Investigating in vitro amyloid peptide 1-42 aggregation: impact of higher molecular weight stable adducts

The self-assembly of amyloid peptides (Aβ), in particular Aβ1–42, into oligomers and fibrils is one of the main pathological events related to Alzheimer’s Disease (AD). Recent studies have demonstrated the ability of carbon monoxide releasing molecules (CORMs) to protect neurons and astrocytes from Aβ1-42 toxicity. In fact, CORMs are able to carry and release controlled levels of CO and are known to exert a wide range of anti-inflammatory and anti-apoptotic activities at physiologically relevant concentrations. In order to investigate the direct effects of CORMs on Aβ1–42, we studied the reactivity of CORM-2 and CORM-3 with Aβ1–42 in vitro and the potential inhibition of its aggregation by mass spectrometry (MS), as well as fluorescence and circular dichroism (CD) spectroscopies. The application of an electrospray ionisation-mass spectrometry (ESI-MS) method allowed the detection of stable Aβ1–42/CORMs adducts, involving the addition of the Ru(CO)2 portion of CORMs at histidine residues on the Aβ1-42 skeleton. Moreover, CORMs showed anti-aggregating properties through formation of stable adducts with Aβ1–42 as demonstrated by a thioflavin T (ThT) fluorescence assay and MS analysis. As a further proof, comparison of the CD spectra of Aβ1–42 recorded in the absence and in the presence of CORM-3 at 1:1 molar ratio showed the ability of CORM-3 to stabilize the peptide in its soluble, unordered conformation, thereby preventing its misfolding and aggregation. This multi-methodological investigation revealed novel interactions between Aβ1–42 and CORMs, contributing new insights into the proposed neuroprotective mechanisms mediated by CORMs and disclosing a new strategy to divert amyloid aggregation and toxicity

Safety issues in nutraceutical exploitation of Chlorella vulgaris, Arthrospira Platensis and Scenedesmus sp. microalgae

Microalgae contain many bioactive compounds, which may be exploited in food and nutraceutical fields. Bisphenol A (BPA) is a contaminant in microalgae that may be released from polymeric plastics. Since it is responsible for toxic effects on humans, the European legislation set the legal BPA limit within foods at 50 µg kg−1 of food weight. In this work, a fast ultrasounds solid-liquid extraction of BPA from commercial microalgal powders of Chlorella vulgaris, Arthrospira Platensis and Scenedesmus sp. was optimized. To increase selectivity, BPA was derivatized by using N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA-TMCS) and it was analysed by GC-MS in selected ion monitoring mode. A design of experiment (DOE) optimization study of the reaction conditions was performed. The analytical method was validated by determining selectivity, linearity (R2= 0.99999 ± 3.3165E-07), precision, accuracy (99.92 ± 9.83E-02 %), recovery (99.65 ± 3.61E-02 %) and sensitivity (LoD= 0.547 ± 9.94E-02 µg kg−1; LoQ= 1.823 ± 3.31E-1 µg kg−1). The overall method proved to be fast, with high recovery and suitable to selectively and sensitively determine the content of BPA, eliminating the interferences from extraction and allowing to control the safety profile of microalgae. Dried microalgae cultivated in a polycarbonate reactor, were found to contain an amount of BPA 6 times exceeding the legal limit

Development of advanced analytical methodologies for Alzheimer’s disease drug discovery

Advanced analytical methodologies were developed to characterize new potential active MTDLs on isolated targets involved in the first stages of Alzheimer’s disease (AD). In addition, the methods investigated drug-protein bindings and evaluated protein-protein interactions involved in the neurodegeneration. A high-throughput luminescent assay allowed the study of the first in class GSK-3β/ HDAC dual inhibitors towards the enzyme GSK-3β. The method was able to identify an innovative disease-modifying agent with an activity in the micromolar range both on GSK-3β, HDAC1 and HDAC6. Then, the same assay reliably and quickly selected true positive hit compounds among natural Amaryllidaceae alkaloids tested against GSK-3β. Hence, given the central role of the amyloid pathway in the multifactorial nature of AD, a multi-methodological approach based on mass spectrometry (MS), circular dichroism spectroscopy (CD) and ThT assay was applied to characterize the potential interaction of CO releasing molecules (CORMs) with Aβ1-42 peptide. The comprehensive method provided reliable information on the different steps of the fibrillation process and regarding CORMs mechanism of action. Therefore, the optimal CORM-3/Aβ1−42 ratio in terms of inhibitory effect was identified by mass spectrometry. CD analysis confirmed the stabilizing effect of CORM-3 on the Aβ1−42 peptide soluble form and the ThT Fluorescent Analysis ensured that the entire fibrillation process was delayed. Then the amyloid aggregation process was studied in view of a possible correlation with AD lipid brain alterations. Therefore, SH-SY5Y cells were treated with increasing concentration of Aß1-42 at different times and the samples were analysed by a RP-UHPLC system coupled with a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode. Each lipid class profiling in SH-SY5Y cells treated with Aß1-42 was compared to the one obtained from the untreated. The approach underlined some peculiar lipid alterations, suitable as biomarkers, that might be correlated to Aß1-42 different aggregation species

Immobilized Enzyme Reactors: an Overview of Applications in Drug Discovery from 2008 to 2018

Abstract: The necessity to develop automated methods for the fast screening of new libraries of compounds and the identification of active entities from natural mixtures has led to an increasing interest in the development of immobilized enzyme reactors (IMERs). This strategy overcomes some drawbacks of the in-solution methods and is, therefore, very attractive in the drug discovery field. This review gives an overview of IMER applications in the last decade. The reported examples concern conventional columns as well as capillary reactors integrated in liquid chromatography or capillary electrophoresis systems, coupled to spectroscopic or mass spectrometry detectors. The experimental setups and main features as well as characterization of new active entities are discussed. As a result of the growing importance of compounds from natural sources in drug discovery, particular attention is given to IMERs developed to be used for the identification of bioactive compounds. Graphical Abstract: [Figure not available: see fulltext.]

Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3β Inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine protein kinase that was originally identified as an enzyme involved in the control of glycogen metabolism. It plays a key role in diverse physiological processes including metabolism, the cell cycle, and gene expression by regulating a wide variety of well-known substances like glycogen synthase, tau-protein, and β-catenin. Recent studies have identified GSK-3β as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3β is one of the most attractive targets for medicinal chemists in the discovery, design, and synthesis of new selective potent inhibitors. In the current study, twenty-eight Amaryllidaceae alkaloids of various structural types were studied for their potency to inhibit GSK-3β. Promising results have been demonstrated by alkaloids of the homolycorine-{9-O-demethylhomolycorine (IC50 = 30.00 ± 0.71 µM), masonine (IC50 = 27.81 ± 0.01 μM)}, and lycorine-types {caranine (IC50 = 30.75 ± 0.04 μM)}