An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds

Ronald S. Boshuizen, Jan P M Langeveld, Mario Salmona, Alun Williams, Rob H. Meloen, Johannes P M Langedijk

Research output: Contribution to journalArticle

Abstract

Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2 mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Aβ (1-42), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 μM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.

Original languageEnglish
Pages (from-to)372-380
Number of pages9
JournalAnalytical Biochemistry
Volume333
Issue number2
DOIs
Publication statusPublished - Oct 15 2004

Keywords

  • Alzheimer's disease
  • Amyloid
  • BSE
  • High-throughput assay
  • Prion
  • PrP106-126
  • Tetracycline
  • Thioflavin S

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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