Double-strand DNA cleavage induced by oxindole-Schiff base copper(II) complexes with potential antitumor activity

Some oxindole-Schiff base copper(II) complexes have already shown potential antitumor activity towards different cells, inducing apoptosis in a process modulated by the ligand, and having nuclei and mitochondria as main targets. Here, three novel copper(II) complexes with analogous ligands were isolated and characterized by spectroscopic techniques, having their reactivity compared to the so far most active complex in this class. Cytotoxicity experiments carried out toward human neuroblastoma SH-SY5Y cells confirmed its proapoptosis property. DNA cleavage studies were then performed in the presence of these complexes, in order to verify the influence of ligand structural features in its nuclease activity. All of them were able to cause double-strand DNA scissions, giving rise to nicked circular Form II and linear Form III species, in the presence of hydrogen peroxide. Additionally, DNA Form II was also detected in the absence of peroxide when the most active complex, [Cu(isaepy)(2)](2+) 1, was used. In an effort to better elucidate their interactions with DNA, solutions of the different complexes titrated with DNA had their absorption spectra monitored. An absorbance hyperchromism observed at 260 nm pointed to the intercalation of these complexes into the DNA structure. Further, investigations of 2-deoxy-D-ribose (DR) oxidation catalyzed by each of those complexes, using 2-thiobarbituric acid reactive species (TBARS) method, and detection of reactive oxygen species (ROS) formation by spin-trapping EPR, suggested that their mechanism of action in performing efficiently DNA cleavage occurs preferentially, but not only by oxidative pathways. (C) 2007 Elsevier Inc. All rights reserved.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

Ligand Exchange Inducing Efficient Incorporation of CisPt Derivatives into Ureasil-PPO Hybrid and Their Interactions with the Multifunctional Hybrid Network

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of the interactions of DNA with the textile dyes Disperse Orange 1 and Disperse Red 1 and their electrolysis products using an electrochemical biosensor

A disposable pencil graphite electrode modified with dsDNA was used in combination with square wave voltammetry in order to evaluate the interaction of DNA with the textile dyes Disperse Orange 1 (DO1) and Disperse Red 1 (DR1), and with the products of their electrolysis. Significant changes in the characteristic oxidation peaks of the guanine and adenine moieties of immobilized dsDNA were observed after incubation of the modified electrode for 180 s in solutions of the dyes in their original forms. The same was observed using the electrolysis products obtained by oxidation and reduction conversions. The oxidation peak currents of the guanine and adenine moieties decreased when the concentrations of DO1 and DR1 were increased up to 5.0 × 10 -6 and 1.0 × 10-6 mol L-1, respectively; the signal decreases were more pronounced after interaction with the oxidized dyes, compared to the reduced compounds. The interactions between DNA and DO1, DR1, and the electrolyzed dyes were further investigated by UV-vis spectrophotometry in solution, and different effects such as hypochromism and hyperchromism were observed in the resulting DNA spectra. The investigated interactions showed clear evidence of changes in the DNA structure, and suggested a predominant intercalation mode leading to damage in the biomolecule. © 2013 Elsevier B.V.

Mitochondrial DNA mapping of social-biological interactions in Brazilian Amazonian African-descendant populations

ABSTRACT: The formation of the Brazilian Amazonian population has historically involved three main ethnic groups, Amerindian, African and European. This has resulted in genetic investigations having been carried out using classical polymorphisms and molecular markers. To better understand the genetic variability and the micro-evolutionary processes acting in human groups in the Brazilian Amazon region we used mitochondrial DNA to investigate 159 maternally unrelated individuals from five Amazonian African-descendant communities. The mitochondrial lineage distribution indicated a contribution of 50.2% from Africans (L0, L1, L2, and L3), 46.6% from Amerindians (haplogroups A, B, C and D) and a small European contribution of 1.3%. These results indicated high genetic diversity in the Amerindian and African lineage groups, suggesting that the Brazilian Amazonian African-descendant populations reflect a possible population amalgamation of Amerindian women from different Amazonian indigenous tribes and African women from different geographic regions of Africa who had been brought to Brazil as slaves. The present study partially mapped the historical biological and social interactions that had occurred during the formation and expansion of Amazonian African-descendant communities.

Influence of perylenediimide–pyrene supramolecular interactions on the stability of DNA-based hybrids: Importance of electrostatic complementarity

Aromatic pi–pi stacking interactions are ubiquitous in nature, medicinal chemistry and materials sciences. They play a crucial role in the stacking of nucleobases, thus stabilising the DNA double helix. The following paper describes a series of chimeric DNA–polycyclic aromatic hydrocarbon (PAH) hybrids. The PAH building blocks are electron-rich pyrene and electron-poor perylenediimide (PDI), and were incorporated into complementary DNA strands. The hybrids contain different numbers of pyrene–PDI interactions that were found to directly influence duplex stability. As the pyrene–PDI ratio approaches 1:1, the stability of the duplexes increases with an average value of 7.5 °C per pyrene–PDI supramolecular interaction indicating the importance of electrostatic complementarity for aromatic pi–pi stacking interactions.

DNA with Hydrophobic Base Substitutes: A Stable, Zipperlike Recognition Motif Based On Interstrand-Stacking Interactions

No Abstract

Role of electrostatic interactions for ligand recognition and specificity of peptide transporters.

BACKGROUND Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.

Hydrolytic behaviour of mono-and dithiolato-bridged dinuclear arene ruthenium complexes and their interactions with biological ligands

The hydrolysis and the reactivity of two dinuclear p-cymene ruthenium monothiolato complexes, [(η6-p-MeC6H4Pri)2Ru2Cl2(µ-Cl)(µ-S-m-9-B10C2H11)] (1) and [(η6-p-MeC6H4Pri)2¬Ru2Cl2(µ-Cl)¬(µ-S¬CH2-p-C6H4-NO2)] (2), and of two dinuclear p-cymene ruthenium dithiolato complexes, [(η6-p-MeC6H4Pri)2Ru2(µ-SCH2CH2Ph)2Cl2] (3) and [(η6-p-Me¬C6H4¬Pri)2¬Ru2(S¬CH2¬C6H4-p-O¬Me)2¬Cl2] (4) towards amino acids, nucleotides, and a single-stranded DNA dodecamer were studied using NMR and mass spectrometry. In aqueous solutions at 37 °C, the monothiolato com¬plexes 1 and 2 undergo rapid hydrolysis, irrespective of the pH value, the predominant species in D2O/acetone-d6 solution at equilibrium being the neutral hydroxo complexes [(η6-p-Me¬C6H4¬Pri)2Ru2(OD)2(µ-OD)(µ-SR)]. The dithiolato complexes 3 and 4 are stable in water under acidic conditions, but undergo slow hydrolysis under neutral and basic conditions. In both cases, the cationic hydroxo complexes [(η6-p-MeC6H4Pri)2Ru2(µ-SR)2¬(OD)¬(CD3CN)]+ are the only spe¬cies observed in D2O/CD3CN at equilibrium. Surprisingly, no adducts are observed upon addition of an excess of L-methionine or L-histidine to the aqueous solutions of the complexes. Upon addition of an excess of L-cysteine, on the other hand, 1 and 2 form the unusual cationic trithiolato complexes [(η6-p-MeC6H4Pri)2¬Ru2{µ-SCH2CH(NH2)COOH}2(µ-SR)]+ containing two bridging cysteinato li¬gands, while 3 and 4 yield cationic trithiolato complexes [(η6-p-MeC6H4Pri)2Ru2[µ-SCH2CH¬(NH2)COOH](µ-SR)2]+ containing one bridging cysteinato ligand. A representative of catio¬nic trithiolato complexes containing a cysteinato bridge of this type, [(η6-p-MeC6H4Pri)2¬Ru2[µ-S¬CH2CH(NH2)COOH](µ-SCH2-p-C6H4-But)2]+ (6) could be synthesised from the di¬thiolato complex [(η6-p-Me¬C6H4¬Pri)2-Ru2(S¬CH2¬C6H4-p-But)2Cl2] (5), isolated as the tetra¬fluo¬ro¬borate salt and fully characterised. Moreover, the mono- and dithiolato complexes 1 - 4 are inert toward nucleotides and DNA, suggesting that DNA is not a target of cytotoxic thiolato-bridged arene ruthenium complexes. In contrast to the trithiolato complexes, monothiolato and dithio¬lato complexes hydrolyse and react with L-cysteine. These results may have im¬portant implications for the mode of action of thiolato-bridged dinuclear arene ruthenium drug candidates, and suggest that their modes of action are different to those of other arene ruthenium complexes.

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^

Searching for the ideal DNA recognition code of covalent ligands

The combitiatorial approach restriction endonuclease protection selection and amplification REPSA was successfully used to determine ideal DNA interactions sites of covalent ligands. Unlike most other combinatorial methods, REPSA is based on inhibition of enzymatic cleavage by specific ligand-DNA complexes, which enables identification of binding sites of various ligands. However, the inherent nature of this technique posses a problem during selection of binding sites of covalent ligands. By modifying the technique according to the nature of the ligand, we demonstrate the flexibility of REPSA in identifying the preferred binding sites for monocovalent ligands, topoisomerase I and tallimustine, and the bicovalent ligand topoisomerase II. From among the preferred binding sites, we identified the consensus binding sequence of camptothecin induced topoisomerase I cleavage as ‘aGWT/Gc’, and tallimustine consensus sequences as ‘GTTCTA’ and ‘TTTTTTC’. We have shown for the first time that preferential binding of tallimustine occurs at sequences not previously reported. Furthermore, our data indicate that tallimustine is a novel DNA minor groove, guanine-specific alkylating agent. ^ Additionally, we have demonstrated in vivo that sequence-specific covalent DNA-binding small molecules have the ability to regulate transcription by inhibiting RNA polymerase II. Tallimustine, binding to its preferred sequences located in the 5′ untranslated region were an effective impediment for transcribing polymerase II. The ability of covalent binding small molecules to target predetermined DNA sequences located downstream of the promoter suggests a general approach for regulation of gene expression. ^

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

A three-hybrid system for detecting small ligand–protein receptor interactions

Small ligand–receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine. We report herein a genetic system, named the yeast three-hybrid system, for detecting ligand–receptor interactions in vivo. This system is adapted from the yeast two-hybrid system with which a third synthetic hybrid ligand is combined. The feasibility of this system was demonstrated using as the hybrid ligand a heterodimer of covalently linked dexamethasone and FK506. Yeast expressing fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and of FKBP12 fused to a transcriptional activation domain activated reporter genes when plated on medium containing the dexamethasone–FK506 heterodimer. The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506. Using this system, we screened a Jurkat cDNA library fused to the transcriptional activation domain in yeast expressing the hormone binding domain of rat glucocorticoid receptor–LexA DNA binding domain fusion protein in the presence of dexamethasone–FK506 heterodimer. We isolated overlapping clones of human FKBP12. These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors.

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.