Development and Application of a Novel Assay for Rapid Identification of Cellular Prion Protein Modulators

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders caused by the misfolding and accumulation of proteins known as prions. Despite decades of research, no effective treatment is available, and the main therapeutic strategy being pursued is to reduce cellular prion protein (PrPC) levels. Antisense oligonucleotides (ASOs) currently represent the only therapeutic strategy in clinical trials, showing promising potential, but further investigations are needed to assess their long-term efficacy. In parallel, the search for novels, effective treatment remains a priority. In this thesis, we present the development and application of an innovative BiFC-based in vitro screening assay, Light-Identification of Protein Suppressors (LIPS), designed for real-time monitoring of PrPC expression and modulation. The LIPS system is based on the Bimolecular Fluorescence Complementation (BiFC) technique, usually used for protein-protein interaction study, and here adapted for protein localization and quantification. LIPS was optimized for high-content screening (HCS) to identify small molecules, natural compounds, and genetic modulators capable of downregulating PrPC levels. A broad range of chemical libraries, including FDA/EMA-approved drugs and computationally selected candidates, were screened, leading to the identification of promising PrP suppressors. Additionally, a dedicated screening campaign of marine natural products was conducted to explore structurally diverse bioactive compounds, leading to the identification of extracts with potential PrPC-suppressing activity. Bioassay-guided fractionation and chemical profiling enabled the characterization of natural hits, further expanding the potential therapeutic candidates. Further applications of LIPS in CRISPR-based genetic screenings revealed novel molecular players involved in PrPC regulation, providing insights into potential therapeutic targets. Furthermore, the LIPS platform was successfully extended beyond prion diseases to investigate novel drug discovery targets, such as Thy-1 and AKT, demonstrating its versatility in identifying modulators of proteins. The results of this thesis establish LIPS as a robust and scalable tool for high-throughput drug discovery, offering new opportunities to identify small molecules and natural compounds with therapeutic potential in prion diseases but also giving new perspectives for the treatment of other diseases by targeting proteins.