Hydrolytic Cleavage Of DNA Research Articles

The Therapeutic Potential in Cancer of Terpyridine-Based Metal Complexes Featuring Group 11 Elements.

Terpyridine-based complexes with group 11 metals emerge as potent metallodrugs in cancer therapy. This comprehensive review focuses on the current landscape of anticancer examples, particularly highlighting the mechanisms of action. While Cu(II) complexes, featuring diverse ancillary ligands, dominate the field, exploration of silver and gold species remains limited. These complexes exhibit significant cytotoxicity against various cancer cell lines with a commendable selectivity for non-tumorigenic cells. DNA interactions, employing intercalation and groove binding, are pivotal and finely tuned through terpyridine ligand functionalization. In addition, copper complexes showcase nuclease activity, triggering apoptosis through ROS generation. Despite silver's high affinity for nitrogen donor atoms, its exploration is relatively sparse, with indications of acting as intercalating agents causing DNA hydrolytic cleavage. Gold(III) compounds, overshadowing gold(I) due to stability concerns, not only intercalate but also induce apoptosis and disrupt the mitochondrial membrane. Further investigations are needed to fully understand the mechanism of action of these compounds, highlighting the necessity of exploring additional biological targets for these promising metallodrugs.

DNA binding, cleavage and anticancer activity of a Zn(II)-Cu(II) hetero-dinuclear carbohydrazone complex containing the secondary ligand 1,10-phenanthroline

A hetero di-nuclear complex [Zn(o-phen)LCu(OAc)] (1) (H3L = o-HOC6H4CH = NNH)2C = O, o-phen = 1,10-phenanthroline) exhibited intercalative DNA binding (Kb = 1.03 × 105 M−1) as revealed from UV–Vis spectral analysis as well as viscosity study and further supported by molecular docking study of 1 with DNA. The apparentbinding constant (Kapp = 21.78 × 105 M−1) obtained from ethidium bromide (EB) displacement assay using fluorescence spectral study further confirmed the efficient binding ability of the complex to DNA. The complex displayed both oxidative as well as hydrolytic DNA cleavage activity. Quantitative hydrolytic cleavage results suggested significant nuclease activity against PUC19 plasmid DNA. Oxidative DNA cleavage experiments done with ROS (reactive oxygen species) scavengers showed the involvement of •OH in the cleavage mechanism. Anticancer activity of 1 was tested by evaluating the cytotoxicity of the complex towards cervical cancer HeLa, cisplatin resistant breast cancer MDA-MB-231 and epidermoid carcinoma A431 cells. IC50 values (11.08 ± 1.1 µM for HeLa, 2.75 ± 0.16 µM for MDA-MB-231 and 1.38 ± 0.07 µM for A431 cells) revealed significant cell death in all three cell lines. Nuclear staining using AO/PI and Hoechst 33,342 suggested cell death occurred due to apoptosis in these cancer cells. 1 induced high level of ROS in HeLa cells. It also caused G2/M phase arrest in HeLa cells as revealed from cell cycle analysis.

Synthesis, Characterization, DNA/HSA Interaction, and Cytotoxic Activity of a Copper(II) Thiolate Schiff Base Complex and Its Corresponding Water-Soluble Stable Sulfinato-O Complex Containing Imidazole as a Co-ligand.

A Cu(II) thiolato complex [CuL(imz)] (1) (H2L = o-HOC6H4C(H)=NC6H4SH-o) and the corresponding water-soluble stable sulfinato-O complex [CuL'(imz)] (2) (H2L' = o-HOC6H4C(H)=NC6H4S(=O)OH) were synthesized and characterized using physicochemical techniques. Compound 2 is found to be a dimer in the solid state as characterized using single-crystal X-ray crystallography. XPS studies clearly showed the differences in the sulfur oxidation states in 1 and 2. Both compounds are found to be monomers in solution as revealed from their four-line X-band electron paramagnetic resonance spectra in CH3CN at room temperature (RT). 1-2 were tested to assess their ability to exhibit DNA binding and cleavage activity. Spectroscopic studies and viscosity experiments suggest that 1-2 bind to CT-DNA through the intercalation mode having moderate binding affinity (Kb ∼ 104 M-1). This is further supported by molecular docking studies of complex 2 with CT-DNA. Both complexes display significant oxidative cleavage of pUC19 DNA. Complex 2 also showed hydrolytic DNA cleavage. The interaction of 1-2 with HSA revealed that they have strong ability to quench the intrinsic fluorescence of HSA by a static quenching mechanism (kq ∼ 1013 M-1 s-1). This is further complemented by Förster resonance energy transfer studies that revealed binding distances of r = 2.85 and 2.75 nm for 1 and 2, respectively, indicating high potential for energy transfer from HSA to complex. 1-2 were capable of inducing conformational changes of HSA at secondary and tertiary levels as observed from synchronous and three-dimensional fluorescence spectroscopy. Molecular docking studies with 2 indicate that it forms strong hydrogen bonds with Gln221 and Arg222 located near the entrance of site-I of HSA. 1-2 showed potential toxicity in human cervical cancer HeLa cells, lung cancer A549 cells, and cisplatin-resistant breast cancer MDA-MB-231 cells and appeared to be most potent against HeLa cells (IC50 = 2.04 μM for 1 and 1.86 μM for 2). In HeLa cells, 1-2 mediated cell cycle arrest in S and G2/M phases, which progressed into apoptosis. Apoptotic features seen from Hoechst and AO/PI staining, damaged cytoskeleton actin viewed from phalloidin staining, and increased caspase-3 activity upon treatment with 1-2 collectively suggested that they induced apoptosis in HeLa cells via caspase activation. This is further supported by western blot analysis of the protein sample extracted from HeLa cells treated with 2.

Equilibrium, DNA binding, docking, and antimicrobial studies of Ni(II) and Cu(II) chelates with 1‐(4′‐nitrobenzoyl)‐3‐thiosemicarbazide

Ni(II) and Cu(II) chelates of 1‐(4′‐nitrobenzoyl)‐3‐thiosemicarbazide (PNBTSC, HL) were synthesized and characterized by liquid chromatography‐mass spectrometry (LC–MS), thermogravimetric analysis (TGA), infra‐red (IR), UV–visible, and electron spin resonance (ESR) spectral techniques, which revealed the tetragonally distorted octahedral geometry around the central metal ion in both the chelates. The kinetic and thermodynamic properties were calculated using the Coats–Redfern relation. Thermal decomposition processes of chelates indicate the thermally stability of the chelates. The optimized molecular geometry, atomic charges, energies of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), and energy gap 0.11203 eV of HL have been studied by density functional theory (DFT) calculations, which revealed the soft nature and high reactivity of the compound. Equilibrium studies of the 1‐(4‐nitrobenzoyl)‐3‐thiosemicarbazide with Ni(II) ion in 70% v/v DMF‐water medium have been evaluated. The pKa value of HL was found to be 7.1, and the values indicated the formation of 1:1 and 1:2 chelates in solution. DNA binding affinities of synthesized metal chelates with calf thymus DNA using UV absorption spectroscopy, spectro‐fluorimetry, and viscosity measurements revealed hypochromism and intercalative mode of binding. The Kb values from the absorption studies are 5.00 × 106 and 3.33 × 106 M−1, and Ksv values from fluorescence studies are 0.108 and 0.395 M−1 for chelates 1 and 2, respectively. The cleavage of supercoiled pBR33 DNA without any additives was studied using gel electrophoresis method which exhibited hydrolytic cleavage of plasmid DNA. Molecular docking studies carried out for the complexes also inferred intercalative mode of binding with DNA. The inhibition constants for HL, 1, and 2 are 107.09, 21.66, and 184.45 μM, respectively. Ligand and its metal chelates screened for antimicrobial activity against Gram positive and Gram negative bacteria revealed higher activity in chelates.

Rational design of an artificial hydrolytic nuclease by introduction of a sodium copper chlorophyllin in L29E myoglobin

Heme proteins have recently emerged as promising artificial metalloenzymes for catalyzing diverse reactions. In this report, L29E Mb, a single mutant of myoglobin (Mb), was reconstituted by replacing the heme with a sodium copper cholorophyllin (CuCP) to form a new green artificial enzyme (named CuCP-L29E Mb). The reconstituted protein CuCP-L29E Mb was found to exhibit hydrolytic DNA cleavage activity, which was not depending on O2. In addition, Mg2+ ion could effectively promote the DNA cleavage activity of CuCP-L29E Mb. Wild-type (WT) Mb reconstituted with CuCP (named CuCP-WT Mb) did not show DNA cleavage activity under the same conditions. This study suggests that both Mg2+ and the ligand Glu29 are critical for the nuclease activity and the artificial nuclease of Mg2+-CuCP-L29E Mb may have potential applications in the future.

Mixed Ligand Mononuclear Copper(II) Complex as a Promising Anticancer Agent: Interaction Studies with DNA/HSA, Molecular Docking, and In Vitro Cytotoxicity Studies.

The isolated copper(II) complex [CuL(o-phen)]·H2O (1) [H2L = o-HO-C6H4C(H)=N-C6H4-SH-o, o-phen = 1,10-phenanthroline] was structurally characterized using single-crystal X-ray crystallography. 1 in CH3CN at liquid nitrogen temperature displayed a characteristic monomeric X-band electron paramagnetic resonance spectrum having a tetragonal character with g∥ = 2.1479 and g⊥ = 2.0691 and A∥ ≈ 18.0 mT and A⊥ ≤ 3.9 mT, respectively. 1 showed a strong binding affinity toward calf thymus DNA as reflected from its intrinsic binding constant (Kb = 7.88 × 105 M–1), and its competitive displacement of ethidium bromide suggested an intercalative DNA-binding mode (Kapp = 1.32 × 106 M–1). This was confirmed from the viscosity study that showed an increase in the viscosity of DNA with an increasing concentration of 1. Complex 1 is highly efficient in promoting oxidative and hydrolytic DNA cleavage (kobs = 1.987 h–1). 1 showed a strong binding affinity with the carrier protein human serum albumin (HSA) (Ka = 5.22 × 105 M–1). A high bimolecular quenching constant kq = 2.29 × 1013 M–1s–1 indicated a static quenching mechanism involved in the fluorescence quenching of HSA by 1. Fluorescence resonance energy transfer theory suggested that the distance (r = 3.52 nm) between 1 and HSA is very close. Molecular docking studies suggested that 1 primarily binds to HSA in subdomain IIA. A protein–ligand interaction profiler was used to visualize hydrophobic, hydrogen bonds, and π–cation interactions between HSA and 1. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using HeLa and MDA-MB-231 cells showed a significant in vitro anticancer activity of 1 (IC50 2.63 and 2.68 μM, respectively). Nuclear staining assays suggested apoptotic cell death in HeLa cells treated with 1. The effect of 1 on the cytoskeletal actin filaments visualized using phalloidin staining showed extensive destruction of actin filaments. Flow cytometric analysis indicated that 1 inhibits the growth of HeLa cells through cell cycle arrest in the S phase. Western blot analysis showed upregulation in the expression of apoptotic marker proteins caspase 3, p53, and Bax. These results collectively indicate that 1 induces apoptosis by promoting DNA damage and has a high potential to act as an anticancer agent.

Pyridine substituted BODIPYs: synthesis, characterization and cholinesterease, α-glucosidase inhibitory, DNA hydrolytic cleavage effects

In this study, the synthesis of new monostyryl (BDPY-2) and distyryl BODIPY dyes (BDPY-4, BDPY-5) containing pyridine groups has been reported for the first time. The acetylcholinesterase from Electrophorus electricus (AChE), butyrylcholinesterase from equine serum (BuChE), α-glucosidase from Saccharomyces cerevisiae and DNA hydrolytic cleavage actions of BDPY-2, BDPY-4, BDPY-5 were investigated using various techniques. The results indicated that the compounds had varying inhibition properties against AChE, BuChE, and α-glucosidase. BDPY-4 was the most potent compound on AChE with IC50 of 54.78 ± 4.51 µM, and Lineweaver–Burk plots indicated that the compound is bound to a site other than the active site as a noncompetitive inhibitor. The compound-protein binding experiment showed that BDPY-4 changed the microenvironment around AChE. On the other hand, the compounds showed lower α-glucosidase inhibition than the positive control. The DNA hydrolytic cleavage effects were not observed on supercoiled plasmid DNA in the presence of the compounds as compared to negative controls. These findings suggested that BDPY-4 might be a promising compound to treat Alzheimer’s diseases.

Fabrication and Characterization of Saffron Stamen Aqueous Extract Controlled Release System as Potential Topical Treatment of Thermal Burn Wounds

AbstractIn our study, we aimed the preparation of Saffron stamen aqueous extract (SSAE) loaded chitosan nanoparticles (CNPs) to be used topically for thermal burns. First, biological activity tests such as DNA binding, DNA cleavage and antibacterial activity were performed to determine the effectiveness of SSAE to be encapsulated. Additionally, a genotoxicity test was performed for the reliability of SSAE. Ionic gelation method was used for encapsulation, and SSAE‐loaded CNPs were obtained. Average particle size, zeta potential, polydispersity index (PdI), morphology, and releasing kinetics of the SSAE‐loaded CNPs and blank CNPs were determined. DNA binding showed that SSAE interacted with DNA electrostatically. It was determined that all doses of SSAE used in hydrolytic and oxidative DNA cleavage experiments cleave DNA. As a result of the Ames / Salmonella test, it was found that 1,25 mg SSAE was not genotoxic. Then, this concentration was used in nanoparticle synthesis. As a result of DLS analysis, it was determined that SSAE‐loaded CNPs have an average size of 57.61±1.234 nm, 0.175±0.008 PdI, and +14±0.755 mV zeta potential. Encapsulation efficiency and loading capacity of SSAE‐loaded CNPs calculated as 71.76 % and 2,64 % respectively. As a result of releasing kinetics, it was determined that 79.54 % of SSAE was released after 168 hours. Antibacterial results revealed that SSAE loaded CNPs were more effective against bacteria than blank SSAE.

Structural and biological characterization of anticancer nickel(II) bis(benzimidazole) complex

In the present study, nickel(II) complex with 2-[2-[2-(1H-benzimidazol-2-yl)ethylsulfanyl]ethyl]-1H-benzimidazole (tebb) of formula [Ni(tebb)2](ClO4)2 has been prepared and its structure was proved by X-ray crystallography. The central nickel atom is in deformed octahedral vicinity. Four nitrogen atoms of two ligands form plane of octahedral and sulfur atoms are in apical positions. Perchlorate anions are outside the coordination sphere. The coordination compound was tested for its biological activities in an array of in vitro assays. It was found that the synthesized complex possesses interesting biological activity, which is most likely related to its cell-type related uptake kinetics. The synthesized complex is readily uptaken by malignant MDA-MB-231 and CACO-2 cells with the lowest uptake by healthy Hs27 fibroblasts. The lowest IC50 values were obtained for MDA-MB-231 cells (5.2–12.7 μM), highlighting exceptional differential cytotoxicity (IC50 values for healthy fibroblasts were 38.6–51.5 μM). Furthermore, it was found the complex is capable to cause hydrolytic DNA cleavage, promotes an efficient DNA fragmentation and to trigger an extensive formation of intracellular reactive oxygen species. Overall, current work presents a synthesis of Ni(II) coordination compound with interesting biological behavior and with a promising potential to be further tested in pre-clinical models.

Development, evaluation and effect of anionic co-ligand on the biological activity of benzothiazole derived copper(II) complexes

Research on development of novel metal based anti-cancer agents continues with its popularity among bioinorganic community. Benzothiazole, an important heterocyclic pharmacophore, was chosen as a valuable and useful scaffold for the synthesis of novel copper(II) complexes. Three new copper(II) complexes obtained from the synthesis of newly synthesized benzothiazole based N-(benzo[d]thiazol-2-ylmethyl)-N-methyl-2-(pyridin-2-yl)ethan-1-amine (btzpy) ligand with CuCl2 [Cu(btzpy)Cl2] (1), Cu(NCS)2 [Cu(btzpy)(NCS)2] (2), and Cu(NO3)2 [Cu(btzpy)(NO3)(H2O)]NO3 (3) were isolated and characterized by physical and spectroscopic measurements, including single-crystal X-ray structures. The interaction of complexes 1 and 3 with calf thymus (CT)-DNA was investigated using ethidium bromide fluorescence quenching assay and weak intercalation with KSV values of 9.8 × 102 M−1 and 8.2 × 102 M−1, respectively was observed. All three complexes have shown DNA cleavage of supercoiled plasmid DNA forming single nicked and double nicked forms in the presence of external reducing agents like 3-mercaptopropionic acid (3-MPA) and ascorbic acid. The water-soluble complexes 1 and 3 also show prominent hydrolytic DNA cleavage. From the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, it was observed that complex 2 also exhibits good antioxidant properties. The cytotoxicity of complexes 1–3 was tested against the lung cancer cell line (A549) and complex 2 with -NCS moiety shows maximum activity in the micromolar range. A rationale for the observed activity is proposed in light of the other properties of these molecules.

Ternary Co(II), Ni(II) and Cu(II) complexes containing dipyridophenazine and saccharin: Structures, reactivity, binding interactions with biomolecules and DNA damage activity

Herein, we report physiochemical aspects, crystal structures, biological reactivity and interaction, DNA damage activity and preliminary antimicrobial properties of three metal complexes, [Co/Ni(dppz)2(sac)(H2O)]ClO4 [Co(II), 1; Ni(II), 2] and [Cu(dppz)2(sac)]ClO4 (3), where dppz is dipyrido[3,2-a:2′,3′-c]phenazine as potential photosensitizer and DNA intercalator and sac is artificial sweetener saccharin (o-sulfobenzimide). The solid-state structural analysis revealed that the bivalent metal centre is in distorted octahedral [MN5(OH2); M = Co(II) (1), M = Ni(II) (2)] and square pyramidal [MN5; M = Cu(II) (3)] geometries arising from N,N donor-dppz and saccharinate (sac) ligands. The complexes showed redox responses with characteristic metal and ligand (dppz) redox couples. The reactivity and speciation of complexes were studied with glutathione (GSH) and sodium L(+) ascorbate (asc) from UV–vis, fluorescence and ESI-MS analyses. The complexes exhibit high binding affinity (K ~ 105–107 M−1) to human serum albumin (HSA) with a preferential binding affinity towards tryptophan (Trp-214) residue. The fluorescence resonance energy transfer (FRET) analysis showed an efficient energy transfer to the complexes. The monocationic complexes strongly bind CT-DNA (Kb ~ 105–106 M−1) by intercalation from planar dppz and stabilized from the electrostatic interactions resulting in unwinding and loss of helicity of DNA double helix. The complexes 1–3 damage supercoiled pUC19 DNA efficiently upon photo-irradiation at UV-A light (λ = 365 nm) to its nicked circular form via generation of hydroxyl radical (OH) and singlet oxygen (1O2) mediated pathways. The hydrolytic DNA cleavage activity of Cu-complex 3 induced by OH radicals and enhanced multifold in the presence of asc, GSH and light indicate involvement of a possible photoredox mechanism. All the complexes show moderate to good anti-microbial activity against Gram-positive Staphylococcus aureus bacterium. The studies showed the active and distinct role of transition metals and ligands for their biological activity.

Octahedral copper(ii)-diimine complexes of triethylenetetramine: effect of stereochemical fluxionality and ligand hydrophobicity on CuII/CuI redox, DNA binding and cleavage, cytotoxicity and apoptosis-inducing ability.

Octahedral copper(ii) complexes of the type [Cu(trien)(diimine)](ClO4)2 (1-4), where trien is triethylenetetramine and diimine is 2,2'-bipyridine (1), 1,10-phenanthroline (2), 5,6-dimethyl-1,10-phenanthroline (3), and 3,4,7,8-tetramethyl-1,10-phenanthroline (4), have been isolated. Single crystal X-ray structures of 1 and 2 reveal that the coordination geometry around Cu(ii) is tetragonally distorted octahedral. The stereochemical fluxionality of the complexes illustrates the observed trend in CuII/CuI redox potentials and DNA binding affinity (Kb: 1, 0.030 ± 0.002 < 2, 0.66 ± 0.01 < 3, 1.63 ± 0.10 < 4, 2.27± 0.20 × 105 M-1), determined using absorption spectral titration. All complexes effect oxidative DNA cleavage more efficiently than hydrolytic DNA cleavage. The bpy complex 1 with stereochemical fluxionality lower than its phen analogue 2 shows a higher cytotoxicity against both A549 lung (IC50, 3.3 μM) and MCF-7 human breast (IC50, 3.9 μM) cancer cells, and induces the generation of the highest amount of ROS in A549 cells. Complex 3 with a higher stereochemical fluxionality and higher ligand hydrophobicity exhibits a higher DNA binding and cleavage ability and higher cytotoxicity (IC50, 2.1 μM) towards MCF-7 cells. Complex 4 with a still higher stereochemical fluxionality displays the highest DNA binding and cleavage ability but a lower cytotoxicity towards both A549 and MCF-7 cell lines due to its tendency to form a five-coordinated complex with the uncoordinated amine group. Annexin V.Cy3 staining and immunoblot analysis demonstrate the mechanism of cell death caused by 1 and 2. The finding of the up-regulation of the pro-apoptotic Bax protein and down-regulation of PARP protein in western blot analysis confirms the induction of apoptosis by these complexes.

Cleaving DNA by nanozymes.

DNA cleavage plays a crucial role in many biological processes such as DNA replication, transcription, and recombination. It is also a powerful tool in gene editing, therapeutics and biosensor design. Nanozymes aim to develop nanomaterial-based enzyme mimics. Compared with natural enzymes, nanozymes offer advantages of higher stability, lower cost, and recyclability. Recently, nanozymes with interesting DNA cleavage activities have emerged, including both hydrolytic and oxidative cleavage. This Perspective starts by introducing DNA cleavage of nanozymes, focusing on recent examples. Some interesting nanozymes include CeO2 nanoparticles for the hydrolytic cleavage of single-stranded DNA oligonucleotides, chiral carbon dots mimicking topoisomerase activity, and light-assisted cleavage of DNA. The corresponding cleavage mechanisms are then discussed along with a few representative applications for DNA repair and as antibacterial agents. Finally, a few future research opportunities are discussed.

Interaction with plasmid DNA of Hoechst-TACN conjugates

ABSTRACTA new family of conjugates between the Hoechst minor groove binder and the TACN metal ion ligand connected through hydrophobic alkyl or more hydrophilic oxyethyl linkers of different length has been prepared. The linkers are connected to the convex side of the Hoechst skeleton thus forcing the TACN ligand to exit the minor groove and interact with the phosphate backbone of DNA. The conjugates preserve the binding mode of Hoechst with an affinity influenced by the nature of the linker, the more hydrophobic being the more efficient. Coordination of Cu(II) or Zn(II) poorly affect these parameters. Nevertheless, the Zn(II) complex bearing a C6 linear alkyl linker induced a modest but reproducible acceleration of the hydrolytic cleavage of DNA which can be ascribed to the ability of the conjugate to deliver the hydrolytic subunit close to the DNA phosphodiester bonds.

Equilibrium, DNA Cleavage and Antimicrobial Studies of Cu(II) and Zn(II) Complexes with Novel 1-Propionyl-4-methyl-3-thiosemicarbazide

Cu(II) and Zn(II) metal complexes with novel Schiff base 1-propionyl-4-methyl-3-thiosemicarbazide (PMTSC) were synthesized and characterized by LC-MS, IR, 1H NMR (D2O exchangeable), 13C NMR and UV-visible spectra, ESR, TGA, molar conductance and magnetic susceptibility measurements. The spectro-analytical studies revealed the composition of complexes as ML2 for Cu(II)-PMTSC and Zn(II)-PMTSC complex. The proton-ligand dissociation constant and metal-ligand formation constants of PMTSC with Zn(II) have been determined in 70 % (v/v) DMF-water medium at 0.1M (KNO3) ionic strength and 303 K using potentiometric Irving-Rossotti titration technique. The results revealed that PMTSC acts as monobasic ligand by releasing proton from amide via enol form and forms 1:2 (Zn-L) complex in solution. The cleavage of plasmid pBR322 DNA without any additives was monitored by gel electrophoresis and these complexes exhibited hydrolytic cleavage of plasmid DNA. The antibacterial activity of the Schiff base and its Cu(II) and Zn(II) complexes were tested against Gram-positive and Gram-negative bacteria.

Peroxidase from proso millet exhibits endonuclease-like activity.

In this study, the mechanism of DNA cleavage by cationic peroxidase from proso millet (PmPOD) was investigated. PmPOD cleaved supercoiled circular DNA into both nicked circular and linear forms via a cleavage mechanism that resembles those of native endonucleases. Inhibition and ligation studies demonstrated that reactive oxygen species and the ferriprotoporphyrin IX moiety in PmPOD are not involved in PmPOD-mediated DNA cleavage. Similar to other endonucleases, Mg ions considerably enhance the DNA cleavage activity of PmPOD. Further studies suggested that PmPOD can disrupt phosphodiester bonds in DNA and mononucleotides, indicating that it is a phosphatase. The phosphatase activity of PmPOD is higher than that of horseradish peroxidase (HRP), but the peroxidase activity of PmPOD was lower than that of HRP. PmPOD-mediated hydrolytic cleavage of DNA observed in this study is different from those reported for heme proteins. This study provides valuable insights into the distinct mechanisms underlying DNA cleavage by heme proteins.

Efficient hydrolytic cleavage of DNA and antiproliferative effect on human cancer cells by two dinuclear Cu(II) complexes containing a carbohydrazone ligand and 1,10-phenanthroline as a coligand.

We report the synthesis, crystal structures and biological activities of two dinuclear Cu(II) complexes [Cu(o-phen)LCu(OAc)] (1) and [Cu(o-phen)LCu(o-phen)](OAc) (2), where o-phen = 1,10-phenanthroline, H3L = o-HOC6H4C(H)=N-NH-C(OH)=N-N=C(H)-C6H4OH-o, and OAc=CH3COO-. Both compounds display strong and broad X-band EPR spectra at RT in their powder state confirming that these are paramagnetic. The intercalative DNA binding of the compounds as revealed from spectrophotometric studies was found to be consistent with the results of fluorescence spectroscopic studies for ethidium bromide displacement assay as well as enhanced viscosity of DNA in the presence of these compounds. The compounds effectively catalyze hydrolytic cleavage of supercoiled pUC19 DNA and show remarkable cytotoxicity toward human lung cancer A549 cell line (IC50 values are 4.34 and 8.46µM for 1 and 2, respectively) and breast cancer MCF7 cell line (IC50 values are 6.50 and 8.68µM for 1 and 2, respectively) and are found to be relatively less toxic toward keratinocyte HaCaT normal cell line (IC50 values are 11.19 and 16.01µM for 1 and 2, respectively). Annexin-V/PI dual staining results analyzed by flow cytometry strongly suggest the induction of apoptotic pathway for the anticancer activity of these complexes. Flow cytometry experiment for cell cycle analysis showed considerable increase in the G2/M phase in both A549 and MCF7 cell lines by these two compounds. On the other hand, compounds 1 and 2 activate reactive oxygen species (ROS) level in A549 cells, but act as scavengers or inhibitors of ROS in MCF7 cell line as analyzed by DCFDA staining using flow cytometry. Two dinuclear Cu(II) complexes exhibit efficient hydrolytic cleavage of DNA and display remarkable cytotoxicity against human lung cancer A549 and breast cancer MCF7cells. The ROS level in A549 cells is activated, but the ROS level in MCF7 cells is decreased in the presence of these complexes. Cell cycle analysis by flow cytometry shows G2/M phase arrest in both these cell lines.

Acceleration of Hydrolytic DNA Cleavage by Dicopper(II) Complexes with p-Cresol-Derived Dinucleating Ligands at Slightly Acidic pH and Mechanistic Insights

Abstract Four dicopper(II) complexes, [Cu2(µ-X)(bcmp)](ClO4)2 [X = OH (1a) and X = Cl (1b)], [Cu2(µ-OH)(Me4bcmp)](ClO4)2 (2), and [Cu2(bcc)](ClO4)3 (3), were synthesized with three p-cresol-derived ligands, 2,6-bis(1,4,7-triazacyclononylmethyl)-4-meth-ylphenol (Hbcmp), 2,6-bis(1,4,7-triaza-4,7-dimethylcyclonon-ylmethyl)-4-methylphenol (HMe4bcmp), and 2,6-bis(1,4,7,10-tetrazacyclododecylmethyl)-4-methylphenol (Hbcc) to study hydrolytic DNA cleavage. Crystal structures of 1a, 1b, 2, and 3 were determined by X-ray analysis. The pH titrations and spectroscopic studies in the complexations of the ligands with copper(II) perchlorate revealed that the dicopper core structures of 1a, 2, and 3 in the solid state are kept at pH 5–9 in an aqueous solution. DNA binding abilities of 1a, 2, and 3 were examined by isothermal titration calorimetry (ITC). DNA cleavage studies were carried out by using supercoiled plasmid pUC19 DNA. 1a largely accelerated hydrolytic DNA cleavage at pH 5–6 but not at pH 7–8. This is the first example of pH-dependent DNA cleavage by a dicopper complex. Inhibition studies with specific DNA binders, 4′,6-diamidino-2-phenylindole and methyl green, suggested that 1a accelerates the DNA cleavage via GC-specific binding. The mechanistic insights into the pH-dependent DNA cleavage are proposed on the basis of the crystal structures, structures in aqueous solutions, DNA binding modes, and DNA cleavage activities of 1a, 1b, 2, and 3.

DNA binding, cleavage and cytotoxicity studies of three mononuclear Cu(II) chloro-complexes containing N-S donor Schiff base ligands.

We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide). 1-3 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92μM for 1, 2 and 3, respectively, as compared to the IC50 value (13 μM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 1-3. Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.

The hydrolysis of 6-phosphogluconolactone in the second step of pentose phosphate pathway occurs via a two-water mechanism

Hydrolysis reaction marks the basis of life yet the mechanism of this crucial biochemical reaction is not completely understood. We recently reported the mechanisms of hydrolysis of nucleoside triphosphate and phosphate monoester. These two reactions hydrolyze P-O-P and P-O-C linkages, respectively. Here, we present the mechanism of hydrolysis of δ-6-phosphogluconolactone, which is an important precursor in the second step of the pentose phosphate pathway. Its hydrolysis requires the cleavage of C-O-C linkage and its mechanism is hitherto unknown.We report three mechanisms of hydrolysis of δ-6-phosphogluconolactone based on density functional computations. In the energetically most favorable mechanism, two water molecules participate in the hydrolysis reaction and the mechanism is sequential, i.e., activation of the attacking water molecule (OH bond breaking) precedes that of the cleavage of the CO bond of the C-O-C linkage. The rate-limiting energy barrier of this mechanism is comparable to the reported experimental free energy barrier. This mechanism has similarities with the mechanism of triphosphate hydrolysis and that of hydrolytic cleavage of DNA in EcoRV enzyme. This two-water sequential hydrolysis mechanism could be the unified mechanism required for the hydrolysis of other hydrolysable species in living cells.