(27aa) Directed Evolution of an Alpha-Synuclein Degrading Enzyme

Synucleinopathies are neurodegenerative disorders characterized by the abnormal aggregation of the neuronal protein alpha-synuclein in the brain, leading to synaptic dysregulation and neuronal death. Currently, there are no effective targeted treatments for these degenerative diseases, including Parkinson’s Disease (PD) and Lewy Body Dementia (LBD). Therefore, we look towards proteases as a modality for degrading the distinctive alpha-synuclein plaques associated with PD and LBD. One such protease that can degrade alpha-synuclein is the serine protease Kallikrein 6, also known as neurosin. Neurosin cleaves alpha-synuclein after lysine at position 80 (K80), resulting in oligomer degradation. However, alpha-synuclein is a poor substrate for neurosin due to neurosin’s preference to cleave after arginine over the lysine cleavage site of alpha-synuclein. This proclivity presents an issue, as protease- activated receptors (PARs) in neurons and endothelial cells are subject to undesirable cleavage by neurosin. Using a high-throughput yeast-based assay, we have identified 6 neurosin variants with switched specificity towards the alpha-synuclein derived sequence VAQKTVEG and away from a highly active AFRFS sequence. We are currently continuing to evolve neurosin to fine-tune its preference for alpha-synuclein cleavage and improve its catalytic activity by optimizing the stringency of our high throughput screen. Our pipeline for characterizing evolved variants involves biochemical characterization of FRET peptides, assays on oligomeric synuclein and their variants, and inhibition of synuclein seeding in SH-SY5Y cells. In parallel, we are building a machine learning-guided continuous evolution platform to select for neurosin variants that can rescue yeast cell growth. We hypothesize that neurosin variants that directly degrade synuclein oligomers may outperform ones that were evolved on synuclein derived peptide sequences and divulge non-catalytic evolutionary pathways.