Research Article
The Library Derived 4554W Peptide Inhibits Primary Nucleation of α-Synuclein

https://doi.org/10.1016/j.jmb.2020.11.005Get rights and content

Highlights

  • Synucleinopathies are caused by aggregation of the protein α-Synuclein (aS).

  • The 4554W peptide inhibits aS aggregation and lowers toxicity.

  • 4554W functions by inhibiting primary nucleation of aS.

  • 4554W does not modulate downstream secondary nucleation or elongation events.

Abstract

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.

Introduction

Synucleinopathies are caused by the misfolding and subsequent aggregation of the protein α-Synuclein (αS), a 140- residue protein, highly expressed in neuronal synapses,1 and the main protein constituent found in Lewy bodies2; the pathogenic hallmark of Parkinson's disease (PD). The αS misfolding pathway is highly complex, and not entirely understood. However in synucleinopathies, αS is observed to ultimately aggregate into extended β-sheet amyloid fibrils with the potential for a number of different polymorphisms.3, 4 Moreover, during the course of amyloidogenesis αS is able to form a variety of pre-fibrillary oligomers, which can be on or off the pathway to fibrils.5, 6, 7, 8, 9 Some of these oligomers and their conformers may be functionally relevant to the disease, with some appearing to be highly toxic to cells.9 Whilst there is increasing evidence regarding precisely which molecular species might be critical for inducing αS toxicity, a number of unanswered questions remain. Pinpointing the precise molecular species responsible for αS driven toxicity and either inhibiting their formation, or sequestering them, represents a promising mechanism towards the treatment of PD and synucleinopathies in general.

Due to the complex nature of protein-protein interactions (PPIs) formed during αS aggregation, ranging from monomeric to a wide variety of conformers and oligomers, it has proven extremely difficult to rationally design effective small molecule inhibitors to modulate the process, leading many to determine this process as undruggable.10 Identification of small molecules has proven difficult for amyloids owing to the requisite number of interactions needed to block these shallow and broad PPIs and therefore to efficiently inhibit the aggregation process. Larger biotherapeutics, such as antibodies, also pose limitations due to their difficulty in traversing the blood-brain barrier (BBB) and other cell membranes, to locate at required sites of action within neurons. An area of emerging interest therefore has been the development of short peptide based molecules that are able to occupy the niche between small molecules and biotherapeutics11, 12, 13; being large enough to form structures that can specifically modulate PPIs by making multiple interactions that can generate the requisite affinity and selectively, and hence distinguish between conformations, or stabilize non-toxic oligomers, while being small enough to be modified to cross biological membranes. Peptide-based therapeutics present a number of advantages over small molecules in that they can (i) make more interactions over the larger surface areas and shallow binding pockets typically found within PPIs, (ii) avoid immunogenicity when short since they fall below the immunogenic threshold, (iii) be more target-specific, due to more interactions, and therefore less toxic, (iv) be quickly synthesized to high purity, (v) be readily modified to prevent the formation of extended strand motifs that are most susceptible to protease degradation, and (vi) be optimized to impart membrane permeability. Limitations such as low cell permeability, potential low affinity binding when highly flexibile, high clearance rates and low oral bioavailability are now being addressed, for example via non-peptidic or cyclic modifications, cell-penetrating peptides (CPP), or lipidic appendages.10

Using an in-cell derived peptide (4554W), capable of inhibiting αS toxicity by modulating aggregation,14 we sought to gain further mechanistic insight by establishing where within the amyloidogenic pathway the peptide functions (Figure 1). To do so, aggregation experiments were performed under carefully designed experimental conditions that sought to individually probe each of the three key processes within the aggregation pathway. In particular, the effect of the peptide was deconvoluted into (i) changes in heterogeneous primary nucleation,15, 16, 17, 18 (ii) changes to fibril elongation,17, 19 and (iii) changes to fibril amplification/secondary nucleation.17, 18, 19, 20 4554W was generated from a library based on preNAC αS45-54; a region within which most early onset SNCA mutations are located, and one that has subsequently been found to feature prominently at the dimeric fibril interface for the majority of αS polymorphs identified.4, 21 Therefore, an improved understanding of the mechanism of action for 4554W could lead towards increased efficacy of treatments for αS driven pathologies, as well as other age-related diseases in which amyloids present.

Section snippets

Results and Discussion

Derivation of peptide 4554W An intracellular Protein-fragment Complementation screening Assay (PCA)14, 22, 23, 24, 25 was previously utilised to generate the peptide inhibitor, 4554W, using wild-type αS45-54 as a design scaffold.14 This 10 residue region within αS was selected owing to the fact that it contained all but one (A30P26) of the then known early onset mutations (E46K,27 H50Q,28 A53T29 and A53E30). Additional G51D/E31 mutants were discovered later, hence D/E residues were not

Protein expression and purification of human wt. αS (140)

Wild type human α-synuclein was recombinantly expressed and purified, based on, and modified from, a previously published method.33, 46 Briefly, the pET21a plasmid containing the human wt. αS (1–140), purchased from Addgene (deposited by the Michal J Fox Foundation MJFF) was transformed into E. coli expression cell line BL21 (DE3). 2xYT overnight cultures containing ampicillin of this human wt. αS (1–140) pET21a BL21 (DE3) E.coli strain were used to inoculate 1 litre 2xYT cultures, containing

Lipid preparation for induced primary nucleation method

The mass of dry DMPS lipid powder was determined using an ultra-micro balance (Sartorius), and dissolved in 20 mM sodium phosphate buffer pH 6.5 to a concentration of 2 mM. This was dissolved by shaking, in a 2 ml Eppendorf tube, on a Thermomixer compact (Eppendorf), at 45 °C, 1400 rpm for 3 hours. The solution was then freeze thawed five times using dry ice and the thermomixer compact (Eppendorf) at 45 °C and 500 rpm. The preparation of the vesicles was carried out by sonication, using a

Seed fibril formation for elongation method

Mature fibrils were produced in a 10 mm Quartz cuvette by incubating 1.5 ml of 400 µM αS monomers in 20 mM sodium phosphate buffer (pH 6.5) for 48 hours at 40 °C maximal stirring (1500 rpm), using a PTFE magnetic stirrer, on an RCT Basic Heat Plate (IKA, Staufen, Germany). The mature fibrils were diluted to 200 µM monomer equivalents using 20 mM sodium phosphate buffer (pH6.5) (1.5 ml) and broken into seeds by 3 rounds of freeze-thawing with liquid N2 followed by 55 °C water bath. The mixture

Author Contributions

RMM KJCW and KJM conducted the experiments and contributed to the experimental design. JMM and RJW directed the research and experimental design. All authors participated in data analysis and writing of the paper.

CRediT authorship contribution statement

Richard M. Meade: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing. Kimberley J. Morris: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. Kathryn J.C. Watt: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft. Robert J. Williams: Data curation, Formal analysis, Funding acquisition, Investigation, Resources,

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JMM is an advisor to Sapience Therapeutics. There are no other financial or commercial conflicts to declare.

Acknowledgements

JMM, RJW and RMM thank BRACE for award of a PhD studentship (BR16/064). The work is supported by a project grant and an equipment grant from Alzheimer’s Research UK (ARUK-PG2018-003; ARUK-EG2018A-008). This work is also supported by the Engineering and Physical Sciences Research Council EP/L016354/1. RMM wishes to thank Michele Vendruscolo and Roxine Staats for assistance, via correspondence, with the primary nucleation ThT assay protocol. Emil Dandanell Agerschou for assistance, via

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