|Maxson, Laura - YALE UNIVERSITY|
|Wong, Cai'ne - IDEXX LABS|
|Caughey, Byron - ROCKY MOUNTAIN LABS|
|Baron, Gerald - ROCKY MOUNTAIN LABS|
Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 31, 2003
Publication Date: September 12, 2003
Citation: Maxson, L., Wong, C., Herrmann, L.M., Caughey, B., Baron, G.S. 2003. A solid-phase assay for identification of modulators of prion protein interactions. Analytical Biochemistry. 323:54-64. Interpretive Summary: Transmissible spongiform encephalopathies (TSEs) or prion diseases include natural sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and chronic wasting disease. The central mechanism in prion diseases is the conversion of an endogenous proteinase K sensitive prion protein to a proteinase K resistant prion protein. A high-throughput, solid-phase assay has been developed based upon the in vitro cell-free conversion reaction. This solid-phase assay allows screening of various compounds for anti-conversion activity. Testing many compounds for anti-conversion activity will lead to possible prion therapeutics and greater understanding of prion disease.
Technical Abstract: The progression of the transmissible spongiform encephalopathies (TSEs) is characterized in part by accumulation of a proteinase K resistant form of the prion protein, which has been converted from the endogenous, proteinase K sensitive form. This conversion reaction provides a target for possible anti-TSE strategies. We have adapted a cell-free conversion reaction to a high throughput, solid-phase format that can be used to screen possible therapeutic compounds for inhibitory activity or to illuminate inhibition and conversion mechanisms. The solid-phase assay was compatible with reactions performed under a variety of conditions. Using this assay, we report that phthalocyanine tetrasulfonate, a known modulator of conversion, inhibited conversion by interfering with binding between the protease-sensitive and protease-resistant forms of the prion protein. A biotinylated form of the protease-sensitive prion protein was successfully converted to the protease-resistant isoform in the solid-phase assay, indicating that biotinylation provides a non-isotopic labeling strategy for large scale screens.