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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

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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.

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Abbreviations

Aβ:

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)

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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.

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Authors and Affiliations

  1. Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Saragossa, Spain

    Alejandro Mahía, María Conde-Giménez, Sandra Salillas, Juan J. Galano-Frutos & Javier Sancho

  2. Biocomputation and Complex Systems Physics Institute (BIFI), Joint Unit IQFR-CSIC-BIFI, Joint Unit EEAD-CSIC-BIFI, Mariano Esquillor s/n, Edificio I+D, 50018, Saragossa, Spain

    Alejandro Mahía, María Conde-Giménez, Sandra Salillas, Juan J. Galano-Frutos & Javier Sancho

  3. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain

    Irantzu Pallarés & Salvador Ventura

  4. Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain

    Irantzu Pallarés & Salvador Ventura

  5. Navarrabiomed-CHN-UPNA, IDISNA, 31008, Pamplona, Spain

    Íñigo Lasa

  6. Instituto de Síntesis Química y Catálisis Homogénea, Departamento de Química Orgánica, CSIC–Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Saragossa, Spain

    María D. Díaz-de-Villegas & José A. Gálvez

  7. Aragon Health Research Institute (IIS Aragón), University of Zaragoza, Avda. San Juan Bosco 13, 50009, Saragossa, Spain

    Javier Sancho

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  1. Alejandro Mahía
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  2. María Conde-Giménez
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Correspondence to Javier Sancho.

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Mahía, A., Conde-Giménez, M., Salillas, S. et al. 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. Anal Bioanal Chem 411, 251–265 (2019). https://doi.org/10.1007/s00216-018-1433-8

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