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Abstract The formation of nuclear biomolecular condensates is often associated with local accumulation of proteins at a site of DNA damage. The key role in the formation of DNA repair foci belongs to PARP1, which is a sensor of DNA damage and catalyzes the synthesis of poly(ADP-ribose) attracting repair factors. We show here that biogenic cations such as Mg2+, Ca2+, Mn2+, spermidine3+, or spermine4+ can induce liquid-like assembly of poly(ADP-ribosyl)ated [PARylated] PARP1 into multimolecular associates (hereafter: self-assembly). The self-assembly of PARylated PARP1 affects the level of its automodification and hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG). Furthermore, association of PARylated PARP1 with repair proteins strongly stimulates strand displacement DNA synthesis by DNA polymerase β (Pol β) but has no noticeable effect on DNA ligase III activity. Thus, liquid-like self-assembly of PARylated PARP1 may play a critical part in the regulation of i) its own activity, ii) PARG-dependent hydrolysis of poly(ADP-ribose), and iii) Pol β–mediated DNA synthesis. The latter can be considered an additional factor influencing the choice between long-patch and short-patch DNA synthesis during repair.

Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF–ERCC1 nuclease forming a damaged 5′-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5′-flap and different size gap to analyze possible repair factor–replication factor interactions. Ternary XPA–FEN1–DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA–FEN1 complex formation in the absence of DNA due to protein–protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA–XPA–FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA–FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1.

Abstract 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.

New imidazolidine-2,4,5-triones with norabietic, nordehydroabietic, and adamantane substituents were synthesized by reacting oxalyl chloride and the corresponding ureas, providing good yields. Bioisosteric replacement of the ureide group with a parabanic acid fragment made it possible to increase the solubility of compounds and conduct biological studies. The compounds inhibit the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 in submicromolar concentrations. Cytotoxic concentrations were also studied on the glioblastoma cell line SNB19.

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1′s regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins—PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1—did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.

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.

Kurgina et al. conduct in vitro characterization of the effect of HPF1 on the catalytic output of PARP1 and PARP2 under several experimental conditions. The authors report that the DNAdependent and DNA-independent autoPARylation of PARP1 and PARP2, as well as the heteroPARylation of histones in complex with the nucleosome are stimulated by HPF1 in a certain range of HPF1 and NAD + concentrations.

Y-box-binding protein 1 (YB-1) is a multifunctional protein involved in the regulation of gene expression. Recent studies showed that in addition to its role in the RNA and DNA metabolism, YB-1 is involved in the regulation of PARP1 activity, which catalyzes poly(ADP-ribose) [PAR] synthesis under genotoxic stress through auto-poly(ADP-ribosyl)ation or protein trans-poly(ADP-ribosyl)ation. Nonetheless, the exact mechanism by which YB-1 regulates PAR synthesis remains to be determined. YB-1 contains a disordered Ala/Pro-rich N-terminal domain, a cold shock domain, and an intrinsically disordered C-terminal domain (CTD) carrying four clusters of positively charged amino acid residues. Here, we examined the functional role of the disordered CTD of YB-1 in PAR binding and in the regulation of PARP1-driven PAR synthesis in vitro. We demonstrated that the rate of PARP1-dependent synthesis of PAR is higher in the presence of YB-1 and is tightly controlled by the interaction between YB-1 CTD and PAR. Moreover, YB-1 acts as an effective cofactor in the PAR synthesis catalyzed by the PARP1 point mutants that generate various PAR polymeric structures, namely, short hypo- or hyperbranched polymers. We showed that either a decrease in chain length or an increase in branching frequency of PAR affect its binding affinity for YB-1 and YB-1–mediated stimulation of PARP1 enzymatic activity. These results provide important insight into the mechanism underlying the regulation of PARP1 activity by PAR-binding proteins containing disordered regions with clusters of positively charged amino acid residues, suggesting that YB-1 CTD-like domains may be considered PAR “readers” just as other known PAR-binding modules.

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.

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.

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.

Poly(ADP-ribose) polymerase 1 (PARP1) is established as a key regulator of the cellular DNA damage response and apoptosis. In addition, PARP1 participates in the global regulation of DNA repair, transcription, telomere maintenance, and inflammation response by modulating various DNA-protein and protein-protein interactions. Recently, it was reported that PARP1 also influences splicing and ribosomal RNA biogenesis. The H/ACA ribonucleoprotein complex is involved in a variety of cellular processes such as RNA maturation. It contains non-coding RNAs with specific H/ACA domains and four proteins: dyskerin (DKC1), GAR1, NHP2, and NOP10. Two of these proteins, DKC1 and GAR1, are targets of poly(ADP-ribosyl)ation catalyzed by PARP1. The H/ACA RNA-binding proteins are involved in the regulation of maturation and activity of the telomerase complex, which maintains telomere length. In this study, we demonstrated that of poly(ADP-ribosyl)ation influences on RNA-binding properties of DKC1 and GAR1 and telomerase assembly and activity. Our data provide the evidence that poly(ADP-ribosyl)ation regulates telomerase complex assembly and activity, in turn regulating telomere length that may be useful for design and development of anticancer therapeutic approaches that are based on the inhibition of PARP1 and telomerase activities.

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.

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.

A series of deoxycholic acid (DCA) amides containing benzyl ether groups on the steroid core were tested against the tyrosyl-DNA phosphodiesterase 1 (TDP1) and 2 (TDP2) enzymes. In addition, 1,2,4- and 1,3,4-oxadiazole derivatives were synthesized to study the linker influence between a para-bromophenyl moiety and the steroid scaffold. The DCA derivatives demonstrated promising inhibitory activity against TDP1 with IC50 in the submicromolar range. Furthermore, the amides and the 1,3,4-oxadiazole derivatives inhibited the TDP2 enzyme but at substantially higher concentration. Tryptamide 5 and para-bromoanilide 8 derivatives containing benzyloxy substituent at the C-3 position and non-substituted hydroxy group at C-12 on the DCA scaffold inhibited both TDP1 and TDP2 as well as enhanced the cytotoxicity of topotecan in non-toxic concentration in vitro. According to molecular modeling, ligand 5 is anchored into the catalytic pocket of TDP1 by one hydrogen bond to the backbone of Gly458 as well as by π–π stacking between the indolyl rings of the ligand and Tyr590, resulting in excellent activity. It can therefore be concluded that these derivatives contribute to the development of specific TDP1 and TDP2 inhibitors for adjuvant therapy against cancer in combination with topoisomerase poisons.

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.

Abstract Background In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. Results Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. Conclusions Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

Background: The genome is always exposed to different kinds of DNA damaging agents. DNA repair systems in cells are responsible for the genome integrity. The damages, which do not make strong disturbance of double stranded DNA structure, are normally processed by the base excision repair (BER) system. To the moment, this system is well characterized, but the details of regulation are still under investigation. DNA compaction additionally complicates the functioning of repair systems. For successful DNA repair, BER stages, as well as the degree of DNA compaction, should be regulated. Poly(ADP-ribose)polymerase1 (PARP1) and poly(ADP-ribose)polymerase2 (PARP2) are key BER regulatory members, which are also known to participate in the regulation of chromatin remodeling. Methods: Polyacrylamide gel electrophoresis. Results: In our study, we focused on the investigation of the influence of PARPs on the activity of major enzymes of the BER system - APE1 and DNA polymerase beta, using both reconstituted nucleosomes and naked DNAs. We obtained the inhibitory effect of PARP1 and PARP2 on the activity of both APE1 and DNA polymerase beta. This effect was attenuated in the presence of NAD+, under conditions of poly(ADP-ribosyl)ation. Conclusion: Our results additionally confirm the currently relevant model for the regulation of the interactions of PARP1 and PARP2 with DNA. In particular, under ADP-ribosylation, autopoly(ADP-ribosyl)ation of these PARPs occurs that contributes to the dissociation of their complexes with the DNA/nucleosome, due to electrostatic repulsion between the DNA and the negatively charged polymer of ADP-ribose, which is covalently attached to PARP. The structural data could clarify the points of interaction and location of PARPs relative to each other or to nucleosome core and BER proteins during repair.

Background: Base excision DNA repair (BER) is very efficient process that involves the action of many enzymes and accessory proteins to correct the most common DNA lesions usually induced by ionizing irradiation and oxidative stress. Coordination of BER depends on assembling protein participants into multicomponent dynamic "repairosome". Knowledge of the oligomeric states of protein complexes is useful for their structural studies. The primary aim of our work was to estimate the oligomeric states of enzymes and coordinating proteins of BER and of their complexes characterized previously by using fluorescence-based approach. Methods: We employed dynamic light scattering (DLS) technique to measure the hydrodynamic sizes of several enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1), present alone or in the equimolar mixtures with each other. DLS measurements were performed in solution under true equilibrium conditions by using a Zetasizer Nano ZS instrument. Glutaraldehyde cross-linking was used to analyze the prevailing complexes in the proteins mixtures. Results: The hydrodynamic radii of the protein particles determined from the volume weighted size distribution profiles have been used to estimate the oligomeric states of proteins and their complexes. All the proteins of different molecular weights and functions in BER have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer (dimer of heterodimers). On the contrary, the oligomerization state of XRCC1 hetero-associated complexes has been shown to vary from the heterodimer (complex with APE1) to the heterotetramer (complexes with Polβ, TDP1 and PARP1). PARP1 was revealed to destabilize strong interaction between Polβ and XRCC1, suggesting modulation of the proteins association mode upon assembling higher order complexes. Conclusion: Our study demonstrates for the first time applicability of the DLS technique for determination of the oligomeric states of various proteins and their complexes, when complemented with fluorescence-based and chemical cross-linking approaches.

Summary: PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates. : Using a single-molecule approach, Singatulina et al. reconstitute the DNA repair system leading to the specific recruitment of FUS, an mRNA-binding protein related to liquid-liquid phase separation biology, to DNA damage sites, thus revealing the capacity of FUS to form dynamic compartments in which damaged DNA is concentrated. Keywords: poly(ADP-ribose) polymerase 1, poly(ADP-ribose), RNA-binding proteins, liquid-liquid phase separation, DNA repair, atomic force microscopy, cancer, neurodegenerative disease

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising target for anticancer therapy due to its ability to counter the effects topoisomerase 1 (Top1) poison, such as topotecan, thus, decreasing their efficacy. Compounds containing adamantane and monoterpenoid residues connected via 1,2,4-triazole or 1,3,4-thiadiazole linkers were synthesized and tested against Tdp1. All the derivatives exhibited inhibition at low micromolar or nanomolar concentrations with the most potent inhibitors having IC50 values in the 0.35–0.57 µM range. The cytotoxicity was determined in the HeLa, HCT-116 and SW837 cancer cell lines; moderate CC50 (µM) values were seen from the mid-teens to no effect at 100 µM. Furthermore, citral derivative 20c, α-pinene-derived compounds 20f, 20g and 25c, and the citronellic acid derivative 25b were found to have a sensitizing effect in conjunction with topotecan in the HeLa cervical cancer and colon adenocarcinoma HCT-116 cell lines. The ligands are predicted to bind in the catalytic pocket of Tdp1 and have favorable physicochemical properties for further development as a potential adjunct therapy with Top1 poisons.

A new type of berberine derivatives was obtained by the reaction of berberrubine with aliphatic sulfonyl chlorides. The new polycyclic compounds have a sultone ring condensed to C and D rings of a protoberberine core. The reaction conditions were developed to facilitate the formation of sultones with high yields without by-product formation. Thus, it was shown that the order of addition of reagents affects the composition of the reaction products: when sulfochlorides are added to berberrubine, their corresponding 9-O-sulfonates are predominantly formed; when berberrubine is added to pre-generated sulfenes, sultones are the only products. The reaction was shown to proceed stereo-selectively and the cycle configuration was confirmed by 2D NMR spectroscopy. The inhibitory activity of the synthesized sultones and their 12-brominated analogs against the DNA-repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important target for a potential antitumor therapy, was studied. All derivatives were active in the micromolar and submicromolar range, in contrast to the acyclic analogs and 9-O-sulfonates, which were inactive. The significance of the sultone cycle and bromine substituent in binding with the enzyme was confirmed using molecular modeling. The active inhibitors are mostly non-toxic to the HeLa cancer cell line, and several ligands show synergy with topotecan, a topoisomerase 1 poison in clinical use. Thus, novel berberine derivatives can be considered as candidates for adjuvant therapy against cancer.

Y-box-binding protein 1 (YB-1) is a multifunctional positively charged protein that interacts with DNA or RNA and poly(ADP-ribose) (PAR). YB-1 is poly(ADP-ribosyl)ated and stimulates poly(ADP-ribose) polymerase 1 (PARP1) activity. Here, we studied the mechanism of YB-1-dependent PAR synthesis by PARP1 in vitro using biochemical and atomic force microscopy assays. PAR synthesis activity of PARP1 is known to be facilitated by co-factors such as Mg2+. However, in contrast to an Mg2+-dependent reaction, the activation of PARP1 by YB-1 is accompanied by overall up-regulation of protein PARylation and shortening of the PAR polymer. Therefore, YB-1 and cation co-factors stimulated PAR synthesis in divergent ways. PARP1 autoPARylation in the presence of YB-1 as well as trans-PARylation of YB-1 are greatly affected by the type of damaged DNA, suggesting that PARP1 activation depends on the formation of a PARP1–YB-1–DNA ternary complex. An unstructured C-terminal part of YB-1 involved in an interaction with PAR behaves similarly to full-length YB-1, indicating that both DNA and PAR binding are involved in the stimulation of PARP1 activity by YB-1. Thus, YB-1 is likely linked to the regulation of PARylation events in cells via an interaction with PAR and damaged DNA.

Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2′,3′,5′-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC50) in the lower micromolar and submicromolar range. 2′,3′,5′-Tri-O-benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 μM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 μM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50–100%). Several O-benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.

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.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC50 values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.

Eleven amide and thioamide derivatives with monoterpene and adamantine substituents were synthesised. They were tested for their activity against the tyrosyl-DNA phosphodiesterase 1 DNA (Tdp1) repair enzyme with the most potent compound 47a, having an IC50 value of 0.64 µM. When tested in the A-549 lung adenocarcinoma cell line, no or very limited cytotoxic effect was observed for the ligands. However, in conjunction with topotecan, a well-established Topoisomerase 1 (Top1) poison in clinical use against cancer, derivative 46a was very cytotoxic at 5 µM concentration, displaying strong synergism. This effect was only seen for 46a (IC50—3.3 µM) albeit some other ligands had better IC50 values. Molecular modelling into the catalytic site of Tdp1 predicted plausible binding mode of 46a, effectively blocking access to the catalytic site.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that mends topoisomerase 1-mediated DNA damage. Tdp1 is a current inhibition target for the development of improved anticancer treatments, as its inhibition may enhance the therapeutic effect of topoisomerase 1 poisons. Here, we report a study on the development of a novel class of Tdp1 inhibitors that is based on the octahydro-2H-chromene scaffold. Inhibition and binding assays revealed that these compounds are potent inhibitors of Tdp1, with IC50 and KD values in the low micromolar concentration range. Molecular modelling predicted plausible conformations of the active ligands, blocking access to the enzymatic machinery of Tdp1. Our results thus help establish a structural-activity relationship for octahydro-2H-chromene-based Tdp1 inhibitors, which will be useful for future Tdp1 inhibitor development work.

An Important task in the treatment of oncological and neurodegenerative diseases is the search for new inhibitors of DNA repair system enzymes. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the DNA repair system enzymes involved in the removal of DNA damages caused by topoisomerase I inhibitors. Thus, reducing the activity of Tdp1 can increase the effectiveness of currently used anticancer drugs. We describe here a new class of semisynthetic small molecule Tdp1 inhibitors based on the bile acid scaffold that were originally identified by virtual screening. The influence of functional groups of bile acids (hydroxy and acetoxy groups in the steroid framework and amide fragment in the side chain) on inhibitory activity was investigated. In vitro studies demonstrate the ability of the semisynthetic derivatives to effectively inhibit Tdp1 with IC50 up to 0.29 µM. Furthermore, an excellent fit is realized for the ligands when docked into the active site of the Tdp1 enzyme.