Preclinical Development of Folding Interfering Degraders Against the Cellular Prion Protein

We have developed a novel experimental paradigm called Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT). The technology builds on the selective degradation of target proteins by stabilizing folding intermediates, recognized by the cellular quality control machinery as improperly folded species. We applied the approach to the cellular prion protein (PrP), a key factor in prion diseases and a mediator of neurotoxic signalling in other neurodegenerative conditions. Our e􀆯ort led to the identification of SM875, a small molecule that dose-dependently reduces PrP expression and suppresses prion propagation in multiple cell lines (Spagnolli et al., 2021). To move SM875 along the drug development pipeline, we recently aimed to optimize it through rational design, validate its mode of action biophysically, and assess its therapeutic potential in vivo. To improve pharmacokinetic properties, we assembled and tested a focused library of SM875 analogues, which included both synthesized compounds and commercially available molecules. Native mass spectrometry confirmed the direct binding of SM875 to a non-native PrP conformer under near-physiological conditions, supporting the predicted mode of action. To evaluate in vivo e􀆯icacy, SM875 was administered intrasplenically to wild-type mice, and western blotting analysis of target tissues confirmed a robust reduction of endogenous PrP levels. These results establish PPI-FIT as a novel targeted protein degradation strategy and validate SM875 as a first-in-class PrP small molecule degrader. The combination of computational modeling, chemical synthesis, biophysical validation, and in vivo testing provides a strong foundation for further developing our PrP-directed folding interfering degraders as therapeutic agents for prion diseases.