2070-DTI,A Topoisomerase Inhibitor Produced by Streptomyces sp. Strain No. 2070

A novel inhibitor of topoisomerase II designated as 2070-DTI was isolated from the culture filtrate of Streptomyces sp. strain No. 2070. The structure was determined to be that of the known soyasaponin I on the basis of spectroscopic methods (NMR and MS). 2070-DTI strongly inhibited the decatenation activity of human placenta topoisomerase II in a noncompetitive manner, and weakly inhibited or was inert towards the relaxation activities of various topoisomerase I's and DNA-related enzymes. 2070-DTI is an inhibitor belonging to the cleavable complex-nonforming type without DNA intercalation.     

Synthesis and biological activity of a photoaffinity etoposide probe.

The epipodophyllotoxin etoposide is a potent and widely used anticancer drug that targets DNA topoisomerase II. The synthesis, photochemical, and biological testing of a photoactivatable aromatic azido analogue of etoposide also containing an iodo group is described. This azido analogue should prove useful for identifying the etoposide interaction site on topoisomerase II. Irradiation of the azido analogue and an aldehyde-containing azido precursor with UV light produced changes in their UV--visible spectra that were consistent with photoactivation. The azido analogue strongly inhibited topoisomerase II and inhibited the growth of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II--DNA covalent complexes were significantly increased subsequent to UV irradiation of drug-treated human leukemia K562 cells as compared to etoposide-treated cells. These results suggest that the photoactivated form of etoposide is a more effective topoisomerase II poison either by interacting directly with the enzyme or with DNA subsequent to topoisomerase II-mediated strand cleavage.     

Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks.

DNA cleavage by eukaryotic type II DNA topoisomerase (EC 5.99.1.3) was strongly inhibited by an oligonucleotide containing 10 dGua residues. Catalytic activities of topoisomerase II, as measured by relaxation and decatenation reactions, were also inhibited by oligo(dG)10. Inhibition was specific to oligo(dG)10; other oligonucleotides, nucleotides, or single-stranded DNAs tested did not influence the activity of topoisomerase II. Oligo(dG)10 did not inhibit other activities such as restriction enzymes. Although the enzyme neither binds nor cleaves oligo(dG)10, inhibition can be explained by the finding that topoisomerase II binds tightly with aggregated oligo(dG) structures (estimated to contain between 20 and 30 molecules of monomeric oligo(dG)10) that form spontaneously prior to addition of enzyme. These aggregated oligo(dG)-topoisomerase complexes are large networks that can be pelleted by a 20-min centrifugation step in a Microfuge. Western blotting with a monoclonal antibody confirmed that topoisomerase II is trapped in these pellets. The ability of the enzyme to form large DNA-protein networks could be a biochemical mechanism by which topoisomerase II might promote or participate in chromosome condensation in vivo prior to mitosis.     

Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus.

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.     

DNA intercalation and inhibition of topoisomerase II. Structure-activity relationships for a series of amiloride analogs

Among its many properties, amiloride is a DNA intercalator and topoisomerase II inhibitor. Previous work has indicated that the most stable conformation for amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine whether the ability of amiloride to intercalate into DNA and to inhibit DNA topoisomerase II was dependent on the ability to assume a cyclized conformation, we studied the structure-activity relationship for 12 amiloride analogs. These analogs contained structural modifications which could be expected to allow or impede formation of a cyclized conformation. Empirical assays consisting of biophysical, biochemical, and cell biological approaches, as well as computational molecular modeling approaches, were used to determine conformational properties for these molecules, and to determine whether they intercalated into DNA and inhibited topoisomerase II. Specifically, we measured the ability of these compounds to 1) alter the thermal denaturation profile of DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA topoisomerase II in intact cells. Results indicated that only those analogs capable of cyclization could intercalate into DNA and inhibit topoisomerase II. Thus, the ability of amiloride and the 12 analogs studied to intercalate into DNA and to inhibit topoisomerase II appears dependent on the ability to exist in a planar, hydrogen-bonded, tricyclic conformation.     

Two specific topoisomerase II inhibitors prevent replication of human cytomegalovirus DNA: an implied role in replication of the viral genome.

In this study, we show that human cytomegalovirus DNA synthesis is inhibited in infected confluent human embryonic lung cells treated with the DNA-intercalative topoisomerase II inhibitor 4-9'-(acridinylamino)methanesulfon-m-anisidide (m-AMSA). Similar inhibitory effects were observed with VM-26, a nonintercalative topoisomerase II inhibitor. This antiviral effect is not attributable to cytotoxic effects per se. Furthermore, m-AMSA appears to have a notably irreversible inhibitory effect on human cytomegalovirus DNA replication. No inhibition of viral DNA synthesis was observed with o-AMSA, a DNA-intercalative isomer of m-AMSA that does not inhibit topoisomerase II.     

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

The effects of short wave ultraviolet (UV)-induced DNA lesions on the catalytic activity of Drosophila melanogaster topoisomerase II were investigated. The presence of these photoproducts impaired the enzyme's ability to relax negatively supercoiled pBR322 plasmid molecules. As determined by DNA photolyase-catalyzed photoreactivation experiments, enzyme inhibition was due to the presence of cyclobutane pyrimidine dimers in the DNA. When 10-20 cyclobutane dimers were present per plasmid, the initial velocity of topoisomerase II-catalyzed DNA relaxation was inhibited approximately 50%. Decreased relaxation activity correlated with an inhibition of the DNA strand passage step of the enzyme's catalytic cycle. In contrast, UV-induced photoproducts did not alter the prestrand passage DNA cleavage/religation equilibrium of topoisomerase II either in the absence or presence of antineoplastic agents. Results of the present study demonstrate that the repair of cyclobutane pyrimidine dimers is important for the efficient catalytic function of topoisomerase II.     

Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.     

DNA topoisomerase II from mammalian mitochondria is inhibited by the antitumor drugs, m-AMSA and VM-26.

A type II DNA topoisomerase has been partially purified from calf thymus mitochondria by a combination of differential centrifugation and column chromatography. The mitochondrial enzyme was inhibited by amsacrine (m-AMSA) slightly at 0.5 microM, significantly at 5.0 microM, and completely at 50 microM. A similar profile was obtained with teniposide (VM-26) although the latter drug was not quite as potent an inhibitor as the former. P4 unknotting assays of the purified nuclear type II topoisomerase in the presence of m-AMSA and VM-26 indicated that the mitochondrial and nuclear enzymes behaved similarly, although the mitochondrial enzyme appeared to be inhibited more strongly.     

Purification of GidA protein,a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate “cleavable complex”, as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.     

Amiloride intercalates into DNA and inhibits DNA topoisomerase II

Amiloride is capable of inhibiting DNA synthesis in mammalian cells in culture. Recent evidence indicates that the enzyme, DNA topoisomerase II, is probably required for DNA synthesis to occur in situ. In experiments to determine the mechanism of inhibition of DNA synthesis by amiloride, we observed that amiloride inhibited both the catalytic activity of purified DNA topoisomerase II in vitro and DNA topoisomerase II-dependent cell functions in vivo. Many compounds capable of inhibiting DNA topoisomerase II are DNA intercalators. Thus, we performed studies to determine if and how amiloride bound to DNA. Results indicated that amiloride 1) shifted the thermal denaturation profile of DNA, 2) increased the viscosity of linear DNA, and 3) unwound circular DNA, all behavior consistent with a DNA intercalation mechanism. Furthermore, quantitative and qualitative measurements of amiloride fluorescence indicated that amiloride (a) bound reversibly to purified DNA under conditions of physiologic ionic strength, and (b) bound to purified nuclei in a highly cooperative manner. Lastly, amiloride did not promote the cleavage of DNA in the presence of DNA topoisomerase II, indicating that the mechanism by which amiloride inhibited DNA topoisomerase II was not through the stabilization of a "cleavable complex" formed between DNA topoisomerase II, DNA, and amiloride. The ability of amiloride to intercalate with DNA and inhibit topoisomerase II is consistent with the proposed planar, hydrogen-bonded, tricyclic nature of amiloride's most stable conformation. Thus, DNA and DNA topoisomerase II must be considered as new cellular targets of amiloride action.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

ICRF-193, an Inhibitor of Topoisomerase II, Demonstrates That DNA Replication in Sperm Nuclei Reconstituted in Xenopus Egg Extracts Does Not Require Chromatin Decondensation

A bis(2,6-dioxopiperazine) derivative, ICRF-193, is a specific inhibitor of topoisomerase II without clearable complex-stabilizing activity. In Xenopus egg extract containing ICRF-193, demembranated sperm head chromatins were inhibited from decondensation. However, nuclear envelope-lamina assembled on the inhibited chromatins. The nuclear envelope-lamina continued to expand even after loss of contact with the chromatin surface. On the other hand, semiconservative DNA replication was initiated as soon as the lamina was assembled onto the surface of condensed chromatin, though the initiation was retarded and its extent was reduced, compared with that in noninhibited chromatins. Thus, it is concluded that topoisomerase II activity is not required for the formation of active DNA replication clusters and the extension of nuclear envelope-lamina on the chromatin, while the nuclear envelope-mediated decondensation of sperm chromatins is dependent on topoisomerase II activity.     

A truncated form of DNA topoisomerase IIbeta associates with the mtDNA genome in mammalian mitochondria.

Despite the likely requirement for a DNA topoisomerase II activity during synthesis of mitochondrial DNA in mammals, this activity has been very difficult to identify convincingly. The only DNA topoisomerase II activity conclusively demonstrated to be mitochondrial in origin is that of a type II activity found associated with the mitochondrial, kinetoplast DNA network in trypanosomatid protozoa [Melendy, T., Sheline, C., and Ray, D.S. (1988) Cell 55, 1083-1088; Shapiro, T.A., Klein, V.A., and Englund, P.A. (1989) J. Biol. Chem.264, 4173-4178]. In the present study, we report the discovery of a type DNA topoisomerase II activity in bovine mitochondria. Identified among mtDNA replicative proteins recovered from complexes of mtDNA and protein, the DNA topoisomerase relaxes a negatively, supercoiled DNA template in vitro, in a reaction that requires Mg2+ and ATP. The relaxation activity is inhibited by etoposide and other inhibitors of eucaryotic type II enzymes. The DNA topoisomerase II copurifies with mitochondria and directly associates with mtDNA, as indicated by sensitivity of some mtDNA circles in the isolated complex of mtDNA and protein to cleavage by etoposide. The purified activity can be assigned to a approximately 150-kDa protein, which is recognized by a polyclonal antibody made against the trypanosomal mitochondrial topo II enzyme. Mass spectrometry performed on peptides prepared from the approximately 150-kDa protein demonstrate that this bovine mitochondrial activity is a truncated version of DNA topoisomerase IIbeta, one of two DNA topoisomerase II activities known to exist in mammalian nuclei.     

Quinolones inhibit DNA religation mediated by Staphylococcus aureus topoisomerase IV. Changes in drug mechanism across evolutionary boundaries

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.