Suchergebnisse

Salvador Ventura and Javier Sancho are supported by SOE4/P1/E831 grant from SUDOE. INTERREG IV B. EUROPEAN UNION. ; An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson's disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer, Parkinson or Type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity towards that diversity of amyloid fibrils, or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here, we show the feasibility of the approach by designing, synthesizing and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The more soluble derivative, compound 1D, stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein or amylin, provides a sensitivity only slightly lower than that of Thioflavin T, displays a large Stokes shift, allows an efficient excitation in the UV spectral region,and it is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by Staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining,S. aureus biofilms containing amyloid forming phenol soluble modulins from those lacking them. ; IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SVis supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government.