Copper, dityrosine cross-links and amyloid-β aggregation

Guillem Vázquez, Ana B. Caballero, Jakub Kokinda, Ana Hijano, Raimon Sabaté, Patrick Gamez

J. Biol. Inorg. Chem. 2019, 24, 1217-1229

Copper is involved in Alzheimer’s disease (AD) where it appears to affect the aggregation of amyloid-β (Aβ) and to catalyze the production of reactive oxygen species (ROS). Oxidative stress apparently produces Aβ dimers that are covalently linked through two tyrosine residues. Such dityrosine cross-links are considered as potential markers of the disease and seem to be implicated in the pathological disorder. In the present study, pure o,o′-dityrosine (diY) was prepared enzymatically (with horseradish peroxidase; HRP), which was subsequently used to construct calibration lines aimed at quantifying nanomolar amounts of diY in reaction mixtures by fluorescence spectroscopy. Hence, diY concentrations down to 67 nM could be determined, which allowed to find that ca. 3% of dityrosine-bridged dimers of Aβ(1–40) were produced after 3 days at 37 °C in the presence of copper and dihydrogen peroxide. These cross-linked dimers in the presence of copper(II) ions completely inhibit the typical aggregation of Aβ, since β sheets could not be detected applying the usual Thioflavin T (ThT) method. Furthermore, the use of a potent Cu(II) chelator, such as the ATCUN tripeptide, l-histidyl-l-alanyl-l-histidine (HAH), efficiently prevented the copper-mediated generation of ROS and the associated dityrosine-bridged Aβ dimers, suggesting that such metal chelators may find future applications in the field of anti-AD drug design.

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Assisted delivery of anti-tumour platinum drugs using DNA-coiling gold nanoparticles bearing lumophores and intercalators: towards a new generation of multimodal nanocarriers with enhanced action

Ana B. Caballero, Lucia Cardo, Sunil Claire, James S. Craig , Nikolas J. Hodges, Anton Vladyka, Tim Albrecht, Luke A. Rochford, Zoe Pikramenou and Michael J. Hannon

Chem. Sci. 2019, 10, 9244-9256

Nanocarriers with unusual DNA binding properties provide enhanced cytotoxic activity beyond that conferred by the platinum agents they release.

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DNA-binding and in vitro cytotoxic activity of platinum(II) complexes of curcumin and caffeine

Valentina Censi, Ana B. Caballero, Marta Pérez-Hernández, Vanessa Soto-Cerrato, Luís Korrodi-Gregório, Ricardo Pérez-Tomás, Maria Michela Dell'Anna, Piero Mastrorilli, Patrick Gamez

J. Inorg. Biochem. 2019, 198, 110749

Three Pt(II) compounds have been prepared that contain the natural ligands curcumin (complexes 1 and 2) or caffeine (complex 3). DNA-intercalating 1 and groove-binding 2 exhibit cytotoxic properties, which can be enhanced through visible-light irradiation.

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Bacterial Inclusion Bodies for Anti-Amyloid Drug Discovery: Current and Future Screening Methods

Ana B. Caballero, Alba Espargaró, Caterina Pont, Maria Antònia Busquets, Joan Estelrich, Diego Muñoz-Torrero, Patrick Gamez, Raimon Sabaté

Curr. Protein Pept. Sc. 2019, 20, 1

Amyloid aggregation is linked to an increasing number of human disorders from nonneurological pathologies such as type-2 diabetes to neurodegenerative ones such as Alzheimer or Parkinson’s diseases. Thirty-six human proteins have shown the capacity to aggregate into pathological amyloid structures. To date, it is widely accepted that amyloid folding/aggregation is a universal process present in eukaryotic and prokaryotic cells. In the last decade, several studies have unequivocally demonstrated that bacterial inclusion bodies – insoluble protein aggregates usually formed during heterologous protein overexpression in bacteria – are mainly composed of overexpressed proteins in amyloid conformation. This fact shows that amyloid-prone proteins display a similar aggregation propensity in humans and bacteria, opening the possibility to use bacteria as simple models to study amyloid aggregation process and the potential effect of both anti-amyloid drugs and pro-aggregative compounds. Under these considerations, several in vitro and in cellulo methods, which exploit the amyloid properties of bacterial inclusion bodies, have been proposed in the last few years. Since these new methods are fast, simple, inexpensive, highly reproducible, and tunable, they have aroused great interest as preliminary screening tools in the search for anti-amyloid (beta-blocker) drugs for conformational diseases. The aim of this mini-review is to compile recently developed methods aimed at tracking amyloid aggregation in bacteria, discussing their advantages and limitations, and the future potential applications of inclusion bodies in anti-amyloid drug discovery.

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Amyloid Pan-inhibitors: One Sole Family of Compounds to Cope with All Conformational Diseases

Alba Espargaró, Caterina Pont, Patrick Gamez, Diego Muñoz-Torrero, Raimon Sabaté

ACS Chem. Neurosci. 2019, 10, 1311-1317.

Amyloids are ubiquitous protein aggregates sharing common internal structural features; they are present in all organisms, from prokaryotes to eukaryotes, where they play physiological or pathological roles. Importantly, amyloids, which are generated by aggregation of a range of distinct proteins, could be a key factor in a number of major human disorders, the so-called conformational diseases. Because all amyloids exhibit similar cross-β motifs, one may envisage that molecules capable of blocking the formation of β-sheet structures could abolish aggregation of all amyloid proteins, albeit with different efficacies. Herein, two different β-sheet blockers were tested against a selection of amyloidogenic proteins, encompassing all the major types of amyloid-based disorders. Analysis of their blocking efficiency, using a simple but contrasted cell-based screening procedure, unequivocally confirms that they indeed behave as aggregation pan-inhibitors. The significant inhibitory effects observed for these compounds against all tested amyloidogenic proteins could spur a broader biological evaluation of other known and new amyloid aggregation inhibitors to further determine the potential use of this class of compounds for the universal treatment of conformational diseases.

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A novel class of multitarget anti-Alzheimer benzohomoadamantane- chlorotacrine hybrids modulating cholinesterases and glutamate NMDA receptors

F. Javier Pérez-Areales, Andreea L. Turcua, Marta Barniol-Xicota, Caterina Pont, Deborah Pivetta, Alba Espargaró, Manuela Bartolini, Angela De Simone, Vincenza Andrisano, Belén Pérez, Raimon Sabaté, Francesc X. Sureda, Santiago Vázquez, Diego Muñoz-Torrero

Eur. J. Med. Chem. 2019, 180, 613-626

The development of multitarget compounds against multifactorial diseases, such as Alzheimer's disease, is an area of very intensive research, due to the expected superior therapeutic efficacy that should arise from the simultaneous modulation of several key targets of the complex pathological network. Here we describe the synthesis and multitarget biological profiling of a new class of compounds designed by molecular hybridization of an NMDA receptor antagonist fluorobenzohomoadamantanamine with the potent acetylcholinesterase (AChE) inhibitor 6-chlorotacrine, using two different linker lengths and linkage positions, to preserve or not the memantine-like polycyclic unsubstituted primary amine. The best hybrids exhibit greater potencies than parent compounds against AChE (IC50 0.33 nM in the best case, 44-fold increased potency over 6-chlorotacrine), butyrylcholinesterase (IC50 21 nM in the best case, 24-fold increased potency over 6-chlorotacrine), and NMDA receptors (IC50 0.89 μM in the best case, 2-fold increased potency over the parent benzohomoadamantanamine and memantine), which suggests an additive effect of both pharmacophoric moieties in the interaction with the primary targets. Moreover, most of these compounds have been predicted to be brain permeable. This set of biological properties makes them promising leads for further anti-Alzheimer drug development.

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Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

Hasier Eraña, Jorge M. Charco, Michele A. Di Bari, Carlos M. Díaz-Domínguez, Rafael López-Moreno, Enric Vidal, Ezequiel González-Miranda, Miguel A. Pérez-Castro, Sandra García-Martínez, Susana Bravo, Natalia Fernández-Borges, Mariví Geijo, Claudia D’Agostino, Joseba Garrido, Jifeng Bian, Anna König, Boran Uluca-Yazgi, Raimon Sabaté, Vadim Khaychuk, Ilaria Vanni, Glenn C. Telling, Henrike Heise, Romolo Nonno, Jesús R. Requena, Joaquín Castilla

Plos Phatogens 2019, 15, Article Number: e1008117

Prion disorders are a group of devastating neurodegenerative diseases caused by an aberrantly folded isoform of the endogenous prion protein. The molecular mechanisms by which this proteinaceous pathogen is able to propagate in the central nervous system and cause neuronal death are poorly understood, partially due to the difficulties elucidating the three-dimensional structure of the aggregation-prone aberrant isoform or prion. Obtaining sufficient amounts of highly pure and homogeneous prions for high-resolution structural studies has been limited until now due to technical reasons. Here, we present a novel method for the production of large amounts of highly infectious recombinant prions suitable for solid state Nuclear Magnetic Resonance imaging, which could help to unveil the molecular pathogenesis of these particular pathogens.

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