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.

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.

A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts.

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 ß, 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.

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.

Mutant-Huntingtin Molecular Pathways Elucidate New Targets for Drug Repurposing.

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.

Methods for Assessment of Nucleotide Excision Repair Efficiency.

Nucleotide excision repair (NER) is responsible for removing a wide variety of bulky adducts from DNA, thus contributing to the maintenance of genome stability. The efficiency with which proteins of the NER system recognize and remove bulky adducts depends on many factors and is of great clinical and diagnostic significance. The review examines current concepts of the NER system molecular basis in eukaryotic cells and analyzes methods for the assessment of the NER-mediated DNA repair efficiency both in vitro and ex vivo.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

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.

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.

FUS RRM regulates poly(ADP-ribose) levels after transcriptional arrest and PARP-1 activation on DNA damage.

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.

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.

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 ZnSO4 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.

Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair.

Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase ß-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.

New 5-Hydroxycoumarin-Based Tyrosyl-DNA Phosphodiesterase I Inhibitors Sensitize Tumor Cell Line to Topotecan.

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 IC50 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.

Inhibition of DNA Repair Enzymes as a Valuable Pharmaceutical Approach.

The DNA repair system plays a crucial role in maintaining the integrity of the genome [...].

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid-liquid phase separation.

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid-liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models.

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.

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.

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.

The XPA Protein-Life under Precise Control.

Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process. XPA interacts with almost all NER participants and organizes the correct NER repair complex. In the absence of XPA's scaffolding function, no repair process occurs. In this review, we briefly summarize our current knowledge about the XPA protein structure and analyze the formation of contact with its protein partners during NER complex assembling. We focus on different ways of regulation of the XPA protein's activity and expression and pay special attention to the network of post-translational modifications. We also discuss the data that is not in line with the currently accepted hypothesis about the functioning of the XPA protein.

PARP1 Activation Controls Stress Granule Assembly after Oxidative Stress and DNA Damage.

DNA damage causes PARP1 activation in the nucleus to set up the machinery responsible for the DNA damage response. Here, we report that, in contrast to cytoplasmic PARPs, the synthesis of poly(ADP-ribose) by PARP1 opposes the formation of cytoplasmic mRNA-rich granules after arsenite exposure by reducing polysome dissociation. However, when mRNA-rich granules are pre-formed, whether in the cytoplasm or nucleus, PARP1 activation positively regulates their assembly, though without additional recruitment of poly(ADP-ribose) in stress granules. In addition, PARP1 promotes the formation of TDP-43- and FUS-rich granules in the cytoplasm, two RNA-binding proteins which form neuronal cytoplasmic inclusions observed in certain neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Together, the results therefore reveal a dual role of PARP1 activation which, on the one hand, prevents the early stage of stress granule assembly and, on the other hand, enables the persistence of cytoplasmic mRNA-rich granules in cells which may be detrimental in aging neurons.

The HPF1-dependent histone PARylation catalyzed by PARP2 is specifically stimulated by an incised AP site-containing BER DNA intermediate.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are DNA-dependent poly(ADP-ribose)transferases localized in nucleus. They have a significant homology in the C-terminal catalytic domain structure but differ in their N-terminal DNA-binding parts. The structural difference has an impact on the interaction of PARP1 and PARP2 with DNA and their DNA-dependent activation. Here, we compare the interaction of PARP1 and PARP2 with free 147 bp nucleosomal DNA and its nucleosome-associated variant (NCP) that contain in one strand a 1-nucleotide gap with 5'-dRP (imitating the intermediate of Base Excision Repair) or no specific damage. The affinity of PARP2 for the DNA strongly depends on the gap presence and to a lesser extent on the association with nucleosomes, while PARP1 interacts primarily with blunt ends of all DNAs and with a lower affinity with the single-strand break. The activities of PARP1 and PARP2 in the autoPARylation reaction and heteromodification of histones are distinctly stimulated by HPF1, depending on the gap presence in activating DNA. The most significant HPF1-induced stimulation of the histone modification in the presence of gapped NCP is a peculiar feature of PARP2. We propose a specific regulatory role of PARP2 in the process of DNA repair in the context of chromatin.