NR ASTE
AU Boshuizen,R.S.; Langeveld,J.P.M.; Salmona,M.; Williams,A.; Meloen,R.H.; Langedijk,J.P.M.
TI An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds
QU Analytical Biochemistry 2004 Oct 15; 333(2): 372-80
PT journal article
AB 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 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. 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.
MH Amino Acid Sequence; Drug Evaluation, Preclinical/*methods; Fluorescent Dyes; Gelatin; Humans; Models, Molecular; Molecular Sequence Data; Neurodegenerative Diseases/*drug therapy/*metabolism/pathology; Neurofibrils/*drug effects/*metabolism; Peptide Fragments/antagonists & inhibitors/chemical; synthesis/chemistry/metabolism; Prions/*antagonists & inhibitors/*chemical synthesis/chemistry/metabolism; Reproducibility of Results; Research Support, Non-U.S. Gov't; Sensitivity and Specificity; Staining and Labeling; Tetracycline; Thiazoles; Time Factors
AD Ronald S. Boshuizen (r.s.boshuizen@pepscan.nl), Rob H. Meloen, Johannes P.M. Langedijk, Pepscan Systems B.V., Edelhertweg 15, 8219 PH Lelystad, The Netherlands; Jan P.M. Langeveld, Central Institute for Animal Disease Control Lelystad, Edelhertweg 15, 8219 PH Lelystad, The Netherlands; Mario Salmona, Istituto Mario Negri, Via Eritrea 62, 20157 Milan, Italy; Alun Williams, Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms, Nr Hatfield, Herts., AL9 7TA, United Kingdom; Rob H. Meloen, Academic Biomedical Centre, University of Utrecht, Yalelaan 1, Utrecht, The Netherlands
SP englisch
PO USA