Introduction

Many neurodegenerative diseases are linked to the misfolding and aggregation of proteins inside brain cells. In Parkinson’s disease and Lewy body dementia, one of the key proteins involved is alpha-synuclein. When alpha-synuclein misfolds, it can form toxic assemblies and larger deposits known as Lewy bodies, which are associated with damage to dopamine-producing neurons and progressive neurological decline.

Researchers at the University of Bath are developing new peptide-based molecules designed to stabilise alpha-synuclein in a non-toxic, native-like state. Rather than trying to remove aggregates after they have formed, the project aims to intervene earlier by preventing the first steps of misfolding and aggregation.

Our approach

The project focuses on small, ultra-stable “constrained peptides”. These are short protein-like molecules that have been chemically locked into a defined shape, allowing them to bind more effectively, resist degradation and potentially enter cells.

Alpha-synuclein is normally a flexible protein, but parts of it can adopt an alpha-helical structure when interacting with biological membranes. This folded state is linked to the normal biological role of alpha-synuclein. The Bath team is exploiting this principle by designing constrained peptides that mimic and stabilise this native folded state, thereby reducing the chance that alpha-synuclein will misfold into toxic aggregates.

A key innovation is the use of intracellular peptide library screening. Instead of designing and testing one molecule at a time, the team screens large peptide libraries inside living cells. This allows the researchers to select molecules that not only bind alpha-synuclein, but also remain soluble, stable and non-toxic in a cellular environment.

The project has also advanced to constrained-peptide discovery using protein-fragment complementation assay screening combined with intracellular crosslinking chemistry. This enables peptides to be chemically constrained during selection, increasing the chance of identifying molecules with the right shape and biological activity from the outset.

Aims and objectives

The project aims to:

develop constrained peptide molecules that bind alpha-synuclein and stabilise its native-like helical state
prevent alpha-synuclein from misfolding into toxic oligomers and aggregates
use intracellular constrained-peptide screening platforms to identify peptides with improved stability, selectivity and cell compatibility
test whether constrained peptides can enter neuronal cells and reduce alpha-synuclein pathology
generate lead molecules that could support future therapeutic development for Parkinson’s disease and Lewy body dementia

Methods

The team uses short peptide sequences derived from the N-terminal region of alpha-synuclein, a region involved in membrane binding and native helical structure formation. These peptides are chemically constrained to hold them in a stable alpha-helical shape.

Candidate peptides are tested using biophysical and structural methods to measure their folding, stability, alpha-synuclein binding and ability to block aggregation. These include circular dichroism, aggregation assays, size-exclusion chromatography, electron microscopy, serum stability testing and binding measurements.

The project also uses intracellular protein-fragment complementation assay screening to discover new alpha-synuclein-binding constrained peptides directly inside cells. This approach allows peptide binding and biological compatibility to be selected simultaneously.

Promising molecules are then tested in neuronal cell systems, including Parkinson’s disease-relevant dopamine neuron models. These experiments assess cell entry, toxicity, co-localisation with alpha-synuclein, reduction of alpha-synuclein pathology and rescue of disease-relevant cellular phenotypes.

Outcomes and impact

A major outcome from this programme has been the identification of a short helix-constrained alpha-synuclein peptide, which showed improved helicity, serum stability, neuronal uptake and ability to inhibit alpha-synuclein aggregation. This work demonstrates that very small, constrained peptides can be used to stabilise a native-like alpha-synuclein state and block downstream toxic aggregation.

More recently, the project has also identified a constrained peptide through PCA screening with intracellular crosslinkers, further supporting the core concept that functional peptide binders can be selected directly inside cells in their constrained form.

In the longer term, this work could provide a new route to therapeutic molecules for diseases driven by alpha-synuclein misfolding. It also establishes a broader drug-discovery strategy for using constrained peptides to stabilise native protein states and prevent pathological protein aggregation.