Tyrosyl-DNA phosphodiesterase 2 (Tdp2) repairs DNA-protein crosslinks and protects against double strand breaks in vivo.

DNA-protein crosslinks pose a significant challenge to genome stability and cell viability. Efficient repair of DPCs is crucial for preserving genomic integrity and preventing the accumulation of DNA damage. Despite recent advances in our understanding of DPC repair, many aspects of this process, especially at the organismal level, remain elusive. In this study, we used zebrafish as a model organism to investigate the role of TDP2 (Tyrosyl-DNA phosphodiesterase 2) in DPC repair. We characterized the two tdp2 orthologs in zebrafish using phylogenetic, syntenic and expression analysis and investigated the phenotypic consequences of tdp2 silencing in zebrafish embryos. We then quantified the effects of tdp2a and tdp2b silencing on cellular DPC levels and DSB accumulation in zebrafish embryos. Our findings revealed that tdp2b is the main ortholog during embryonic development, while both orthologs are ubiquitously present in adult tissues. Notably, the tdp2b ortholog is phylogenetically closer to human TDP2. Silencing of tdp2b, but not tdp2a, resulted in the loss of Tdp2 activity in zebrafish embryos, accompanied by the accumulation of DPCs and DSBs. Our findings contribute to a more comprehensive understanding of DPC repair at the organismal level and underscore the significance of TDP2 in maintaining genome stability.

Cheminformatic Identification of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) Inhibitors: A Comparative Study of SMILES-Based Supervised Machine Learning Models.

BACKGROUND: Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs damages in DNA induced by abortive topoisomerase 1 activity; however, maintenance of genetic integrity may sustain cellular division of neoplastic cells. It follows that Tdp1-targeting chemical inhibitors could synergize well with existing chemotherapy drugs to deny cancer growth; therefore, identification of Tdp1 inhibitors may advance precision medicine in oncology. OBJECTIVE: Current computational research efforts focus primarily on molecular docking simulations, though datasets involving three-dimensional molecular structures are often hard to curate and computationally expensive to store and process. We propose the use of simplified molecular input line entry system (SMILES) chemical representations to train supervised machine learning (ML) models, aiming to predict potential Tdp1 inhibitors. METHODS: An open-sourced consensus dataset containing the inhibitory activity of numerous chemicals against Tdp1 was obtained from Kaggle. Various ML algorithms were trained, ranging from simple algorithms to ensemble methods and deep neural networks. For algorithms requiring numerical data, SMILES were converted to chemical descriptors using RDKit, an open-sourced Python cheminformatics library. RESULTS: Out of 13 optimized ML models with rigorously tuned hyperparameters, the random forest model gave the best results, yielding a receiver operating characteristics-area under curve of 0.7421, testing accuracy of 0.6815, sensitivity of 0.6444, specificity of 0.7156, precision of 0.6753, and F1 score of 0.6595. CONCLUSIONS: Ensemble methods, especially the bootstrap aggregation mechanism adopted by random forest, outperformed other ML algorithms in classifying Tdp1 inhibitors from non-inhibitors using SMILES. The discovery of Tdp1 inhibitors could unlock more treatment regimens for cancer patients, allowing for therapies tailored to the patient's condition.

From the TOP: Formation, recognition and resolution of topoisomerase DNA protein crosslinks.

Since the report of "DNA untwisting" activity in 1972, ∼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2ß, TOP3α, TOP3ß and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3'-phosphotyrosyl (TDP1) and 5'-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways.

TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes.

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

Synthesis of furanotriterpenoids from betulin and evaluation of Tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitory properties of new semi-synthetic triterpenoids.

For the first time, a synthetic route for preparing lupane and oleanane derivatives with a hydrogenated furan ring as a cycle A of triterpene scaffold is described. Most of the synthesized compounds, furanoterpenoids and their synthetic intermediates, were non-toxic against the tested cancer and non-cancerous cell lines, and evinced significant inhibitory activity with IC50 1.0-9.0 µM in the tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibition test. Lupane derivatives - 1-oxime 7, 1,10-seco-hydroxynitrile 11 and furanoterpenoid 14 - were selected as those expected to be the most promising compounds. The results of molecular modeling evinced the strongest binding of compound 11 to the active site of Tdp1 compared to the reference drug. Simultaneously, only compound 11 at subtoxic concentration (10 µM) produced a synergetic effect on the topotecan activity against HeLa-V cells.

Two new types of structures from soft coral-associated epiphytic fungus Aspergillus versicolor CGF9-1-2.

Global Natural Products Social (GNPS) molecular networking platform was applied to discovery the undescribed compounds from the common marine fungi Aspergillus versicolor CGF9-1-2, ultimately resulting in isolation of four new polyketides, decumbenone E (1), decumbenone F (2), 2'-epi-8-O-methylnidurufin (6), (-)-phomoindene A (7), one new nucleoside, 3-methyl-9-(2-methylbutene)-xanthine (8), and five known analogues. Their structures were elucidated based on 1D/2D NMR spectroscopic and HRESIMS data analyses, meanwhile, the absolute configurations of new compounds were established based on the X-ray crystallographic experiments, as well as the electronic circular dichroism (ECD) analysis. All compounds were predicted pharmaceutical chemistry with ten commonly disease-related proteins by molecular docking. In addition, all compounds against TDP1 were performed in vitro, which was consistent with the docking result, and compound 6 shown a weak inhibitory activity.

TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Transcriptomic analysis of HEK293A cells with a CRISPR/Cas9-mediated TDP1 knockout.

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.

Zymolyase Treatment of Saccharomyces cerevisiae Affects Cellular Proteins and Degrades Tyrosyl-DNA Phosphodiesterase I.

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.

New Dual Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 and 2 Based on Deoxycholic Acid: Design, Synthesis, Cytotoxicity, and Molecular Modeling.

Deoxycholic acid derivatives containing various heterocyclic functional groups at C-3 on the steroid scaffold were designed and synthesized as promising dual tyrosyl-DNA phosphodiesterase 1 and 2 (TDP1 and TDP2) inhibitors, which are potential targets to potentiate topoisomerase poison antitumor therapy. The methyl esters of DCA derivatives with benzothiazole or benzimidazole moieties at C-3 demonstrated promising inhibitory activity in vitro against TDP1 with IC50 values in the submicromolar range. Furthermore, methyl esters 4d-e, as well as their acid counterparts 3d-e, inhibited the phosphodiesterase activity of both TDP1 and TDP2. The combinations of compounds 3d-e and 4d-e with low-toxic concentrations of antitumor drugs topotecan and etoposide showed significantly greater cytotoxicity than the compounds alone. The docking of the derivatives into the binding sites of TDP1 and TDP2 predicted plausible binding modes of the DCA derivatives.

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.

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.

Usnic Acid Derivatives Inhibit DNA Repair Enzymes Tyrosyl-DNA Phosphodiesterases 1 and 2 and Act as Potential Anticancer Agents.

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 IC50 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.

Identification and optimization of TDP1 inhibitors from anthraquinone and chalcone derivatives: consensus scoring virtual screening and molecular simulations.

Virtual screening aims to identify and rank compounds with drug/lead-like properties based on their affinity for the protein target. We developed a methodology that integrates structure- and ligand-based screening approaches to enhance hit rates against the TDP1 protein within a database of anthraquinone and chalcone derivatives, followed by evaluation of prioritized compounds through molecular simulations. This technique is particularly useful for training set imbalances. Four screening methods were used: QSAR, pharmacophore, shape similarity, and docking. Each method was individually trained to score compounds, and the scores were fused to create parallel Z-score fusion. The QSAR models exhibited satisfactory R2 values (0.84 to 0.75), whereas the pharmacophoric and shape similarity models demonstrated excellent performance (ROC:0.82-0.88). Docking enrichment analysis identified 6N0D as the optimal TDP1 crystal structure (ROC = 0.73). Remarkably, the consensus scoring method surpassed other screening methods, achieving the highest ROC value of 0.98. Docking screening prioritized compounds with binding modes resembling the co-crystallized ligands, whereas MMGBSA, consensus, and docking produced dynamic simulations that were as stable as the co-crystallized ligands. Additionally, the QSAR-selected compounds exhibited binding modes similar to those of commercially available TDP1 inhibitors. In this study, a strong correlation was found between the inhibitory concentrations and binding energy values of commercialized TDP1 inhibitors, indicating that the top-ranked compounds are expected to have potent inhibitory effects in the nano-/micromolar range. The results of this study establish that consensus scoring can be used as an adaptable mainstay virtual screening methodology, pending subsequent experimental validation for affirmation.Communicated by Ramaswamy H. Sarma.

Repair of topoisomerase 1-induced DNA damage by tyrosyl-DNA phosphodiesterase 2 (TDP2) is dependent on its magnesium binding.

Topoisomerases are enzymes that relax DNA supercoiling during replication and transcription. Camptothecin, a topoisomerase 1 (TOP1) inhibitor, and its analogs trap TOP1 at the 3'-end of DNA as a DNA-bound intermediate, resulting in DNA damage that can kill cells. Drugs with this mechanism of action are widely used to treat cancers. It has previously been shown that tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs TOP1-induced DNA damage generated by camptothecin. In addition, tyrosyl-DNA phosphodiesterase 2 (TDP2) plays critical roles in repairing topoisomerase 2 (TOP2)-induced DNA damage at the 5'-end of DNA and in promoting the repair of TOP1-induced DNA damage in the absence of TDP1. However, the catalytic mechanism by which TDP2 processes TOP1-induced DNA damage has not been elucidated. In this study, we found that a similar catalytic mechanism underlies the repair of TOP1- and TOP2-induced DNA damage by TDP2, with Mg2+-TDP2 binding playing a role in both repair mechanisms. We show chain-terminating nucleoside analogs are incorporated into DNA at the 3'-end and abort DNA replication to kill cells. Furthermore, we found that Mg2+-TDP2 binding also contributes to the repair of incorporated chain-terminating nucleoside analogs. Overall, these findings reveal the role played by Mg2+-TDP2 binding in the repair of both 3'- and 5'-blocking DNA damage.

Development of an LC-MS/MS-based method for quantification and pharmacokinetics study on SCID mice of a dehydroabietylamine-adamantylamine conjugate, a promising inhibitor of the DNA repair enzyme.

Earlier, it was found that the agent KS-389, a conjugate of dehydroabietylamine and 1-aminoadamantane, possess inhibiting activity with regard to Tdp1. It this study, LC-MS/MS-based methods of quantification of KS-389 in mice blood and several organs (brain, liver and kidney) were developed and validated. Validation of the methods was performed according to the guidelines of U.S. Food and Drug Administration and European Medicines Agency in terms of selectivity, linearity, accuracy, precision, recovery, matrix effect, stability and carry-over. Dried blood spots (DBS) method was used for blood sample preparation. HPLC separation was performed on a reversed-phase column; the total analysis time was 12 min. Mass spectral detection was performed on a 6500 QTRAP mass spectrometer in multiple reaction monitoring mode. Transitions 463.5→135.1/107.2 and 336.2→332.2/176.2 were scanned for KS-389 and 2,5-bis(4-diethylaminophenyl)-1,3,4-oxadiazole used as the internal standard, respectively. Pharmacokinetics of the compound as well as its distribution in the organs were studied on SCID mice after intraperitoneal administration of the substance at a dose of 5 mg/kg, and it was found that its maximum concentration in blood is reached in 1-1.5 h and was 80 ng/mL. The maximum concentration in all organs is reached after the same time and is approximately 1500 ng/g and 1100 ng/g in liver and kidney, respectively. This is the first report on the pharmacokinetics of Tdp1 inhibitor based on dehydroabietylamine and 1-aminoadamantane after a single administration to mice. Also, the substance was found to be able to penetrate the blood-brain barrier which is important for, and its maximum concentration was c.a. 25-30 ng/g. These results are important for glioma treatment and make it promising for this purpose.

MUS81 cleaves TOP1-derived lesions and other DNA-protein cross-links.

BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

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.

Natural Products and Their Derivatives as Inhibitors of the DNA Repair Enzyme Tyrosyl-DNA Phosphodiesterase 1.

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.

[Effect of Usnic Acid-Derived Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Used as Monotherapy or in Combination with Olaparib on Transplanted Tumors In Vivo].

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.

Novel TDP1 Inhibitors: Disubstituted Thiazolidine-2,4-Diones Containing Monoterpene Moieties.

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 IC50 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.