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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. Synopsis: Tyrosyl-DNA phosphodiesterase 1 (TDP1) supports repair of trapped Top1-DNA covalent complexes. This study shows that TDP1 is phosphorylated at its residue S61 by CDK1 during mitosis, countering DNA damage, mitotic abnormalities, and chromosomal instability upon replication stress. CDK1 phosphorylates TDP1 at S61 during mitosis. TDP1 defective for S61 phosphorylation (TDP1-S61A) is retained on the mitotic chromosomes, triggering DNA damage and mitotic defects. A MUS81-dependent pathway is predominantly responsible for mitotic clearing of trapped Top1-DNA covalent complexes. MUS81 depletion rescues chromosomal abnormalities in TDP1-S61A-expressing cells. Blocking CDK1-mediated TDP1 phosphorylation results in increased levels of DNA damage and genomic instability upon replication stress. [ABSTRACT FROM AUTHOR]

Abstract Background A higher number of tumor buds in the invasive front of colorectal cancer (CRC) specimens has been shown to contribute to a poor prognosis in CRC patients. Because macrophages (Mφs) have been demonstrated to alter the phenotype of cancer cells, we hypothesized that the phenotype of CRC cells in the tumor budding (TB) area might be changed by the interaction between CRC cells and Mφs. Methods We assessed the expression of topoisomerase 1 in CRC cells to estimate the acquisition of chemoresistance in CRC. To demonstrate the tumor–stromal interaction between CRC cells and Mφs, we assessed two histological findings, the number of Mφs per single CRC cell and the proximity between CRC cells and Mφs by histological spatial analysis using HALO software. Results The expression levels of topoisomerase 1 in CRC cells were decreased in deeper areas, especially in the TB area, compared to the surface area. Our histological spatial analysis revealed that 2.6 Mφs located within 60 μm of a single CRC cell were required to alter the phenotype of the CRC cell. Double-immunofluorescence staining revealed that higher Mφs were positive for interleukin-6 (IL-6) in the TB area and that AE1/AE3-positive CRC cells were also positive for phospho-STAT3 (pSTAT3) in the TB area; thus, the IL-6 receptor (IL-6R)/STAT3 signaling pathway in CRC cells was upregulated by IL-6 derived from neighboring Mφs. Conclusion IL-6 secreted from the neighboring Mφs would alter the phenotype of CRC cells via IL-6R/STAT3 signaling pathway.

Photodynamic therapy (PDT) is an important oncologic intervention option for combating drug resistance of current chemotherapy in cancer. To improve spatiotemporal control, the development of subcellular organelle‐targeting photosensitizers is critical due to the diffusion limits of short‐lived ROS. Mitochondrial topoisomerase 1 (TOP1MT), exclusively localized in mitochondria, is emerging as a promising target for anticancer treatment. In this work, we designed and synthesized mitochondria‐targeting photosensitizers via Top1 inhibition activity of IQ6 and visualized the covalent adducts between the engaged proteins and the synthesized photoaffinity labeling probes (IQ‐Ps). Positively charged derivatives (IQ‐Ss) were also synthesized and their photo‐induced cytotoxicity was assessed to select IQ‐S1. Based on the cellular accumulation of IQ‐S1 in mitochondria and the enhanced PDT activity, we demonstrated that IQ‐S1 functions as a photosensitizer with sequential targeting of mitochondria and nucleus depending on irradiation times. Our findings suggest that the translocation of IQ‐S1 is related to the damage to mitochondrial Top1. The synthesized IQ‐S1 may contribute to the identification and tracking of cellular target proteins along with the PDT progression. [ABSTRACT FROM AUTHOR]

In the modern world with climate changes and increasing pollution, different types of stress are becoming an increasing challenge. Hence, the identification of reliable biomarkers of stress and accessible sensors to measure such biomarkers are attracting increasing attention. In the current study, we demonstrate that the activity, but not the expression, of the ubiquitous enzyme topoisomerase 1 (TOP1), as measured in crude cell extracts by the REEAD sensor system, is markedly reduced in response to thermal stress in both fruit flies (Drosophila melanogaster) and cultivated human cells. This effect was observed in response to both mild-to-moderate long-term heat stress and more severe short-term heat stress in D. melanogaster. In cultivated HeLa cells a reduced TOP1 activity was observed in response to both cold and heat stress. The reduced TOP1 activity appeared dependent on one or more cellular pathways since the activity of purified TOP1 was unaffected by the utilized stress temperatures. We demonstrate successful quantitative measurement of TOP1 activity using an easily accessible chemiluminescence readout for REEAD pointing towards a sensor system suitable for point-of-care assessment of stress responses based on TOP1 as a biomarker. [ABSTRACT FROM AUTHOR]

Abstract 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.

Circular RNA (circRNA) dysregulation has been linked to osteoarthritis (OA). The present study investigated the involvement of hsa_circ_0072568 (circ0072568) in OA. The expression of circ0072568 was detected in OA tissues and interleukin (IL)-1ß-stimulated human chondrocytes. After performing dual-luciferase reporter and RNA immunoprecipitation assays, MTT, enzyme-linked immunosorbent assay and western blot analysis were used to assess the functions of circ0072568 in IL-1ß-induced inflammation in chondrocytes in vitro. Circ0072568 was inhibited in OA tissues and the cell model in vitro. Circ0072568 overexpression protected the chondrocytes against IL-1ß-induced inflammation and extracellular matrix (ECM) breakdown. Circ0072568 directly attached to microRNA (miR)-382-5p and enhanced the production of topoisomerase 1 (TOP1). Furthermore, miR-382-5p overexpression or TOP1 knockdown attenuated the effects of circ0072568 in IL-1ß-stimulated human chondrocytes. On the whole, the present study demonstrates that the Circ0072568/miR-382-5p/TOP1 axis is involved in inflammation and ECM degradation in OA. These findings may contribute to the development of potential therapeutic strategies for OA. [ABSTRACT FROM AUTHOR]

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. [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 cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1's functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70's functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70's interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions. [ABSTRACT FROM AUTHOR]

In the modern world with climate changes and increasing pollution, different types of stress are becoming an increasing challenge. Hence, the identification of reliable biomarkers of stress and accessible sensors to measure such biomarkers are attracting increasing attention. In the current study, we demonstrate that the activity, but not the expression, of the ubiquitous enzyme topoisomerase 1 (TOP1), as measured in crude cell extracts by the REEAD sensor system, is markedly reduced in response to thermal stress in both fruit flies (Drosophila melanogaster) and cultivated human cells. This effect was observed in response to both mild-to-moderate long-term heat stress and more severe short-term heat stress in D. melanogaster. In cultivated HeLa cells a reduced TOP1 activity was observed in response to both cold and heat stress. The reduced TOP1 activity appeared dependent on one or more cellular pathways since the activity of purified TOP1 was unaffected by the utilized stress temperatures. We demonstrate successful quantitative measurement of TOP1 activity using an easily accessible chemiluminescence readout for REEAD pointing towards a sensor system suitable for point-of-care assessment of stress responses based on TOP1 as a biomarker.

DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody–drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs. [ABSTRACT FROM AUTHOR]

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The acquisition of multidrug resistance (MDR) to chemotherapy is a major obstacle to successful cancer treatment. Aiming to improve the potency of anthraquinone-derived antitumor compounds against MDR cancer cells, we employed a rational design approach to develop new heteroarene-fused anthraquinones. Shifting the carboxamide group in the naphtho[2,3- f ]indole scaffold from the 3-position to 2 increased the lipophilicity and P-glycoprotein (P-gp) binding of the derivatives, potentially enhancing their ability to circumvent P-gp-mediated MDR. To validate the computations, we developed a scheme for heterocyclization into esters of naphtho[2,3- f ]indole-2-carboxylic acid, based on the 5- endo -dig cyclization of 2-alkynyl-3-amino-1,4-dimethoxyanthraquinone under mild basic conditions using tetra- n -butylammonium fluoride (TBAF). The synthesized naphthoindole-2-carboxamides, particularly compound 1a bearing (S)-3-aminopyrrolidine in the carboxamide fragment, demonstrated the highest antiproliferative activity. Most importantly, 1a suppressed the growth of the P-gp-positive K562/4 leukemia tumor cell line (resistance index = 2.4), while its 3-isomer LCTA-2640 and Dox did not (RI = 125 and 140, respectively). Studies of intracellular uptake and distribution showed that 1a , unlike its 3-substituted isomer, effectively accumulated in resistant tumor cells, confirming the correlation between in silico and experimental data. The lead compound 1a interacts with DNA duplex and inhibits topoisomerase 1 but does not induce oxidative stress. Treatment with 1a increases the population of apoptotic cells in both K562 and K562/4 sublines, regardless of the cell cycle phase. Taken together, this work provides an interesting example of how a little modification in chemical structure can lead to striking differences in antitumor properties. In conclusion, we have identified a potent class of compounds that offer distinct advantages in combating resistant tumor cells. [Display omitted] • A scheme for synthesis of naphtho[2,3- f ]indole-2-carboxylic acid has been developed. • Naphthoindole-2-carboxamides have increased lipophilicity, cellular accumulation and P-gp binding. • Compound 1a interacts with DNA duplex and inhibit topoisomerase 1 (Top1) inducing apoptosis of tumor cells. • Position of pharmacophore is critical for circumvention of P-gp resistance of tumor cells. [ABSTRACT FROM AUTHOR]

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]

Mutation in Werner (WRN) RECQL helicase is associated with premature aging syndrome (Werner syndrome, WS) and predisposition to multiple cancers. In patients with solid cancers, deficiency of the WRN RECQL helicase is paradoxically associated with enhanced overall survival in response to treatment with TOP1 inhibitors, which stabilize pathological TOP1‐DNA‐covalent‐complexes (TOP1cc) on the genome. However, the underlying mechanism of WRN in development of chemoresistance to TOP1 inhibitors is not yet explored. Our whole‐genome transcriptomic analysis for ~25,000 genes showed robust activation of NF‐κB‐dependent prosurvival genes in response to TOP1cc. CRISPR‐Cas9 knockout, shRNA silencing, and under‐expression of WRN confer high‐sensitivity of multiple cancers to TOP1 inhibitor. We demonstrated that WRN orchestrates TOP1cc repair through proteasome‐dependent and proteasome‐independent process, unleashing robust ssDNA generation. This in turn ensues signal transduction for CHK1 mediated NF‐κB‐activation through IκBα‐degradation and nuclear localization of p65 protein. Intriguingly, our site‐directed mutagenesis and rescue experiments revealed that neither RECQL‐helicase nor DNA‐exonuclease enzyme activity of WRN (WRNE84A, WRNK577M, and WRNE84A‐K577M) were required for TOP1cc removal, ssDNA generation and signaling for NF‐κB activation. In correlation with patient data and above results, the TOP1 inhibitor‐based targeted therapy showed that WRN‐deficient melanoma tumors were highly sensitive to TOP1 inhibition in preclinical in vivo mouse model. Collectively, our findings identify hitherto unknown non‐enzymatic role of WRN RECQL helicase in pathological mechanisms underlying TOP1cc processing and subsequent NF‐κB‐activation, offering a potential targeted therapy for WRN‐deficient cancer patients. [ABSTRACT FROM AUTHOR]

Excessive neuroinflammation and neuronal loss contribute to mechanisms of spinal cord injury (SCI). Accumulating evidence has suggested that topoisomerase 1 (Top1) inhibition can suppress exacerbated immune responses and protect against lethal inflammation. Pyroptosis is a recently identified pro‐inflammatory programmed mode of cell death. However, the effects and underlying mechanisms of Top1 inhibition in SCI remains unclear. Locomotor functional recovery in mice was evaluated through Basso Mouse Scale (BMS). Neuronal loss was evaluated by immunochemistry staining of NeuN. Pyroptosis was determined by immunofluorescence staining, western blot, flow cytometry, cell viability, and cytotoxicity assays. In the present study, we estimated the effects of chemical inhibition of Top1 in an SCI model. Administration of Top1 inhibitor camptothecin (CPT) to mice significantly improved locomotor functional recovery after SCI. Moreover, CPT reduced Top1 level, inhibited nucleotide‐binding oligomerization domain‐like receptor 3 (NLRP3) inflammasome activation and pyroptosis, attenuated proinflammatory cytokines levels, diminished the number of neutrophil and neuronal loss in mice. Furthermore, CPT in oxygen‐glucose deprivation neurons down‐regulated Top1 level, attenuated NLRP3 inflammasome activation, and suppressed pyroptosis and inflammatory response. Together, our findings indicate that inhibition of Top1 with CPT can inhibit pyroptosis, control neuroinflammation, and improve functional recovery after SCI. [ABSTRACT FROM AUTHOR]

Leishmania donovani, a unicellular protozoan parasite, causes a wide range of human diseases including fatal visceral leishmaniasis. Tyrosyl DNA‐phosphodiesterase 1 (TDP1) hydrolyzes the phosphodiester bond between DNA 3′‐end and a tyrosyl moiety of trapped topoisomerase I‐DNA covalent complexes (Top1cc). We have previously shown Leishmania harbors a TDP1 gene (LdTDP1), however, the biological role of TDP1 remains largely unknown. In the present study, we have generated TDP1 knockout L. donovani (LdTDP1−/−) promastigotes and have shown that LdTDP1−/− parasites are deficient in 3′‐phosphodiesterase activities and were hypersensitive to Top1‐poison like camptothecin (CPT), DNA alkylation agent like methyl methanesulfonate, and oxidative DNA lesions generated by hydrogen peroxide but were not sensitive to etoposide. We also detected elevated levels of CPT‐induced reactive oxygen species triggering cell cycle arrest and cell death in LdTDP1−/− promastigotes. LdTDP1−/− promastigotes accumulate a significant change in the membrane morphology with the accumulation of membrane pores, which is associated with oxidative stress and lipid peroxidation. To our surprise, we detected that LdTDP1−/− parasites were hypersensitive to antileishmanial drugs like amphotericin B and miltefosine, which could be rescued by complementation of wild‐type TDP1 gene in the LdTDP1−/− parasites. Notably, multidrug‐resistant L. donovani clinical isolates showed a marked reduction in TDP1 expression and were sensitive to Top1 poisons. Taken together, our study provides a new role of LdTDP1 in protecting L. donovani parasites from oxidative stress‐induced DNA damage and resistance to amphotericin B and miltefosine. [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]

Topoisomerases regulate DNA topology, organization of the intracellular DNA, the transmission of genetic materials, and gene expressions. Other than the nuclear genome, mitochondria also harbor the small, circular DNA (mtDNA) that encodes a critical subset of proteins for the production of cellular ATP; however, mitochondria are solely dependent on the nucleus for all the mitochondrial proteins necessary for mtDNA replication, repair, and maintenance. Mitochondrial genome compiles topological stress from bidirectional transcription and replication, therefore imports four nuclear encoded topoisomerases (Top1mt, Top2α, Top2β, and Top3α) in the mitochondria to relax mtDNA supercoiling generated during these processes. Trapping of topoisomerase on DNA results in the formation of protein-linked DNA adducts (PDAs), which are widely exploited by topoisomerase-targeting anticancer drugs. Intriguingly mtDNA is potentially exposed to DNA damage that has been attributed to a variety of human diseases, including neurodegeneration, cancer, and premature aging. In this review, we focus on the role of different topoisomerases in the mitochondria and our current understanding of the mitochondrial DNA damage through trapped protein-DNA complexes, and the progress in the molecular mechanisms of the repair for trapped topoisomerase covalent complexes (Topcc). Finally, we have discussed how the pathological DNA lesions that cause mtDNA damage, trigger mitochondrial fission and mitophagy, which serve as quality control events for clearing damaged mtDNA. [ABSTRACT FROM AUTHOR]

DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody–drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs.

Objective: To explore chemotherapy sensitivity of irinotecan and its relationship with topoisomerase 1 level in the patients with rectal cancer. Methods: Eighty patients with rectal cancer who received irinotecan chemotherapy in the Hospi - tai Affiliated to Jiangnan University from January 2017 to June 2019 were included in the study. Clinical data including gender, age, clinical stage, differentiation degree and pathological type were collected for study. After 8 cycles of chemotherapy, observations were made in the short-term effects of the patients. Those who failed to respond to the treatment were regarded as insensitive, and those who responded to the treatment were regarded as sensitive, and they were respectively designated as the insensitive and sensitive groups. On the 2nd day after admission, the pathological tissues of the patients were collected and the expres - sion levels of TOPO-1 were detected and compared between the patients of the two groups. The receiver operating characteristic (ROC) curve of the patients was plotted to analyze the evaluation value of TOPO-1 in chemotherapy sensitivity of irinotecan. Serum carbo hydrate antigen 19-9 (CA19-9) and car cino embryonic antigen (CEA) levels were measured at admission and after 8 cycles of chemotherapy, and the serum indexes were compared between the two groups. The correlation between TOPO-1 level and serum levels of CA19-9 and CEA was analyzed by Pearson linear correlation. Results: Of the 80 patients, 32 (40.00%) were partially rem it tent, 26 patients (32.50%) were stable and 22 patients (27.50%) were without remission. The expression level of TOPO-1 in the sensitive group was significantly higher than that in the insensitive group [(16.39i3.26vs (11.28i 2.11外＜0 .05). The area under the curve of TOPO-1 expression for the evaluation of chemotherapy sensitivity to irinotecan was 0 .78O(standard error --0 .054, P

DNA topoisomerase 1 (Top1) is a prime target of chemotherapy agents and a crucial enzyme that maintains DNA topology, during transcription, replication, repair, and recombination by relaxing DNA torsional strain through reversible DNA single-strand breaks process. Top1 is a selective target of camptothecin (CPT), a natural alkaloid compound and an important class of anti-cancer drugs, that reversibly bind the covalent DNA–Top1 complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. However, the use of CPTs was hampered by several drawbacks. In the present study, we have performed a receptor-ligand molecular docking analysis of a set of 738 camptothecin-like molecules in order to identify new potential derivative structures of camptothecin, a more potent anticancer agent with better efficacy, less toxicity, and side effects. MolDock score and hydrogen bonding interactions are the parameters used for docking studies. The inhibition phenomenon is mainly governed by various non-covalent interactions, including hydrogen bonds, steric interactions, and van der Waals interactions between the DNA basis and ligand. Among the top 10 ranked molecules, all the CPT similar ligands were found to have higher binding affinity in comparison with the CPT of the human DNA–Top1. The first ranked molecule (21882279) showed the highest docking score value (− 229.993 kcal/mol) which it binds to the cavity by Arg364 residue. These results suggest that similar ligands may serve as a leading compound for the development of anticancer agents. [ABSTRACT FROM AUTHOR]

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.

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]

RNA/DNA hybrids are processed by RNases H1 and H2, while single ribonucleoside-monophosphates (rNMPs) embedded in genomic DNA are removed by the error-free, RNase H2-dependent ribonucleotide excision repair (RER) pathway. In the absence of RER, however, topoisomerase 1 (Top1) can cleave single genomic rNMPs in a mutagenic manner. In RNase H2-deficient mice, the accumulation of genomic rNMPs above a threshold of tolerance leads to catastrophic genomic instability that causes embryonic lethality. In humans, deficiencies in RNase H2 induce the autoimmune disorders Aicardi–Goutières syndrome and systemic lupus erythematosus, and cause skin and intestinal cancers. Recently, we reported that in Saccharomyces cerevisiae, the depletion of Rnr1, the major catalytic subunit of ribonucleotide reductase (RNR), which converts ribonucleotides to deoxyribonucleotides, leads to cell lethality in absence of RNases H1 and H2. We hypothesized that under replicative stress and compromised DNA repair that are elicited by an insufficient supply of deoxyribonucleoside-triphosphates (dNTPs), cells cannot survive the accumulation of persistent RNA/DNA hybrids. Remarkably, we found that cells lacking RNase H2 accumulate ~ 5-fold more genomic rNMPs in absence than in presence of Rnr1. When the load of genomic rNMPs is further increased in the presence of a replicative DNA polymerase variant that over-incorporates rNMPs in leading or lagging strand, cells missing both Rnr1 and RNase H2 suffer from severe growth defects. These are reversed in absence of Top1. Thus, in cells lacking RNase H2 and containing a limiting supply of dNTPs, there is a threshold of tolerance for the accumulation of genomic ribonucleotides that is tightly associated with Top1-mediated DNA damage. In this mini-review, we describe the implications of the loss of RNase H2, or RNases H1 and H2, on the integrity of the nuclear genome and viability of budding yeast cells that are challenged with a critically low supply of dNTPs. We further propose that our findings in budding yeast could pave the way for the study of the potential role of mammalian RNR in RNase H2-related diseases. [ABSTRACT FROM AUTHOR]

In our pursuit of developing novel analogs of anthracyclines with enhanced antitumor efficacy and safety, we have designed a synthesis scheme for 4,11-dihydroxy-5,10-dioxocyclopenta[ b ]anthracene-2-carboxamides. These newly synthesized compounds exhibit remarkable antiproliferative potency against various mammalian tumor cell lines, including those expressing activated mechanisms of multidrug resistance. The structure of the diamine moiety in the carboxamide side chain emerges as a critical determinant for anticancer activity and interaction with key targets such as DNA, topoisomerase 1, and ROS induction. Notably, the introduced modification to the doxorubicin structure results in significantly increased lipophilicity, cellular uptake, and preferential distribution in lysosomes. Consequently, while maintaining an impact on anthracyclines targets, these novel derivatives also demonstrate the potential to induce cytotoxicity through pathways associated with lysosomes. In summary, derivatives of cyclic diamines, particularly 3-aminopyrrolidine, can be considered a superior choice compared to aminosugars for incorporation into natural and semi-synthetic anthracyclines or new anthraquinone derivatives, aiming to circumvent efflux-mediated drug resistance. [Display omitted] • • New cyclopentane-fused anthraquinone derivatives have been designed and synthesized. • • Selected compounds have strong antiproliferative potency and circumvent Pgp-mediated efflux. • • Derivative 9 has increased lipophilicity and faster cellular uptake than doxorubicin. • • Terminal amino group in the side chain is crucial for DNA binding and Top1 inhibition. [ABSTRACT FROM AUTHOR]

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

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.

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.

Featured Application: Inhibition of Tdp1 has the potential to increase the potency of the anticancer drugs topotecan and irinotecan, topoisomerase 1 poisons. Furthermore, Tdp1 inhibitors can facilitate the therapeutic use of these drugs in other cancer types. 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. [ABSTRACT FROM AUTHOR]

Tyrosyl‐DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of the phosphodiester bond in the DNA‐topoisomerase 1 (Top1) covalent complex and repairs some other 3′‐end DNA adducts. Currently, Tdp1 functions as an important target in cancer drug design owing to its ability to break down various DNA adducts induced by chemotherapeutics. Tdp1 inhibitors may sensitize tumor cells to the action of Top1 poisons, thereby potentiating their effects. This mini‐review summarizes findings from studies reporting the combined inhibition of Top1 and Tdp1. Two different approaches have been considered for developing such drug precursors. [ABSTRACT FROM AUTHOR]

Abstract Derivatives of the anthraquinone (anthracene-9,10-dione) such as doxorubicin, mitoxantrone and others have proved great clinical efficacy for decades. Currently the search in this exceptionally productive chemical class is aimed at optimization of antitumor properties including circumvention of drug resistance. Previously we have reported that heteroarene-fused anthraquinones fused to a 5-membered heterocyclic ring are advantageous in killing drug resistant tumor cells. Herein we present the synthesis and antitumor properties of a series of new anthra[2,3- b ]furan-2-carboxamides. Vast majority of new derivatives were similarly cytotoxic to wild type tumor cell lines and their isogenic sublines with P-glycoprotein overexpression and/or p53 inactivation. Comparison of structurally close derivatives varying in their position relative to the furan moiety, that is, furan-3-carboxamide 1 vs furan-2-carboxamides 5 and 6 , revealed fundamental differences in the cytotoxicity profiles, formation of drug-DNA complexes, efficacy of topoisomerase 1 inhibition and mechanisms of tumor cell death. Together with previous SAR data on the role of individual substituents, these results provide evidence that regioisomerization of anthra[2,3- b ]furancarboxamides generates the practically perspective derivatives whose properties may vary significantly. Graphical abstract Image 1 Highlights • A series of new anthra[2,3- b ]furan-2-carboxamides was synthesized and evaluated. • Individual compounds were potent against wild type and drug resistant tumor cells. • Anthra[2,3- b ]furan-2-carboxamides inhibit topoisomerase 1-mediated DNA unwinding and induce apoptosis. • Position of the carboxamide moiety is important for antitumor properties. [ABSTRACT FROM AUTHOR]

Combinatorial inhibition of Topoisomerase 1 (TOP1) and Poly (ADP-ribose) polymerase 1 (PARP1) is an attractive therapeutic strategy which is under active investigation to address chemoresistance to TOP1 inhibitors. However, this combinatorial regimen suffers from severe dose limiting toxicities. Dual inhibitors often offer significant advantages over combinatorial therapies involving individual agents by minimizing toxicity and providing conducive pharmacokinetic profiles. In this study, we have designed, synthesized and evaluated a library of 11 candidate conjugated d ual i nhibitors for P ARP1 and T OP1, named as DiPT-1 to DiPT-11. Our extensive screening showed that one of the hits i.e. DiPT-4 has promising cytotoxicity profile against multiple cancers with limited toxicities towards normal cells. DiPT-4 induces extensive DNA double stand breaks (DSBs), cell cycle arrest and apoptosis in cancer cells. Mechanistically, DiPT-4 has the propensity to bind catalytic pockets of TOP1 and PARP1, leading to significant inhibition of both TOP1 and PARP1 at in vitro and cellular level. Interestingly, DiPT-4 causes extensive stabilization of TOP1-DNA covalent complex (TOP1cc), a key lethal intermediate associated with induction of DSBs and cell death. Moreover, DiPT-4 inhibited p oly (A DP- r ibosylation) i.e. PARylation of TOP1cc, leading to long lived TOP1cc with a slower kinetics of degradation. This is one of the important molecular processes which helps in overcoming resistance in cancer in response to TOP1 inhibitors. Together, our investigation showed DiPT-4 as a promising dual inhibitor of TOP1 and PARP1, which may have the potential to offer significant advantages over combinatorial therapy in clinical settings. [Display omitted] • Design and synthesis of dual inhibitors of PARP-1 and TOP1 was achieved. • One of the dual inhibitors, DiPT-4 showed promising cytotoxicity against multiple cancers. • DiPT-4 inhibits both TOP1 and PARP1 activities at in vitro and cellular level. • DiPT-4 induces extensive DNA double stand breaks, cell cycle arrest and apoptosis. • DiPT-4 causes prolonged stabilization of TOP1-DNA covalent complex (TOP1cc) through inhibition of PARP-1. [ABSTRACT FROM AUTHOR]

Mammalian topoisomerase 1 (TOP1) is an essential enzyme for normal development. TOP1 relaxes supercoiled DNA to remove helical constraints that can otherwise hinder DNA replication and transcription and thus block cell growth. Unfortunately, this exact activity can covalently trap TOP1 on the DNA that could lead to cell death or mutagenesis, a precursor for tumorigenesis. It is therefore important for cells to find a proper balance between the utilization of the TOP1 catalytic activity to maintain DNA topology and the risk of accumulating the toxic DNA damages due to TOP1 trapping that prevents normal cell growth. In an apparent contradiction to the negative attribute of the TOP1 activity to genome stability, the detrimental effect of the TOP1-induced DNA lesions on cell survival has made this enzyme a prime target for cancer therapies to kill fast-growing cancer cells. In addition, cumulative evidence supports a direct role of TOP1 in promoting transcriptional progression independent of its topoisomerase activity. The involvement of TOP1 in transcriptional regulation has recently become a focus in developing potential new treatments for a subtype of autism spectrum disorders. Clearly, the impact of TOP1 on human health is multifold. In this review, we will summarize our current understandings on how TOP1 contributes to human diseases and how its activity is targeted for disease treatments.

Camptothecin and its derivatives (CPTs) have strong toxicity to eukaryotic cells by targeting their DNA topoisomerase 1 (Top1) protein and have been increasingly explored as potential pesticides for plant protection. However, the detailed structure-binding mechanism of the interactions between CPTs and the insect Top1 protein remains unclear, which significantly hinders the development of novel CPTs as new insecticides. Herein, a series of 7-amide camptothecin analogs based on the binding mode of camptothecin in complex with Top1 (Sf Top1)-DNA from Spodoptera frugiperda cultured cell line Sf9 were designed and synthesized. Fifteen of these compounds exhibited excellent cytotoxic activity (values of IC50 from 2.01 to 6.78 μM) compared with camptothecin (29.47 μM). The molecular simulations revealed the binding mechanism when the camptothecin parent rings were inserting parallel to DNA bases and stabling the ternary complex by π-π stacked and hydrogen-bond interactions, and further suggested that introduction of lipophilic and some electron-withdrawing groups on the amide linkage of camptothecin could be beneficial to its activity via some non-covalent interactions. Furthermore, almost all the synthesized compounds could inhibit the growth of Spodoptera litura larvae strongly (Inhibition rate from 50.20 to 79.05%), superior or comparable to camptothecin (55.69%) after 8 days of exposure. In particular, the compounds 4c, 4d, 4f, and 4j, which presented more than 70% inhibitory activities, were deserved to be developed as potential biorational pesticides. The information described here would be useful for the further design and development of potentially effective pesticides in the field of plant protection.

Human topoisomerase 1B is a ubiquitous and essential enzyme involved in relaxing the topological state of supercoiled DNA to allow the progression of fundamental DNA metabolism. Its enzymatic catalytic cycle consists of cleavage and religation reaction. A ternary fluorescence resonance energy transfer biosensor based on a suicide DNA substrate conjugated with three fluorophores has been developed to monitor both cleavage and religation Topoisomerase I catalytic function. The presence of fluorophores does not alter the specificity of the enzyme catalysis on the DNA substrate. The enzyme-mediated reaction can be tracked in real-time by simple fluorescence measurement, avoiding the use of risky radioactive substrate labeling and time-consuming denaturing gel electrophoresis. The method is applied to monitor the perturbation brought by single mutation on the cleavage or religation reaction and to screen the effect of the camptothecin anticancer drug monitoring the energy transfer decrease during religation reaction. Pathological mutations usually affect only the cleavage or the religation reaction and the proposed approach represent a fast protocol for assessing chemotherapeutic drug efficacy and analyzing mutant's properties. [ABSTRACT FROM AUTHOR]

Chemical modifications of the anthraquinone scaffold are aimed at optimization of this exceptionally productive class of antitumor drugs. In particular, our previously reported anthra[2,3- b ]furan-3-carboxamides demonstrated a high cytotoxic potency in cell culture and in vivo. In this study, we expanded our series of anthra[2,3- b ]furan-3-carboxamides by modifying the key functional groups and dissected the structure-activity relationship within this chemotype. The majority of new compounds inhibited the growth of mammalian tumor cell lines at submicromolar to low micromolar concentrations. We found that 4,11-hydroxy groups as well as the carbonyl moiety in the carboxamide fragment were critical for cytotoxicity whereas the substituent at the 2-position of anthra[2,3- b ]furan was not. Importantly, the new derivatives were similarly potent against wild type cells and their variants resistant to doxorubicin due to P-glycoprotein (Pgp) expression or p53 inactivation. The most cytotoxic derivatives 6 and 9 attenuated plasmid DNA relaxation by topoisomerase 1. Finally, we demonstrated that 6 and 9  at 1 μM induced intracellular oxidative stress, accumulation in G2/M phase of the cell cycle, and apoptosis in gastric carcinoma cell lines regardless of their p53 status. These results further substantiate the potential of anthra[2,3- b ]furan-3-carboxamides as antitumor drug candidates. [ABSTRACT FROM AUTHOR]

ABSTRACT Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans.

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.

High levels of transcription stimulate mutation rates in microorganisms, and this occurs primarily through an enhanced accumulation of DNA damage. The major source of transcription-associated damage in yeast is Topoisomerase I (Top1), an enzyme that removes torsional stress that accumulates when DNA strands are separated. Top1 relieves torsional stress by nicking and resealing one DNA strand, and some Top1-dependent mutations are due to trapping and processing of the covalent cleavage intermediate. Most, however, reflect enzyme incision at ribonucleotides, which are the most abundant noncanonical component of DNA. In either case, Top1 generates a distinctive mutation signature composed of short deletions in tandem repeats; in the specific case of ribonucleotide-initiated events, mutations reflect sequential cleavage by the enzyme. Top1-dependent mutations do not require highly activated transcription, but their levels are greatly increased by transcription, which partially reflects an interaction of Top1 with RNA polymerase. Recent studies have demonstrated that Top1-dependent mutations exhibit a strand bias, with the nature of the bias differing depending on the transcriptional status of the underlying DNA. Under low-transcription conditions, most Top1-dependent mutations arise in the context of replication and reflect incision at ribonucleotides incorporated during leading-strand synthesis. Under high-transcription conditions, most Top1-dependent events arise when the enzyme cleaves the non-transcribed strand of DNA. In addition to increasing genetic instability in growing cells, Top1 activity in transcriptionally active regions may be a source of mutations in quiescent cells. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 ( rnh201-RED ; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase ε ( pol2-M644G ) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 ( rnh201 Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2–5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single ribonucleotides incorporated during DNA replication. [ABSTRACT FROM AUTHOR]

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair. [Display omitted] • SLX4 assembles μm-sized globular clusters of nanocondensates on chromatin • Site-specific interactions yield SLX4 condensates that are regulated by SENP6 and RNF4 • SLX4 compartmentalizes and activates the SUMO/RNF4 pathway • SLX4 condensation triggers TOP1-DPCs chromatin extraction and nascent DNA degradation In this study, Alghoul et al. show that the Fanconi anemia protein SLX4 forms globular clusters of condensates on chromatin that compartmentalize DNA damage response proteins. Compartmentalization of the SUMO/RNF4 pathway by SLX4 triggers the extraction of topoisomerase 1-DNA-protein cross-links from chromatin and the degradation of nascent DNA. [ABSTRACT FROM AUTHOR]