Mithramycin forms a stable dimeric complex by chelating with Fe(II): DNA-interacting characteristics, cellular permeation and cytotoxicity

Mith (mithramycin) forms a 2:1 stoichiometry drug–metal complex through the chelation with Fe(II) ion as studied using circular dichroism spectroscopy. The binding affinity between Mith and Fe(II) is much greater than other divalent metal ions, including Mg(II), Zn(II), Co(II), Ni(II) and Mn(II). The [(Mith)₂–Fe(II)] complex binds to DNA and induces a conformational change of DNA. Kinetic analysis of surface plasmon resonance studies revealed that the [(Mith)₂–Fe(II)] complex binds to DNA duplex with higher affinity compared with the [(Mith)₂–Mg(II)] complex. A molecular model of the Mith-DNA–Metal(II) complex is presented. DNA-break assay showed that the [(Mith)₂–Fe(II)] complex was capable of promoting the one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide. Intracellular Fe(II) assays and fluorescence microscopy studies using K562 indicated that this dimer complex maintains its structural integrity and permeates into the inside of K562 cells, respectively. The [(Mith)₂–Fe(II)] complex exhibited higher cytotoxicity than the drug alone in some cancer cell lines, probably related to its higher DNA-binding and cleavage activity. Evidences obtained in this study suggest that the biological effects caused by the [(Mith)₂–Fe(II)] complex may be further explored in the future.     

Elucidation of the DNA-interacting properties and anticancer activity of a Ni(II)-coordinated mithramycin dimer complex

Mithramycin (Mith) forms a drug-metal complex with a 2:1 stoichiometry by chelation with a Ni(II) ion, which was determined using circular dichroism spectroscopy. Mith exhibits an increased affinity (~55 fold) for Ni(II) in the presence of DNA compared to the absence of DNA, suggesting that DNA acts as an effective template to facilitate chelation. Also, we characterized the DNA-acting properties of a Ni(II) derivative of Mith. Kinetic analysis using surface plasmon resonance and UV melting studies revealed that Ni^II(Mith)₂ binds to duplex DNA with a higher affinity compared to Mg^II(Mith)₂. The thermodynamic parameters revealed a higher free energy of formation for duplex DNA in the presence of Ni^II(Mith)₂ compared to duplex DNA in the presence of Mg^II(Mith)₂. The results of a DNA-break assay indicated that Ni^II(Mith)₂ is capable of promoting one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide; the DNA cleavage rate of Ni^II(Mith)₂ was calculated to be 4.1 × 10^?4 s^?1. In cell-based experiments, Ni^II(Mith)₂ exhibited a more efficient reduction of c-myc and increased cytotoxicity compared to Mith alone because of its increased DNA-binding and cleavage activity. The evidence obtained in this study suggests that the biological effects of Ni^II(Mith)₂ require further investigation in the future.     

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.     

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A3

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co^II(Chro)₂ complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6 mM concentration of spermine completely abolished the DNA-binding activity of Fe^II(Chro)₂ and Cu^II(Chro)₂ and interfered with the associative binding of Co^II(Chro)₂ complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2 mM) promoted DNA strand cleavage by Cu^II(Chro)₂, whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co^II(Chro)₂ or Fe^II(Chro)₂. Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe^II(Chro)₂. Despite the fact that Cu^II(Chro)₂ and Fe^II(Chro)₂ demonstrated lower DNA-binding activity than Co^II(Chro)₂ in the absence of spermine, while Cu^II(Chro)₂ and Fe^II(Chro)₂ exhibited greater cytoxicity against HepG2 cells than Co^II(Chro)₂, possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.     

The effects of loop size on Sac7d-hairpin DNA interactions

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1-L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA.     

Structures, spectra, and DNA-binding properties of mixed ligand copper(II) complexes of iminodiacetic acid: the novel role of diimine co-ligands on DNA conformation and hydrolytic and oxidative double strand DNA cleavage

The coordination geometry around copper(II) in [Cu(imda)(phen)(H2O)] (1) (H2imda = iminodiacetic acid, phen = 1,10-phenanthroline) is described as distorted octahedral while those in [Cu(imda)(5,6-dmp)] (2) (5,6-dmp = 5,6-dimethyl-1,10-phenanthroline) and [Cu(imda)(dpq)] (3) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) as trigonal bipyramidal distorted square-based pyramidal with the imda anion facially coordinated to copper(II). Absorption spectral (Kb: 1, 0.60+/-0.04x10(3); 2, 3.9+/-0.3x10(3); 3, 1.7+/-0.5x10(4) M(-1)) and thermal denaturation studies (deltaTm: 1, 5.70+/-0.05; 2, 5.5+/-10; 3, 10.6+/-10 degrees C) and viscosity measurements indicate that 3 interacts with calf thymus DNA more strongly than 1 and 2. The relative viscosities of DNA bound to 1 and 3 increase while that of DNA bound to 2 decreases indicating formation of kinks or bends and/or conversion of B to A conformation as revealed by the decrease in intensity of the helicity band in the circular dichroism spectrum of DNA. While 1 and 3 are bound to DNA through partial intercalation, respectively, of phen ring and the extended planar ring of dpq with DNA base stack, the complex 2 is involved in groove binding. All the complexes show cleavage of pBR322 supercoiled DNA in the presence of ascorbic acid with the cleavage efficiency varying in the order 3 > 1 > 2. The highest oxidative DNA cleavage of dpq complex is ascribed to its highest Cu(II)/Cu(I) redox potential. Oxidative cleavage studies using distamycin reveal minor groove binding for the dpq complex but a major groove binding for the phen and 5,6-dmp complexes. Also, all the complexes show hydrolytic DNA cleavage activity in the absence of light or a reducing agent with cleavage efficiency varying in the order 1 > 3 > 2.     

Effect of DNA local conformation on the affinity and binding specificity of bis-netropsins to DNA

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group (<--Nt-Pt(NH3)-Nt-->) or aliphatic pentamethylene chain (<--Nt-(CH2)5-Nt-->), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of <--Nt-(CH2)5-Nt--> with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for <--Nt-Pt(NH3)-Nt--> and <--Nt-(CH2)5-Nt--> binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.     

Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration

The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents.     

Binding of mismatch repair protein MutS to mispaired DNA adducts of intercalating ruthenium(II) arene complexes

The present study was performed to examine the affinity of Escherichia coli mismatch repair (MMR) protein MutS for DNA damaged by an intercalating compound. We examined the binding properties of this protein with various DNA substrates containing a single centrally located adduct of ruthenium(II) arene complexes [(eta(6)-arene)Ru(II)(en)Cl][PF(6)] [arene is tetrahydroanthracene (THA) or p-cymene (CYM); en is ethylenediamine]. These two complexes were chosen as representatives of two different classes of monofunctional ruthenium(II) arene compounds which differ in DNA-binding modes: one that involves combined coordination to G N7 along with noncovalent, hydrophobic interactions, such as partial arene intercalation (tricyclic-ring Ru-THA), and the other that binds to DNA only via coordination to G N7 and does not interact with double-helical DNA by intercalation (monoring Ru-CYM). Using electrophoretic mobility shift assays, we examined the binding properties of MutS protein with various DNA duplexes (homoduplexes or mismatched duplexes) containing a single centrally located adduct of ruthenium(II) arene compounds. We have shown that presence of the ruthenium(II) arene adducts decreases the affinity of MutS for ruthenated DNA duplexes that either have a regular sequence or contain a mismatch and that intercalation of the arene contributes considerably to this inhibitory effect. Since MutS initiates MMR by recognizing DNA lesions, the results of the present work support the view that DNA damage due to intercalation is removed from DNA by a mechanism(s) other than MMR.     

Drug binding by branched DNA: selective interaction of tetrapyridyl porphyrins with an immobile junction

The differential binding of a number of water-soluble cationic porphyrins to a branched DNA molecule is reported. Tetrakis(4-N-methylpyridiniumyl)porphine (H2TMpyP-4) interacts near the branch point with an immobile DNA junction formed from four 16-mer strands. Its Cu(II) and Ni(II) derivatives show stronger preferential binding in the neighborhood of the branch point. Axially liganded derivatives, Zn, Co, and Mn, also interact near this branch point, but in a different way. We use the reagents methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and bis(o-phenanthroline)copper(I) [(OP)2Cu(I)] to cleave complexes of DNA duplex controls and the junction with these porphyrins. The resulting cleavage patterns are consistent with previous evidence that the branch point provides a strong site for intercalative binding agents, which is not available in unbranched duplexes of identical sequence. The preferential scission by (OP)2Cu(I) in the presence of Ni and Cu porphyrins near the branch point exceeds that seen for any agents we have studied. This hyperreactivity is not seen in the case of porphyrins with axial ligands, ZnTMpyP-4, CoTMpyP-4, and MnTMpyP-4, although these also interact near the branch point. The Zn derivative tends to protect sites close to the branch point from cutting, while the Co and Mn porphyrins moderately enhance cleavage of sites in this region.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.     

DNA topoisomerase II selects DNA cleavage sites based on reactivity rather than binding affinity

DNA topoisomerase II modulates DNA topology by relieving supercoil stress and by unknotting or decatenating entangled DNA. During its reaction cycle, the enzyme creates a transient double-strand break in one DNA segment, the G-DNA. This break serves as a gate through which another DNA segment is transported. Defined topoisomerase II cleavage sites in genomic and plasmid DNA have been previously mapped. To dissect the G-DNA recognition mechanism, we studied the affinity and reactivity of a series of DNA duplexes of varied sequence under conditions that only allow G-DNA to bind. These DNA duplexes could be cleaved to varying extents ranging from undetectable (<0.5%) to 80%. The sequence that defines a cleavage site resides within the central 20bp of the duplex. The DNA affinity does not correlate with the ability of the enzyme to cleave DNA, suggesting that the binding step does not contribute significantly to the selection mechanism. Kinetic experiments show that the selectivity interactions are formed before rather than subsequent to cleavage. Presumably the binding energy of the cognate interactions is used to promote a conformational change that brings the enzyme into a cleavage competent state. The ability to modulate the extent of DNA cleavage by varying the DNA sequence may be valuable for future structural and mechanistic studies that aim to determine topoisomerase structures with DNA bound in pre- and post-cleavage states and to understand the conformational changes associated with DNA binding and cleavage.     

DNA binding, cleavage, and cytotoxic activity of the preorganized dinuclear zinc(II) complex of triazacyclononane derivatives

A preorganized cleft dinuclear zinc(II) complex of 2,6-bis(1-methyl-1,4,7-triazacyclonon-1-yl)pyridine 1 as an artificial nuclease was prepared via an improved method. The interactions of 1, 2 [1,4,7-triazacyclononane (TACN)], and their zinc(II) complexes with calf thymus DNA were studied by spectroscopic techniques, including fluorescence and CD spectroscopy. The results indicate that the DNA binding affinities of these compounds are in the following order: Zn(II)2 -1 > Zn(II) -2 > 1 > 2. The binding constants of the Zn (II)2 -1 and Zn(II)-2 complexes are 3.57 x 10(6) and 1.43 x 10(5) M(-1), respectively. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of the dinuclear Zn (II)2 -1 complex, which exhibits powerful DNA cleavage efficiency. Kinetic data for DNA cleavage promoted by the Zn(II)2 -1 complex under physiological conditions give the observed rate constant ( k obs) of 0.136 h(-1), which shows an 10(7)-fold rate acceleration over uncatalyzed supercoiled DNA. The comparison of the dinuclear Zn(II)2 -1 complex with the mononuclear zinc(II) complex of 1,4,7-triazacyclononane indicates that the DNA cleavage acceleration promoted by the Zn(II)2 -1 complex is due to the efficient cooperative catalysis of the two proximate zinc(II) cation centers. A hydrolytic mechanism of the cleavage process was suggested, and a preliminary study of the antitumor activity was also conducted.     

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Synthesis, crystal structure and DNA cleavage activities of copper(II) complexes with asymmetric tridentate ligands

Two asymmetric tridentate copper(II) complexes, [Cu(dppt)Cl(2)].0.25H(2)O (1) (dppt=3-(1,10-phenanthrolin-2-yl)-5,6-diphenyl-as-triazine) and [Cu(pta)Cl(2)] (2) (pta=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]acenaphthylene), have been prepared and characterized by elemental analysis, IR and Fast atomic bombardment mass spectra. Complex 1 has also been structurally characterized. The complexes exist as distorted square pyramid with five co-ordination sites occupied by the tridentate ligand and the two chlorine anions. DNA interaction studies suggest that the ligand planarity of the complex has a significant effect on DNA binding affinity increasing in the order [Cu(dppt)Cl(2)]< [Cu(pta)Cl(2)]. In the presence of ascorbate or glutathione, the two complexes are found to cause significant cleavage of double-strand pBR 322 DNA and [Cu(pta)Cl(2)] exhibited the higher cleaving efficiency.     

Efficient double-strand scission of plasmid DNA by quaternized-chitosan zinc complex

N-[(2-Hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTACC) was prepared to construct a chitosan-based zinc complex (HTACC-Zn(II)) as a catalyst with good water solubility for rapid DNA cleavage. Results indicated that the observed rate constant (k(obs)) of plasmid DNA cleaved by HTACC-Zn(II) could be enhanced by 10(7)-fold compared with that of uncatalyzed DNA cleavage. The kinetic behavior of HTACC-Zn(II) for DNA cleavage is well fitted by Michaelis-Menten model. The results of gel electrophoresis suggested that HTACC-Zn(II) preferentially perform double-strand break of plasmid DNA.     

DNA cleavage promoted by trigonal-bipyramidal zinc(II) and copper(II) complexes formed by asymmetric tripodal tetradendate 2-[bis(2-aminoethyl)amino]ethanol

Asymmetric trigonal-bipyramidal Zn(II) complex 1 formed by 2-[bis(2-aminoethyl)amino]ethanol (L) was found to be able to promote the cleavage of supercoiled plasmid DNA pBR322 to the nicked and linear DNA via a hydrolytic manner but only in neutral Tris-HCl buffer, no cleavage was observed in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. However, the copper complex 2 of L, possessing the similar coordination geometry, can only promote DNA cleavage via an oxidative mechanism in the presence of ascorbic acid. ESI-MS study implies that complex 1 exist mainly as [Zn(L)]²⁺/[Zn(L-H)]⁺ in neutral Tris-HCl buffer. Moreover, there is no discriminable species for complex 1 in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. A phosphate activation mechanism via phosphate coordinating to Zn(II) center of [Zn(L)]²⁺/[Zn(L-H)]⁺ to form the stable trigonal-bipyramidal structure is proposed for the hydrolytic cleavage promote by complex 1. For complex 2, the abundance of [Cu(L)Cl]⁺ is higher than that of [Cu(L)]²⁺/[Cu(L-H)]⁺ in Tris-HCl buffer. The lower phosphate binding/activating ability of Cu(II) in complex 2 may be the origin for its incapability to promote the hydrolytic DNA cleavage. However, the readily accessible redox potential of Cu(II) makes complex 2 promote the oxidative DNA cleavage. Although the DNA cleavage promoted by complex 1 has no specificity, trigonal-bipyramidal Zn(II) complexes formed by asymmetric tripodal polyamine with ethoxyl pendent should be a novel potential model for practical artificial nuclease.