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.