43 results on '"Dyrkheeva NS"'
Search Results
2. Transcriptomic analysis of HEK293A cells with a CRISPR/Cas9-mediated TDP1 knockout.
- Author
-
Dyrkheeva NS, Zakharenko AL, Malakhova AA, Okorokova LS, Shtokalo DN, Medvedev SP, Tupikin AA, Kabilov MR, and Lavrik OI
- Subjects
- Humans, HEK293 Cells, Gene Knockout Techniques methods, Transcriptome genetics, Gene Expression Profiling, DNA Repair genetics, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, CRISPR-Cas Systems
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
3. A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts.
- Author
-
Khodyreva SN, Ilina ES, Dyrkheeva NS, Kochetkova AS, Yamskikh AA, Maltseva EA, Malakhova AA, Medvedev SP, Zakian SM, and Lavrik OI
- Subjects
- Humans, Cell Extracts, Cell Line, X-ray Repair Cross Complementing Protein 1 genetics, DNA Repair, Excision Repair, Poly (ADP-Ribose) Polymerase-1 genetics
- Abstract
Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.
- Published
- 2024
- Full Text
- View/download PDF
4. Enhancement of the Antitumor and Antimetastatic Effect of Topotecan and Normalization of Blood Counts in Mice with Lewis Carcinoma by Tdp1 Inhibitors-New Usnic Acid Derivatives.
- Author
-
Kornienko TE, Chepanova AA, Zakharenko AL, Filimonov AS, Luzina OA, Dyrkheeva NS, Nikolin VP, Popova NA, Salakhutdinov NF, and Lavrik OI
- Subjects
- Animals, Mice, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Esterases, Topotecan pharmacology, Topotecan therapeutic use, Carcinoma, Benzofurans
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme and one of the causes of tumor resistance to topoisomerase 1 inhibitors such as topotecan. Inhibitors of this Tdp1 in combination with topotecan may improve the effectiveness of therapy. In this work, we synthesized usnic acid derivatives, which are hybrids of its known derivatives: tumor sensitizers to topotecan. New compounds inhibit Tdp1 in the micromolar and submicromolar concentration range; some of them enhance the effect of topotecan on the metabolic activity of cells of various lines according to the MTT test. One of the new compounds (compound 7 ) not only sensitizes Krebs-2 and Lewis carcinomas of mice to the action of topotecan, but also normalizes the state of the peripheral blood of mice, which is disturbed in the presence of a tumor. Thus, the synthesized substances may be the prototype of a new class of additional therapy for cancer.
- Published
- 2024
- Full Text
- View/download PDF
5. Pharmacokinetic study of Tdp1 inhibitor resulted in a significant increase in antitumor effect in the treatment of Lewis lung carcinoma in mice by its combination with topotecan.
- Author
-
Okhina AA, Kornienko TE, Rogachev AD, Luzina OA, Popova NA, Nikolin VP, Zakharenko AL, Dyrkheeva NS, Pokrovsky AG, Salakhutdinov NF, and Lavrik OI
- Abstract
We have previously shown that the Tdp1 inhibitor, enamine derivative of usnic acid, the agent OL9-116, enhances the antitumor activity of topotecan. In the present study, we developed and validated LC-MS/MS method for the quantification of OL9-116 in mouse whole blood and studied pharmacokinetics of the agent. The substance OL9-116 was shown to be stable in the whole blood in vitro. Sample preparation included two steps: mixing 10 µL of a blood sample with 10 µL of 0.2 M ZnSO
4 aqueous solution, followed by protein precipitation with 100 µL of acetonitrile containing internal standard. Quantification of the compound was performed using SCIEX 6500 QTRAP mass spectrometer in MRM mode following chromatographic separation on a C8 reversed-phase column. The method was validated in terms of selectivity, linearity, accuracy, precision, recovery, and stability of the prepared sample. When the agent OL9-116 was administered intragastrically at a dose of 150 mg/kg, the maximum concentration in the blood (about 5000 ng/mL) was reached after 2-4 h followed by the distribution and elimination of the compound. A study of the antitumor activity of a combination of OL9-116 and topotecan against Lewis lung carcinoma revealed that administration of topotecan 3 h after OL9-116 resulted in the most pronounced antitumor effect compared to simultaneous or individual administration of both compounds., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 Elsevier B.V. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
6. Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.
- Author
-
Maltseva EA, Vasil'eva IA, Moor NA, Kim DV, Dyrkheeva NS, Kutuzov MM, Vokhtantsev IP, Kulishova LM, Zharkov DO, and Lavrik OI
- Subjects
- Humans, DNA Repair, DNA Damage, DNA genetics, DNA metabolism, DNA Breaks, RNA, CRISPR-Cas Systems, Poly(ADP-ribose) Polymerases genetics, Poly(ADP-ribose) Polymerases metabolism
- Abstract
CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Maltseva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2023
- Full Text
- View/download PDF
7. Mutant-Huntingtin Molecular Pathways Elucidate New Targets for Drug Repurposing.
- Author
-
Makeeva VS, Dyrkheeva NS, Lavrik OI, Zakian SM, and Malakhova AA
- Subjects
- Humans, Drug Repositioning, Drug Development, Huntingtin Protein genetics, Mutation, Huntington Disease drug therapy, Huntington Disease genetics, Huntington Disease metabolism, Neurodegenerative Diseases
- Abstract
The spectrum of neurodegenerative diseases known today is quite extensive. The complexities of their research and treatment lie not only in their diversity. Even many years of struggle and narrowly focused research on common pathologies such as Alzheimer's, Parkinson's, and other brain diseases have not brought cures for these illnesses. What can be said about orphan diseases? In particular, Huntington's disease (HD), despite affecting a smaller part of the human population, still attracts many researchers. This disorder is known to result from a mutation in the HTT gene, but having this information still does not simplify the task of drug development and studying the mechanisms of disease progression. Nonetheless, the data accumulated over the years and their analysis provide a good basis for further research. Here, we review studies devoted to understanding the mechanisms of HD. We analyze genes and molecular pathways involved in HD pathogenesis to describe the action of repurposed drugs and try to find new therapeutic targets.
- Published
- 2023
- Full Text
- View/download PDF
8. Usnic Acid Derivatives Inhibit DNA Repair Enzymes Tyrosyl-DNA Phosphodiesterases 1 and 2 and Act as Potential Anticancer Agents.
- Author
-
Zakharenko AL, Dyrkheeva NS, Luzina OA, Filimonov AS, Mozhaitsev ES, Malakhova AA, Medvedev SP, Zakian SM, Salakhutdinov NF, and Lavrik OI
- Subjects
- Etoposide, Dimethyl Sulfoxide, Phosphoric Diester Hydrolases genetics, DNA Repair Enzymes, DNA-Binding Proteins genetics, Antineoplastic Agents pharmacology
- Abstract
Tyrosyl-DNA phosphodiesterase 1 and 2 (Tdp1 and Tdp2) are DNA repair enzymes that repair DNA damage caused by various agents, including anticancer drugs. Thus, these enzymes resist anticancer therapy and could be the reason for resistance to such widely used drugs such as topotecan and etoposide. In the present work, we found compounds capable of inhibiting both enzymes among derivatives of (-)-usnic acid. Both (+)- and (-)-enantiomers of compounds act equally effectively against Tdp1 with IC
50 values in the range of 0.02-0.2 μM; only (-)-enantiomers inhibited Tdp2 with IC50 values in the range of 6-9 μM. Surprisingly, the compounds protect HEK293FT wild type cells from the cytotoxic effect of etoposide (CC50 3.0-3.9 μM in the presence of compounds and 2.4 μM the presence of DMSO) but potentiate it against Tdp2 knockout cells (CC50 1.2-1.6 μM in the presence of compounds against 2.3 μM in the presence of DMSO). We assume that the sensitizing effect of the compounds in the absence of Tdp2 is associated with the effective inhibition of Tdp1, which could take over the functions of Tdp2.- Published
- 2023
- Full Text
- View/download PDF
9. New 5-Hydroxycoumarin-Based Tyrosyl-DNA Phosphodiesterase I Inhibitors Sensitize Tumor Cell Line to Topotecan.
- Author
-
Khomenko TM, Zakharenko AL, Kornienko TE, Chepanova AA, Dyrkheeva NS, Artemova AO, Korchagina DV, Achara C, Curtis A, Reynisson J, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Humans, Phosphodiesterase Inhibitors pharmacology, Phosphodiesterase Inhibitors chemistry, Structure-Activity Relationship, Phosphoric Diester Hydrolases metabolism, Cell Line, Tumor, Topotecan pharmacology, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry
- Abstract
Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is an important enzyme in the DNA repair system. The ability of the enzyme to repair DNA damage induced by a topoisomerase 1 poison such as the anticancer drug topotecan makes TDP1 a promising target for complex antitumor therapy. In this work, a set of new 5-hydroxycoumarin derivatives containing monoterpene moieties was synthesized. It was shown that most of the conjugates synthesized demonstrated high inhibitory properties against TDP1 with an IC
50 in low micromolar or nanomolar ranges. Geraniol derivative 33a was the most potent inhibitor with IC50 130 nM. Docking the ligands to TDP1 predicted a good fit with the catalytic pocket blocking access to it. The conjugates used in non-toxic concentration increased cytotoxicity of topotecan against HeLa cancer cell line but not against conditionally normal HEK 293A cells. Thus, a new structural series of TDP1 inhibitors, which are able to sensitize cancer cells to the topotecan cytotoxic effect has been discovered.- Published
- 2023
- Full Text
- View/download PDF
10. Natural Products and Their Derivatives as Inhibitors of the DNA Repair Enzyme Tyrosyl-DNA Phosphodiesterase 1.
- Author
-
Zakharenko AL, Luzina OA, Chepanova AA, Dyrkheeva NS, Salakhutdinov NF, and Lavrik OI
- Subjects
- Phosphoric Diester Hydrolases metabolism, DNA Repair Enzymes genetics, DNA Topoisomerases, Type I metabolism, DNA Repair, DNA, Biological Products pharmacology, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important repair enzyme that removes various covalent adducts from the 3' end of DNA. Particularly, covalent complexes of topoisomerase 1 (TOP1) with DNA stabilized by DNA damage or by various chemical agents are an examples of such adducts. Anticancer drugs such as the TOP1 poisons topotecan and irinotecan are responsible for the stabilization of these complexes. TDP1 neutralizes the effect of these anticancer drugs, eliminating the DNA adducts. Therefore, the inhibition of TDP1 can sensitize tumor cells to the action of TOP1 poisons. This review contains information about methods for determining the TDP1 activity, as well as describing the inhibitors of these enzyme derivatives of natural biologically active substances, such as aminoglycosides, nucleosides, polyphenolic compounds, and terpenoids. Data on the efficiency of combined inhibition of TOP1 and TDP1 in vitro and in vivo are presented.
- Published
- 2023
- Full Text
- View/download PDF
11. Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.
- Author
-
Dyrkheeva NS, Malakhova AA, Zakharenko AL, Okorokova LS, Shtokalo DN, Pavlova SV, Medvedev SP, Zakian SM, Nushtaeva AA, Tupikin AE, Kabilov MR, Khodyreva SN, Luzina OA, Salakhutdinov NF, and Lavrik OI
- Subjects
- CRISPR-Cas Systems, DNA, DNA Repair, DNA Topoisomerases, Type I genetics, DNA Topoisomerases, Type I metabolism, Esterases metabolism, Proteasome Endopeptidase Complex metabolism, Transcriptome, Poly (ADP-Ribose) Polymerase-1 metabolism, Phosphoric Diester Hydrolases metabolism, Topotecan pharmacology
- Abstract
Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.
- Published
- 2023
- Full Text
- View/download PDF
12. [Effect of Usnic Acid-Derived Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Used as Monotherapy or in Combination with Olaparib on Transplanted Tumors In Vivo].
- Author
-
Kornienko TE, Zakharenko AL, Ilina ES, Chepanova AA, Zakharova OD, Dyrkheeva NS, Popova NA, Nikolin VP, Filimonov AS, Luzina OA, Salakhutdinov NF, and Lavrik OI
- Subjects
- Humans, DNA, Phosphoric Diester Hydrolases genetics, Neoplasms drug therapy, Neoplasms genetics
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that removes various adducts from the 3' end of DNA. Such adducts are formed by enzymes that introduce single-strand breaks in DNA during catalysis (for example, topoisomerase 1) and a number of anticancer drugs with different mechanisms of action. Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme that catalyzes posttranslational modification (PARylation) of various targets and thus controls many cell processes, including DNA repair. Tdp1 is a PARP1 target, and its PARylation attracts Tdp1 to the site of DNA damage. Olaparib is a PARP1 inhibitor used in clinical practice to treat homologous recombination-deficient tumors. Olaparib inhibits PARylation and, therefore, DNA repair. The Tdp1 inhibitor OL7-43 was used in combination with olaparib to increase the antitumor effect of the latter. Olaparib cytotoxicity was found to increase in the presence of OL7-43 in vitro. OL7-43 did not exert a sensitizing effect, but showed its own antitumor and antimetastatic effects in Lewis and Krebs-2 carcinoma models.
- Published
- 2023
- Full Text
- View/download PDF
13. Novel TDP1 Inhibitors: Disubstituted Thiazolidine-2,4-Diones Containing Monoterpene Moieties.
- Author
-
Ivankin DI, Kornienko TE, Mikhailova MA, Dyrkheeva NS, Zakharenko AL, Achara C, Reynisson J, Golyshev VM, Luzina OA, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Humans, Models, Molecular, Monoterpenes pharmacology, Topotecan pharmacology, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Thiazolidinediones pharmacology
- Abstract
Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a promising target for antitumor therapy; the use of TDP1 inhibitors with a topoisomerase 1 poison such as topotecan is a potential combination therapy. In this work, a novel series of 3,5-disubstituted thiazolidine-2,4-diones was synthesized and tested against TDP1. The screening revealed some active compounds with IC
50 values less than 5 μM. Interestingly, compounds 20d and 21d were the most active, with IC50 values in the submicromolar concentration range. None of the compounds showed cytotoxicity against HCT-116 (colon carcinoma) and MRC-5 (human lung fibroblasts) cell lines in the 1-100 μM concentration range. Finally, this class of compounds did not sensitize cancer cells to the cytotoxic effect of topotecan.- Published
- 2023
- Full Text
- View/download PDF
14. Influence of Tyrosyl-DNA Phosphodiesterase 1 Inhibitor on the Proapoptotic and Genotoxic Effects of Anticancer Agent Topotecan.
- Author
-
Chepanova AA, Zakharenko AL, Dyrkheeva NS, Chernyshova IA, Zakharova OD, Ilina ES, Luzina OA, Salakhutdinov NF, and Lavrik OI
- Subjects
- Animals, Mice, DNA, DNA Damage, Phosphoric Diester Hydrolases metabolism, Topotecan pharmacology, Topotecan therapeutic use, Apoptosis, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, Carcinoma, Lewis Lung drug therapy
- Abstract
To date, various strategies have been proposed to increase the efficiency of cancer therapy. It is known that the action of DNA repair system can determine the resistance of cancer cells to DNA-damaging chemotherapy and radiotherapy, and one of these ways to increase therapeutic efficiency is the search for inhibitors of enzymes of the DNA repair system. Inhibition of the DNA repair enzyme tyrosyl-DNA phosphodiesterase1 (Tdp1) leads to an increase in the effectiveness of the topoisomerase 1 (Top1) inhibitor, the anticancer drug topotecan. Covalent complexes Top1-DNA, which are normally short-lived and are not a threat to the cell, are stabilized under the influence of topotecan and lead to cell death. Tdp1 eliminates such stabilized complexes and thus weaken the effect of topotecan therapy. We have previously shown that the use of the usnic acid hydrazonothiazole derivative OL9-119 in combination with topotecan increased the antitumor and antimetastatic efficacy of the latter in a mouse model of Lewis lung carcinoma. In this work, it was shown that the combined use of topotecan and Tdp1 inhibitor, the hydrazonothiazole derivative of usnic acid OL9-119, leads to an increase in the DNA-damaging effect of topotecan which is used in the clinic for the treatment of cancer. The study of the proapoptotic effect of the compound OL9-119 showed that the compound itself does not induce apoptosis, but increases the proapoptotic effect of topotecan. The results of the study could be used to improve the effectiveness of anticancer therapy and/or to reduce the therapeutic dose of topotecan and, therefore, the severity of side effects., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
15. PARP1 Gene Knockout Suppresses Expression of DNA Base Excision Repair Genes.
- Author
-
Zakharenko AL, Malakhova AA, Dyrkheeva NS, Okorokova LS, Medvedev SP, Zakian SM, Kabilov MR, Tupikin AA, and Lavrik OI
- Subjects
- Humans, HEK293 Cells, Gene Knockout Techniques, DNA Repair, DNA, Poly (ADP-Ribose) Polymerase-1 genetics, Poly (ADP-Ribose) Polymerase-1 metabolism, Poly(ADP-ribose) Polymerases genetics, DNA Glycosylases genetics, DNA Glycosylases metabolism
- Abstract
The effect of PARP1 knockout in HEK293 cells on the gene expression of DNA base excision repair (BER) proteins was studied. It was shown that the expression of all differentially expressed genes (DEGs) of BER was reduced by knockout. The expression of the DNA glycosylase gene NEIL1, which is considered to be one of the common "hubs" for binding BER proteins, has changed the most. The expression of genes of auxiliary subunits of DNA polymerases δ and ε is also significantly reduced. The PARP1 gene knockout cell line obtained is an adequate cell model for studying the activity of the BER process in the absence of PARP1 and testing drugs aimed at inhibiting repair processes. It has been found for the first time that knockout of the PARP1 gene results in a significant change in the level of expression of proteins responsible for ribosome biogenesis and the functioning of the proteasome., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
16. The Lipophilic Purine Nucleoside-Tdp1 Inhibitor-Enhances DNA Damage Induced by Topotecan In Vitro and Potentiates the Antitumor Effect of Topotecan In Vivo.
- Author
-
Chernyshova IA, Zakharenko AL, Kurochkin NN, Dyrkheeva NS, Kornienko TE, Popova NA, Nikolin VP, Ilina ES, Zharkov TD, Kupryushkin MS, Oslovsky VE, Drenichev MS, and Lavrik OI
- Subjects
- Animals, Mice, Humans, Phosphodiesterase Inhibitors pharmacology, Purine Nucleosides, Structure-Activity Relationship, Topoisomerase I Inhibitors pharmacology, Esterases metabolism, DNA Damage, DNA, DNA Topoisomerases, Type I metabolism, Topotecan pharmacology, Phosphoric Diester Hydrolases metabolism
- Abstract
The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug. We hypothesize that Tdp1 inhibition would sensitize cells towards the effect of Tpc. Herein, we report the synthesis and study of lipophilic derivatives of purine nucleosides that efficiently suppress Tdp1 activity, with IC
50 values in the 0.3-22.0 μM range. We also showed that this compound class can enhance DNA damage induced by topotecan in vitro by Comet assay on human cell lines HeLa and potentiate the antitumor effect of topotecan in vivo on a mice ascitic Krebs-2 carcinoma model. Thereby, this type of compound may be useful to develop drugs, that sensitize the effect of topotecan and reduce the required dose and, as a result, side effects.- Published
- 2022
- Full Text
- View/download PDF
17. Monoterpene substituted thiazolidin-4-ones as novel TDP1 inhibitors: Synthesis, biological evaluation and docking.
- Author
-
Ivankin DI, Dyrkheeva NS, Zakharenko AL, Ilina ES, Zarkov TO, Reynisson J, Luzina OA, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Esterases metabolism, HeLa Cells, Humans, Monoterpenes pharmacology, Structure-Activity Relationship, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism
- Abstract
Tyrosyl-DNA phosphodiesterase 1(TDP1) is a promising target for a new therapy in oncological disease as an adjunct to topoisomerase 1 (TOP1) drugs. In this paper, novel thiazolidin-4-one derivatives with a benzyl and monoterpene substituents were synthesized. Compounds with a monoterpene fragment attached via a phenyloxy linker were active against TDP1 with IC
50 values in the 1 ÷ 3 μM range, while direct attachment of monoterpene moiety to the thiazolidin-4-one fragment had no activity. Molecular modelling predicted two plausible binding modes of the active compounds both effectively blocking access to the catalytic site of TDP. At non-toxic concentrations the active ligands potentiated the efficacy of the TOP1 poison topotecan in human cervical cancer HeLa cells, but not in non-cancerous HEK293A cells., (Copyright © 2022 Elsevier Ltd. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
18. Adamantane-Monoterpenoid Conjugates Linked via Heterocyclic Linkers Enhance the Cytotoxic Effect of Topotecan.
- Author
-
Munkuev AA, Dyrkheeva NS, Kornienko TE, Ilina ES, Ivankin DI, Suslov EV, Korchagina DV, Gatilov YV, Zakharenko AL, Malakhova AA, Reynisson J, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Camphor, Monoterpenes pharmacology, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Topotecan pharmacology, Adamantane pharmacology, Antineoplastic Agents pharmacology
- Abstract
Inhibiting tyrosyl-DNA phosphodiesterase 1 (TDP1) is a promising strategy for increasing the effectiveness of existing antitumor therapy since it can remove the DNA lesions caused by anticancer drugs, which form covalent complexes with topoisomerase 1 (TOP1). Here, new adamantane-monoterpene conjugates with a 1,2,4-triazole or 1,3,4-thiadiazole linker core were synthesized, where (+)-and (-)-campholenic and (+)-camphor derivatives were used as monoterpene fragments. The campholenic derivatives 14a - 14b and 15a - b showed activity against TDP1 at a low micromolar range with IC
50 ~5-6 μM, whereas camphor-containing compounds 16 and 17 were ineffective. Surprisingly, all the compounds synthesized demonstrated a clear synergy with topotecan, a TOP1 poison, regardless of their ability to inhibit TDP1. These findings imply that different pathways of enhancing topotecan toxicity other than the inhibition of TDP1 can be realized.- Published
- 2022
- Full Text
- View/download PDF
19. In Vitro and In Silico Studies of Human Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) Inhibition by Stereoisomeric Forms of Lipophilic Nucleosides: The Role of Carbohydrate Stereochemistry in Ligand-Enzyme Interactions.
- Author
-
Dyrkheeva NS, Chernyshova IA, Ivanov GA, Porozov YB, Zenchenko AA, Oslovsky VE, Zakharenko AL, Nasyrova DI, Likhatskaya GN, Mikhailov SN, Lavrik OI, and Drenichev MS
- Subjects
- Humans, Ligands, Nucleosides pharmacology, Phosphoric Diester Hydrolases chemistry
- Abstract
Inhibition of human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) by different chiral lipophilic nucleoside derivatives was studied. New Tdp1 inhibitors were found in the series of the studied compounds with IC
50 = 2.7-6.7 μM. It was shown that D-lipophilic nucleoside derivatives manifested higher inhibition activity than their L-analogs, and configuration of the carbohydrate moiety can influence the mechanism of Tdp1 inhibition.- Published
- 2022
- Full Text
- View/download PDF
20. New Deoxycholic Acid Derived Tyrosyl-DNA Phosphodiesterase 1 Inhibitors Also Inhibit Tyrosyl-DNA Phosphodiesterase 2.
- Author
-
Salomatina OV, Dyrkheeva NS, Popadyuk II, Zakharenko AL, Ilina ES, Komarova NI, Reynisson J, Salakhutdinov NF, Lavrik OI, and Volcho KP
- Subjects
- Binding Sites, Cell Line, Chemical Phenomena, Chemistry Techniques, Synthetic, Deoxycholic Acid pharmacology, Enzyme Activation drug effects, Humans, Models, Molecular, Molecular Conformation, Molecular Structure, Phosphodiesterase Inhibitors chemical synthesis, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Protein Binding, Recombinant Proteins chemistry, Structure-Activity Relationship, Deoxycholic Acid analogs & derivatives, Deoxycholic Acid chemistry, Phosphodiesterase Inhibitors chemistry, Phosphoric Diester Hydrolases chemistry
- Abstract
A series of deoxycholic acid (DCA) amides containing benzyl ether groups on the steroid core were tested against the tyrosyl-DNA phosphodiesterase 1 (TDP1) and 2 (TDP2) enzymes. In addition, 1,2,4- and 1,3,4-oxadiazole derivatives were synthesized to study the linker influence between a para -bromophenyl moiety and the steroid scaffold. The DCA derivatives demonstrated promising inhibitory activity against TDP1 with IC
50 in the submicromolar range. Furthermore, the amides and the 1,3,4-oxadiazole derivatives inhibited the TDP2 enzyme but at substantially higher concentration. Tryptamide 5 and para -bromoanilide 8 derivatives containing benzyloxy substituent at the C-3 position and non-substituted hydroxy group at C-12 on the DCA scaffold inhibited both TDP1 and TDP2 as well as enhanced the cytotoxicity of topotecan in non-toxic concentration in vitro. According to molecular modeling, ligand 5 is anchored into the catalytic pocket of TDP1 by one hydrogen bond to the backbone of Gly458 as well as by π-π stacking between the indolyl rings of the ligand and Tyr590, resulting in excellent activity. It can therefore be concluded that these derivatives contribute to the development of specific TDP1 and TDP2 inhibitors for adjuvant therapy against cancer in combination with topoisomerase poisons.- Published
- 2021
- Full Text
- View/download PDF
21. New Hybrid Compounds Combining Fragments of Usnic Acid and Thioether Are Inhibitors of Human Enzymes TDP1, TDP2 and PARP1.
- Author
-
Dyrkheeva NS, Filimonov AS, Luzina OA, Orlova KA, Chernyshova IA, Kornienko TE, Malakhova AA, Medvedev SP, Zakharenko AL, Ilina ES, Anarbaev RO, Naumenko KN, Klabenkova KV, Burakova EA, Stetsenko DA, Zakian SM, Salakhutdinov NF, and Lavrik OI
- Subjects
- Benzofurans pharmacology, Cell Line, Cell Survival drug effects, DNA Topoisomerases, Type I metabolism, DNA-Binding Proteins metabolism, Enzyme Inhibitors chemical synthesis, Humans, Poly (ADP-Ribose) Polymerase-1 metabolism, Poly(ADP-ribose) Polymerases metabolism, Structure-Activity Relationship, Sulfides pharmacology, Sulfoxides chemistry, Sulfoxides pharmacology, Topoisomerase I Inhibitors pharmacology, Topotecan pharmacology, Benzofurans chemistry, DNA-Binding Proteins antagonists & inhibitors, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Poly (ADP-Ribose) Polymerase-1 antagonists & inhibitors, Sulfides chemistry
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3' phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA-TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC
50 values in the 1.4-25.2 μM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.- Published
- 2021
- Full Text
- View/download PDF
22. New Hybrid Compounds Combining Fragments of Usnic Acid and Monoterpenoids for Effective Tyrosyl-DNA Phosphodiesterase 1 Inhibition.
- Author
-
Dyrkheeva NS, Filimonov AS, Luzina OA, Zakharenko AL, Ilina ES, Malakhova AA, Medvedev SP, Reynisson J, Volcho KP, Zakian SM, Salakhutdinov NF, and Lavrik OI
- Subjects
- Benzofurans pharmacology, HEK293 Cells, Humans, Monoterpenes pharmacology, Benzofurans chemistry, Monoterpenes chemistry, Phosphodiesterase Inhibitors chemical synthesis, Phosphodiesterase Inhibitors chemistry, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism
- Abstract
Usnic acid (UA) is a secondary metabolite of lichens that exhibits a wide range of biological activities. Previously, we found that UA derivatives are effective inhibitors of tyrosyl-DNA phosphodiesterase 1 (TDP1). It can remove covalent complex DNA-topoisomerase 1 (TOP1) stabilized by the TOP1 inhibitor topotecan, neutralizing the effect of the drugs. TDP1 removes damage at the 3' end of DNA caused by other anticancer agents. Thus, TDP1 is a promising therapeutic target for the development of drug combinations with topotecan, as well as other drugs for cancer treatment. Ten new UA enamino derivatives with variation in the terpene fragment and substituent of the UA backbone were synthesized and tested as TDP1 inhibitors. Four compounds, 11a - d , had IC
50 values in the 0.23-0.40 μM range. Molecular modelling showed that 11a - d , with relatively short aliphatic chains, fit to the important binding domains. The intrinsic cytotoxicity of 11a - d was tested on two human cell lines. The compounds had low cytotoxicity with CC50 ≥ 60 μM for both cell lines. 11a and 11c had high inhibition efficacy and low cytotoxicity, and they enhanced topotecan's cytotoxicity in cancerous HeLa cells but reduced it in the non-cancerous HEK293A cells. This "protective" effect from topotecan on non-cancerous cells requires further investigation.- Published
- 2021
- Full Text
- View/download PDF
23. Novel Tdp1 Inhibitors Based on Adamantane Connected with Monoterpene Moieties via Heterocyclic Fragments.
- Author
-
Munkuev AA, Mozhaitsev ES, Chepanova AA, Suslov EV, Korchagina DV, Zakharova OD, Ilina ES, Dyrkheeva NS, Zakharenko AL, Reynisson J, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Adamantane chemistry, Carbon-13 Magnetic Resonance Spectroscopy, Cell Line, Tumor, Humans, Ligands, Mass Spectrometry, Proton Magnetic Resonance Spectroscopy, Structure-Activity Relationship, Adamantane pharmacology, Monoterpenes chemistry, Phosphoric Diester Hydrolases drug effects
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising target for anticancer therapy due to its ability to counter the effects topoisomerase 1 (Top1) poison, such as topotecan, thus, decreasing their efficacy. Compounds containing adamantane and monoterpenoid residues connected via 1,2,4-triazole or 1,3,4-thiadiazole linkers were synthesized and tested against Tdp1. All the derivatives exhibited inhibition at low micromolar or nanomolar concentrations with the most potent inhibitors having IC
50 values in the 0.35-0.57 µM range. The cytotoxicity was determined in the HeLa, HCT-116 and SW837 cancer cell lines; moderate CC50 (µM) values were seen from the mid-teens to no effect at 100 µM. Furthermore, citral derivative 20c , α-pinene-derived compounds 20f , 20g and 25c, and the citronellic acid derivative 25b were found to have a sensitizing effect in conjunction with topotecan in the HeLa cervical cancer and colon adenocarcinoma HCT-116 cell lines. The ligands are predicted to bind in the catalytic pocket of Tdp1 and have favorable physicochemical properties for further development as a potential adjunct therapy with Top1 poisons.- Published
- 2021
- Full Text
- View/download PDF
24. [Antitumor Activity of the Combination of Topotecan and Tyrosyl-DNA-Phosphodiesterase 1 Inhibitor on Model Krebs-2 Mouse Ascite Carcinoma].
- Author
-
Dyrkheeva NS, Zakharenko AL, Novoselova ES, Chepanova AA, Popova NA, Nikolin VP, Luzina OA, Salakhutdinov NF, Ryabchikova EI, and Lavrik OI
- Subjects
- Animals, Ascites, DNA, Mice, Phosphoric Diester Hydrolases genetics, Carcinoma, Krebs 2, Topotecan pharmacology
- Abstract
Topotecan is a cytostatic drug from the camptothecin group, it acts by inhibiting topoisomerase 1 (TOP1). Tyrosyl-DNA phosphodiesterase 1 (TDP1) is capable of interfering with the action of TOP1 inhibitors, reducing their therapeutic efficacy. Suppression of TDP1 activity may enhance the effects of topotecan. In this work, we investigated the effect of the antitumor drug topotecan alone and in combination with a TDP1 inhibitor, a hydrazinothiazole derivative of usnic acid, on Krebs-2 mouse ascites tumors. We have previously shown that this derivative efficiently inhibits TDP1. In the present work, we show that both topotecan and the TDP1 inhibitor have an antitumor effect when evaluated separately. The combination of topotecan and the TDP1 inhibitor additively reduces both the weight of the ascites tumor and the number of cells in ascites. In mice, the TDP1 inhibitor alone or in combination with topotecan eliminated the tumor cells. After the combined intraperitoneal administration of these two compounds, we observed cells in which lipid droplets occupied almost the entire cytoplasm and the accumulation of cell detritus, which was absent in the samples collected from mice treated with each compound separately.
- Published
- 2021
- Full Text
- View/download PDF
25. Deoxycholic acid as a molecular scaffold for tyrosyl-DNA phosphodiesterase 1 inhibition: A synthesis, structure-activity relationship and molecular modeling study.
- Author
-
Salomatina OV, Popadyuk II, Zakharenko AL, Zakharova OD, Chepanova AA, Dyrkheeva NS, Komarova NI, Reynisson J, Anarbaev RO, Salakhutdinov NF, Lavrik OI, and Volcho KP
- Subjects
- Humans, Structure-Activity Relationship, Models, Molecular, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry, Antineoplastic Agents chemical synthesis, Molecular Structure, Cell Line, Tumor, Drug Screening Assays, Antitumor, Dose-Response Relationship, Drug, Cell Proliferation drug effects, Phosphoric Diester Hydrolases metabolism, Deoxycholic Acid chemistry, Deoxycholic Acid pharmacology, Deoxycholic Acid chemical synthesis, Phosphodiesterase Inhibitors pharmacology, Phosphodiesterase Inhibitors chemistry, Phosphodiesterase Inhibitors chemical synthesis
- Abstract
Para-Bromoanilides of deoxycholic acid with various functional groups on the steroid scaffold were designed as promising tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitors. Tdp1 is a DNA repair enzyme, involved in removing DNA damage caused by topoisomerase I poisons; an important class of anticancer drugs. Thus, reducing the activity of Tdp1 can increase the efficacy of anticancer drugs in current use. Inhibitory activity in the low micromolar and submicromolar concentrations was observed with 3,12-dimethoxy para-bromoanilide 17 being the most active with an IC
50 value of 0.27 μM. The activity of N-methyl para-bromoanilides was 3-4.8 times lower than of the corresponding para-bromoanilides. Increased potency of the ligands was seen with higher molecular weight and log P values. The ligands were evaluated for their cytotoxic potential in a panel of tumor cell lines; all were nontoxic to the A549 pulmonary adenocarcinoma cell line. However, derivatives containing a hydroxyl group at the 12th position were more toxic than their 12-hydroxyl group counterparts (acetoxy-, oxo- and methoxy- group) against HCT-116 human colon and HepG2 hepatocellular carcinomas. In addition, an N-methyl substitution led to an increase in toxicity for the HCT-116 and HepG2 cell lines. The excellent activity as well as low cytotoxicity, derivative 17 can be considered as a lead compound for further development., (Copyright © 2020. Published by Elsevier Inc.)- Published
- 2021
- Full Text
- View/download PDF
26. The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair Enzyme.
- Author
-
Gladkova ED, Nechepurenko IV, Bredikhin RA, Chepanova AA, Zakharenko AL, Luzina OA, Ilina ES, Dyrkheeva NS, Mamontova EM, Anarbaev RO, Reynisson J, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Antineoplastic Agents, Phytogenic metabolism, Antineoplastic Agents, Phytogenic pharmacology, Berberine chemistry, Berberine pharmacology, Binding Sites, DNA Repair drug effects, Drug Combinations, Drug Design, Drug Synergism, HeLa Cells, Humans, Inhibitory Concentration 50, Molecular Docking Simulation, Phosphodiesterase Inhibitors metabolism, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Protein Binding, Protein Conformation, Structure-Activity Relationship, Topoisomerase I Inhibitors chemistry, Topotecan chemistry, Antineoplastic Agents, Phytogenic chemical synthesis, Berberine analogs & derivatives, Phosphodiesterase Inhibitors chemical synthesis, Phosphoric Diester Hydrolases chemistry, Topoisomerase I Inhibitors pharmacology, Topotecan pharmacology
- Abstract
A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure-activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.
- Published
- 2020
- Full Text
- View/download PDF
27. Inhibition of Tyrosyl-DNA Phosphodiesterase 1 by Lipophilic Pyrimidine Nucleosides.
- Author
-
Zakharenko AL, Drenichev MS, Dyrkheeva NS, Ivanov GA, Oslovsky VE, Ilina ES, Chernyshova IA, Lavrik OI, and Mikhailov SN
- Subjects
- Dose-Response Relationship, Drug, Enzyme Inhibitors toxicity, HeLa Cells, Humans, Pyrimidine Nucleosides toxicity, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Hydrophobic and Hydrophilic Interactions, Phosphoric Diester Hydrolases metabolism, Pyrimidine Nucleosides chemistry, Pyrimidine Nucleosides pharmacology
- Abstract
Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2',3',5'-tri- O -benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC
50 ) in the lower micromolar and submicromolar range. 2',3',5'-Tri- O -benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 μM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 μM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50-100%). Several O -benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.- Published
- 2020
- Full Text
- View/download PDF
28. Design, Synthesis, and Biological Investigation of Novel Classes of 3-Carene-Derived Potent Inhibitors of TDP1.
- Author
-
Il'ina IV, Dyrkheeva NS, Zakharenko AL, Sidorenko AY, Li-Zhulanov NS, Korchagina DV, Chand R, Ayine-Tora DM, Chepanova AA, Zakharova OD, Ilina ES, Reynisson J, Malakhova AA, Medvedev SP, Zakian SM, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- CRISPR-Cas Systems, Cell Proliferation drug effects, Cell Survival drug effects, Drug Synergism, Gene Knockout Techniques, HCT116 Cells, HEK293 Cells, HeLa Cells, Humans, Inhibitory Concentration 50, Phosphodiesterase Inhibitors chemistry, Phosphoric Diester Hydrolases genetics, Topotecan pharmacology, Bicyclic Monoterpenes chemistry, Drug Design, Phosphodiesterase Inhibitors chemical synthesis, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Signal Transduction drug effects
- Abstract
Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC
50 value of 0.65 μM. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k , a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 -/- cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.- Published
- 2020
- Full Text
- View/download PDF
29. Identification of novel inhibitors for the tyrosyl-DNA-phosphodiesterase 1 (Tdp1) mutant SCAN1 using virtual screening.
- Author
-
Mamontova EM, Zakharenko AL, Zakharova OD, Dyrkheeva NS, Volcho KP, Reynisson J, Arabshahi HJ, Salakhutdinov NF, and Lavrik OI
- Subjects
- Antineoplastic Agents chemistry, Antineoplastic Agents metabolism, Antineoplastic Agents pharmacology, Binding Sites, Cell Line, Cell Survival drug effects, Coumarins chemistry, Coumarins metabolism, Coumarins pharmacology, Drug Design, Drug Synergism, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Humans, Inhibitory Concentration 50, Ligands, Mutation, Phosphoric Diester Hydrolases chemistry, Phosphoric Diester Hydrolases genetics, Protein Structure, Tertiary, Topotecan chemistry, Topotecan metabolism, Topotecan pharmacology, Enzyme Inhibitors chemistry, Molecular Docking Simulation, Phosphoric Diester Hydrolases metabolism
- Abstract
Spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1) is a debilitating neurological disease that is caused by the mutation the Tyrosyl-DNA phosphodiesterase 1 (TDP1) DNA repair enzyme. The crucial His493 in TDP1's binding site is replaced with an arginine amino acid residue rendering the enzyme dysfunctional. A virtual screen was performed against the homology model of SCAN1 and seventeen compounds were identified and tested in a novel SCAN1 specific biochemical assay. Six compounds showed activity with IC
50 values between 3.5 and 25.1 µM. The most active ligand 5 (3.5 µM) is a dicoumarin followed by a close structural analogue 6 at 6.0 µM. A less potent series of β-carbolines (14 and 15) was found with potency in the mid-teens. According to molecular modelling an excellent fit for the active ligands into the binding pocket is predicted. To the best of our knowledge, data on inhibitors of the mutant form of TDP1 has not been reported previously. The virtual hits were also tested for wild type TDP1 activity and all six SCAN1 inhibitors are potent for the former, e.g., ligand 5 has a measured IC50 at 99 nM. In the last decade, TDP1 is considered as a promising target for adjuvant therapy against cancer in combination with Topoisomerase 1 poisons. The active ligands are mostly non-toxic to cancer cell lines A-549, T98G and MCF-7 as well as the immortalized WI-38 human fetal lung cells. Furthermore, ligands 5 and 7, show promising synergy in conjunction with topotecan, a clinically used topoisomerase 1 anticancer drug. The active ligands 5, 7, 14 and 15 have a good balance of the physicochemical properties required for oral bioavailability making the excellent candidates for further development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
30. New Hydrazinothiazole Derivatives of Usnic Acid as Potent Tdp1 Inhibitors.
- Author
-
Filimonov AS, Chepanova AA, Luzina OA, Zakharenko AL, Zakharova OD, Ilina ES, Dyrkheeva NS, Kuprushkin MS, Kolotaev AV, Khachatryan DS, Patel J, Leung IKH, Chand R, Ayine-Tora DM, Reynisson J, Volcho KP, Salakhutdinov NF, and Lavrik OI
- Subjects
- Cell Survival drug effects, Dose-Response Relationship, Drug, Humans, Models, Molecular, Molecular Conformation, Molecular Structure, Phosphoric Diester Hydrolases, Protein Binding, Structure-Activity Relationship, Benzofurans chemistry, Benzofurans pharmacology, Phosphodiesterase Inhibitors chemistry, Phosphodiesterase Inhibitors pharmacology, Thiazoles chemistry, Thiazoles pharmacology
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC
50 values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.- Published
- 2019
- Full Text
- View/download PDF
31. DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.
- Author
-
Krumkacheva OA, Shevelev GY, Lomzov AA, Dyrkheeva NS, Kuzhelev AA, Koval VV, Tormyshev VM, Polienko YF, Fedin MV, Pyshnyi DV, Lavrik OI, and Bagryanskaya EG
- Subjects
- Base Sequence, Binding Sites, Cloning, Molecular, DNA genetics, DNA metabolism, DNA Damage, DNA-(Apurinic or Apyrimidinic Site) Lyase genetics, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Electron Spin Resonance Spectroscopy, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Humans, Molecular Dynamics Simulation, Nucleic Acid Conformation, Oligonucleotides metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, DNA chemistry, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, Oligonucleotides chemistry, Spin Labels chemical synthesis
- Abstract
A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
- Full Text
- View/download PDF
32. Novel group of tyrosyl-DNA-phosphodiesterase 1 inhibitors based on disaccharide nucleosides as drug prototypes for anti-cancer therapy.
- Author
-
Komarova AO, Drenichev MS, Dyrkheeva NS, Kulikova IV, Oslovsky VE, Zakharova OD, Zakharenko AL, Mikhailov SN, and Lavrik OI
- Subjects
- Antineoplastic Agents chemical synthesis, Antineoplastic Agents chemistry, Cell Proliferation drug effects, Cells, Cultured, Disaccharides chemical synthesis, Disaccharides chemistry, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Humans, Molecular Structure, Nucleosides chemical synthesis, Nucleosides chemistry, Phosphodiesterase Inhibitors chemical synthesis, Phosphodiesterase Inhibitors chemistry, Structure-Activity Relationship, Topotecan chemical synthesis, Topotecan chemistry, Antineoplastic Agents pharmacology, Disaccharides pharmacology, Nucleosides pharmacology, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism, Topotecan pharmacology
- Abstract
A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors based on disaccharide nucleosides was identified. TDP1 plays an essential role in the resistance of cancer cells to currently used antitumour drugs based on Top1 inhibitors such as topotecan and irinotecan. The most effective inhibitors investigated in this study have IC
50 values (half-maximal inhibitory concentration) in 0.4-18.5 µM range and demonstrate relatively low own cytotoxicity along with significant synergistic effect in combination with anti-cancer drug topotecan. Moreover, kinetic parameters of the enzymatic reaction and fluorescence anisotropy were measured using different types of DNA-biosensors to give a sufficient insight into the mechanism of inhibitor's action.- Published
- 2018
- Full Text
- View/download PDF
33. [Excision of Carbohydrate-Modified dNMP Analogues from DNA 3' end by Human Apurinic/Apyrimidinic Endonuclease 1 (APE1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1)].
- Author
-
Dyrkheeva NS, Lebedeva NA, Sherstyuk YV, Abramova TV, Silnikov VN, and Lavrik OI
- Subjects
- Carbohydrates chemistry, Humans, DNA chemistry, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, Phosphoric Diester Hydrolases chemistry
- Abstract
We have studied the excision efficiency of human apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) on matched or mismatched bases located at the 3' end of DNA primers. We have used model DNA duplexes, which mimic DNA structures that occur during either replication (DNA with a 3' recessed end) or repair (DNA with a single-strand break). Both APE1 and TDP1 are more efficient in removing ribose-modified dNMP residues from mismatched pairs rather than canonical pairs. Thus, both of these enzymes may act as proofreading factors during the repair synthesis catalyzed by DNA polymerases including DNA polymerase β (Polβ). The design of new DNA polymerase inhibitors, which act as DNA or RNA chain terminators, is one of the main strategies in the development of antiviral agents. The excision efficacy of APE1 and TDP1 has also been studied for 3'-modified DNA duplexes that contain ddNMP or phosphorylated morpholino nucleosides (MorB) commonly used as terminators in the DNA synthesis. We have also investigated the insertion of ddNTP and morpholino nucleotides catalyzed by Polβ and human immunodeficiency virus reverse transcriptase. This experiment has pointed to MorCyt, cytosine-containing morpholino nucleoside, as a potential antiviral agent.
- Published
- 2018
- Full Text
- View/download PDF
34. Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1.
- Author
-
Kuznetsov NA, Lebedeva NA, Kuznetsova AA, Rechkunova NI, Dyrkheeva NS, Kupryushkin MS, Stetsenko DA, Pyshnyi DV, Fedorova OS, and Lavrik OI
- Subjects
- Apurinic Acid chemistry, Apurinic Acid metabolism, Binding Sites genetics, DNA chemistry, DNA genetics, DNA, Single-Stranded chemistry, DNA, Single-Stranded genetics, DNA, Single-Stranded metabolism, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, DNA-(Apurinic or Apyrimidinic Site) Lyase genetics, Fluorescence Resonance Energy Transfer, Humans, Hydrolysis, Kinetics, Mutation, Nucleic Acid Conformation, Phosphoric Diester Hydrolases chemistry, Phosphoric Diester Hydrolases genetics, Polynucleotides chemistry, Polynucleotides metabolism, Protein Binding, Substrate Specificity, DNA metabolism, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Phosphoric Diester Hydrolases metabolism
- Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3'-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3'-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3'-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.
- Published
- 2017
- Full Text
- View/download PDF
35. [Contamination of exosome preparations, isolated from biological fluids].
- Author
-
Grigor'eva AE, Dyrkheeva NS, Bryzgunova OE, Tamkovich SN, Chelobanov BP, and Ryabchikova EI
- Subjects
- Adenocarcinoma blood, Animals, Biomarkers metabolism, Breast Neoplasms blood, Cell Fractionation, Cell-Derived Microparticles metabolism, Cell-Derived Microparticles ultrastructure, Dogs, Exosomes ultrastructure, Female, Gene Expression, Humans, Lipoproteins ultrastructure, MCF-7 Cells, Madin Darby Canine Kidney Cells, Male, Microscopy, Electron, Transmission, Particle Size, Prostatic Neoplasms urine, Tetraspanin 29 genetics, Tetraspanin 29 metabolism, Tetraspanin 30 genetics, Tetraspanin 30 metabolism, Ultracentrifugation, Adenocarcinoma chemistry, Artifacts, Breast Neoplasms chemistry, Exosomes metabolism, Lipoproteins chemistry, Prostatic Neoplasms chemistry
- Abstract
The aim of our study was to attract the attention of researchers at the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 ("JEOL", Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and culture fluid (MDCK, MDA-MB и MCF-7 cells). All exosome preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations contained contaminating structures: distinct particles of low electron density without limiting membrane ("non-vesicles"). Two main kinds of the "non-vesicles" species were found in exosome preparations: 20-40 nm in size, representing 10-40% of all structures in the preparations; and 40-100 nm in size (identical to exosomes by size). Morphology of the "non-vesicles" allowed to identify them as lipoproteins of intermediate and low density (20-40 nm), and very low density (40-100 nm). The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparation by electron microscopy and take into account the presence of contaminating structures in analysis of experimental data.
- Published
- 2017
- Full Text
- View/download PDF
36. AP Endonuclease 1 as a Key Enzyme in Repair of Apurinic/Apyrimidinic Sites.
- Author
-
Dyrkheeva NS, Lebedeva NA, and Lavrik OI
- Subjects
- Humans, DNA genetics, DNA metabolism, DNA Breaks, Single-Stranded, DNA Repair physiology, DNA-(Apurinic or Apyrimidinic Site) Lyase genetics, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism
- Abstract
Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety. APE1 exhibits 3'-phosphodiesterase, 3'-5'-exonuclease, and 3'-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system.
- Published
- 2016
- Full Text
- View/download PDF
37. Capturing snapshots of APE1 processing DNA damage.
- Author
-
Freudenthal BD, Beard WA, Cuneo MJ, Dyrkheeva NS, and Wilson SH
- Subjects
- Binding Sites, Catalytic Domain, Crystallography, X-Ray, Humans, Models, Molecular, Protein Binding, Protein Conformation, DNA chemistry, DNA metabolism, DNA Damage, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism
- Abstract
DNA apurinic-apyrimidinic (AP) sites are prevalent noncoding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA-repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive, owing in part to limited structural information. We report multiple high-resolution human APE1-DNA structures that divulge new features of the APE1 reaction, including the metal-binding site, the nucleophile and the arginine clamps that mediate product release. We also report APE1-DNA structures with a T-G mismatch 5' to the AP site, representing a clustered lesion occurring in methylated CpG dinucleotides. These structures reveal that APE1 molds the T-G mismatch into a unique Watson-Crick-like geometry that distorts the active site, thus reducing incision. These snapshots provide mechanistic clarity for APE1 while affording a rational framework to manipulate biological responses to DNA damage.
- Published
- 2015
- Full Text
- View/download PDF
38. Base excision repair of tandem modifications in a methylated CpG dinucleotide.
- Author
-
Sassa A, Çağlayan M, Dyrkheeva NS, Beard WA, and Wilson SH
- Subjects
- Base Pair Mismatch, Base Pairing, Base Sequence, Binding Sites, DNA chemistry, DNA Glycosylases antagonists & inhibitors, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Guanine analogs & derivatives, Guanine metabolism, Humans, Thymine DNA Glycosylase metabolism, CpG Islands genetics, DNA genetics, DNA metabolism, DNA Methylation, DNA Repair
- Abstract
Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
39. Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication.
- Author
-
Dyrkheeva NS, Khodyreva SN, and Lavrik OI
- Subjects
- Animals, Azides chemistry, Cell Extracts, DNA chemistry, DNA Polymerase beta chemistry, DNA Polymerase beta genetics, DNA Primers chemistry, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, DNA-(Apurinic or Apyrimidinic Site) Lyase genetics, DNA-Binding Proteins metabolism, Deoxycytosine Nucleotides chemistry, Mice, Oligonucleotides metabolism, Photoaffinity Labels, Recombinant Proteins chemistry, Recombinant Proteins metabolism, X-ray Repair Cross Complementing Protein 1, DNA metabolism, DNA Polymerase beta metabolism, DNA Repair, DNA Replication, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism
- Abstract
Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase beta with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase beta was accomplished by DNA containing photoreactive group at the 3 -end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase beta depending on the nature of the 5 -group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5 -phosphate group in the nick/gap) or long-patch (DNA containing 5 -sugar phosphate or 5 -flap) base excision repair (BER) intermediates. Other DNA duplexes (3 -recessed DNA and DNA with the 5 -hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5 -sugar phosphate group in the nick/gap, whereas DNA polymerase beta binds to DNA duplex with a mononucleotide gap flanked by the 5 -p group. When APE1 and DNA polymerase beta are both present, a ternary complex APE1-DNA polymerase beta-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5 -flap or mononucleotide-gapped DNA with 5 -p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase beta, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3 -5 exonuclease activity on 3 -recessed DNA duplex.
- Published
- 2008
- Full Text
- View/download PDF
40. [Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues].
- Author
-
Dyrkheeva NS, Khodyreva SN, and Lavrik OI
- Subjects
- DNA Breaks, Single-Stranded, Humans, Hydrogen-Ion Concentration, Kinetics, Nucleic Acid Heteroduplexes chemistry, Oligonucleotides chemistry, Osmolar Concentration, Photochemistry, Structure-Activity Relationship, DNA chemistry, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, Deoxycytidine Monophosphate analogs & derivatives, Deoxycytidine Monophosphate chemistry, Exonucleases chemistry, Thymidine Monophosphate chemistry
- Abstract
Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, the cleavage of DNA 5' to the AP site, it displays other weak enzymatic activities. One of them is 3'-5' exonuclease activity, which is most effectively pronounced for DNA duplexes containing modified or mismatched nucleotides at the 3' end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase beta during the base excision repair process. We determined the quantitative parameters of the 3'-5' exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3' terminus of single-strand DNA and from photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.
- Published
- 2008
- Full Text
- View/download PDF
41. [Multifunctional human apurinic/apyrimidinic endonuclease 1: the role of additional functions].
- Author
-
Dyrkheeva NS, Khodyreva SN, and Lavrik OI
- Subjects
- Apoptosis physiology, Base Pairing, Catalysis, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase biosynthesis, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Deoxyribose metabolism, Exonucleases metabolism, Humans, Phosphoric Diester Hydrolases metabolism, Phosphoric Monoester Hydrolases metabolism, DNA-(Apurinic or Apyrimidinic Site) Lyase physiology
- Abstract
Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is multifunctional enzyme. APEI is involved in the DNA base excision repair process (BER). APE1 participates in BER by cleaving the DNA adjacent to the 5' side of an AP site to produce a hydroxyl group at the 3' terminus of an unmodified nucleotide upstream of the nick and a 5' deoxyribose phosphate moiety downstream. In addition to its AP-endonucleolytic function, APE1 possesses 3' phosphodiesterase, 3'-5' exonuclease and 3' phosphatase activities. Independently of being characterized as DNA repair protein, APE1 was identified as redox-factor (Ref-1). Our own and literature data on the role of APE1 additional functions in cell metabolism and on interactions of APE1 with DNA and other proteins that participate in BER are analyzed in this review.
- Published
- 2007
42. Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3' terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes.
- Author
-
Dyrkheeva NS, Lomzov AA, Pyshnyi DV, Khodyreva SN, and Lavrik OI
- Subjects
- Drug Stability, Hot Temperature, Humans, Nucleic Acid Heteroduplexes metabolism, Oligodeoxyribonucleotides metabolism, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Exodeoxyribonucleases metabolism, Nucleotides metabolism
- Abstract
Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3' phosphodiesterase and 3'-5' exonuclease activities. The 3'-5' exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase beta. Here, we examine the removal of matched and mismatched dNMP from the 3' terminus of the 3'-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3' terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3' terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3'-5' exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5' margin of the nick. The 3'-5' exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5' margin of the nick as well as for flapped and recessed DNAs.
- Published
- 2006
- Full Text
- View/download PDF
43. 3'-5' exonuclease activity of human apurinic/apyrimidinic endonuclease 1 towards DNAs containing dNMP and their modified analogs at the 3 end of single strand DNA break.
- Author
-
Dyrkheeva NS, Khodyreva SN, Sukhanova MV, Safronov IV, Dezhurov SV, and Lavrik OI
- Subjects
- Animals, Base Pair Mismatch, Cytidine Triphosphate analogs & derivatives, Cytidine Triphosphate chemistry, Cytidine Triphosphate metabolism, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, Deoxyribonucleotides metabolism, Exodeoxyribonucleases metabolism, Humans, Nucleic Acid Heteroduplexes chemistry, Nucleic Acid Heteroduplexes metabolism, Photochemistry, Rats, Thymine Nucleotides metabolism, DNA Damage, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry, Deoxyribonucleotides chemistry, Exodeoxyribonucleases chemistry, Nucleotides metabolism
- Abstract
Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.
- Published
- 2006
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.