Single-strand Scissions Research Articles

Insight into the in vitro anticancer screening, molecular docking and biological efficiency of pyridine-based transition metal(II) complexes

The undesirable effects caused by the chemotherapeutic drugs are already in use, mainly the platinated compounds requiring amendments for which many potential scaffolds resembling the activity of cisplatin are explored. Herein, the DNA binding, cleavage, molecular docking, antimicrobial proficiency, and cytotoxic nature of four non-platinated transition metal(II) complexes incorporating pyridine moiety were investigated. The complexes adopted octahedral geometry, and the mode of interaction with DNA was explored by absorption spectroscopy, fluorescence spectroscopy, electrochemical technique, and viscosity measurements. These studies indicate a groove-binding mode of the complexes to CT DNA. The stability of the synthesized complexes was investigated at physiological pH. All the complexes exhibited single-strand scission of the supercoiled pBR322 DNA where copper(II) complex (1) showed double-strand DNA prominently by converting the supercoiled DNA to linear form. The antimicrobial screening of the complexes yielded expected results. The complexes selectively showed activity against cancer cell lines and less toxicity toward the noncancerous NHDF cell line. Overall, 1 showed superior activity in the biological investigation. These studies reveal that the coordination of transition metal(II) ion with the ligand plays a pivotal role in the enhancement of the biological potential of the complexes.

Antioxidant, DNA interaction, molecular docking and cytotoxicity studies of aminoethylpiperazine‐containing macrocyclic binuclear copper(II) complexes

A series of new macrocyclic binuclear copper(II) complexes of the type [Cu2L1–5(ClO4)](ClO4) (1–5) were synthesized by template condensation between precursor compounds 2,6‐bis(4‐aminoethylpiperazin‐1‐ylmethyl)‐4‐substituted phenols and 2,6‐diformyl‐4‐substituted phenols. The synthesized precursors and complexes were characterized using regular physicochemical techniques. The rate constant values obtained for the hydrolysis of 4‐nitrophenylphosphate were in the range 1.83 × 10−2–4.19 × 10−2 min−1. Antioxidant studies against 2,2′‐diphenyl‐1‐picrylhydrazyl revealed the antioxidant potency of the synthesized complexes. Binding studies of the complexes with calf thymus DNA were conducted using electronic, viscometric and voltammetric techniques, and the obtained results suggested a non‐covalent groove mode of binding. The oxidative cleavage of pBR322 DNA in the presence of co‐reactant H2O2 and radical scavengers showed single strand scission and involvement of H2O2 radical in the cleavage process. Molecular docking studies were performed to insert complexes into the crystal structures of 1BNA and VEGFR kinase at active sites to determine the possible binding mode and predominant binding interactions. In vitro cytotoxicity of the complexes was tested against human epidermoid carcinoma cells (A431) by MTT assay, which revealed the effective anticancer activity of the complexes. Live cell and fluorescent imaging of A431 cells showed that the complexes induce cell death through apoptosis.

DFT investigations of phosphotriesters hydrolysis in aqueous solution: a model for DNA single strand scission induced by N-nitrosoureas

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

An Extracellular S1-Type Nuclease of Marine Fungus Penicillium melinii

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5-8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3'-5' mode to produce 5'-oligonucleotides and 5'-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

A Versatile DNA Nanochip for Direct Analysis of DNA Base‐Excision Repair

Direct observation of enzymes interacting with DNA should be one of the ultimate technologies for investigating the mechanical behavior of the enzymes during the reactions. Atomic force microscopy (AFM) enables observation of biomolecules at a nanoscale spatial resolution; however, for a stable analysis, a scaffold to observe the reaction should be explored. DNA origami has recently been developed for the construction of a wide variety of multidimensional nanostructures, which can be used as scaffolds to incorporate various functionalities at specific positions. We intended to construct an AFM-based analysis system for DNA repair using a DNA origami scaffold carrying various substrate double-stranded DNAs (dsDNA). We employed DNA baseexcision repair (BER) enzymes 8-oxoguanine glycosylase (hOgg1) and T4 pyrimidine dimer glycosylase (PDG) to analyze the reaction on the DNA scaffold (Figure 1a). In the repair process, hOgg1 removes 8-oxoguanine (oxoG) to prevent G:C!T:A transversion during replication by DNA glycosylase activity and endonuclease activity for apurine– apyrimidine (AP) sites (AP-lyase activity) (Figure 1a). PDG removes photodamaged pyrimidine dimer including cis–syn cyclobutane thymine dimer (T T) by DNA glycosylase/AP-lyase activity. BER enzymes often require structural changes of the target DNA strands, such as DNA bending, for the reaction to proceed. The enzyme hOgg1 bends double-helix DNA by about 708 with flipping out of the oxoG for procession of the excision. The enzyme PDG bends double-helix DNA by 608 with flipping out of the 3’-side of A in the opposite strand of T T. The glycosylase/AP-lyase activity of these enzymes leads to single-strand scission at the damaged nucleotide (Figure 1b). In addition, the reaction intermediates of both reactions form a covalent bond with the enzyme by reduction with NaBH4. [5,10] We recently developed a framelike DNA origami scaffold to incorporate two different substrate dsDNAs. If the above-mentioned enzymatic and chemical reactions

Photochemical Activities of N‐Nitroso Carboxamides and Sulfoximides and Their Application to DNA Cleavage

N-Nitroso compounds containing benzene, fluorene or fluorenone rings were synthesized. Photolysis of these compounds with 312-nm UV light provided the NO(*) species, the presence of which was corroborated by use of an EPR method and of 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO) as a trapping agent. During irradiation of N-methyl-N-nitroso-9-fluorenone carboxamide (14 c) in the absence of PTIO, it underwent decomposition followed by recombination to give the heterocyclic nitric oxide radical 15. Incorporation of intercalating moieties endowed the N-nitroso compounds with DNA-cleaving ability through single-strand scission upon UV irradiation in a phosphate buffer (pH 5.0-8.0) under aerobic conditions.

Chartreusin, elsamicin A and related anti-cancer antibiotics.

Chartreusin and elsamicin A are structurally related antibiotics that bind to GC-rich tracts in DNA, with a clear preference for B-DNA over Z-DNA. They inhibit RNA synthesis and cause single-strand scission of DNA via the formation of free radicals. Elsamicin A can also be regarded as the most potent inhibitor of topoisomerase II reported so far. It can inhibit the formation of several DNA-protein complexes. Elsamicin A binding to the P1 and P2 promoter regions of the c-myc oncogene inhibits the binding of the Sp1 transcription factor, thus inhibiting transcription. Despite the pharmacological interest in chartreusin, elsamicin A and their derivatives, there is no experimental data on the structure of their complexes with DNA. This shortcoming has been partially solved by a theoretical approach, which provided some details about the DNA-elsamicin A interaction, and the thermodynamic characterization of the binding of chartreusin and elsamicin A to DNA. Elsamicin A but not chartreusin is being developed clinically as an anti-cancer agent. IST-622 (6-O-(3-ethoxypropylonyl)-3',4'-O-exo-benzylidene-chartreusin), a novel semi-synthetic derivative of chartreusin, which has shown a promising anti-cancer activity in a phase II study, appears to be a pro-drug with a more suitable pharmacokinetic profile than chartreusin.

Synthetic Studies on Novel Leinamycin Derivatives

AbstractFor Abstract see ChemInform Abstract in Full Text.

Interaction of aminoglycosides and their copper(ii) complexes with nucleic acids: implication to the toxicity of these drugs

Cupric complexes of eight aminoglycosidic antibiotics were screened for their specific behavior towards tRNAPhe, both in oxidative and neutral surrounding. Without H2O2, the cleavage efficiency was dependent on the resultant charge of the molecule. A comparative assay using tRNAPhe devoid of the natural hypermodification in the anticodon loop proved that hypermodification is indispensable for site recognition and subsequent cleavage. The intensity of single and double strand scissions in plasmid DNA also proceeded in a charge-dependent manner. Unlike free antibiotics, their cupric complexes in the presence of H2O2, facilitated plasmid linearisation and degradation. The participation of ROS in those processes was confirmed using NDMA as a reporter molecule, whose consumption was influenced by the protonation state of the complex.

Oxime esters of anthraquinone as photo-induced DNA-cleaving agents for single- and double-strand scissions

Anthraquinone- O-9-(4-cyanobenzoyl)oxime ( 13 ) with binding constant of 4.49×10 4 M −1 exhibited single-strand scission of DNA at the concentration of 10 μM and double-strand scission at 50 μM upon UV irradiation.

Synthesis of Intramolecularly Activated Lactenediynes and Evaluation of Their Activity Against Plasmid DNA

The synthesis of new “selectively activated” enediyne prodrugs is reported. These compounds, belonging to the “lactenediyne” family, each possess a protected primary amine tethered at the β-lactam nitrogen. This, once deblocked, can act as a trigger, provoking a cascade of events probably terminating with Bergman cycloaromatization of the enediyne moiety. In vitro experiments on plasmid DNA have shown that the protected compounds are inactive, while the unprotected amine is able to provoke single-strand scissions. These results open the way towards development of enzymatically activated lactenediyne prodrugs. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

新規レイナマイシン誘導体の合成研究

Leinamycin, a novel antitumor antibiotic, was isolated from a culture broth of Streptomyces sp. The unique structural features of leinamycin include the 1-oxo-1, 2-dithiolan-3-one moiety which is fused in a spiro fashion to an 18-membered lactam with an extensively conjugated thiazole ring. Leinamycin causes single strand scission of plasmid DNA in the presence of thiol cofactor. Isolation of a guanine-leinamycin adduct revealed the unprecedented chemical reactions which would be responsible for the thiol-mediated DNA cleavage by leinamycin. As a part of our program aimed at discovering clinically useful leinamycin derivatives, chemical modification of natural leinamycin have been investigated. In this review, synthesis and chemistry of novel leinamycin derivatives with potent antitumor activity are described. Thioester derivatives with 3-isothiazolidinone 1-oxide moiety are more stable than leinamycin and serve as prodrugs that could be converted into active form in biological media.

Studies on single-strand scissions to cell-free plasmid DNA by the dopaminergic neurotoxin ‘TaClo’ (1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline)

The DNA-damaging effect of the potent dopaminergic neurotoxin ‘TaClo’ (1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline) is reported. After irradiation (300 nm, 350 nm) of cell-free pBR322 DNA in the presence of TaClo, single-strand breaks were observed by gel electrophoresis. It seems likely that TaClo undergoes a light-induced formation of reactive species (probably radicals). In comparison with TaClo, the bromo derivative exhibits an increased toxic action while the fluoro derivative shows a weaker effect towards DNA. In the presence of Cu(II) at 37°C in the dark, DNA strand scissions were found to occur, too. The formation of Cu(I), as detected by UV-absorption at 480 nm using the Cu(I) complexing ligand BCS, hints at the formation of a reactive species by a Cu(II)/Cu(I) mediated redox cycle. Until now, the chemical nature of this reactive species has not been elucidated.

Synthesis, structural characterization and potentiometric studies of divalent metal complexes with an octadentate tetraazamacrocyclic ligand and their DNA cleavage ability

Four new metal complexes with an octadentate macrocyclic ligand [L=1,4,7,10-tetrakis(2-carbamoylethyl)-1,4,7,10-tetraazacyclododecane] were synthesized and characterized by elemental analyses, IR and MS. These complexes have the general formula: M(L)(NO 3) 2· nH 2O [M=Co ( 1), Ni ( 2), n=2; M=Cu ( 3), Zn ( 4), n=1]. The structures of complexes 1, 2 and 3 have been determined by X-ray diffraction. [Co(L)(NO 3)](NO 3)·2H 2O ( 1) crystallizes in the triclinic space group P1̄. The refinement converges with R=0.045 and R w=0.057 for 3233 reflections with ( I>2.5 σ( I)). The Co has an octahedral geometry, and was coordinated with four amine nitrogen atoms of macrocyclic ligand and two or one acylate oxygen atoms derived from amide pendant groups. Two water molecules crystallize in the lattice cell and two kinds of hydrogen bonds exist between the coordinated acylate group and the water molecules: N(6)H(27)⋯O(11) 2.158, N(8)H(40)⋯O(12) 2.158 Å or exist between the uncoordinated acylate group and the nitrate ion: N(5)H(21)⋯O(8) 2.452, N(7)H(33)⋯O(5) 2.256 Å. [Ni(L)(NO 3)](NO 3)·2H 2O ( 2) crystallizes in the monoclinic space group C2/ c. The refinement converges with R=0.044 and R w=0.059 for 2726 reflections with ( I>3 σ( I)). The crystal of complex 2 has high symmetry with a C 2 v symmetric operation. The central Ni atom is coordinated to four amine nitrogen atoms of macrocyclic skeleton and two acylate oxygen atoms of pendant amide groups. The polyhedron of the central atom can be described as a pseudo-octahedron. Two water molecules crystallized in the cell lattice. [Cu(L)](NO 3) 2·H 2O ( 3) crystallizes in the monoclinic space group P2 1/ n. The refinement converges with R=0.037 and R w=0.048 for 3174 reflections with ( I>2.5 σ( I)). Complex 3 has a five-coordinated pyramid. The bond lengths of CoO (av.) 2.088(3), NiO 2.089(2) Å are almost equal, and are shorter than CuO 2.137(3) Å. On the other hand, the bond lengths of CoN (av.) and NiN (av.) are 2.194(3) and 2.153(2) Å, respectively. Both of them are longer than CuN (av.) 2.0465(3) Å. The potentiometric experiments in aqueous solution showed that the stability constants of three complexes were in the range of log K ML=17.11–15.00. The DNA cleavage results showed that the cobalt complex can effectively cleave supercoiled DNA to form nicked or linear DNA by performing single strand and double strand scissions under aerobic conditions in the presence of hydrogen peroxide as co-oxidant. Electron paramagnetic resonance results with the aid of DMPO as trapping agent demonstrated that the cleavage mechanism possibly occurred via hydroxy radical attacking.

Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 μm CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2′-, 4,4′-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of themale than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrationsof 5 (> 5-fold), 50 (> 2-fold) and 500 μm (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

Radiation-Induced DNA Damage in Tumors and Normal Tissues: V. Influence of pH and Nutrient Depletion on the Formation of DNA-Protein Crosslinks in Irradiated Partially and Fully Hypoxic Tumor Cells

It has been demonstrated that the yield of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) appears to depend predominantly on the oxygen concentration in the microenvironment around the DNA of mammalian cells (Radiat. Res. 142, 163-168, 1995). Consequently, these DNA lesions have the potential to be used to detect hypoxic cells or estimate the hypoxic fraction of solid tumors. Although it has been demonstrated that physiological factors (e.g. pH, temperature, nutrient depletion, etc.) have no influence on the induction and repair of both DNA strand breaks and DPCs in irradiated oxygenated cells (Radiat. Res. 140, 321-326, 1994), there are no data to suggest how these physiological factors influence the induction and repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic cells. In the present study, the influence of pH (6.6-7.3) and nutrient depletion on the formation and/or repair of radiation-induced DNA strand breaks and DPCs in partially hypoxic (O2 concentration in the gas phase of 2.0-8.0%) or fully hypoxic (O2 concentration in the gas phase of 0.3-0.4%) populations of exponential (day 3) and plateau-phase (day 5) 9L rat brain tumor cells in culture was determined. Each population of cells was irradiated at 37 degrees C with 15 Gy and trypsinized at 4 degrees C after one half-time of strand break repair at 37 degrees C, and the DNA damage was measured using our alkaline elution technique, with or without proteinase K (PK) in the lysis solution. An analysis of the DNA-protein crosslink factor as a function of the oxygen concentration and a statistical comparison of the single-strand scission factor measured at eluted fraction 7 (SSSF7), with or without PK in the lysis solution, indicate that pH and nutrient depletion do not significantly influence the formation and/or repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic 9L cells. The data also demonstrate that the DNA lesions produced in irradiated partially hypoxic cells will cause the hypoxic fraction or fractional hypoxic volume to be overestimated by this DNA damage assay, not underestimated. However, this potential overestimation of the hypoxic fraction or fractional hypoxic volume should not limit the usefulness of this DNA damage assay in most laboratory studies, because it will take the contribution of a large cohort of partially hypoxic cells to overcome the dominance of the signal from a relatively small cohort of fully hypoxic cells.

Synthesis, characterization and dna binding study of Co(III) polypyridyl mixed-ligand complexes

Two complexes [Co(phen)2TATP]3 and [Co(bipy)2TATP]3, where TATP is the planar ligand 1,4,8,9-tetra-aza-triphenylene, were synthesized and characterized. The binding of Co(III) polypyridyl mixed-ligand complexes to calf thymus DNA was investigated using absorption spectroscopy, fluorescence spectroscopy, viscosity measurement, and DNA cleavage assay. The absorption spectroscopic studies showed that the two new complexes [Co(phen)2TATP]3 and [Co(bipy)2TATP]3 exihibited apparently higher intercalating efficiency to DNA base pairs than [Co(phen)3]3 and [Co(bipy)3]3. The fluorescence of DNA helix was quenched by Co(III) complexes very strongly. From the viscosity experiment, we inferred that the binding of [Co(phen)3]3 to DNA may be not by classical intercalation. When irradiated at 302 nm, [Co(phen)2TATP]3 induced single-strand scissions and [Co(bipy)2TATP]3 induced double-strand scissions in supercoiled DNA.

Enhanced DNA Photocleavage by a Subnanomolar Amount of Mercury(II) Porphyrin

Abstract Mercury (II)-porphyrin complex as low as 3 × 10−7 mol dm−3 significantly accelerated single and double strand scissions of pBluescript plasmid DNA upon visible photoirradiation with the formation of singlet oxygen, hydroxyl radical, and other oxygen radicals which were confirmed by EPR measurement.

DNA degradation by the copper(II) complex with tripodal-ligands containing peptide group

Copper-based transition metal complexes which perform single-strand scission of DNA in a site-specific manner have been designed. The complexes Cu(dpg) + and Cu(dpgt)Cl + (H(dpg) and (dpgt) represent bis(2-pyridylmethyl) derivatives of glycylglycine and glycylglycylglycine, respectively, were prepared in this study and its crystal structures were determined. We have found that Cu(dpgt)Cl + and Cu(dpg) + complexes convert supercoiled plasmid DNA into a mixture of nicked and linear forms in the presence of hydrogen peroxide, whereas related Cu(II) complexes, which lack a peptide group, produce complete degradation of DNA under the same experimental conditions.

DNA Binding and Cleaving Activity of Antitumor Metal Complex: Sodium trans-dinitrobis(2,4-pentanedionato)cobalt(III)

Abstract The antitumor metal complex, sodium trans-dinitrobis(2,4-pentanedionato)cobalt(III) bound to adenine rich regions of DNA helix and caused single strand scission under the irradiation of light.