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Identification of Anti-prion Compounds using a Novel Cellular Assay.

ABSTRACT: Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (?105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.

SUBMITTER: Imberdis T

PROVIDER: S-EPMC5207084 | biostudies-other | 2016 Dec

REPOSITORIES: biostudies-other

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Identification of Anti-prion Compounds using a Novel Cellular Assay.

Imberdis Thibaut T Heeres James T JT Yueh Han H Fang Cheng C Zhen Jessie J Rich Celeste B CB Glicksman Marcie M Beeler Aaron B AB Harris David A DA

The Journal of biological chemistry 20161101 50

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrP<sup>C</sup>) into the infectious isoform (PrP<sup>Sc</sup>). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected wi ...[more]

PMID: 27803163

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Project description: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.

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Identification of Anti-prion Compounds using a Novel Cellular Assay.

Publications

Identification of Anti-prion Compounds using a Novel Cellular Assay.

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