Human Topoisomerase IIalpha Research Articles

Tumor growth-arrest effect of tetrahydroquinazoline-derivative human topoisomerase II-alpha inhibitor in HPV-negative head and neck squamous cell carcinoma

Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. There is a persisting demand for new approaches to establish more efficient strategies for their treatment. In this regard, the human topoisomerase II (topoII) enzyme is a validated chemotherapeutics target, as topoII regulates vital cellular processes such as DNA replication, transcription, recombination, and chromosome segregation in cells. TopoII inhibitors are currently used to treat some neoplasms such as breast and small cells lung carcinomas. Additionally, topoII inhibitors are under investigation for the treatment of other cancer types, including oral cancer. Here, we report the therapeutic effect of a tetrahydroquinazoline derivative (named ARN21934) that preferentially inhibits the alpha isoform of human topoII. The treatment efficacy of ARN21934 has been evaluated in 2D cell cultures, 3D in vitro systems, and in chick chorioallantoic membrane cancer models. Overall, this work paves the way for further preclinical developments of ARN21934 and possibly other topoII alpha inhibitors of this promising chemical class as a new chemotherapeutic approach for the treatment of oral neoplasms.

Exploring substituted 3,4-dihydropyrimidinone and thione derivatives as anti-prostate cancer agents: Computational screening, synthesis, characterization, and in vitro efficacy assessment

Despite decades of research, anticancer medication therapy is still largely constrained to prevent growth and limit the spread of tumour cells. To speed up the process of finding anticancer drugs, exploring novel therapeutic macromolecules and identifying the right small molecules using an integration of computational and experimental approaches is a recent tactic adopted by investigators. In the present study, we designed and created a library of 200 ligands with 3,4-dihydropyrimidinones or their thiones linked with imidazolidinones through amide linkage and explored the extent of their in vitro anticancer activity towards human topoisomerase IIalpha (TOP2α or TOP2A) using DU-145 and PC-3 prostate cancer cell lines using the SRB assay method. This was accomplished by the molecular docking and synthesis of the titled compounds using a series of organic nucleophilic addition reactions, including the Biginelli one-pot condensation reaction. The preliminary physical character evaluation was employed for the synthesized compounds, and the structural parameters were analyzed using FTIR, proton NMR, 13C NMR, and LC-MS/MS. The anti-prostate cancer activity of the prepared Biginelli analogues was evaluated against cancer cell lines and mild-to-moderate reduction in the viability of cell lines was observed with compounds 5, 6, 9, 14, 15, 16, 17, and 19. Among them, compounds 5, 14, 15, and 16 exhibited significant potent activity against TOP2α or TOP2A. This result indicated that the derivatives of Biginelli (3,4-dihydropyrimidinones/thiones) could be promising anti-prostate cancer drug candidates.

Nostocine A Derivatives as Human DNA Topoisomerase II-alpha Inhibitor

Abstract: Introduction: Due to heavy morbidity and mortality from cancer, the designing of newer drugable molecules against breast cancer is the call of day. As, Schiff-base sulfonamides have been widely used in tumor treatments. Methods: Nostocine-sulfonamide (NS) Schiff-base molecules were designed with tools of bioinformatics against the target enzyme, human topoisomerase II-alpha (topo IIa) against breast cancer. The designed NS conjugates were assessed by RO5, ADMET and molecular docking. Results: Herein, these analogues, NS-20b (Nostocine A-sulfaphenazole), 12a (Nostocine A-sulfisoxazole) and 16b (Nostocine A-sulfamethazineare) are N-heteroaryl substituted sulfonamide moieties linked with pyrazolo[4,3-e][1,2,4]triazine of Nostocine A. Conclusion: These derivatives would act as potent inhibitors of topo IIa for breast cancer. Key words: Pyrazolotriazine, Nostocine A, Cyanobacterium, Nostoc spongiforium, Breast cancer, Docking.

Studies on Non-synonymous Polymorphisms Altering Human DNA Topoisomerase II-Alpha Interaction with Amsacrine and Mitoxantrone: An In Silico Approach.

DNA topoisomerase II-α (Top2-α), an essential enzyme for the management of DNA during replication, transcription, recombination, and chromatin remodeling, is one of the most important anticancer targets. Numerous molecules have been designed as Top2-α inhibitors. However, several studies have shown that polymorphisms and mutations in Top2 have conferred resistance to most of these anticancer drugs. The aim of this study was to computationally examine the mechanisms by which genomic variations in Top2-α could affect its resistance to Amsacrine and Mitoxantrone as important inhibitors of the enzyme. The results showed that variants K529E, R568H, R568G and T530M could affect Top2-α inhibition by Amsacrine causing possible drug-resistant. Moreover, R487K, and Y481C variants could change the response of the enzyme to Mitoxantrone. These results could facilitate the prediction and development of more effective drugs for Top2-α variants, making the cancer chemotherapy more effectiv.

Carbonic anhydrase IX is a predictive marker of doxorubicin resistance in early-stage breast cancer independent of HER2 and TOP2A amplification

Background:In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin.Methods:We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification.Results:Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m−2 of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio.Conclusion:We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.

Metal Ion Interactions in the DNA Cleavage/Ligation Active Site of Human Topoisomerase IIα

Human topoisomerase IIalpha utilizes a two-metal-ion mechanism for DNA cleavage. One of the metal ions (M(1)(2+)) is believed to make a critical interaction with the 3'-bridging atom of the scissile phosphate, while the other (M(2)(2+)) is believed to interact with a nonbridging oxygen of the scissile phosphate. Based on structural and mutagenesis studies of prokaryotic nucleic acid enzymes, it has been proposed that the active site divalent metal ions interact with type II topoisomerases through a series of conserved acidic amino acid residues. The homologous residues in human topoisomerase IIalpha are E461, D541, D543, and D545. To address the validity of these assignments and to delineate interactions between individual amino acids and M(1)(2+) and M(2)(2+), we individually mutated each of these acidic amino acid residues in topoisomerase IIalpha to either cysteine or alanine. Mutant enzymes displayed a marked loss of catalytic and DNA cleavage activity as well as a reduced affinity for divalent metal ions. Additional experiments determined the ability of wild-type and mutant topoisomerase IIalpha enzymes to cleave an oligonucleotide substrate that contained a sulfur atom in place of the 3'-bridging oxygen of the scissile phosphate in the presence of Mg2+, Mn2+, or Ca2+. On the basis of the results of these studies, we conclude that the four acidic amino acid residues interact with metal ions in the DNA cleavage/ligation active site of topoisomerase IIalpha. Furthermore, we propose that M(1)(2+) interacts with E461, D543, and D545 and M(2)(2+) interacts with E461 and D541.

Topoisomerase II α Status in Renal Medullary Carcinoma: Immuno-Expression and Gene Copy Alterations of a Potential Target of Therapy

Renal medullary carcinoma is an aggressive renal neoplasm without currently available effective therapy to our knowledge. Topoisomerase II alpha is a gyrase involved in cell proliferation, and DNA maintenance and repair. Topoisomerase II alpha is a target of inhibiting agents such as anthracyclines. Triggered by a recent response to topoisomerase II alpha inhibitors in a patient with renal medullary carcinoma, we evaluated topoisomerase II alpha expression in relation to the proliferation index and topoisomerase II alpha gene copy number status in a larger series of patients with renal medullary carcinoma. Archival tissues from 14 renal medullary carcinomas were retrieved from our 3 institutions. Immunohistochemistry was performed using monoclonal antibodies for topoisomerase II alpha and Ki67. The percent of cells with positive nuclear staining was assessed in the highest area of expression for each marker. A previously suggested greater than 5% cutoff was used for topoisomerase II alpha over expression. The topoisomerase II alpha gene copy number was evaluated using fluorescence in situ hybridization. Locus specific topoisomerase II alpha gene and chromosome 17 centromere probes were used. The total number of topoisomerase II alpha and chromosome 17 centromere signals was counted in 150 cells per tumor and a topoisomerase II alpha-to-chromosome 17 centromere signal ratio was calculated in each tumor. A topoisomerase II alpha-to-chromosome 17 centromere ratio of 2.0 or greater and less than 0.8 was used as a cutoff for amplification and deletion, respectively. The percent of tumor cells with polysomic, eusomic or monosomic chromosome 17 status was also determined. On immuno-expression analysis topoisomerase II alpha immunohistochemistry was technically inconclusive in 1 renal medullary carcinoma. Topoisomerase II alpha was over expressed in 11 of 13 renal medullary carcinomas (85%) (median 50%, range 1% to 80%). As expected, a high Ki67 proliferation index was noted in 13 of 14 tumors (median 87.5%, range 2% to 100%). Ki67 expression was greater than topoisomerase II alpha expression in all 13 informative tumors. A strong, statistically significant correlation was found for topoisomerase II alpha and Ki67 expression (pairwise CC 0.9, p = 0.0000). Topoisomerase II alpha over expression was associated with shorter survival (p = 0.000). On fluorescence in situ hybridization no topoisomerase II alpha amplification was detected in any of the 14 renal medullary carcinomas, including the 11 with topoisomerase II alpha over expression. Topoisomerase II alpha gene deletions were noted in 4 tumors. Two of 4 deletions were associated with chromosome 17 monosomy and 2 were in eusomic chromosome 17 tumors. Topoisomerase II alpha is over expressed in 85% of renal medullary carcinomas, potentially supporting the use of topoisomerase II alpha inhibitor agents to treat this aggressive renal tumor. Our findings suggest that topoisomerase II alpha over expression in our renal medullary carcinoma cohort was not due to gene amplification, but rather to transcriptional or post-transcriptional modifications. The significance of the incidentally found topoisomerase II alpha deletions in 28% of renal medullary carcinomas requires further evaluation.

The QTK Loop Is Essential for the Communication between the N-Terminal ATPase Domain and the Central Cleavage−Ligation Region in Human Topoisomerase IIα

We have characterized a human topoisomerase IIalpha enzyme with a deletion of the conserved QTK loop, which extends from the transducer domain to the ATP-binding pocket in the GHKL domain. The loop has been suggested to play a role for interdomain communication in type II topoisomerases. The mutant enzyme performs only very low levels of strand passage, although it is able to cleave and ligate DNA as well as close the N-terminal clamp. Cleavage is nearly unaffected by ATP and ATP analogues relative to the wild-type enzyme. Although the enzyme is able to close the clamp, the clamp has altered characteristics, allowing trapping of DNA also in the absence of an ATP analogue. The enzyme furthermore retains intrinsic levels of ATPase activity, but the activity is not stimulated by DNA. Our observations demonstrate that the QTK loop is an important player for the interdomain communication in human topoisomerase IIalpha. First, the loop seems to play a role in keeping the N-terminal clamp in an open conformation when no nucleotide is present. Once the nucleotide binds, it facilitates clamp closure, although it is not essential for this event. The QTK loop, in contrast, is essential for the DNA-stimulated ATPase activity of human topoisomerase IIalpha.

Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases

All type II topoisomerases require divalent metal ions to cleave and ligate DNA. To further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIbeta was characterized and compared to that of topoisomerase IIalpha. This study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3'-bridging oxygen or the nonbridging oxygens of the scissile phosphate. On the basis of time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal ion mechanism to support DNA cleavage. Second, an interaction between one divalent metal ion and the 3'-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3'-oxygen. Third, there is an important interaction between a divalent second metal ion and a nonbridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIbeta. If this interaction exists in topoisomerase IIalpha, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIalpha and IIbeta.

In silico studies with human DNA topoisomerase-II alpha to unravel the mechanism of in vitro genotoxicity of benzene and its metabolites

Exposure of humans to benzene present in environment may lead to adverse chronic effects—even at the genetic level. However, the mechanism of its genotoxicity is not well understood. In the present study, in vitro genotoxicity of benzene (BZ) and its major metabolites [p-benzoquinone (BQ), hydroquinone (HQ), catechol (CT), 1,2,4-benzenetriol (BT) and trans–trans muconic acid (MA)] at concentrations 0.5–50 μM, was assessed in Chinese hamster ovary (CHO) cells employing the alkaline Comet assay, cytokinesis blocked micronucleus (CBMN) assay, flow cytometric analysis of micronucleus (flow MN) and chromosome aberration (CA) test. The data revealed significant ( P < 0.05) concentration-dependent response in all end points. HQ was found to be the most potent DNA damaging metabolite in the Comet assay followed by BQ > BT > CT > BZ > MA. Both CBMN and flow MN assays revealed a good correlation in their results, where BQ and MA exhibited maximum and minimum micronucleus induction respectively. Significant chromosomal aberrations were induced mainly by BQ, BT and HQ, with moderate response shown by CT and BZ and least by MA. The results demonstrated the utility of sensitive techniques like Comet assay and flow cytometry for determination of MN, to quantify in vitro genotoxicity at low levels and also suggested that partly non-repaired DNA damage could cause adverse health effects in human population exposed to benzene. In silico studies using different endpoints of genotoxicity and molecular docking studies with human topoisomerase-II alpha, a major DNA repair enzyme were also conducted. These corroborated the results obtained from the in vitro data, pointing to a direct relationship of the observed genotoxicity with the structural properties and various interactions of metabolites with the enzyme. This comprehensive study demonstrated that genotoxicity of benzene in mammalian cells is mainly due to the inhibition of topoisomerase by the metabolites.

The Efficacy of Topoisomerase II-Targeted Anticancer Agents Reflects the Persistence of Drug-Induced Cleavage Complexes in Cells

Genistein, a widely consumed bioflavonoid with chemopreventative properties in adults, and etoposide, a commonly prescribed anticancer drug, are well-characterized topoisomerase II poisons. Although both compounds display similar potencies against human topoisomerase IIalpha and IIbeta in vitro and induce comparable levels of DNA cleavage complexes in cultured human cells, their cytotoxic and genotoxic effects differ significantly. As determined by assays that monitored cell viability or the phosphorylation of histone H2AX, etoposide was much more toxic in CEM cells than genistein. Further studies that characterized the simultaneous treatment of cells with genistein and etoposide indicate that the differential actions of the two compounds are not related to the effects of genistein on cellular processes outside of its activity against topoisomerase II. Rather, they appear to result from a longer persistence of cleavage complexes induced by etoposide as compared to genistein. Parallel in vitro studies with purified type II enzymes led to similar conclusions regarding cleavage complex persistence. Isoform-specific differences were observed in vitro and in cells treated with etoposide. To this point, the t 1/2 of etoposide-induced DNA cleavage complexes formed with topoisomerase IIalpha in CEM cells was approximately 5 times longer than those formed with topoisomerase IIbeta. The cytotoxicity of etoposide following four treatment-recovery cycles was similar to that induced by continuous exposure to the drug over an equivalent time period. Taken together, these findings suggest that it may be possible to preferentially target topoisomerase IIalpha with etoposide by employing a schedule that utilizes pulsed drug treatment-recovery cycles.

Human topoisomerase II uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen.

Dietary Polyphenols as Topoisomerase II Poisons: B Ring and C Ring Substituents Determine the Mechanism of Enzyme-Mediated DNA Cleavage Enhancement

Dietary polyphenols are a diverse and complex group of compounds that are linked to human health. Many of their effects have been attributed to the ability to poison (i.e., enhance DNA cleavage by) topoisomerase II. Polyphenols act against the enzyme by at least two different mechanisms. Some compounds are traditional, redox-independent topoisomerase II poisons, interacting with the enzyme in a noncovalent manner. Conversely, others enhance DNA cleavage in a redox-dependent manner that requires covalent adduction to topoisomerase II. Unfortunately, the structural elements that dictate the mechanism by which polyphenols poison topoisomerase II have not been identified. To resolve this issue, the activities of two classes of polyphenols against human topoisomerase IIalpha were examined. The first class was a catechin series, including (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC). The second was a flavonol series, including myricetin, quercetin, and kaempferol. Compounds were categorized into four distinct groups: EGCG and EGC were redox-dependent topoisomerase II poisons, kaempferol and quercetin were traditional poisons, myricetin utilized both mechanisms, and ECG and EC displayed no significant activity. On the basis of these findings, a set of rules is proposed that predicts the mechanism of bioflavonoid action against topoisomerase II. The first rule centers on the B ring. While the C4'-OH is critical for the compound to act as a traditional poison, the addition of -OH groups at C3' and C5' increases the redox activity of the B ring and allows the compound to act as a redox-dependent poison. The second rule centers on the C ring. The structure of the C ring in the flavonols is aromatic and planar and includes a C4-keto group that allows the formation of a proposed pseudo ring with the C5-OH. Disruption of these elements abrogates enzyme binding and precludes the ability to function as a traditional topoisomerase II poison.

Substituents on Etoposide That Interact with Human Topoisomerase IIα in the Binary Enzyme−Drug Complex: Contributions to Etoposide Binding and Activity

Etoposide is a widely prescribed anticancer agent that stabilizes topoisomerase II-mediated DNA strand breaks. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. A recent study that focused on yeast topoisomerase II demonstrated that the H15 geminal protons of the etoposide A-ring, the H5 and H8 protons of the B-ring, and the H2', H6', 3'-methoxyl, and 5'-methoxyl protons of the E-ring contact topoisomerase II in the binary enzyme-drug complex [ Wilstermann et al. (2007) Biochemistry 46, 8217-8225 ]. No interactions with the C4 sugar were observed. The present study used DNA cleavage assays, saturation transfer difference [ (1)H] NMR spectroscopy, and enzyme-drug binding studies to further define interactions between etoposide and human topoisomerase IIalpha. Etoposide and three derivatives that lacked the C4 sugar were analyzed. Except for the sugar, 4'-demethyl epipodophyllotoxin is identical to etoposide, epipodophyllotoxin contains a 4'-methoxyl group on the E-ring, and 6,7- O, O-demethylenepipodophyllotoxin replaces the A-ring with a diol. Results suggest that etoposide-topoisomerase IIalpha binding is driven by interactions with the A- and B-rings and potentially by stacking interactions with the E-ring. We propose that the E-ring pocket on the enzyme is confined, because the addition of bulk to this ring adversely affects drug function. The A- and E-rings do not appear to contact DNA in the enzyme-drug-DNA complex. Conversely, the sugar moiety subtly alters DNA interactions. The identification of etoposide substituents that contact topoisomerase IIalpha in the binary complex has predictive value for drug behavior in the enzyme-etoposide-DNA complex.

Mechanism of Inhibition of DNA Gyrase by ES‐1273, a Novel DNA Gyrase Inhibitor

We investigated the mode of action of ES-1273, a novel DNA gyrase inhibitor obtained by optimization of ES-0615, which was found by screening our chemical library using anucleate cell blue assay. ES-1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone- and coumarin-resistance as that against a susceptible strain. In addition, ES-1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES-1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES-1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES-1273. ES-1273 antagonized quinolone-induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase-DNA complex was observed in the presence of ES-1273. Taken together, these results indicate that ES-1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES-1273 interacts with DNA. Therefore, the interaction between ES-1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES-1273 inhibits topoisomerase IV and human topoisomerase IIalpha, but not human topoisomerase I. These findings indicate that ES-1273 is a type II topoisomerase specific inhibitor.

HMGB1 interacts with human topoisomerase IIalpha and stimulates its catalytic activity.

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme and its unique decatenation activity has been implicated in many aspects of chromosome dynamics such as chromosome replication and segregation during mitosis. Here we show that chromatin-associated protein HMGB1 (a member of the large family of HMG-box proteins with possible functions in DNA replication, transcription, recombination and DNA repair) promotes topo IIα-mediated catenation of circular DNA, relaxation of negatively supercoiled DNA and decatenation of kinetoplast DNA. HMGB1 interacts with topo IIα and this interaction, like the stimulation of the catalytic activity of the enzyme, requires both HMG-box domains of HMGB1. A mutant of HMGB1, which cannot change DNA topology stimulates DNA decatenation by topo IIα indistinguishably from the wild-type protein. Although HMGB1 stimulates ATP hydrolysis by topo IIα, the DNA cleavage is much more enhanced. The observed abilities of HMGB1 to interact with topo IIα and promote topo IIα binding to DNA suggest a mechanism by which HMGB1 stimulates the catalytic activity of the enzyme via enhancement of DNA cleavage.

Bioflavonoids as Poisons of Human Topoisomerase IIα and IIβ

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.

Quinone-Induced Enhancement of DNA Cleavage by Human Topoisomerase IIα: Adduction of Cysteine Residues 392 and 405

Several quinone-based metabolites of drugs and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein and appear to increase levels of enzyme-DNA cleavage complexes by at least two potentially independent mechanisms. Treatment of topoisomerase IIalpha with quinones inhibits DNA religation and blocks the N-terminal gate of the protein by cross-linking its two protomer subunits. It is not known whether these two effects result from adduction of quinone to the same amino acid residue(s) in topoisomerase IIalpha or whether they are mediated by modification of separate residues. Therefore, this study identified amino acid residues in human topoisomerase IIalpha that are modified by quinones and determined their role in the actions of these compounds as topoisomerase II poisons. Four cysteine residues were identified by mass spectrometry as sites of quinone adduction: Cys170, Cys392, Cys405, and Cys455. Mutations (Cys --> Ala) were individually generated at each position. Only mutations at Cys392 or Cys405 reduced sensitivity ( approximately 50% resistance) to benzoquinone. Top2alphaC392A and top2alphaC405A displayed faster rates ( approximately 2-fold) of DNA religation than wild-type topoisomerase IIalpha in the presence of the quinone. In contrast, as determined by DNA binding, protein clamp closing, and protomer cross-linking experiments, mutations at Cys392 and Cys405 did not affect the ability of benzoquinone to block the N-terminal gate of topoisomerase IIalpha. These findings indicate that adduction of Cys392 and Cys405 is important for the actions of quinones against the enzyme and increases levels of cleavage complexes primarily by inhibiting DNA religation.

Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: bimodal recognition of DNA geometry by type II enzymes.

Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIalpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIalpha and topoisomerase IIbeta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage.

A Three-Dimensional Quantitative Structure-Activity Relationship Study of the Inhibition of the ATPase Activity and the Strand Passing Catalytic Activity of Topoisomerase IIα by Substituted Purine Analogs

Based on the topoisomerase IIalpha catalytic inhibitory activity of a previous hit compound, NSC35866, we screened 40 substituted purines or purine-like compounds from the National Cancer Institute repository for their ability to inhibit the ATPase activity of human topoisomerase IIalpha. Several compounds, including NSC348400, NSC348401 and NSC348402, were inhibitory at submicromolar concentrations. Three-dimensional quantitative structure-activity relationship models using comparative molecular field and comparative molecular similarity indices analyses were constructed using 24 of these compounds. The ability of 10 selected compounds to inhibit the complete DNA strand passage reaction of topoisomerase IIalpha correlated well with their potency as ATPase inhibitors. None of the 40 compounds significantly increased levels of the topoisomerase IIalpha-DNA covalent complex, suggesting that they functioned as catalytic topoisomerase II inhibitors and not as topoisomerase II poisons. Although some of these compounds could antagonize the effect of etoposide on the level of topoisomerase IIalpha-DNA covalent complex formation in vitro, in contrast to NSC35866, they were not capable of antagonizing etoposide-induced cytotoxicity and DNA strand breaks in cells. Two independently selected human SCLC cell lines with reduced topoisomerase IIalpha expression displayed cross-resistance to NSC348400, NBSC348401, and NSC348402, whereas an MDR1 line was fully sensitive. These results suggest that topoisomerase IIalpha is a functional cellular target for most of these substituted purine compounds and that these compounds do not display MDR1 liability.