A Bimolecular Fluorescence Complementation Assay for the Rapid Identification of Compounds Suppressing the Cellular Prion Protein

Prion diseases are fatal neurodegenerative disorders caused by the misfolding of the cellular prion protein (PrP) into its pathogenic form (PrPSc). PrP plays a pivotal role in propagating the disease and its associated neurotoxicity. It may also act as a receptor for β-amyloid and α-synuclein oligomers, thereby potentially linking it to Alzheimer's and Parkinson's diseases. Given this central role, suppressing PrP is a promising therapeutic strategy. However, there are currently no chemical compounds ready for clinical application, highlighting the need for innovative drug discovery approaches. To address this issue, we have developed LIPS (Light Identification of Protein Suppressors), a phenotypic, high-content screening platform designed to rapidly identify PrP-suppressing compounds via imaging-based readouts. LIPS is based on a split superfolder GFP system: GFP11 is fused to the C-terminus of PrP, while GFP1–10 is targeted to specific organelles, enabling fluorescence reconstitution upon PrP biosynthesis. The initial implementation, LIPS-PrP^ER, targets the endoplasmic reticulum and provides high sensitivity, specificity, and spatial resolution. We screened approximately 2,000 compounds, as well as our own library of over 150 natural product extracts and sgRNA libraries, to identify PrP genetic modulators. Cardiac glycosides emerged as potent PrP suppressors among the compounds at sub-micromolar concentrations. Their effects were validated by Western blot, and their activity in humans is now being investigated in an ongoing observational clinical study. To assess selectivity, we developed LIPS-THY1^ER, a counter-screen targeting the GPI-anchored protein Thy-1. We are further expanding LIPS to monitor PrP across cellular compartments to create a versatile tool for therapeutic discovery.