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A strategy for designing inhibitors of -synuclein aggregation and toxicity as a novel treatment for Parkinson’s disease and related disorders

OMAR M. A. EL-AGNAF^*,1, KATERINA E. PALEOLOGOU^*, BRETT GREER, ABDULMAWALA M. ABOGREIN^*, JENNY E. KING^*, SULTAN A. SALEM^*, NIGEL J. FULLWOOD^*, FIONA E. BENSON^*, REBECCA HEWITT^*, KIRSTIE J. FORD^*, FRANCIS L. MARTIN^*, PATRICK HARRIOTT, MARK R. COOKSON and DAVID ALLSOP^*

^* Department of Biological Sciences, Lancaster University, Lancaster, UK;
School of Biology and Biochemistry, Queen’s University of Belfast, Belfast, UK; and
Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA.

¹ Correspondence: E-mail: o.el-agnaf{at}lancaster.ac.uk

SPECIFIC AIMS

The aim of this work was to design short synthetic peptides that could inhibit aggregation of the -synuclein (-syn) protein, implicated in Parkinson’s disease (PD) and related disorders, into toxic oligomers. First, we identified the critical binding region in -syn responsible for its self-association. It was thought that short synthetic peptides homologous to this region would interact with the same region in the full-length -syn molecule and block its assembly into oligomers and amyloid fibrils. To identify the appropriate binding region, we synthesized a library of overlapping short peptides spanning the entire -syn primary sequence. Peptides capable of binding to full-length -syn were modified and tested for their ability to inhibit aggregation and toxicity of -syn.

PRINCIPAL FINDINGS

1. Identification of the binding region in -syn responsible for its self-association
We synthesized an overlapping library of 7-mer peptides, corresponding to residues 1–7, 2–8, 3–9, ... , 132–138, 133–139, 134–140 of -syn. These peptides were tagged with a biotin group, which was used to detect any binding of peptides to full-length -syn immobilized on a microtiter plate. Peptides corresponding to the hydrophobic region located in the central part of -syn (amino acid residues 64–100) produced the highest binding to full-length -syn. Peptides corresponding to the N-terminal region (residues 1–60) of -syn had low binding levels, whereas peptides corresponding to the C-terminal region (residues 101–140) of -syn showed no binding. Peptides which gave the highest level of binding to -syn were found to correspond to the N-terminal half (residues 64–86 of -syn) of the non-Aß component region (NAC). NAC corresponds to residues 61–95 of -syn.

2. Designing peptides as inhibitors of -syn aggregation
To design compounds to inhibit -syn aggregation, we made small peptides from the binding region linked to further solubilizing amino acids. For the binding sequence, we chose hydrophobic residues 68–72 of -syn (GAVVT). The strategy we adopted for the solubilizing component was to place one or two hydrophilic arginine residues at the N- and/or the C terminus of this GAVVT peptide. This could decrease interaction between the peptide and monomeric -syn, since the targeted binding region in -syn does not contain these arginine residues. Glycine is also a hydrophilic residue and is the most conformationally unrestrained amino acid, and generally breaks secondary structure. Placing glycine residues as spacers between arginine and binding residues GAVVT should result in the solubilizing component of the inhibitor peptide, (i.e., the first two (RG) and/or last two residues (GR) ), not partaking in binding between the inhibitor and -syn. Based on these criteria, we designed several peptides, referred to here as -syn inhibitors (ASI), for evaluation (Table 1 ). Since we found that peptides corresponding to the N-terminal region of -syn had low binding to the -syn molecule, we included some peptides from this region as a control (Table 1) .

View this table: [in this window] [in a new window]   Table 1. Primary structure of the native sequence -syn(68-75) and ASI peptides that were synthesized and studied

3. Inhibition of -syn fibril formation
Incubation of recombinant -syn at 50 µM with continuous shaking for 96 h at 37°C in phosphate buffered saline (PBS), pH 7.4, led to aggregation of the protein and formation of amyloid-like fibrils, as confirmed by Thioflavin T (Th-T) assay (Fig. 1 ) and by negative stain electron microscopy (EM). When -syn was incubated with ASI1 (RGGAVVTGR-NH₂) peptide at 2:1; 1:1 and 1:2 (-syn: peptide molar ratio) with a constant -syn concentration of 50 µM, complete inhibition of amyloid fibril formation was achieved (Fig. 1) . Shorter peptides, truncated at N- and C-termini of GAVVT, were also synthesized (Table 1) . RG and GR residues of the peptides at N- and C-terminal ends, respectively, were again introduced to increase solubility. Since ASI peptides were designed from the region of -syn covering amino acid residues 68–72, we also included the unmodified fragment of -syn corresponding to residues 68–75 as a control. Effects of ASI peptides on -syn aggregation over 96 h were then tested using Th-T assay (Fig. 1) . The shortest peptide that still retained ability to inhibit -syn aggregation had the central sequence AVVT, corresponding to -syn(69–72). Peptides -syn(68–75), ASI4 (RGAVVGR) and ASI5 (RGVVTGR) did not show any significant inhibition of -syn fibrillogenesis (Fig. 1) . Control NTC peptides also showed no inhibition of -syn fibrillogenesis.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of ASI peptides on -syn fibrillogenesis. -Syn samples were incubated for 4 days with continuous shaking at 37°C in presence of various concentrations of ASI and NAC(8-15) peptides and amyloid fibril formation was measured by Th-T binding assay. Data are presented as percentages of the signal obtained from -syn incubated in absence of ASI peptides. Data shown are representative of at least three independent experiments. Assays were performed in triplicate, and mean +/– SD are shown.

We also used EM to examine negatively stained preparations made from -syn solutions that were either aged alone or with ASI peptides. The -syn solution incubated alone revealed a dense network of fibrils averaging 10 nm in diameter and extending for several hundred nanometers in length. In contrast, the sample containing peptide ASI1 showed only a thinly scattered distribution of small irregular globular aggregates ranging from less than a nanometer up to a few tens of nanometers in size. Samples containing both ASI2 and ASI3 peptides revealed a sparse scattering of irregular aggregates and truncated fibrils. The sample containing ASI4 showed a dense network of highly abnormal fibrils, whereas the sample containing peptide ASI5 had fibrils similar in size and appearance to the control. Appearance and distribution of fibrils in the sample containing -syn 68–75 appeared to be similar to the control.

All peptides that were active inhibitors of -syn aggregation also inhibited NAC fibrillogenesis, and inactive peptides failed to inhibit NAC aggregation.

4. Inhibition of early -syn oligomerization
We also tested effects of ASI peptides on -syn oligomerization. The same fresh and aged samples of -syn solutions alone or with inhibitor peptides were tested by a novel ELISA developed in our laboratory that recognizes only oligomeric forms of -syn. All peptides that inhibited formation of -syn fibrils (as demonstrated by EM and Th-T) also inhibited formation of -syn oligomers, as detected by this ELISA. We also investigated effects of peptide inhibitors on -syn aggregation as detected by immunoblotting. Freshly dissolved -syn migrated predominantly as a band of 16 kDa, corresponding to the monomer, whereas -syn that had been aged for 4 days showed a much less intense monomer band and large amounts of a dimer at 34 kDa and a high molecular weight species that did not enter the separating gel. Samples of -syn aged in presence of active ASI peptides contained predominantly monomeric and dimeric species.

5. Effects of inhibitors in a cell model of -syn toxicity
Neuronal cells transfected with mutant forms of -syn responsible for familial PD (A30P or A53T), which are associated with increased -syn oligomerization, exhibit an enhanced susceptibility to apoptotic cell death by various toxic insults, including Fe(II) exposure. We developed a cell permeable inhibitor of -syn aggregation (ASI1D), using the polyarginine peptide delivery system. This ASI1D peptide was able to enter cells and inhibit DNA damage induced by Fe(II) in BE(2)-M17 human dopaminergic neuroblastoma cells transfected with -syn(A53T) (M17-A53T), as assessed by Comet assay. ASI peptides without this delivery system did not reverse levels of Fe(II)-induced DNA damage. ASI1D peptide increased (P<0.0005) the number of cells positive for Bcl-2, while significantly (P<0.05) decreasing the number of cells stained positive for Bax.

CONCLUSIONS

There is substantial evidence from molecular genetics, transgenic animal studies, and aggregation/toxicity studies with recombinant -syn (and various peptide fragments derived from it) to suggest that conversion of this protein from soluble monomers to aggregated insoluble forms in the brain is a key event in the pathogenesis of PD and related diseases. It seems increasingly likely that early ‘soluble oligomers’ are actually toxic species responsible for neurodegeneration and neuronal cell death. This suggests that inhibition of -syn oligomerization may be a viable strategy for therapeutic intervention in these diseases. -Syn is normally a soluble unfolded protein, with little or no ordered structure under normal physiological conditions, but it has an inbuilt tendency to self-oligomerise and to assemble into fibrillar aggregates. In this study we attempted to inhibit this aggregation process by designing small peptides that can specifically interact with the region of -syn responsible for its self-aggregation. To identify this critical region, we synthesized an extensive library of overlapping -syn peptides. The binding sequence identified using the peptide library was located in the central hydrophobic region of -syn, corresponding to amino acid residues 64–100. Our attempt to design compounds as potential inhibitors to arrest aggregation of -syn was based on peptides taken from the hydrophobic binding sequence -syn(68–72) linked to a solubilizing component. Using this new strategy, we designed several peptides as -syn inhibitors (ASI) (Table 1) . Our results show that some of these ASI peptides could inhibit formation of early and late -syn aggregates, as detected by EM, Th-T binding, immunoblotting, and oligomer-specific ELISA. These results support the idea that these peptides bind specifically to their homologous native sequence in -syn and so inhibit its self-oligomerization (Fig. 2 ).

View larger version (29K): [in this window] [in a new window]   Figure 2. Schematic diagram showing possible mechanisms for -syn aggregation. -Syn molecules interact during oligomerization leading to formation of protofibrils and then to mature amyloid fibrils, the formation of which involves interaction between the hydrophobic binding region (amino acid residues 64–100 (gray diamonds) of -syn molecules (A). ASI peptides (black inverted triangles) that can interact with the binding region of -syn should inhibit -syn oligomerization and toxicity (B).

Problems associated with peptide therapy include transport through the blood-brain barrier, generation of an immune response, and high sensitivity of peptides to proteolytic degradation. These difficulties can be minimized by reducing peptide length and by use of D-amino acids or N-methylated amino acid derivatives. We have shown that ASI peptide fused to an arginine-rich carrier (ASI1D) can enter M17-A53T cells and inhibit DNA damage induced by Fe(II) in these cells. It would be of great interest to test effects of ASI1D on animal models of PD. Since the peptides we identified here are relatively small, they could also represent the starting point for designing peptidomimetic molecules more suitable as new drugs for chronic therapy of PD and related disorders in the future.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1346fje; doi: 10.1096/fj.03-1346fje

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