HYPE-mediated AMPylation as a Novel Therapeutic for Neurodegeneration

The heat shock protein HSPA5 or BiP serves as a sentinel for ER (endoplasmic reticulum) stress and regulates proteostasis by chaperoning unfolded and misfolded proteins. We and others recently discovered an additional level of proteostatic control, whereby the Fic protein, HYPE/FicD, regulates BiP’s chaperoning capacity by the reversible, post-translational addition of AMP to BiP. Wild-type (WT) HYPE is predominantly a de-AMPylating enzyme that is intrinsically inhibited for AMPylation and shows only basal adenylyltransferase activity relative to a constitutively active mutant (E234G HYPE). 

Proteostatic dysfunction is often seen in neurodegenerative diseases associated with protein aggregation. Accordingly, we observed that HYPE can directly AMPylate the misfolded presynaptic protein αSynuclein (αSyn) implicated in neurodegenerative phenotypes associated with Parkinson’s disease (PD). Interestingly, HYPE-mediated AMPylation ameliorates many of the in vitro neurotoxic phenotypes of αSyn such as αSyn fibrillation and increased membrane permeability. These potentially cytoprotective phenomena conferred by HYPE’s adenylyltransferase activity make it an attractive point of intervention for PD therapy and for interrogating proteostasis. To this end, we conducted a pilot screen of FDA-approved, natural, and semi-synthetic small-molecule compound libraries towards the identification of activators of WT HYPE and inhibitors of E234G HYPE AMPylation. Employing fluorescence polarization of a labelled ATP analogue on a 384-well microplate platform, we’ve developed a robust, high-throughput screening (HTS) assay suitable for monitoring changes in AMPylation. First-pass selection of our pilot screen yielded multiple compounds capable of manipulating AMPylation in vitro. After expanding our HTS to over 30,000 novel compounds (including those with blood brain barrier penetrance to target PD), we obtained several promising lead candidates. Ongoing cellular validations of these lead compounds to determine their efficacy against intracellular AMPylation of HYPE substrates will be discussed. Challenging neuronal cell culture models of PD with these hits will provide molecular insights into αSyn-induced neurotoxicity, paving the path for novel therapeutic strategies in combating PD during early stages of disease onset.