Shefali N. Ramteke

Ruthenium(II) polypyridyl complex as inhibitor of acetylcholinesterase and Aβ aggregation

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-β peptide (Aβ) 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 Aβ (1-40) aggregation is investigated using Thioflavin T fluorescence and Transmission Electron Microscopy. Both complexes efficiently inhibit Aβ (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 Aβ aggregation.

Mass spectral studies reveal the structure of Aβ1-16-Cu2+ complex resembling ATCUN motif.

In Alzheimers disease, copper binds to amyloid beta (Aβ) peptide and generates oxidative stress. The coordination of histidine (His) residues to Cu(2+) is still uncertain. We studied Cu(2+) binding to Aβ1-16 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 Aβ1-16 peptide resembles the ATCUN motif of human serum albumin.

Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity

Aggregation of amyloid β peptide (Aβ) is an important event in the progression of Alzheimers disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aβ aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimers disease. Thiosemicarbazone compounds are known for their variety of biological activities. However, the potential of thiosemicarbazone compounds towards inhibition of Aβ 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 Aβ(1-42) peptide aggregation. Our results indicate that 3-acetyl coumarin thiosemicarbazone inhibits Aβ(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 Aβ-induced cytotoxicity in SH-SY5Y cell line. These findings indicate that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties.

Effects of oxidation on copper-binding properties of Aβ1-16 peptide: A pulse radiolysis study

Abstract The reaction of hydroxyl radicals (•OH) with Aβ1-16 peptide was carried out using pulse radiolysis to understand the effect of oxidation of peptide on its copper-binding properties. This reaction produced oxidized, dimeric and trimeric Aβ1-16 peptide species. The formation of these products was established with the help of fluorescence spectroscopy and mass spectrometry. The mass spectral data indicate that the major site of oxidation is at His6, while the site for dimerization is at Tyr10. Diethyl pyrocarbonate-treated Aβ1-16 peptide did not produce any trimeric species upon oxidation with •OH. The quantitative chemical modification studies indicated that one of the three histidine residues is covalently modified during pulse radiolysis. The copper-binding studies of the oxidized peptide revealed that it has similar copper-binding properties as the unoxidized peptide. Further, the cytotoxicity studies point out that both oxidized and unoxidized Aβ1-16 peptide are equally efficient in producing free radicals in presence of copper and ascorbate that resulted in comparable cell death.

Fluorescent Copper Probe Inhibiting Aβ1–16-Copper(II)-Catalyzed Intracellular Reactive Oxygen Species Production

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 β (Aβ) peptide-copper complex. The data showed that OBEP-CS1 completely inhibits the copper-catalyzed oxidation as well as decarboxylation/deamination of Aβ1-16. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aβ-CuII-catalyzed reactive oxygen species production in SH-SY5Y cells. We also demonstrated that Aβ1-16 peptide can bind intracellular copper and thereby exert oxidative stress.

Effects of oxidation on redox and cytotoxic properties of copper complex of Aβ1–16 peptide

Abstract The effect of oxidation on redox and cytotoxic properties of copper complex of amyloid beta (Aβ) peptide was studied by gamma radiolysis. The oxidation of Aβ1–16 and Aβ1–16/Cu(II) complex was carried out using hydroxyl (•OH) radicals produced by gamma radiolysis and the products were analyzed using mass spectrometry. The presence of Cu(II) was found to enhance the oxidation of Aβ1–16 peptide. The oxidation of residues Asp1, His6, and His13 was enhanced due to their involvement in copper binding. The oxidation of His residues of Aβ1–16 peptide, which are chiefly responsible for copper binding, resulted in altered redox properties and subsequently in higher cytotoxicity of the Aβ1–16 peptide in SH-SY5Y cells.

One-pot synthesis and evaluation of novel 3-aryl-6-ethoxycarbonyl-4-hydroxy-2H-pyran-2-one as a potent cytotoxic agent

A series of new open chain analogs of Phelligridin J were synthesized by a clean one-pot approach. These compounds were evaluated for their in vitro cytotoxicity against normal and breast cancer cell lines. All the compounds exhibited potent cytotoxic activity in the lower micro molar range. Compound 5o exhibited the maximum cytotoxic activity with IC50 values of 12.49 and 13.76 μM, whereas compound 5a showed two-fold selectivity viz. IC50 values of 21.80 to 43.40 μM against breast cancer (MCF7) and normal fibroblast (NIH3T3) cell lines, respectively.

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1–16 peptide

Abstract The binding of metal ions to Aβ 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 Cu2+ on redox properties and cytotoxicity of Aβ peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aβ peptide has higher propensity of H2O2 generation. The oxidation of Aβ1–16 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 Aβ1–16–Cu2+ (1:2) complex.

A concave microwell array fabricated using the ommatidium of the common fruit fly for efficient cell culture

Microfluidics technology holds great promise for designing state-of-the-art culture chips due to its ability to incorporate the nanoliter liquid transport. We employed soft lithography to reproducibly replicate the structure of the ommatidium with nanoscale fidelity. MCF-7 cells cultured in these chips, occupy individual concave wells, which augment the capability of the chip for cell growth and single-cell analyses and generation of tumor-like structures.

Fluorescent zinc(II) complexes for gene delivery and simultaneous monitoring of protein expression

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.