Treatment with 1, 10 Phenanthroline-5-Amine Reduced Amyloid Burden in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent age-related dementia, and, despite numerous attempts to halt or reverse its devastating progression, no effective therapeutics have yet been confirmed clinically. However, one class of agents that has shown promise is certain metal chelators. OBJECTIVE: For the novel assessment of the effect of oral administration of 1,10-phenanthroline-5-amine (PAA) on the severity of amyloid plaque load, we used a transgenic (Tg) mouse model with inserted human autosomally dominant (familial) AD genes: amyloid-ß protein precursor (AßPP) and tau. METHODS: AßPP/Tau transgenic mice that model AD were allotted into one of two groups. The control group received no treatment while the experimental group received PAA in their drinking water starting at 4 months of age. All animals were sacrificed at 1 year of age and their brains were stained with two different markers of amyloid plaques, Amylo-Glo+ and HQ-O. RESULTS: The control animals exhibited numerous dense core plaques throughout the neo- and allo- cortical brain regions. The experimental group treated with PAA, however, showed 62% of the amyloid plaque burden seen in the control group. CONCLUSIONS: Oral daily dosing with PAA will significantly reduce the amyloid plaque burden in transgenic mice that model AD. The underlying mechanism for this protection is not fully known; however, one proposed mechanism involves inhibiting the "metal-seeding" of Aß.

Induction of apoptosis in SGC-7901 cells by iridium(III) complexes via endoplasmic reticulum stress-mitochondrial dysfunction pathway.

This study was intended to evaluate the anticancer activity of three newly synthesized iridium(III) complexes [Ir(ppy)2(PEIP)](PF6) (1) (ppy = 2-phenylpyridine, PEIP = 2-phenethyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(SIP)](PF6) (2) (SIP = (E)-2-styryl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(PEYIP)](PF6) (3) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]phenanthroline). The cytotoxic activity in vitro against A549, SGC-7901, HepG2, HeLa and normal NIH3T3 cells was investigated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. We found that the complexes 1, 2 and 3 significantly inhibited cell proliferation, in particular, complexes 2 and 3 show high cytotoxic effect on SGC-7901 cells with an IC50 value of 5.8 ± 0.7 and 4.4 ± 0.1 µM. Moreover, cell cycle assay revealed that the complexes could block G2/M phase of the cell cycle. Apoptotic evaluation by Annexin V/PI staining indicated that complexes 1-3 can induce apoptosis in SGC-7901 cells. In addition, microscopy detection suggested that disruption of mitochondrial functions, characterized by increased generation of intracellular ROS and Ca2+ as well as decrease of mitochondrial membrane potential. Western blot analysis shows that the complexes upregulate the expression of pro-apoptotic Bax and downregulate the expression of anti-apoptotic Bcl-2, which further activates caspase-3 and prompts the cleavage of PARP. Taken together, these results demonstrated that complexes 1-3 exert a potent anticancer effect on SGC-7901 cells via ROS-mediated endoplasmic reticulum stress-mitochondrial apoptotic pathway and have a potential to be developed as novel chemotherapeutic agents for human gastric cancer. Three new iridium(III) complexes [Ir(ppy)2(PEIP)](PF6) (1) (ppy = 2-phenylpyridine, PEIP = 2-phenethyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(SIP)](PF6) (2) (SIP = 2-styryl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(PEYIP)](PF6) (3) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized. The anticancer activity in vitro was investigated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The results show that the complexes induce apoptosis via ROS-mediated endoplasmic reticulum stress-mitochondrial dysfunction pathway.

New iron(III) anti-cancer aminobisphenolate/phenanthroline complexes: Enhancing their therapeutic potential using nanoliposomes.

Malignant melanoma is an aggressive and deadly form of skin cancer and novel and improved therapeutic options are needed. A promising strategy involves the use of metallodrugs combined with liposomes for targeted delivery to cancer cells. In this work, a family of iron(III) complexes was synthesized bearing a trianionic aminobisphenolate ligand (L) and phenanthroline-type co-ligands (NN). Four ternary iron complexes of general formula [Fe(L)(NN)] were obtained: [Fe(L)(amphen)] (1), [Fe(L)(phen)] (2), [Fe(L)(Clphen)] (3), and [Fe(L)(Mephen)] (4), as well as a fifth complex [Fe(L)(NEt3)(H2O)] (5) without the bidentate co-ligand. All complexes were characterized by analytic and spectroscopic techniques and demonstrated to be stable in aqueous environment. Complexes 1 and 2 were able to bind DNA and presented high cytotoxic activity towards human cancer cells. Complex 1 (IronC) was selected for incorporation into different liposomal formulations, which were fully characterized and screened against murine melanoma cells. The IronC liposomal formulation with the highest incorporation efficiency (∼95%) and a low IC50 value (7.1 ± 0.7 µM) was selected for in vivo evaluation. In a syngeneic murine melanoma model the liposomal formulation of IronC yielded the highest impairment on tumour progression when compared with the control, temozolomide, and with the iron complex in free form.

A quantitative method for the assessment of homocysteine thiolactonase enzyme activity.

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)22+) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)22+) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)2+) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)2+) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H+ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.

Interaction of the potent antitumoral compounds Casiopeinas® with blood serum and cellular bioligands.

Casiopeinas® are among the few CuII compounds patented for their antitumor activity, but their mode of action has not been fully elucidated yet. One of them, Cas II-gly, is formed by 4,7-dimethyl-1,10-phenanthroline (Me2phen) and glycinato (Gly). In blood and cells, Cas II-gly can keep its identity or form mixed species with serum or cytosol bioligands (bL or cL) with composition CuII-Me2phen-bL/cL, CuII-Gly-bL/cL, or CuII-bL/cL. In this study, the binding of Cas II-gly with low molecular mass bioligands of blood serum (citric, L-lactic acid, and L-histidine) and cytosol (reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide (NADH), adenosine triphosphate (ATP), and l-ascorbic acid) was examined through the application of instrumental (ElectroSpray Ionization-Mass Spectrometry and Electron Paramagnetic Resonance) and computational (Density Functional Theory) methods. The results indicated that mixed species CuII-Me2phen-bL/cL are formed, with the bioligands replacing glycinato. The formation of these adducts may participate in the copper transport toward the target organs and facilitate the cellular uptake or, in constrast, preclude it. In the systems with GSH, NADH and L-ascorbate, a redox reaction occurs with the partial oxidation of cL to the corresponding oxidized form (GSSG, NAD+ and dehydroascorbate) which interact with CuII. Formed CuI ion does not give complexation reactions with reduced or oxidized form of bioligands for its 'soft' character and low affinity for oxygen and nitrogen donors compared to CuII. However, CuI could promote Fenton-like reactions with production of reactive oxygen species (ROS) related to the antitumor activity of Casiopeinas®.

An in silico investigation of the binding modes and pathway of APTO-253 on c-KIT G-quadruplex DNA.

The stability of c-KIT G-quadruplex DNA via ligands has been a significant concern in the growing field of cancer therapy. Thus, it is very important to understand the mechanism behind the high binding affinity of the small drug molecules on the c-KIT G-quadruplex DNA. In this study, we have investigated the binding mode and pathway of the APTO-253 ligand on the c-KIT G-quadruplex DNA employing a total of 10 µs all atom molecular dynamics simulations and further 8.82 µs simulations via the umbrella sampling method using both OL15 and BSC1 latest force fields for DNA structures. From the cluster structure analysis, mainly three binding pathways i.e., top, bottom and side loop stacking modes are identified. Moreover, RMSD, RMSF and 2D-RMSD values indicate that the c-KIT G-quadruplex DNA and APTO-253 molecules are stable throughout the simulation run. Furthermore, the number of hydrogen bonds in each tetrad and the distance between the two central K+ cations confirm that the c-KIT G-quadruplex DNA maintains its conformation in the process of complex formation with the APTO-253 ligand. The binding free energies and the minimum values in the potential of mean forces suggest that the binding processes are energetically favorable. Furthermore, we have found that the bottom stacking mode is the most favorable binding mode among all the three modes for the OL15 force field. However, for the BSC1 force field, both the top and bottom binding modes of the APTO-253 ligand in c-KIT G-quadruplex DNA are comparable to each other. To investigate the driving force for the complex formation, we have noticed that the van der Waals (vdW) and π-π stacking interactions are mainly responsible. Our detailed studies provide useful information for the discovery of novel drugs in the field of stabilization of G-quadruplex DNAs.

Use of neomycin as a structured amino-containing side chain motif for phenanthroline-based G-quadruplex ligands and telomerase inhibitors.

In this paper, we report the synthesis of a phenanthroline and neomycin conjugate (7). Compound 7 binds to a human telomeric G-quadruplex (G1) with a higher affinity compared with its parent compounds (phenanthroline and neomycin), which is determined by several biophysical studies. Compound 7 shows good selectivity for G-quadruplex (G4) DNA over duplex DNA. The binding of 7 with G1 is predominantly enthalpy-driven, and the binding stoichiometry of 7 with G1 is one for the tight-binding event as determined by ESI mass spectrometry. A plausible binding mode is a synergistic effect of end-stacking and groove interactions, as indicated by docking studies. Compound 7 can inhibit human telomerase activity at low micromolar concentrations, which is more potent than previously reported 5-substituted phenanthroline derivatives.

Copper Complexes with 1,10-Phenanthroline Derivatives: Underlying Factors Affecting Their Cytotoxicity.

The interpretation of in vitro cytotoxicity data of Cu(II)-1,10-phenanthroline (phen) complexes normally does not take into account the speciation that complexes undergo in cell incubation media and its implications in cellular uptake and mechanisms of action. We synthesize and test the activity of several distinct Cu(II)-phen compounds; up to 24 h of incubation, the cytotoxic activity differs for the Cu complexes and the corresponding free ligands, but for longer incubation times (e.g., 72 h), all compounds display similar activity. Combining the use of several spectroscopic, spectrometric, and electrochemical techniques, the speciation of Cu-phen compounds in cell incubation media is evaluated, indicating that the originally added complex almost totally decomposed and that Cu(II) and phen are mainly bound to bovine serum albumin. Several methods are used to disclose relationships between structure, activity, speciation in incubation media, cellular uptake, distribution of Cu in cells, and cytotoxicity. Contrary to what is reported in most studies, we conclude that interaction with cell components and cell death involves the separate action of Cu ions and phen molecules, not [Cu(phen)n] species. This conclusion should similarly apply to many other Cu-ligand systems reported to date.

Chiral Ru(ii) complexes act as a potential non-viral gene carrier for directional transportation to the nucleus and cytoplasm.

Guanine-rich DNA sequences can spontaneously fold into four-stranded structures called G-quadruplexes (G4s). G4s have been identified extensively in the promoter regions of several proto-oncogenes, including c-myc, as well as telomeres. G4s have attracted an increasing amount of attention in the field of nanotechnology because of their use as versatile building blocks of DNA-based nanostructures. In this study, we report the self-assembly of c-myc G-quadruplex DNA controlled by a pair of chiral ruthenium(ii) complexes coordinated by 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline (PBEPIP), Λ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Λ-RM0627, bpy = bipyridine) and Δ-[Ru(bpy)2(PBEPIP)](ClO4)2 (Δ-RM0627). Λ-RM0627 could promote the high-order self-assembly of c-myc G-quadruplex DNA into a nanowire structure, whereas Δ-RM0627 could induce DNA condensation into G-quadruplex aggregates. Moreover, in vitro studies on human liver carcinoma HepG2 cells showed that the nanowire of c-myc G-quadruplex DNA promoted by Λ-RM0627 could be localized in the nuclei of cells, whereas the nanoparticle of c-myc G-quadruplex DNA generated by Δ-RM0627 was taken up and localized in the cytoplasm. This study provides examples of the enantioselective self-assembly of G4 DNA molecules controlled by chiral ruthenium(ii) complexes and suggests the potential applications of assembled nanostructures as non-viral DNA vectors for gene therapy.

Biological activity and photocatalytic properties of a naphthyl-imidazo phenanthroline (HNAIP) ligand and its [Ir(ppy)2(HNAIP)]Cl and [Rh(ppy)2(HNAIP)]Cl complexes.

The synthesized 2-(hydroxy-1-naphtyl)imidazo-[4,5-f][1,10]phenanthroline (HNAIP) ligand and its new iridium ([Ir(ppy)2(HNAIP)]Cl) and rhodium ([Rh(ppy)2(HNAIP)]Cl) complexes, being ppy = 2-phenylpiridinate, show cytotoxic effects in SW480 (colon adenocarcinoma) and A549 (epithelial lung adenocarcinoma) cells. They all are cytotoxic in the tested cell lines. HNAIP and [Rh(ppy)2(HNAIP)]+ are the most cytotoxic, whereas [Ir(ppy)2(HNAIP)]+ displays negligible cytotoxicity towards A549 cells and moderate activity towards SW480. The interaction of all three compounds with Bovine Serum Albumin (BSA), l-glutathione reduced (GSH), nicotinamide adenine dinucleotide (NADH) and DNA was studied to explain the differences found in terms of cytotoxicity. None of them are able to interact with BSA, thus excluding bioavailability due to plasma protein interaction as the possible differentiating factor in their biological activity. By contrast, small differences have been observed regarding DNA interaction. In addition, taking advantage of the emission properties of these molecules, they have been visualized in the cytoplasmic region of A549 cells. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show, in turn, that the internalization ability follow the sequence [Rh(ppy)2(HNAIP)]+ > [Ir(ppy)2(HNAIP)]+ > cisplatin. Therefore, it seems clear that the cellular uptake by tumour cells is the key factor affecting the different cytotoxicity of the metal complexes and that this cellular uptake is influenced by the hydrophobicity of the studied complexes. On the other hand, preliminary catalytic experiments performed on the photo-oxidation of GSH and some amino acids such as l-methionine (Met), l-cysteine (Cys) and l-tryptophan (Trp) provide evidence for the photocatalytic activity of the Ir(III) complex in this type of reactions.

Anti-MUC1 Aptamer as Carrier Tool of the Potential Radiosensitizer 1,10 Phenanthroline in MCF-7 Breast Cancer Cells.

BACKGROUND: Proteins overexpressed in malignant tissues form important targets in the development of targeted therapeutics, and aptamers comprise an important affinity agent for therapy and drug delivery. In this study, aberrantly expressed mucin 1 glycoprotein was investigated as a therapeutic target in a breast cancer model. MATERIALS AND METHODS: In order to determine the feasibility of using an aptamer against mucin 1 (aptA) as carrier of the cytotoxic compound 1,10-phenanthroline to MCF-7 cells, as a potential radiosensitizer, was studied in experiments using circular dichroism and rhodamine labelling by fluorescent microscopy and flow cytometry. RESULTS: 1,10-Phenanthroline can be intercalated within aptA when complexed with Fe(II) ions, with dissociation constant (Kd) of 30 µM. The complex was subsequently capable of binding to and being internalised in MCF-7 breast cancer cells. CONCLUSION: aptA can carry 1,10-phenanthroline to cancer cells specifically and this complex represents a potential target-directed anticancer therapy.

Dinuclear platinum(II) complexes of imidazophenanthroline-based bridging ligands as potential anticancer agents: synthesis, characterization, and in vitro cytotoxicity studies.

The synthesis and characterization of the dinucleating ligands 1,2-bis(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)ethane (L1) and 1,2-bis(2-(1H-imidazo[4,5-f][1, 10]phenanthrolin-2-yl)phenoxy)hexane (L2) and their dinuclear complexes [Pt2(L1)Cl4] (1) and [Pt2(L2)Cl4] (2) and the in vitro cytotoxicity of the complexes against HeLa, HepG2, and MCF-7 cell lines are reported. Ligand L1 crystallizes in the orthorhombic system with the space group Pbca. The complexes 1 and 2 undergo aquation following first-order kinetics. The MTT and trypan blue assays indicate higher cytotoxicity of the complexes towards the HepG2 and MCF-7 cell lines compared to cisplatin. The AO/EB assay and flow cytometry by Annexin V alexa fluor®488/PI double staining assay demonstrate distinct morphological changes of apoptosis in a dose dependent manner. The cell cycle analysis shows a marked decrease in the DNA content in the G0/G1 phase with an increase in the G2/M phase on increasing the concentration of the complexes. The potential of the complexes as anticancer agents is demonstrated by their antiproliferative activity on the cell lines. The complexes interact with the major groove of DNA through H-bonding between the imidazole N-H protons and the nucleotide residues DC`21/N4 (cytosine) for complex 1 and DT`7/O2 (thymine) and DT`19/O2 (thymine) for complex 2, with the binding energy of - 1.98 and - 4.45 kcal/mol, respectively. Dinuclear Pt(II) complexes of imidazophenanthroline-based dinucleating ligands exhibit antiproliferative activity against HeLa, HepG2, and MCF-7 cell lines.

Redox-cycling and intercalating properties of novel mixed copper(II) complexes with non-steroidal anti-inflammatory drugs tolfenamic, mefenamic and flufenamic acids and phenanthroline functionality: Structure, SOD-mimetic activity, interaction with albumin, DNA damage study and anticancer activity.

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O')2(phen)] (1), [Cu(mef-O,O')2(phen)] (2), [Cu(fluf-O,O')2(phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1-3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~106). The anticancer activities of complexes 1-3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface.

Electronic and fluorescent studies on the interaction of DNA and BSA with a new ternary praseodymium complex containing 2,9-dimethyl 1,10-phenanthroline and antibacterial activities testing.

In this study, fluorescence emission spectra, UV-vis absorption spectra, ethidium bromide (EB)-competition experiment, and iodide quenching experiment were used for the interaction study of the Fish salmon DNA (FS-DNA) with [Pr(dmp)2Cl3(OH2)] where dmp is 2,9-dimethyl 1,10-phenanthroline. The binding constant and the number of binding sites of the complex with FS-DNA were 6.09 ± 0.04 M-1 and 1.18, respectively. The free energy, enthalpy, and entropy changes (ΔG°, ΔH°, and ΔS°) in the binding process of the Pr(III) complex with FS-DNA were -8.02 kcal mol-1, +39.44 kcal mol-1, and +159.56 cal mol-1 K-1, respectively. Based on these results, the interaction process between FS-DNA with [Pr(dmp)2Cl3(OH2)] was spontaneous and the main binding interaction force was groove binding mode. Also, Fluorescence and electronic absorption spectroscopy were used in order to evaluate the binding characteristics, stoichiometry, and interaction mode of praseodymium(III) (Pr(III)) complex with bovine serum albumin (BSA). Title complex showed good binding propensity to BSA presenting moderately high Kb values. The fluorescence quenching of BSA by Pr(III) complex has been observed to be the static process. The positive ΔH° and ΔS° values showed that the hydrophobic interaction is the main force in the binding of Pr(III) complex and BSA. Eventually, the average aggregation number, , of BSA potentially induced by title complex confirmed the 1:1 stoichiometry for title complex-BSA adducts. In vitro, antimicrobial activity of title complex was indicated that the complex is more active against both Escherichia coli and Enterococcus faecalis bacterial strains than Staphylococcus aureus, and Pseudomonas aeruginosa. Communicated by Ramaswamy H. Sarma.

Correlation of intracellular oxygen and cell metabolism by simultaneous PLIM of phosphorescent TLD1433 and FLIM of NAD(P)H.

During photodynamic therapy (PDT), disruption of cell respiration and metabolic changes could be one of the first events. Photophysical characteristics of the photosensitizer (PS) and its specific redox potential define consumption of molecular oxygen followed by generation of reactive oxygen species. The potential PS TLD1433 is based on transition metal Ru(II) and possess an oxygen-dependent luminescence. This enables the study of oxygen consumption by PS-phosphorescence lifetime imaging (PLIM) and simultaneously changes the cellular metabolic state by nicotinamide adenine dinucleotide (NAD(P)H)-fluorescence lifetime imaging (FLIM). Within this study, localization and cellular function of TLD1433 is investigated in bladder carcinoma cells using time-resolved and confocal laser scanning microscopy. Simultaneous FLIM/PLIM of NAD(P)H and TLD1433 during PDT correlated oxygen consumption, redox state and cellular energy metabolism. Our investigations aimed to provide a personalized protocol in theranostic PDT procedures and demonstrate the potential use of TLD1433 PDT also under hypoxic conditions, which are otherwise difficult to treat.

Design, synthesis, and antiprotozoal evaluation of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives.

A series of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives was synthesized, and the compounds were screened in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiparasitic activity with IC50 values in the µm range. The in vitro cytotoxicity of these molecules was assessed by incubation with human HepG2 cells; for some derivatives, cytotoxicity was observed at significantly higher concentrations than antiparasitic activity. The 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline 1h was identified as the most potent antimalarial candidate with ratios of cytotoxic-to-antiparasitic activities of 107 and 39 against a chloroquine-sensitive and a chloroquine-resistant strain of P. falciparum, respectively. As the telomeres of the parasite P. falciparum are the likely target of this compound, we investigated stabilization of the Plasmodium telomeric G-quadruplexes by our phenanthroline derivatives through a FRET melting assay. The ligands 1f and 1m were noticed to be more specific for FPf8T with higher stabilization for FPf8T than for the human F21T sequence.

Synergistic cytotoxicity caused by forming a complex of copper and 2,9-dimethyl-1,10-phenanthroline in cultured vascular endothelial cells.

Organic-inorganic hybrid molecules, which are composed of organic-ligand(s) and metal(s), are indispensable as synthetic reagents in chemistry, but they have made very little in the way of contributions to biological research. Previously, we reported that the cytotoxicity of organic-inorganic hybrid molecules in vascular endothelial cells depends on interactions between the intramolecular metal and ligand, but remains independent of the hydrophobicity of the intramolecular metal(s). Herein, we show a synergistic cytotoxicity produced by forming a complex of copper and 2,9-dimethyl-1,10-phenanthroline in vascular endothelial cells that depends on the intracellular accumulation of copper.

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis.

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Šfrom the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

Phenanthroline-bis-oxazole ligands for binding and stabilization of G-quadruplexes.

BACKGROUND: G-quadruplexes (G4) are found at important genome regions such as telomere ends and oncogene promoters. One prominent strategy to explore the therapeutic potential of G4 is stabilized it with specific ligands. METHODS: We report the synthesis of new phenanthroline, phenyl and quinoline acyclic bisoxazole compounds in order to explore and evaluate the targeting to c-myc and human telomeric repeat 22AG G4 using FRET-melting, CD-melting, NMR, fluorescence titrations and FID assays. RESULTS: The design strategy has led to potent compounds (Phen-1 and Phen-2) that discriminate different G4 structures (human telomeric sequences and c-myc promoter) and selectively stabilize G4 over duplex DNA. CD studies show that Phen-2 binds and induces antiparallel topologies in 22AG quadruplex and also binds c-myc promotor, increasing their Tm in about 12°C and 30°C respectively. In contrast, Phen-1 induces parallel topologies in 22AG and c-myc, with a moderate stabilization of 4°C for both sequences. Consistent with a CD melting study, Phen-2 binds strongly (K=106 to 107M-1) to c-myc and 22AG quadruplexes. CONCLUSIONS: Phen-1 and Phen-2 discriminated among various quadruplex topologies and exhibited high selectivity for quadruplexes over duplexes. Phen-2 retains antiparallel topologies for quadruplex 22AG and does not induce conformational changes on the parallel c-myc quadruplex although Phen-1 favors the parallel topology. NMR studies also showed that the Phen-2 binds to the c-myc quadruplex via end stacking. GENERAL SIGNIFICANCE: Overall, the results suggest the importance of Phen-2 as a scaffold for the fine-tuning with substituents in order to enhance binding and stabilization to G4 structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

New alkalophilic ß-galactosidase with high activity in alkaline pH region from Teratosphaeria acidotherma AIU BGA-1.

A ß-d-galactosidase exhibiting high activity in the alkaline pH region was purified from Teratosphaeria acidotherma AIU BGA-1, which we previously isolated as a unique fungal producer of three acidophilic and one alkalophilic ß-d-galactosidases (Isobe et al., J. Biosci. Bioeng., 116, 171-174, 2013). The enzyme was stable in the pH range 7.5-10.0 and exhibited optimal activity at pH 8.0 and 60°C. The enzyme hydrolyzed 2-nitrophenyl ß-d-galactopyranoside, 4-nitrophenyl ß-d-galactopyranoside, and lactose, and the Km values were estimated to be 0.349 mM, 0.488 mM, and 701 mM, respectively. Chelating reagents (EDTA and o-phenanthroline) and metals (Cu2+and Ni2+) inhibited the enzyme activity, and Mn2+ was a good activator. The enzyme also exhibited transgalactosylation activity for lactose. The enzyme's molecular mass was estimated to be 180 kDa, and its structure was monomeric. Thus, the enzymatic and physicochemical characteristics of the alkalophilic ß-galactosidase in this study clearly differed from those of the previously known alkalophilic ß-d-galactosidases.