Relaxation Of Supercoiled Plasmid DNA Research Articles

Dicobalt(ii) helices kill colon cancer cells via enantiomer-specific mechanisms; DNA damage or microtubule disruption.

Highly diastereoselective self-assembly reactions give both enantiomers (Λ and Δ) of anti-parallel triple-stranded bimetallic Co(ii) and Co(iii) cationic helices, without the need for resolution; the first such reaction for Co. The complexes are water soluble and stable, even in the case of Co(ii). Studies in a range of cancer and healthy cell lines indicate high activity and selectivity, and substantial differences between enantiomers. The oxidation state has little effect, and correspondingly, Co(iii) compounds are reduced to Co(ii) e.g. by glutathione. In HCT116 colon cancer cells the Λ enantiomer induces dose-dependent G2-M arrest in the cell cycle and disrupts microtubule architectures. This Co(ii) Λ enantiomer is ca. five times more potent than the isostructural Fe(ii) compound. Since the measured cellular uptakes are similar this implies a higher affinity of the Co system for the intracellular target(s); while the two systems are isostructural they have substantially different charge distributions as shown by calculated hydrophobicity maps. In contrast to the Λ enantiomer, Δ-Co(ii) induces G1 arrest in HCT116 cells, efficiently inhibits the topoisomerase I-catalyzed relaxation of supercoiled plasmid DNA, and, unlike the isostructural Fe(ii) system, causes DNA damage. It thus seems very likely that redox chemistry plays a role in the latter.

Trans-Dichloro(triphenylarsino)(N,N-dialkylamino)platinum(II) Complexes: In Search of New Scaffolds to Circumvent Cisplatin Resistance.

The high incidence of the resistance phenomenon represents one of the most important limitations to the clinical usefulness of cisplatin as an anticancer drug. Notwithstanding the considerable efforts to solve this problem, the circumvention of cisplatin resistance remains a challenge in the treatment of cancer. In this work, the synthesis and characterization of two trans-dichloro(triphenylarsino)(N,N-dialkylamino)platinum(II) complexes (1 and 2) were described. The trypan blue exclusion assay demonstrated an interesting antiproliferative effect for complex 1 in ovarian carcinoma-resistant cells, A2780cis. Quantitative analysis performed by ICP-AES demonstrated a scarce ability to platinate DNA, and a significant intracellular accumulation. The investigation of the mechanism of action highlighted the ability of 1 to inhibit the relaxation of supercoiled plasmid DNA mediated by topoisomerase II and to stabilize the cleavable complex. Cytofluorimetric analyses indicated the activation of the apoptotic pathway and the mitochondrial membrane depolarization. Therefore, topoisomerase II and mitochondria could represent possible intracellular targets. The biological properties of 1 and 2 were compared to those of the related trans-dichloro(triphenylphosphino)(N,N-dialkylamino)platinum(II) complexes in order to draw structure–activity relationships useful to face the resistance phenotype.

Copper(II) and silver(I)-1,10-phenanthroline-5,6-dione complexes interact with double-stranded DNA: further evidence of their apparent multi-modal activity towards Pseudomonas aeruginosa

Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (− 7.9 kcal/mol) or minor (− 7.2 kcal/mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M−1) than Ag-phendione (Kapp = 2.79 × 105 M−1) and phendione (Kapp = 1.33 × 105 M−1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa.Graphical abstract

Complete labelling of pneumococcal DNA-binding proteins with seleno-L-methionine

Streptococcus pneumoniae is a pathogenic and opportunistic Gram-positive bacterium that is the leading cause of community-acquired respiratory diseases, varying from mild- to deathly- infections. The appearance of antibiotic-resistant isolates has prompted the search for novel strategies and targets to tackle the bacterial resistances. One of the most promising approaches is the structure-based knowledge of possible targets in conjunction with rational design and docking of inhibitors of the chosen targets. A useful technique that helps to solve protein structures is to label them with an amino acid derivative like seleno-methionine that facilitates tracing of some of the amino acid residues. We have chosen two pneumococcal DNA-binding proteins, namely the relaxase domain of MobM protein from plasmid pMV158, and the RelB-RelE antitoxin-toxin protein complex. Through several changes that improve substantially a previous protocol (Budisa et al., 1995), we have used seleno-L-methionine to incorporate selenium into the amino acid sequence of the selected proteins. We have achieved 100% labelling of the proteins and could demonstrate that the labelled proteins retained full activity as judged from the relaxation of supercoiled plasmid DNA and from gel-retardation assays.

Multi-targeted effects of G4-aptamers and their antiproliferative activity against cancer cells

We selected and investigated nine G-quadruplex (G4)-forming aptamers originally designed against different proteins involved in the regulation of cellular proliferation (STAT3, nucleolin, TOP1, SP1, VEGF, and SHP-2) and considered to be potential anticancer agents. We showed that under physiological conditions all the aptamers form stable G4s of different topology. G4 aptamers designed against STAT3, nucleolin and SP1 inhibit STAT3 transcriptional activity in human breast adenocarcinoma MCF-7 cells, and all the studied aptamers inhibit TOP1-mediated relaxation of supercoiled plasmid DNA. STAT3 inhibition by G4 aptamer designed against SP1 protein provides a new explanation for the SP1 and STAT3 crosstalk described recently. We found some correlation between G4-mediated inhibition of the DNA replication and TOP1 activity. Four G4 aptamers from our dataset that appeared to be the strongest TOP1 inhibitors most efficiently decreased de novo DNA synthesis, by up to 79–87%. Seven G4 aptamers demonstrated significantly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells than on immortalized mammary epithelial MCF-10A cells. Pleiotropic properties of G4 aptamers and their high specificity against cancer cells observed for the majority of the studied G4 aptamers allowed us to present them as promising candidates for multi-targeted cancer therapy.

In vitro antiproliferative/cytotoxic activity of 2,3ʹ-biindole againstvarious cancer cell lines

2,3'-Biindole (2) was synthesized via bromination of indole (1) with molecular bromine and underwent simultaneous dimerization. Antiproliferative and cytotoxic activity of 2 was investigated in vitro on C6 (rat brain tumor), HeLa (human cervix carcinoma), and HT29 (human colon carcinoma) cells lines by using BrdU cell proliferation ELISA and lactate dehydrogenase (LDH) assays. In contrast to 5-fluorouracil (5-FU), 2,3'-biindole (2) significantly inhibited proliferation of HeLa and HT29 cell lines. According to LDH assay, the cytotoxicity of compound 2 was low on HT29 cell lines and high on HeLa and C6 cell lines. Moreover, 2 did not cause any DNA laddering on the DNA of tested cells; therefore, it is suggested that the mechanism of action of this compound may not involve apoptosis. In addition, 2 inhibited relaxation of supercoiled plasmid DNA by topoisomerase activity. Results of the present study indicates that biindole (2) may have promising anticancer and antitopoisomerase potential with an unknown mechanism of action.

P-Thiophenylalanine-Induced DNA Cleavage and Religation Activity of a Modified Vaccinia Topoisomerase IB

Vaccinia DNA topoisomerase IB is the smallest of the type IB topoisomerases. Because of its small size (314 amino acids) and target site specificity (5'(C/T)CCTTp(↓) sites), it constitutes an excellent model for studying the interaction of type IB enzymes with duplex DNA. In this study, p-thiophenylalanine was incorporated into the enzyme active site (position 274) by in vitro translation in the presence of a chemically misacylated tRNA. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate (k(cl) = 1 × 10(-4) s(-1)) was 30 times lower than for the wild-type enzyme (k(cl) = 3 × 10(-3) s(-1)). In contrast, thiophenylalanine-induced DNA religation was faster than that of the wild-type enzyme. We propose that the change in kinetics reflects the difference in bond energies between the O-P and S-P bonds being formed and broken in the reactions catalyzed by the wild-type and modified enzymes. We also studied the effect of adding Mg(2+) and Mn(2+) to the wild-type and modified topoisomerases I. Divalent metal ions such as Mg(2+) and Mn(2+) increased DNA relaxation activity of the wild-type and modified enzymes. However, the pattern of increases failed to support the possibility that metal ion-heteroatom interaction is required for catalysis.

Time-resolved chloroquine-induced relaxation of supercoiled plasmid DNA

Herein, we report on the in vitro change of DNA conformation of plasmids bound to a 3-aminopropyl-modified mica surface and monitoring the events by atomic force microscopy (AFM) imaging under near physiological conditions. In our study, we used an intercalating drug, chloroquine, which is known to decrease the twist of the double helix and thus altered the conformation of the whole DNA. During our experiments, a chloroquine solution was added while imaging a few highly condensed plasmid nanoparticles in solution. AFM images recorded after the drug addition clearly show a time-resolved relaxation of these bionanoparticles into a mixture of loose DNA strands.

Topoisomerase I/II inhibition by a novel naphthoquinone containing a modified anthracycline ring system

In an attempt to create more effective chemotherapeutic compounds, the naphthoquinone adduct 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was synthesized. Cell viability studies revealed TU100 is specific for eukaryotes and induces cell death. Based on its structural similarities to the anthracyclines and isoquinolines, the ability of TU100 to inhibit topoisomerase I and II was examined. TU100 was an effective inhibitor of both enzymes, as indicated by its ability to prevent topoisomerase-mediated relaxation of supercoiled plasmid DNA. The mechanism of action does not involve TU100 intercalation into DNA, unlike anthracyclines. Pre-incubation of topoisomerase with TU100 dramatically decreased the IC(50), suggesting the drug is a novel slow acting topoisomerase inhibitor that works in the absence of DNA. Taken together these results indicate the novel naphthoquinone adduct TU100 is a dual topoisomerase I/II inhibitor with a unique mechanism of action and chemotherapeutic potential.

Bis-phenanthroline copper(ii) phthalate complexes are potent in vitro antitumour agents with ‘self-activating’ metallo-nuclease and DNA binding properties

Three, structurally characterised, bis-phen Cu(II) complexes of the phthalate isomers display rapid, low micromolar in vitro cytotoxicity against a range of epithelial tumour cells. The complexes induce relaxation of supercoiled plasmid DNA in the absence of external reducing agents and display efficient CT-DNA, Poly[d(A-T)](2) and Poly[d(G-C)](2) binding.

Click Chelators – The Behavior of Platinum and Palladium Complexes in the Presence of Guanosine and DNA

AbstractTriazole chelators, synthesized by click chemistry, are convenient ligands for palladium(II) and platinum(II). Conformational changes induced by the complexation of these PdII and PtII complexes with guanosine are similar to those of cisplatin. In addition, these complexes behave like cisplatin with regard to the relaxation of supercoiled plasmid DNA.

Protection of DNA and membrane from γ-radiation induced damage by the extract of Acorus calamus Linn.: An in vitro study

Acorus calamus, an ethnomedicinally important plant, was investigated for its protecting activity against radiation induced DNA and membrane damage. The in vitro free radical scavenging activity of the extract (water:ethanol, 1:1) of A. calamus was studied by parameters viz DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activity, hydroxyl radical scavenging activity, and superoxide radical scavenging activity. Membrane damage due to radiation exposure was measured as the peroxidation of lipids in terms of thiobarbituric acid reacting substance (TBARS). The in vitro DNA damage was monitored by assessing the radiation induced relaxation of supercoiled plasmid DNA (pBR322). Damage to cellular DNA induced by γ-radiation (6 Gy) was monitored by alkaline single cell gel electrophoresis or comet assay in murine cells and human peripheral blood leukocytes. Enhancement of DNA repair mechanism was also monitored. The extract effectively scavenged free radicals in a concentration dependent manner. Presence of A. calamus extract during irradiation prevented peroxidation of membrane lipids in mouse liver homogenate. It helped to reduce the disappearance of the covalently closed circular (ccc) form of plasmid DNA following exposure to γ-radiation. Also the A. calamus extract effectively protected DNA from radiation induced strand breaks and enhanced the DNA repair process. Hence A. calamus extract can be used as a good source of natural radioprotecting agent.

Fenton-Like Reaction Catalyzed by the Rare Earth Inner Transition Metal Cerium

Cerium (Ce) is a rare earth metal that is not known to have any biological role. Cerium oxide materials of several sizes and shapes have been developed in recent years as a scaffold for catalysts. Indeed even cerium oxide nanoparticles themselves have displayed catalytic activities and antioxidant properties in tissue culture and animal models. Because of ceria's ability to cycle between the +3 and +4 states at oxygen vacancy sites, we investigated whether cerium metal would catalyze a Fenton-like reaction with hydrogen peroxide. Indeed, cerium chloride did exhibit radical production in the presence of hydrogen peroxide, as assessed by relaxation of supercoiled plasmid DNA. Radical production in this reaction was also followed by production of radical cation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Radical scavengers and spin traps were capable of competing with ABTS for radicals produced in this cerium dependent Fenton-like reaction. Electron paramagnetic resonance experiments reveal both hydroxyl radical and superoxide anion in a reaction containing cerium and hydrogen peroxide. Based on these results we propose that cerium is capable of redox-cycling with peroxide to generate damaging oxygen radicals.

The role of topoisomerases and RNA transcription in the action of the antitumour benzonaphthyridine derivative SN 28049

SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a DNA intercalating drug that binds selectively to GC-rich DNA and shows curative activity against the Colon 38 adenocarcinoma in mice. We wished to investigate the roles of topoisomerase (topo) I, topo II and RNA transcription in the action of SN 28049. We used clonogenic assays to study the cytotoxicity of SN 28049; RNA interference and enzyme assays to examine the role of topo I in SN 28049 action; 3H uridine incorporation and reporter assays to study its effects on transcription; and RT-PCR to examine its ability to reduce endogenous h-TERT expression. In clonogenic assays, SN 28049 showed a biphasic cytotoxic dose response curve in H460 cells typical of acridine derivatives such as N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) although it was approximately 16-fold more potent. Down-regulation of topo IIalpha in HTETOP cells reduced the cytotoxicity of SN 28049, establishing its action as a topo IIalpha poison. Surprisingly, down-regulation of topo I in H460 cells by RNA interference sensitised them to the actions of SN 28049 and other topo II poisons. SN 28049 also inhibited topo I-mediated relaxation of supercoiled plasmid DNA. SN 28049 was also an inhibitor of transcription in HEK293 cells and was more potent at reducing luciferase expression from a GC-rich SP-1 binding promoter than from a non-GC-rich AP-1 binding promoter. The drug also reduced luciferase reporter gene expression driven by the SP-1-binding survivin promoter as well as reducing endogenous h-TERT expression in HEK293 cells whose promoter also contains SP-1 binding sites. We conclude that SN 28049 has a complex action that may involve poisoning of topo IIalpha, suppression of topo I and inhibition of gene transcription from promoters with SP-1 sites. These actions may contribute to the promising experimental solid tumour anticancer activity of SN 28049.

A Copper‐Metalated, Hybrid Inorganic–Organic Polymer as an Oxidative Nuclease

AbstractThis study describes nucleolytic activity of a copper‐metalated, multisite‐coordinating, hybrid polymer CPPL‐Cu under oxidative conditions. Rapid relaxation of supercoiled plasmid DNA was observed and mechanistic probing by chemical and enzymatic assays revealed involvement of singlet‐oxygen‐derived reactive species. The heterogeneous nature of the catalyst allowed a facile recycling of the artificial nuclease. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

(+)-Myristinin A, a Naturally Occurring DNA Polymerase β Inhibitor and Potent DNA-Damaging Agent

The first stereoselective total synthesis of the naturally occurring flavan myristinin A has been accomplished, as well as its biochemical evaluation. This synthesis verified the structural assignment and allowed for the determination of the absolute stereochemistry. Myristinin A exhibits biochemical activity both as a potent DNA-damaging agent and DNA polymerase beta inhibitor. Relaxation of supercoiled plasmid DNA was observed at picomolar concentrations, in addition to inhibition of polymerase beta at low micromolar concentrations.

Biocatalysis by metallated cyclotriphosphazenes: L2Zn(NO3)2 {L = spiro-N3P3[O2C12H8][N(CH3)NH2]} as a synthetic phosphoesterase and nuclease

Catalytic activity of [L2.Zn][NO3]2] (L = spiro-N3P3[O2C12H8][N(CH3)NH2]) towards the hydrolysis of two phosphodiesters, [bis(p-nitrophenyl)phosphate, bNPP] and [2-(hydroxypropyl)-p-nitrophenyl phosphate, hNPP] has been examined. While the rate of hydrolysis of the former is accelerated over a million-fold, the rate of hydrolysis of the latter also is enhanced considerably. Detailed kinetic evaluation of these reactions has been carried out and all the kinetic parameters including the Michaelis-Menten parameters are reported. The catalyst [L2.Zn][NO3]2] has also been found to be an effective nuclease. Relaxation of supercoiled plasmid DNA, pBR322, occurs in presence of [L2.Zn][NO3]2 without the need for any exogenous reagents.

A Dihydroflavonol Glucoside from Commiphora africana that Mediates DNA Strand Scission

A crude CH(2)Cl(2)-MeOH extract prepared from Commiphora africana was found to mediate Cu(2+)-dependent relaxation of supercoiled plasmid DNA. Bioassay-guided fractionation of this extract was carried out and was monitored by the use of an in vitro DNA strand scission assay. The dihydroflavonol glucoside phellamurin (1) was identified as the active principle responsible for the DNA cleavage activity of the crude extract.

Solid-phase synthesis and biochemical evaluation of conformationally constrained analogues of deglycobleomycin A5

Deglycobleomycin binds to and degrades the self-complementary oligonucleotide d(CGCTAGCG)2 in a sequence selective fashion. A previous modeling study [J. Am. Chem. Soc. 120, (1998), 7450] had shown that, during binding to double stranded DNA, the conformation of the methylvalerate domain of deglycoBLM approximated that of S-proline. In the belief that an analogue of deglycoBLM structurally constrained to mimic the DNA-bound conformation might exhibit facilitated DNA binding and cleavage, an analogue of deglycoBLM was prepared in which the methylvalerate moiety was replaced by S-proline. This deglycoBLM analogue, as well as the related analogue containing R-proline, was synthesized on a TentaGel resin. Both of the analogues were found to be capable of binding Fe2+ and activating O2 for transfer to styrene. However, both deglycoBLM analogues exhibited diminished abilities to effect the relaxation of supercoiled plasmid DNA, and neither mediated sequence selective DNA cleavage.

Antitumor triptycene bisquinones: a novel synthetic class of dual inhibitors of DNA topoisomerase I and II activities.

Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin in the nanomolar range in vitro, but have the advantage of blocking nucleoside transport and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these lead antitumor drugs were tested for their ability to trigger the DNA topoisomerase (Topo) inhibitions responsible for the initial and massive high-molecular-weight cleavage of DNA required for tumor cells to commit apoptosis. Interestingly, antitumor TTs have the unusual ability to inhibit, in a concentration-dependent manner, the relaxation of supercoiled plasmid DNA catalyzed by both purified human Topo I and II enzymes. However, if there is a relationship between the ability of TT analogs to inhibit Topo activities and their quinone functionality and cytotoxicity, it is far from perfect, suggesting that other molecular targets may be involved in the mechanism of action of these antitumor drugs. Moreover, one of the most cytotoxic TT bisquinone, 6-bromo-7-methoxy- or 7-bromo-6-methoxy-2-N-methylamino-1 H,4 H,5 H,8H-9,10-dihydro-9,10-[1',2']benzenoanthracene-1,4,5,8-tetraone (TT24), inhibits Topo II activity more effectively than amsacrine (m-AMSA) and matches the Topo I inhibitory effect of camptothecin (CPT). The dual inhibitory activity of TT24 is substantiated by the findings that TT24 mimics the action of m-AMSA in the Topo II assay, where the Topo I inhibitor CPT is ineffective, and also mimics the action of CPT in the Topo I assay, where the Topo II inhibitor etoposide is ineffective. Because of their ability to target nucleoside transport and topoisomerase activities, synthetic TT bisquinones might represent a novel class of bifunctional drugs valuable to develop new means of polychemotherapy and circumvent MDR.