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)

By contrast with other DNA minor groove binders, Hoechst 33258 inhibited topoisomerase-mediated activity in intact cells. To determine whether specific structural alterations could modify the topoisomerase reactivity of this drug, a series of analogs of Hoechst 33258 (compound 1) was examined. When the relative DNA binding affinities (Ka) of these agents were determined, compound 1 had the highest Ka while agents with substitutions in either of the benzimidazole moieties showed reduced affinity. Whether these changes in DNA binding correlated with topoisomerase inhibitory potency was next examined. In isolated nuclei, 25 microM of agents 1, 5 and 7 reduced VM-26 induced cross-links by 64, 65 and 83%, compared with 15 to 25% reductions by agents 2, 3, 4 and 6, respectively. The structural modification common to the less active compounds was the substitution of an oxygen for nitrogen at either position 1 or 2. On the basis of these results, agents 1, 2, 3 and 7, representing a range of inhibitory potency, were chosen for further analyses. Cross-link induction by m-AMSA and camptothecin in isolated nuclei, as well as by VM-26 in intact cells, was inhibited to a greater extent by agents 1 and 7 than 2 or 3. Additionally, all four drugs inhibited relaxation of pBR 322 DNA induced by both topoisomerases, although topoisomerase I was 2 to 5-fold more sensitive than topoisomerase II. A linear correlation was observed between the logarithms of the Ka value of compounds 1, 2 and 3 and their IC25 values for both topoisomerases, suggesting a strong dependence on DNA binding affinity for enzyme inhibition. Nevertheless, agent 7, despite having less affinity for calf thymus DNA than 1, was the most potent topoisomerase inhibitor tested in intact cells and in isolated enzyme systems. Thus, retention of nitrogen at positions 1 and 2 as well as the addition of nitrogen at position 16 was associated with increased topoisomerase inhibitory potency.
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PMID:Effects of analogs of the DNA minor groove binder Hoechst 33258 on topoisomerase II and I mediated activities. 137 46

Although the stabilization of topoisomerase II cleavable complexes by etoposide (VP-16) has been recognized to be important for cell killing, the lethal events following the formation of cleavable complexes remain to be elucidated. In an attempt to characterize the biochemical requirements for VP-16-induced cytotoxicity, we examined the effects of calcium depletion in Chinese hamster DC3F cells. Four-hour preincubation in calcium-free medium or in complete medium containing 5 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) protected against the cytotoxicity of VP-16. Under these same conditions, the VP-16-induced DNA single-strand break frequency in calcium-depleted cells remained similar to that of control cells. Cell-cycle analysis and thymidine pulse incorporation indicated that calcium depletion did not alter DNA synthesis and cell cycle distribution. Drug-induced cytotoxicity was restored progressively within 4-8 hr after calcium-depleted cells were refed with calcium-containing medium. Calcium depletion also protected against the cytotoxicity of camptothecin, hyperthermia and, to a lesser extent, nitrogen mustard and gamma radiation in DC3F cells. Similar results were obtained in human colon carcinoma HT-29 cells. Our results suggest that topoisomerase II-mediated DNA breaks are only potentially lethal and that calcium-dependent cellular processes are required for the cytotoxicity of topoisomerase inhibitors.
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PMID:Cell death induced by topoisomerase inhibitors. Role of calcium in mammalian cells. 164 24

Cytotoxic effects and topoisomerase II-mediated DNA breaks induced in vitro by ellipticine derivatives were examined in connection with 1H NMR and circular dichroism (CD) studies on molecular structures and interactions of drugs with DNA. The compounds included four 9-hydroxyellipticine and two 7-hydroxyisoellipticine derivatives. Structure-activity relationships indicated that a change in nitrogen atom position in the pyridinic ring greatly affected drug effects both on topoisomerase II action and cytotoxicity to L1210 cells. The four 9-hydroxyellipticine derivatives yielded bell-shaped curves in in vitro topoisomerase II-mediated DNA break assays, whereas the two 7-hydroxyisoellipticine derivatives demonstrated an almost linear increase at the same concentration (0-10 microM). In both cases, the intensity of cleavage was modulated by the position and the degree of methylation on the pyridinic ring, and results were correlated with cytotoxic activity expressed as the in vitro ID50 values for L1210 leukemia cells. 1H NMR experiments performed on free drug molecules in solution revealed that the two protons (alpha and beta) contiguous to the biologically important hydroxy group were sensitive to changes in electron distribution produced by the distant chemical modifications and methylations of the pyridinic ring. A linear relationship was observed between the differences in chemical shifts of alpha and beta protons (delta delta alpha-beta) versus ID50 values. CD experiments indicated that, at weak ionic strength I = 0.02 and at pH 7, drugs interact with the poly[d(A-T)] duplex according to a "three-mode binding model" which is governed by the drug structure and the drug to DNA ratio. The intercalation mode was related to the induction of topoisomerase II-mediated DNA cleavage, while the external binding mode consecutive to intercalation was related to cleavage suppression. These two modes concerned the good intercalators 9-hydroxyellipticines. The third was found for the weak intercalators 7-hydroxyisoellipticines and was characterized by self-stacked molecules bound "outside" DNA, presumably in the minor groove. Ligands either could be intercalated partially or linked at the edge of bases with a small number of molecules filling intercalation sites, for the second alternative. In addition to having different binding modes, 9-hydroxyellipticines were better inducers of DNA distortions than 7-hydroxyisoellipticines. The incidence of the drug binding modes on DNA-topoisomerase II recognition was discussed in connection with the in vitro cytotoxic activity exhibited by the drugs.
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PMID:DNA-drug recognition and effects on topoisomerase II-mediated cytotoxicity. A three-mode binding model for ellipticine derivatives. 184 65

A human Burkitt's lymphoma cell line (Raji-HN2) made resistant to nitrogen mustard, a bifunctional alkylating agent, was used to study the mechanism of resistance to nitrogen mustard. A comparative study of Raji-HN2 and the parental sensitive Raji cell lines revealed the following: (1) The DNA of Raji-HN2 cells was crosslinked by nitrogen mustard to a lower extent than Raji DNA; (2) once interstrand crosslinks were formed, they were repaired at the same rate in both cell lines; (3) DNA crosslink formation in Raji-HN2, but not in Raji cells, was enhanced by novobiocin, a topoisomerase II inhibitor; (4) Raji-HN2 cells had elevated topoisomerase II activity and were hypersensitive to topoisomerase inhibitors (amsacrine, novobiocin, teniposide); (5) similar amounts of topoisomerase I were found in both cell lines; and (6) the chromatin of Raji-HN2 but not of Raji cells, was hypersensitive to DNase I digestion. The relationship between DNA repair, topoisomerase II activity, chromatin structure and drug resistance is discussed.
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PMID:Elevated topoisomerase II activity and altered chromatin in nitrogen mustard-resistant human cells. 281 39

A human Burkitt lymphoma cell line, Raji-HN2, made 10-fold more resistant to nitrogen mustard (HN2) than the parental Raji cell line, exhibited the following characteristics when compared to the parental Raji cells: (i) decreased HN2-induced DNA interstrand crosslinking; (ii) increased (3-fold) DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] activity; (iii) increased (4- to 11-fold) sensitivity to topoisomerase II inhibitors; (iv) increased (2-fold) glutathione content; and (v) increased (2-fold) cell doubling time. The resistant phenotype was unstable and was maintained by weekly treatment of the cells with HN2. Growing the resistant cells in the absence of HN2 resulted in a time-dependent decrease in both resistance to HN2 and topoisomerase II activity and an increase in DNA interstrand crosslinking induced by HN2. We hypothesize that HN2 resistance is due to enhanced monoadduct repair with resultant decreased DNA crosslinking and that this process is mediated by topoisomerase II.
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PMID:Elevated DNA topoisomerase II activity in nitrogen mustard-resistant human cells. 282 70

A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP-hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription.
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PMID:Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. 301 47

2-(Diethylamino-2-ethyl)9-hydroxyellipticinium-chloride, HCl (DHE), a new congener of the antitumor agent elliptinium acetate (Celiptium) (NMHE), has recently been selected for phase I clinical trials. NMHE has a methyl group at nitrogen 2 on the ellipticine ring while DHE possesses a basic diethylaminoethyl chain at this position. Compared to NMHE, the presence of the diethylaminoethyl side chain results in the following: a significant increase in the lipophilicity of the drug; no significant modification in either the binding constant values to DNA or the ability to intercalate between DNA base pairs; a marked decrease in the unwinding angle value of supercoiled DNA; and no significant change in the alteration of the catalytic activity of topoisomerase II in vitro. DHE appears to act as a simple reversible intercalating agent as shown by the selective mutagenic effect on Salmonella TA 1977 tester strain and by its inability to induce the SOS functions in a sfiA lac fusion containing Escherichia coli strain. From a pharmacological point of view, the presence of the diethylaminoethyl chain results in a 2-fold increase in the cytotoxicity to L1210 cultured cells, a strong increase in the antitumor efficiency on experimental murine tumors such as L1210 and P388 leukemia, B16 melanoma, M 5076 reticulosarcoma, and colon 38 adenocarcinoma, and finally an objective decrease in the acute and subacute toxicity in mice, rat, and macaque. The absence of significant differences in the interaction of NMHE and DHE with their potential targets in vitro leads to the hypothesis that the superiority of DHE in terms of cytotoxicity and antitumor efficiency may be due to an increase in the diffusion across cellular membrane and a more favorable biodistribution in vivo.
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PMID:Physicochemical and pharmacological properties of the antitumor ellipticine derivative 2-(diethylamino-2-ethyl)9-hydroxy ellipticinium-chloride, HCl. 367 74

Cyclophosphamide, an alkylating agent belonging to the family of nitrogen mustards, is commonly used to treat progressive autoimmune diseases in humans. At the molecular level, its cytotoxicity results from DNA double strand crosslinks and, at higher concentrations, from DNA strand breaks. At the cellular level, cyclophosphamide may selectively affect mature lymphocytes with relative sparing of the respective precursor cells. In this study, we show that 4-hydroxycyclophosphamide (4-OH-CP), the active metabolite of cyclophosphamide, induces apoptosis in mature human lymphocytes at concentrations that are achieved in vivo. Since cyclophosphamide requires enzymatic conversion in the liver to yield its active metabolite, 4-OH-CP was generated in vitro by non-enzymatic hydrolysis of mafosfamide. Apoptotic cell death of lymphocytes was characterized by typical morphological changes, nucleosomal DNA fragmentation, and quantified by 3'-OH end labeling of fragmented DNA. The percentage of apoptotic cells both depended on drug concentration and time of exposure. Cycloheximide or ZnSO4 did not suppress 4-OH-CP induced apoptosis. Etoposide, a topoisomerase II inhibitor known to induce apoptosis in human tumor cell lines like 4-OH-CP, did induce detectable DNA fragmentation in only a minor proportion of T-lymphocytes but suppressed T-cell proliferation.
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PMID:Mafosfamide induces DNA fragmentation and apoptosis in human T-lymphocytes. A possible mechanism of its immunosuppressive action. 759 14

A series of analogs based on a novel template, 11-aza-(20S)-camptothecin, were obtained from total synthesis and tested as potential anticancer drugs in the topoisomerase I enzyme cleavable complex assay. The parent compound 11-aza-(20S)-camptothecin (8) was derived from a Friedlander condensation between the known aminopyridine derivative 3-(3-amino-4-picolylidene)-p-toluidine and optically active tricyclic ketone 7. Compound 8 had activity approximately twice that of (20S)-camptothecin in the calf thymus topoisomerase I cleavable complex assay. Compounds were prepared wherein the 11-aza nitrogen atom was quaternized as either the corresponding N-oxide or methyl iodide. Compounds with quaternized N-11 showed improved water solubility and were equipotent to the clinically investigated camptothecin analog topotecan in the cleavable complex assay. These compounds were evaluated in vivo in nude mice bearing HT-29 human colon carcinoma xenografts. The analog 11-aza-(20S)-camptothecin 11-N-oxide was found to significantly retard tumor growth when compared to untreated controls. Finally, 7,10-disubstituted 11-azacamptothecin analogs were synthesized using Pd(0) coupling reactions of 10-bromo-7-alkyl-11-aza-(20S)-camptothecins 19 and 20, which in turn were available from a Friedlander condensation of the novel bromopyridine derivatives 17a and 17b with 7. Among the 10-substituted series, a number of analogs displayed extremely high in vitro potency against topoisomerase I and improved aqueous solubility. A significant number of the compounds were found to be active in whole cell cytotoxicity assays and several were evaluated in nude mice bearing the HT-29 tumor xenografts. The most effective of these proved to be (S)-11-aza-7-ethyl-10-(aminohydroximinomethyl)camptothecin trifluoracetic acid salt (27), a potent topoisomerase I inhibitor which demonstrated excellent efficacy in both short term and in extended in vivo assays. A comparison between in vitro enzyme data and in vivo data from nude mouse studies in other compounds in this series revealed a poor overall correlation between topoisomerase inhibition in vitro and antitumor efficacy in vivo.
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PMID:Synthesis, topoisomerase I inhibitory activity, and in vivo evaluation of 11-azacamptothecin analogs. 770 14

Terpentecin and UCT4B are new family of antitumor antibiotics with topoisomerase II mediated DNA cleavage activity. Based on the taxonomic studies, the producing strain S-464 was identified as Streptomyces sp. This strain is different from Kitasatosporia griseola which had been identified as the terpentecin-producing strain in 1988. Fermentation studies showed that natural nitrogen sources supported higher titer of terpentecin, and the synthetic medium with inorganic nitrogen sources supported selective production of UCT4B. An improved isolation method was developed for the large scale purification of terpentecin.
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PMID:Terpentecin and ECT4B, new family of topoisomerase II targeting antitumor antibiotics produced by Streptomyces: producing organism, fermentation and large scale purification. 773 Jan 54


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