Adaptation of the Protein Misfolding Cyclic Amplification (Pmca) Technique for the Screening of Anti-Prion Compounds

Authors

Katherine Do
Rebeca Benavente
Celso S G Catumbela
Uffaf Khan
Carlos Kramm
Claudio Soto
Rodrigo Morales

Publication Date

7-31-2024

Journal

The FASEB Journal

Abstract

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc 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 PrPSc 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 PrPSc 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 PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc 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

Animals, Protein Folding, PrPSc Proteins, Mice, Prion Diseases, Prions, anti-prion molecules, in vitro screening, prion strains, prions, protein misfolding cyclic amplification (PMCA)

Comments

Supplementary Materials

Data Availability Statement

PMID: 39072789