Friday, May 9, 2025
5/9/2025
Mechanism of Action of Prion Protein-Lowering Small Molecules - PROJECT SUMMARY Prion disease is a rapidly fatal neurodegenerative disease that arises from a single protein, the native prion protein (PrP), which is capable of post-translational conversion into a self-templating and deadly conformer termed a prion. The centrality of PrP to this process, as the essential substrate for disease, has long led to the therapeutic hypothesis of PrP reduction in the brain. However, despite strong genetic evidence and pharmacological proof of concept using antisense oligonucleotides, whole-brain PrP reduction has been difficult to achieve at scale with large gene, biologic or even oligonucleotide therapeutics. We have recently identified two small molecules with PrP-lowering properties that may point towards more therapeutically facile ways to achieve this end, while simultaneously illuminating new facets of PrP’s biology and basal regulation. Following their discovery through a high-throughput immunofluorence-based phenotypic screen, we have now confirmed both potency and considerable proteomic selectivity with PrP among the top two downregulated proteins in cells following treatment with either compound. We now seek to take the following steps to deeply understand the relevance of these molecules to prion biology, both by identifying the mechanisms by which they are lowering PrP and by exploring their ability to do so in vivo. 1) Targeted cell biological and genetic follow-up of PrP-lowering compounds. We will use molecular biology tools to probe changes to PrP’s processing, localization, synthesis rate and degradation rate following treatment. We will assess the drug responsiveness of a minimal ectopic expression cellular system. We will also perform targeted knockdown and overexpression of the non-PrP proteins most effected by compound treatment according to tandem mass tag (TMT) proteomics. 2) Unbiased approaches to discover the mechanism of PrP-lowering compounds. As a complement to proteomics, we will perform RNA sequencing on compound treated and untreated cells. To refine the specificity of our proteomic profiles of drug activity, we will perform TMT in a range of paradigms leveraging inactive analogs of lead compounds, inactive compounds with the same annotated mechanism of action, PrP knockout cells, and multiple treatment timepoints. Finally, we will perform an immunofluorescence- based, pooled genome-wide CRISPR screen to discover effectors and sensitizers of drug-mediated PrP reduction. 3) In vivo pharmacokinetics and target engagement. Leveraging the strong pharmacokinetic profiles of our lead compounds, we will dose mice to achieve accumulation in known PrP-expressing tissues, including PrP-expressing peripheral tissues for robustness against the possibility of low brain uptake. We will then assess the relationship between drug accumulation and PrP levels. Altogether, this work will both uncover one or more biological pathways capable of regulating PrP levels and provide much-needed new directions for translational efforts against this fatal and currently untreatable disease, potentially offering clues to aid development of the first PrP-lowering small molecule drug.
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