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Biomedical subjects

G Capranico

Publications and source records attributed to G Capranico.

At least 19 recordsLinked to original sources
PubMed2001

The topoisomerase II poison clerocidin alkylates non-paired guanines of DNA: implications for irreversible stimulation of DNA cleavage.

Clerocidin, a diterpenoid with antibacterial and antitumor activity, stimulates in vitro DNA cleavage mediated by mammalian and bacterial topoisomerase (topo) II. Different from the classical topoisomerase poisons, clerocidin-stimulated breaks at guanines immediately preceding the sites of DNA cleavage are not resealed upon heat or salt treatment. To understand the mechanism of irreversible trapping of the topo II-cleavable complex, we have investigated the reactivity of clerocidin per se towards DNA. We show here that the drug is able to nick negatively supercoiled plasmids. DNA cleavage by clerocidin in enzyme-free medium is due to the ability of the drug to form covalent adducts with guanines. Indeed, clerocidin was able to specifically react with short oligonucleotides when the guanines were unpaired and exposed as in bulges or in the single-strand form. The clerocidin epoxy group attacks the nitrogen at position 7 of guanines, leading to strand scission at the modified site. Our findings also demonstrate that trapping of topoisomerases by clerocidin is specific for type II enzymes. The guanine-alkylating ability of clerocidin suggests an unprecedented mechanism of topo II poisoning, according to which the enzyme renders the drug reactive toward DNA by distorting the double-helical structure of the nucleic acid at the cleavage site.

Alkylation↗
PubMed2001

A comparative study of cellular and molecular pharmacology of doxorubicin and MEN 10755, a disaccharide analogue.

MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in -1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drug's efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.

Antineoplastic Agents↗
PubMed2001

Directed evolution to increase camptothecin sensitivity of human DNA topoisomerase I.

BACKGROUND: Human DNA topoisomerase I (top1) relaxes DNA supercoiling during basic nuclear processes. The enzyme is the main target of antitumor agents, such as camptothecins (CPT), that transform top1 into a DNA-damaging agent. RESULTS: By directed evolution of a C-terminal portion, we selected human top1 mutants that were 22-28-fold more CPT-sensitive than wild-type top1 in Saccharomyces cerevisiae cells. The evolved enzymes showed unique mutation patterns and were more processive in plasmid relaxation assays. A top1 mutant had only two amino acid changes in the linker domain, one of which may change a linker/core domain contact surface. The mutant stimulated DNA cleavage to higher levels than the wild-type enzyme and was more sensitive to CPT in a cleavage assay. Moreover, the mutant was more CPT-sensitive than wild-type top1 in a repair-deficient yeast strain. CONCLUSIONS: Mutations in the linker domain can affect DNA binding and CPT sensitivity of human top1. Such drug-hypersensitive topoisomerases may be useful in developing DNA cutters with high cell lethality and in new drug discovery programs.

Blotting, Western↗
PubMed2001

A novel 9-aza-anthrapyrazole effective against human prostatic carcinoma xenografts.

OBJECTIVES: Systematic investigation of a novel series of intercalating agents, 9-aza-anthrapyrazoles, has led to the identification of a promising analogue, BBR 3438. This study describes the antitumour efficacy of the novel compound in human prostate carcinoma models and the molecular/cellular basis of its activity. METHODS AND RESULTS: The novel 9-aza-anthrapyrazole BBR 3438 was significantly more effective than doxorubicin and losoxantrone (DuP-941) in two of the three tested prostate carcinoma models. The superior activity was more evident in PC3 tumour, since BBR 3438 produced an appreciable rate of complete tumour regressions. Under these conditions, the drug-induced antiproliferative activity paralleled delayed apoptosis. Tumour response to in vivo drug treatment was associated with an early down-regulation of Bcl-2, which was somewhat more marked for the aza compound. In fact, the 9-aza-anthrapyrazole induced DNA cleavage in vitro with isolated DNA topoisomerase II (isoform alpha) and DNA strand breaks in prostatic carcinoma cells. Although the molecular effects of losoxantrone and the 9-aza analogue on the enzyme target were comparable, the cytotoxic effects of BBR 3438 could be enhanced by long-term exposure as a consequence of favourable cellular accumulation and prominent DNA-binding affinity. In addition, a lower reduction potential of the 9-aza-anthrapyrazole in comparison with classical anthrapyrazoles suggests an increased ability of the drug to induce oxidative stress following free radical production, which may be a contributing factor in determining the long-term response (i.e. delayed cell death) to genotoxic damage. CONCLUSIONS: BBR 3438 exhibited a unique profile of preclinical activity with a superior efficacy against prostatic carcinoma models compared to reference compounds (doxorubicin and losoxantrone). The antitumour efficacy of BBR 3438 against prostatic carcinoma could be the result of a combination of favourable events, including enhanced intracellular accumulation and an increased DNA-binding affinity favouring the accumulation of multiple sublethal or lethal damage. In spite of its enhanced cytotoxic potency, the 9-aza compound was better tolerated in vivo than losoxantrone, thus improving the therapeutic index. The preclinical profile of efficacy against prostatic carcinoma, a tumour resistant to conventional antitumour drugs, makes the novel 9-aza-anthrapyrazole BBR 3438 a promising candidate for clinical evaluation.

Adenocarcinoma↗
PubMed2001

Anthracyclines: selected new developments.

Anthracycline antibiotics play an important role in cancer chemotherapy. The need for an improvement of their therapeutic index has stimulated an ongoing search for anthracycline analogues with improved properties. Analogue development was originally limited by a lack of information on the cellular drug target, nevertheless almost 20 years ago the mechanism of action of doxorubicin and daunorubicin was revealed and DNA topoisomerase II was recognised to be their main cellular target. Several anthracyclines interfere with topoisomerase II functions by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to tyrosine residues of the enzyme. Investigations on the sequence specificity of doxorubicin in vitro and in nuclear chromatin of living cell have led to a molecular model of drug receptor on the topoisomerase II-DNA complex. Anthracyclines are likely placed at the interface between the DNA cleavage site and the active site of the enzyme, forming a DNA-drug-enzyme ternary complex. Moreover, a quite detailed structure-function relationship has been established for anthracyclines. First, drug intercalation is necessary but not sufficient for topoisomerase II poisoning; second, the removal of the 4-methoxy and 3'-amino substituents greatly increases the drug activity and third, the 3' substituent of the sugar moiety markedly influences the sequence selectivity of anthracycline-stimulated DNA cleavage. These relationships have been exploited during the last decade by several groups, including ours, in the search for new anthracycline drugs with lower side effects and higher activity against resistant cancer cells. This review will focus on areas of the anthracycline field including synthesis of new analogues, new strategies of synthesis and recent developments in the area of drug delivery.

Animals↗
PubMed2000

Loss of drug-stimulated topoisomerase II DNA breaks in living cells is different at two unrelated loci.

Topoisomerase II (top2) has been implicated in the initial steps of chromosomal translocations leading to leukemias and lymphomas, since it can generate DNA cleavage. To evaluate the effects of chromatin structure on enzyme-mediated cleavage, we determined the kinetics of loss of double-stranded DNA breaks stimulated by top2 poisons in Drosophila melanogaster Kc cells at two genomic regions that differ in chromatin structure. Moreover, cleavage loss was determined at 25 degrees C as well as after heat shock. Kinetics were dependent on the poison, nevertheless, loss rate overall was slow at the histone gene cluster, an active chromatin domain. At the repressed satellite III DNA, loss of cleavage was much faster and complete after 5 min in drug-free medium. In addition, differences were noted among sites that were closely spaced and equally intense. Following heat shock at 37 degrees C, we observed reduced cleavage levels and faster loss of breaks at the histone gene cluster. In vitro reversal could only partially explain the in vivo kinetics. Thus, the chromatin context of DNA breaks might play a role in the loss of top2 DNA breaks. The present findings suggest that irreversible cuts may more likely occur in active than silent loci.

Animals↗
PubMed2000

In vivo site specificity and human isoenzyme selectivity of two topoisomerase II-poisoning anthracyclines.

Anthracyclines exert antitumor activity by stimulating site-selective DNA cleavage by topoisomerase II (top2). DNA cleavage sites stimulated by two anthracycline analogues, dh-EPI and da-IDA, were investigated at the histone gene cluster of cultured Drosophila Kc cells. The two agents stimulated analogue-specific patterns of double-stranded DNA cleavage in Kc cell chromatin. Analyses of 47 base sequences of dh-EPI sites showed that the analogue largely followed the in vitro selectivity rule, the requirement of (5')TA at 3' ends of cleaved strands. da-IDA was more selective than dh-EPI, and thus fewer sites could be collected. Nevertheless, base sequences were consistent with its in vitro base preferences. DNA cleavage was then studied in vitro with Drosophila and human top2 isoforms. The tested drugs stimulated distinct in vitro patterns that corresponded to the in vivo patterns. Human top2alpha promoted cleavage patterns that were much more similar to those of Drosophila top2 (both in vitro and in vivo) than human top2beta. Moreover, da-IDA showed a marked site-dependent preference for human top2beta. Thus, DNA site selection in vivo is different for the test anthracyclines, and together with a degree of beta-form specificity, may affect drug activity in human cells.

Animals↗
PubMed1999

Tethering a type IB topoisomerase to a DNA site by enzyme fusion to a heterologous site-selective DNA-binding protein domain.

Topoisomerase IB (Top1) has essential functions in higher eukaryotes, but effective anticancer agents can transform it into a lethal DNA-cleaving toxin. Fusion of the yeast Gal4 DNA-binding protein domain (amino acids 1-105; Gal4DBD) to the NHz terminus of full-length human Top1 results in a GalTop chimera that maintains basic properties of the two parent proteins. DNA cleavage and binding activities of GalTop were then compared to Top1 to establish whether the fusion protein had altered site specificity. Under conditions of reduced binding of Top1 to DNA, Gal4DBD was able to selectively anchor the chimera on a template containing a Gal4 consensus motif, thus bringing Top1 to cleave 20-40-bp sequences close to the Gal4 motif with high specificity. Footprinting analyses showed that the chimera protected a DNA region that was wider than that protected by a Gal4DBD protein fragment, consistent with the cleavage results. The data demonstrate that a Top1 can be targeted to a specific DNA site by protein fusion to a heterologous DNA-binding domain. Such hybrid topoisomerase-derived enzymes may be useful for directing Top1 activity to specific genomic loci in living cells.

Binding Sites↗
PubMed1999

Configurational requirements of the sugar moiety for the pharmacological activity of anthracycline disaccharides.

The amino sugar is recognized to be a critical determinant of the activity of anthracycline monosaccharides related to doxorubicin and daunorubicin. In an attempt to improve the pharmacological properties of such agents, novel anthracycline disaccharides have been designed in which the amino sugar, daunosamine, is separated from the aglycone by another carbohydrate moiety. In the present study, we examined the influence of the orientation of the second sugar residue on drug biochemical and biological properties in a series of closely related analogs. This structure-activity relationship study showed that the substitution of the daunosamine for the disaccharide moiety dramatically reduced the cytotoxic potency of the drug in the 4-methoxy series (daunorubicin analogs). In contrast, in the 4-demethoxy series (idarubicin analogs), the C-4 axial, but not the equatorial, configuration conferred a cytotoxic potency and antitumor activity comparable to that of doxorubicin. The configuration also influenced the drug's ability to stimulate topoisomerase II alpha-mediated DNA cleavage. Indeed, the glycosides with the equatorial orientation were ineffective as topoisomerase II poisons, whereas the compounds with axial orientation were active, although the daunorubicin analog exhibited a lower activity than the idarubicin analog. It is conceivable that the axial orientation allows an optimal interaction of the drug with the DNA-enzyme complex only in the absence of the methoxy group. Our results are consistent with a critical role of the sugar moiety in drug interaction with the target enzyme in the ternary complex.

Animals↗
PubMed1999

Genomic sites of topoisomerase II activity determined by comparing DNA breakage enhanced by three distinct poisons.

To define the sites of topoisomerase II activity in two genomic regions of Drosophila melanogaster Kc cells, we have investigated in vivo DNA cleavage sites stimulated by three poisons with diverse sequence specificity, clerocidin, VM-26 and dh-EPI (an anthracycline analog). DNA cleavage was studied by PFGE (pulse-field gel electrophoresis), standard gel electrophoresis, and by genomic primer extension. Poisons stimulated specific intensity patterns of cleavage in the two genomic regions studied. At the centromeric satellite III DNA, fragments of about 270-310 and 385-430 kb could be detected specifically after treatment with clerocidin, suggesting a complex DNA loop organisation, which may correspond with a centromere-specific higher-order chromatin structure. Clerocidin-dependent DNA fragmentation was detectable by PFGE but not by standard agarose gel electrophoresis; while VM-26-dependent cleavage was detected with either method, dh-EPI was ineffective at this locus. Thus, clerocidin DNA cleavage sites were rarer than those of VM-26 at the satellite locus. In the histone H2A-H2B intergenic region, clerocidin and dh-EPI stimulated cleavage whereas VM-26 was only weakly effective. Some clerocidin cleavage sites did not undergo spontaneous reversion, indicating that this agent can stimulate irreversible cleavage in vivo. Direct genomic sequencing showed that many clerocidin and dh-EPI sites, although distinct, mapped to the transcription start and to the proximal promoter of the H2A gene, suggesting that the region is highly accessible to topoisomerase II. Thus, the enzyme may play a role in maintaining a highly accessible chromatin structure under normal cell growth conditions, possibly mediated by specialised protein complexes. This study demonstrates that the use of distinct poisons greatly improves the definition of genomic sites of topoisomerase II activity.

Animals↗
PubMed1999

Topoisomerase poisoning activity of novel disaccharide anthracyclines.

Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I.

Antibiotics, Antineoplastic↗
PubMed1999

Drugs acting on DNA topoisomerases: recent advances and future perspectives.

DNA-topoisomerases, a family of DNA-processing enzymes, represent the pharmacological target of major clinically useful chemotherapeutic agents. These drugs essentially act by trapping a topoisomerase-DNA cleavable complex, an intermediate in the enzyme s catalytic cycle. Research activity in this field continues to grow exponentially, resulting in a wealth of new information on the functional role and the biochemical and structural properties of the enzymes. In addition, the drug pharmacophores have been further characterized, along with their sequence preferences, and key interactions with the target macromolecules are being unveiled. This review will discuss the recent advances in elucidating the mode of action of DNA-topoisomerases and of topoisomerase-targeted anticancer agents.

Animals↗
PubMed1998

DNA sequence selectivity of topoisomerases and topoisomerase poisons.

Chemical agents able to interfere with DNA topoisomerases are widespread in nature, and some of them have clinical efficacy as antitumor or antibacterial drugs. Drugs which have as a target DNA topoisomerases could be divided into two categories: poisons and catalytic inhibitors. Classical topoisomerase poisons stimulate cleavage in a sequence-selective manner, yielding drug-specific cleavage intensity pattern. The mechanisms of drug interaction with DNA topoisomerases, the DNA sequence selectivity of the action of topoisomerase II poisons and the identification of structural determinants of their activity have suggested that topoisomerase II poisons may fit into a specific pharmacophore, constituted by a planar ring system with DNA intercalation or intercalation-like properties, and protruding side chains interfering with the protein side of the covalent enzyme-DNA complex. The complete definition of the diverse pharmacophores of topoisomerase II poisons will certainly be of value for the design of new agents directed to specific genomic sites, and more effective in the treatment of human cancer.

Antineoplastic Agents↗
PubMed1998

Mapping drug interactions at the covalent topoisomerase II-DNA complex by bisantrene/amsacrine congeners.

To identify structural determinants for the sequence-specific recognition of covalent topoisomerase II-DNA complexes by anti-cancer drugs, we investigated a number of bisantrene congeners, including a 10-azabioisoster, bearing one or two 4, 5-dihydro-1H-imidazol-2-yl hydrazone side chains at positions 1, 4, or 9 of the anthracene ring system. The studied bisantrene/amsacrine (m-AMSA) hybrid and bisantrene isomers were able to poison DNA topoisomerase II with an intermediate activity between those of bisantrene and m-AMSA. Moving the side chain from the central to a lateral ring (from C-9 to C-1/C-4) only slightly modified the drug DNA affinity, whereas it dramatically affected local base preferences of poison-stimulated DNA cleavage. In contrast, switching the planar aromatic systems of bisantrene and m-AMSA did not substantially alter the sequence specificity of drug action. A computer-assisted steric and electrostatic alignment analysis of the test compounds was in agreement with the experimental data, since a common pharmacophore was shared by bisantrene, m-AMSA, and 9-substituted analogs, whereas the 1-substituted isomer showed a radically changed pharmacophoric structure. Thus, the relative space occupancy and electron distribution of putative DNA binding (aromatic rings) and enzyme binding (side chains) moieties are fundamental in directing the specific action of topoisomerase II poisons and in determining the poison pharmacophore.

Amsacrine↗
PubMed1998

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues.

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.

Antibiotics, Antineoplastic↗