α-Synuclein (45-54) (human)

Parkinson's Disease (PD) is characterized by the accumulation of Lewy bodies in dopaminergic neurons. The main protein component of Lewy bodies, α-synuclein (αS), is also firmly linked to PD through the identification of a number of single point mutations that are autosomal dominant for early-onset disease. Consequently, the misfolding and subsequent aggregation of αS is thought to be a key stage in the development and progression of PD. Therefore, modulating the aggregation pathway of αS is an attractive therapeutic target. Owing to the fact that all but one of the familial mutations is located in the preNAC 45-54 region of αS, we previously designed a semi-rational library using this sequence as a design scaffold. The 45-54 peptide library was screened using a protein-fragment complementation assay approach, leading to the identification of the 4554W peptide. The peptide was subsequently found to be effective in inhibiting primary nucleation of αS, the earliest stage of the aggregation pathway. Here, we build upon this previous work by screening the same 45-54 library against five of the known αS single-point mutants that are associated with early-onset PD (A30P, E46K, H50Q, G51D, and A53T). These point mutations lead to a rapid acceleration of PD pathology by altering either the rate or type of aggregates formed. All ultimately lead to earlier disease onset and were therefore used to enforce increased assay stringency during the library screening process. The ultimate aim was to identify a peptide that is effective against not only the familial αS variant from which it has been selected but that is also effective against WT αS. Screening resulted in five peptides that shared common residues at some positions, while deviating at others. All reduced aggregation of the respective target, with several also identified to be effective at reducing aggregation when incubated with other variants. In addition, our results demonstrate that a previously optimized peptide, 4554W(N6A), is highly effective against not only WT αS but also several of the single-point mutant forms and hence is a suitable baseline for further work toward a PD therapeutic.

Aggregation of α-Synuclein (αS) is widely regarded as a key factor in neuronal cell death, leading to a wide range of synucleinopathies, including Parkinson's Disease. Development of therapeutics has therefore focused on inhibiting aggregation of αS into toxic forms. One such inhibitor, based on the preNAC region αS45-54 (4554W), was identified using an intracellular peptide library screen, and subsequently shown to both inhibit formation of αS aggregates while simultaneously lowering toxicity. Subsequent efforts have sought to determine the mode of 4554W action. In particular, and consistent with the fact that both target and peptide are co-produced during library screening, we find that the peptide inhibits primary nucleation of αS, but does not modulate downstream elongation or secondary nucleation events. These findings hold significant promise towards mechanistic understanding and development of molecules that can module the first steps in αS aggregation towards novel treatments for Parkinson's disease and related synucleinopathies.

Misfolding and aggregation of alpha-synuclein (αS) within dopaminergic neurons is a key factor in the development and progression of a group of age-related neurodegenerative diseases, termed synucleinopathies, that include Parkinson's disease (PD). We previously derived a peptide inhibitor from a 209,952-member intracellular library screen by employing the preNAC region (45-54) as a design template. At least six single-point mutations firmly linked to early-onset Parkinson's disease (E46K, H50Q, G51D, A53T/E/V) are located within this region, strongly implicating a pathogenic role within αS that leads to increased cytotoxicity. A library-derived ten residue peptide, 4554W, was consequently shown to block αS aggregation at the point of primary nucleation via lipid induction, inhibiting its conversion into downstream cytotoxic species. Here we couple truncation with a full alanine scan analysis, to establish the effect upon the αS aggregation pathway relative to 4554W. This revealed the precise residues responsible for eliciting inhibitory interaction and function, as well as those potentially amenable to modification or functionalisation. We find that modification N6A combined with N-terminal truncation results in a peptide of significantly increased efficacy. Importantly, our data demonstrate that the peptide does not directly disrupt αS lipid-binding, a desirable trait since antagonists of αS aggregation and toxicity should not impede association with small synaptic neurotransmitter vesicles, and thus not disrupt dopaminergic vesicle fusion and recycling. This work paves the way toward the major aim of deriving a highly potent peptide antagonist of αS pathogenicity without impacting on native αS function.