Search

DNA–protein crosslinks (DPC) are common DNA lesions induced by various external and endogenous agents. One of the sources of DPC is the apurinic/apyrimidinic site (AP site) and proteins interacting with it. Some proteins possessing AP lyase activity form covalent complexes with AP site‐containing DNA without borohydride reduction (suicidal crosslinks). We have shown earlier that tyrosyl‐DNA phosphodiesterase 1 (TDP1) but not AP endonuclease 1 (APE1) is able to remove intact OGG1 from protein–DNA adducts, whereas APE1 is able to prevent the formation of DPC by hydrolyzing the AP site. Here we demonstrate that TDP1 can remove intact PARP2 but not XRCC1 from covalent enzyme–DNA adducts with AP‐DNA formed in the absence of APE1. We also analyzed an impact of APE1 and TDP1 on the efficiency of DPC formation in APE1−/− or TDP1−/− cell extracts. Our data revealed that APE1 depletion leads to increased levels of PARP1–DNA crosslinks, whereas TDP1 deficiency has little effect on DPC formation. [ABSTRACT FROM AUTHOR]

A number of new conjugates of dehydroabiethylamine and adamantane was obtained using a three-step synthetic scheme in order to study the effect of linker type and length on biological activity. The inhibitory activity of the synthesized compounds toward the DNA repair enzyme Tyrosyl-DNA phosphodiesterase 1 was studied. The compounds are potent inhibitors, exhibiting activity in micromolar concentrations. [ABSTRACT FROM AUTHOR]

DNA–protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of double-strand breaks, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, ageing and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare tyrosyl-DNA phosphodiesterase 1 (TDP1) orthologues between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, we have found increased TDP2 expression in TDP1-deficient cells and embryos. Understanding the role of TDP1 in DPCR at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.

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. [ABSTRACT FROM AUTHOR]

The tyrosyl-DNA phosphodiesterase 1 (TDP1) enzyme hydrolyzes the phosphodiester bond between a tyrosine residue and the 3′-phosphate of DNA in the DNA–topoisomerase I (TopI) complex, being involved in different DNA repair pathways. A small TDP1 gene subfamily is present in plants, where TDP1α has been linked to genome stability maintenance, while TDP1β has unknown functions. This work aimed to comparatively investigate the function of the TDP1 genes by taking advantage of the rich transcriptomics databases available for the Arabidopsis thaliana model plant. A data mining approach was carried out to collect information regarding gene expression in different tissues, genetic backgrounds, and stress conditions, using platforms where RNA-seq and microarray data are deposited. The gathered data allowed us to distinguish between common and divergent functions of the two genes. Namely, TDP1β seems to be involved in root development and associated with gibberellin and brassinosteroid phytohormones, whereas TDP1α is more responsive to light and abscisic acid. During stress conditions, both genes are highly responsive to biotic and abiotic treatments in a time- and stress-dependent manner. Data validation using gamma-ray treatments applied to Arabidopsis seedlings indicated the accumulation of DNA damage and extensive cell death associated with the observed changes in the TDP1 genes expression profiles. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 IC50 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. [ABSTRACT FROM AUTHOR]

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs stalled type I topoisomerase (TOP1)-DNA complexes by hydrolyzing the phosphodiester bond between the TOP1 Y723 residue and the 3′-phosphate of its DNA substrate. Although TDP1 antagonists could potentially reduce the dose of TOP1 inhibitors needed to achieve effective anticancer effects, the development of validated TDP1 inhibitors has proven to be challenging. This may, in part, be due to the open and extended nature of the TOP1 substrate binding region. We have previously reported imidazopyrazines and imidazopyridines that can inhibit TDP1 catalytic function in vitro. We solved the TDP1 crystal structures with bound inhibitors of this class and found that the dicarboxylic acid functionality within the N-(3,4-dicarboxyphenyl)-2-diphenylimidazo [1,2-a]pyridin-3-amine platform overlaps with aspects of phosphoryl substrate recognition. Yet phosphonic acids could potentially better-replicate cognate TOP1-DNA substrate binding interactions than carboxylic acids. As reported herein, we designed phosphonic acid-containing variants of our previously reported carboxylic acid-containing imidazopyrazine and imidazopyridine inhibitors and effected their synthesis using one-pot Groebke–Blackburn–Bienayme multicomponent reactions. We obtained crystal structures of TDP1 complexed with a subset of inhibitors. We discuss binding interactions of these inhibitors within the context of phosphate-containing substrate and carboxylic acid-based inhibitors. These compounds represent a new structural class of small molecule ligands that mimic aspects of the 3′-processed substrate that results from TDP1 catalysis.

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. • Transcriptomic analysis of HEK293A cells with a knockout of the TDP1 gene (TDP1-KO cells) was performed for the first time. • Among DEGs common to three biological replicates of TDP1-KO cells no DNA repair enzymes or factors were found. • We identified DEGs of cell adhesion and communication, spermatogenesis, a cytokine response, the MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

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 IC50 ~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. [ABSTRACT FROM AUTHOR]

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 IC50 = 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. [ABSTRACT FROM AUTHOR]

• TDP1 activity and protein amount is upregulated in the tumor tissue in NSCLC. • TOP1 activity and protein amount is upregulated in the tumor tissue in NSCLC. • TDP1 and TOP1 activity and protein correlates in normal and tumor tissue in NSCLC. • TOP1 and TDP1 upregulation and correlation is independent of clinical parameters. • Dual targeting of TDP1 and TOP1 may be preferable over TOP1 drugs alone. Topoisomerase 1 (TOP1) is a drug target used in anticancer treatment of various cancer types. The effect of the TOP1 drugs can be counteracted by the enzymatic activity of tyrosyl-DNA phosphodiesterase 1 (TDP1). Thus, to elucidate the relevance of combining TDP1 and TOP1 as drug targets for anticancer treatment in NSCLC, TDP1 and TOP1 was for the first time quantified in a large cohort of paired normal and tumor tissue from NSCLC patients, and data were correlated between the two enzymes and to clinical data. TDP1 and TOP1 activity and protein concentration were measured in paired normal and tumor tissue from 150 NSCLC patients using TDP1 and TOP1 specific biosensors and ELISA. TDP1 and TOP1 activity and protein concentration were correlated to clinical data. TDP1 and TOP1 activity and protein concentration were significantly upregulated from normal to tumor tissue for the individual patients, but did not correlate to any of the clinical data. TDP1 and TOP1 activity were upregulated in 89.3% and 82.7% of the patients, respectively, and correlated in both normal and tumor tissue. The same tendency was observed for protein concentration with an upregulation of TDP1 and TOP1 in 73.0% and 84.4% of the patients, respectively. The activity and protein concentration correlated in normal and tumor tissue for both TDP1 and TOP1. The upregulations of TDP1 and TOP1 from normal to tumor tissue combined with the observation that TDP1 and TOP1 did not correlate to any of the clinical data indicate that both proteins are important for development or maintenance of the tumor cells in NSCLC. Correlations between TDP1 and TOP1 indicate a biological dependency and potential co-regulation of the enzymes. These observations is encouraging in relation to using TOP1 and TDP1 as targets in anticancer treatment of NSCLC. [ABSTRACT FROM AUTHOR]

Introduction: Tyrosyl-DNA phosphodiesterase 1 (TDP1) can repair oxidative damage-caused 3′-phosphoglycolates and promote cancer progression. However, the clinical significance of TDP1 and its correlation with microRNAs (miRNAs) in gastric cancer (GC) remains unknown. Methods: The relationship of TDP1 or let-7c-5p with the clinical outcomes of GC was determined by a tissue microarray and TCGA dataset. Cell viability and invasion were assessed by MTT and Transwell assays. Pearson correlation analysis, luciferase gene report, qRT-PCR, and Western blot analyses were used to analyze the interaction between TDP1 and let-7c-5p in GC tissues and cells. Results: We found that TDP1 expression was elevated in GC tissues and associated with the dysregulation of let-7c-5p. Knockdown of TDP1 inhibited GC cell proliferation and invasion. let-7c-5p could be found to bind with TDP1, reduce its expression levels, and represent a predictive marker in GC. Conclusion: Our findings demonstrated that dysregulation of let-7c-5p/TDP1 axis could predict a poor prognosis in GC. [ABSTRACT FROM AUTHOR]

Introduction: Tyrosyl-DNA phosphodiesterase 1 (TDP1) can repair oxidative damage-caused 30-phosphoglycolates and promote cancer progression. However, the clinical significance of TDP1 and its correlation with microRNAs (miRNAs) in gastric cancer (GC) remains unknown. Methods: The relationship of TDP1 or let-7c-5p with the clinical outcomes of GC was determined by a tissue microarray and TCGA dataset. Cell viability and invasion were assessed by MTT and Transwell assays. Pearson correlation analysis, luciferase gene report, qRT-PCR, and Western blot analyses were used to analyze the interaction between TDP1 and let-7c-5p in GC tissues and cells. Results: We found that TDP1 expression was elevated in GC tissues and associated with the dysregulation of let-7c-5p. Knockdown of TDP1 inhibited GC cell proliferation and invasion. let-7c-5p could be found to bind with TDP1, reduce its expression levels, and represent a predictive marker in GC. Conclusion: Our findings demonstrated that dysregulation of let-7c-5p/TDP1 axis could predict a poor prognosis in GC. [ABSTRACT FROM AUTHOR]

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a DNA repair enzyme that plays a key role in repairing damage caused by various antitumor drugs. It is a promising target in medicinal chemistry for the creation of cancer adjuvant therapy. Inhibition of this enzyme together with the use of anticancer chemotherapy enhances the effect of the latter. The natural mutant of TDP1, TDP1(H493R), causes severe neurodegenerative disease spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1). Inhibition of TDP1(H493R) appears to be useful in containment the progression of the disease. A library of compounds was synthesized starting from dehydroabietylamine including heterocyclic pharmacophore groups in the core. To obtain the desired products, the starting dehydroabietylamine was introduced sequentially in reaction with isothiocyanate and ethyl bromoacetate. Different classes of heterocyclic derivatives—2-iminothiazolidin-4-ons and 2-thioxoimidazolidin-4-ones—were obtained depending on the addition order of reagents. 2-Iminothiazolidin-4-thiones were obtained from 2-iminothiazolidin-4-ones under the action of the Lawesson's reagent. Effective TDP1 inhibitors were found among the obtained compounds that work in submicromolar concentrations. The inhibitor of TDP1(H493R) was also detected. [ABSTRACT FROM AUTHOR]

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 IC50 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.

One of the challenges that living organisms face is to promptly respond to genotoxic stress to avoid DNA damage. To this purpose, all organisms, including plants, developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms and need to be finely regulated. In this scenario, microRNAs (miRNAs) are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells whereas studies in plants are still scarce. To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC), targeting distinct components of DDR, namely topoisomerase I (TopI) and tyrosyl-DNA phosphodiesterase 1 (TDP1), were used. Phenotypic (germination percentage and speed, seedling growth) and molecular (cell death, DNA damage, and gene expression profiles) analyses demonstrated that the imposed treatments impact DDR. Our results show that these treatments do not influence the germination process but rather inhibit seedling development, causing an increase in cell death and accumulation of DNA damage. Moreover, treatment-specific changes in the expression of suppressor of gamma response 1 (SOG1), master-regulator of plant DDR, were observed. Additionally, the expression of multiple genes playing important roles in different DNA repair pathways and cell cycle regulation were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identified from our previous bioinformatics approaches as putatively targeting genes involved in DDR processes were investigated alongside their targets. The obtained results indicate that under most conditions when a miRNA is upregulated the corresponding candidate target gene is downregulated, providing an indirect evidence of miRNAs action over these targets. Hence, the present study extends the present knowledge on the information available regarding the roles played by miRNAs in the post-transcriptional regulation of DDR in plants.

Bifunctional 8-oxoguanine-DNA glycosylase (OGG1), a crucial DNA-repair enzyme, removes from DNA 8-oxo-7,8-dihydroguanine (8-oxoG) with following cleavage of the arising apurinic/apyrimidinic (AP) site. The major enzyme in eukaryotic cells that catalyzes the cleavage of AP sites is AP endonuclease 1 (APE1). Alternatively, AP sites can be cleaved by tyrosyl-DNA phosphodiesterase 1 (TDP1) to initiate APE1-independent repair, thus expanding the ability of the base excision repair (BER) process. Poly(ADP-ribose) polymerase 1 (PARP1) is a regulatory protein of DNA repair. PARP2 is also activated in response to DNA damage and can be regarded as the BER participant. Here we analyze PARP1 and PARP2 interactions with DNA intermediates of the initial stages of the BER process (8-oxoG and AP-site containing DNA) and their interplay with the proteins recognizing and processing these DNA structures focusing on OGG1. OGG1 as well as PARP1 and PARP2 form covalent complex with AP site-containing DNA without borohydride reduction. AP site incision by APE1 or TDP1 removal of protein adducts but not proteins’ PARylation prevent DNA-protein crosslinks.

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 IC50 ~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.

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 IC50 = 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.

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.

One of the challenges that living organisms face is to promptly respond to genotoxic stress to avoid DNA damage. To this purpose, all organisms, including plants, developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms and need to be finely regulated. In this scenario, microRNAs (miRNAs) are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells whereas studies in plants are still scarce. To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC), targeting distinct components of DDR, namely topoisomerase I (TopI) and tyrosyl-DNA phosphodiesterase 1 (TDP1), were used. Phenotypic (germination percentage and speed, seedling growth) and molecular (cell death, DNA damage, and gene expression profiles) analyses demonstrated that the imposed treatments impact DDR. Our results show that these treatments do not influence the germination process but rather inhibit seedling development, causing an increase in cell death and accumulation of DNA damage. Moreover, treatment-specific changes in the expression of suppressor of gamma response 1 (SOG1), master-regulator of plant DDR, were observed. Additionally, the expression of multiple genes playing important roles in different DNA repair pathways and cell cycle regulation were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identified from our previous bioinformatics approaches as putatively targeting genes involved in DDR processes were investigated alongside their targets. The obtained results indicate that under most conditions when a miRNA is upregulated the corresponding candidate target gene is downregulated, providing an indirect evidence of miRNAs action over these targets. Hence, the present study extends the present knowledge on the information available regarding the roles played by miRNAs in the post-transcriptional regulation of DDR in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human tyrosyl-DNA phosphodiesterase 1 (TDP1) belongs to the phospholipase D superfamily, whose members contain paired catalytic histidine and lysine residues within two conserved motifs and hydrolyze phosphodiester bonds. TDP1 is a DNA repair enzyme that processes 3′ DNA end blocking lesions and a wide range of synthetic DNA adducts as a substrate. TDP1 hydrolyzes DNA-adducts via two coordinated SN2 nucleophilic attacks mediated by the action of two histidine residues and leads to the formation of the covalent intermediate. Hydrolysis of this intermediate is proposed to be carried out by a water molecule that is activated by the His493 residue acting as a general base. It was known that phospholipase D enzymes are able to catalyze not only hydrolysis but also a transphosphatidylation reaction in the presence of primary alcohols in which they transfer the substrate to the alcohol instead of water. Here, we first demonstrated that TDP1 is able to undergo a “transphosphooligonucleotidation” reaction, transferring the substrate residue to the alcohol, thus inducing the formation of covalent DNA adducts with different primary alcohol residues. Such adducts can be accumulated in the conditions of high concentration of alcohol. We demonstrated that glycerol residue was efficiently cleaved from the 3′-end by TDP1 but not by its mutant form associated with the disease spinocerebellar ataxia with axonal neuropathy. Therefore, the second reaction step can be carried out not only by a water molecule but also by the other small nucleophilic molecules, e.g., glycerol and ethanol. Thus, in some cases, TDP1 can be regarded not only as a repair enzyme but also as a source of DNA damage especially in the case of mutation. Such damages can make a negative contribution to the stability of cell vitality.

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 IC50 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.

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 IC50 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.

DNA sensors can be used as robust tools for high-throughput drug screening of small molecules with the potential to inhibit specific enzymes. As enzymes work in complex biological pathways, it is important to screen for both desired and undesired inhibitory effects. We here report a screening system utilizing specific sensors for tyrosyl-DNA phosphodiesterase 1 (TDP1) and topoisomerase 1 (TOP1) activity to screen in vitro for drugs inhibiting TDP1 without affecting TOP1. As the main function of TDP1 is repair of TOP1 cleavage-induced DNA damage, inhibition of TOP1 cleavage could thus reduce the biological effect of the TDP1 drugs. We identified three new drug candidates of the 1,5-naphthyridine and 1,2,3,4-tetrahydroquinolinylphosphine sulfide families. All three TDP1 inhibitors had no effect on TOP1 activity and acted synergistically with the TOP1 poison SN-38 to increase the amount of TOP1 cleavage-induced DNA damage. Further, they promoted cell death even with low dose SN-38, thereby establishing two new classes of TDP1 inhibitors with clinical potential. Thus, we here report a dual-sensor screening approach for in vitro selection of TDP1 drugs and three new TDP1 drug candidates that act synergistically with TOP1 poisons.

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 IC50 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.

DNA-protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of double-strand breaks, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, ageing and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare tyrosyl-DNA phosphodiesterase 1 (TDP1) orthologues between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, we have found increased TDP2 expression in TDP1-deficient cells and embryos. Understanding the role of TDP1 in DPCR at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.

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 IC50 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. [ABSTRACT FROM AUTHOR]

Six imines including four new ones were synthesized via the reaction of monoterpenoid aldehydes with aminoadamantanes. The inhibitory activities of the synthesized compounds against purified human recombinant DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) fell in the range IC50 = 5.5-7.1 μM. [ABSTRACT FROM AUTHOR]

DNA repair is a complicated process that occurs due to a network of different pathways and mechanisms for correction of DNA damages during the normal DNA biosynthesis and under the influence of external and internal factors. The study of these mechanisms and their regulation is closely related to both diagnostics and the search for ways of treating various diseases, including oncological ones. Malignant neoplasms are one of the three most widespread diseases in the world, which unfortunately often become the reason for a lethal outcome. Traditional cancer therapy aims to the DNA damage in malignant cells, and its result depends on the efficiency of the repair systems. In many cancer cells, the individual DNA repair enzymes are overexpressed, which promotes the resistance of these tumors to the therapy. On the other hand, defects in the DNA repair systems in cancer cells allow researchers to find both appropriate biomarkers for diagnostics and targets for the development of the specific and effective therapy. Currently, DNA repair inhibitors are being actively developed in order to increase the sensitivity of the tumor cells to traditional chemotherapy. The principle of synthetic lethality is used to create cell-specific drugs and to improve the effectiveness of the treatment. In this review, we discuss the current state of research and prospects for the development of inhibitors for five important DNA repair enzymes. [ABSTRACT FROM AUTHOR]

Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3′ and 5′ terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-Sc eI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81. [ABSTRACT FROM AUTHOR]

The synthesis of (+)-usnic acid derivatives is described. The derivatives contain one or two cyano groups, connected to the acetophenone fragment of dibenzofuran core by linkers of different length and character, or some other modifications. The influence of these compounds on the activity of recombinant human tyrosyl-DNA phosphodiesterase 1 and MCF-7 tumor cells' viability has been estimated. The data indicate a distinct dependence of functional characteristics of the compounds on their structure. [ABSTRACT FROM AUTHOR]