ABSTRACT
A variety of fluorescent probes are proposed to monitor the intracellular copper content. So far, none of the probes have been evaluated for their potential to inhibit copper-associated intracellular oxidative stress. Herein, we studied the ability of a fluorescent copper probe, OBEP-CS1, to inhibit intracellular oxidative stress associated with an amyloid ß (Aß) peptide-copper complex. The data showed that OBEP-CS1 completely inhibits the copper-catalyzed oxidation as well as decarboxylation/deamination of Aß1-16. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aß-CuII-catalyzed reactive oxygen species production in SH-SY5Y cells. We also demonstrated that Aß1-16 peptide can bind intracellular copper and thereby exert oxidative stress.
Subject(s)
Amyloid beta-Peptides/metabolism , Chelating Agents/pharmacology , Copper/metabolism , Fluorescent Dyes/pharmacology , Peptide Fragments/metabolism , Reactive Oxygen Species/antagonists & inhibitors , Amyloid beta-Peptides/chemistry , Catalysis , Cell Line, Tumor , Chelating Agents/chemistry , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Copper/chemistry , Fluorescent Dyes/chemistry , Humans , Oxidation-Reduction , Oxidative Stress/drug effects , Peptide Fragments/chemistry , Reactive Oxygen Species/chemistry , Reactive Oxygen Species/metabolismABSTRACT
Two new zinc(ii) complexes, [Zn(l-His)(NIP)]+(1) and [Zn(acac)2(NIP)](2) (where NIP is 2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, acac = acetyl acetone), have been synthesized and characterized by elemental analysis, UV-vis, fluorescence, IR, 1H NMR and electron spray ionization mass spectroscopies. Gel retardation assay, atomic force microscopy and dynamic light scattering studies show that 1 and 2 can induce the condensation of circular plasmid pBR322 DNA into nanometer size particles under ambient conditions. Treatment of 2 with 5 mM EDTA restored 30% of the supercoiled form of DNA, revealing partial reversibility of DNA condensation. The in vitro transfection experiment demonstrates that the complexes can be used to deliver pCMV-tdTomato-N1 plasmid which expresses red fluorescent protein. The confocal studies show that the fluorescent nature of complexes is advantageous for visualizing the intracellular delivery of metal complexes as well as transfection efficiency using two distinct emission windows.
Subject(s)
Coordination Complexes/chemistry , DNA, Circular/administration & dosage , Fluorescent Dyes/chemistry , Plasmids/administration & dosage , Transfection/methods , Zinc/chemistry , DNA, Circular/genetics , Gene Expression , Gene Transfer Techniques , Humans , Imidazoles/chemistry , Luminescent Proteins/genetics , MCF-7 Cells , Microscopy, Confocal , Naphthalenes/chemistry , Optical Imaging , Phenanthrolines/chemistry , Plasmids/genetics , Red Fluorescent ProteinABSTRACT
The synthesis, spectral and electrochemical characterization of the complexes of the type [Ru(NN)2(txbg)](2+) where NN is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), dipyrido [3,2-d:2',3f] quinoxaline (dpq) (3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz) (4) which incorporate the tetra-xylene bipyridine glycoluril (txbg) as the ancillary ligand are described in detail. Crystal structures of ligand txbg and complex 2 were solved by single crystal X-ray diffraction. Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM) results indicated that at micromolar concentration all complexes exhibit significant potential of Aß aggregation inhibition, while the ligand txbg displayed weak activity towards Aß aggregation. Complex 1 showed relatively low inhibition (70%) while complexes 2-4 inhibited nearly 100% Aß aggregation after 240 h of incubation. The similar potential of complexes 2-4 and absence of any trend in their activity with the planarity of polypyridyl ligands suggests there is no marked effect of planarity of coligands on their inhibitory potential. Further studies on acetylcholinesterase (AChE) inhibition indicated very weak activity of these complexes against AChE. Detailed interactions of Aß with both ligand and complex 2 have been studied by molecular modeling. Complex 2 showed interactions involving all three polypyridyl ligands with hydrophobic region of Aß. Furthermore, the toxicity of these complexes towards human neuroblastoma cells was evaluated by MTT assay and except complex 4, the complexes displayed very low toxicity.
Subject(s)
Amyloid beta-Peptides/chemistry , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Peptide Fragments/chemistry , Protein Aggregates/drug effects , Ruthenium/chemistry , Acetylcholinesterase/metabolism , Alkynes/chemistry , Cell Line, Tumor , Humans , Hydrophobic and Hydrophilic Interactions , Imidazoles/chemistry , Ligands , Models, Molecular , Protein ConformationABSTRACT
The binding of metal ions to Aß peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu(2+) on redox properties and cytotoxicity of Aß peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aß peptide has higher propensity of H2O2 generation. The oxidation of Aß1-16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aß1-16-Cu(2+) (1:2) complex.
Subject(s)
Amyloid beta-Peptides/chemistry , Coordination Complexes/pharmacology , Copper/chemistry , Cytotoxins/pharmacology , Histidine/chemistry , Hydrogen Peroxide/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Ascorbic Acid/pharmacology , Cell Line, Tumor , Coordination Complexes/chemistry , Cytotoxins/chemistry , Electrochemical Techniques , Gamma Rays , Humans , Hydrolysis , Neurons/chemistry , Neurons/cytology , Neurons/drug effects , Oxidation-Reduction , Protein BindingABSTRACT
Aggregation of amyloid ß peptide (Aß) is an important event in the progression of Alzheimer's disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aß aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimer's disease. Thiosemicarbazone compounds are known for their variety of biological activities. However, the potential of thiosemicarbazone compounds towards inhibition of Aß peptide aggregation and the subsequent toxicity is little explored. Herein, we report synthesis and x-ray crystal structure of novel compound 3-acetyl coumarin thiosemicarbazone and its efficacy toward inhibition of Aß(1-42) peptide aggregation. Our results indicate that 3-acetyl coumarin thiosemicarbazone inhibits Aß(1-42) peptide aggregation up to 80% compared to the parent 3-acetyl coumarin which inhibits 52%. Further, 3-acetyl coumarin thiosemicarbazone provides neuroprotection against Aß-induced cytotoxicity in SH-SY5Y cell line. These findings indicate that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties.
Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/toxicity , Coumarins/chemistry , Peptide Fragments/chemistry , Peptide Fragments/toxicity , Protein Aggregates/drug effects , Thiosemicarbazones/chemistry , Thiosemicarbazones/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Conformation , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacologyABSTRACT
Two ruthenium(II) polypyridyl complexes [Ru(phen)3](2+) (1) and [Ru(phen)2(bxbg)](2+) (2) (where phen = 1,10 phenanthroline, bxbg = bis(o-xylene)bipyridine glycoluril) have been evaluated for acetylcholinesterase (AChE) and Amyloid-ß peptide (Aß) aggregation inhibition. Complex 2 exhibits higher potency of AChE inhibition and kinetics and molecular modeling studies indicate that ancillary ligand plays significant role in inhibitory potency exhibited by complex 2. The inhibitory effect of these complexes on Aß (1-40) aggregation is investigated using Thioflavin T fluorescence and Transmission Electron Microscopy. Both complexes efficiently inhibit Aß (1-40) aggregation and are negligibly toxic to human neuroblastoma cells. This is the first demonstration that ruthenium(II) polypyridyl complexes simultaneously inhibit AChE and Aß aggregation.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Ruthenium/chemistry , Ruthenium/pharmacology , Acetylcholinesterase/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/ultrastructure , Cell Line, Tumor , Humans , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Phenanthrolines/chemistry , Phenanthrolines/pharmacology , Pyridines/chemistry , Pyridines/pharmacologyABSTRACT
In Alzheimer's disease, copper binds to amyloid beta (Aß) peptide and generates oxidative stress. The coordination of histidine (His) residues to Cu(2+) is still uncertain. We studied Cu(2+) binding to Aß1-16 peptide using the diethyl pyrocarbonate (DEPC) assay and mass spectrometry. Our results show that only one His is involved in Cu(2+) coordination, which is identified as His6 using mass spectral studies. Novel nickel displacement studies have further supported the proposal that the Cu(2+) binding site of Aß1-16 peptide resembles the ATCUN motif of human serum albumin.
