Silybins inhibit human IAPP amyloid growth and toxicity through stereospecific interactions

Type 2 Diabetes is a major public health threat, and its prevalence is increasing worldwide. The abnormal accumulation of islet amyloid polypeptide (IAPP) in pancreatic β-cells is associated with the onset of the disease. Therefore, the design of small molecules able to inhibit IAPP aggregation represents a promising strategy in the development of new therapies. Here we employ in vitro, biophysical, and computational methods to inspect the ability of Silybin A and Silybin B, two natural diastereoisomers extracted from milk thistle, to interfere with the toxic self-assembly of human IAPP (hIAPP). We show that Silybin B inhibits amyloid aggregation and protects INS-1 cells from hIAPP toxicity more than Silybin A. Molecular dynamics simulations revealed that the higher efficiency of Silybin B is ascribable to its interactions with precise hIAPP regions that are notoriously involved in hIAPP self-assembly i.e., the S20-S29 amyloidogenic core, H18, the N-terminal domain, and N35. These results highlight the importance of stereospecific ligand-peptide interactions in regulating amyloid aggregation and provide a blueprint for future studies aimed at designing Silybin derivatives with enhanced drug-like properties. Keywords: Aggregation; Diabetes; Inhibitors; Molecular dynamics; Peptid

Etinilación y propargilación de nitronas cíclicas y su aplicación en la síntesis de glicomiméticos

Máster Universitario en Química molecular y Catálisis Homogénea.Las transglicosilasas son enzimas de membrana cruciales para la remodelación de la pared celular fúngica y bacteriana, imprescindibles para mantener la integridad estructural de estos organismos. En el caso de los hongos, el componente principal de la pared celular, el β-(1,3)-D-glucano, no está presente en las células humanas y, por lo tanto, inhibir selectivamente los enzimas responsables de la síntesis y la remodelación de la pared celular de los hongos combatiría las infecciones fúngicas sin interaccionar con las células humanas. Estudios recientes del grupo de investigación con el que se colabora en este proyecto muestran la primera estructura cristalina de una transglicosilasa, Gas2 de Saccharomyces cerevisiae (ScGas2). El sitio activo de Gas2 se conserva totalmente con la familia Gel de Aspergillosis fumigatus y con las familias Ph y Pga de Candida Albicans; al no existir una estructura cristalográfica de estos enzimas, Gas2 nos proporciona un modelo adecuado para desarrollar inhibidores de especies patógenas causantes de aspergilosis o candidiasis. En este trabajo se han sintetizo potenciales inhibidores de ScGas2 que contienen una parte glicosídica, esencial para el reconocimiento por parte del enzima, y que incorporan pirrolidinas polihidroxiladas capaces de mimetizar azúcares, proporcionando además la capacidad de interaccionar con el entorno aniónico de la proteína cercano al sitio activo. La interacción de los glicomiméticos con el enzima se ha evaluado mediante experimentos STD-RMN (Saturation Transfer Difference- NMR) mostrando resultados positivos para algunos de los ligandos sintetizados.Peer Reviewe

Stereoselective ethynylation and propargylation of chiral cyclic nitrones: application to the synthesis of glycomimetics

Ethynylation and propargylation of chiral nonracemic polyhydroxylated cyclic nitrones with Grignard reagents are efficient methods for preparing building blocks containing an alkyne moiety to be used in copper-catalyzed azide alkyne cycloaddition click chemistry. Whereas ethynylation takes place with excellent diastereoselectivity, propargylation afforded mixtures of diastereomers in some cases. The use of (trimethylsilyl)propargyl bromide as precursor of the Grignard reagent is necessary to avoid the formation of undesired allene derivatives. DFT calculations explain, within the experimental error, the observed behavior. Cycloaddition of the obtained pyrrolidinyl alkynes with sugar azides derived from β-(1,3)-glucans provides glycomimetics suitable to be used against fungal transglycosylases.This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) (project number CTQ2013-44367-C2-1-P), by the Fondos Europeos para el Desarrollo Regional (FEDER), and the Gobierno de Aragón (Zaragoza, Spain, Bioorganic Chemistry Group, E-10).Peer Reviewe

Synthesis of new transglycosylases ligands and study of the glycomimetics-ScGas2 interactions

Resumen del póster presentado a la VII International Conference on Molecular Recognition, celebrada en Zaragoza (España) del 1 al 3 de febrero de 2016.Transglycosylases are crucial membrane-bound enzymes for the remodeling of the bacterial and fungal cell wall, which are indispensable to maintain the structural integrity of those microorganisms. Among all fungal transglycosylases, Gas2 of Saccharomyces cerevisiae (ScGas2) is the only one whose structure has been described so far, and is an optimum model for a new class of antimicotic drugs due to its active site is totally conserved with other enzymes in pathogen fungi, such as Aspergillus fumigatus and Candida albicans. In this communication, we will report the design and synthesis of pyrrolidine-containing ligands for ScGas2; which are formed by a glycosidic part that is essential to the enzyme recognition, and a polihydroxylated pyrrolidine part, which is able to mimic sugars and could also provide the possibility of interacting with the anionic residues in the active site of the enzyme. The interaction of the glycomimetics with the protein has been evaluated by Saturation Transfer Difference NMR experiments (STD-NMR) and molecular docking calculations. Recognition effects of modifications in the ligands structure (length of carbohydrate moiety, flexibility of pyrrolidine ring) will be discussed.Peer Reviewe

Recent advances in the preparation of enantiomerically pure hydroxylamines from nitrones

This review covers the recent advances in the synthesis of enantiomerically pure hydroxylamine’s employing nitrones as starting materials. Nucleophilic additions of organometallic reagents to nitrones are the most common way for introducing a hydroxyamino group into carbon skeletons with the concomitant formation of a new carbon-carbon bond. Addition of nucleophiles derived from enolates, cyanide or fluorinated derivatives allows the preparation of complex structures. Radical additions and, in particular samarium diiodidemediated reductive coupling of nitrones with carbonyl compounds and α,β-unsaturated esters have also been considered. All these approaches provide efficient methods of preparation of enantiomerically pure hydroxylamine’s that are valuable synthetic intermediates.We thank for their support of our programs: MINECO (Madrid, Spain) and FEDER Program (Project CTQ2013-44367-C2-1-P) and the Government of Aragon (Group E-10). H.H. thanks UE (Erasmus program) for a mobility grant. M.G. thanks MECD for a FPU pre-doctoral grant.Peer Reviewe

Study of melamine-formaldehyde/phase change material microcapsules for the preparation of polymer films by extrusion

This article belongs to the Special Issue Innovative and Functional Polymer Membranes: Development and Applications.n-Eicosane-melamine formaldehyde microcapsules of an average size of 1.1 μm and latent heat of fusion of 146.2 ± 5.3 J/g have been prepared. They have been characterized by scanning electron microscopy, FTIR spectroscopy, calorimetric techniques, and thermogravimetric analyses. Under processing conditions, the microcapsules apparently preserved their properties, also maintaining their n-eicosane loading and heat storage capacity under washing conditions (water with detergent at 60 °C). The microcapsules synthesis has been scaled up for the fabrication of functional films by extrusion. For that, polymer films containing 10 wt.% of microcapsules were prepared at a pilot plant level. In those films, even though a fraction of the n-eicosane loading was lost during the extrusion process, the microcapsules showed good compatibility within the polyamide. The percentage of PCM in the polyamide 6 films was estimated by TGA, verifying also the heat storage capacity predicted by DSC (2.6 ± 0.7 J/g).This research was funded by Ministry of Economy and Competitiveness of Spain (MCIN/AEI/10.13039/501100011033), grant number RTC-2015-3503-5 and by European Regional Development Fund (ERDF, “A way of making Europe”).Peer reviewe

Multi-target strategy in Alzheimer’s disease and type II diabetes mellitus. The role of silybins A and B.

In the last decade, a large number of evidences points to the misfolding, aggregation and accumulation of structurally abnormal proteins, termed amyloid, as a common pathogenic mechanism of a range of increasingly common human disorders, including Alzheimer`s Disease (AD) and type II diabetes mellitus (T2DM). In particular, AD and T2DM are characterized by the accumulation of the amyloid-β (Aβ) peptide in neuronal tissues and islet amyloid polypeptide (IAPP) in pancreatic cells, respectively (1), implicitly suggesting that targeting protein misfolding and self-assembly would cure the diseases. However, the failure of all clinical trials focusing on anti-aggregating drugs has clearly demonstrated that a deeper understanding of the phenomena involved in proteome maintenance is needed. It is widely known that cells express an integrated array of proteolytic machineries that control protein homeostasis (proteostasis). As a matter of fact, the latest studies suggest that pathological conditions occur when the equilibrium between the production and the clearance of the involved protein results unbalanced. The ubiquitin–proteasome system (UPS) is the major quality control pathway responsible for cellular homeostasis; it is the primary proteolytic route for misfolded proteins and ubiquitination is utilized as a degradation signal (2). Many researchers have screened a number of molecules using the whole UPS as target (proteasome activators, inhibitors of deubiquitinating enzymes DUBs or ubistatins); however, very few molecules, if any, selected by using this strategy have shown to have the potential to be pipelined to clinical trials. But, due to the multifactorial nature of protein diseases, it may be difficult to find an effective drug by screening molecules on a single target. This study evaluates the ability of silybins A and B, components of the natural product silymarin, to rescue the proteostatic balance of the amyloidogenic peptides Aβ and IAPP against several targets: proteasome, polyubiquitination, membrane protection, amyloid aggregation and oxidative stress. The obtained results evidence the important role of the stereochemistry, demonstrating the high potential of these natural compounds in anti-AD therapeutics. Our multi-target strategy to rescue proteostasis opens the door to a new approach to face these important pathologies

Substitution of the Native Zn(II) with Cd(II), Co(II) and Ni(II) Changes the Downhill Unfolding Mechanism of Ros87 to a Completely Different Scenario

The structural effects of zinc replacement by xenobiotic metal ions have been widely studied in several eukaryotic and prokaryotic zinc-finger-containing proteins. The prokaryotic zinc finger, that presents a bigger beta beta beta alpha alpha domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interaction with metallo-proteins. Several studies have been conducted on Ros87, the DNA binding domain of the prokaryotic zinc finger Ros, and have demonstrated that the domain appears to structurally tolerate Ni(II), albeit with important structural perturbations, but not Pb(II) and Hg(II), and it is in vitro functional when the zinc ion is replaced by Cd(II). We have previously shown that Ros87 unfolding is a two-step process in which a zinc binding intermediate converts to the native structure thorough a delicate downhill folding transition. Here, we explore the folding/unfolding behaviour of Ros87 coordinated to Co(II), Ni(II) or Cd(II), by UV-Vis, CD, DSC and NMR techniques. Interestingly, we show how the substitution of the native metal ion results in complete different folding scenarios. We found a two-state unfolding mechanism for Cd-Ros87 whose metal affinity K-d is comparable to the one obtained for the native Zn-Ros87, and a more complex mechanism for Co-Ros87 and Ni-Ros87, that show higher K-d values. Our data outline the complex cross-correlation between the protein-metal ion equilibrium and the folding mechanism proposing such an interplay as a key factor in the proper metal ion selection by a specific metallo-protein

New Phosphate-Linked Tyrosol Dimers: Synthesis, antioxidant activity, metal chelating capacity and effect on Aβ aggregation

Phenylpropanoids are secondary metabolites widely distributed in plants. They are believed to protect human cells against oxidative stress and in turn, to prevent various diseases associated with it, such as cancer, cardiovascular, and neurodegenerative disorders as well [2,3]. Recently, many natural phenylpropanoids have emerged as a particularly promising class of small molecules able to inhibit β-amyloid aggregation and disrupt preformed amyloid fibrils that represent important targets in the development of pharmacological treatments of Alzheimer’s disease (AD) [4]. Unfortunately, natural phenylpropanoids often have poor pharmacokinetic properties and low bioavailability. In this frame an interesting approach in the design of new bioactive compounds could be the combination of two or more polyphenolic "fragments". This "natural-fragment-based drug-discovery" approach would allow the assembly, also in a combinatorial manner, of libraries based on complex polyphenols in a few steps. This strategy provides us with the possibility of easily modifying both scaffold and decorations and modulating pharmacodynamic and pharmacokinetic properties [5]. Therefore, we planned to join two "tyrosol-fragments" by a phosphodiester bridge to obtain a new class of phosphate-linked compounds [6], which are potentially more bioactive than the corresponding precursors. We report here the general synthetic strategy to obtain new phosphate-linked tyrosol dimers in good yield and their full characterization. Since oxidative stress, as well as metal ion dyshomeostasis, are known to play important roles in AD pathogenesis, preliminary studies are focused on the evaluation of their antioxidant activity, metal chelating ability and their ability to inhibit β-amyloid aggregation