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)

Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.
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PMID:Biochemistry of topoisomerase I and II inhibition by anthracenyl-amino acid conjugates. 759 37

We tested the potential impact of tyrosine phosphorylation on the expression of the c-myc gene in two colon cancer cell lines, HCT8 and SW837. We found that the protein tyrosine kinase inhibitor genistein causes a decrease in the abundance of c-myc RNA and an inhibition of proliferation with a similar dose response. Geldanamycin, a mechanistically different tyrosine kinase inhibitor, also causes a decrease in both the expression of c-myc RNA and proliferation. Genistein has also been found to inhibit topoisomerase II, but the topoisomerase II inhibitor novobiocin did not lower the expression of c-myc. The most likely interpretation is that inhibition of protein tyrosine kinase activity caused a decrease in c-myc expression in these cells. The impact of tyrosine phosphorylation on the expression of the c-myc gene is further supported by the finding that inhibition of phosphotyrosine phosphatase using orthovanadate causes an increase in the level of c-myc RNA. The effect of genistein on HCT8 cells is not dependent on the synthesis of new protein and does not involve an alteration in the stability of the message. Analysis of transcription in the c-myc gene reveals a more complicated picture with a decrease in initiation and an increase in elongation but no net change in transcription. We speculate that the genistein induced reduction in myc expression is the result of a posttranscriptional intranuclear event(s).
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PMID:Influence of protein tyrosine phosphorylation on the expression of the c-myc oncogene in cancer of the large bowel. 764 26

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein.DNA complex at sites containing a conserved sequence element 5'-CCCTT decreases. The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive "hinge." The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino-terminal region is punctuated by a trypsin-sensitive "bridge" at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.
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PMID:Proteolytic footprinting of vaccinia topoisomerase bound to DNA. 774 4

The pH dependences of the internal equilibrium (Kcl) and rate constants for site-specific DNA strand cleavage (kcl) and resealing (kr) catalyzed by Vaccinia DNA topoisomerase I have been investigated using single-turnover conditions in the pH range 4.6-9.8 at 20 degrees C. The pH dependence of the rate constant for strand cleavage (kcl) shows a bell-shaped profile with apparent pKa values of 6.3 +/- 0.2 and 8.4 +/- 0.2, suggesting base catalysis of the attack of the active site Tyr-274 on the phosphodiester phosphorus, and acid catalysis of the expulsion of the 5'-deoxyribose oxygen. A low pKa (i.e., 6.3) for Tyr-274 in the free enzyme is ruled out by NMR titration from pH 5.1 to 8.8 monitoring the C-zeta chemical shift of [zeta-13C]-tyrosine-enriched topoisomerase. The dependence of the internal equilibrium constant (Kcl) on pH reveals very similar pKa values as kcl (5.8 +/- 0.2 and 8.6 +/- 0.2). However, kr is found to be independent of pH. The differing response of kcl and kr to pH rules out a simple two-state internal cleavage equilibrium and suggests that a conformational change occurs following formation of the covalent complex which retains the correct protonation state for strand religation. A conformation step is further indicated by a 4.6-fold "thio effect" on kcl upon substitution of the nonbridging oxygen atom of the attacked phosphoryl group by sulfur [Stivers, J. T., Shuman, S., & Mildvan, A. S. (1994) Biochemistry 33, 327], and the absence of such an effect on kr, (krphos/krthio = 0.9 +/- 0.2), indicating the rates of cleavage and religation to be limited by covalent chemistry and a conformational step, respectively. The rate constant of this conformational change in the direction of religation agrees with the average rate constant for supercoil release from plasmid substrates, suggesting this conformational change to be a part of the topoisomerization step. Although the general acid and general base catalysts have not yet been identified, the quantitative roles of these and other residues in catalysis are discussed.
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PMID:Vaccinia DNA topoisomerase I: kinetic evidence for general acid-base catalysis and a conformational step. 780 9

A mutant yeast type II topoisomerase was generated by in vitro mutagenesis followed by selection in vivo for resistance to the quinolone CP-115,953. The resulting mutant enzyme had a single point mutation which converted His1012 to Tyr (top2H1012Y). top2H1012Y was overexpressed in yeast, purified, and characterized in vitro. The mutant type II topoisomerase was slightly less active than the wild type enzyme, apparently due to a decreased affinity for DNA. The affinity of the mutant enzyme for ATP was similar to that of wild type topoisomerase II. As determined by DNA cleavage assays, top2H1012Y was resistant to CP-115,953 and etoposide both prior to and following the DNA strand-passage event. In marked contrast, the mutant enzyme displayed wild type sensitivity to amsacrine and was severalfold hypersensitive to ellipticine. A similar pattern of resistance was observed in yeast cells harboring the top2H1012Y allele. Thus, it appears that the mutant type II topoisomerase can distinguish between nonintercalative and intercalative agents. Finally, the His1012-->Tyr mutation defines a potential new drug resistance-conferring region on eukaryotic topoisomerase II.
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PMID:A yeast type II topoisomerase selected for resistance to quinolones. Mutation of histidine 1012 to tyrosine confers resistance to nonintercalative drugs but hypersensitivity to ellipticine. 782 29

Vaccinia virus (VV) and Shope fibroma virus (SFV), representatives of the orthopox and leporipox genera, respectively, encode type I DNA topoisomerases. Here we report that the 957-nt F4R open reading frame of orf virus (OV), a representative of the parapox genus, is predicted to encode a 318-aa protein with extensive homology to these enzymes. The deduced amino acid sequence of F4R has 54.7 and 50.6% identity with the VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The F4R protein was expressed in Escherichia coli under the control of an inducible T7 promoter, partially purified, and shown to be a bona fide type I topoisomerase. Like the VV enzyme, the OV enzyme relaxed negatively supercoiled DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SDS-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PCR used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at nt 677 that was predicted to replace a highly conserved Tyr residue with a Cys. This allele directed the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. Incubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an indistinguishable set of DNA cleavage fragments, although the relative abundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV enzyme, we demonstrated that the OV enzyme also cleaved efficiently immediately downstream of the sequence CCCTT.
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PMID:Identification and characterization of the orf virus type I topoisomerase. 783 75

Although data from epidemiological studies and cancer models suggest that genistein plays an important role in cancer prevention, the biochemical target(s) of genistein action is (are) not known. Genistein is a potent in vitro inhibitor of protein tyrosine kinase (PTK) activity, especially that of the epidermal growth factor receptor (EGF-R), having little effect on serine/threonine kinases. This led to the suggestion that genistein might exert its anti-cancer effects through inhibiting the activity of EGF-R PTK, or other crucial PTK's in vivo. Subsequent studies on intact tumor cell lines demonstrated that EGF-R and other growth factor receptors are able to transmit mitogenic signals in the presence of genistein. In fact, it is difficult to detect decreases in the tyrosine phosphorylation of discrete proteins after genistein treatment. Other mechanisms for the effect of genistein have been suggested from in vitro and cell culture data. Genistein not only inhibits the activity of purified topoisomerase II in vitro, but also leads to the accumulation of protein-associated single strand breaks in whole cells. Genistein also inhibits the production of reactive oxygen species which may lead to tissue damage and DNA modification. Additionally, genistein acts as a weak estrogen, modifies cellular differentiation programs, inhibits angiogenesis. modulates cell cycle events and may precipitate apoptosis. However, few of the above mechanisms in tumor cells are sensitive to the physiological serum concentrations of genistein (< 18.5 mumol/L, or < 5 micrograms/mL). Primary, nontransformed human mammary epithelial cells, which have a much greater sensitivity to genistein, would be a better system for the study of these mechanisms.
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PMID:Evaluation of the biochemical targets of genistein in tumor cells. 788 65

The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex. In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance. To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons. Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells. This correlated with a significant reduction in drug-induced DNA cleavage in vitro. However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II. The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin. In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin. These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.
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PMID:A camptothecin-resistant DNA topoisomerase I mutant exhibits altered sensitivities to other DNA topoisomerase poisons. 789 Jul 48

We have already established a human leukemia sub-line resistant to the growth-inhibitory effect of TPA (12-O-tetradecanoylphorbol 13-acetate) (K562/TPA) derived from K562. K562/TPA was found to be a non-P-glycoprotein-mediated multidrug-resistant cell line, in which intracellular drug accumulation was not reduced. In K562/TPA, adriamycin (ADM) was distributed mainly in the cytoplasm and was scarcely observed in the nucleus. We determined the relative levels of multidrug-resistance-associated protein (MRP), which was recently identified as the novel transporter. The relative levels of MRP in K562/TPA were the same as in K562. Although the catalytic activity of K562/TPA topoisomerase II was about half that of the parental cells, resistance to other drugs could not be explained by topoisomerase-II activity. To elucidate the mechanism of drug resistance in K562/TPA, we tried to find chemicals that would reverse the drug resistance. Tyrosine-kinase inhibitors enhanced the cytotoxicity of anti-neoplastic drugs against K562/TPA. Therefore we examined the modification of nuclear ADM accumulation in K562/TPA by one of these tyrosine-kinase inhibitors, genistein. Although the amount of ADM was decreased in the nuclei of K562/TPA cells, it was significantly increased after incubation in the presence of genistein. The formation of DNA single-strand breaks by ADM, etoposide, and ACNU was significantly lower in K562/TPA than in K562, but was significantly increased in the presence of genistein. These results suggest that genistein could overcome drug resistance by enhancing the accumulation of drug into the nuclear fraction of K562/TPA.
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PMID:Reversal of multidrug resistance by tyrosine-kinase inhibitors in a non-P-glycoprotein-mediated multidrug-resistant cell line. 790 94

Stabilization of the topoisomerase-cleavable complexes is the common initial event leading to the cytotoxicity of topoisomerase I and II (top1 and top2) inhibitors. Using saintopin (STP), a poison of both topoisomerases, we studied top1- and top2-cleavable complexes (Yamashita, Y., Kawada, S.-Z., Fujii, N., and Nakano, H. (1991) Biochemistry 30, 5838-5845). top1 and top2 sites induced in the presence of STP showed the same preferences for the base located 3' to the topoisomerase-induced DNA break (position +1): preference for G and not C. A camptothecin-resistant top1 with a mutation (Asn722-->Ser) next to the catalytic tyrosine (Tyr723) was cross-resistant to STP, suggesting that both STP and camptothecin interact with the protein near the catalytic tyrosine. These results are consistent with a dual interaction of the drug with the enzyme and the DNA and provide further evidence for the "drug-stacking" model. This model proposes that topoisomerase inhibitors bind, possibly through hydrogen bonding and/or stacking, with one of the bases flanking the DNA termini (guanine at position +1 in the case of STP) and within the enzyme catalytic pocket, most likely by stacking with the catalytic tyrosine.
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PMID:Saintopin, a dual inhibitor of DNA topoisomerases I and II, as a probe for drug-enzyme interactions. 796 22


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