NR AOQI
AU Bertsch,U.; Giese,A.; Bieschke,J.G.; Weber,P.; Levin,J.; Kretzschmar,H.A.
TI Screening for potential prion disease therapeutics using a single-molecule fluorescence assay of PrPc association to prion rods in vitro
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - IV-16
PT Konferenz-Poster
AB
Potential chemical therapeutics for prion disease to date have been identified by serendipitous discovery. An assay suitable for systematic high-throughput screening of substance libraries for such therapeutics has yet been missing.
Based on a fluorescence correlation spectroscopy set-up in combination with the SIFT-technique (Scanning for Intensely Fluorescent Targets) we were able to develop an assay that allows to determine the ability of a substance to interfere with the association of monomeric recombinant PrPc to preformed prion aggregates isolated from natural sources. This assay can be fully automated, performed in a total sample volume of only 20 µl, and requires a measurement time of 75 seconds per substance.
The assay system has been validated using model substances that had previously been known to interfere with the generation of PrPsc and the infectious process in vivo. One potential prion therapeutic, the polycationic lipid 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), was found to inhibit the association of fluorescently labelled recombinant PrP with prion aggregates dramatically at low micromolar concentrations. In controls other lipids without effect on prion propagation and infectivity in vivo, like dioleoyl-L-phosphatidylethanolamine (DOPE), did not influence the aggregation, as was expected.
This test system therefore represents an assay for prion therapeutics that has the potential to perform systematic high-throughput and high-content screenings for potential drugs against prion diseases in vitro.
AD Uwe Bertsch, Armin Giese, Jan Bieschke, Petra Weber, Johannes Levin, Hans A. Kretzschmar, Ludwig-Maximilians-Universität München, Germany
SP englisch
PO Deutschland