Prion diseases are caused by the conversion of normal cellular
prion proteins (PrP) into lethal
prion aggregates. These
prion aggregates are composed of
proteinase K (PK) resistant fibrils and comparatively PK-sensitive oligomers. Currently there are no anti-
prion pharmaceuticals available to treat or prevent
prion disease. Methods of discovering anti-
prion molecules rely primarily on relatively complex cell-based, tissue slice or animal-model assays that measure the effects of small molecules on the formation of PK-resistant
prion fibrils. These assays are difficult to perform and do not detect the compounds that directly inhibit oligomer formation or alter
prion conversion kinetics. We have developed a simple cell-free method to characterize the impact of anti-
prion fibril compounds on both the oligomer and fibril formation. In particular, this assay uses shaking-induced conversion (ShIC) of recombinant PrP in a 96-well format and resolution enhanced native acidic gel electrophoresis (RENAGE) to generate, assess and detect PrP fibrils in a high throughput fashion. The end-point PrP fibrils from this assay can be further characterized by PK analysis and negative
stain transmission electron microscopy (TEM). This cell-free, gel-based assay generates metrics to assess anti-
prion fibril efficacy and kinetics. To demonstrate its utility, we characterized the action of seven well-known anti-
prion molecules:
Congo red,
curcumin, GN8,
quinacrine, chloropromazine,
tetracycline, and
TUDCA (taurourspdeoxycholic
acid), as well as four suspected anti-
prion compounds:
trans-resveratrol,
rosmarinic acid,
myricetin and
ferulic acid. These findings suggest that this in vitro assay could be useful in identifying and comprehensively assessing novel anti-
prion fibril compounds. Abbreviations: PrP,
prion protein; PK,
proteinase K; ShIC, shaking-induced conversion; RENAGE, resolution enhanced native acidic gel electrophoresis; TEM, transmission electron microscopy;
TUDCA, taurourspdeoxycholic
acid;
BSE, bovine spongiform encephalopathy; CWD,
chronic wasting disease;
CJD, Creutzfeldt Jakob disease; GSS, Gerstmann-Sträussler-Scheinker syndrome; FFI,
fatal familial insomnia; PrPc, cellular
prion protein; recPrPC, recombinant monomeric
prion protein; PrPSc, infectious particle of misfolded
prion protein; RT-QuIC, real-time quaking-induced conversion; PMCA,
Protein Misfolding Cyclic Amplification; LPS,
lipopolysaccharide; EGCG,
epigallocatechin gallate; GN8, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-
acetamide;
DMSO,
dimethyl sulfoxide; ScN2A,
scrapie infected
neuroblastoma cells; IC50, inhibitory concentration for 50% reduction; recMoPrP 23-231, recombinant full-length mouse
prion protein residues 23-231;
EDTA; PICUP, photo-induced cross-linking of unmodified
protein; BSA,
bovine serum albumin;; PMSF,
phenylmethanesulfonyl fluoride.