Self-association and unique DNA binding properties of the anti-cancer agent TAS-103, a dual inhibitor of topoisomerases I and II

The objective of our study was to investigate the self-association and DNA-binding properties of the DNA topoisomerases I (Topo I) and II (Topo II) dual inhibitor: 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinoline-7-one dihydrochloride (TAS-103), by means of 1H-NMR and 31P-NMR spectroscopy, structure computation techniques, thermal melting study, and UV-Visible spectroscopy. In aqueous solution, all chemical shifts of TAS-103 underwent upfield shifts depending with an increase in concentration. The two-dimensional (2D)-NMR spectra and structure computations indicated that TAS-103 self-associated through pi-pi stacking and hydrophobic interactions of the aromatic chromophores. Thermal melting indicated that the binding of TAS-103 to DNA with a potency equal to that of ethidium bromide (EtBr). The UV-Visible spectra of TAS-103 titrated by several DNA exhibited hypochromic and hypsochromic effects. The 31P-NMR spectrum of the 6:1 TAS-103/d(CGCGAATTCGCG)(2) complex showed two broadening signals. 2D-NMR spectra of the 1:1 TAS-103/d(CGCGAATTCGCG)(2) complex indicated that the chemical shift differences of the DNA are very small. However, those of the terminal region are relatively large. The chemical shift differences of TAS-103 showed that the proton resonances except H2 underwent downfield shifts. From these observations, we conclude that TAS-103 binds to DNA by two modes. The major binding mode is on the surface (outside binding) and the minor binding mode by intercalation.     

Chiral ruthenium(II) anthraquinone complexes as dual inhibitors of topoisomerases I and II

Abstract  

DNA topoisomerases (I and II) have been one of the excellent targets in anticancer drug development. Here two chiral ruthenium(II) anthraquinone complexes, Δ- and Λ-[Ru(bpy)₂(ipad)]²⁺, where bpy is 2,2′-bipyridine and ipad is 2-(anthracene-9,10-dione-2-yl)imidazo[4,5-f][1,10]phenanthroline, were synthesized and characterized. As expected, both of the Ru(II) complexes intercalate into DNA base pairs and possess an obviously greater affinity with DNA. Topoisomerase inhibition and DNA strand passage assay confirmed that the two complexes are efficient dual inhibitors of topoisomerases I and II by interference with the DNA religation. In MTT cytotoxicity studies, two Ru(II) complexes exhibited antitumor activity against HeLa, MCF-7, HepG2 and BEL-7402 tumor cell lines. Flow cytometry analysis shows an increase in the percentage of cells with apoptotic morphological features in the sub-G1 phase for Ru(II) complexes. Nuclear chromatin cleavage has also been observed from AO/EB staining assay and alkaline single-cell gel electrophoresis (comet assay). The results demonstrated that Δ- and Λ-[Ru(bpy)₂(ipad)]²⁺ act as dual inhibitors of topoisomerases I and II, and cause DNA damage that can lead to cell cycle arrest and/or cell death by apoptosis.     

Ungeremine effectively targets mammalian as well as bacterial type I and type II topoisomerases

From the methanol extract of the bulbs of Pancratium illyricum L., three phenanthridine type alkaloids, ungeremine (1), (-)-lycorine (2) and (+)-vittatine (3) were isolated. For the evaluation of their anticancer and antibacterial potential, compounds 1-3 were tested against human (I, IIα) and bacterial (IA, IV) topoisomerases. Our data demonstrated that ungeremine impairs the activity of both, human and bacterial topoisomerases. Remarkably, ungeremine was found to largely increments the DNA cleavage promoted by bacterial topoisomerase IA, a new target in antimicrobial chemotherapy.     

Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage

DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.     

Epitope distribution of randomly established monoclonal antibodies against human type II DNA topoisomerases

The epitopes of about 100 monoclonal antibodies against human type II DNA topoisomerase were mapped along the enzyme molecules. Although they were randomly and independently established, epitope sites were unevenly distributed the toward N-terminal or C-terminal region. We suggest that the central catalytic domain is hidden inside the molecule and inaccessible to the antigen recognition sites. Using antibodies, we demonstrate the distinct localization of isoforms of Topo II in cultured cells. Some particularly useful antibodies are listed.     

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins.

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of approximately 100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact beta/alpha fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by site-directed mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg2+that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.     

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.     

Both DNA topoisomerases I and II relax 2 micron plasmid DNA in living yeast cells

Measurements at various temperatures of the linking number of yeast 2 microns plasmid DNA in wild-type cells and in cells bearing mutations in the DNA topoisomerase I and II genes show that bulk 2 microns plasmid minichromosome are maintained in a relaxed state by the combined action of topoisomerases I and II. Bulk 2 microns minichromosomes are not under torsional stress in vivo and are not substrates for a putative gyrase-like topoisomerase.     

Synthesis and inhibition mechanism of Delta lac-acetogenins, a novel type of inhibitor of bovine heart mitochondrial complex I

We have synthesized Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring and suggested that their inhibition mechanism may be different from that of common acetogenins [Hamada et al. (2004) Biochemistry 43, 3651-3658]. To elucidate the inhibition mechanism of Deltalac-acetogenins in more detail, we carried out wide structural modifications of original Deltalac-acetogenins and characterized the inhibitory action with bovine heart mitochondrial complex I. In contrast to common acetogenins, both the presence of adjacent bis-THF rings and the stereochemistry around the hydroxylated bis-THF rings are important structural factors required for potent inhibition. The inhibitory potency of a derivative possessing an n-butylphenyl ether structure (compound 7) appeared to be superior to that of the original Deltalac-acetogenins and equivalent to that of bullatacin, one of the most potent natural acetogenins. Double-inhibitor titration of steady-state complex I activity showed that the extent of inhibition of compound 7 and bullatacin is not additive, suggesting that the binding sites of the two inhibitors are not identical. Competition tests using a fluorescent ligand indicated that the binding site of compound 7 does not overlap with that of other complex I inhibitors. The effects of compound 7 on superoxide production from complex I are also different from those of other complex I inhibitors. Our results clearly demonstrate that Deltalac-acetogenins are a novel type of inhibitor acting at the terminal electron-transfer step of bovine complex I.     

Irreversible inhibition of type I dehydroquinase by substrates for type II dehydroquinase

Mechanistic differences between type I and type II dehydroquinases have been exploited in the design of type specific inhibitors. (2R)-2-Bromo-3-dehydroquinic acid (3), (2R)-2-fluoro-3-dehydroquinic acid (5) and 2-bromo-3-dehydroshikimic acid (4), all excellent substrates for type II dehydroquinase, are shown to be irreversible inhibitors of type I dehydroquinase.     

Induction of chromosomal aberrations and SCE by camptothecin, an inhibitor of mammalian topoisomerase I

The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with camptothecin (CM), an inhibitor of mammalian topoisomerase I. While no chromosome-type aberrations were found in G1-treated cells, instead there was a dose-dependent induction of chromatid-type aberrations. These types of chromosomal alteration were not induced during the treatment itself but during the S phase, as CM is not efficiently removed with the normal washing procedure after treatment.     

Genotoxic profile of inhibitors of topoisomerases I (camptothecin) and II (etoposide) in a mitotic recombination and sex-chromosome loss somatic eye assay of Drosophila melanogaster

Genotoxic carcinogens which interact with DNA may produce double-strand breaks as normal intermediates of homologous mitotic recombination, and may give rise to structural chromosome aberrations and inter-chromosomal deletion-recombination. The genotoxic profile of two inhibitors of DNA topoisomerases were evaluated using an in vivo somatic w/w+ eye assay of Drosophila melanogaster for the detection of loss of heterozygosity (LOH) by homologous mitotic recombination, intra-chromosomal recombination and structural chromosomal aberrations. We studied camptothecin (CPT) as a topoisomerase-I-interactive agent and etoposide (ETOP) as a topoisomerase II inhibitor. These drugs act by stabilizing a ternary complex consisting of topoisomerases covalently linked to DNA at single-strand or at double-strand breaks, thereby preventing the relegation step of the breakage/rejoining reaction mediated by the enzyme. The genotoxic profiles were determined from the appearance of eye tissue in adult flies, in which LOH and expression of the reporter gene white produced light clones. The results demonstrated that both compounds were significantly genotoxic, with CPT being more effective than ETOP. Inter-chromosomal mitotic recombination was the major mechanism responsible for the induction of light spots by both compounds in XX females. Loss of the ring X chromosome (rX), was significantly enhanced by CPT, and this topoisomerase blocker also produced intra-chromosomal recombination (XY males).     

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.     

Tetrahydroindazole inhibitors of bacterial type II topoisomerases. Part 2: SAR development and potency against multidrug-resistant strains

We have previously reported a novel class of tetrahydroindazoles that display potency against a variety of Gram-positive and Gram-negative bacteria, potentially via interaction with type II bacterial topoisomerases. Herein are reported SAR investigations of this new series. Several compounds possessing broad-spectrum potency were prepared. Further, these compounds exhibit activity against multidrug-resistant Gram-positive microorganisms equivalent to that against susceptible strains.     

Purification and characterization of Plasmodium berghei DNA topoisomerases I and II: drug action, inhibition of decatenation and relaxation, and stimulation of DNA cleavage

It has recently been suggested that topoisomerases could be important targets for drugs used in several diseases. This prompted us to purify and characterize the topoisomerases I and II present in the erythrocytes of protozoan parasites of the genus Plasmodium, the causative agent of malaria, in order to later use these enzymatic systems in antimalarial drug assays. The topoisomerases were purified from Plasmodium berghei, a parasite of mouse red cells. The Plasmodium topoisomerase II consists of two subunits with a molecular weight of about 160K. The enzyme is ATP- and Mg2+-dependent. The conditions for the reactions of relaxation, unknotting, decatenation, and catenation were found to be similar to those observed with enzymes from other eukaryotic cells. The Plasmodium topoisomerase I is a monomeric enzyme with a Mr of 70K-100K. It is ATP-independent and K+- or Na-dependent. Mg2+ is not required for relaxation but stimulates the reaction. Topoisomerase II was more sensitive to drug action than topoisomerase I. The most active drugs were the ellipticine derivatives. The antimalarial drugs, currently used in human clinical therapy, were poor inhibitors. Some antitumoral drugs stimulated the double-stranded DNA cleavage activity of Plasmodium topoisomerase II, like that of mammalian topoisomerases II. Antimalarial drugs had no stimulating activity. It is therefore suggested that Plasmodium topoisomerases are not good targets for antimalarial drugs.     

Comparison of effects of fostriecin, novobiocin, and camptothecin, inhibitors of DNA topoisomerases, on DNA replication and repair in human cells.

Fostriecin causes a delayed inhibition of replicative DNA synthesis in human cells, consistent with a role for DNA topoisomerase II (its target enzyme) at a late stage in replication. Fostriecin does not inhibit UV-induced excision repair. The less specific inhibitor novobiocin blocks repair in permeabilised cells given a low dose of UV, presumably through a mechanism other than the inhibition of topoisomerase II. Its effect cannot be accounted for by a depletion of the ATP required for incision. Camptothecin, an inhibitor of DNA topoisomerase I, blocks replicative DNA synthesis immediately but incompletely, suggesting a participation of topoisomerase I at the replication fork, but it, too, has no influence on DNA repair. We thus find no evidence for involvement of either topoisomerase I or II in the response of cells to UV damage.     

Cadmium is a catalytic inhibitor of DNA topoisomerase II

Cadmium (Cd²⁺) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.