title: A fast and specific method to screen for intracellular amyloid inhibitors using bacterial model systems.
abstract: The aggregation of a large variety of amyloidogenic proteins is linked to the onset of devastating human disorders. Therefore, there is an urgent need for effective molecules able to modulate the aggregative properties of these polypeptides in their natural environment, in order to prevent, delay or halt the progression of such diseases. On the one hand, the complexity and cost of animal models make them inefficient at early stages of drug discovery, where large chemical libraries are usually screened. On the other hand, in vitro aggregation assays in aqueous solutions hardly reproduce (patho)physiological conditions. In this context, because the formation of insoluble aggregates in bacteria shares mechanistic and functional properties with amyloid self-assembly in higher organisms, they have emerged as a promising system to model aggregation in the cell. Here we show that bacteria provide a powerful and cost-effective system to screen for amyloid inhibitors using fluorescence spectroscopy and flow cytometry, thanks to the ability of the novel red fluorescent ProteoStat dye to detect specifically intracellular amyloid-like aggregates. We validated the approach using the Alzheimer's linked Aβ40 and Aβ42 peptides and tacrine- and huprine-based aggregation inhibitors. Overall, the present method bears the potential to replace classical in vitro anti-aggregation assays.
authors.affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain. Electronic address: Susanna.Navarro.Cantero@uab.cat.
authors.forename: Susanna
authors.initials: S
authors.lastname: Navarro
authors.affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.
authors.forename: Anita
authors.initials: A
authors.lastname: Carija
authors.affiliation: Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia, and Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Joan XXIII, 27-31, E-08028, Barcelona, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: Muñoz-Torrero
authors.affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain. Electronic address: salvador.ventura@uab.es.
authors.forename: Salvador
authors.initials: S
authors.lastname: Ventura
mesh_terms: D000682:Amyloid; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D003362:Cost-Benefit Analysis; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects; D002479:Inclusion Bodies / Q000187:drug effects / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry*; D066329:Protein Aggregates / Q000187:drug effects*; D013619:Tacrine / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D066329:Protein Aggregates; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D013619:Tacrine
keywords: Amyloid; Inclusion bodies; Protein aggregation inhibitors; ProteoStat; Screening