Journal of Molecular Biology
Volume 432, Issue 4, 14 February 2020, Pages 967-977
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Structural Optimization of Inhibitors of α-Synuclein Fibril Growth: Affinity to the Fibril End as a Crucial Factor

https://doi.org/10.1016/j.jmb.2019.11.019Get rights and content

Highlights

  • Approaches for improving the efficiency of inhibitors of α-synuclein fibril growth are described.

  • Tuning charge of the inhibitor enhances the inhibition efficiency.

  • Position of the bulky group and its distance to the fibril end affect the inhibitor activity.

  • Binding of β-sheet-rich bulky group to the second filament of fibril decreases Kd.

  • Inhibitor efficiency linearly correlates with its affinity to the fibril end.

Abstract

Background

Misfolding of the neuronal protein α-synuclein into amyloid fibrils is a pathological hallmark of Parkinson's disease, a neurodegenerative disorder that has no cure. Inhibition of the fibril growth is considered a promising therapeutic approach. However, the majority of the existing inhibitors are either unspecific or work at high micromolar concentrations. Earlier, we created a protein-based inhibitor of α-synuclein fibril growth that consists of an α-synuclein moiety and a bulky group. It specifically binds to α-synuclein fibril ends and blocks them by creating steric hindrance to subsequent monomer binding.

Results

In this work, we prepared a series of inhibitors with modified α-synuclein moieties and bulky groups of different structure, size, and position. We studied the structure-activity relationship of these inhibitors and optimized them by improving affinity to the fibril end and blocking efficiency. The inhibitors were tested in a Thioflavin T-based kinetic assay, and their affinity to the fibril ends was measured by fluorescence anisotropy. We showed that decrease in electrostatic repulsion between inhibitor and fibril end improved the inhibitor efficiency. Inhibitors with rigid β-sheet-rich bulky groups bind to fibril ends stronger than monomeric α-synuclein and therefore have a high inhibition efficiency, showing a linear correlation between Kd and IC50.

Significance

We determined which properties of inhibitor molecules are the most important for good performance and found that the inhibitor affinity to the fibril end is a key feature that determines its inhibition efficiency. Applying this knowledge, we improved existing inhibitors and reached IC50 value of 300 nM.

Introduction

α-Synuclein (AS) is a small 140 aa neuronal protein that is involved in the regulation of synaptic vesicle-mediated protein trafficking and in mediating vesicle interactions [[1], [2], [3]]. In its native form, AS is an intrinsically disordered monomer that forms an α-helix upon interaction with membranes. However, it can also exist in a pathological fibrillar form.

Misfolding of AS into the pathological parallel β-sheet amyloid form is involved in the development of Parkinson's disease (PD) [4,5]. PD is a progressive neurodegenerative disorder characterized by the presence of AS-containing inclusions (Lewy bodies) and the loss of dopaminergic neurons [6]. An effective cure for PD is not developed yet, and the currently used therapeutic approaches mostly include maintaining dopamine levels or using neuroprotective agents [[7], [8], [9]].

There is evidence that during PD development, AS fibrils are responsible for cytotoxicity and cell-to-cell spreading of the pathology [10]. Addition of preformed AS fibrils to cultured neuronal cells causes structural and functional defects [11]. Moreover, cell-to-cell propagation of the PD pathology and progressive neurodegeneration are observed in animal brains after inoculation of preformed fibrils obtained in vitro [[12], [13], [14]] or derived from postmortem brains of PD patients [15] or synucleinopathy patients [16]. Inhibition of fibril growth and seeding capacity is considered to be a potent approach for preventing AS toxicity and PD-related neurodegeneration [9,10,17].

Misfolding of AS into amyloid fibrils occurs with the formation of multiple hydrogen bonds between neighboring protein molecules in the fibril. This makes the fibrillization process almost irreversible. Fibrils contain several thousands of monomers and reach several (~2–20) micrometers in length. They grow via binding of monomeric protein to the fibril ends [18,19], which serve as a template for monomer folding into the amyloid conformation. Therefore, the rate of fibrillization is proportional to the concentration of the fibril ends. Formation of new ends upon fibril breaking or due to other secondary nucleation mechanisms accelerates the fibrillization [19].

Several approaches for inhibition of AS fibrillization have been reported up to now. Stabilizing the monomeric form of AS by DNA aptamers [20] or engineered peptide β-wrapin AS69 [21] has been shown to inhibit AS fibrillization. Different types of antibodies were reported to slow down AS fibrillization by binding to the oligomeric form of AS [22,23]. Several small aromatic molecules (mannitol-based [24], EGCG, Dopamine, Amph [25], SynuClean-D [26]) destabilize AS amyloid fibrils by intercalation. However, all inhibitors that target abundant species are effective only at concentrations comparable to the AS concentration (up to 22 μM in neurons [1]).

More promising are inhibitors that block centers of fibril amplification, namely late on-pathway oligomers or fibril ends, and thus act at much lower concentrations. For example, aforementioned β-wrapin AS69 was shown to inhibit AS aggregation at substoichiometric concentrations by blocking prefibrillar species [21]. Inhibitors that bind to the ends of AS fibrils and prevent their elongation (heat shock proteins [[27], [28], [29]] and AS di-tyrosine dimers [30]) also work by principle of targeting rare species. However, although rational design of inhibitors blocking AS oligomers is not possible yet due to the absence of thorough knowledge about this wide class of species, such a drawback is not relevant for inhibitors targeting fibril ends. Recently, we developed a chimeric protein AS-2L (Inh-β) that consists of two parts: (i) an AS molecule, which works as the fibril binding domain and (ii) a bulky protein group 2L (consisting of two β-sheet-rich globular L domains), which creates steric hindrance to further binding of monomers to the blocked fibril end (Fig. 1A). AS-2L efficiently inhibits AS fibrillization at 1:50 ratio [31], showing an IC50 of ~1 μM.

In this work, we studied the relationship between structure and activity of protein-based inhibitors that block AS fibril ends. We created 14 new inhibitor molecules, which are based on AS-2L, determined their efficiency in a seeded aggregation kinetic assay, and found a linear correlation between the activity of inhibitors and their affinity to the fibril ends.

Section snippets

Optimization of the fibril binding domain

Two processes that limit the inhibitor efficiency are the dissociation of the inhibitor molecule from the blocked fibril end and further binding of monomers to the blocked fibril end (Fig. 1B) [31]. Suppression of both processes is necessary for improvement of the inhibition efficacy. We decided to optimize the fibril binding region to decrease inhibitor dissociation from the fibril end, and to optimize the bulky group to create more steric hindrance and prevent undesirable binding of monomers

Discussion

Inhibition of amyloid fibril spreading between cells is a promising approach to retard the progression of Parkinson's disease. The first rationally designed protein-based inhibitor of AS fibril growth (reported earlier as Inh-β, or AS-2L [31]), despite its selectivity to fibril ends, is still far from being optimal. In this work, we explored the structure–activity relationship of different AS-2L derivatives to develop molecules with improved inhibition properties.

With the aim to enhance binding

Conclusions

In summary, we created a set of protein inhibitors of AS fibril growth based on AS-2L, the most efficient inhibitor of α-synuclein fibril growth reported to date. We studied the structure–activity relationship of these molecules and found that the best approaches for improving the inhibitor efficiency are (i) decreasing the electrostatic repulsion by modifying fibril binding moiety; (ii) optimizing the bulky group position with respect to the fibril core by optimizing distance to the fibril

Protein expression and purification

Plasmids for expression of the inhibitors were prepared by molecular cloning; for more details, see S-1. Expression and purification of proteins was performed as described in our previous work [38]. Shortly, AS was expressed in Escherichia coli, purified by ion-exchange FPLC, and stored in 10 mM Tris–HCl buffer, pH 7.4 at ~220 μM protein concentration at −20 °C. Inhibitors were expressed in E. coli, purified using Ni-NTA resin (His-tag purification, cleaved with proteases listed in Table S3),

Acknowledgments

Dr. Lucie Bednarova is acknowledged for providing support with CD measurements. The authors thank Shubhra Sachan for preparing AS(A18C)-L mutant and Yevhenii Kyriukha for labeling it with SRhB.

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