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A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aβ1–42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms

  • Alejandro Mahía
  • María Conde-Giménez
  • Sandra Salillas
  • Irantzu Pallarés
  • Juan J. Galano-Frutos
  • Íñigo Lasa
  • Salvador Ventura
  • María D. Díaz-de-Villegas
  • José A. Gálvez
  • Javier Sancho
Research Paper
  • 40 Downloads

Abstract

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward 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 most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (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 efficient excitation in the UV spectral region, and 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, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them.

Graphical abstract

Keywords

Amyloid fibrils Aggregation inhibitor Pyrene Stokes shift Fluorescent probes Biofilm matrix 

Abbreviations

Amyloid beta

AD

Alzheimer’s disease

BSA

Bovine serum albumin

CC50

Half-maximal cytotoxic concentration;

DAPI

4′,6-Diamidine-2′-phenylindole

DLS

Dynamic light scattering

DMSO

Dimethyl sulfoxide

PBS

Phosphate-buffered saline

PSM

Phenol-soluble modulin

PNG

Peptone, NaCl, and glucose

TEM

Transmission electron microscopy

ThT

Thioflavin T

TSBg

Tryptic soy broth (0.6 g/L) supplemented with glucose (1.5 g/L)

Notes

Acknowledgements

IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SV is 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. The authors acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, University of Zaragoza. We thank N. Cremades for her generous gift of α-synuclein and V. Fernández-Moreira and M.C. Gimeno for the measurement of lifetimes and quantum yields.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2018_1433_MOESM1_ESM.pdf (4.4 mb)
ESM 1 (PDF 4534 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Alejandro Mahía
    • 1
    • 2
  • María Conde-Giménez
    • 1
    • 2
  • Sandra Salillas
    • 1
    • 2
  • Irantzu Pallarés
    • 3
    • 4
  • Juan J. Galano-Frutos
    • 1
    • 2
  • Íñigo Lasa
    • 5
  • Salvador Ventura
    • 3
    • 4
  • María D. Díaz-de-Villegas
    • 6
  • José A. Gálvez
    • 6
  • Javier Sancho
    • 1
    • 2
    • 7
  1. 1.Departamento de Bioquímica y Biología Molecular y Celular, Facultad de CienciasUniversidad de ZaragozaSaragossaSpain
  2. 2.Biocomputation and Complex Systems Physics Institute (BIFI), Joint Unit IQFR-CSIC-BIFI, Joint Unit EEAD-CSIC-BIFISaragossaSpain
  3. 3.Institut de Biotecnologia i de BiomedicinaUniversitat Autònoma de BarcelonaBellaterraSpain
  4. 4.Departament de Bioquímica i Biologia MolecularUniversitat Autònoma de BarcelonaBellaterraSpain
  5. 5.Navarrabiomed-CHN-UPNA, IDISNAPamplonaSpain
  6. 6.Instituto de Síntesis Química y Catálisis Homogénea, Departamento de Química OrgánicaCSIC–Universidad de ZaragozaSaragossaSpain
  7. 7.Aragon Health Research Institute (IIS Aragón)University of ZaragozaSaragossaSpain

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