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).