Thymus DNA Topoisomerase Research Articles

New spiro-acridines: DNA interaction, antiproliferative activity and inhibition of human DNA topoisomerases

Two new spiro-acridines were synthesized by introducing cyano-N-acylhydrazone between the acridine and phenyl rings followed by spontaneous cyclization. The final compounds (E)-1⿲-(benzylideneamino)-5⿲-oxo-1⿲,5⿲-dihydro-10H-spiro[acridine-9,2⿲-pyrrole]-4⿲-carbonitrile (AMTAC-01) and (E)-1⿲-((4-methoxybenzylidene)amino)-5⿲-oxo-1⿲,5⿲-dihydro-10H-spiro[acridine-9,2⿲-pyrrole]-4⿲-carbonitrile (AMTAC-02) were evaluated for their interactions with calf thymus DNA, antiproliferative and human topoisomerase I and IIα inhibitory activities. Both compounds presented ability to bind DNA. The binding constant determined by UV⿿vis spectroscopy was found to be 104M⿿1. Antiproliferative assay demonstrated that AMTAC-01 and AMTAC-02 were most active against prostate and melanoma tumor cell lines, respectively. The compound did not present Topo I inhibitory activity. However, both derivatives displayed topoisomerase IIα inhibitory activity comparable to amsacrine, and AMTAC-02 was more potent than AMTAC-01 with methoxy substituent group on phenyl ring. This study demonstrates that the new derivatives are promising molecules with topoisomerase IIα inhibitory and antiproliferative activities.

81 A new series of biologically active DNA minor groove binders based on bisbenzimidazole and benzimidazole-pyrrole motives

As we reported earlier, dimeric bisbenzimidazoles DB(n) (Ivanov et al., 2011a) were able to noncovalently bind in the DNA minor groove and displayed inhibitory activity at low concentrations toward three different DNA-dependent enzymes (Susova et al., 2010; Ivanov et al., 2011b; Cherepanova et al., 2011; Tunitskaya et al., 2011). However, these compounds had a substantial drawback: a low solubility in water solutions, which prevent their potential use in vivo as biologically active preparations. Therefore, for the new minor groove binder series, additional groups were added into the molecule structures for the sake of hydrophility and DNA affinity improvement. A DBP(n) series contains piperazine cycle in the oligomethylene linker which connects bisbenzimidazole fragments of molecule together. A DBPy(n) series have a pyrrole and benzimidazole cycles combination in their structure (Figure 1). DBP(n) (n = 1–4) compounds were studied as inhibitors of calf thymus DNA topoisomerase I (topo-I). All of them inhibited topo-I activity at 1–5 μM concentrations, besides, at concentrations over 5 μM, the inhibitory activities of DBP(n) were few times higher than that of DB(n). It was found that dimeric bisbenzoimidazoles DBP(1–3) were not mutagenic. They were inactive in the point mutagenesis assessment using the Ames test (with Salmonella Typhimurium bacteria as the tested object) and did not display mutagenic or recombinogenic properties in the Drosophila wing somatic mutation and recombination test (SMART). All the compounds of series were evaluated for cytotoxicity in the MTT test on breast cancer MCF-7, large bowel adenocarcinoma HCT-116, noncancer HEK-293 cell lines and on primary murine fibroblasts. All the tested DBP( n ) were not toxic up to concentrations of 100 μM. Contrary, the one tested compound from DBPy(n) series, DBPy(4) (n = 4), showed substantial cytotoxicity against cancer HCT-116 and human leukemic K-562 cell lines with an IC50 of 1–2 μM. Judging by the combination of such criterions as water solubility, topo-I inhibition activity and mutagenicity, it would be safe to assume that dimeric bisbenzoimidazoles of DBP(n) series are more perspective minor groove binders than previously synthesized DB( n ).

Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.

G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

Inhibition of DNA Topoisomerase I by Dihydrotanshinone I, Components of a Medicinal Herb Salvia miltiorrhiza Bunge.

Dihydrotanshinone I induced topoisomerase I-mediated DNA cleavage in vitro as strongly as camptothecin, but topoisomerase II-mediated DNA cleavage was not affected. In a DNA relaxation assay using calf thymus DNA topoisomerase I and supercoiled pBR322 plasmid DNA, dihydrotanshinone I reduced topoisomerase I-mediated DNA relaxation in a dose-dependent manner. Heat treatment (65°C) of the reaction mixture containing dihydrotanshinone I and topoisomerase I resulted in a substantial reduction in DNA cleavage, suggesting topoisomerase I and dihydrotanshinone I may form a reversible cleavable complex to induce DNA damage. A DNA unwinding assay using T4 DNA ligase showed that dihydrotanshinone I is a very weak DNA intercalator. These results suggest that dihydrotanshinone I inhibits the catalytic activity of topoisomerase I by the formation of a cleavable complex and at least in part through the intercalation into DNA.

Evolution of the adenosine triphosphate site and design of new inhibitors of DNA topoisomerases II

DNA topoisomerase II is required for chromosome segregation. The enzyme expresses itsbiological activity upon ATP hydrolysis into ADPand inorganic phosphate. Both ATP and dATP areequivalent substrates, but the 8-azidoadenosine5′-triphosphate is a poor substrate comparing toATP for calf thymus DNA topoisomerase II. Thisresult is interesting and can be used for thedesign of new inhibitors or new bettersubstrates and furthermore for biocaptors.

Psychotropic drugs and local anesthetics restore cardiolipin-inhibited mammalian DNA topoisomerase I activity.

The activity of mammalian topoisomerase I is inhibited by acidic phospholipids. We investigated the effect of psychotropic drugs and local anesthetics on cardiolipin-inhibited calf thymus DNA topoisomerase I activity. Chlorpromazine, promethazine, and imipramine, which interact with phospholipids, suppressed the inhibitory effect of cardiolipin. The present results suggest that relaxation of DNA supercoiling is involved in the cytotoxic action of these drugs.

Microbial transformation of azacarbazoles X: Regioselective hydroxylation of 5,11-dimethyl-5H-indolo [2,3-b]quinoline, a novel DNA topoisomerase II inhibitor, by Rhizopus arrhizus

Microbial transformation of cytotoxic 5,11-dimethyl-5H-indolo[2,3-b]quinoline (a compound displaying antitumor activity and affecting the activity of calf thymus DNA topoisomerase II) was performed by the Rhizopus arrhizus strain and yielded a 9-hydroxy derivative. The metabolite obtained displayed a stronger cytotoxity against KB cells than the parent compound (ID50=0.001 μmol/mL), and stimulated also the formation of calf thymus topoisomerase II mediated pSP65 DNA cleavage in vitro at the concentration of 3 μM. Being analogous to 9-hydroxyellipticine (which is an antitumor alkaloid), this novel indolo[2,3-b] quinoline derivative can be regarded as a novel potential antitumor agent.

DNA topoisomerase I inhibitors: cytotoxic flavones from Lethedon tannaensis.

From ethyl acetate and methanolic extracts of Lethedon tannaensis leaves, which were cytotoxic against murine leukemia (P-388) and human nasopharynx carcinoma (KB) cells, one new and six known 5-hydroxy-7-methoxyflavones variously substituted on the B ring were isolated and their structures determined by spectral analysis. Compounds active against KB cells were velutin (4) (IC50 4.8 microM), 7,3',5'-tri-O-methyltricetin (2) (IC50 22.2 microM), genkwanin (6) (IC50 30.6 microM), and the novel compound, 7,3',4'-tri-O-methyltricetin, named lethedocin (1) (IC50 47.6 microM). These flavones required the presence of hydroxyl groups at C-5 and C-4' and methoxyl groups at C-7 and C-3' for inhibition of calf thymus DNA topoisomerase I activity.

Rescue of Schizosaccharomyces pombe from camptothecin-mediated death by a DNA topoisomerase I inhibitor, TAN-1518 A

TAN-1518 A is a cytotoxic agent with suppressive activity against Meth A fibrosarcoma in vivo. This compound inhibits calf thymus DNA topoisomerase I (Topo I) but does not stimulate cleavable complex formation in the nuclei of Chinese hamster ovary (CHO)-K1 cells, suggesting that it inhibits Topo I in a manner different from that of camptothecin (CPT). To clarify the mode of action of TAN-1518 A, we examined its effects on the eukaryotic microorganism Schizosaccharomyces pombe (S. pombe), which does not require Topo I as an essential factor for growth. TAN-1518 A inhibited purified S. pombe Topo I as potently as did CPT. TAN-1518 A, unlike CPT, did not stimulate Topo I-induced DNA cleavage; instead, it inhibited CPT-induced cleavable complex formation. We constructed a S. pombe strain, IR9, that produced excess Topo I. IR9 was hypersensitive to CPT, but its growth was not affected by TAN-1518 A. The CPT-mediated death of IR9 cells was reduced dramatically in the presence of TAN-1518 A. These findings clearly demonstrate that TAN-1518 A is a specific inhibitor of Topo I in eukaryotic cells and also suggest that this agent inhibits some earlier step(s) that occurs before the formation of cleavable complex on DNA strands in the catalytic cycle of this enzyme.

Marine natural products. XXXII. Absolute configurations of C-4 of the manoalide family, biologically active sesterterpenes from the marine sponge Hyrtios erecta.

Cytotoxic sesterterpenes, manoalide 25-acetals (1a, 1b), seco-manoalide (2), (E)-neomanoalide (3), (Z)-neomanoalide (4), and heteronemin (6), were isolated from the marine sponge Hyrtios erecta (collected at Amami Island, Kagoshima Prefecture, Japan) by bioassay-guided separation and the absolute configurations of these manoalide family members have been determined. Manoalide 25-acetals (1a, 1b) were shown to exhibit in vivo antitumor activity and to inhibit the DNA-relaxing activity of mouse DNA topoisomerase I and the DNA-unknotting activity of calf thymus DNA topoisomerase II.

Topoisomerase II mediated DNA strand cleavage and antitumor activities against murine leukemia P388 of novel acridines

In order to clarify the mechanism of topoisomerase II mediated DNA cleavage activity caused by intercalators (9-anilinoacridines), antitumor activities against murine P388 and calf thymus DNA topoisomerase II mediated DNA cleavage activities of compounds m-AMSA, A4P73, A3P56, and A3P166 have been studied. A4P73 was found to have potent antitumor and DNA cleavage activities.

New naphthacenecarboxamide antibiotics, TAN-1518 A and B, have inhibitory activity against mammalian DNA topoisomerase I.

New naphthacenecarboxamide antibiotics, TAN-1518 A and B, were isolated from a culture broth of Streptomyces sp. AL-16012. Their structures were elucidated from their reactions and from spectroscopic analyses. The relaxation of supercoiled pBR322 DNA by calf thymus DNA topoisomerase I was inhibited by these metabolites as potently as by camptothecin. However, the decatenation of kinetoplast DNA by calf thymus DNA topoisomerase II was little affected by these agents. The major metabolite, TAN-1518 A, strongly suppressed the growth of various murine and human tumor cells, inducing apoptosis. Unlike camptothecin, TAN-1518 A did not stimulate cleavable complex formation in the nuclei of CHO-K1 cells and had weak activity in intercalating into DNA strands. This metabolite arrested the growth of human tumor cell lines in G1 phase of the cell cycle. These results suggest that TAN-1518 A and B are novel antitumor agents targeting topoisomerase I.

Specific regulatory role of phosphorylation of calf thymus DNA-topoisomerase I smaller forms on the relaxational activity expression. Phosphorylation role on Topo I smaller forms activity.

Calf thymus Topo I is found to be associated with three active breakdown products, resolved from intact enzyme, which do not appear to be unique to one extraction procedure. They are phosphoproteins, whose enzymatic activity can be modulated through changes in phosphorylation, and which can be phosphorylated 'in vitro', by N II protein kinase, in the same five sites as the intact enzyme. Different amounts of 32P incorporated are observed however, in the corresponding sites. We conclude: 1. proteolysis is probably an 'in vivo' phenomenon, as the Topo I smaller species are observed, during isolation from the earlier crude fractions, and as a minimum of them is always present, even if precautions are taken to minimize proteolysis; 2. a specific regulatory role in the DNA relaxational activity might be played by N II protein kinase phosphorylation, indeed, in the smaller species; 3. the different degrees of 32P incorporation, in analogous phosphorylation sites, might represent a different signal for modulating the gene expression.

2',5'-oligoadenylates inhibit relaxation of supercoiled DNA by calf thymus DNA topoisomerase I.

DNA topoisomerases interconvert various topological isomers of DNA and play key roles in replication and gene expression. The possible involvement of the 2',5'-oligoadenylates (2-5A) system in cell growth, regulation, and cell differentiation led us to investigate the effects of 2-5A on mammalian topoisomerases. We found that the calf thymus type I topoisomerase was inhibited by a variety of 2-5A compounds. The level of inhibition was dependent upon the number of residues and the degree of phosphorylation at the 5' terminus. The 5'-triphosphorylated 2',5' hexamer, ppp(Ap)5A, was the most effective, strongly reducing relaxation at less than micromolar concentrations. These results raise the possibility that physiological concentrations of 2-5A of sufficient chain length may be capable of regulating gene expression by virtue of a direct inhibition of DNA topoisomerase I.

Optimum DNA relaxation reaction conditions for calf thymus DNA-Topoisomerase I are determined by specific enzyme features

Reactivity and chemical properties of calf thymus topoisomerase I have been investigated with respect to enzyme ability to relax supercoiled DNA. The relaxation rate has been analyzed at optimum and relatively high salt concentration. Catalysis is processive at optimum salt concentration and distributive at a higher one; camptothecin decreases the initial rate of reaction in both salt conditions, but more so at the higher one. We conclude that: 1. calf thymus topoisomerase I requires, for its maximum reactivity, specific and characteristic reaction conditions; 2. salt concentration affects DNA processing, indeed influencing the initial rate of DNA relaxation and directly reflecting the salt-dependence for the enzyme-duplex DNA binding; 3. topoisomerase I, from various sources, maybe individually responds to alteration of assay parameters such as pH, Mg++ and NaCl concentrations, indicating that individual criteria could be responsible for the catalytic activity optimum.

Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: Comparison with other type II enzymes

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila , yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme α. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.

Role of calf thymus DNA-Topoisomerase I phosphorylation on relaxation activity expression and on DNA-protein interaction

Calf thymus DNA-Topoisomerase I activity was found to be altered by changing in phosphorylation: it was completely inhibited upon dephosphorylation by alkaline phosphatase, but incubation with N II protein kinase and ATP restored the relaxation activity to a level higher than that observed prior to dephosphorylation. The calf thymus Topoisomerase I-mediated DNA cleavage, induced by camptothecin, also proved to be inhibited by dephosphorylation, which, apparently, stabilizes the initial enzyme-substrate complex. We conclude that: the native protein is partially phosphorylated, the phosphorylation involvement is essential for the activity expression and also for DNA-protein interaction, changes in the degree of phosphorylation might be involved in the regulation of DNA processing; that evokes some properties of chromatinic peptide models, which bind DNA only when phosphorylated and leads to the assumption that they represent the minimum functional substrate for N II protein kinase.

Phosphorylation sites for type n ii protein kinase in DNA-topoisomerase i from calf thymus

1. 1. Calf thymus DNA-topoisomerase I has been isolated, in an improved preparation, nearly to SDS-PAGE homogeneity, as a single major protein (100 kDa). 2. 2. In vitro labeling experiments, which employed the purified enzyme [γ- 32P]ATP and N II protein kinase, also showed that the calf thymus topoisomerase I became phosphorylated. 3. 3. Phosphorylation was accompanied by an increase in topoisomerase I activity. 4. 4. Phosphoaminoacid analysis indicated that only serine residues became phosphorylated. 5. 5. Tryptic peptides mapping, by HV electrophoresis, indentified five major [ 32P]peptides. This number is higher than that reported for topoisomerase I from Novikoff hepatoma cells. 6. 6. Separation of each spot, by reverse phase HPLC, resulted in their elution at fractions 1, 2, 3, 4 and 5 with 9, 11, 16, 27 and 28% acetonitrile, respectively. 7. 7. Isolated phosphopeptides will be subjected to sequencing, to DNA-binding and transcription regulation tests; then, it will be speculated whether type N II protein kinase may contribute to the physiological regulation of DNA-topoisomerase I activity from calf thymus, as well.

In vitropreferential topoisomerization of bent DNA

The steps of the topoisomerization reaction by calf thymus DNA topoisomerase I on a DNA domain containing an intrinsically bent DNA sequence have been analyzed. High preferentiality of binding, cleavage and topoisomerization on the bent segment relative to the rest of the DNA domain was observed. These studies show the importance of the local DNA conformation in the reaction of eukaryotic DNA topoisomerase I and the interest of the use of this enzyme as a tool for the analysis of DNA conformation and DNA dynamics.