It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.
The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.
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 α 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.
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