinones and prepared a series of G4 ligands carrying amino- or guanidinoalkylamino side chains. The heterocyclic core and structure of the terminal groups strongly affect on binding to G4-forming oligonucleotides, cellular accumulation and antitumor potency of compounds. In particular, thiadiazole- and selenadiazole- but not triazole-based ligands inhibit the proliferation of tumor cells (e.g. K562 leukemia) and stabilize primarily telomeric and c-MYC G4s. Anthraselenadiazole derivative 11a showed a good affinity to c-MYC G4 in vitro and down-regulated expression of c-MYC oncogene in cellular conditions. Further studies revealed that anthraselenadiazole 11a provoked cell cycle arrest and apoptosis in a dose- and time-dependent manner inhibiting K562 cells growth. Taken together, this work gives a valuable example that the closely related heterocycles may cause a significant difference in biological properties of G4 ligands. [Display omitted] • A method for synthesis of azole-fused anthraquinone derivatives has been developed. • Selenadiazole- and thiadiazole-based ligands effectively stabilize G4s. • Guanidination of the aminoalkyl side chains increases the thermal shifts of G4s melting. • In vitro anticancer activity of the ligands correlates to G4s binding and intracellular uptake. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
236. Transcription-facilitating histone chaperons interact with genomic and synthetic G4 structures.
- Author
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Pavlova, Iulia I., Tsvetkov, Vladimir B., Isaakova, Ekaterina A., Severov, Vyacheslav V., Khomyakova, Ekaterina A., Lacis, Ivan A., Lazarev, Vassilii N., Lagarkova, Maria A., Pozmogova, Galina E., and Varizhuk, Anna M.
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BINDING site assay , *CELL survival , *CHROMATIN-remodeling complexes , *MOLECULAR models , *CELL lines , *ANTINEOPLASTIC agents - Abstract
Affinity for G-quadruplex (G4) structures may be a common feature of transcription-facilitating histone chaperons (HCs). This assumption is based on previous unmatched studies of HCs FACT, nucleolin (NCL), BRD3, and ATRX. We verified this assumption and considered its implications for the therapeutic applications of synthetic (exogenous) G4s and the biological significance of genomic G4s. First, we questioned whether exogenous G4s that recognize cell-surface NCL and could trap other HCs in the nucleus are usable as anticancer agents. We performed in vitro binding assays and selected leading multi-targeted G4s. They exhibited minor effects on cell viability. The presumed NCL-regulated intracellular transport of G4s was inefficient or insufficient for tumor-specific G4 delivery. Next, to clarify whether G4s in the human genome could recruit HCs, we compared available HC ChIP-seq data with G4-seq/G4-ChIP-seq data. Several G4s, including the well-known c-Myc quadruplex structure, were found to be colocalized with HC occupancy sites in cancer cell lines. As evidenced by our molecular modeling data, c-Myc G4 might interfere with the HC function of BRD3 but is unlikely to prevent the BRD3-driven assembly of the chromatin remodeling complex. The c-Myc case illustrates the intricate role of genomic G4s in chromatin remodeling, nucleosome remodeling, and transcription. Unlabelled Image [ABSTRACT FROM AUTHOR]
- Published
- 2020
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- View/download PDF
237. DNA complexes with Ni nanoparticles: structural and functional properties.
- Author
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Tatarinova, Olga, Smirnov, Igor, Safenkova, Irina, Varizhuk, Anna, and Pozmogova, Galina
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METAL nanoparticles , *DNA , *COMPLEX compounds , *MOLECULAR structure , *SUPRAMOLECULAR chemistry , *BIOPOLYMERS , *MAGNETIC nanoparticles , *MASS spectrometry , *ELECTROPHORESIS - Abstract
Supramolecular complexes of biopolymers based on magnetic nanoparticles play an important role in creation of biosensors, implementation of theragnostic and gene therapeutic methods and biosafety evaluation. We investigated the impact of DNA interactions with nanoparticles of nickel (nNi) on the integrity and functionality of DNA. Data obtained by mass spectrometry, electrophoresis, TEM and AFM microscopy techniques, bacterial transformation, and real-time PCR provide evidence that ssDNA and plasmid DNA (pDNA) efficiently form complexes with nNi. AFM data suggest that the complexes are necklace-type structures, in which nanoparticles are randomly distributed along the DNA chains, rather than highly entangled clot-type structures. After desorption, observed DNA characteristics in bioanalytical and biological systems remain unchanged. Only supercoiled pDNA was nicked, but remained, as well as a plasmid-nNi complex, active in expression vector assays. These results are very important for creation of new methods of DNA immobilization and controlled manipulation. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
238. DNA G-Quadruplexes Contribute to CTCF Recruitment.
- Author
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Tikhonova, Polina, Pavlova, Iulia, Isaakova, Ekaterina, Tsvetkov, Vladimir, Bogomazova, Alexandra, Vedekhina, Tatjana, Luzhin, Artem V., Sultanov, Rinat, Severov, Vjacheslav, Klimina, Ksenia, Kantidze, Omar L., Pozmogova, Galina, Lagarkova, Maria, and Varizhuk, Anna
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HIGH mobility group proteins , *DNA , *QUADRUPLEX nucleic acids , *HUMAN genome , *BINDING site assay , *CHROMATIN - Abstract
G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
239. Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges.
- Author
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Turaev, Anton V., Isaakova, Ekaterina A., Severov, Vjacheslav V., Bogomazova, Alexandra N., Zatsepin, Timofei S., Sardushkin, Makar V., Aralov, Andrey V., Lagarkova, Maria A., Pozmogova, Galina E., and Varizhuk, Anna M.
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DNA structure , *DETECTORS , *CELL nuclei , *CELL imaging , *DNA - Abstract
We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools. • A novel 'genome-inspired' approach for designing pH sensors was probed. • A robust method for selecting 'fast' sensors based on melting assays was proposed. • New sensors were obtained using selected genomic i-motifs and fluorescent labels. • The lead sensor detected rapid and slight pH changes within the physiological range. • The sensor is useable for pH imaging in living cells and localizes in cell nuclei. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
240. Efficient silica synthesis from tetra(glycerol)orthosilicate with cathepsin- and silicatein-like proteins.
- Author
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Povarova, Natalia V., Barinov, Nikolay A., Baranov, Mikhail S., Markina, Nadezhda M., Varizhuk, Anna M., Pozmogova, Galina E., Klinov, Dmitry V., Kozhemyako, Valery B., and Lukyanov, Konstantin A.
- Abstract
Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L - a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins - silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
241. NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.
- Author
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Barykina, Natalia V., Doronin, Danila A., Subach, Oksana M., Sotskov, Vladimir P., Plusnin, Viktor V., Ivleva, Olga A., Gruzdeva, Anna M., Kunitsyna, Tatiana A., Ivashkina, Olga I., Lazutkin, Alexander A., Malyshev, Aleksey Y., Smirnov, Ivan V., Varizhuk, Anna M., Pozmogova, Galina E., Piatkevich, Kiryl D., Anokhin, Konstantin V., Enikolopov, Grigori, and Subach, Fedor V.
- Abstract
The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
242. Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics.
- Author
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Doronin, D. A., Sotskov, V. P., Plusnin, V. V., Barykina, N. V., Subach, O. M., Subach, F. V., Ivleva, O. A., Isaakova, E. A., Enikolopov, G. N., Anokhin, K. V., Pozmogova, G. E., Varizhuk, A. M., Malyshev, A. Y., Smirnov, I. V., and Piatkevich, K. D.
- Subjects
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CALCIUM in the body , *TROPONIN , *FLUORESCENT proteins , *CALCIUM ions , *PROTEIN engineering , *CHROMOPHORES - Abstract
Background: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC’s high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. Results: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. Conclusions: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
243. Synthesis and Biological Evaluation of Benzo [4,5]- and Naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone Derivatives.
- Author
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Kamzeeva P, Dagaev N, Lizunova S, Khodarovich Y, Sogomonyan A, Kolchanova A, Pokrovsky V, Alferova V, Chistov A, Eshtukov-Shcheglov A, Eshtukova-Shcheglova E, Belyaev E, Skvortsov D, Varizhuk A, and Aralov A
- Subjects
- Animals, Mice, Apoptosis, Cell Line, Tumor, G2 Phase Cell Cycle Checkpoints, Cell Proliferation, Molecular Structure, Antineoplastic Agents therapeutic use, Lung Neoplasms drug therapy
- Abstract
Azacarbazoles have attracted significant interest due to their valuable properties, such as anti-pathogenic and antitumor activity. In this study, a series of structurally related tricyclic benzo[4,5]- and tertacyclic naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone derivatives with one or two positively charged tethers were synthesized and evaluated for anti-proliferative activity. Lead tetracyclic derivative 5b with two amino-bearing arms inhibited the metabolic activity of A549 lung adenocarcinoma cells with a CC
50 value of 3.6 μM, with remarkable selectivity (SI = 17.3) over VA13 immortalized fibroblasts. Cell-cycle assays revealed that 5b triggers G2/M arrest without signs of apoptosis. A study of its interaction with various DNA G4s and duplexes followed by dual luciferase and intercalator displacement assays suggests that intercalation, rather than the modulation of G4-regulated oncogene expression, might contribute to the observed activity. Finally, a water-soluble salt of 5b was shown to cause no acute toxic effects, changes in mice behavior, or any decrease in body weight after a 72 h treatment at concentrations up to 20 mg/kg. Thus, 5b is a promising candidate for studies in vivo; however, further investigations are needed to elucidate its molecular target(s).- Published
- 2023
- Full Text
- View/download PDF
244. Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication.
- Author
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Knizhnik E, Chumakov S, Svetlova J, Pavlova I, Khodarovich Y, Brylev V, Severov V, Alieva R, Kozlovskaya L, Andreev D, Aralov A, and Varizhuk A
- Subjects
- Humans, Phosphorothioate Oligonucleotides pharmacology, SARS-CoV-2 metabolism, RNA, Viral metabolism, Antiviral Agents pharmacology, DNA metabolism, Virus Replication, Nucleic Acid Conformation, Frameshifting, Ribosomal, COVID-19
- Abstract
Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA-DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.
- Published
- 2023
- Full Text
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245. Phenotypic Test of Benzo[4,5]imidazo[1,2-c]pyrimidinone-Based Nucleoside and Non-Nucleoside Derivatives against DNA and RNA Viruses, Including Coronaviruses.
- Author
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Kamzeeva P, Petushkov I, Knizhnik E, Snoeck R, Khodarovich Y, Ryabukhina E, Alferova V, Eshtukov-Shcheglov A, Belyaev E, Svetlova J, Vedekhina T, Kulbachinskiy A, Varizhuk A, Andrei G, and Aralov A
- Subjects
- Humans, Antiviral Agents pharmacology, Antiviral Agents chemistry, RNA, Viral, Pandemics, SARS-CoV-2, DNA, Nucleosides pharmacology, Nucleosides chemistry, RNA Viruses
- Abstract
Emerging and re-emerging viruses periodically cause outbreaks and epidemics around the world, which ultimately lead to global events such as the COVID-19 pandemic. Thus, the urgent need for new antiviral drugs is obvious. Over more than a century of antiviral development, nucleoside analogs have proven to be promising agents against diversified DNA and RNA viruses. Here, we present the synthesis and evaluation of the antiviral activity of nucleoside analogs and their deglycosylated derivatives based on a hydroxybenzo[4,5]imidazo[1,2-c]pyrimidin-1(2H)-one scaffold. The antiviral activity was evaluated against a panel of structurally and phylogenetically diverse RNA and DNA viruses. The leader compound showed micromolar activity against representatives of the family Coronaviridae , including SARS-CoV-2, as well as against respiratory syncytial virus in a submicromolar range without noticeable toxicity for the host cells. Surprisingly, methylation of the aromatic hydroxyl group of the leader compound resulted in micromolar activity against the varicella-zoster virus without any significant impact on cell viability. The leader compound was shown to be a weak inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase. It also inhibited biocondensate formation important for SARS-CoV-2 replication. The active compounds may be considered as a good starting point for further structure optimization and mechanistic and preclinical studies.
- Published
- 2023
- Full Text
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246. Genome-Wide Transcriptional Response of Mycobacterium smegmatis MC 2 155 to G-Quadruplex Ligands BRACO-19 and TMPyP4.
- Author
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Shitikov E, Bespiatykh D, Malakhova M, Bespyatykh J, Bodoev I, Vedekhina T, Zaychikova M, Veselovsky V, Klimina K, Ilina E, and Varizhuk A
- Abstract
G-quadruplexes (G4s) are non-canonical DNA structures that could be considered as potential therapeutic targets for antimicrobial compounds, also known as G4-stabilizing ligands. While some of these ligands are shown in vitro to have a stabilizing effect, the precise mechanism of antibacterial action has not been fully investigated. Here, we employed genome-wide RNA-sequencing to analyze the response of Mycobacterium smegmatis to inhibitory concentrations of BRACO-19 and TMPyP4 G4 ligands. The expression profile changed (FDR < 0.05, log
2 FC > |1|) for 822 (515↑; 307↓) genes in M. smegmatis in response to BRACO-19 and for 680 (339↑; 341↓) genes in response to TMPyP4. However, the analysis revealed no significant ligand-induced changes in the expression levels of G4-harboring genes, genes under G4-harboring promoters, or intergenic regions located on mRNA-like or template strands. Meanwhile, for the BRACO-19 ligand, we found significant changes in the replication and repair system genes, as well as in iron metabolism genes which is, undoubtedly, evidence of the induced stress. For the TMPyP4 compound, substantial changes were found in transcription factors and the arginine biosynthesis system, which may indicate multiple biological targets for this compound., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shitikov, Bespiatykh, Malakhova, Bespyatykh, Bodoev, Vedekhina, Zaychikova, Veselovsky, Klimina, Ilina and Varizhuk.)- Published
- 2022
- Full Text
- View/download PDF
247. 16S rRNA gene sequencing data of the upper respiratory tract microbiome in the SARS-CoV-2 infected patients.
- Author
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Galeeva J, Babenko V, Bakhtyev R, Baklaushev V, Balykova L, Bashkirov P, Bespyatykh J, Blagonravova A, Boldyreva D, Fedorov D, Gafurov I, Gaifullina R, Galova E, Gospodaryk A, Ilina E, Ivanov K, Kharlampieva D, Khromova P, Klimina K, Kolontarev K, Kolyshkina N, Koritsky A, Kuropatkin V, Lazarev V, Manolov A, Manuvera V, Matyushkina D, Morozov M, Moskaleva E, Musarova V, Ogarkov O, Orlova E, Pavlenko A, Petrova A, Pozhenko N, Pushkar D, Rumyantsev A, Rumyantsev S, Rumyantsev V, Rychkova L, Samoilov A, Shirokova I, Sinkov V, Solovieva S, Starikova E, Tikhonova P, Trifonova G, Troitsky A, Tulichev A, Udalov Y, Varizhuk A, Vasiliev A, Veselovsky V, Vereshchagin R, Volnukhin A, Yusubalieva G, and Govorun V
- Abstract
The SARS-CoV-2 pandemic is a big challenge for humanity. The COVID-19 severity differs significantly from patient to patient, and it is important to study the factors protecting from severe forms of the disease. Respiratory microbiota may influence the patient's susceptibility to infection and disease severity due to its ability to modulate the immune system response of the host organism. This data article describes the microbiome dataset from the upper respiratory tract of SARS-CoV-2 positive patients from Russia. This dataset reports the microbial community profile of 335 human nasopharyngeal swabs collected between 2020-05 and 2021-03 during the first and the second epidemic waves. Samples were collected from both inpatients and outpatients in 4 cities of the Russian Federation (Moscow, Kazan, Irkutsk, Nizhny Novgorod) and sequenced using the 16S rRNA gene amplicon sequencing of V3-V4 region. Data contains information about the patient such as age, sex, hospitalization status, percent of damaged lung tissue, oxygen saturation (SpO2), respiratory rate, need for supplemental oxygen, chest computer tomography severity score, SARS-CoV-2 lineage, and also information about smoking and comorbidities. The amplicon sequencing data were deposited at NCBI SRA as BioProject PRJNA751478., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier Inc.)
- Published
- 2022
- Full Text
- View/download PDF
248. EGCG as an anti-SARS-CoV-2 agent: Preventive versus therapeutic potential against original and mutant virus.
- Author
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Tsvetkov V, Varizhuk A, Kozlovskaya L, Shtro A, Lebedeva O, Komissarov A, Vedekhina T, Manuvera V, Zubkova O, Eremeev A, Shustova E, Pozmogova G, Lioznov D, Ishmukhametov A, Lazarev V, and Lagarkova M
- Subjects
- Animals, Catechin pharmacology, Chlorocebus aethiops, Molecular Docking Simulation, Molecular Dynamics Simulation, SARS-CoV-2 chemistry, Vero Cells, Viral Proteins chemistry, Viral Proteins metabolism, Virus Internalization drug effects, Antiviral Agents pharmacology, Catechin analogs & derivatives, Mutation, SARS-CoV-2 drug effects, SARS-CoV-2 genetics, COVID-19 Drug Treatment
- Abstract
In the search for anti-SARS-CoV-2 drugs, much attention is given to safe and widely available native compounds. The green tea component epigallocatechin 3 gallate (EGCG) is particularly promising because it reportedly inhibits viral replication and viral entry in vitro. However, conclusive evidence for its predominant activity is needed. We tested EGCG effects on the native virus isolated from COVID-19 patients in two independent series of experiments using VERO cells and two different treatment schemes in each series. The results confirmed modest cytotoxicity of EGCG and its substantial antiviral activity. The preincubation scheme aimed at infection prevention has proven particularly beneficial. We complemented that finding with a detailed investigation of EGCG interactions with viral S-protein subunits, including S2, RBD, and the RBD mutant harboring the N501Y mutation. Molecular modeling experiments revealed N501Y-specific stacking interactions in the RBD-ACE2 complex and provided insight into EGCG interference with the complex formation. Together, these findings provide a molecular basis for the observed EGCG effects and reinforce its prospects in COVID-19 prevention therapy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)
- Published
- 2021
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249. Aureolic Acid Group of Agents as Potential Antituberculosis Drugs.
- Author
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Bespyatykh J, Bespiatykh D, Malakhova M, Klimina K, Bespyatykh A, Varizhuk A, Tevyashova A, Nikolenko T, Pozmogova G, Ilina E, and Shitikov E
- Abstract
Mycobacterium tuberculosis is one of the most dangerous pathogens. Bacterial resistance to antituberculosis drugs grows each year, but searching for new drugs is a long process. Testing for available drugs to find active against mycobacteria may be a good alternative. In this work, antibiotics of the aureolic acid group were tested on a model organism Mycobacterium smegmatis . We presumed that antibiotics of this group may be potential G4 ligands. However, this was not confirmed in our analyses. We determined the antimicrobial activity of these drugs and revealed morphological changes in the cell structure upon treatment. Transcriptomic analysis documented increased expression of MSMEG_3743/soj and MSMEG_4228/ftsW , involved in cell division. Therefore, drugs may affect cell division, possibly disrupting the function of the Z-ring and the formation of a septum. Additionally, a decrease in the transcription level of several indispensable genes, such as nitrate reductase subunits ( MSMEG_5137/narI and MSMEG_5139/narX ) and MSMEG_3205/hisD was shown. We concluded that the mechanism of action of aureolic acid and its related compounds may be similar to that bedaquiline and disturb the NAD+/NADH balance in the cell. All of this allowed us to conclude that aureolic acid derivatives can be considered as potential antituberculosis drugs.
- Published
- 2020
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250. G4 Aptamers: Trends in Structural Design.
- Author
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Varizhuk A, Ilyinsky N, Smirnov I, and Pozmogova G
- Subjects
- Animals, Humans, Aptamers, Nucleotide chemical synthesis, Aptamers, Nucleotide chemistry, G-Quadruplexes
- Abstract
Many potent DNA aptamers are known to contain a G-quadruplex (G4) core. Structures and applications of the majority of such aptamers have been reviewed previously. The present review focuses on the design and optimization of G4 aptamers. General features of bioactive G4s are analyzed, and the main strategies for construction of aptamers with desired properties and topologies, including modular assembly, control of an aptamer folding and some others, are outlined. Chemical modification as a method for post-SELEX G4 aptamer optimization is also discussed, and the effects of loop and core modifications are compared. Particular attention is paid to the emerging trends, such as the development of genomic G4- inspired aptamers and the combinatorial approaches which aim to find a balance between rational design and selection.
- Published
- 2016
- Full Text
- View/download PDF
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