Synthesis and biological evaluation of a new series of ebselen derivatives as glutathione peroxidase (GPx) mimics and cholinesterase inhibitors against Alzheimer’s disease

A series of ebselen derivatives were designed, synthesised and evaluated as inhibitors of cholinesterases (ChEs) and glutathione peroxidase (GPx) mimics. Most of the compounds were found to be potent against AChEs and BuChE, compounds 5e and 5i, proved to be the most potent against AChE with IC₅₀ values of 0.76 and 0.46 μM, respectively. Among these hybrids, most of the compounds were found to be good GPx mimics compare with ebselen. The selected compounds 5e and 5i were also used to determine the catalytic parameters and in vitro hydrogen peroxide scavenging activity. The results indicate that compounds 5e and 5i may be excellent multifunctional agents for the treatment of AD.     

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine

Peroxidase is a commonly used catalyst in luminol–H₂O₂ chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol–H₂O₂ CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol–H₂O₂ CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of ¹O₂ on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.     

Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of foodborne pathogenic bacteria

Rapid, sensitive and point-of-care detection of foodborne pathogenic bacteria is essential for food safety. In this study, we found that hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers), as solid mimic peroxidase, could catalyze oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H₂O₂ to a green-colored product. HCH nanoflowers, integrating the essential functions of both biological recognition and signal amplification, meet the requirements of signal labels for colorimetric immunoassay of bacteria. In view of the excellent peroxidase mimetic catalytic activity of HCH nanoflowers, a colorimetric biosensing platform was newly constructed and applied for sensitive detection of foodborne Escherichia coli O157:H7 (E. coli O157:H7). The corresponding detection limits was as low as 4.1?CFU/mL with wide linear ranges (10¹–10⁶?CFU/mL).     

Double-stranded microRNA mimics can induce length- and passenger strand–dependent effects in a cell type–specific manner

MicroRNAs are short (17–26) noncoding RNAs driving or modulating physiological and pathological cellular events. Overexpression of miR-155 is pathogenic in B-cell malignancy but was also reported in a number of solid tumors—in particular, in breast cancer, where its role remains unclear and often contradictory. Using representative cell line models, we sought to determine whether the discrepant miR-155 effects in breast cancer could be explained by the heterogeneity of the disease. The growth of six breast cancer cell lines transfected with several miRNA mimics was analyzed. We found MCF-7 cell growth to be inhibited by miR-155 and miR-145 mimics, both 23-nt long, but not by a number of shorter mimics, including a universal commercial negative control. Microarray and Western blot analyses revealed induction of apoptosis, associated with interferon-β after activation of the double-stranded RNA sensor pathway. 3′ Trimming of the miRNA mimics to 21 nt substantially reduced their growth-inhibitory potency. Mutating the canonical seed of the miR-155 mimic had no effect on the induced inhibition, which was abolished by mutating the miRNA seed of the artificial passenger strand. A panel of breast cancer cell lines showed a wide range of sensitivities to 23-mer mimics, broadly consistent with the sensitivity of the cell lines to Poly (I:C). We demonstrate two sources for nonspecific in vitro effects by miRNA mimics: duplex length and the artificial passenger strand. We highlight the danger of a universal 21-mer negative control and the importance of using matched seed mutants for reliable interpretation of phenotypes.     

Interaction of ‘toxic’ and ‘immunogenic’ A‐gliadin peptides with a membrane‐mimetic environment

Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in ‘toxic’ and ‘immunogenic’ classes, which elicit an innate immune response and an HLA‐mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31–43 (P31–43) and the 56–68 (P56–68) A‐gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane‐mimetic medium. We show that P31–43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56–68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD. Copyright © 2009 John Wiley & Sons, Ltd.     

Computational design of proteins stereochemically optimized in size, stability, and folding speed

Artificial proteins potentially barrier-free in the folding kinetics are approached computationally under the guidance of protein-folding theories. The smallest and fastest folding globular protein triple-helix-bundle (THB) is so modified as to minimize or eliminate its presumed barriers in folding speed. As the barriers may reside in the ordering of either secondary or tertiary structure, the elements of both secondary and tertiary structure in the protein are targeted for prenucleation with suitable stereochemically constrained amino acid residues. The required elements of topology and sequence for the THB are optimized independently; first the topology is optimized with simulated annealing in polypeptides of highly simplified alphabet; next, the sequence in side chains is optimized using the standard inverse design methods. The resultant three best-adapted THBs, variable in topology and distinctive in sequences, are assessed by comparing them with a few benchmark proteins. The results of mainly molecular dynamics (MD) comparisons, undertaken in explicit water at different temperatures, show that the designed sequences are favorably placed against the chosen benchmarks as THB proteins potentially thermostable in the native folds. Folding simulation experiments with MD establish that the designed sequences are rapid in the folding of individual helices, but not in the evolution of tertiary structure; energetic cum topological frustrations remain but could be the artifacts of the starting conformations that were chosen in the THBs in the folding simulations. Overall, a practical high-throughput approach for de novo protein design has been developed that may have fruitful application for any type of tertiary structure.     

Improved access to 2-O-monobenzyl ethers of beta-cyclodextrin as precursors of catalysts for organophosphoryl esters hydrolysis

A comparative study of reaction conditions was performed for the synthesis of a 2-O-monobenzyl ether of cyclomaltoheptaose (beta-CD). Optimal conditions involved sodium ethoxide in Me(2)SO and benzyl bromide. The methodology was extended to the preparation of various 2(I)-O-iodobenzyl and 2(I)-O-carboxymethylbenzyl derivatives of beta-CD including a 3-carboxymethyl-4-iodobenzyl derivative of interest as precursor of an enzyme mimic to degrade the organophosphoryl ester diethyl 4-nitrophenyl phosphate (paraoxon).     

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.     

Assessing the impact of inductive electronic effects on the metrical parameters and reactivity of a series of ferrous complexes

Reported are four iron(II) complexes with N-benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^H) and three electronically modified derivatives: N-(4-methoxy)benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^OMe), N-(4-chloro)benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^Cl), and N-(4-nitro)benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^NO₂). The four ligands react with FeCl₂ to form a series of mononuclear species with the general formula [Fe(L^R)Cl₂]. The cis-α conformation of the ligand places the amine N-donors trans to the Fe-Cl bonds. The identity of the 4-benzyl substituent has profound influences on the lengths of the iron-ligand bonds, the optical spectra, and the redox activities of the [Fe(L^R)Cl₂] compounds.     

Designed molecules that fold to mimic protein secondary structures

Molecules that fold to mimic protein secondary structures have emerged as important targets of bioorganic chemistry. Recently, a variety of compounds that mimic helices, turns, and sheets have been developed, with notable advances in the design of beta-peptides that mimic each of these structures. These compounds hold promise as a step toward synthetic molecules with protein-like properties and as drugs that block protein-protein interactions.