bioRxiv2022
Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood-brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4-dehydro-oleandrin to engage with its cognate binding pocket within the NKA subunit. The improved brain bioavailability of C4-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases. AUTHOR SUMMARYPrion diseases are fatal neurodegenerative diseases for which there is no effective treatment. An abundance of data indicates that reducing the levels of a specific protein, termed the cellular prion protein (PrPC), would not only be safe but would delay disease onset and extend prion disease survival. This project builds on our recent discovery that PrPC binds to NKAs, specific cellular transport proteins that use energy to electrify cellular membranes by pumping charged potassium and sodium metals in and out of cells. We showed that targeting NKAs with their natural inhibitors, cardiac glycosides (CGs), causes brain cells to internalize and degrade NKAs, and that PrPC, on account of residing next to NKAs, gets co-degraded. Natural CGs act primarily on the heart. Here, we used computational modeling to identify a CG, termed KDC203, that is predicted to have favorable characteristics for brain applications. We show that KDC203 reduces PrPC levels by 84% in immortalized human brain-like cells grown in the dish. Moreover, we show that KDC203 exhibits relatively low toxicity, predominantly targets the brain when subcutaneously injected into mice, and has other promising pharmacological characteristics that recommend it for further evaluation for the treatment of prion diseases.