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C Sissi

Publications and source records attributed to C Sissi.

At least 19 recordsLinked to original sources
PubMed2005

The effects of metal ions on the structure and stability of the DNA gyrase B protein.

The effects of mono- and divalent metal ions on the DNA gyrase B subunit, on its 43 kDa and 47 kDa domains, and on two mutants in the Toprim domain (D498A and D500C) were investigated by means of circular dichroism and protein melting experiments. Both types of metal ion, with the notable exception of Mn2+, did not affect the conformational properties of the enzyme subunit at room temperature, but were able to produce selective and differential effects on protein stability. In particular, monovalent (K+) ions increased the stability of the gyrase B structure, whereas destabilising effects were most prominent using Mn2+ as the metal ion. Ca2+ and Mg2+ produced comparable changes in the gyrase B melting profile. Additionally, we found that monovalent (K+) ions were more effective in the 43 kDa N-terminal domain where ATP binding occurs, whereas divalent ions caused large modifications in the conformational stability of the 47 kDa C-terminal domain. Our results on gyrase B mutants indicate that D498 interacts with Mn2+, whereas it has little effect on the binding of the other ions tested. A D500C mutation, in contrast, effectively impairs Mg2+ affinity, suggesting effective contacts between this ion and D500 in the wild-type enzyme. Hence, the sites of metal ion complexation within the Toprim domain are modulated by the nature of the ion species. These results suggest a double role played by metal ions in the catalytic steps involving DNA gyrase B. One has to do with direct involvement of cations complexed to the Toprim domain in the DNA cutting-rejoining process, the other, until now overlooked, is connected to the dramatic changes in protein flexibility produced by ion binding, which reduces the energy required for the huge conformational changes essential for the catalytic cycle to occur.

Binding Sites↗
PubMed2001

Quantitation of camptothecin and related compounds.

Camptothecin and congeners represent a clinically very useful class of anticancer agents. Proper identification and quantitation of the original compounds and their metabolites in biological fluids is fundamental to assess drug metabolism and distribution in animals and in man. In this paper we will review the recent literature available on the methods used for separation and quantitative determination of the camptothecin family of drugs. Complications arise from the fact that they are chemically labile, and the pharmacologically active lactone structure can undergo ring opening at physiological conditions. In addition, a number of metabolic changes usually occur, producing a variety of active or inactive metabolites. Hence, the conditions of extraction, pre-treatment and quantitative analysis are to be carefully calibrated in order to provide meaningful results.

Camptothecin↗
PubMed2001

Anthracyclines: recent developments in their separation and quantitation.

Anthracyclines are among the most widely used anticancer agents. Notwithstanding the large efforts to develop new drugs with a better pharmaceutical profile, daunorubicin, doxorubicin, epirubicin and idarubicin are still the most used in clinical practice. Many efforts are now ongoing to reduce the side effects by using pharmaceutical formulations able to release the drug in the most appropriate way and monitoring the quantity of anthracyclines and their metabolites in the body fluids or tissues frequently and in every patient to maintain the drug concentration within the expected range. This review describes the most recent developments in the separation and quantitation of the above clinically useful drugs, together with their principal metabolites. Some less widely used derivatives will also be considered.

Antibiotics, Antineoplastic↗
PubMed2001

Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions.

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59. The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA. We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.

Amino Acid Substitution↗
PubMed2001

Effects of sulfation on antithrombin-thrombin/factor Xa interactions in semisynthetic low molecular weight heparins.

Most of the biological effects of heparin and low molecular weight (LMW) heparins are related to their ability to bind to many different proteins. To gain insight into structure-activity relationships, we investigated quantitatively the interactions of a series of sulfated LMW heparins of similar molecular weights (derived from statistical desulfation of a supersulfated heparin) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation of the protease inhibitor against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the degree of sulfation of the LMW heparins was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the heparin derivatives in activating AT toward the proteases is generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated LMW heparin showed serpin activation ability higher than expected from the affinity studies. These results indicate that chemical modification of the sulfation pattern of LMW heparin can be used to efficiently modulate binding affinity and activity toward biological targets.

Antithrombins↗
PubMed2001

DNA-interactive anticancer aza-anthrapyrazoles: biophysical and biochemical studies relevant to the mechanism of action.

The physicochemical and DNA-binding properties of anticancer 9-aza-anthrapyrazoles (9-aza-APs) were investigated and compared with the carbocyclic analogs losoxantrone (LX) and mitoxantrone (MX). Unlike their carbocyclic counterparts, the tested 9-aza-APs do not undergo self-aggregation phenomena. The pyridine nitrogen at position 9, missing in the carbocyclic derivatives, is involved in protonation equilibria at physiological pH. In addition, 9-aza-APs are electrochemically reduced at a potential intermediate between LX and MX. These data fully agree with quantum mechanical calculations. Binding to nucleic acids was examined by spectroscopic, chiroptical, and DNase I footprinting techniques as a function of ionic strength and base composition. The 9-aza-APs exhibit prominent affinity for DNA, with an important electrostatic contribution to the binding free energy. A very remarkable sequence preference pattern dramatically favors GC steps in double-helical DNA, whereas the carbocyclic reference compounds show a substantially lower selectivity for GC. A common DNA complexation geometry, considerably differing from that of MX, characterizes all anthrapyrazoles. Hence, bioisosteric substitution and ring-hydroxy deletion play an important role in defining the physicochemical properties and in modulating the affinity of anthrapyrazoles for the nucleic acid, the geometry of the intercalation complex, and the sequence specific contacts along the DNA chain. Drug stimulation of topoisomerase II-mediated DNA cleavage is remarkably attenuated in the aza-bioisosteric derivatives, suggesting that other non-enzyme-mediated cytotoxic mechanism(s), possibly connected with free radical production, are responsible for efficient cell killing. The biophysical and biochemical properties exhibited by 9-aza-APs contribute to clarifying the peculiar pharmacological profile of this family of compounds.

Animals↗
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↗
PubMed2000

DNA phosphodiester bond hydrolysis mediated by Cu(II) and Zn(II) complexes of 1,3,5,-triamino-cyclohexane derivatives.

The hydrolytic activity of the 1,3,5-triaminocyclohexane derivatives TACH, TACI and TMCA complexed to Zn(II) and Cu(II) towards a model phosphoric ester and plasmid DNA has been evaluated by means of spectroscopic and gel-electrophoresis techniques. At conditions close to physiological, a prominent cleavage effect mediated by the nature of the ligand and metal ion was generally observed. TACI complexes are the most active in relaxing supercoiled DNA, the effect being explained by the affinity of the hydroxylated ligand for the nucleic acid. As indicated by the dependence of cleavage efficiency upon pH, Zn(II)-complexes act by a purely hydrolytic mechanism. In the case of Cu(II)-complexes, although hydrolysis should be prominent, involvement of an oxidative pathway cannot be completely ruled out.

Cations↗
PubMed2000

6-hydroxy derivative as new desfluoroquinolone (DFQ): synthesis and DNA-binding study.

A new 6-desfluoroquinolone derivative, characterized by the presence of a 6-hydroxyl group instead of the usual fluorine atom at the C-6 position, was synthesized with the aim to better understand the mechanistic role of the C-6 substituent in the quinolone/DNA/DNA-gyrase interaction. The antibacterial activity unambiguously shows that the hydroxyl group is a good substitute for the C-6 fluorine atom, especially against Gram-positive bacteria. On the contrary, it is a very weak inhibitor of the target DNA gyrase, displaying the highest IC50 value observed for all the C-6 substituted analogues. This behaviour could be explained on the basis of its DNA binding properties.

Anti-Infective Agents↗
PubMed1999

Interaction of calicheamicin gamma1(I) and its related carbohydrates with DNA-protein complexes.

We report studies of the contribution of DNA structure, holding the sequence constant, to the affinity of calicheamicin gamma(1)(I) and its aryltetrasaccharide moiety for DNA. We used polynucleotide chains as models of known protein-binding sequences [the catabolite activator protein (CAP) consensus sequence, AP-1 and cAMP response element (CRE) sites] in their free and protein-bound forms. The proteins were selected to provide examples in which the minor-groove binding site for the carbohydrate is (CAP) or is not (GCN4) covered by the protein. Additionally, peptides related to the GCN4 and CREB families, which have different bending effects on their DNA-binding sites, were used. We observe that proteins of the CREB class, which induce a tendency to bend toward the minor groove at the center of the site, inhibit drug-cleavage sites located at the center of the free AP-1 or CRE DNA sites. In the case of GCN4, which does not induce DNA bending, there is no effect on calicheamicin cleavage of the CRE site, but we observe a GCN4-induced rearrangement of the cutting pattern in the AP-1 site. This effect may arise from either a subtle local conformational rearrangement not accompanied by bending or a localized reduction in DNA flexibility. Whereas GCN4 binding is not inhibited by the calicheamicin aryltetrasaccharide, binding of CAP to its DNA target is significantly inhibited, and calicheamicin cutting of DNA at the center of the CAP-DNA complex site is strongly reduced by protein binding. This result probably reflects steric inhibition of drug binding by the protein.

Aminoglycosides↗
PubMed1999

Design and synthesis of modified quinolones as antitumoral acridones.

The bacterial topoisomerase II (DNA gyrase) and the mammalian topoisomerase II represent the cellular targets for quinolone antibacterials and a wide variety of anticancer drugs, respectively. In view of the mechanistic similarities and sequence homologies exhibited by the two enzymes, tentative efforts to selectively shift from an antibacterial to an antitumoral activity was made by synthesizing a series of modified tricyclic quinolones, in which the essential 3-carboxylic function is surrogated by phenolic OH and the classic C-6 fluorine atom is replaced by a NH2 group. The resulting 7-amino-9-acridone derivatives were assayed for their antibacterial as well as cytotoxic activities. No antibacterial activity was found. On the other hand, many derivatives showed significant cytotoxic activity against both HL-60 and P388 leukemias and a wide panel of human and rodent solid tumor cells, derivatives 25 and 26 displaying the best overall antiproliferative activity. Against the LoVo cell line, derivative 25 exhibited higher cytotoxic effects than etoposide.

Acridines↗
PubMed1999

Binding of bis-substituted 2-aza-anthracenedione regioisomers to DNA: effects of the relative positioning of the side chains.

The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) derivatives bearing two 3-dimethylaminopropylamino side chains at different (6,9, 7,9 and 8,9) positions of the planar ring system have been investigated. The affinity for the nucleic acid is dramatically affected by the substitution pattern, the 6,9-regioisomer being substantially more effective than the 7,9- or the 8,9-congeners. This cannot be ascribed to different binding mechanisms, as all compounds are shown to intercalate into the double helix. Instead, the geometry of intercalation into DNA and the site specificity are extensively affected by the substitution pattern. The site preference is CA (or AC) for the 6,9-regioisomer, whereas it is TA (or AT) for the 8,9-congener, the 7,9-analogue lying in between. Molecular modeling studies are in agreement with the experimental results. Although the 6,9-regioisomer was remarkably cytotoxic, it stimulated topoisomerase II-mediated cleavage of DNA very poorly. Hence, a different mechanism of DNA damage is probably operating in 2-aza-anthracenediones as the main cell-killing event. Changes in affinity for DNA, intercalation geometry and sequence specificity can explain the different cytotoxic responses exhibited by the test drugs.

Animals↗
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

Mg(2+)-mediated binding of 6-substituted quinolones to DNA: relevance to biological activity.

The interaction of a number of novel 6-substituted quinolone derivatives with DNA in the presence/absence of magnesium ions has been investigated by fluorometric techniques. The drug-single-stranded nucleic acid interaction is invariantly mediated by the metal ion. In all cases optimal complex formation is found at physiological Mg2+ concentration. From titrations at different [Mg2+] the binding constant for the ternary drug-DNA-Mg2+ complex (KT) has been evaluated. Interestingly, a good relationship is found between KT and gyrase poisoning activity of the test quinolones (IC50), which confirms that DNA-affinity of the quinolone, modulated by Mg2+, plays an important role in poisoning the cleavable gyrase-DNA complex and, consequently, in eliciting antibacterial activity in this family of drugs. The results obtained with different 6-substituted compounds supports the idea that position 6 of the drug, besides playing a pharmacokinetic role, is involved in recognition of the enzyme pocket. Our data do not support a mechanism of action based upon quinolone intercalation into B-DNA.

Binding Sites↗
PubMed1998

DNA-binding preferences of bisantrene analogues: relevance to the sequence specificity of drug-mediated topoisomerase II poisoning.

To elucidate structure-activity relationships for drugs that are able to poison or inhibit topoisomerase II, we investigated the thermodynamics and stereochemistry of the DNA binding of a number of anthracene derivatives bearing one or two 4, 5-dihydro-1H-imidazol-2-yl-hydrazone side chains (characteristic of bisantrene) at different positions of the planar aromatic system. An aza-bioisostere, which can be considered a bisantrene-amsacrine hybrid, was also tested. The affinity for nucleic acids in different sequence contexts was evaluated by spectroscopic techniques, using various experimental conditions. DNA-melting and DNase I footprinting experiments were also performed. The location and number of the otherwise identical side chains dramatically affected the affinity of the test compounds for the nucleic acid. In addition, the new compounds exhibited different DNA sequence preferences, depending on the locations of the dihydroimidazolyl-hydrazone groups, which indicates a major role for the side-chain position in generating specific contacts with the nucleic acid. Molecular modeling studies of the intercalative binding of the 1- or 9-substituted isomers to DNA fully supported the experimental data, because a substantially more favorable recognition of A-T steps, compared with G-C steps, was found for the 9-substituted derivative, whereas a much closer energy balance was found for the 1-substituted isomer. These results compare well with the alteration of base specificity found for the topoisomerase II-mediated DNA cleavage stimulated by the isomeric drugs. Therefore, DNA-binding specificity appears to represent an important determinant for the recognition of the topoisomerase-DNA cleavable complex by the drug, at least for poisons belonging to the amsacrine-bisantrene family.

Animals↗
PubMed1997

Physicochemical properties, cytotoxic activity and topoisomerase II inhibition of 2,3-diaza-anthracenediones.

The physicochemical, cytotoxic and pharmacological properties of 2,3-diaza-anthracenedione derivatives were examined to gain insight into the structure-activity relationships in this class of compounds. Spectrophotometric, chiroptical and voltammetric measurements were performed, along with cell cytotoxicity, alkaline elution, topoisomerase II-mediated DNA cleavage and cellular drug-uptake determination. In comparison with classic anthracenediones such as mitoxantrone and ametantrone, the aza derivatives were characterized by less negative reduction potentials, lower affinity for DNA and modified geometry of intercalation. The biological effects of the new compounds were also profoundly affected by bioisosteric N for C replacement. Stimulation of topoisomerase II-mediated DNA cleavage was not observed, whereas other mechanisms of cell cytotoxicity, possibly involving oxidative DNA damage appeared to be operative. The inability to generate protein-associated strand breaks could be explained by an unfavorable orientation of the drug in the intercalation complex rather than by a reduced binding to DNA. Geometry of drug intercalation may have a critical influence on the formation of the ternary complex. In turn, the onset of a different DNA-damaging pathway is likely to be related to easy redox cycling of the 2,3-diaza-substituted anthracenedione derivatives, which could produce radical species to a remarkably greater extent than could the carbocyclic parent drugs.

Anthraquinones↗
PubMed1997

Preferred interaction of D-peptidyl-anthraquinones with double-stranded B-DNA.

The quest for more specific drugs in antitumor chemotherapy led us to the design of anthraquinone-peptide conjugates capable of selective recognition of the nucleic acid. We present here the DNA binding characteristics, sequence specificity and geometry of interaction of a pair of enantiomers containing the lysine-glycine dipeptide in the side chains. The D enantiomer binds right handed double stranded DNA more efficiently than the L form under all conditions tested. The source of higher binding affinity is not electrostatic in nature and rests in the more favorable hydrophobic contacts of the D-lysyl side chains in the drug-DNA complex. Both derivatives exhibit preference for alternating GC base sequences and intercalate into DNA in a threading mode as suggested by chiroptical and theoretical studies. The D enantiomer, being a peptidyl derivative that contains a non-natural amino acid, has the considerable advantage of being less susceptible to enzymatic hydrolysis and could therefore represent a lead compound for further development.

Anthraquinones↗