Synthesis of a novel linear polymer of a macrocyclic polyamine copper (II) complex and its interaction with plasmid DNA

The novel linear polymer of a macrocyclic polyamine copper (II) complex, which has many cyclen groups linked by epichlorohydrin, has been synthesized as a DNA cleavage agent. The structure of the polymer 3 was identified by ¹HNMR and IR and its molecular weight was measured by GPC. The result of agarose gel electrophoresis assay showed that Cu-(II) complex 4 could act as a powerful catalyst for the cleavage of plasmid DNA under physiological conditions.     

Formation of Cu(II)-brazilin complex in the presence of DNA and its activities as chemical nuclease

Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)-brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)-brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)-brazilin complex and DNA bases.     

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.     

Chlorella virus Marburg topoisomerase II: high DNA cleavage activity as a characteristic of Chlorella virus type II enzymes

Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria chlorella virus-1 (PBCV-1) that has an exceptionally high DNA cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA cleavage is critical to the physiological functions of chlorella virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA cleavage specificity/site utilization. These findings suggest that robust DNA cleavage is intrinsic to the viral enzyme and imply that chlorella virus topoisomerase II plays a physiological role beyond the control of DNA topology.     

Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II.

The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.     

Inhibition of topoisomerase II catalytic activity by two ruthenium compounds: a ligand-dependent mode of action

The ability of two structurally different ruthenium complexes to interfere with the catalytic activity of topoisomerase II was studied to elucidate their molecular mechanism of action and relative antineoplastic activity. The first complex, [RuCl2(C6H6)(dmso)], could completely inhibit DNA relaxation activity of topoisomerase II and form a drug-induced cleavage complex. This strongly suggests that the drug interferes with topoisomerase II activity by cleavage complex formation. The bi-directional binding of [RuCl2(C6H6)(dmso)] to DNA and topoisomerase II was verified by immunoprecipitation experiments which confirmed the presence of DNA and ruthenium in the cleavage complex. The second complex, Ruthenium Salicylaldoxime, could not inhibit topoisomerase II relaxation activity appreciably and also could not induce cleavage complex formation, though its DNA-binding characteristics and antiproliferation activity were almost comparable to those of [RuCl2(C6H6)(dmso)]. The results suggest that the difference in ligands and their orientation around a metal atom may be responsible for topoisomerase II poisoning by the first complex and not by the second. A probable mechanism is proposed for [RuCl2(C6H6)(dmso)], where the ruthenium atom interacts with DNA and ligands of the metal atom form cross-links with topoisomerase II. This may facilitate the formation of a drug-induced cleavage complex.     

DNA binding, cleavage, and cytotoxic activity of the preorganized dinuclear zinc(II) complex of triazacyclononane derivatives

A preorganized cleft dinuclear zinc(II) complex of 2,6-bis(1-methyl-1,4,7-triazacyclonon-1-yl)pyridine 1 as an artificial nuclease was prepared via an improved method. The interactions of 1, 2 [1,4,7-triazacyclononane (TACN)], and their zinc(II) complexes with calf thymus DNA were studied by spectroscopic techniques, including fluorescence and CD spectroscopy. The results indicate that the DNA binding affinities of these compounds are in the following order: Zn(II)2 -1 > Zn(II) -2 > 1 > 2. The binding constants of the Zn (II)2 -1 and Zn(II)-2 complexes are 3.57 x 10(6) and 1.43 x 10(5) M(-1), respectively. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of the dinuclear Zn (II)2 -1 complex, which exhibits powerful DNA cleavage efficiency. Kinetic data for DNA cleavage promoted by the Zn(II)2 -1 complex under physiological conditions give the observed rate constant ( k obs) of 0.136 h(-1), which shows an 10(7)-fold rate acceleration over uncatalyzed supercoiled DNA. The comparison of the dinuclear Zn(II)2 -1 complex with the mononuclear zinc(II) complex of 1,4,7-triazacyclononane indicates that the DNA cleavage acceleration promoted by the Zn(II)2 -1 complex is due to the efficient cooperative catalysis of the two proximate zinc(II) cation centers. A hydrolytic mechanism of the cleavage process was suggested, and a preliminary study of the antitumor activity was also conducted.     

Mechanism of bleomycin action: in vitro studies

The cytotoxic activity of bleomucin results from DNA cleavage, which is also accomplished $\text{in vitro}$ by reaction mixtures containing Fe(II), drug and O₂. Bleomycin forms a complex with Fe(II) and O₂ in the presence or absence of DNA. The species attacking DNA forms rapidly from this complex. The nature of the attacking species and of the primary lesion(s) to DNA are not yet known, but two major insults to DNA have been characterized. They are the release of free bases from their glycosidic linkages and, at other residues, the cleavage of the polymer backbone at the deoxyribose C3-C4 bond.     

DNA cleavage study using copper (II)-GlyAibHis: a tripeptide complex based on ATCUN peptide motifs

Development of new chemical nucleases is a matter of great interest because of their extensive use in biotechnology and as therapeutic agents. The ATCUN (amino terminal Cu(II) and Ni(II) binding) is a peptide motif that occurs naturally in the serum albumins. The similar peptide motif (GlyAibHis) having unnatural amino acid Aib (alpha-aminoisobutyric acid) was synthesized and its Cu(II) complex was characterized by ESI-MS and spectrophotometry studies. The reactivity of this complex toward DNA cleavage has been investigated. Cu(II)-GlyAibHis shows the DNA cleavage only in presence of mild oxidizing agents like ascorbate by an oxidative mechanism rather than hydrolytic and follows the pseudo first order kinetics (K obs = 0.085 min(-1)). The non-hydrolytic mechanism was further supported by the hydrolysis of pNPP which followed the pseudo first order kinetics (K obs = 1.98 x 10(-2) min(-1)) having no pH effect.     

Efficient double-strand scission of plasmid DNA by quaternized-chitosan zinc complex

N-[(2-Hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTACC) was prepared to construct a chitosan-based zinc complex (HTACC-Zn(II)) as a catalyst with good water solubility for rapid DNA cleavage. Results indicated that the observed rate constant (k(obs)) of plasmid DNA cleaved by HTACC-Zn(II) could be enhanced by 10(7)-fold compared with that of uncatalyzed DNA cleavage. The kinetic behavior of HTACC-Zn(II) for DNA cleavage is well fitted by Michaelis-Menten model. The results of gel electrophoresis suggested that HTACC-Zn(II) preferentially perform double-strand break of plasmid DNA.     

Studies of sequence-specific DNA binding, DNA cleavage, and topoisomerase I inhibition by the dimeric chromomycin A3 complexed with Fe(II)

Chromomycin A3 (Chro) has been evidenced to exhibit much higher binding affinity toward Fe(II) by forming a highly stable 2:1 drug/metal complex, compared to its structural analogue, mithramycin (Mith). Different properties of the [(Chro)2-Fe(II)] complex acting on DNA, such as sequence specificity, DNA cleavage, and topoisomerase I (TopI) inhibition were studied. Kinetic analyses of surface plasmon resonance showed that the affinity of the [(Chro)2-Fe(II)] complex upon binding to hairpin DNA duplexes containing various tetranucleotide sequences follows the order: GGCC > CGCG > CCGG approximately GCGC > AGCT > ACGT > TGCA > TCGA. According to circular dichroism (CD) studies, most hairpin DNA duplexes appeared to retain their B-type conformations in the presence of the [(Chro)2-Fe(II)] complex, except the duplex containing the GGCC sequence, which exhibited the features of both A- and B-type DNA. In DNA-cleavage assays, the [(Chro) 2-Fe(II)] complex was shown to cause single-stranded cleavage of plasmid DNA because of a Fenton-type reaction. DNA cleavage activity of the [(Chro) 2-Fe(II)] complex was increased at low pH. Moreover, the complex was capable of inhibiting TopI activity. The [(Chro)2-Fe(II)] complex exhibited higher cytotoxicity than the [(Mith) 2-Fe(II)] complex in several cancer cell lines, most likely owing to its more stable dimeric structure and higher DNA-binding affinity. Our results provide significant evidence that the [(Chro)2-Fe(II)] complex could be promising in terms of its biological applications in the future.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide

Beyond its essential physiological functions, topoisomerase II is the primary cellular target for a number of clinically relevant antineoplastic drugs. Although the chemotherapeutic efficacies of these drugs correlate with their abilities to stabilize the covalent topoisomerase II-DNA cleavage complex, their molecular mechanism of action has yet to be described. In order to characterize the drug-induced stabilization of this enzyme-DNA complex, the effect of etoposide on the DNA cleavage/religation reaction of Drosophila melanogaster topoisomerase II was studied. Under the conditions employed, etoposide increased levels of enzyme-mediated double-stranded DNA cleavage 5-6-fold and single-stranded cleavage approximately 4-fold. Maximal stimulation was observed at 80-100 microM etoposide with 50% of the maximal effect at approximately 15 microM drug. By employing a topoisomerase II mediated DNA religation assay [Osheroff, N. & Zechiedrich, E.L. (1987) Biochemistry 26, 4303-4309], etoposide was found to stabilize the enzyme-DNA cleavage complex (at least in part) by inhibiting the enzyme's ability to religate cleaved DNA. Moreover, in order for the drug to affect religation, it has to be present at the time of DNA cleavage.     

Topoisomerase II poisoning and antineoplastic action by DNA-nonbinding diacetyl and dicarboxaldoxime derivatives of ferrocene

Topoisomerase II is a major molecular target for a number of DNA-binding anticancer drugs. In the present study, we report topoisomerase II inhibition and anticancer activity by four substituted ferrocene derivatives which do not bind to DNA. The first derivative, acetyl-substituted ferrocene (monoacetylferrocene), showed a minor inhibition of topoisomerase II activity along with a consequent inhibition of cancer cell proliferation. The second derivative (diacetylferrocene) showed a higher potency of action compared to the monosubstituted derivative. The third and fourth derivatives, with mono- and disubstituted carboxaldoxime groups (ferrocenecarboxaldoxime and ferrocenedicarboxaldoxime), showed a higher anticancer action and stronger topoisomerase II inhibition. To understand their molecular mechanism of action, cleavage assays were carried out to monitor the drug-induced, topoisomerase II mediated DNA cleavage. The results show that diacetylferrocene and ferrocenedicarboxaldoxime could form an enzyme-drug-DNA ternary complex, called a "cleavage complex," resulting in DNA cleavage. These results along with those of an immunoprecipitation assay indicate that the two compounds interact with topoisomerase II alone and poison its activity by trapping the enzyme and enzyme-cleaved DNA in the covalently closed cleavage complex. The formation of such a complex has numerous genetic implications, which ultimately results in neoplastic cell death.     

Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil

Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and (1)H NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were subsequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes. The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.     

DNA cleavage by novel copper (II) complex and the role of beta-cyclodextrin in promoting cleavage

A new cyclen derivative N-1-naphthyl-[4-amino-5-oxo-5-(1,4,7,10-tetraazacyclododecan-1-yl)]valeramide and the copper (II) complex were synthesized and characterized. The copper (II) complex showed DNA cleavage ability without the existence of other additives. The pUC19 plasmid DNA was cleaved to linear form by 0.71 microM of complex under physiological conditions. beta-Cyclodextrin was used to investigate the relationship of nuclease activity and DNA binding ability. The addition of beta-cyclodextrin exhibited an unexpected ability to promote the cleavage of DNA. The role of the beta-cyclodextrin in DNA cleavage process was studied by (1)H NMR and fluorescence spectrum. According to the data of viscosity measurement, it was confirmed that the binding of complex with DNA should be a groove binding model. All the results suggested that the increasing of the DNA cleavage ability was attributed to the interaction between beta-cyclodextrin and the naphthyl moieties. beta-Cyclodextrin could include the naphthyl moieties and keep it from the minor/major groove of DNA and decreased the DNA binding ability, therefore, the copper (II) center was activated to generate more reactive oxygen species, which was responsible for DNA cleavage.     

Synthesis and biological evaluation of the Zn (II)-IDB complexes appended with oligopolyamide as potent artificial nuclease

The zinc (II) complexes, which contain oligopolyamide and bis(2-benzimidazolylmethyl)amine (IDB) conjugated by flexible linker, have been successfully synthesized, characterized, and evaluated as DNA cleavage agents. The cleavage activity of these complexes on DNA was studied by electrophoresis. The results showed that the cleavage activity of zinc (II) complexes was enhanced comparing with those without oligopolyamide. Specially, at a high reaction concentration (1.2mM), Zn (II) complex can cleave the plasmid DNA bearing some selectivity. Further, the spectroscopic data suggested that Zn (II) complexes with oligopolyamide backbone possessed A-T (adenine and thymine) rich sequences preference.     

Amsacrine as a topoisomerase II poison: importance of drug-DNA interactions

Amsacrine (m-AMSA) is an anticancer agent that displays activity against refractory acute leukemias as well as Hodgkin's and non-Hodgkin's lymphomas. The drug is comprised of an intercalative acridine moiety coupled to a 4'-amino-methanesulfon-m-anisidide headgroup. m-AMSA is historically significant in that it was the first drug demonstrated to function as a topoisomerase II poison. Although m-AMSA was designed as a DNA binding agent, the ability to intercalate does not appear to be the sole determinant of drug activity. Therefore, to more fully analyze structure-function relationships and the role of DNA binding in the action of m-AMSA, we analyzed a series of derivatives for the ability to enhance DNA cleavage mediated by human topoisomerase IIα and topoisomerase IIβ and to intercalate DNA. Results indicate that the 3'-methoxy (m-AMSA) positively affects drug function, potentially by restricting the rotation of the headgroup in a favorable orientation. Shifting the methoxy to the 2'-position (o-AMSA), which abrogates drug function, appears to increase the degree of rotational freedom of the headgroup and may impair interactions of the 1'-substituent or other portions of the headgroup within the ternary complex. Finally, the nonintercalative m-AMSA headgroup enhanced enzyme-mediated DNA cleavage when it was detached from the acridine moiety, albeit with 100-fold lower affinity. Taken together, our results suggest that much of the activity and specificity of m-AMSA as a topoisomerase II poison is embodied in the headgroup, while DNA intercalation is used primarily to increase the affinity of m-AMSA for the topoisomerase II-DNA cleavage complex.     

Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26.

Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.     

Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein.

Escherichia coli omega protein is found to form a complex with single-stranded DNA. The complex is stable in buoyant CsCl or Cs2SO4 density gradients. Addition of Mg(II) to the concentrated salt solutions, however, leads to the dissociation of the complex, even in the presence of EDTA in molar excess over Mg(II). The dissociated omega retains its enzymatic activity; the DNA recovered from the dissociated complex is indistinguishable from the original DNA. Exposure of the complex to alkali results in the cleavage of the DNA. This cleavage generates a 3'-hydroxyl DNA terminus, and the omega protein is found linked to the 5'-terminus, presumably covalently. Pronase digestion of the complex results initially in the removal of approximately 30% of the protein. A significant fraction of the residual complex is still stable in concentrated salt solutions, and can be dissociated by Mg(II). Extensive digestion with pronase results in the removal of the protein and the cleavage of the DNA chain.