RESUMEN
The human telomeric G-quadruplex structural motif of DNA has come to be known as a new and stimulating target for anticancer drug discovery. Small molecules that interact with G-quadruplex structures in a selective way have gained impressive interest in recent years as they may serve as potential therapeutic agents. Here, we show how circular dichroism, UV resonance Raman and small angle X-ray scattering spectroscopies can be effectively combined to provide insights into structural and molecular aspects of the interaction between human telomeric quadruplexes and ligands. This study focuses on the ability of berberine and palmatine to bind with human telomeric quadruplexes and provides analysis of the conformational landscape visited by the relevant complexes upon thermal unfolding. With increasing temperature, both free and bound G-quadruplexes undergo melting through a multi-state process, populating different intermediate states. Despite the structural similarity of the two ligands, valuable distinctive features characterising their interaction with the G-quadruplex emerged from our multi-technique approach.
Asunto(s)
Alcaloides de Berberina/metabolismo , Berberina/metabolismo , ADN/metabolismo , G-Cuádruplex , Berberina/química , Alcaloides de Berberina/química , Dicroismo Circular , ADN/química , ADN/genética , Humanos , Ligandos , Dispersión del Ángulo Pequeño , Espectrometría Raman , Difracción de Rayos XRESUMEN
The binding properties toward the human telomeric G-quadruplex of the two natural alkaloids coptisine and chelerythrine were studied using spectroscopic techniques, molecular modeling, and X-ray diffraction analysis. The results were compared with reported data for the parent compounds berberine and sanguinarine. Spectroscopic studies showed modest, but different rearrangements of the DNA-ligand complexes, which can be explained considering particular stereochemical features for these alkaloids, in spite of the similarity of their skeletons. In fact, the presence of a dioxolo moiety rather than the two methoxy functions improves the efficiency of coptisine and sanguinarine in comparison to berberine and chelerythrine, and the overall stability trend is sanguinarine > chelerythrine ≈ coptisine > berberine. Accordingly, the X-ray diffraction analysis confirmed the involvement of the benzodioxolo groups in the coptisine/DNA binding by means of π···π, O···π, and CH···O interactions. Similar information is provided by modeling studies, which, additionally, evidenced reasons for the quadruplex vs double-helix selectivity shown by these alkaloids. Thus, the analyses shed light on the key role of the benzodioxolo moieties in strengthening the interaction with the G4-folded human telomeric sequence and indicated the superior G4 stabilizing properties of the benzophenanthridine scaffold with respect to the protoberberine one and conversely the better G4 vs dsDNA selectivity profile of coptisine over the other alkaloids.
Asunto(s)
Alcaloides/química , Benzodioxoles/química , Benzofenantridinas/química , Berberina/análogos & derivados , ADN/química , Berberina/química , Alcaloides de Berberina/química , Cristalografía por Rayos X/métodos , G-Cuádruplex , Humanos , Isoquinolinas/química , LigandosRESUMEN
The interactions of three representative gold(III) complexes with human telomeric DNA sequences were analysed using a variety of biophysical methods, including DNA melting, circular dichroism, SPR and ESI MS; remarkable interactions were highlighted for all tested complexes, although they were associated to significantly different binding profiles. The most interesting compound was Auoxo6, a dinuclear gold(III) complex, which beyond manifesting a conspicuous binding affinity for the G-quadruplex conformation, turned out to be very effective in inducing a non-canonical secondary structure. These findings may clear the way for novel biological and pharmacological applications of this class of metal compounds.
Asunto(s)
Complejos de Coordinación/química , G-Cuádruplex , Oro/química , Dicroismo Circular , Complejos de Coordinación/síntesis química , Humanos , Conformación de Ácido Nucleico , Espectrometría de Masa por Ionización de Electrospray , Resonancia por Plasmón de SuperficieRESUMEN
Platinum-complexes represent some of the most successful groups of clinically used anticancer drugs. Their mechanism of action relies on the formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7) and involving one or two spatially close residues. The formation of stable DNA adducts is recognized as a DNA damaging event and, ultimately, drives cells to death. Nevertheless, nucleobases are not the only reliable targets of these drugs and other biomolecules can be involved. Among them large interest has been devoted to proteins since they contain several potential reactive sites for platinum (His, Met, and Cys) and, in particular, because the reaction of the metal with sulfur containing groups is a kinetically favored process. As a result, the occurrence of protein adducts and DNA-protein cross-links must be further taken into account in order to fully define cisplatin mechanism of action. Herein, we will summarize the most recent experimental evidence collected so far on protein-cisplatin adduct formation to better dissect its correlation with the drug pharmacological profile. Indeed, in addition to modulation of drug bioavailability and toxicity, the potential role of proteins as reaction intermediates or reservoir systems in platinum drugs can be envisaged. Additionally, the effects of Pt-coordinating groups on the chemical reactivity of the metal complexes will be reviewed. From all these outcomes a general model for Pt-based drugs mechanism of action can be drawn which is more articulate than the one currently supported. It claims proteins as reactive intermediates for DNA platination and it defines them as relevant to fully describe the clinical potential of this class of anticancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Compuestos Organoplatinos/farmacología , Proteínas/metabolismo , Animales , Antineoplásicos/química , Cisplatino/química , Humanos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Compuestos Organoplatinos/química , Proteínas/químicaRESUMEN
Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.
Asunto(s)
G-Cuádruplex , Compuestos Organometálicos/química , Fenantrolinas/química , Dicroismo Circular , ADN/efectos de los fármacos , ADN/metabolismo , División del ADN/efectos de los fármacos , Humanos , Ligandos , Metales/química , Estructura Molecular , Compuestos Organometálicos/síntesis química , Fenantrolinas/síntesis química , Fenantrolinas/farmacología , Telomerasa/antagonistas & inhibidores , Telomerasa/química , Telomerasa/metabolismoRESUMEN
Simocyclinone D8, a coumarin derivative isolated from Streptomyces antibioticus Tü 6040, represents an interesting new antiproliferative agent. It was originally suggested that this drug recognizes the GyrA subunit and interferes with the gyrase catalytic cycle by preventing its binding to DNA. To further characterize the mode of action of this antibiotic, we investigated its binding to the reconstituted DNA gyrase (A(2)B(2)) as well as to its GyrA and GyrB subunits and the individual domains of these proteins, by performing protein melting and proteolytic digestion studies as well as inhibition assays. Two binding sites were identified, one (anticipated) in the N-terminal domain of GyrA (GyrA59) and the other (unexpected) at the C-terminal domain of GyrB (GyrB47). Stabilization of the A subunit appears to be considerably more effective than stabilization of the B subunit. Our data suggest that these two distinct sites could cooperate in the reconstituted enzyme.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/química , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Dicroismo Circular , Cumarinas/farmacología , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/metabolismo , ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Glicósidos/farmacología , Termodinámica , Inhibidores de Topoisomerasa IIRESUMEN
Aptamers are short DNA- or RNA-based oligonucleotides selected from large combinatorial pools of sequences for their capacity to efficiently recognize targets ranging from small molecules to proteins or nucleic acid structures. Like antibodies, they exhibit high specificity and affinity for target binding. As a result, they may display effective interference in biological processes, which renders them not only valuable diagnostic tools, but also promising therapeutic agents. In fact, one aptamer that inhibits human VEGF already received approval for the treatment of age-related macular degeneration, while several others are undergoing clinical trials. Aptamers display a large number of structural arrangements, which accounts for their binding efficiency and selectivity for unrelated targets. Among several architectures, the G-quadruplex (G-4) is adopted by several aptamers, the most popular of which shows inhibitory properties against thrombin, a pharmacologically relevant protein. G-4 structures consist of planar arrays of four guanines, each guanine pairing with two neighbours by Hoogsteen bonding. Recent work shows that G-4 arrangement is highly polymorphic and therefore represents a large family of stable structures with a common overall fold, but with well differentiated recognition elements that allow prominent diversity to be explored. Conformational plasticity consents fine tuning of target recognition as obtained by aptamer selection. Here, we will review the present knowledge on aptamers based on the G-4 structures and assess their diagnostic and therapeutic potential as biotech drugs for the detection and treatment of severe pathologies including vascular, cancer and viral diseases.
Asunto(s)
Diseño de Fármacos , G-Cuádruplex , Neoplasias , Enfermedades Vasculares , Virosis , Anticoagulantes/química , Anticoagulantes/uso terapéutico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Antirreumáticos/química , Antirreumáticos/uso terapéutico , Antivirales/química , Antivirales/uso terapéutico , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/uso terapéutico , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Enfermedades Vasculares/diagnóstico , Enfermedades Vasculares/tratamiento farmacológico , Virosis/diagnóstico , Virosis/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Girasa de ADN/química , Metales/farmacología , Sitios de Unión , Catálisis , Cationes Bivalentes/farmacología , Cationes Monovalentes/farmacología , Dicroismo Circular , Girasa de ADN/genética , Estabilidad de Enzimas/efectos de los fármacos , Proteínas de Escherichia coli/química , Calor , Mutación Missense , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , TermodinámicaRESUMEN
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.
Asunto(s)
Antitrombinas/metabolismo , Factor Xa/metabolismo , Heparina de Bajo-Peso-Molecular/química , Sulfatos/farmacología , Trombina/metabolismo , Interacciones Farmacológicas , Inhibidores del Factor Xa , Humanos , Concentración 50 Inhibidora , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Trombina/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Etanolaminas/uso terapéutico , Sustancias Intercalantes/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Pirazoles/uso terapéutico , Pirazolonas , Animales , Antraquinonas/uso terapéutico , Antígenos de Neoplasias , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Neoplasias/efectos de los fármacos , Proteínas de Unión al ADN , Doxorrubicina/uso terapéutico , Etanolaminas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes bcl-2 , Humanos , Sustancias Intercalantes/farmacología , Masculino , Ratones , Ratones Desnudos , Estructura Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Pirazoles/farmacología , Inducción de Remisión , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antiinfecciosos/farmacología , Cationes Bivalentes/farmacología , Ciprofloxacina/farmacología , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Magnesio/farmacología , Sustitución de Aminoácidos/genética , Calcio/farmacología , Cromatografía de Afinidad , Dicroismo Circular , ADN-Topoisomerasas de Tipo II/genética , Farmacorresistencia Microbiana , Estabilidad de Enzimas/efectos de los fármacos , Peso Molecular , Mutación/genética , Concentración Osmolar , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Subunidades de Proteína , Sales (Química)/farmacología , Temperatura , TermodinámicaRESUMEN
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.
Asunto(s)
Antraquinonas/farmacología , Antineoplásicos/farmacología , ADN/efectos de los fármacos , Mitoxantrona/farmacología , Pirazoles/farmacología , Pirazolonas , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Clostridium perfringens/genética , ADN/metabolismo , Huella de ADN , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , Desoxirribonucleasa I/metabolismo , Fluorometría , Sustancias Intercalantes , Micrococcus/genética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Plásmidos/genética , Timo/químicaRESUMEN
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.
Asunto(s)
Camptotecina/análogos & derivados , Camptotecina/análisis , Inhibidores Enzimáticos/análisis , Topotecan/análisis , Humanos , Irinotecán , Inhibidores de Topoisomerasa IRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/análisis , Antibióticos Antineoplásicos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Daunorrubicina/análisis , Daunorrubicina/aislamiento & purificación , Doxorrubicina/análisis , Doxorrubicina/aislamiento & purificación , Electroforesis Capilar , Epirrubicina/análisis , Epirrubicina/aislamiento & purificación , Idarrubicina/análisis , Idarrubicina/aislamiento & purificación , Espectrofotometría UltravioletaRESUMEN
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.
Asunto(s)
Cobre/farmacología , Ciclohexanoles/farmacología , Ciclohexilaminas/farmacología , Zinc/farmacología , Cationes/farmacología , Ciclohexanoles/química , Ciclohexilaminas/química , ADN Bacteriano/efectos de los fármacos , ADN Superhelicoidal/efectos de los fármacos , Hidrólisis , Cinética , Estructura Molecular , Organofosfatos/metabolismo , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antiinfecciosos/síntesis química , Inhibidores Enzimáticos/síntesis química , Quinolonas/síntesis química , Tetrahidroisoquinolinas , Inhibidores de Topoisomerasa II , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Flúor/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Magnesio/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinolonas/química , Quinolonas/metabolismo , Quinolonas/farmacología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antraquinonas/metabolismo , ADN/metabolismo , Animales , Antraquinonas/química , Antraquinonas/farmacología , Compuestos Aza/química , Compuestos Aza/metabolismo , Compuestos Aza/farmacología , Bovinos , ADN/química , ADN Superhelicoidal/metabolismo , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Sustancias Intercalantes/farmacología , Modelos Moleculares , Espectrofotometría , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por Sustrato , Inhibidores de Topoisomerasa IIRESUMEN
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.
Asunto(s)
Aminoglicósidos , Antibacterianos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Antibacterianos/química , Secuencia de Bases , Unión Competitiva , ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Enediinos , Escherichia coli/genética , Proteínas Fúngicas/farmacología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacáridos/química , Polisacáridos/genética , Polisacáridos/farmacología , Proteínas Quinasas/farmacología , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
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.