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1.  The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II 
Biochemical pharmacology  2012;84(12):1617-1626.
Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC and topoisomerase II (EC inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.
PMCID: PMC3501595  PMID: 23041231
Dovitinib; Anticancer; DNA; Topoisomerase II; Topoisomerase I
2.  The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα 
Biochemical Pharmacology  2012;84(1):52-58.
The thiosemicarbazones Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and triapine have potent antiproliferative activity and have been evaluated as anticancer agents. While these compounds strongly bind iron and copper, their mechanism(s) of action are incompletely understood. A recent report (Rao et al., Cancer Research 69:948-957, 2009) suggested that Dp44mT may, in part, exert its cytotoxicity through poisoning of DNA topoisomerase IIα. In the present report, a variety of assays were used to determine whether Dp44mT and triapine target topoisomerase IIα. Neither compound inhibited topoisomerase IIα decatenation or induced cleavage of pBR322 DNA in the presence of enzyme. In cells, Dp44mT did not stabilize topoisomerase IIα covalent binding to DNA using an immunoblot band depletion assay, an ICE (immunodetection of complexes of enzyme-to-DNA) assay, and a protein-DNA covalent complex forming assay. Dp44mT did not display cross resistance to etoposide resistant K562 cells containing reduced topoisomerase IIα levels. Synchronized Dp44mT-treated CHO cells did not display a G2/M cell cycle block expected of a topoisomerase II inhibitor. A COMPARE analysis of Dp44mT using the NCI 60-cell line data indicated that inhibition of cell growth was poorly correlated with DNA topoisomerase IIα mRNA levels. In summary, we found no support for the conclusion that Dp44mT inhibits cell growth through the targeting of topoisomerase IIα. Since clinical trials of triapine are underway, it will be important to better understand the intracellular targeting and mechanisms of action of the thiosemicarbazones to support forward development of these agents and newer analogs.
PMCID: PMC3348365  PMID: 22503743
Dp44mT; triapine; topoisomerase IIα; cell cycle analysis; iron; thiosemicarbazone
3.  3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I 
Journal of Antimicrobial Chemotherapy  2012;67(12):2882-2891.
Antibiotic resistance in bacterial pathogens is a serious clinical problem. Novel targets are needed to combat increasing drug resistance in Escherichia coli. Our objective is to demonstrate that 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2yl]-1H-benzimidazole (DMA) inhibits E. coli DNA topoisomerase I more strongly than human topoisomerase I. In addition, DMA is non-toxic to mammalian cells at antibiotic dosage level.
In the present study, we have established DMA as an antibacterial compound by determining MICs, post-antibiotic effects (PAEs) and MBCs for different standard as well as clinical strains of E. coli. We have described the differential catalytic inhibitory mechanism of bis-benzimidazole, DMA, for human and E. coli topoisomerase I and topoisomerase II by performing different assays, including relaxation assays, cleavage–religation assays, DNA unwinding assays, ethidium bromide displacement assays, decatenation assays and DNA gyrase supercoiling assays.
DMA significantly inhibited bacterial growth at a very low concentration, but did not affect human cell viability at higher concentrations. Activity assays showed that it preferentially targeted E. coli topoisomerase I over human topoisomerase I, topoisomerase II and gyrase. Cleavage–religation assays confirmed DMA as a poison inhibitor of E. coli topoisomerase I. This study illuminates new properties of DMA, which may be further modified to develop an efficient topoisomerase inhibitor that is selective towards bacterial topoisomerase I.
This is the first report of a bis-benzimidazole acting as an E. coli topoisomerase I inhibitor. DMA is a safe, non-cytotoxic molecule to human cells at concentrations that are needed for antibacterial activity.
PMCID: PMC3494844  PMID: 22945915
Hoechst 33342; DMA; MICs; MBCs; PAEs
4.  Drugging Topoisomerases: Lessons and Challenges 
ACS chemical biology  2013;8(1):82-95.
Topoisomerases are ubiquitous enzymes that control DNA supercoiling and entanglements. They are essential during transcription and replication and topoisomerase inhibitors are among the most effective and most commonly used anticancer and antibacterial drugs. This review consists in two parts. In the first part (“Lessons”), it gives background information on the catalytic mechanisms of the different enzyme families (6 different genes in humans and 4 in most bacteria), describes the “interfacial inhibition” by which topoisomerase-targeted drugs act as topoisomerase poisons and describes clinically relevant topoisomerase inhibitors. It generalizes the interfacial inhibition principle, which was discovered from the mechanism of action of topoisomerase inhibitors, and discusses how topoisomerase inhibitors kill cells by trapping topoisomerases on DNA rather than by classical enzymatic inhibition. Trapping protein-DNA complexes extends to a novel mechanism of action of PARP inhibitors and could be applied to the targeting of transcription factors. The second part of the review focuses on the challenges for discovery and precise use of topoisomerase inhibitors, including targeting topoisomerase inhibitors using chemical coupling and encapsulation for selective tumor delivery, use of pharmacodynamic biomarkers to follow drug activity, complexity of the response determinants for anticancer activity and patient selection, prospects of rational combinations with DNA repair inhibitors targeting tyrosyl-DNA-phosphodiesterases 1 and 2 (TDP1 and TDP2) and PARP, and the unmeet need to develop inhibitors for type IA enzymes.
PMCID: PMC3549721  PMID: 23259582
5.  Voreloxin Is an Anticancer Quinolone Derivative that Intercalates DNA and Poisons Topoisomerase II 
PLoS ONE  2010;5(4):e10186.
Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research.
Methods/Principal Findings
Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more potent.
As a first-in-class anticancer quinolone derivative, voreloxin is a toposiomerase II-targeting agent with a unique mechanistic signature. A detailed understanding of voreloxin's molecular mechanism, in combination with its evolving clinical profile, may advance our understanding of structure-activity relationships to develop safer and more effective topoisomerase II-targeted therapies for the treatment of cancer.
PMCID: PMC2855444  PMID: 20419121
6.  Topoisomerase Assays 
Current Protocols in Pharmacology  2012;CHAPTER:Unit3.3.
Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro.
PMCID: PMC3397423  PMID: 22684721
Topoisomerase; Topoisomerase I; Topoisomerase II; camptothecin; etoposide; topoisomerase poison
7.  Molecular Characterization of Recombinant Pneumocystis carinii Topoisomerase I: Differential Interactions with Human Topoisomerase I Poisons and Pentamidine 
The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The P. carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg2+ requirement. Cleavage assays reveal that both the human and P. carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4′,6′-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that P. carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme, P. carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of P. carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with P. carinii topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.
PMCID: PMC127280  PMID: 12069967
8.  Novel DNA Topoisomerase IIα Inhibitors from Combined Ligand- and Structure-Based Virtual Screening 
PLoS ONE  2014;9(12):e114904.
DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, “poisons”, binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the β-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the β-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors.
PMCID: PMC4260913  PMID: 25489853
9.  Novobiocin and Additional Inhibitors of the Hsp90 C-Terminal Nucleotide-binding Pocket 
Current medicinal chemistry  2008;15(26):2702-2717.
The 90 kDa heal shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer.
Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol.
The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site (~700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones.
Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site.
EGCG is one of the active ingredients found in green tea EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding.
Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimies the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones. Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis.
This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity.
PMCID: PMC2729083  PMID: 18991631
10.  DNA topoisomerases from pathogenic fungi: targets for the discovery of antifungal drugs. 
Antimicrobial Agents and Chemotherapy  1992;36(12):2778-2784.
DNA topoisomerases, a class of enzymes that change the topological structure of DNA, have been shown to be the target of many therapeutic agents, including antibacterial agents (quinolones) and anticancer agents. These drugs inhibit the enzyme in a unique way so that the enzyme is converted into a cellular poison. Candida albicans and Aspergillus niger are two major opportunistic fungal pathogens. Our results show that these fungi have high levels of both type I and type II topoisomerases (with a minimum of 5 x 10(5) ATP-independent relaxation units and 2 x 10(5) P-4 unknotting units per liter of wild-type C. albicans). The ATP-dependent type II topoisomerase (termed C. albicans topoisomerase II) was purified by approximately 2,000-fold from C. albicans cells by using a simple isolation scheme that consists of three column procedures: hydroxylapatite, phosphocellulose, and heparin-agarose chromatographies. The responses of the Candida and the calf thymus topoisomerase II to some known topoisomerase II inhibitors were measured. Etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide, compounds known to inhibit catalysis and to enhance DNA breakage by mammalian topoisomerase II, and A-80198, an etoposide derivative, enhanced cleavage by both enzymes at similar concentrations of these compounds, with the response of the calf thymus topoisomerase II from slightly to fourfold higher in magnitude than the response of the Candida enzyme in the same concentration range. In contrast, A-75272 (a cytotoxic tricyclic quinolone) shows a slightly stronger DNA cleavage enhancement effect with the Candida enzyme than with the mammalian counterpart. The abundance of the enzyme in cells and the different drug responses of the host enzyme and the fungal enzyme suggest that the fungal topoisomerase may serve as a target for the discovery of effective and safe antifungal agents.
PMCID: PMC245544  PMID: 1336349
11.  DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia 
The protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.
Author Summary
Giardia lamblia becomes infective by differentiation into water-resistant cysts. During encystation, cyst wall proteins (CWPs) are highly synthesized and are targeted to the cyst wall. However, little is known about the regulation mechanisms of these genes. DNA topoisomerases can resolve the topological problems and are needed for a variety of key cellular functions, including cell proliferation, cell differentiation and organ development in higher eukaryotes. We found that giardial Topo II was highly expressed during encystation. Topo II is present in Giardia nuclei and is associated with the encystation-induced cwp gene promoters. Topo II has typical DNA cleavage activity of type II topoisomerases. Interestingly, overexpression of Topo II can induce cwp gene expression and cyst formation. Addition of a type II topoisomerase inhibitor, etoposide, significantly decreased the levels of cwp gene expression and cyst formation. Etoposide also has growth inhibition effect on Giardia. Our results suggest that Topo II plays an important role in induction of encystation by up-regulation of the cwp gene expression. Our results provide insights into the function of Topo II in parasite differentiation into cysts and help develop ways to interrupt the parasite life cycle.
PMCID: PMC3656124  PMID: 23696909
12.  The Impact of the C-Terminal Domain on the Interaction of Human DNA Topoisomerase II α and β with DNA 
PLoS ONE  2011;6(2):e14693.
Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction.
Methodology/Principal Findings
We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes.
The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme's KD for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.
PMCID: PMC3040172  PMID: 21358820
13.  CFS-1686 Causes Cell Cycle Arrest at Intra-S Phase by Interference of Interaction of Topoisomerase 1 with DNA 
PLoS ONE  2014;9(12):e113832.
CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.
PMCID: PMC4252032  PMID: 25460368
14.  B-Ring-Aryl Substituted Luotonin A Analogues with a New Binding Mode to the Topoisomerase 1-DNA Complex Show Enhanced Cytotoxic Activity 
PLoS ONE  2014;9(5):e95998.
Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate-catalyzed Friedländer reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors.
PMCID: PMC4022624  PMID: 24830682
15.  Novel acridine-based compounds that exhibit an anti-pancreatic cancer activity are catalytic inhibitors of human topoisomerase II 
European journal of pharmacology  2008;602(2-3):223-229.
We have identified a small library of novel substituted 9-aminoacridine derivatives that inhibit cell proliferation of pancreatic cancer cell lines by inducing apoptosis. (Goodell, J.R. et al., 2008. J. Med. Chem. 51, 179–182.). To further investigate their antiproliferative activities, we have assessed the antiproliferative activity of these acridine-based compounds against several pancreatic cancer cell lines. All four compounds used in this study inhibited the proliferation of pancreatic cancer cell lines in vitro. In addition, we have employed a xenograft tumor model and found that these compounds also inhibit the proliferation of pancreatic cancer in vivo. In light of the potential importance of the anticancer activity of these acridine-based compounds, we have conducted a series of biochemical assays to determine the effect of these compounds on human topoisomerase II. Unlike amsacrine, these compounds do not poison topoisomerase II. Similar to amsacrine, however, these compounds intercalate into DNA in a way that they would alter the apparent topology of the DNA substrate. Thus, inhibition of the relaxation activity of topoisomerase II by these compounds has been reexamined using a DNA strand passage assay. We have found that these compounds, indeed, inhibit the catalytic activity of topoisomerase II. Thus, these novel acridine-based compounds with anti-pancreatic cancer activity are catalytic inhibitors, not poisons, of human topoisomerase II.
PMCID: PMC2637346  PMID: 19071108
acridine derivatives; anticancer drug; cancer; catalytic inhibitor; DNA intercalation; topoisomerase
16.  The Use of Divalent Metal Ions by Type II Topoisomerases 
Type II topoisomerases are essential enzymes that regulate DNA under- and overwinding and remove knots and tangles from the genetic material. In order to carry out their critical physiological functions, these enzymes utilize a double-stranded DNA passage mechanism that requires them to generate a transient double-stranded break. Consequently, while necessary for cell survival, type II topoisomerases also have the capacity to fragment the genome. This feature of the prokaryotic and eukaryotic enzymes, respectively, is exploited to treat a variety of bacterial infections and cancers in humans. All type II topoisomerases require divalent metal ions for catalytic function. These metal ions function in two separate active sites and are necessary for the ATPase and DNA cleavage/ligation activities of the enzymes. ATPase activity is required for the strand passage process and utilizes the metal-dependent binding and hydrolysis of ATP to drive structural rearrangements in the protein. Both the DNA cleavage and ligation activities of type II topoisomerases require divalent metal ions and appear to utilize a novel variant of the canonical two-metal-ion phosphotransferase/hydrolase mechanism to facilitate these reactions. This article will focus primarily on eukaryotic type II topoisomerases and the roles of metal ions in the catalytic functions of these enzymes.
PMCID: PMC2918885  PMID: 20703329
Topoisomerase IIα; divalent metal ion; divalent cation; ATP hydrolysis; DNA cleavage; DNA ligation; topoisomerase II poisons
17.  A functional type I topoisomerase from Pseudomonas aeruginosa 
BMC Molecular Biology  2009;10:23.
Pseudomonas aeruginosa encodes a putative topoisomerase with sequence similarity to the eukaryotic type IB topoisomerase from Vaccinia virus. Residues in the active site are conserved, notably Tyr292 which would be predicted to form the transient covalent bond to DNA.
The gene encoding the P. aeruginosa topoisomerase I was cloned and expressed in E. coli. The enzyme relaxes supercoiled DNA, while a mutant containing a Tyr292 to Phe substitution at the active site was found to be catalytically inert. This is consistent with the role of Tyr in forming the covalent intermediate. Like Vaccinia topoisomerase, the P. aeruginosa topoisomerase relaxes DNA in the absence of ATP, but unlike Vaccinia topoisomerase, P. aeruginosa topoisomerase does not relax supercoiled DNA without MgCl2 present. In addition, high concentration of NaCl is not able to substitute for MgCl2 as seen for Vaccinia topoisomerase. A truncated derivative of the topoisomerase lacking residues 1–98 relaxes DNA, with both full length and truncated enzyme exhibiting equivalent requirements for divalent cations and the ability to relax DNA to completion, suggesting a shared domain organization. DNA-binding assays suggest an only modest preference for the CCCTT pentameric sequence required for transesterification by Vaccinia topoisomerase IB.
P. aeruginosa encodes a functional topoisomerase with significant similarity to the type IB enzyme encoded by poxviruses. In contrast to the Vaccinia-encoded homolog, the P. aeruginosa-encoded enzyme requires divalent cations for catalytic activity, relaxes DNA to completion, and does not exhibit a strong preference for the pentameric sequence stringently required by the Vaccinia-encoded homolog. A comparison with the structure of poxviral topoisomerase in complex with DNA suggests that bacterial homologs of the eukaryotic type IB topoisomerase may exhibit a relaxed sequence preference due to the lack of conservation of certain residues involved in sequence-specific DNA contacts, and that interaction with an only modestly preferred sequence may result in suboptimal positioning of catalytic residues.
PMCID: PMC2666729  PMID: 19317906
18.  Dihydrobetulinic Acid Induces Apoptosis in Leishmania donovani by Targeting DNA Topoisomerase I and II: Implications in Antileishmanial Therapy 
Molecular Medicine  2003;9(1-2):26-36.
Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 μM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.
PMCID: PMC1430381  PMID: 12765337
19.  Aberrant DNA topoisomerase II activity, radioresistance and inherited susceptibility to cancer. 
British Journal of Cancer  1991;63(1):29-36.
Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.
PMCID: PMC1971654  PMID: 1846552
20.  Use of in vitro topoisomerase II assays for studying quinolone antibacterial agents. 
Antimicrobial Agents and Chemotherapy  1989;33(10):1697-1703.
Several quinolones and antitumor compounds were tested as inhibitors of purified calf thymus topoisomerase II in unknotting, catenation, radiolabeled DNA cleavage, and quantitative nonradiolabeled cleavage assays. The antitumor agents VP-16 (demethylepipodophyllotoxin ethylio-beta-D-glucoside) and ellipticine demonstrated drug-enhanced topoisomerase II DNA cleavage (the concentration of drug that induced 50% of the maximal DNA cleavage in the test system [CC50]) at levels of less than or equal to 5 micrograms/ml. Nalidixic acid, norfloxacin, and oxolinic acid did not induce significant topoisomerase II DNA cleavage, whereas ciprofloxacin did induce some cleavage above background levels. CP-67,015, a new 6,8-difluoro-7-pyridyl 4-quinolone which possesses potent antibacterial activity, inhibited bacterial DNA gyrase at 0.125 micrograms/ml in a nonradioactive DNA cleavage assay. Unlike other quinolones characterized to date, CP-67,015 was shown to strongly enhance topoisomerase II-induced radiolabeled DNA cleavage with a CC50 of 33 micrograms/ml and demonstrated cleavage in a nonradiolabeled DNA cleavage assay with a CC50 of 73 micrograms/ml. The topoisomerase II-mediated cleavage of DNA by CP-67,015 is consistent with its reported clastogenic effect on DNA in cell culture and its positive mutagenic response in mouse lymphoma cells. In vitro topoisomerase II catalytic and cleavage assays are useful for gaining preliminary information concerning the possible interaction(s) of some quinolones with eucaryotic topoisomerase II which may relate directly to their safety (mutagenicity, clastogenicity, or both) in human and veterinary medicinal usage.
PMCID: PMC172740  PMID: 2556075
21.  Crystal Structures of Escherichia coli Topoisomerase IV ParE Subunit (24 and 43 Kilodaltons): a Single Residue Dictates Differences in Novobiocin Potency against Topoisomerase IV and DNA Gyrase 
Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Å resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Å resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 μM). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP Km increased ≈5-fold and the Vmax decreased ≈30%. In contrast, the topoisomerase IV ATP Km decreased by a factor of 6, and the Vmax increased ≈2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.
PMCID: PMC400558  PMID: 15105144
22.  Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases. 
Antimicrobial Agents and Chemotherapy  1996;40(12):2714-2720.
Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.
PMCID: PMC163609  PMID: 9124828
23.  Differential induction of Leishmania donovani bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I 
Nucleic Acids Research  2006;34(4):1121-1132.
Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.
PMCID: PMC1373691  PMID: 16488884
24.  Dual Role of Topoisomerase II in Centromere Resolution and Aurora B Activity 
PLoS Biology  2008;6(8):e207.
Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.
Author Summary
Successful cell division requires that chromosomes are properly condensed and that each sister chromatid is self-contained by the time the sister pairs are segregated into separate daughter cells. It is also essential that the kinetochores at the centromeres of each pair of sister chromatids bind microtubules from opposite spindle poles. Topoisomerase II is a highly conserved enzyme that removes interlinks from DNA and is known to be essential to proper chromosome segregation during cell division. In this work, we have used state-of-the-art four-dimensional fluorescent microscopy to follow progression through mitosis in living cells depleted of topoisomerase II. We find that when the enzyme is absent, the two sister centromeres do not separate, and chromosomes missegregate. Moreover, the inappropriate centromere structure that results prevents the correct activation of the Aurora B kinase, which forms part of a regulatory mechanism that monitors correct segregation of chromosomes; as a result, cells exit mitosis abnormally.
Analysis of cells lacking topoisomerase II reveals that the enzyme has an essential role in the segregation of chromosomes, and specifically centromeres, at anaphase-telophase of mitosis: it prevents non-disjunction and allows activation of the Aurora B kinase, so as to correct improper attachments between microtubules and the kinetochore.
PMCID: PMC2525683  PMID: 18752348
25.  Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. 
Journal of Virology  1984;49(1):1-8.
Vaccinia virus cores contain a type I topoisomerase which promotes the relaxation of superhelical DNA of either handedness (Bauer et al., Proc. Natl. Acad. Sci. U.S.A. 74:1841-1845, 1977). The activity of partially purified vaccinia virus topoisomerase (VV-Topo I) was determined in the presence of ATP, dATP, GTP, ADP, and ATP analogs in which hydrolysis of the alpha, beta or beta, gamma phosphate bond is restricted. Topoisomerase activity was stimulated 2.5-fold by the addition of 2 to 4 mM ATP or dATP to standard assay mixtures; 2 mM GTP produced no significant effect on enzyme activity. The addition of 2 mM beta, gamma-imido ATP or 2 mM gamma-thiophosphate ATP reduced VV-Topo I activity by 80 and 65%, respectively. In contrast, 4 mM alpha, beta-methylene ATP produced no significant change in topoisomerase activity compared to ATP itself. Assays performed in the presence of 4 mM ADP exhibited an 80% reduction in enzyme activity. The preparations of VV-Topo I used for these studies showed, however, no detectable DNA-dependent or -independent ATPase activity. The activity of VV-Topo I was similarly measured in the presence of the antibiotics novobiocin and coumermycin A1, which inhibited enzyme activity by 50% at concentrations of 180 and 40 microM, respectively. Comparable inhibition of VV-Topo I activity was observed in the presence of 1 mM beta, gamma-imido ATP. We determined that novobiocin inhibits vaccinia core transcription at the same concentrations which inhibit vaccinia core topoisomerase I activity. These results suggest that the vaccinia DNA topoisomerase may play a role in the ATP-dependent transcription of viral genes from intact core particles.
PMCID: PMC255417  PMID: 6317884

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