A Survey of Antiprion Compounds Reveals the Prevalence of Non-PrP Molecular Targets

Susan J St Martin,Sina Ghaemmaghami,Stanley B Prusiner,Olga V Bogatova,Guillaume Poncet-Montange,Brian K Shoichet

doi:10.1074/jbc.m111.234393

Susan J St Martin, Sina Ghaemmaghami + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.234393

Copy DOI

Abstract

Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrP(Sc)) of the prion protein (PrP(C)). Cell-based screens have identified several compounds that induce a reduction in PrP(Sc) levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrP(Sc) to proteolysis, or altered the cellular localization or expression level of PrP(C). None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrP(Sc) in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts.

Highlights

27718 JOURNAL OF BIOLOGICAL CHEMISTRY been identified, including pentosan polysulfate, dextran sulfate, HPA-23, Congo red, suramin, dendritic polyamines, 2-aminothiazoles, and quinacrine [9]
To investigate whether antiprion compounds identified in prion-infected neuronal cell lines have a tendency to interact with PrPC, PrPSc, or other targets, we screened a library of 2,160 known drugs and natural products and identified 206 compounds that cleared PrPSc in neuroblastoma (N2a) cell lines at a concentration of less than 1 ␮M
We screened a chemical library of 2,160 compounds containing known drugs and natural products (Microsource) for antiprion activity in ScN2a cells

Summary

EXPERIMENTAL PROCEDURES

Chemical Library—The chemical library of 2,160 compounds screened in both cell-based and direct-binding assays was obtained from the MicroSource Discovery System (MSDI, Gaylordsville, CT), and includes known drugs, bioactives, and natural products. 4 ϫ 104 ScN2a cells were treated with the compound of interest for 5 days at 1 ␮M final concentration. DMSO was added to the protein sample to reach a final concentration of 1%. After treatment and cell lysis, normalized crude extracts were analyzed by Western immunoblotting using conjugated D13-HRP antibody to detect PrP. Proteolytic digestions were stopped by the addition of PMSF (2 mM final concentration), and samples were analyzed by Western immunoblotting using conjugated D13-HRP antibody to detect PrP. Cells were washed successively 3 times with PBS for 10 min, and incubated in the dark at room temperature for 2 h with a FITC-conjugated goat anti-human IgG (H&L) polyclonal antibody (5 ␮g/ml diluted in 10% normal goat serum, Jackson ImmunoResearch). Cells were lysed and residual PrPC levels were evaluated by Western immunoblotting

Identification of Compounds with Antiprion Activity

Identification of Compounds That Interact with PrP

Identification of Compounds That Reduce PrPC Expression Levels

Identification of Compounds That Disrupt Lipid Rafts and PrPC Localization

Effect of Amcinonide on PrPC Expression and Degradation

DISCUSSION