A solid-phase assay for identification of modulators of prion protein interactions.
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 the 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 nonisotopic labeling strategy for large-scale screens.
Maxson
Laura
L
Rocky Mountain Laboratories, NIAID, NIH, 903 S. 4th St., Hamilton, MT 59840, USA.
Wong
Caíne
C
Herrmann
Lynn M
LM
Caughey
Byron
B
Baron
Gerald S
GS
D000818:Animals; D019921:Biotinylation; D001921:Brain; D006224:Cricetinae; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019286:Endopeptidase K / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D017096:Prion Diseases / Q000188:drug therapy; D011328:Prions / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000032:analysis
D007211:Indoles; D011328:Prions; D020033:Protein Isoforms; C028418:tetrasulfophthalocyanine; D019286:Endopeptidase K