Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ciprofloxacin is an important and commonly used member of the fluoroquinolone group of antibiotics. Ciprofloxacin inhibits DNA topoisomerase II and DNA topoisomerase IV activities, eventually leading to bacterial cell death. In addition, an increase of reactive oxygen species in the bacterial cells in response to ciprofloxacin has been shown. We investigated the role of reactive oxygen species in the antibacterial action of ciprofloxacin by studying the effects of different antioxidant compounds on ciprofloxacin susceptibility of Escherichia coli. Among the antioxidants checked, glutathione and ascorbic acid provided substantial protection against ciprofloxacin. The involvement of superoxide anion (O2-) and hydrogen peroxide (H2O2) in the antibacterial action of ciprofloxacin was analyzed using superoxide dismutase, catalase, and alkyl hydroperoxide reductase knockout strains of E. coli. The effects of multicopy sod genes on ciprofloxacin susceptibility of E. coli were also analyzed. On the basis of our results, we conclude that O2- and H2O2 may be involved in antibacterial action of ciprofloxacin. Our findings that glutathione gave protection against other fluoroquinolones and not against nonfluoroquinolone antibiotics imply that reactive oxygen species may have a similar role in the antibacterial action of all these fluoroquinolones and that glutathione-mediated protection is not a general phenomenon but specific to fluoroquinolones. These observations are of significance, as fluoroquinolones are important antibiotics with immense therapeutic value, and the effectiveness of treatment by these drugs may be affected by dietary intake and cellular levels of these antioxidants.
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PMID:Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli. 1649 56

Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase-DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.
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PMID:Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I. 1658 4

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Since we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts, this anticancer drug is considered to function as a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its enzymatic activation in target tissues. Here, we demonstrate that ellipticine is also oxidized by peroxidases, which are abundantly expressed in several target tumor tissues. Lactoperoxidase, myeloperoxidase and horseradish peroxidase were used as models. Peroxidases in the presence of hydrogen peroxide oxidize ellipticine to an ellipticine dimer and N(2)-oxide of ellipticine as the major and minor metabolite, respectively. Inhibition of the peroxidase-mediated ellipticine oxidation by radical scavengers ascorbate, glutathione and NADH suggests a one-electron mechanism of the oxidation. The implication of the oxidation of ellipticine by peroxidases in its mechanism of action is discussed.
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PMID:Oxidation of an antitumor drug ellipticine by peroxidases. 1660 8

The 14-3-3 protein family consists of seven isoforms, most of which are expressed abundantly in neurons and glial cells, although the sigma isoform, a p53 target gene originally identified as an epithelium-specific marker, has not been identified in the human central nervous system. Here, we show that human astrocytes in culture expressed 14-3-3sigma under stress conditions. By Western blot, the expression of 14-3-3sigma, p53 and p21 was coordinately upregulated in astrocytes following exposure to hydrogen peroxide, 4-hydroxy-2-nonenal (4-HNE) or etoposide, a topoisomerase II inhibitor. 14-3-3sigma was induced by treatment with 5-aza-2'-deoxycytidine, suggesting a hypermethylated status of the gene promoter in astrocytes. In vivo, a small subset of hypertrophic reactive astrocytes, often showing a multinucleated morphology, expressed 14-3-3sigma in active demyelinating lesions of multiple sclerosis (MS) and ischemic lesions of cerebral infarction, where the expression of 4-HNE and 8-hydroxy-2'-deoxyguanosine was enhanced in reactive astrocytes. Microarray analysis of etoposide-treated astrocytes verified upregulation of p53-responsive genes and concurrent downregulation of mitotic checkpoint-regulatory genes. These observations suggest that 14-3-3sigma might serve as a marker of oxidative and DNA-damaging stresses inducing the mitotic checkpoint dysfunction in reactive astrocytes under pathological conditions.
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PMID:Human astrocytes express 14-3-3 sigma in response to oxidative and DNA-damaging stresses. 1679 59

A series of 13 anthrapyrazole compounds that are analogues of piroxantrone and losoxantrone were synthesized, and their cell growth inhibitory effects, DNA binding, topoisomerase IIalpha mediated (EC 5.99.1.3) cleavage of DNA, and inhibition of DNA topoisomerase IIalpha decatenation catalytic activities were determined. Cell growth inhibitory activity was well-correlated with DNA binding, suggesting that these compounds may act by targeting DNA. However, cell growth inhibition was not well-correlated with the inhibition of topoisomerase IIalpha catalytic activity, suggesting that these anthrapyrazoles did not act solely by inhibiting the catalytic activity of topoisomerase II. Most of the analogues were able to induce DNA cleavage, and thus, it was concluded that they acted, at least in part, as topoisomerase II poisons. Structure-based three-dimensional quantitative structure-activity analyses (3D-QSAR) were carried out on the aligned structures of the anthrapyrazoles docked into DNA using comparative molecular field analysis (CoMFA) and comparative molecular similarity index (CoMSIA) analyses in order to determine the structural features responsible for their activity. Both CoMFA and CoMSIA yielded statistically significant models upon partial least-squares analyses. The 3D-QSAR analyses showed that hydrogen-bond donor interactions and electrostatic interactions with the protonated amino side chains of the anthrapyrazoles led to high cell growth inhibitory activity.
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PMID:A structure-based 3D-QSAR study of anthrapyrazole analogues of the anticancer agents losoxantrone and piroxantrone. 1685 14

Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. gamma-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2beta knock-out mouse embryonic fibroblasts and Top2beta small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIalpha was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIalpha-DNA and TopIIbeta-DNA adducts. By contrast, hydrogen peroxide-induced gamma-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage.
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PMID:A protease pathway for the repair of topoisomerase II-DNA covalent complexes. 1697 21

Six new bimetallic complexes of the type CuCu, CuCo, CuNi, CuZn and CuMn were prepared. The structures of these complexes and the ligand have been proposed on the basis of FAB mass, elemental analysis, UV-vis, IR, EPR and CV studies. All the complexes completely cleave pBS (SK-) DNA at a concentration of 10 microM; however, even at lower concentrations of 2 microM and 0.1 microM, the complexes (I and Ia) showed partial cleavage. The results of the fluorescence binding studies of the metal complexes with CT-DNA showed that the presence of aliphatic ligands added additional binding effects including electrostatic, hydrogen binding and vander Waals interactions. Complexes (I, Ia) showed 50% inhibition of COX-1 and COX-2 activities at as low a concentration as 12.5 microM, 13.5 microM, 14 microM and 14.5 microM. Inhibition assay of top I and top II by different complexes in mutant yeast strains (JN394, JN394 t(-1) and JN394 t(2-5)) with all the complexes showed significant inhibition of topoisomerase at 5 microM concentration. Complexes I and Ia exhibited good anti-microbial activities against all human pathogens tested except Klebsiella pneumoniae. The following studies showed that among the synthesized bimetallic complexes, complexes I and Ia seem to be promising candidates possessing DNA cleavage activities besides anti-microbial and anti-inflammatory properties to serve as chemical nucleases and chemotherapeutic agents.
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PMID:Synthesis, characterization and in vitro biological activity studies of Cu-M (M = Cu2+, Co2+, Ni2+, Mn2+, Zn2+) bimetallic complexes. 1701 70

Vaccinia DNA topoisomerase (vTopo) catalyzes highly specific nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5C4+C3+T+2T+1p \N-1 using an active-site tyrosine nucleophile, thereby expelling a 5' hydroxyl leaving group of the DNA. Here, we report the energetic effects of subtle modifications to the major-groove hydrogen-bond donor and acceptor groups of the 3'-GGGAA-5' consensus sequence of the nonscissile strand in the context of duplexes in which the scissile strand length was progressively shortened. We find that the major-groove substitutions become energetically more damaging as the scissile strand is shortened from 32 to 24 and 18 nucleotides, indicating that enzyme interactions with the duplex region present in the 32-mer but not the 24- or 18-mer weaken specific interactions with the DNA major groove. Regardless of strand length, the destabilizing effects of the major-groove substitutions increase as the reaction proceeds from the Michaelis complex to the transition state for DNA cleavage and, finally, to the phosphotyrosine-DNA covalent complex. These length-dependent anticooperative interactions involving the DNA major groove and duplex regions 3' to the cleavage site indicate that the major-groove binding energy is fully realized late during the reaction for full-length substrates but that smaller more flexible duplex substrates feel these interactions earlier along the reaction coordinate. Such anticooperative binding interactions may play a role in strand exchange and supercoil unwinding activities of the enzyme.
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PMID:Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I. 1719 89

Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3' damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu(96) (changed to Gln) and Asp(210) (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue beta-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.
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PMID:A dominant-negative form of the major human abasic endonuclease enhances cellular sensitivity to laboratory and clinical DNA-damaging agents. 1725 46

Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.
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PMID:Topoisomerase IIbeta mediated DNA double-strand breaks: implications in doxorubicin cardiotoxicity and prevention by dexrazoxane. 1787 25


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