The Design and Biophysical Analysis of Constrained Peptide Inhibitors of Alpha-Synuclein Aggregation

: (Alternative Format Thesis)

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder pathologically defined by the misfolding and aggregation of alpha-synuclein (αSyn) into toxic oligomers and fibrils that accumulate as Lewy body inclusions. These pathogenic processes remain untreatable, highlighting the need for disease-modifying strategies that can modulate αSyn aggregation. This study explores the use of short, conformationally constrained peptides to inhibit αSyn aggregation, combining structural, biophysical, biochemical and cellular approaches to develop and characterise novel peptide-based inhibitors.
An initial focus was placed on understanding previously reported inhibitors. Using high-resolution nuclear magnetic resonance (NMR), the mechanisms of the 4654w(N6A) and αS1-25 peptides were studied. 4654w(N6A) was found to stabilise low-order oligomers rather than monomeric αSyn, while αS1-25 was found to inhibit lipid-induced aggregation by displacing αSyn from vesicle surfaces. Subsequent structural studies of the αS1-25 and its lactamised derivative, αS2-12(L6), revealed both peptides adopt well-defined amphipathic α-helices that underpin their lipid-targeting activity.
Building on these insights, a genetically encoded intracellular cyclised peptide library was screened, leading to the identification of a potent cyclic peptide inhibitor, αS2-12W. This peptide adopts an α-helical conformation, interacts with the C-terminal region of αSyn and potently inhibits lipid-induced aggregation at substoichiometric concentrations. αS2-12W displayed strong cellular uptake, low toxicity and rescued a C. elegans PD model from αSyn-related pathology. To extend these findings, αS2-12W was evaluated against several pathologically relevant αSyn species including phosphorylated, C-terminally truncated and familial mutant forms. While some inhibitory activity was retained against most variants, it was abolished for the C-terminally truncated version, confirming the importance of the C-terminal binding site for activity. From here, a panel of αS2-12W-based proteolysis targeting chimeras (PROTACs) were synthesised, which maintained inhibitory function but, under the tested conditions, did not induce clear degradation of αSyn. However, this has established a precedent to facilitate the optimisation of linkers and sequences to degrade αSyn.
Overall, this work identifies αS2-12W as a potent, cell-permeable inhibitor of αSyn aggregation, suggests a mechanism of action and lays the groundwork for future peptide-based degradation strategies in PD and related synucleinopathies.

Date of Award	12 Nov 2025
Original language	English
Awarding Institution	University of Bath
Supervisor	Jody Mason (Supervisor), Robert Williams (Supervisor) & Matthew Crump (Supervisor)