Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds

Abstract

Prion diseases result from the misfolding of the physiological prion protein (PrP^C) to a pathogenic conformation (PrP^Sc). Compelling evidence indicates that prevention and/or reduction of PrP^Sc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrP^Sc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrP^Sc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrP^Sc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrP^Sc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrP^Sc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.

Keywords: anti‐prion molecules; in vitro screening; prion strains; prions; protein misfolding cyclic amplification (PMCA).

FIGURE 1

Representative panels of optimized 96wp-PMCA assays. In vitro replication of (A) RML or (B) SSLOW prions. Left panels explain how seeded (black circles) and unseeded (white circles) reactions were positioned across the 96 well plate. Right panels demonstrate actual results. All samples shown in this experiment were PK treated before visualized in dot blots, as described in Methods.

FIGURE 2

Specific activity of small molecules of known anti-prion and anti-amyloid activities in mouse prion strains. Sixteen small molecules with proven anti-prion or anti-amyloid activities (dissolved in DMSO) were tested at four different concentrations for their anti-prion activities against the RML (A), 301C (B) and ME7 (C) prion strains using the 96wp-PMCA. Each molecule was tested in concentrations ranging from 100 to 0.1 μM. Controls for solvent representative for all strains, positive samples and negative samples without compounds were included to monitor the assay. Dot blots were modified for labeling. The panels shown in this figure are representative from three independent assays.

FIGURE 3

Lowest concentration of molecules able to halt the in vitro replication of three mouse prion strains.

FIGURE 4

Specific activity of small molecules of known anti-prion and anti-amyloid activity in Syrian hamster prion strains. Sixteen small molecules with proven anti-prion or anti-amyloid activity (dissolved in DMSO) were tested at four different concentrations for their anti-prion activities against the HY (A), SSLOW (B) and 263K (C) prion strains using the 96wp-PMCA. Each molecule was tested in concentrations ranging from 100 to 0.1 μM. Controls for solvent representative for all strains, positive samples and negative samples without compounds were included to monitor the assay. Dot blots were modified for labeling. The panels shown in this figure are representative from three independent assays.

FIGURE 5

Lowest concentration of molecules able to halt the in vitro replication of three Syrian hamster prion strains.

FIGURE 6

Screening of a small compound library to identify molecules active against CWD prions. A small library of compounds (described in Methods, n = 204 molecules) was tested against CWD prions. All molecules were tested at a concentration of 100 μM. Unseeded PMCA reactions, used as negative controls, are marked noted in the red rectangles. Blacked rectangles depict PMCA reactions were no molecules were added (positive controls). Compounds identified with anti-prion activity in this assay were circled and include clorgyline hydrochloride (blue), ZNL005 (green), zingerone (orange), (−)-securine (pink), bemestron (yellow), UM-164 (light blue) and Hoechst 34580 (terracotta).