Structure-based peptide inhibitors of alpha-synuclein aggregation

Abstract

The present invention relates to inhibitory peptides that bind to alpha-synuclein molecules and inhibit alpha-synuclein amyloid aggregation, alpha-synuclein cytotoxicity, and alpha-synuclein diffusion. Methods of making and using the inhibitory peptides are described (e.g., for treating a subject having a condition or disease mediated by alpha-synuclein, such as Parkinson's disease, Lewy body dementia, or MSA).

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 356,410, filed June 29, 2016, entitled “STRUCTURE-BASED PEPTIDEINHIBITORS OF ALPHA-SYNUCLEIN AGGREGATION”, the contents of which are incorporated herein by reference.

[0003] Government Support Statement

[0004] This invention was developed with government support, pursuant to approval number AG029430 granted by the National Institutes of Health. The government owns certain rights to this invention.

[0005] sequence list

[0006] This application includes a sequence list that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. Background Technology

[0007] Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA) are collectively classified as synucleinopathies, a group of neurodegenerative diseases characterized by the pathological accumulation of the protein α-synuclein in neuronal cells. These together constitute the second most common form of neurodegenerative disease. The presynaptic protein α-synuclein, found in soluble and membrane-associated parts of the brain, accumulates in diseases such as Parkinson's (PD). These aggregates are a major component of Lewy bodies (a defining histological feature of this neurodegenerative disease) and have been shown to accompany neuronal damage. 7 Not wanting to be bound by any particular mechanism, some have suggested that this observation, along with two other observations, indicates that aggregated α-synuclein is a molecular cause of Parkinson's disease. 8 Firstly, families with hereditary PD carry α-syn mutations (such as A53T) and abundant Lewy bodies. 9, 10, 11 Secondly, families with repetitive or triple genes encoding α-syn develop early-onset PD, possibly because α-syn is forced into amyloid protein at high local concentrations. 12, 13 .

[0008] Based on structural studies, many different α-syn fibrils models have been proposed. Limited proteolytic and NMR studies have shown that the fibril core consists of 30-100 residues (Miake H (2002) J Biol Chem 277(21):19213–19219). Crystal structure and NMR studies suggest different α-syn fibrils models. In one model based on short-segment crystal structures, two monomers of each fold form an extended stereozipper (Rodriguez JA et al. (2015) Nature 525(7570):486-490). In a second model based on ssNMR, a Greek key pattern topology with one monomer per amyloid layer has been shown (Tuttle MD et al. (2016) Nat Struct Mol Biol 23(5):409-415). In summary, these studies indicate that α-syn can form polymorphic fibril structures. Fragments 68-78 of α-syn, called NACore, can form the core of α-syn fibrils. NACore is located within the 35-residue NAC (non-amyloid β-fraction) domain found in amyloid deposits (Rodriguez JA et al. (2015) Nature 525(7570):486-490). NACore readily aggregates, and the aggregates exhibit properties similar to full-length α-syn, such as diffraction patterns and cytotoxicity. Furthermore, β-synuclein (a homolog) lacks residues 74-84 and is not found in amyloid deposits, and removal of residues 71-82 has previously been shown to reduce aggregation and toxicity in vitro and in Drosophila models (Giasson et al. (2001) J Biol Chem 276(4):2380-2386 and Periquet et al. (2007) J Neurosci 27(12):3338-3346). Additionally, modification at Thr72 prevents its aggregation tendency.

[0009] The inventors recently demonstrated that short amino acid inhibitors designed to specifically “cap” growing aggregates may effectively prevent the aggregation of the Alzheimer’s disease-associated protein Tau and the sperm-derived HIV infection enhancer factor (SEVI) (Sievers et al.). 36 US Patent No. 8,754,034 57 They also demonstrated that the same design strategy can lead to designed cell-penetrating peptides that inhibit p53 amyloid formation, rescue mutant p53 function in cancer cell lines, and reduce tumor proliferation (US Patent Application 2014 / 037387). 58Therefore, there exists an attractive therapeutic window that targets α-synuclein amyloid aggregation, a major component of intracellular deposits in the form of Lewy bodies (LB) in Parkinson's disease and related neurodegenerative diseases.

[0010] It is necessary to identify agents that prevent and / or inhibit α-synuclein aggregation and / or cytotoxicity. Summary of the Invention

[0011] Despite extensive characterization of α-synamyloid formation, progress in developing therapeutics capable of inhibiting α-syn aggregation or reducing the prion-like spread of α-syn aggregates (“seeds”) between cells has been limited. Promising approaches include antibodies that chelate α-syn aggregates and small-molecule stabilizers that bind α-syn monomers (see, for example, Mandler M et al. (2015) Mol Neurodegener 10(1). doi:10.1186 / s13024-015-0008-9; Wrasidlo W et al. (2016) Brain:aww238). Using the atomic structure of NACore as a template, we developed a new class of inhibitors, namely peptides that bind α-syn seeds and prevent their growth and elongation. As shown below, these inhibitors inhibit the formation and seeding of α-synuclein fibrils, as well as the spread of α-synuclein aggregates, in many model systems.

[0012] The invention disclosed herein has many embodiments. One embodiment of the invention is a composition comprising at least one repressive peptide that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein (SEQ ID NO:1). In a typical embodiment of the invention, the repressive peptide comprises the sequence GAVVWGVTAVKK (SEQ ID NO:3) or RAVVTGVTAVAE (SEQ ID NO:4). Optionally, the repressive peptide comprises the sequence GAVVWGVTAVKKKKK (SEQ ID NO:5), GAVVWGVTAVKKGRKKRRQRRRPQ (SEQ ID NO:6), or YGRKKRRQRRRAVVTGVTAVAE (SEQ ID NO:7). In some embodiments of the invention, the composition comprises multiple repressive peptides. Typically, the repressive peptide is 6 to 30 amino acids in length.

[0013] In the inhibitory peptide compositions of the present invention, at least one amino acid in the inhibitory peptide may comprise a non-naturally occurring amino acid (e.g., a D-amino acid or an amino acid containing an N-methyl moiety); and / or the inhibitory peptide is coupled to a heteropeptide tag. Such heteropeptide tags comprise amino acid sequences that increase peptide solubility; or amino acid sequences that facilitate monitoring or manipulating the peptide; or amino acid sequences that promote peptide entry into mammalian cells. Optionally, these peptide compositions comprise a pharmaceutically acceptable carrier and a peptide stabilizing excipient.

[0014] Another embodiment of the invention is an expression vector encoding a repressive peptide that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein. A related embodiment is a kit comprising a peptide or an expression vector encoding such a peptide that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein (SEQ ID NO: 1). Embodiments of the invention also include methods for preparing the peptides disclosed herein by chemical synthesis or recombinant generation. Another embodiment of the invention is a complex comprising α-synuclein and a peptide that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein.

[0015] Another embodiment of the invention is a method for reducing or inhibiting α-synuclein (SEQ ID NO:1) aggregation, comprising contacting α-synuclein amyloid fibrils with an inhibitory peptide disclosed herein in an amount sufficient to reduce or inhibit α-synuclein aggregation. Optionally, in this method, the α-synuclein amyloid fibrils are in an in vivo environment. Alternatively, in this method, the α-synuclein amyloid fibrils are in an in vitro environment. A related embodiment of the invention is a method for regulating the size or growth rate of α-synuclein amyloid fibrils, comprising contacting the fibrils with an amount of at least one inhibitory peptide in an environment that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein (SEQ ID NO:1), wherein the inhibitory peptide contacts residues 68-78 of α-synuclein in such an environment that the contacted α-synuclein amyloid fibrils exhibit a regulated size or growth rate.

[0016] Another embodiment of the invention is a method for observing the presence or absence of α-synuclein amyloid fibrils in a biological sample, comprising combining the biological sample with a peptide that binds to residues 68-78 of α-synuclein, such that the peptide binds to α-synuclein amyloid fibrils that may be present in the biological sample, and then monitoring the presence of a complex formed between the α-synuclein amyloid fibrils and the peptide; wherein the presence of the complex indicates the presence of α-synuclein amyloid fibrils in the biological sample. In this embodiment, one of our inhibitors can be combined with imaging agents such as radiolabeled, radiopaque, fluorescent dyes, fluorescent proteins, colorimetric labels, etc. (e.g., thereby facilitating imaging methods such as MRI or PET), and our inhibitor will bind to α-synuclein fibrils in the patient's brain, thereby allowing imaging of the fibrils for diagnosis of subsequent disease progression.

[0017] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. However, it should be understood that while the detailed description and specific examples illustrate some embodiments of the invention, they are given by way of illustration rather than limitation. Many changes and modifications can be made within the scope of the invention without departing from its spirit, and the invention encompasses all such modifications. Attached Figure Description

[0018] The patent or application document contains at least one color drawing.

[0019] Figure 1 Design of α-syn inoculation inhibitors. (A) Structure-based design of α-syn aggregation inhibitors. The NACore structure consists of two self-complementary β-sheets forming a stereo zipper. Three types of inhibitors (brownish-red, orange, and cyan) binding to one or both ends were identified. (B) Binding energies of the different inhibitors calculated by Rossetta show that S37 and S62 bind to both interfaces, while S71 was expected to bind to only one interface. All three inhibitors exhibited high shape complementarity. (C, D, E) Thiosyl T assays measuring α-syn aggregation and the effects of the three inhibitors. 50 μM of α-syn and a 5-fold molar excess of the inhibitor were added.

[0020] Figure 2α-syn aggregates formed in the presence of inhibitors are not capable of inoculation. (A) Experimental design for cell culture inoculation assay. α-syn aggregates in the presence of inhibitors, and the mixture is transfected into HEK293 cells expressing YFP-labeled WT α-syn or A53T α-syn (green). A53T is a family variant of α-synuclein that causes early-onset Parkinson's disease. After transfection, endogenous α-syn forms fluorescent spots (red). (B) NACore does not affect the inoculation ability of α-syn fibrils. (C, D, E) As measured by counting the total number of particles formed per well in cells expressing WT and A53T, 50 μM α-syn aggregates in 10, 5, 2, and 1-fold excesses of S37, S61, and S62 are not capable of inoculation. (F) As measured by counting the total number of granules formed per well in cells expressing WT and A53T, 50 μM α-syn accumulated in excess S71 was not capable of inoculation. Results are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA.

[0021] Figure 3 Inhibitors prevent seeding in cell culture. (A) Experimental design for cell culture seeding assay. 125 nM recombinant α-syn fibrils were transfected with different amounts of inhibitors, and aggregation was monitored over time. (B) NACore does not affect seeding capacity. (C, D, E, F) S37, S61, S62, and S71 reduce the seeding capacity of α-syn. All data are reported as particle counts per well and normalized to particle counts in buffer-treated wells. Results are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA.

[0022] Figure 4 : Filaments extracted from PD brain tissue were seeded with α-syn to aggregate in vitro and in cell culture. (A) Protocol used to extract sarcosyl-insoluble protein filaments from PD brain tissue. (B, C, D) 2% seed from 3 different subjects induced rapid α-syn aggregation, with a 4-10 fold increase in ThT fluorescence. (E, F, G) α-syn seeded with filaments from PD subjects induced more particles than α-syn alone. All data are reported as particle counts per well and normalized to particle counts in buffer-treated wells. Results are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA.

[0023] Figure 5The inhibitors prevented filament seeding from PD tissue extracts. Filament seeding α-syn aggregation was tested from four different subjects and monitored by ThT assay. S61 and S62 were effective for all tested seeds. S71 was effective for seeds B and D.

[0024] Figure 6 α-syn fibrils formed in the presence of PD filament seeds and inhibitors were not capable of inoculation. (A) Experimental design of cell culture inoculation assays (B, C, D) tested the inhibitory effects of the inhibitors on inoculation of three different PD brain extracts. S71 was effective for all three seeds, while S61 was effective for seed B. All data are reported as particle counts per well, normalized to particle counts in buffer-treated wells. Results are presented as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA.

[0025] Figure 7 Inhibitors reduced the seeding of α-syn fibrils from PD-associated filaments in cell culture. (A) Experimental design for cell culture seeding assay. (B, C, D) α-syn fibrils formed in the presence of two different PD-associated filaments were transfected into YFP-α-syn HEK cells, and fluorescence was measured for 6 days thereafter. S37 prevented seeding at concentrations ranging from 12.5 μM to 1.25 μM. S61 prevented seeding, with a significant effect on day 2. S71 reduced seeding at a concentration of 6.25 μM. All data are reported as particle counts per well, normalized to particle counts on day 0 before transfection. Results are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA. Detailed Implementation

[0026] Unless otherwise defined, all specialized terms, symbols, and other scientific terms or expressions used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, for clarity and / or convenience of reference, terms with their commonly understood meanings are defined herein, and the inclusion of these definitions herein should not be construed as indicating a material difference from those commonly understood in the art. Many techniques and procedures described or referenced herein are well understood by one of ordinary skill in the art and can generally be practiced using conventional methods. All publications, patents, and patent applications referenced herein are incorporated herein in their entirety by reference for all purposes. In the description of preferred embodiments, reference may be made to the accompanying drawings, which form a part thereof, and specific embodiments in which the invention can be practiced are illustrated by way of illustration. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention.

[0027] Alpha-synuclein (a 140-amino acid protein) is found in amyloid deposits in neurons during disease states. The causal link between α-synuclein amyloid formation and disease progression is supported by the following findings: gene duplication and familial mutations that increase amyloid load also cause early-onset PD, and over 90% of patients with sporadic PD are positive for α-synuclein deposit staining. The invention disclosed herein provides a structure-based approach to halt α-synuclein aggregation. We hypothesize that the atomic structures of NACore and PreNAC are preserved within the α-synuclein amyloid seed and recruit endogenous proteins into a zipper conformation. Using the atomic structure of NACore, we developed inhibitors that inhibit fibril formation and tested their efficacy in vitro and in cell culture. The same procedure can be used to develop PreNAC-based inhibitors. The inhibitors are optimized to cap the ends of fibrils, preventing further monomer addition. We used Rosetta software to design peptide sequences that preferentially interact with the NACore fragment. Rosetta used energy functions that considered electrostatic interactions, hydrogen bonds, and van der Waals forces to assess binding energies. Once a specific residue was confirmed to bind favorably at a particular position on the designed peptide, it was immobilized and prevented from further design, while the rest of the sequence was further refined. We repeated this immobilization and redesign process until an optimal set of sequences was determined. This rational design process allowed for the computational sampling of inhibitor sequences on an order of magnitude greater than experimentally feasible.

[0028] We screened crystals of peptide segments within the NAC domain and adjacent regions to seek structural information regarding the molecular basis of α-syn aggregation and toxicity. The ZipperDB algorithm was used. 55 We identified crystallizable amyloid-forming segments within the NAC domain. Specifically, we identified and focused on the central segment of α-synuclein, residues 68-78, referred to herein as NACore due to its crucial role in α-synuclein aggregation and cytotoxicity. NACore is the protofilament-forming core of the 35-residue NAC (non-amyloid-β component) domain (residues 61-95) of the complete 140-residue α-synuclein. In some studies, we utilized subfractions of NACore consisting of residues 69-77. In other studies, we crystallized and determined the structure of residues 47-56 of the α-synuclein, termed PreNAC, which forms another 3D zipper. The identified segments were chemically synthesized and crystallized, and their three-dimensional structures were determined by microcrystallization and microelectron diffraction (MicroED).

[0029] To find the most effective inhibitor, nearly 100 different designs were tested empirically, and the inhibitor design was continuously optimized over multiple rounds. For example, we observed that the position of the tag on the N or C end affects its efficacy. Additionally, the type of modification added also affects efficacy. In our case, only Trp substitution at Thr72 was effective, while Arg substitution was ineffective. Although the computational method is insufficient to identify a successful design, it can narrow down our search for candidate inhibitors, which can then be optimized through a reasonable design.

[0030] The efficacy of the capping inhibitor in preventing inoculation was tested using a cell-based assay. In this system, transfection with nanomolar amounts of α-syn seeds induced aggregation of endogenous proteins. The aggregates exhibited amyloid-forming properties—binding to small, amyloid-specific molecules, precise translocation during cell division, and significant specificity (Sanders DW et al. (2014) Neuron 82(6):1271–1288). For example, α-syn fibrils can only inoculate α-syn proteins into the aggregates. Notably, no acute cell death was observed during aggregate formation in this system; only a slight slowing of cell proliferation was observed. Our inhibitor prevented spot formation in this system, and a single application of the inhibitor was effective for 2–3 days.

[0031] In the case of amyloid diseases, inoculation is the transfer of pathological protein aggregates along the connected tissue sequence. This process contributes to the progression and severity of neurodegenerative diseases. To date, in part due to a lack of information on the structural characteristics of pathological seeds, there are no therapeutic agents specifically targeting inoculation. This application relates to, for example, the design, synthesis, and functional characterization of peptides that specifically bind to α-synuclein (α-syn) aggregates and block, inhibit, and / or reduce α-syn aggregation and / or α-syn cytotoxicity. Peptide inhibitors specifically “cap” the growth aggregates of α-synuclein. In some embodiments, the peptides are fused with cell-penetrating peptides that enhance their delivery into cells.

[0032] In addition to the spontaneous aggregation of intracellular α-syn into amyloid fibrils, a second phenomenon contributing to disease progression is the prion-like spread of α-syn aggregates (Goedert M (2015). Science 349(6248):1255555-1–1255555-9). Braak staging has shown that the pathology spreads gradually over time through connected brain regions, and cell culture and animal models have shown that small amounts of α-syn aggregates can act as seeds and induce native protein aggregation (see, for example, Braak H et al. (2003) Neurobiol Aging 24(2):197–211; Braak et al. (2009) Adv Anat EmbryolCell Biol 201:1–119; Masuda-Suzukake M et al. (2013) Brain 136(4):1128–1138; Desplats P et al. (2009) Proc Natl Acad Sci USA 106(31):13010–13015; Luk KC et al. (2009) Proc Natl Acad Sci 106(47):20051–20056). Although different from typical prions that can spread from person to person, this 'inoculation' phenomenon appears to be a driving factor in disease progression.

[0033] We observed that the inoculation capacity of fibrils extracted from patients varied and exhibited characteristics of similar strains. In vitro, fibrils extracted from PD patients caused a significant increase in α-syn aggregation, and in cell culture models, inoculated samples showed increased spot formation. Notably, unlike previous reports of α-syn filament seeding in cell culture from patients, we did not observe inoculation in cell culture in our assays (Prusiner SB et al. (2015) Proc Natl Acad Sci 112(38):E5308–E5317; and Woerman AL et al. (2015) Proc Natl Acad Sci 112(35):E4949–E4958). Previous reports utilized substantia nigra tissue, while we used frontal and temporal lobe tissues, where inoculation efficacy may differ. In fact, previous reports have confirmed that the inoculation ability of protofibrils extracted from different brain regions is similar to that of different strains (Prusiner SB et al. (2015) Proc Natl Acad Sci 112(38):E5308–E5317). Furthermore, different inhibitors have different efficacies on different seeds. For example, S61 is effective only on seeds A and B, while S71 is effective on seeds B and D. Recently, the NMR structure of a full-length α-syn protofibril was reported, in which the NACore segment was not found in the extended zipper conformation, but segments 68–78 were found in the core of the protofibril (Tuttle MD et al. (2016) Nat Struct Mol Biol 23(5):409–415). Alternatively, the inhibitors described therein may bind to the NACore conformation in the NMR structure. It is also conceivable that the NMR structure and the stereo zipper structure are different polymorphs. In the absence of diagnostic methods for different polymorphs in human subjects, it is theoretically possible to use a mixture of different inhibitors that target different polymorphs.

[0034] We used a combination of computational methods and rational design to develop a series of inhibitors that target and prevent the spread of α-synuclein aggregates. Our approach was made possible only by determining the atomic structure of the α-synuclein amyloid fibrillary nucleus, which can then be used to inoculate other diseases that play a role in disease progression. The inhibitors prevent α-synuclein aggregation in in vitro and cell culture models. The inhibitors also showed efficacy in preventing patient-derived α-synuclein fibrillary inoculation in in vitro and cell culture models. Our results provide evidence that the pathological seeding of α-synuclein contains a stereozipper and propose a therapeutic approach targeting its spread and progression applicable to PD and related synucleinopathies. Similarly, our inhibitors could be applied to diagnostic methods for PD and related synucleinopathies.

[0035] We hypothesize that mutations, overexpression, or other cytokines can destabilize the structure of native α-synuclein, exposing a sticky, three-dimensional zipper-like segment that has been proposed as a fundamental building block of amyloid aggregates. 18, 19 Therefore, we generated a high-resolution view of the amyloid spikes of α-synuclein aggregates. We then applied a Rosetta-based method. 36 The α-synuclein 69-77 or α-synuclein 68-78 structures are used as templates to design specific "terminal capping" α-synuclein growth aggregates, thereby disrupting and inhibiting the further aggregation of α-synuclein.

[0036] We used the atomic structure of NACore [68-GAVVTGVTAVA-78] (SEQ ID NO: 46) as a template and designed peptide inhibitors using computational and structure-based methods. The atomic structure of NACore revealed a pair of self-complementary β-sheets forming a stereozipper (Sawaya MR et al. (2007) Nature 447(7143):453–457). Inhibitors were predicted using a Rossetta-based computational model to bind to the stereozipper interface and 'end-cap' the protofibrils. We identified three candidate inhibitors; S37, S61, and S71 (which bind favorably to one or both ends of the zipper) Figure 1 The binding energy and shape complementarity of the three inhibitors are also advantageous. Figure 1 B). All inhibitors retain most of the residues of the native NACore sequence but contain one or more modified residues. Rodriguez et al. showed that the smaller 9-residue segment in NACore [69-AVVTGVTAV-77] (SEQ ID NO: 48) aggregates more slowly than NACore and has a similar structure. Rodriguez et al. (2015) also described a second segment of a-syn with residues 47-56 of the sequence GVVHGVTTV, termed PreNAC. Inhibitors designed with this sequence may also inhibit fibrillation of a-syn.

[0037] To prevent self-aggregation of our designed inhibitors, we used shorter segments along with one or more modifications. S37 has a W mutation at Thr72 and an additional polylysine tag at the C-terminus to induce charge-charge repulsion. It is expected to bind to both tips of the stereozipper fibrils. S61 and S62 retain the same inhibitor sequence as S37, but instead of the polylysine tag, a TAT tag is added to aid dissolution and prevent self-aggregation. S71 has a methylated glycine at Gly73 that weakens the hydrogen bonds along the β-sheet and an additional TAT tag for dissolution and cell penetration.

[0038] We tested the efficacy of the inhibitors in an in vitro aggregation assay. Recombinant purified α-syn aggregated in the presence of the inhibitors, and the aggregation was monitored by measuring the fluorescence of the amyloid-binding dye thiosulfate T. All three inhibitors prevented aggregation, with a significant reduction in ThT fluorescence. Figure 1 C, 1D, 1E).

[0039] We tested the efficacy of the inhibitor in preventing aggregation in a cell culture model. For these assays, we used two types of HEK293T cells stably expressing YFP-tagged full-length WT α-syn and A53T α-syn (Sanders DW et al. (2014) Neuron 82(6):1271–1288). In this model, lipofectamine-mediated transfection of recombinant fibrils resulted in the aggregation of endogenous YFP-tagged proteins, which were observed as fluorescent spots. Furthermore, the size and number of these spots increased over time. This aggregation over time indicates a 'seedling' phenomenon, whereby a small amount of amyloid fibrils induces the aggregation of endogenous proteins. First, we tested the parent peptide segment NACore to examine its effect on seeding. α-syn aggregated in the presence of a molar excess of NACore ( Figure 2 B). The mixture was transfected into cells and spot formation was visualized, with the number of spots counted as particles per well. As expected, NACore did not result in a significant reduction in spot formation in either cell line. Next, we allowed 50 μM α-syn to accumulate in the presence of 500 μM, 250 μM, 100 μM, and 50 μM inhibitors corresponding to 10, 5, 2, and 1-fold overdoses, respectively. The mixture was then transfected into cells, and accumulation was monitored over time for up to 3 days by fluorescence imaging. Figure 2 A). In WT and A53T expressing cell lines, S37 resulted in a significant reduction in seeding time for up to 2 days. Similar to S37, S61 ( Figure 2 D) also led to reduced spot formation, with the greatest efficacy in both cell lines at 2, 5, and 10-fold overdoses. Aggregates formed in the presence of S62 (… Figure 2 E) also lacked inoculation ability, forming significantly fewer particles at all inhibitor concentrations. S71, tested at equimolar and substoichiometric ratios, was found to reduce the inoculation efficacy of aggregates. Figure 2 F). These results indicate that the inhibitors prevent the formation of inoculated aggregates.

[0040] We tested the efficacy of the inhibitor in preventing inoculation in a cell culture model. Figure 3 A). We transfected α-synuclein fibrils together with different inhibitors. NACore, the parental aggregate peptide, did not affect the inoculation efficacy of α-synuclein fibrils ( Figure 3B). At concentrations of 12.5 μM and 6.25 μM, S37 significantly reduced seeding in both cell lines, lasting for up to 2 days. Figure 3 C). Concentrations of S61 at 2.5 μM and 1.25 μM resulted in reduced inoculation duration, lasting up to 6 days in WT α-syn cells and 2 days in A53T α-syn HEK cells. Figure 3 D). In both cell lines, S62 was effective at concentrations of 12.5 μM and 6.25 μM. Figure 3 E). S71 is effective at a low concentration of 1.25 μM ( Figure 3 F). Interestingly, we observed that adding higher concentrations of these inhibitors did not prevent inoculation, suggesting that the critical concentration range has maximum efficacy. Together, these results indicate that the inhibitors can cap protofibrillary seeds and prevent their elongation.

[0041] We extracted insoluble protein aggregates from frozen autopsy PD brain tissue. We obtained tissue from four different subjects, including the substantia nigra and frontal region from one subject and the temporal and frontal regions from other subjects. Using a previously described protocol (Goedert et al. (1992) Neuron 8(1):159–168), including precipitation with the ionic detergent sarcosyl, we extracted the insoluble protein aggregates (…). Figure 4 A). All samples were robustly seeded with α-syn aggregates in vitro and in our cell culture model. In vitro addition of 2% seed increased ThT fluorescence by 4 to 10 times. Figure 4 (B, 4C, 4D) and lag time were slightly reduced. The inoculated samples were then transfected into HEK cells. Consistent with ThT assays, all inoculated samples induced rapid spot formation ( Figure 4 (E, 4F, 4G). Therefore, protofibrils extracted from PD brain tissue were inoculated with recombinant proteins, and the aggregates formed upon inoculation induced rapid spot formation in cell cultures.

[0042] We tested the effects of different inhibitors on preventing α-syn aggregation in the presence of PD-extracted seeds. As measured by ThT fluorescence assay, S71 and S62 showed the most effective efficacy against all seeds. Figure 5 S61 also reduced the aggregation of two different seeds. Figure 5 B, 5D), while S37 showed a marginal decrease in ThT fluorescence (B ...). Figure 5 D). Next, we tested the inoculation efficacy of α-syn aggregates formed in the presence of PD seeds and different inhibitors (D). Figure 6 A). Aggregates formed in the presence of S71 ( Figure 6B, 6C, and 6D) did not induce spot formation in HEK cells expressing WT and A53T. S61 also showed efficacy ( Figure 6 C). Consistent with in vitro assays, S37 was ineffective in reducing the inoculation efficacy of aggregates. These results together indicate that S62 and S71 can prevent the formation of inoculated fibrils.

[0043] We tested the efficacy of inhibitors in preventing PD fibrillation seeding in cell culture. α-syn aggregates formed in the presence of PD fibrillation were transfected into WT HEK cells along with different inhibitors. Figure 7 A). S37 prevented spot formation in two different PD filaments at concentrations of 12.5 μM–1.25 μM for up to 2 days. Figure 7 B). Similar to S37, S61 also showed efficacy for up to 2 days at concentrations of 12.5 μM–1.25 μM, and S71 also prevented seeding at similar concentrations. These results indicate that the inhibitors robustly prevent seeding in cell culture.

[0044] The above disclosure describes work relating to many embodiments of the present invention. The invention disclosed herein provides such inhibitory peptides; pharmaceutical compositions comprising the inhibitory peptide of the present invention and a pharmaceutically acceptable carrier; methods for blocking, inhibiting, and / or preventing α-synuclein aggregation and / or α-synuclein cytotoxicity using the inhibitory peptide, the method comprising contacting an α-synuclein molecule (e.g., monomer, small aggregate, oligomer, or fibril) with an effective amount of the peptide inhibitor of the present invention, or administering an effective amount of the peptide inhibitor of the present invention to a subject; and computer-related embodiments, such as methods for designing and obtaining inhibitory peptides or small molecules based on the structures described herein.

[0045] The advantages of the inhibitory peptides of the present invention include: (1) the synthetic peptides are not expensive. (2) due to their composition and small size, cell penetration and protein stability are not challenging. In addition, the peptides can be fused with cell-penetrating peptides, which enhance their delivery into cells. (3) they are unexpectedly stable: they do not proteolytically degrade and exhibit a sufficiently long half-life to function in vivo (e.g., within the body). (4) peptide inhibitors are specific to their targets, thus providing fewer side effects compared to, for example, small molecules that may bind to many targets.

[0046] The invention disclosed herein has many embodiments. One embodiment of the invention is a composition comprising at least one inhibitory peptide that inhibits the aggregation of α-synuclein (SEQ ID NO: 1) by binding to residues 68-78 of α-synuclein. Working embodiments of these peptides, as disclosed in the examples below, include S37, S62, and S71, as shown in Table 1 below:

[0047]

[0048] In the table, N-me-Gly represents glycine with a methylated amino group. Inhibitor sequences are shown in bold. Non-bold elements indicate tags, dissolution tags, or adapters.

[0049] In a typical embodiment of the invention, the repressive peptide comprises the sequence GAVVWGVTAVKK (SEQ ID NO:3) or RAVVTGVTAVAE (SEQ ID NO:4). Optionally, the repressive peptide comprises the sequence GAVVWGVTAVKKKKK (SEQ ID NO:5), GAVVWGVTAVKKGRKKRRQRRRPQ (SEQ ID NO:6), or YGRKKRRQRRRAVVTGVTAVAE (SEQ ID NO:7). In some embodiments of the invention, the composition comprises multiple repressive peptides. Typically, the repressive peptide is 6 to 30 amino acids in length. Active variants of any of these repressive peptides are also included. Repressive peptides having the foregoing sequences, including active variants, are sometimes referred to herein as "the repressive peptide of the invention".

[0050] In the inhibitory peptide compositions of the present invention, at least one amino acid in the inhibitory peptide comprises a non-naturally occurring amino acid (e.g., a D-amino acid or an amino acid containing an N-methyl moiety); and / or the inhibitory peptide is coupled to a heteropeptide tag. Such heteropeptide tags include amino acid sequences that increase peptide solubility in vivo or in vitro (e.g., multiple arginine residues); or amino acid sequences that facilitate the monitoring or manipulation of the peptide in vivo or in vitro (multiple lysine or histidine amino acids); or amino acid sequences that facilitate peptide entry into mammalian cells (e.g., cell-penetrating peptide sequences). Another aspect of the invention is a pharmaceutical composition comprising the inhibitory peptide of the present invention and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are sometimes referred to herein as “pharmaceutical compositions of the present invention.” Optionally, the peptide compositions disclosed herein comprise a pharmaceutically acceptable carrier and a peptide stabilizing excipient.

[0051] Another embodiment of the invention is an expression vector encoding a repressive peptide that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein. Optionally, the expression vector is a vector (such as a lentivirus) for delivering the polypeptide to mammalian cells. In this regard, another embodiment of the invention is a method for delivering DNA encoding a repressive peptide that inhibits α-synuclein aggregation in mammalian cells (e.g., in vitro or in vivo cells) by contacting the mammalian cells with a vector transducing the cells, thereby expressing the DNA within the cells.

[0052] Another embodiment is a kit comprising a peptide or expression vector encoding α-synuclein (SEQ ID NO: 1) that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein. Embodiments of the invention also include methods for preparing the peptides disclosed herein by chemical synthesis or recombinant generation. Another embodiment of the invention comprises an α-synuclein and a peptide that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein.

[0053] Another implementation is a peptide designed on the PreNAC structure (residues 47-56 of α-synuclein: GVVHGVTTVA) to inhibit the formation of α-synuclein fibrils.

[0054] Another embodiment of the invention is a method for reducing or inhibiting α-synuclein (SEQ ID NO:1) aggregation, comprising contacting α-synuclein amyloid fibrils with an inhibitory peptide disclosed herein in an amount sufficient to reduce or inhibit α-synuclein aggregation. Optionally, in this method, the α-synuclein amyloid fibrils are in an in vivo environment. Alternatively, in this method, the α-synuclein amyloid fibrils are in an in vitro environment. A related embodiment of the invention is a method for regulating the size or growth rate of α-synuclein amyloid fibrils, comprising contacting the fibrils with an amount of at least one inhibitory peptide in an environment that inhibits α-synuclein aggregation by binding to residues 68-78 of α-synuclein (SEQ ID NO:1), wherein the inhibitory peptide contacts residues 68-78 of α-synuclein in such an environment that the contacted α-synuclein amyloid fibrils exhibit a regulated size or growth rate.

[0055] The peptide inhibitors disclosed herein can be used as a therapy to halt the spread of Parkinson's disease in the brain. Alternatively, the peptide inhibitors disclosed herein can be used as diagnostic probes to identify pathological aggregation seeds of the protein α-synuclein in diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. In this regard, embodiments of the invention include a method for observing aggregation seeds of the protein α-synuclein in a biological sample, the method comprising contacting the biological sample with a peptide that binds to residues 68-78 of α-synuclein, and then observing whether the peptide, if present in the biological sample, binds to the aggregation seed of the protein α-synuclein. Typically in these methods, a heterologous peptide tag is coupled to an inhibitory peptide to facilitate observation of the peptide. Optionally, in these methods, the biological sample is derived from an individual suspected of having Parkinson's disease, Lewy body dementia, or multiple system atrophy.

[0056] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. For example, in the foregoing case, the pharmaceutical composition may comprise one or more inhibitory peptide molecules of the present invention, which may be the same or different.

[0057] Another aspect of the invention is a complex comprising an α-synuclein molecule (e.g., α-synuclein monomer, small aggregate, oligomer, or fibril) and the repressive peptide of the invention. These molecules can be bound to, coupled to, or otherwise associated with each other. The α-synuclein and the repressive peptide can be covalently or non-covalently linked.

[0058] Other aspects of the invention include a polynucleotide encoding the inhibitory peptide of the invention; an expression vector containing the polynucleotide; cells transfected with the polynucleotide or the expression vector; and a method of preparing the peptide, comprising expressing the peptide in the transfected cells, culturing the cells, and harvesting the resulting peptide.

[0059] Another aspect of the invention is a method for inhibiting (preventing, terminating) the aggregation of α-synuclein molecules (e.g., monomers, small aggregates, oligomers, or fibrils of α-synuclein), comprising contacting α-synuclein molecules with an effective amount of the inhibitory peptide or pharmaceutical composition of the invention. The α-synuclein molecules may be in solution or in cells, with the cells in a culture or in a subject. In one embodiment, contacting α-synuclein molecules as monomers, oligomers, or small aggregates prevents the aggregation of α-synuclein molecules (oligomerization, further oligomerization, and / or fibril formation). In another embodiment, contacting aggregated forms of α-synuclein or fibrils prevents further aggregation of the aggregated forms or fibrils (fibrilization).

[0060] In one embodiment of this method, the α-synuclein protein molecules are contacted in subjects suffering from diseases or conditions mediated by the presence of fibrillated α-synuclein (sometimes referred to herein as α-synuclein-mediated diseases or conditions, or synucleinopathy), such as Parkinson's disease (PD), Lewy body dementia, and multiple system atrophy. α-synuclein pathology is also seen in other related neurodegenerative diseases, such as sporadic and familial Alzheimer's disease.

[0061] Another aspect of the invention is a method for treating a subject suffering from an α-synuclein-mediated disease or condition, such as Parkinson's disease, Lewy body dementia, or multiple system atrophy, comprising administering to the subject an effective amount of the inhibitory peptide or pharmaceutical composition of the invention. This treatment can result in the blocking (prevention) or inhibition of α-synuclein aggregation and / or α-synuclein cytotoxicity and pathological spread (inoculation) in the subject.

[0062] Another aspect of the invention is a computer-implemented method for identifying peptides that inhibit α-synuclein aggregation and / or α-synuclein cytotoxicity as described herein. Another aspect of the invention is a kit comprising the inhibitory peptide of the invention, optionally packaged in a container. Another aspect of the invention is a method for preparing the inhibitory peptide of the invention, comprising chemically synthesizing the inhibitory peptide or recombinantly generating the inhibitory peptide.

[0063] Another embodiment of the invention is a method for observing the presence or absence of α-synuclein amyloid fibrils in a biological sample, comprising combining the biological sample with a peptide that binds to residues 68-78 of α-synuclein, such that the peptide binds to α-synuclein amyloid fibrils that may be present in the biological sample, and then monitoring the presence of a complex formed between the α-synuclein amyloid fibrils and the peptide; wherein the presence of the complex indicates the presence of α-synuclein amyloid fibrils in the biological sample. Optionally, in this method, the presence of the complex formed between the α-synuclein amyloid fibrils and the peptide is monitored using a detectable tag (e.g., a heteropeptide tag) conjugated to the peptide. In an illustrative embodiment of the invention, the peptide comprises the sequence GAVVWGVTAVKK (SEQ ID NO: 3) or RAVVTGVTAVAE (SEQ ID NO: 4). Typically, this method is performed on biological samples obtained from individuals suspected of having Parkinson's disease. Such embodiments of the present invention can be used, for example, in diagnostic methods designed to observe the presence or status of PD, such as for detecting diseases that begin before clinical symptoms and for tracking the effectiveness (or lack thereof) of therapeutic treatments.

[0064] The peptide inhibitors of this invention specifically (selectively, preferentially) bind to α-synuclein rather than unintended proteins. The proteins bound by the peptide inhibitors can be, for example, monomers, small aggregates, oligomers, or fibrils. For example, the binding can be 2-fold, 5-fold, 10-fold, 100-fold, or 200-fold stronger, or binding to unintended targets may be completely undetectable. Binding specificity can be determined using conventional methods, such as competitive binding assays or other suitable analytical methods.

[0065] This also includes active variants of the aforementioned repressive peptides. An "active variant" is a variant that retains at least one property of the repressive peptide described herein (e.g., the ability to bind to α-synuclein and / or block, inhibit, or prevent α-synuclein fibrillation (aggregation) and / or α-synuclein cytotoxicity). As used herein, fibrillation refers to the formation of filaments or fibrils, such as amyloid fibrils.

[0066] Suitable active variants include peptide-mimicking compounds (any compound containing non-peptide structural elements that is capable of mimicking the biochemical and / or biological effects of naturally occurring peptide-mimicking compounds, including, for example, those compounds designed to mimic the structure and / or binding activity (e.g., hydrogen bonding and hydrophobic packaging interactions) of said peptides according to the methods disclosed herein). The inhibitory peptides of the present invention, including their active variants, are sometimes referred to herein as “peptide compounds” or “compounds”.

[0067] In one embodiment, the active variant of the repressive peptide shortens the original repressive peptide by 1-3 (e.g., 1, 2, or 3) amino acids at the N-terminus, C-terminus, or both. In another embodiment, the active variant extends the original repressive peptide by 1, 2, 3, or 4 amino acids at the C-terminus, for example, at the positions where the amino acid residues appear in α-synuclein.

[0068] This includes various other types of active variants. In some embodiments, amino acids other than those described above are substituted. These amino acids can help protect the peptide inhibitor from proteolysis or otherwise stabilize the peptide, and / or otherwise promote desired pharmacodynamic properties. In some embodiments, non-natural amino acids allow the inhibitor to bind more tightly to the target because the side chain optimizes hydrogen bonding and / or its nonpolar interactions. Additionally, non-natural amino acids provide the opportunity to introduce detectable biomarkers, such as strongly fluorescent biomarkers, for example, to measure values ​​such as the inhibition constant. Peptide mimics are also included, such as peptide-like substances, β-amino acids, N-ethylated amino acids, and small molecule mimics.

[0069] In one embodiment, a non-natural amino acid replaces an amino acid in the sequence. More than 100 non-natural amino acids are commercially available. These include, for example,

[0070]

[0071] In another embodiment, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) L-amino acids are replaced with D-amino acids.

[0072] In another embodiment, the peptide includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) N-methylated residues.

[0073] The inhibitory peptides of the present invention may comprise, for example, L-amino acids, D-amino acids, non-natural amino acids, or combinations thereof.

[0074] Active variants include molecules containing various tags at the N-terminus or C-terminus of the peptide. For example, the repressive peptide of the present invention may contain, as tags, the following at its N-terminus and / or its C-terminus: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more lysine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more arginine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more glutamate residues; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more aspartic acid residues; combinations of these amino acid residues; or other polar tags obvious to those skilled in the art. Other active variants include mutations in the α-synuclein sequence that increase the affinity of the repressive peptide for α-synuclein.

[0075] In one implementation, the inhibitor is provided by the method described by Jiang et al. 63 (This is incorporated herein by reference, particularly regarding the method), small molecules designed using the atomic structure of the fibrous-forming segment of α-synuclein described herein as a basis for designing inhibitors. Suitable small molecules that can be identified by this method of Jiang et al. will be readily apparent to those skilled in the art.

[0076] In one embodiment of the invention, the peptide of the invention is modified such that one, two, or three amino acids are replaced by amino acids having non-naturally occurring side chains, such as the non-natural amino acids discussed above, or by amino acids having side chains produced by Jiang et al. 63 The amino acid substitutions of the side chains are designed with small molecule crosslinks (e.g., via the ε-amino group of Lys residues). Some representative fibronectin-binding molecules are shown below. These active variants not only cap the growth aggregates of α-synuclein but also enhance the inhibitory activity of the peptide by binding (clamping) the sides of the stereozipper through the modified side chains.

[0077] By Jiang et al. 63 The designed fiber-bonded compounds include:

[0078]

[0079]

[0080] In one embodiment of the invention, the inhibitory peptide of the invention is isolated or purified using conventional techniques such as those described herein. “Isolation” means separation from components that normally associate with it, such as… ， The components present after peptide synthesis are separated. The separated peptide can be a protein cleavage product containing the peptide sequence. The purity of the "purified" repressed peptide can be, for example, higher than 90%, 95%, 98%, or 99%.

[0081] In one embodiment, to enhance the cell permeability of the inhibitory peptide of the present invention, the peptide is fused with any one of a variety of cell-penetrating peptides (CPPs). CPPs generally have the following amino acid composition, which contains a high relative abundance of positively charged amino acids such as lysine or arginine, or has a sequence with an alternating pattern of polar / charged amino acids and non-polar hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are hydrophobic peptides containing only non-polar residues with low net charge, or having hydrophobic amino acid groups that are critical for cellular uptake. Some typical CPPs that can be fused with the inhibitory peptides of the present invention are provided in Table 2.

[0082] Table 2

[0083] ● Name Sequence

[0084] Reference – Original or Review

[0085] ● Poly-ARG nR where 4<n<17 (e.g., n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) (SEQ ID NO:8)

[0086] Wender, P.A., Mitchell, D.J., Pattabiraman, K., Pelkey, E.T., Steinman, L. and Rothbard, J.B. (2000). The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U. S. A. 97, 13003-8.

[0087] ● Poly-LYS nK where 4<K<17 (e.g., K=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)

[0088] ● D-Poly-ARG nR where 4<n<17 (e.g., n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)

[0089] ● D-Poly-LYS nK where 4<K<17 (e.g., K=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)

[0090] ● SynB1 RGGRLSYSRRRFSTSTGR (SEQ ID NO:9)

[0091] ● SynB3 RRLSYSRRRF (SEQ ID NO:10)

[0092] ● Documentation RQIKIWFQNRRMKWKK (SEQ ID NO:11)

[0093] Derossi , D. , Joliot , AH , Chassaing , G. and Prochiantz , A. (1994). The third helix of the antennapedia homeodomain translocates through biological memory branes. J. Biol. Chem. Rev. 269, 10444–50.

[0094] ● PenArg RQNRRMRWRR (SEQ ID NO:12)

[0095] ● PenLight KQIKIWFQNKKMKWKK (SEQ ID NO:13)

[0096] ● TatP59W GRKKRRQRRRPWQ (SEQ ID NO:14)

[0097] ● Tat (48-60) GRKKRRQRRRPPQ (SEQ ID NO:15)

[0098] Vives , E. , Brodin , P. and Lebleu , B. (1997). A truncated HIV-1 Tatprotein basic domain rapidly translocates through the plasma membrane andaccumulates in the cell nucleus. J. Biol. Chem. 272 , 16010–7

[0099] ● R9-Tat GRRRRRRRRRPPQ (SEQ ID NO:16)

[0100] Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245, 1–7.

[0101] ● Tat YGRKKRRQRRR (SEQ ID NO:17)

[0102] Vives, E., Brodin, P. and Lebleu, B. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-7.

[0103] ● D-Tat GRKKRRQRRRPPQ (SEQ ID NO:18)

[0104] Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245, 1–7.

[0105] ● BMV Gag(7-25) KMTRAQRRAAARRNRWTAR (SEQ ID NO:19)

[0106] Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245, 1–7.

[0107] ● FHVCoat(35-49) RRRRNRTRRNRRRVR (SEQ ID NO:20)

[0108] Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245, 1–7.

[0109] ● HTLV-II Rex(4-16) TRRQRTRRARRNR (SEQ ID NO:21)

[0110] Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245, 1–7.

[0111] ● P22 N-(14–30) NAKTRRHERRRKLAIER (SEQ ID NO:22)

[0112] ● pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO:23)

[0113] Elmquist, A., Lindgren, M., Bartfai, T. and Langel, Ü. (2001). VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions. Exp. Cell Res. 269, 237-44.

[0114] ● Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:24)

[0115] Pooga, M., Hällbrink, M., Zorko, M. and Langel, Ü. (1998). Cell penetration by transportan. FASEB J. 12, 67-77.

[0116] ● TP10 AGYLLGKINLKALAALAKKIL (SEQ ID NO:25)

[0117] Soomets, U., Lindgren, M., Gallet, X., Hällbrink, M., Elmquist, A., Balaspiri, L., Zorko, M., Pooga, M., Brasseur, R. and Langel, Ü. (2000). Deletion analogues of transportan. Biochim. Biophys. Acta 1467, 165-76.

[0118] ● PTD-4 PIRRRKKLRRLK (SEQ ID NO:26)

[0119] ● PTD-5 RRQRRTSKLMKR (SEQ ID NO:27)

[0120] ● Pep-1 ac-KETWWETWWTEWSQPKKKRKV-cya (SEQ ID NO:28)

[0121] ● Pep-2 ac-KETWFETWFTEWSQPKKKRKV-cya (SEQ ID NO:29)

[0122] Morris, MC, Chaloin, L, Choob, M, Archdeacon, J, Heitz, F and Divita, G (2004). Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression. Gene Ther 11: 757–764.

[0123] ● Pep-3 ac-KWFETWFTEWPKKRK-cya (SEQ ID NO:30);

[0124] Morris , MC , Gross , E , Aldrian-Herrada , G , Choob , M , Archdeacon , J ,Heitz , F Person(2007). A non-covalent peptide-based carrier for in vivo deliveryof DNA mimics. Nucleic Acids Res 35:e49.

[0125] ● EN(1–22) WITH MDAQTRRRERRAEKQAQWKAAN (SEQ ID NO:31)

[0126] ● B 21 N-(12–29) TAKTRYKARRAELIAERR (SEQ ID NO:32)

[0127] ● U2AF(142–153) SQMTRQARRLYV (SEQ ID NO:33)

[0128] ● PRP6(129–144) TRRNKRNRIQEQLNRK (SEQ ID NO:34)

[0129] ● MAP KLALKLALKLALALKLA (SEQ ID NO:35)

[0130] ● SBP MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO:36)

[0131] ● FBP GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:37)

[0132] ● MPG ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya (SEQ ID NO:38)

[0133] Morris, MC, Vidal, P, Chaloin, L, Heitz, F and Divita, G (1997). A new peptide vector for efficient delivery of oligonucleotides into mammaliancells. Nucleic Acids Res 25: 2730–2736.

[0134] ● MPG(ΔNLS) ac- GALFLGFLGAAGSTMGAWSQPKSKRKV-cya (SEQ ID NO:39)

[0135] ● REV(34-50) TRQARRNRRRRWRERQR (SEQ ID NO:40)

[0136] Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245, 1–7.

[0137] ● From Jiang et al., PNAS 2004, ACPP - lowercase indicates D-aa. The symbol "__" in some of these sequences indicates a position that can be inserted into any of a variety of protease cleavage sites recognized in the art:

[0138] EEEEEDDDDK_AXRRRRRRRRRXC (SEQ ID NO:41)

[0139] EEEEEDDDDK_ARRRRRRRRRRXC (SEQ ID NO:42)

[0140] EDDDDK_AXRRRRRRRRRXC (SEQ ID NO:43)

[0141] EEDDDDK_ARXRRXRRXRRXRRXC (SEQ ID NO:44)

[0142] DDDDDDK_ARRRRRRRRXC (SEQ ID NO:45)

[0143] In another implementation, CPP is poly(D-)1-16 ).

[0144] Typically, it is desirable for CPPs to be relatively short, for example, less than about 30 amino acids, so as to improve stability and pharmacodynamic properties once the molecule enters the cell.

[0145] In some embodiments, the CPP is directly linked (fused) to the peptide of the present invention. In other embodiments, it is desirable to use a linker to separate the highly charged CPP from the inhibitory peptide so that the inhibitor retains its activity. Any of a variety of linkers can be used. The size range of the linker can be, for example, 1 to 7 or even more amino acids (e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids). For example, the linker can be an N-terminal QVTNVG and a C-terminal QKTVEG or a truncated form thereof, the truncated form having 1, 2, 3, 4 or 5 consecutive amino acids at the N-terminus linked to the inhibitory peptide.

[0146] In embodiments of the invention, the inhibitory peptide is detectably labeled. Labeled peptides can be used, for example, to better understand the mechanism of action and / or cellular localization of the inhibitory peptide. Suitable labels that enable detection (e.g., provide a detectable signal, or can be detected) are conventional and well-known to those skilled in the art. Suitable detectable labels include, for example, radioactive agents, fluorescent labels, etc. Methods for attaching such labels to proteins, or assays for detecting their presence and / or amount, are conventional and well-known.

[0147] The repressive peptide of the present invention can be synthesized by any of a variety of methods recognized in the art (e.g., chemical synthesis or recombinant expression in a suitable host cell). To produce a sufficient quantity of the repressive peptide for use in the methods of the present invention, an practitioner can, for example, use conventional techniques to generate a nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, wherein the sequence is controlled by an expression control sequence such as a promoter or enhancer, which can then direct the synthesis of the peptide. For example, DNA can be synthesized de novo with suitable adapters at the ends to clone it into a vector; (b) the entire DNA sequence can be cloned into a vector; or (c) starting with overlapping oligonucleotides, ligating them using conventional PCR-based gene synthesis methods, and inserting the resulting DNA into a vector. Suitable expression vectors (e.g., plasmid vectors, viruses (including bacteriophages), vectors, artificial vectors, yeast vectors, eukaryotic vectors, etc.) will be apparent to those skilled in the art, as will methods for preparing vectors, targeting insert sequences, expressing proteins encoded by nucleic acids, and isolating or purifying the expressed proteins.

[0148] Another aspect of the invention is a pharmaceutical composition comprising one or more inhibitory peptides and a pharmaceutically acceptable carrier. Components of the pharmaceutical composition may be detectably labeled, for example, having radioactive or fluorescent labels or labels suitable for detection by positron emission tomography (PET) or magnetic resonance imaging (MRI). For example, the peptides of the invention may be coupled to detectable labels selected from radioactive labels, radiopaque labels, fluorescent dyes, fluorescent proteins, colorimetric labels, etc. In some embodiments, the inhibitory peptide is present in an effective amount to achieve the desired effect.

[0149] "Pharmaceutical acceptable" means that it can be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, including pharmaceutical compositions acceptable for veterinary and human use. For example, a "pharmaceutical acceptable salt" of a compound means a salt that is pharmaceutically acceptable as defined herein and has the desired pharmacological activity of the parent compound.

[0150] Another aspect of the invention is a polynucleotide encoding the repressive peptide of the invention. In embodiments of the invention, the polynucleotide is operatively linked to a regulatory control sequence (e.g., a promoter or enhancer) to facilitate the production of the encoded protein upon introduction (e.g., by transfection) into suitable cells. Other embodiments include cells comprising an expression vector; and methods for preparing the repressive peptide of the invention, comprising culturing cells and harvesting the resulting peptide.

[0151] As used throughout this application, “about” means 5% of the value plus or minus.

[0152] Another aspect of the invention is a kit for performing any of the methods described herein. The kit may contain an appropriate amount of the inhibitory peptide of the invention; reagents for generating said peptide; reagents for assaying its function or activity; and so on. The kit of the invention may include instructions for performing the method. Other optional elements of the kit of the invention include suitable buffer solutions, culture medium components, etc.; a computer or computer-readable medium providing a structural schematic diagram of the crystal structure described herein; a container; or packaging material. Reagents for performing suitable controls may also be included. The reagents in the kit may be in a container, wherein the reagents are stable, for example, in lyophilized form or a stable liquid. The reagents may also be in a single-use form, for example, administered to the subject in a single-reaction form.

[0153] The characterization of the candidate inhibitory peptides of the present invention can be performed by any of a variety of conventional methods. For example, the ability of the peptide to reduce or inhibit α-synuclein aggregation, cytotoxicity, or intercellular diffusion can be determined. Assays can be performed in vitro or in vivo. Suitable assays will be apparent to those skilled in the art; some suitable assays are described herein.

[0154] One aspect of the invention is a method for reducing or inhibiting α-synuclein aggregation, comprising contacting α-synuclein profilaments with an effective amount of one or more inhibitory peptides of the invention. Such methods can be carried out in solution or in cells (e.g., cultured cells or cells in a subject's body).

[0155] Another aspect of the invention is a method for treating a subject suffering from a disease or condition mediated by the presence of fibrillating α-synuclein (sometimes referred to herein as α-synuclein-mediated disease or condition), comprising administering to the subject an effective amount of the inhibitory peptide or pharmaceutical composition of the invention. Examples of such diseases or conditions include Parkinson's disease (PD), Lewy body dementia, or multiple system atrophy. Another aspect of the invention is a method for preventing the onset of such diseases or conditions (e.g., PD), or for treating a subject in the early stages of such diseases or conditions, or those developing such diseases or conditions, in order to prevent or inhibit the progression of the condition or disease.

[0156] The inhibitory peptides or pharmaceutical compositions of the present invention are sometimes referred to herein as "inhibitors".

[0157] The “effective amount” of the inhibitor of the present invention is the amount that can produce a measurable amount of the desired result, such as the amount that inhibits α-synuclein aggregation or cytotoxicity; for diagnostic assays, it is the amount that can detect the target, such as α-synuclein aggregates; or in therapeutic treatments, it is the amount that can reduce or improve the symptoms of the disease or symptom being treated in a measurable amount.

[0158] "Subject" can be any subject (patient) with aggregated (fibrillated) α-synuclein molecules associated with a symptom or disease that can be treated by the methods of the present invention. In one embodiment of the invention, the subject suffers from PD. Typical subjects include vertebrates, such as mammals, including laboratory animals, dogs, cats, non-human primates, and humans.

[0159] The inhibitors of the present invention can be formulated into various forms of pharmaceutical compositions suitable for a chosen route of administration, such as oral, nasal, intraperitoneal or parenteral, intravenous, intramuscular, local or subcutaneous, or by injection into tissues.

[0160] Suitable oral forms for administering inhibitors include lozenges, sugar tablets, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention time, oral patches, and sublingual tablets.

[0161] The inhibitors of the present invention can be administered systemically, for example, orally in combination with pharmaceutically acceptable media such as inert diluents or assimilated food carriers, or by inhalation or blowing. They can be encapsulated in coated or uncoated hard or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the patient's diet. For oral therapeutic administration, the compounds can be combined with one or more excipients and used in the form of ingestible tablets, lozenges, tablets, capsules, elixirs, suspensions, syrups, rice paper capsules, etc. For compositions suitable for human administration, the term "excipient" is intended to include, but is not limited to, those components described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2006) (hereinafter Remington's).

[0162] Inhibitors can be combined with inert, finely powdered carriers and administered by inhalation or blowing into the subject. Such compositions and formulations should contain at least 0.1% of the compound. Of course, the percentage of the composition and formulation can vary and can conveniently range from about 2% to about 60% of the weight of a given unit dosage form.

[0163] Tablets, lozenges, pills, capsules, etc., may also contain the following substances: binders such as gum arabic, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and may contain sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavorings. When the unit dosage form is a capsule, in addition to the above types of substances, it may also contain a liquid carrier, such as vegetable oil or polyethylene glycol. Syrups or elixirs may contain active compounds, sucrose or fructose as sweeteners, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavorings.

[0164] Various other substances can exist as coatings or otherwise alter the physical form of solid dosage forms. For example, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar. Of course, any substance used to prepare any dosage form should be pharmaceutically acceptable and substantially non-toxic at the amounts used.

[0165] Additionally, inhibitors can be incorporated into sustained-release formulations and devices. For example, inhibitors can be incorporated into timed-release capsules, timed-release tablets, and timed-release pills. In some embodiments, the composition is administered using a dosage form selected from effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention time, oral patches, and sublingual tablets.

[0166] Inhibitors can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of inhibitors can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof, and oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth.

[0167] Suitable drug dosage forms for injection or infusion may include sterile aqueous solutions, dispersions, or sterile powders containing compounds suitable for the ad hoc preparation of sterile injectable or infusion solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, fluid, and stable under production and storage conditions. The liquid carrier or medium may be a solvent or liquid dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. For example, appropriate flowability can be maintained by forming liposomes, by maintaining the desired particle size in the case of dispersions, or by using surfactants.

[0168] Sterile injection solutions are prepared by incorporating the required amount of the compound, along with various other ingredients listed above (as needed), into a suitable solvent, followed by sterilization through filtration. In the case of sterile powders used to prepare sterile injection solutions, a preferred preparation method is vacuum drying and freeze-drying techniques, producing a powder containing the active ingredient present in the previously sterile filtered solution plus any other desired ingredients.

[0169] For topical application, the compounds can be applied in their pure form. However, it is generally desirable to apply them to the skin as a composition or formulation together with a dermatologically acceptable carrier, which can be solid or liquid.

[0170] Useful solid carriers include finely crushed solids such as talc, clay, microcrystalline cellulose, silica, and alumina. Other solid carriers include conventional non-toxic polymer nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols, or water / alcohol / glycol blends, wherein the compound can be effectively dissolved or dispersed horizontally, optionally with the aid of a non-toxic surfactant. Adjuvants such as fragrances and other antimicrobial agents can be added to optimize the properties for a given application. The resulting liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

[0171] The effective dose of the peptides or pharmaceutical compositions of the present invention can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art.

[0172] For example, the concentration of the compound in a liquid composition such as a detergent may be about 0.1-25% by weight or about 0.5-10% by weight. The concentration in a semi-solid or solid composition such as a gel or powder may be about 0.1-5% by weight or about 0.5-2.5% by weight.

[0173] The effective dosage and route of administration of the agents of this invention are conventional. The exact amount of the agent (effective dosage) will vary from subject to subject, depending on factors such as the subject's species, age, weight, and general or clinical condition, the severity or mechanism of any condition being treated, the specific agent or carrier used, the method and timing of administration, etc. The therapeutically effective dosage can be determined empirically using conventional methods known to those skilled in the art. See, for example, *The Pharmacological Basis of Therapeutics*, edited by Goodman and Gilman, Macmillan Publishing Co., New York. For example, the effective dosage may initially be estimated in cell culture assays or in suitable animal models. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine the useful dosage and route of administration in humans. The therapeutic dosage may also be selected similarly to the dosage of an equivalent therapeutic agent.

[0174] The attending clinician will consider the details of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic) to select the specific administration mode and dosing regimen. Treatment may include daily or multiple daily doses of the compound over a period of days to months or even years.

[0175] However, generally speaking, a suitable dose will be in the range of about 0.001 to about 100 mg / kg, for example, about 0.01 to about 100 mg / kg body weight per day, such as above about 0.1 mg / kg, or in the range of about 1 to about 10 mg / kg body weight per day. For example, a suitable dose could be about 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, or 30 mg / kg body weight per day.

[0176] Inhibitors can be conveniently administered in unit dosage forms; for example, each unit dosage form contains 0.05 to 10,000 mg, 0.5 to 10,000 mg, 5 to 1,000 mg, or about 100 mg of the active ingredient. In some embodiments, dosage units contain about 0.1 mg, about 0.5 mg, about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg of the active ingredient.

[0177] Example

[0178] Example I - Toxic nucleus of α-synuclein in Parkinson's disease: derived from an invisible crystal structure

[0179] We screened crystals of peptide segments within the NAC domain and adjacent regions to seek structural information about the molecular basis of α-syn aggregation and toxicity.

[0180] For the two segments, namely NACore, residues 68 GAVVTGVTAVA 78 (SEQ ID NO:46) and PreNAC, 47 GVVHGVTTVA 56 Extensive crystal screening of (SEQ ID NO:47) resulted in amorphous, non-crystalline aggregates on the surface. However, upon examination with electron microscopy, we discovered that the aggregates were elongated nanocrystal clusters with cross-sections of only 50-300 nm, making them invisible under conventional optical microscopes. We confirmed the ordered crystallinity of NACore using SACLA and LCLS free-electron lasers. We also discovered a 9-residue fragment of NACore, which we termed SubNACore. 69 AVVTGVTAV 77 (SEQ ID NO:48) produces crystals with a volume 1,000-10,000 times that of NACore nanocrystals. Therefore, we are able to apply synchrotron methods... 18, 19 This was applied to these larger crystals to determine the structure of amyloid fibrils. Although this 9-residue fragment was missing only two residues compared to NACore, it did not have the same toxicity. 20 This provides insights into the toxicity of α-syn, as described below.

[0181] To determine the structures of the invisible NACore and PreNAC crystals, we turned to MicroED. 21 In MicroED, an extremely low-dose electron beam is guided onto a nanocrystal under cryogenic conditions within a transmission electron microscope, producing a diffraction pattern. In our experiment, a very small wavelength (0.025 Å) was used at 200 keV, and the Ewald spheres were essentially flat, resulting in diffraction patterns very similar to 2D slices through 3D reciprocal space. A series of such diffraction patterns were collected as the crystal was continuously rotated within the beam. 5 Diffraction data collected from multiple crystals will be scaled together to generate a complete 3D diffraction dataset. MicroED has been successfully applied to chicken egg white lysozyme. 6, 5 Bovine liver catalase 22 and Ca2+-ATPase 23The structures are well-known. However, NACore and PreNAC are the first previously unknown structures identified by MicroED.

[0182] For NACore and PreNAC, we collect electron diffraction patterns from nanocrystals preferentially oriented and laid flat on the surface of a porous carbon Quantifoil grating in a freeze-hydrated state. The grating is first screened to obtain crystals of appropriate size, and candidate crystals are then selected for diffraction. We collect data using crystals exhibiting strong diffraction by continuously rotating unidirectionally about a fixed axis, acquiring a series of diffraction frames at fixed time intervals. 5 Needle-shaped crystals typically exceed the length required for diffraction; those that are unbent and 100 to 300 nm wide produce the best diffraction patterns. Data from multiple crystals is integrated, scaled, and merged together.

[0183] Phase determination was performed using the SubNACore atomic model and the ideal β-chain model as probes via molecular substitution for polycrystalline NACore and PreNAC datasets, respectively. Differential density maps of the diffraction phases calculated from the SubNACore probe structure and the NACore structure factor clearly revealed the positions of missing residues, two water molecules, and several hydrogen atoms after subsequent refinement. Refinement of the full NACore and PreNAC models against MicroED data yielded structures with an acceptable R-factor at 1.4 Å resolution. Electron scattering factors were used for refinement calculations. 24 .

[0184] The NACore peptide chain has an almost fully extended β-chain structure. This can be achieved through site-directed spin labeling. 25, 26 As predicted, these NACore chains align and stack to form β-sheets. As is common in amyloid spines, the folds are paired, and these paired folds form typical stereozipper filaments, previously thought to be the spines in many amyloid protofilaments formed from short segments of protofilament-forming proteins. An unusual feature of this stereozipper is that the 11-residue width of the zipper is longer than previously observed, and each pair of folds contains two water molecules, each associated with a threonine side chain, within the interface, rather than being completely dry. Furthermore, in our NACore crystals, each fold forms two tight interfaces: interface A, with each chain having an embedded accessible surface area of ​​268 Ų, which is more extensive and likely stronger than interface B (167 Ų), because the terminal residues of the opposing folds bend towards each other. The PreNAC structure reveals peptide chains forming β-chains kinked at residue 51 of glycine. These chains are arranged in paired β-sheets that, like the NACore structure, interweave to form stereozipper filaments. Figure 3 Of particular note is the five-residue segment of PreNAC ().51 GVTTV 55 (SEQ ID NO:49) and the segment of five residues of NACore ( 73 GVTAV 77 (SEQ ID NO:50) differ by only one residue, their main chains and identical side chains closely overlap, and the α-carbon RMS deviation is 1.5 Å. This implies that the weaker interface B of NACore simulates the hypothetical interface between NACore and PreNAC. Below, we propose the importance of the possible contact between these two α-syn segments.

[0185] The correlation between the structure of NACore and that of full-length α-syn fibrils was determined by the similarity of their diffraction patterns. Specifically, full-length and N-terminal acetylated NACore fibrils... 27 The fiber diffraction patterns of α-syn protein-aligned protofibrils show the same main peak as those of aligned NACore nanocrystals. All three protofibrils show a strong reflectance of 2.4 Å in their diffraction patterns. This reflectance in NACore is due to the translation of one β-sheet of the stereo zipper along the fiber axis relative to another β-sheet by 2.4 Å, half the 4.8 Å spacing between β-chains, allowing the two folds to intersect each other closely. All three exhibit a strong reflectance of 4.6 Å, which in NACore is caused by the stacking of β-chains and the interlacing between adjacent β-sheets of the stereo zipper, while the common reflectance near 8.2 Å is likely due to the distance between adjacent β-sheet pairs constituting the α-syn protofibrils. This strong comparison of the fiber diffraction patterns strongly suggests that the structure of NACore is similar to the spines of our complete α-syn toxic protofibrils.

[0186] The combined structure of NACore and PreNAC allows us to construct a speculative model of most of the ordered segments of the A53T early-onset mutant α-syn. Experimental support for this model comes from the consistency between simulated fiber diffraction and measured diffraction patterns of α-syn and N-acetyl-α-syn protofibrils, as well as the alignment of NACore nanocrystals. Above, we mentioned that the weaker interface B of NACore simulates the hypothetical interaction between PreNAC and NACore. In fact, the interacting side chains (G73, T75, and V77) in the weaker NACore interface B are identical to the interacting side chains (G51, T53, and V55) in the hypothetical interface between PreNAC and NACore.

[0187] The identity and structure of cytotoxic amyloid proteins formed by α-syn remain a subject of in-depth research. 17, 28, 29, 30, 31, 32Over the past decade, the weighting of evidence has shifted the scientific view from fully developed amyloid fibrils found in Lewy bodies, as toxic entities, towards smaller transient amyloid oligomers. However, more recently, quantitative arguments have been put forward in favor of fibrils. 33 Not wishing to be bound by any particular mechanism, our experiments on the cytotoxicity of NACore to neuroblastoma cells are suggested to be consistent with the view that fibrils are toxic: we found that NACore aggregated and shaken for 72 hours exhibited abundant fibrils, was more toxic than freshly dissolved NACore, and was comparable in toxicity to similarly aggregated, intact α-syn. We also found that NACore was more cytotoxic than SubNACore, which is shortened by two residues. This is consistent with the fact that NACore forms fibrils faster than SubNACore. These observations do not preclude the formation of non-fibril-like oligomer assemblies present but not visible in our NACore and α-syn aggregated samples. Of course, NACore is merely a fragment of the full-length α-syn and lacks most of the membrane-binding motifs at the N-terminus of proteins associated with membrane disruption. 34, 35 However, it is clear that NACore is the minimal entity that encompasses all characteristics of full-length a-syn aggregation and toxicity.

[0188] The extremely small size of NACore is characteristic of amyloid crystals and various other target biocrystals. For amyloid crystals, our hypothesis is that the tiny size is a result of the natural twisting of the β-sheets that form protofilaments. The lattice suppresses this twisting, generating strain that increases with crystal growth in these crystals. Ultimately, this strain prevents further addition of β-chains, limiting the thickness of the needle-like crystals. Based on our experience, longer segments (e.g., 11 residues compared to 9 residues) limit crystal growth even more; in the cases of 11-residue NACore and 10-residue PreNAC, strain produces nanocrystals invisible to optical microscopy. These crystals are too small for fixation and conventional synchrotron data acquisition but are well-suited for MicroED analysis. Our NACore and PreNAC structures demonstrate that MicroED can determine novel and accurate biomaterial structures at atomic resolution. This discovery paves the way for applying MicroED to other important biomaterials in which only nanocrystals can grow. In our specific application, we have been able to understand the atomic arrangement of the core of the key NAC domain. This provides an opportunity for the design of structure-based α-synamyloid formation inhibitors. 36 .

[0189] method

[0190] crystallization

[0191] SubNACore69 AVVTGVTAV 77 Microcrystals of (SEQ ID NO:48) were grown from a synthetic peptide purchased from CS Bio. Crystals were grown at room temperature by hanging drop evaporation. The lyophilized peptide was dissolved in water at a concentration of 2.9 mg / ml in 48 mM lithium hydroxide. The peptide was mixed with a reservoir containing 0.9 M ammonium phosphate and 0.1 M sodium acetate at pH 4.6 at a 2:1 ratio.

[0192] NACore 68 GAVVTGVTAV 78 Nanocrystals of (SEQ ID NO:46) were grown from a synthetic peptide purchased from CS Bio. Ten batches of the synthetic peptide (CSBio) at a concentration of 1 mg / ml in sterile water were shaken overnight at 37°C on a Torrey Pines orbital mixing plate at speed set to 9. Insoluble substances were washed in 30% (w / v) glycerol and then stored in water at room temperature before diffraction. The samples contained a mixture of fibrils and crystals.

[0193] PreNAC 47 GVVHGVTTVA 56 Nanocrystals of (SEQ ID NO:47) were grown from a synthetic peptide purchased from InnoPep. Crystallization experiments of the synthetic peptide were prepared in batches. The peptide was weighed and dissolved at a concentration of 5 mg / ml in sterile filtered 50 mM phosphate buffer (pH 7.0) containing 0.1% DMSO. The solution was then shaken overnight at 37°C on a Torrey Pines orbital mixing plate at a speed of 9.

[0194] Data collection and processing

[0195] X-ray diffraction data from SubNACore microcrystals were collected using synchrotron radiation at the Advanced Photon Source, Northeast Collaborative Access Team (DCT) 24-ID-E microfocusing beamline. The beamline was equipped with an ADSC Quantum 315 CCD detector. Data from the single crystal was collected at 0.9791 Å wavelength using a 5 μm beam diameter in a 5° wedge. Data from three different cross-sections along the needle axis were used. The crystal was cryogenically cooled (100 K) for data collection. The Denzo / Scalepack from the HKL program kit was used. 37 Process and restore data.

[0196] X-ray diffraction data from NACore nanocrystals were collected using XFEL radiation on a CXI instrument (Coherent X-ray Imaging) at a Linear Coherent Light Source (LCLS)-SLAC. The photon energy of the X-ray pulses was 8.52 keV (1.45 Å). Considering a beamline transmittance of 60%, each 40 fs pulse contained up to 6 x 10¹¹ photons at the sample location. The beam diameter was approximately 1 μm. We used approximately 25 μl of particulate material suspended in 1 mL of water at a specific concentration. A liquid jet injector and a pneumatic virtual nozzle were used. 38 The sample was injected into the XFEL beam. The microjet width was approximately 4 μm, and the flow rate was 40 μl / min. The sample caused significant sputtering of the liquid jet. (Using cctbx.xfel) 39, 40 Process XFEL data.

[0197] Electron diffraction data from NACore and PreNAC nanocrystals were collected using MicroED technology. 5, 6 These nanocrystals typically clump together. To break up the clumps, approximately 100 μL of nanocrystals were placed in an ultrasonic bath for 30 minutes. After appropriate dilution, the nanocrystals were deposited as 2–3 μl droplets onto a quantifoil porous carbon EM grid, optimized for crystal density on the grid. All grids were then vitrified by inserting a Vitrobot Mark IV (FEI) into liquid ethane and then transferred to liquid nitrogen for storage. The frozen hydrated grid was transferred to a cryogenic TEM using a Gatan 626 cryostat. Diffraction patterns and crystal images were collected using a FEI Tecnai F20 TEM equipped with a FEG operating at 200 kV and recorded using a bottom-mounted TVIPS F416 CMOS camera with a sensor size of 4000 square pixels, each square pixel being 15.6 μm in size. The diffraction patterns were recorded by operating the detector in rolling shutter mode at 2 x 2 pixel areas, resulting in a final image of 2000 square pixels. Using a selected area aperture with an illumination spot size of approximately 1 micrometer, individual image frames were captured with exposure times of 3–4 seconds per image. This geometry corresponds to an electron dose of less than 0.1 e⁻ / Ų per second. During each exposure, the crystal continuously rotates within the beam at a rate of 0.3° per second, equivalent to a 1.2° wedge per frame. Diffraction data were collected from several crystals, each with a different orientation relative to the rotation axis. These datasets each span a wedge in reciprocal lattice space ranging from 40° to 80°.

[0198] Calibration of the sample-to-detector distance was performed using a polycrystalline gold standard and by referencing significant reflections in electron diffraction experiments and corresponding reflections in XFEL data. This was achieved using cctbx.xfel data specifically for thermophilic protein datasets.39 Refine the tile positions of optical measurements to perform x / y position calibration of the 64-tile CSPAD detector.

[0199] To achieve compatibility with traditional X-ray data processing procedures, diffraction images were converted from TIFF or TVIPS format to SMV crystal format. We used XDS to index the diffraction images. 41 XSCALE is used to merge and scale datasets from different crystals. For NACore, data from four crystals are merged, while for PreNAC, data from three crystals are merged to compile the final dataset.

[0200] Structural determination

[0201] Molecular substitution solutions of SubNACore were obtained using the Phaser 42 program. The search model consisted of a geometrically ideal β-chain composed of nine alanine residues. The REFMAC program was used. 43 Crystallographic refinement was performed. Molecular substitution solutions of NACore were obtained using the program Phaser 42. The search model consisted of the previously determined SubNACore structure. The Phenix program was used for further refinement. 44 Perform crystallographic refinement. Use the program Phaser. 42 Obtain a molecular substitution solution of PreNAC. The search model consists of a geometrically ideal β-chain, which comprises six residues with the sequence GVTTVA. The Phenix program was used for this purpose. 44 Crystallographic refinement was carried out.

[0202] Use COOT 45 Modeling was performed for all segments. The coordinates and structural factors of the final model have been stored in the Protein Data Bank (PDB). The PDB codes 4RIK for SubNACore, 4RIL for NACore, and 4ZNN for PreNAC. PyMold was used. 46 Explain the structure.

[0203] α-synuclein

[0204] Previously, human wild-type α-syn constructs were characterized. 47 (pRK172, ampicillin, T7 promoter), its sequence is:

[0205] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHG

[0206] VATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 1).

[0207] α-synuclein purification

[0208] According to the publicly available plan 31 Full-length α-syn was purified. The α-syn construct was transformed into the *E. coli* expression cell line BL21 (DE3) gold (Agilent Technologies, Santa Clara, CA) for wild-type α-syn protein expression. Single colonies were incubated in 100 mL of LB Miller broth (Fisher Scientific, Pittsburgh, PA) supplemented with 100 μg / mL ampicillin (Fisher Scientific, Pittsburgh, PA) and grown overnight at 37°C. 1 L of LB Miller broth supplemented with 100 μg / mL ampicillin was incubated with 10 mL of the overnight culture in a 2 L shake flask and grown at 37°C until the culture reached an OD600 of ~0.6-0.8 as measured by a BioPhotometer UV / VIS spectrophotometer (Eppendorf, Westbury, NY). Add IPTG (isopropyl β-D-1-thiogalactopyranoside) to a final concentration of 0.5 mM and incubate at 30 °C for 4–6 hours. Harvest cells by centrifugation at 5,500 x g for 10 minutes at 4 °C. Freeze and store cell clumps at -80 °C.

[0209] Cell clumps were thawed on ice and resuspended in lysis buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA pH 8.0) and lysed by sonication. The crude cell lysate was clarified by centrifugation at 15,000 x g for 30 minutes at 4 °C. The clarified cell lysate was boiled and centrifuged to remove cell debris. HCl was added dropwise to the protein solution on ice while stirring to precipitate the proteins in the supernatant at pH 3.5, followed by centrifugation at 15,000 x g for 30 minutes at 4 °C. The supernatant was dialyzed against buffer A (20 mM Tris-HCl, pH 8.0). After dialyzing, the solution was filtered through a 0.45 μm syringe (Corning, NY 14831) and then loaded onto a 20 mL HiPrep Q HP 16 / 10 column (GE Healthcare, Piscataway, NJ). The Q-HP column was washed with 5 column volumes of buffer A, and proteins were eluted using a 100% linear gradient in 5 column volumes of buffer B (20 mM Tris-HCl, 1 M NaCl, pH 8.0). Proteins were eluted in approximately 50-70% buffer B; peak fractions were collected. The collected sample was concentrated approximately 10-fold using an Amicon Ultra-15 centrifuge filter. Approximately 5 ml of the concentrated sample was loaded onto a HiPrep 26 / 60 Sephacryl S-75 HR column equilibrated with filtration buffer (25 mM sodium phosphate, 100 mM NaCl, pH 7.5). Peak fractions from the gel filtration column were collected and dialyzed against 5 mM Tris-HCl (pH 7.5) to concentrate to 3 mg / ml. These were then filtered through a 0.2 μm pore size filter (Corning, NY 14831) and stored at 4°C.

[0210] Based on the scheme of Der-Sarkissian et al. 25 The full-length α-syn of recombinant expression with N-terminal acetylation was prepared and purified in the following manner.

[0211] The α-syn plasmid was co-expressed with a heterodimeric protein acetylation complex from *Schizosaccharomyces pombe* to induce N-terminal (pACYC-DUET, chloramphenicol, T7 promoter) acetylation. 48The two vectors were co-transformed into *E. coli* BL21(DE3) using a medium containing ampicillin and chloramphenicol. Cell cultures were grown in TB medium containing ampicillin and chloramphenicol and induced overnight at 25°C with 0.5 mM IPTG to express α-syn. Cells were harvested by centrifugation, and cell clumps were resuspended in lysis buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA pH 8.0, and 1 mM benzyl sulfonyl fluoride) and lysed using an Emulsiflex homogenizer (Avestin). Lysates were boiled and debris removed by centrifugation. Protein fractions were also removed by precipitation on ice at low pH followed by centrifugation. The remaining supernatant was pH-adjusted by titration and dialyzed with buffer A (20 mM Tris-HCl pH 8.0, 1 mM DTT, 1 mM EDTA pH 8.0). The resulting protein solution was loaded onto a 5 mL Q-Sepharose FF column (GE Healthcare) equilibrated with buffer A and eluted with a linear gradient of buffer B (1 M NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT, 1 mM EDTA pH 8.0). Fractions containing α-syn were identified by SDS-PAGE, collected, concentrated, and further purified by size exclusion method (Sephacryl S-100 16 / 60, GE Healthcare) in 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA. Fraction purity was assessed by SDS-PAGE.

[0212] via LC-MS27 49 Characterizing acetylated proteins. Expected mean mass: 14460.1 Da for α-synuclein and 14502.1 Da for acetylated α-synuclein. Observed mean mass: 14464.0 Da for α-synuclein and 14506.0 Da for acetylated α-synuclein. The 4 Da deviation between the observed and expected mean masses is due to instrumental error.

[0213] Inhibitor Synthesis

[0214] The inhibitor was commercially available from Genscript Inc. with a purity greater than 98%, and dissolved in 100% DMSO at a concentration of 10 mM. The dissolved inhibitor was filtered through a 0.1 μM filter and aliquoted into 20 μL aliquots at -20°C for storage until further use.

[0215] Thiamine T assay

[0216] Fibrillation assays were performed at a protein concentration of 50 μM under the same conditions as those used for inoculation assays of aggregated proteins (but with the addition of ThT). All assays were performed on black Nunc 96-well optical plates (Thermo Scientific). The plates were shaken at 600 rpm with a 3 mm rotation diameter in a Varioskan microplate reader (Thermo) at 37 °C. λ ex =444nm, λ em =482nm, fluorescence measurement was recorded every 30 minutes, and the integration time was 200μs.

[0217] Aggregation determination

[0218] The purified α-synuclein was dialyzed in 0.1 M sodium sulfate and 25 mM sodium phosphate, and then aggregated for 5-6 days by shaking at 50 μM and speed 9 in a Torrey Pine shaker at 37 °C.

[0219] Sarcosyl-insoluble protein filaments were extracted from PD human brain tissue.

[0220] Human frozen brain tissue was obtained from the UCLA Brain Tumor Translational Resource Center (BTTR). Creatyl-insoluble proteins were extracted using a previously described protocol. Briefly, the frozen tissue was homogenized in ice-cold PBS at 200 mg / mL using a Dunns homogenizer. The homogenate was then diluted to 50 mg / mL in buffer A (10 mM Tris 7.4, 800 mM NaCl, 1 mM EGTA, 10% sucrose) to a total volume of 1 mL and incubated at 4°C. Centrifuge at 20,700g for 20 minutes at C. Collect the supernatant in an ultracentrifuge tube, resuspend the clump in 0.5mL of buffer A, and then centrifuge at 4°C. Centrifuge at 20,700g for 20 minutes at C. Collect the supernatants together. Then add 150 μl of 10% creatine amide (w / v) in Millipore water and incubate at 700 rpm for 1 hour at room temperature on a flatbed rotary shaker. Then centrifuge the solution at 4°C using an SW 55 rotor (Beckman Coulter). Centrifuge at 100,000g for 1 hour at C. Discard the supernatant and wash the clumps with 5 mL of buffer A, then centrifuge at 4°C. Centrifuge at 100,000g for 20 minutes at C. Resuspend the clump in 100 μl of 50 mM Tris Ph 7.4.

[0221] HEK293T cell culture

[0222] HEK293T cells stably expressing YFP-tagged WT α-syn and A53T α-syn were a generous gift from Dr. Marc Diamond. Cells were grown in a humidified incubator at 37°C and 5% CO2 in Durbeco modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (HyClone), 1% penicillin / streptomycin (Gibco), and 1% glutamax (Gibco).

[0223] Seedling HEK293T cells

[0224] 10,000 cells in 90 μL of medium were seeded in 96-well black-walled plates (Cat #3660) and allowed to adhere overnight. α-syn was transfected at a final monomer concentration of 125 nM. Lipofectamine 2000 was diluted in OptiMEM medium (2.5 ± 17.5 μL) and incubated at room temperature for 5 min. Protein aggregates were diluted in OptiMEM medium (1:20) and sonicated with low pulses in a water bath sonicator for 3 min. The diluted lipofectamine and protein sample were then mixed 1:1 and incubated at room temperature for 20 min, followed by adding 10 μL to each well. All samples were added in triplicate, and the experiment was repeated at least twice. To co-transfect α-syn fimbriae with the inhibitor, the fimbriae and inhibitor were diluted in OptiMEM and incubated for 3 h, followed by sonication.

[0225] Measurement of intracellular spots in cells

[0226] Spot formation and cell growth were measured using a Celigo imaging cell counter that allows for unbiased measurements. Wells were imaged using a fluorescent GFP channel, and confluence was measured using Celigo analysis software. Images of the entire well were taken, and particle counts were performed using ImageJ via particle analysis. Wells on a plate were analyzed on all days using the same settings. The total particle count in each well was normalized relative to confluence and reported as the particle count per well.

[0227] fibril formation and detection

[0228] α-syn was purified to a concentration of 500 μM in 50 mM Tris, 150 mM KCl, and pH 7.5 by shaking in a Torey Pine shaker at 37 °C. To form fibrous samples of SubNACore and NACore, the lyophilized peptides were dissolved in 5 mM lithium hydroxide, 20 mM sodium phosphate, pH 7.5, and 0.1 M NaCl to achieve a final concentration of 500 μM. All samples were shaken in a Torey Pine shaker at 37 °C for 72 hours. Freshly dissolved samples were prepared by dissolving the lyophilized peptides and immediately added to cells for assay.

[0229] Turbidity measurements were used to compare NACore and SubNACore aggregation. Peptide samples were freshly dissolved in sample buffer containing 5 mM LiOH and 1% DMSO to a final concentration of 1.6 mM, and then filtered through a PVDF filter (Millipore, 0.1 μm). Measurements were performed using black NUNC 96-well plates with 200 μL sample / well (3–4 replicates per sample). The plates were shaken at 300 rpm with a 3 mm diameter rotation at 37°C in a Varioskan microplate reader (Thermo). Absorbance readings were recorded every 3–15 minutes at 340 nm.

[0230] Negative staining transmission electron microscopy

[0231] The presence of fibrils in cytotoxic samples was evaluated using electron microscopy. In short, 5 μL of sample was directly spotted onto a newly glow-discharged carbon-coated electron microscope grid (Ted Pella, Redding, CA). After incubation for 4 minutes, the grid was rinsed twice with 5 μL of distilled water and stained with 2% uranium acetate for 1 minute. The samples were then examined under a FEI T12 electron microscope.

[0232] fibril diffraction

[0233] Protofibrils, both with and without N-terminal acetylation, formed from purified α-syn were concentrated by centrifugation, washed, and simultaneously oriented while drying between two glass capillaries. Similarly, NACore nanocrystals were also concentrated, washed, and simultaneously oriented while drying between two glass capillaries. The aligned protofibrils or nanocrystals were fixed onto cryopinions and diffracted using 1.54 Å X-rays generated by a Rigaku rotating anode generator equipped with an HTC imaging plate and a built-in X-Stream cryogenic cooling system. All spectra were collected at a distance of 180 mm and analyzed using the Advxv software package.

[0234] Cytotoxicity assay

[0235] Adhesive PC12 cells were cultured in RPMI 1640 medium (ATCC; catalog number 30-2001) supplemented with 10% horse serum and 5% fetal bovine serum, and seeded at 10,000 cells per well for a final volume of 90 μL. All MTT assays were performed using the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Kit (MTT, Promega catalog number 4100). Cells were cultured in 96-well plates at 37°C and 5% CO2 for 20 h, followed by sample addition (Costar catalog number 3596). 10 μL of sample was added to each well containing 90 μL of medium, and incubated at 37°C and 5% CO2 for 24 h. Then, 15 μL of dye solution (Promega catalog number 4102) was added to each well, and incubated at 37°C and 5% CO2 for 4 h. Following this, 100 μL of solubilization / stop mixture (Promega catalog number 4101) was added to each well. After incubation at room temperature for 12 hours, absorbance was measured at 570 nm. Background absorbance was recorded at 700 nm. Data for cells treated with 1% (w / v) SDS were normalized to 0% decrease, and data for cells treated with sample buffer were normalized to 100% decrease.

[0236] Lactose dehydrogenase assays were performed using the CytoTox-ONE™ Homogeneous Membrane Integrity (Promega, catalog number G7890) according to the manufacturer's instructions. In short, cells were seeded at 10,000 cells per well in 96-well black-walled, clear-bottomed (Fisher Cat #07-200-588) tissue culture plates to a final volume of 90 μL. Cells were incubated at 37°C and 5% CO2 for 20 hours before adding the sample. Next, 10 μL of sample was added to each well, and the cells were incubated for another 24 hours. 100 μL of reagent was added to each well and incubated at room temperature for 15 minutes. The reaction was terminated by adding 50 μL of stop solution. Fluorescence was measured at excitation and emission wavelengths of 560 nm and 590 nm, respectively. Data were normalized using buffer-treated cells as 0% release and cells treated with 0.1% Triton X-100 as 100% release.

[0237] The atomic coordinates of the structure of NACore: GAVVTGVTAVA (SEQ ID NO:46) are shown in Table 3. The atomic coordinates of PreNAC are shown in Table 4.

[0238] The atomic structure of NACore reveals amyloid protofibrils formed by self-complementary paired β-sheets that fit together to form a stereozipper. Using Rossetta-based computational modeling, each inhibitor was designed to bind to the tip of the stereozipper, thus 'closing' the protofibril and preventing the addition of other α-synuclein molecules into it. We designed three candidate inhibitors: S37, S61, and S71, which bind favorably to one or both ends of the zipper. The calculated binding energy and shape complementarity of each of the three inhibitors also favored binding. Each inhibitor retained most of the residues of the native NACore sequence but also contained one or more modified residues. Rodriquez et al. showed that smaller 9-residue segments in NACore [69-77] aggregated more slowly than NACore and were structurally similar. To prevent self-aggregation of our designed inhibitors, we used shorter segments along with one or more modifications. S37 has a W mutation at Thr72 and an additional polylysine tag at the C-terminus to induce charge-charge repulsion. It is expected to bind to both tips (i.e., the top and bottom) of the stereozipper fibril. S61 and S62 retain the same inhibitor sequence as S37, but instead of the polylysine tag, a TAT tag is added to aid dissolution and prevent self-aggregation. S71 has a methylated glycine at Gly73 that weakens the hydrogen bonds along the β-sheet and an additional TAT tag for dissolution and cell penetration.

[0239] Example II: Computational Design

[0240] Based on the atomic structure of the fibrous formation segment of α-synuclein, we designed a series of inhibitors to suppress aggregation. We designed peptide inhibitors to specifically "cap" the growth aggregates of α-synuclein. The ZipperDB algorithm (Goldschmidt et al.) was used. 55 This study identified a crystallizable amyloid-forming segment within the NAC domain. This domain has been reported to be both necessary and sufficient for the aggregation and toxicity of α-synuclein (Bodles et al.). 20 The identified segment (the α-synuclein segment consisting of residues 69-77 and 68-78) was chemically synthesized and crystallized; its three-dimensional structure was determined by microcrystalline structure and microelectron diffraction (MicroED). We then followed the steps outlined in the Rosetta-based method (Sievers et al.). 36 Using α-synuclein 69-77 or α-synuclein 68-78 structures as templates, inhibitors that disrupt α-synuclein aggregation were designed. Hypothetical inhibitors were identified and selected for experimental characterization.

[0241] Example III - Second Round Design - Rational Design Based on Sequence

[0242] In another design, we started with several α-synuclein amyloid-forming segments: 47-56, 68-78, and 84-94. N-methylation was introduced at two independent residues in the native amino acid sequence. In these N-methyl amino acids, an additional methyl group replaced an amine hydrogen. This blocked hydrogen bonds and prevented the addition of peptide monomers at their growth ends. In addition to N-methylation, it was thought that the extra polar / charged amino acid extension from the native α-synuclein sequence would improve the overall solubility of the peptide inhibitor.

[0243] Table 3

[0244] Title: Electron Diffraction Structure of the Nuclear Part of Parkinson's Virus

[0245] Title 2 Electron diffraction structure of α-synuclein amyloid protein, GAVVTGVTAVA

[0246] Compound MOL_ID: 1;

[0247] Compound 2 molecule: α-synuclein;

[0248] Compound 3, chain: A;

[0249] Compound 4 Synonym: Non-Aβ component of AD amyloid protein,

[0250] Compound 5 is the non-A4 component NACP of the amyloid precursor;

[0251] Compound 6 engineered: Yes

[0252] Source MOL_ID: 1;

[0253] Source 2 Synthetic: Yes;

[0254] Source 3: Biology_Science: Homo sapiens;

[0255] Source 4: Biology - Common: Human;

[0256] Source 5, Biology_TAXID: 9606;

[0257] Source 6 Other_Details: Synthetic peptide GAVVTGVTAVA corresponds to

[0258] Source 7: Segment 68-78 of human α-synuclein

[0259] Keywords: amyloid protein, α-synuclein, Parkinson's disease, toxic nucleus, NACORE

[0260] Keyword 2: Lipid-binding protein

[0261] EXPDTA Electron Crystallography

[0262] Authors JARODRIGUEZ, M.IVANOVA, MRSAWAYA, D.CASCIO, F.REYES, D.SHI, L.JOHNSON,

[0263] Author 2 E.GUENTHER, S.SANGWAN, J.HATTNE, B.NANNENGA, ASBREWSTER,

[0264] Author 3 M.MESSERSCHMIDT, S.BOUTET, NKSAUTER, T.GONEN, DSEISENBERG

[0265] Journal authors: JARODRIGUEZ, M. IVANOVA, MRSAWAYA, D. CASCIO, F. REYES, D. SHI

[0266] Journal authors 2: L. JOHNSON, E. GUENTHER, S. SANGWAN, J. HATTNE, B. NANNENGA,

[0267] Journal authors 3: Asbury, M. Messerschmidt, S. Bouette, K. K. Sauter, T. Gonn.

[0268] Journal author 4 DSEISENBERG

[0269] Journal Title: Microstructure of the Toxicity Nucleus of α-Synuclein

[0270] Journal Title 2 Amyloid Protein

[0271] Journal Title 3 Proteins Associated with the Development of Parkinson's Disease

[0272] Journal references to be published

[0273] Journal References

[0274] Note 2

[0275] Note 2: Resolution. 1.43 angstroms.

[0276] Note 3

[0277] Note 3: Detailed.

[0278] Note 3: Program: BUSTER

[0279] Note 3 Authors: BRICOGNE, BLANC, BRANDL, FLENSBURG, KELLER

[0280] Note 3: PACIOREK, ROVERSI, SMART, VONRHEIN, WOMACK

[0281] Note 3: MATTHEWS, TEN EYCK, TRONRUD

[0282] Note 3

[0283] Note 3: Data for further refinement.

[0284] Note 3: High resolution range (angstroms): 1.43

[0285] Note 3: Low resolution range (angstroms): 16.43

[0286] Note 3: Data cutoff value (∑(F)): 0.000

[0287] Note 3: Scope integrity (%): 87.9

[0288] Note 3: Number of reflections: 1073

[0289] Note 3

[0290] Note 3 is suitable for detailed data.

[0291] Note 3: Cross-validation: from beginning to end

[0292] Note 3: Free R-value test set selection: random

[0293] Note 3: R-value (working set + test set): 0.251

[0294] Note 3: R-value (working set): 0.248

[0295] Note 3: Free R-value: 0.275

[0296] Note 3: Free R-value test set size (%): 11.840

[0297] Note 3: Free R-value test set count: 127

[0298] Note 3: Free R-value estimation error: zero

[0299] Note 3

[0300] Note 3: Suitable for the highest resolution libraries.

[0301] Note 3: Total number of face values ​​used: 5

[0302] Note 3: High-resolution library range (angstroms): 1.43

[0303] Note 3: Low-resolution library range (angstroms): 1.60

[0304] Note 3: Surface integrity (working + testing) (%): 87.88

[0305] Note 3: Surface element reflection (working + test set): 286

[0306] Note 3: R-value of surface area (working + test set): 0.2642 1

[0308] Note 3: Surface element reflection (working set): 245

[0309] Note 3: Surface R value (working set): 0.2532

[0310] Note 3: Surface element free R-value: 0.3310

[0311] Note 3: Surface element free R-value test set size (%): 14.34

[0312] Note 3: Number of free R-value test set items for surface elements: 41

[0313] Note 3: Error in estimating the free R-value of the surface element: zero.

[0314] Note 3

[0315] Note 3: Number of non-hydrogen atoms used for refinement

[0316] Note 3: Protein atoms: 66

[0317] Note 3: Nucleic acid atoms: 0

[0318] Note 3: Heterogeneous hybrid atoms: 0

[0319] Note 3 Solvent atoms: 2

[0320] Note 3

[0321] Note 3: B value.

[0322] Note 3 is from Wilson plotting method (A 2): 10.33

[0323] Note 3: Mean B value (population, A) 2): 12.75

[0324] Note 3: Overall anisotropy B value.

[0325] Note 3 B11 (A 2): -3.98760

[0326] Note 3 B22 (A 2): 3.49080

[0327] Note 3 B33 (A) 2): 0.49680

[0328] Note 3 B12 (A 2): 0.00000

[0329] Note 3 B13 (A 2): -1.55090

[0330] Note 3 B23 (A 2): 0.00000

[0331] Note 3

[0332] Note 3: Estimated coordinate error.

[0333] Note 3: Estimated value (A) from the LUZZATI plotting method: 0.305

[0334] Note 3: DPI (BLOW EQ-10) is based on R value (A): zero.

[0335] Note 3: DPI (BLOW EQ-9) is based on the free R value (A): zero.

[0336] Note 3: DPI (CRUICKSHANK) is based on R value (A): 0.108

[0337] Note 3: DPI (CRUICKSHANK) is based on the free R value (A): zero.

[0338] Note 3

[0339] Note 3 Reference: BLOW, D. (2002) ACTA CRYST D58, 792-797

[0340] Note 3 CRUICKSHANK, DWJ (1999) ACTA CRYST D55, 583-601

[0341] Note 3

[0342] Note 3: Correlation coefficient.

[0343] Note 3: Correlation coefficient FO-FC: 0.913

[0344] Note 3: Free correlation coefficient FO-FC: 0.900

[0345] Note 3

[0346] Note 3: Number of defined geometric function terms: 15

[0347] Note 3: Weighted counting function.

[0348] Note 3: Bond length: 65; 2.000; harmonics

[0349] Note 3: Key angle: 90°; 2.000°; Harmonic

[0350] Note 3: Torsion angle: 16°; 2.000°; sine wave

[0351] Note 3: Triangular carbon plane: 1; 2.000; harmonics

[0352] Note 3: General plane: 10; 5.000; harmonics

[0353] Note 3: Isotropic thermodynamic factor: 65; 20,000; harmonics

[0354] Note 3: Poor nonbonded contact: zero; zero; zero

[0355] Note 3: Improper reversal: zero; zero; zero

[0356] Note 3: False rotation angle: zero; zero; zero

[0357] Note 3: Chiral improper torsion: 11; 5.000; half harmonic

[0358] Note 3: Total usage: Zero; Zero; Zero

[0359] Note 3: Utility distance: zero; zero; zero

[0360] Note 3 Utility angle: zero; zero; zero

[0361] Note 3: Utility reversal: zero; zero; zero

[0362] Note 3 Ideal distance contact term: 76; 4,000; half harmonic

[0363] Note 3

[0364] Note 3: RMS deviation from the ideal value.

[0365] Note 3: Bond length (A): 0.010

[0366] Note 3: Key angle (degrees): 1.65

[0367] Note 3: Peptide ω twist angle (degrees): 4.08

[0368] Note 3: Other torsion angles (degrees): 6.49

[0369] Note 3

[0370] Note 3: TLS Details

[0371] Note 3: Number of TLS groups: zero 2

[0373] Note 3

[0374] Note 3: Other detailed notes: None

[0375] Note 4

[0376] Note 4: 4RIL follows format V. 3.30, July 13, 2011.

[0377] Note 100

[0378] Note 100: This entry was processed by the RCSB on October 9, 2014.

[0379] Note 100: RCSB ID code is RCSB087390.

[0380] Note 240

[0381] Note 240 Experimental Details

[0382] Note 240 Refactoring method: None

[0383] Note 240 Sample type: None

[0384] Note 240 Sample type: None

[0385] Note 240: Data Acquisition

[0386] Note 240: Data collection date: August 28, 2014

[0387] Note 240 Temperature (Kelvin): 100.0

[0388] Note 240 PH: Zero

[0389] Note 240: Number of crystals used: 4

[0390] Note 240: Microscope model: TECNAI F20 TEM

[0391] Note 240: Detection type: TVIPS F416 CMOS camera

[0392] Note 240 Accelerating voltage (kV): Zero

[0393] Note 240: Unique Reflection Count: 1073

[0394] Note 240: High resolution range (A): 1.430

[0395] Note 240: Low resolution range (A): 16.430

[0396] Note 240: Data conversion software: XSCALE

[0397] Note 240: Range integrity (%): 89.9

[0398] Note 240: Data redundancy: 4.400

[0399] Note 240 is located within the highest resolution housing.

[0400] Note 240: Highest resolution housing, high range (A): 1.43

[0401] Note 240: Highest resolution housing, low range (A): 1.60

[0402] Note 240: Shell integrity (%): 82.5

[0403] Note 240: Shell data redundancy: 4.40

[0404] Note 240: Shell R combined (I): 0.56500

[0405] Note 240: Methods used for determining structure: Molecular substitution

[0406] Note 240: Software used: PHASER

[0407] Note 240 Startup Mode: 4RIK

[0408] Note 290

[0409] Note 290 Crystallographic symmetry

[0410] Note 290 Symmetry operator of space group: C 1 2 1

[0411] Note 290

[0412] Note 290 SYMOP symmetry

[0413] Note 290 NNNMMM operator

[0414] Note 290 1555 X, Y, Z

[0415] Note 290 2555 -X, Y, -Z

[0416] Note 290 3555 X+1 / 2, Y+1 / 2, Z

[0417] Note 290 4555 -X+1 / 2, Y+1 / 2, -Z

[0418] Note 290

[0419] Note 290 NNN -> Number of operators

[0420] Note 290 MMM -> Translation vector

[0421] Note 290

[0422] Note 290 Crystallographic symmetry transformation

[0423] Note 290: The following conversions apply to atoms / HETATM.

[0424] Note 290: The record in this entry was produced by crystallization.

[0425] Note 290 Related molecules.

[0426] Note 290 SMTRY1 1 ​​1.000000 0.000000 0.000000 0.00000

[0427] Note 290 SMTRY2 1 0.000000 1.000000 0.000000 0.00000

[0428] Note 290 SMTRY3 1 0.000000 0.000000 1.000000 0.00000

[0429] Note 290 SMTRY1 2 -1.000000 0.000000 0.000000 0.00000

[0430] Note 290 SMTRY2 2 0.000000 1.000000 0.000000 0.00000

[0431] Note 290 SMTRY3 2 0.000000 0.000000 -1.000000 0.00000

[0432] Note 290 SMTRY1 3 1.000000 0.000000 0.000000 35.40500

[0433] Note 290 SMTRY2 3 0.000000 1.000000 0.000000 2.41000

[0434] Note 290 SMTRY3 3 0.000000 0.000000 1.000000 0.00000

[0435] Note 290 SMTRY1 4 -1.000000 0.000000 0.000000 35.40500

[0436] Note 290 SMTRY2 4 0.000000 1.000000 0.000000 2.41000 3

[0438] Note 290 SMTRY3 4 0.000000 0.000000 -1.000000 0.00000

[0439] Note 290

[0440] Note 290: None

[0441] Note 300

[0442] Note 300 Biomolecules: 1, 2

[0443] Note 300: See Note 350 for the provided author and / or program.

[0444] Note 300 refers to the generated entry.

[0445] Note 300: Component information for the structure. Notes may also provide relevant information.

[0446] Note 300: Hides information about the surface area.

[0447] Note 300: This biological unit is part of a β-sheet. A fold is...

[0448] Note 300 is composed of chain A and a unit cell translated along B.

[0449] Note: Size 300. Other folds are achieved by using a symmetrical partner - X+1 / 2.

[0450] Note: 300 Y+1 / 2, -Z and unit cell translation along B constitute the composition.

[0451] Note 350

[0452] Note 350: Representative molecules can be generated by applying the BIOMT conversion given below.

[0453] Note 350: Known biologically important oligomeric states

[0454] Note 350: Complete polymer

[0455] The coordinates of 350 are noted. The coordinates are also provided.

[0456] Note 350: Amorphous and crystallographic operations.

[0457] Note 350

[0458] Note 350 Biomolecules: 1

[0459] Note 350: Biological unit identified by the authors: tetramer

[0460] Note 350 applies the following to the chain: A

[0461] Note: 350 BIOMT1 1 1.000000 0.000000 0.000000 0.00000

[0462] Note: 350 BIOMT2 1 0.000000 1.000000 0.000000 0.00000

[0463] Note: 350 BIOMT3 1 0.000000 0.000000 1.000000 0.00000

[0464] Note: 350 BIOMT1 2 1.000000 0.000000 0.000000 0.00000

[0465] Note: 350 BIOMT2 2 0.000000 1.000000 0.000000 4.82000

[0466] Note: 350 BIOMT3 2 0.000000 0.000000 1.000000 0.00000

[0467] Note: 350 BIOMT1 3 -1.000000 0.000000 0.000000 35.40500

[0468] Note: 350 BIOMT2 3 0.000000 1.000000 0.000000 2.41000

[0469] Note: 350 BIOMT3 3 0.000000 0.000000 -1.000000 0.00000

[0470] Note: 350 BIOMT1 4 -1.000000 0.000000 0.000000 35.40500

[0471] REMARK 350 BIOMT2 4 0.000000 1.000000 0.000000 7.23000

[0472] REMARK 350 BIOMT3 4 0.000000 0.000000 -1.000000 0.00000

[0473] REMARK 350

[0474] REMARK 350 BIOMOLECULE: 2

[0475] REMARK 350 AUTHOR DETERMINED BIOLOGICAL UNIT: MONOMER

[0476] REMARK 350 APPLY THE FOLLOWING TO CHAIN: A

[0477] REMARK 350 BIOMT1 1 1.000000 0.000000 0.000000 0.00000

[0478] REMARK 350 BIOMT2 1 0.000000 1.000000 0.000000 0.00000

[0479] REMARK 350 BIOMT3 1 0.000000 0.000000 1.000000 0.00000

[0480] DBREF 4RIL A 1 11 UNP P37840 SYUA_HUMAN 68 78

[0481] SEQRES 1 A 11 GLY ALA VAL VAL THR GLY VAL THR ALA VAL ALA

[0482] FORMUL 2 HOH 2(H2 O)

[0483] CRYST1 70.810 4.820 16.790 90.00 105.68 90.00 C 1 2 1 4

[0484] ORIGX1 1.000000 0.000000 0.000000 0.00000

[0485] ORIGX2 0.000000 1.000000 0.000000 0.00000

[0486] ORIGX3 0.000000 0.000000 1.000000 0.00000

[0487] Ratio 1 0.014122 0.000000 0.003964 0.00000

[0488] Ratio 2 0.000000 0.207469 0.000000 0.00000

[0489] Ratio 3 0.000000 0.000000 0.061861 0.00000

[0490] Atom 1 N GLY A 1 -1.664 2.302 6.349 1.00 23.58 N

[0491] Atom 2 CA GLY A 1 -1.194 2.605 5.007 1.00 23.02 C

[0492] Atom 3 C GLY A 1 0.105 1.901 4.702 1.00 21.89 C

[0493] Atom 4 O GLY A 1 0.121 0.676 4.579 1.00 20.14 O

[0494] Atomic 5 N ALA A 2 1.197 2.666 4.558 1.00 16.28 N

[0495] Atom 6 CA ALA A 2 2.509 2.094 4.221 1.00 15.41 C

[0496] Atom 7 C ALA A 2 3.664 2.731 5.005 1.00 12.05 C

[0497] Atom 8 O ALA A 2 3.668 3.942 5.227 1.00 10.77 O

[0498] Atom 9 CB ALA A 2 2.764 2.186 2.712 1.00 15.67 C

[0499] Atom 10 N VAL A 3 4.640 1.905 5.417 1.00 5.98 N

[0500] Atom 11 CA VAL A 3 5.852 2.304 6.129 1.00 3.93 C

[0501] Atom 12 C VAL A 3 7.023 1.700 5.344 1.00 5.93 C 4

[0503] Atomic 13 O VAL A 3 7.217 0.485 5.388 1.00 6.66 O

[0504] Atom 14 CB VAL A 3 5.856 1.891 7.629 1.00 7.66 C

[0505] Atom 15 CG1 VAL A 3 7.148 2.337 8.330 1.00 6.70 C

[0506] Atom 16 CG2 VAL A 3 4.661 2.494 8.333 1.00 6.73 C

[0507] Atom 17 N VAL A 4 7.702 2.522 4.533 1.00 3.26 N

[0508] Atom 18 CA VAL A 4 8.782 2.087 3.638 1.00 4.61 C

[0509] Atom 19 C VAL A 4 10.065 2.743 4.088 1.00 4.32 C

[0510] Atomic 20 O VAL A 4 10.149 3.973 4.071 1.00 3.00 O

[0511] Atom 21 CB VAL A 4 8.458 2.457 2.171 1.00 8.53 C

[0512] Atom 22 CG1 VAL A 4 9.486 1.874 1.188 1.00 9.58 C

[0513] Atom 23 CG2 VAL A 4 7.041 2.046 1.790 1.00 9.20 C

[0514] Atom 24 N THR A 5 11.066 1.952 4.438 1.00 3.32 N

[0515] Atom 25 CA THR A 5 12.360 2.426 4.896 1.00 3.88 C

[0516] Atom 26 C THR A 5 13.504 1.769 4.092 1.00 5.43 C

[0517] Atom 27 O THR A 5 13.567 0.539 4.039 1.00 6.20 O

[0518] Atom 28 CB THR A 5 12.540 2.051 6.351 1.00 6.84 C

[0519] Atom 29 OG1 THR A 5 11.414 2.503 7.130 1.00 9.13 O

[0520] Atom 30 CG2 THR A 5 13.891 2.524 6.943 1.00 10.98 C

[0521] Atom 31 N GLY A 6 14.417 2.588 3.546 1.00 6.04 N

[0522] Atom 32 CA GLY A 6 15.571 2.100 2.793 1.00 6.46 C

[0523] Atom 33 C GLY A 6 16.874 2.688 3.292 1.00 7.38 C

[0524] Atomic 34 O GLY A 6 16.959 3.896 3.501 1.00 6.32 O

[0525] Atom 35 N VAL A 7 17.913 1.864 3.436 1.00 4.11 N

[0526] Atom 36 CA VAL A 7 19.223 2.311 3.884 1.00 5.04 C

[0527] Atom 37 C VAL A 7 20.243 1.730 2.870 1.00 4.68 C

[0528] Atomic 38 O VAL A 7 20.244 0.519 2.678 1.00 5.69 O

[0529] Atom 39 CB VAL A 7 19.532 1.802 5.294 1.00 9.15 C

[0530] Atom 40 CG1 VAL A 7 21.007 1.991 5.629 1.00 9.39 C

[0531] Atom 41 CG2 VAL A 7 18.606 2.431 6.367 1.00 9.75 C

[0532] Atom 42 N THR A 8 21.105 2.570 2.279 1.00 3.68 N

[0533] Atom 43 CA THR A 8 22.201 2.169 1.357 1.00 4.35 C

[0534] Atom 44 C THR A 8 23.468 2.783 1.930 1.00 6.01 C

[0535] Atom 45 O THR A 8 23.530 4.005 2.031 1.00 6.48 O

[0536] Atom 46 CB THR A 8 21.874 2.496 -0.083 1.00 12.43 C

[0537] Atom 47 OG1 THR A 8 20.601 1.921 -0.394 1.00 14.76 O

[0538] Atom 48 CG2 THR A 8 22.929 1.954 -1.075 1.00 13.32 C

[0539] Atomic 49 N ALA A 9 24.453 1.977 2.339 1.00 7.04 N

[0540] Atom 50 CA ALA A 9 25.628 2.542 2.994 1.00 9.57 C

[0541] Atom 51 C ALA A 9 26.967 1.849 2.849 1.00 11.59 C

[0542] Atomic 52 O ALA A 9 27.052 0.628 2.756 1.00 8.18 O

[0543] Atom 53 CB ALA A 9 25.328 2.729 4.478 1.00 10.30 C

[0544] Atom 54 N VAL A 10 28.017 2.646 2.956 1.00 10.58 N

[0545] Atom 55 CA VAL A 10 29.405 2.206 3.043 1.00 12.39 C

[0546] Atom 56 C VAL A 10 29.896 2.880 4.314 1.00 18.51 C

[0547] Atomic 57 O VAL A 10 30.171 4.075 4.301 1.00 16.08 O

[0548] Atom 58 CB VAL A 10 30.273 2.561 1.816 1.00 17.83 C

[0549] Atom 59 CG1 VAL A 10 31.744 2.243 2.085 1.00 18.89 C

[0550] Atom 60 CG2 VAL A 10 29.778 1.831 0.567 1.00 17.39 C

[0551] Atomic 61 N ALA A 11 29.916 2.136 5.427 1.00 17.81 N

[0552] Atom 62 CA ALA A 11 30.340 2.647 6.726 1.00 23.47 C

[0553] Atom 63 C ALA A 11 31.643 1.988 7.097 1.00 51.68 C

[0554] Atomic 64 O ALA A 11 31.622 0.916 7.734 1.00 58.63 O

[0555] Atom 65 CB ALA A 11 29.289 2.350 7.778 1.00 24.49 C

[0556] Atom 66 OXT ALA A 11 32.691 2.485 6.653 1.00 74.50 O

[0557] TER 67 ALA A 11

[0558] HETATM 68 O HOH A 101 9.830 4.990 7.085 1.00 11.92 O

[0559] HETATM 69 O HOH A 102 18.548 3.873 0.260 1.00 20.36 O

[0560] The main 203 0 0 0 0 0 0 6 68 1 0 1

[0561] Finish 5

[0563] The atomic coordinates of the structure of PreNAC:GVVHGVTTVA (SEQ ID NO:47) are shown in Table 4.

[0564] Table 4

[0565] Header----27-APR-15 xxxx

[0566] Compound---

[0567] Note 3

[0568] Note 3: Detailed.

[0569] Note 3: Program: REFMAC 5.8.0073

[0570] Note 3 Authors: Murshhudov, Skubak, Lebedev, Pannu

[0571] Note 3: STEINER, NICHOLLS, WINN, LONG, VAGIN

[0572] Note 3

[0573] Note 3: Refine the objective: Maximum likelihood

[0574] Note 3

[0575] Note 3: Data for further refinement.

[0576] Note 3: High resolution range (angstroms): 1.41

[0577] Note 3: Low resolution range (angstroms): 32.94

[0578] Note 3: Data cutoff value (∑(F)): None

[0579] Note 3: Scope completeness (%): 86.73

[0580] Note 3: Number of reflections: 1006

[0581] Note 3

[0582] Note 3 is suitable for detailed data.

[0583] Note 3: Cross-validation: from beginning to end

[0584] Note 3: Free R-value test set selection: random

[0585] Note 3: R-value (working set + test set): 0.26104

[0586] Note 3: R-value (working set): 0.25243

[0587] Note 3: Free R-value: 0.33523

[0588] Note 3: Free R-value test set size (%): 10.0

[0589] Note 3: Free R-value test set count: 112

[0590] Note 3

[0591] Note 3: Suitable for the highest resolution libraries.

[0592] Note 3: Total number of face values ​​used: 20

[0593] Note 3: High-resolution area of ​​facets: 1.409

[0594] Note 3: Low-resolution library range: 1.446

[0595] Note 3: Reflection (working set) in library 3: 49

[0596] Note 3: Library completeness (working + testing) (%): 51.43

[0597] Note 3: Surface area R-value (working set): 0.388

[0598] Note 3: Count of free R-value sets for surface elements: 5

[0599] Note 3: Surface element free R-value: 0.528

[0600] Note 3

[0601] Note 3: Number of non-hydrogen atoms used for refinement

[0602] Note 3: All atoms: 68

[0603] Note 3

[0604] Note 3: B value.

[0605] Note 3 is from Wilson plotting method (A 2): None

[0606] Note 3: Mean B value (population, A) 2): 19.367

[0607] Note 3: Overall anisotropy B value.

[0608] Note 3 B11 (A 2): -0.23

[0609] Note 3 B22 (A 2): -0.23

[0610] Note 3 B33 (A) 2): 0.49

[0611] Note 3 B12 (A 2): 0.00

[0612] Note 3 B13 (A 2): -0.30

[0613] Note 3 B23 (A 2): -0.00

[0614] Note 3

[0615] Note 3: Estimate the overall coordinate error.

[0616] Note 3: ESU(A) based on R value: 0.118

[0617] Note 3: ESU(A) based on the free R value: 0.133

[0618] Note 3: ESU(A) based on maximum likelihood: 0.155

[0619] Note 3: ESU(A) based on the maximum likelihood B value 2): 4.934

[0620] Note 3

[0621] Note 3: Correlation coefficient.

[0622] Note 3: Correlation coefficient FO-FC: 0.950

[0623] Note 3: Free correlation coefficient FO-FC: 0.895

[0624] Note 3

[0625] Note 3: RMS weighting for RMS deviation from the ideal value.

[0626] Note 3: Bond length refinement atoms (A): 66; 0.017; 0.019

[0627] Note 3: Other bond lengths (A): 69; 0.010; 0.020

[0628] Note 3: Bond angle refinement (degrees): 91; 1.876; 1.905 1

[0630] Note 3: Other bond angles (degrees): 155; 0.648; 3.000

[0631] Note 3: Torsion angle, period 1 (degree): 9; 5.684; 5.000

[0632] Note 3: Torsion angle, period 2 (degrees): 1; 75.981; 20.000

[0633] Note 3: Torsion angle, period 3 (degrees): 7; 7.001; 15.000

[0634] Note 3: Chiral center limit (A) 3): 14; 0.098; 0.200

[0635] Note 3: General planar refinement of atoms (A): 73; 0.005; 0.020

[0636] Note 3 Other general planes (A): 13; 0.001; 0.020

[0637] Note 3

[0638] Note 3: Limits for isotropic thermodynamic factors. Calculate RMS weights.

[0639] Note 3: Main chain bond refinement atoms (A) 2): 39; 6.545; 1.715

[0640] Note 3 Other atoms in the main chain (A) 2): 38; 3.948; 1.607

[0641] Note 3: Main chain corner refinement atoms (A) 2): 41; 8.395; 2.554

[0642] Note 3 Other atoms at the main chain angle (A) 2): 42; 8.555; 2.605

[0643] Note 3: Side chain bond refinement atoms (A) 2): 27; 5.989; 2.165

[0644] Note 3: Other atoms in the side chain (A) 2): 27; 5.243; 2.077

[0645] Note 3: Other atoms at the side chain angle (A) 2): 44; 8.091; 3.056

[0646] Note 3: Long-range B refines atoms (A) 2): 54; 9.624; 14.765

[0647] Note 3: Long range B other refined atoms (A) 2): 55; 11.399; 15.221

[0648] Note 3

[0649] Note 3: NCS Restrictions on Statistical Data

[0650] Note 3: Number of NCS groups: zero

[0651] Note 3

[0652] Note 3: Twin Details

[0653] Note 3: Number of twin domains: zero

[0654] Note 3

[0655] Note 3

[0656] Note 3: TLS Details

[0657] Note 3: Number of TLS groups: zero

[0658] Note 3

[0659] Note 3

[0660] Note 3: Modeling of bulk solvents.

[0661] Note 3: Method used: concealment

[0662] Note 3: Mask calculation parameters

[0663] Note 3: VDW probe radius: 1.20

[0664] Note 3: Ion probe radius: 0.80

[0665] Note 3: Shrinkage radius: 0.80

[0666] Note 3

[0667] Note 3: Other detailed notes:

[0668] Note 3: Hydrogen has been added to the placement location.

[0669] Note 3: U-value: Refined separately

[0670] Note 3

[0671] Crystal 1 17.930 4.710 33.030 90.00 94.33 90.00 P 1 21 1

[0672] Scale 1 0.055772 0.000000 0.004219 0.00000

[0673] Scale 2 -0.000000 0.212314 0.000000 0.00000

[0674] Scale 3 0.000000 -0.000000 0.030362 0.00000

[0675] Atom 1 N GLY A 47 -7.911 -0.639 18.278 1.00 20.19 N

[0676] Atom 2 CA GLY A 47 -6.901 0.239 18.839 1.00 16.21 C

[0677] Atom 3 C GLY A 47 -5.632 -0.528 19.111 1.00 16.40 C

[0678] Atomic 4 O GLY A 47 -5.600 -1.727 19.011 1.00 22.51 O

[0679] Atom 5 N VAL A 48 -4.568 0.169 19.444 1.00 17.59 N

[0680] Atom 6 CA VAL A 48 -3.313 -0.460 19.729 1.00 15.77 C

[0681] Atom 7 CB VAL A 48 -2.269 -0.088 18.637 1.00 17.32 C

[0682] Atom 8 CG1 VAL A 48 -0.855 -0.462 19.056 1.00 17.64 C

[0683] Atom 9 CG2 VAL A 48 -2.659 -0.731 17.326 1.00 18.74 C

[0684] Atom 10 C VAL A 48 -2.906 0.080 21.062 1.00 13.82 C

[0685] Atomic 11 O VAL A 48 -3.040 1.262 21.327 1.00 18.65 O

[0686] Atom 12 N VAL A 49 -2.365 -0.782 21.895 1.00 16.57 N

[0687] Atom 13 CA VAL A 49 -1.779 -0.342 23.136 1.00 16.11 C

[0688] Atom 14 CB VAL A 49 -2.592 -0.887 24.317 1.00 16.48 C

[0689] Atom 15 CG1 VAL A 49 -1.861 -0.622 25.628 1.00 20.39 C

[0690] Atom 16 CG2 VAL A 49 -4.002 -0.324 24.303 1.00 20.73 C

[0691] Atom 17 C VAL A 49 -0.366 -0.882 23.250 1.00 13.58 C

[0692] Atomic 18 O VAL A 49 -0.178 -2.043 23.137 1.00 20.25 O

[0693] Atom 19 N HIS A 50 0.614 -0.063 23.557 1.00 16.22 N 2

[0695] Atom 20 CA HIS A 50 2.002 -0.524 23.593 1.00 15.91 C

[0696] Atom 21 CB HIS A 50 2.612 -0.176 22.220 1.00 18.19 C

[0697] Atom 22 CG HIS A 50 4.036 -0.543 22.024 1.00 23.24 C

[0698] Atomic 23 ND1 HIS A 50 4.728 -1.390 22.851 1.00 32.41 N

[0699] Atom 24 CE1 HIS A 50 5.964 -1.539 22.399 1.00 29.90 C

[0700] Atom 25 NE2 HIS A 50 6.088 -0.839 21.291 1.00 34.03 N

[0701] Atom 26 CD2 HIS A 50 4.892 -0.215 21.028 1.00 33.27 C

[0702] Atom 27 C HIS A 50 2.680 0.171 24.736 1.00 14.51 C

[0703] Atom 28 O HIS A 50 2.686 1.396 24.776 1.00 21.97 O

[0704] Atom 29 N GLY A 51 3.213 -0.580 25.689 1.00 15.69 N

[0705] Atom 30 CA GLY A 51 3.877 0.021 26.829 1.00 11.67 C

[0706] Atom 31 C GLY A 51 2.957 0.800 27.740 1.00 12.93 C

[0707] Atomic 32 O GLY A 51 2.967 2.022 27.786 1.00 20.51 O

[0708] Atom 33 N VAL A 52 2.174 0.081 28.498 1.00 15.10 N

[0709] Atom 34 CA VAL A 52 1.315 0.692 29.514 1.00 14.31 C

[0710] Atom 35 CB VAL A 52 -0.162 0.399 29.278 1.00 14.02 C

[0711] Atom 36 CG1 VAL A 52 -1.002 0.803 30.489 1.00 16.11 C

[0712] Atom 37 CG2 VAL A 52 -0.626 1.146 28.077 1.00 15.58 C

[0713] Atom 38 C VAL A 52 1.778 0.094 30.802 1.00 13.92 C

[0714] Atom 39 O VAL A 52 1.742 -1.133 30.969 1.00 20.14 O

[0715] Atom 40 N THR A 53 2.253 0.954 31.698 1.00 17.96 N

[0716] Atom 41 CA THR A 53 2.886 0.481 32.943 1.00 18.30 C

[0717] Atom 42 CB THR A 53 4.381 0.885 33.010 1.00 20.92 C

[0718] Atom 43 OG1 THR A 53 5.064 0.409 31.840 1.00 29.94 O

[0719] Atom 44 CG2 THR A 53 5.063 0.255 34.175 1.00 22.40 C

[0720] Atom 45 C THR A 53 2.116 1.037 34.132 1.00 14.85 C

[0721] Atom 46 O THR A 53 1.778 2.193 34.103 1.00 21.57 O

[0722] Atom 47 N THR A 54 1.840 0.202 35.151 1.00 15.66 N

[0723] Atom 48 CA THR A 54 1.239 0.607 36.427 1.00 14.08 C

[0724] Atom 49 CB THR A 54 -0.184 0.059 36.602 1.00 18.69 C

[0725] Atom 50 OG1 THR A 54 -1.031 0.491 35.534 1.00 35.75 O

[0726] Atom 51 CG2 THR A 54 -0.768 0.525 37.872 1.00 23.36 C

[0727] Atom 52 C THR A 54 2.061 -0.047 37.500 1.00 12.83 C

[0728] Atom 53 O THR A 54 2.023 -1.258 37.626 1.00 22.40 O

[0729] Atom 54 N VAL A 55 2.821 0.725 38.262 1.00 14.38 N

[0730] Atom 55 CA VAL A 55 3.639 0.203 39.339 1.00 13.58 C

[0731] Atom 56 CB VAL A 55 5.099 0.641 39.138 1.00 15.65 C

[0732] Atom 57 CG1 VAL A 55 5.915 0.347 40.358 1.00 16.16 C

[0733] Atom 58 CG2 VAL A 55 5.696 -0.078 37.939 1.00 17.66 C

[0734] Atom 59 C VAL A 55 3.100 0.756 40.673 1.00 14.57 C

[0735] Atomic 60 O VAL A 55 2.916 1.968 40.780 1.00 16.23 O

[0736] Atomic 61 N ALA A 56 2.809 -0.130 41.639 1.00 17.51 ​​N

[0737] Atom 62 CA ALA A 56 2.466 0.268 43.022 1.00 26.29 C

[0738] Atom 63 CB ALA A 56 1.113 -0.279 43.465 1.00 21.51 C

[0739] Atom 64 C ALA A 56 3.547 -0.222 43.971 1.00 38.92 C

[0740] Atomic 65 O ALA A 56 3.757 -1.425 44.111 1.00 69.25 O

[0741] Atom 66 OXT ALA A 56 4.249 0.549 44.634 1.00 54.03 O

[0742] HETATM 67 O HOH B 1 4.715 2.482 29.991 1.00 27.67 O

[0743] HETATM 68 O HOH B 2 -1.034 3.065 33.500 1.00 32.66 O 3

[0745] References

[0746]

[0747]

[0748]

[0749]

[0750]

[0751] From the foregoing description, those skilled in the art can readily identify the essential features of the invention, and changes and modifications can be made to adapt it to various uses and conditions and to maximize its utilization without departing from its spirit and scope. The foregoing embodiments should be interpreted as merely illustrative and do not limit the scope of the invention in any way. The entire disclosure of all applications, patents, and publications cited herein and in the accompanying drawings is hereby incorporated by reference, in its entirety, and particularly with respect to the information cited therein.

Claims

1. A composition comprising at least one inhibitory peptide, said inhibitory peptide inhibiting α-synuclein (SEQ ID NO:1) aggregation by binding to residues 68-78 of α-synuclein; wherein: The inhibitory peptide consists of the following sequence: GAVVWGVTAVKKKKK (SEQ ID NO: 5); GAVVWGVTAVKKGRKKRRQRRRPQ (SEQ ID NO: 6); or YGRKKRRQRRRAVVT{N-me-Gly}VTAVAE.

2. The composition of claim 1, wherein the composition comprises a plurality of inhibitory peptides.

3. A pharmaceutical composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier, said pharmaceutically acceptable carrier comprising a peptide stabilizing excipient.

4. A complex comprising α-synuclein and the peptide composition of claim 1.

5. An expression vector encoding the repressor peptide of claim 1.

6. A method for preparing the peptide of claim 1, comprising chemically synthesizing the peptide or recombinantly generating the peptide.

7. A modified peptide, which is: GAVVWGVTAVKKKKK (SEQ ID NO: 5); GAVVWGVTAVKKGRKKRRQRRRPQ (SEQ ID NO: 6); or YGRKKRRQRRRAVVT{N-me-Gly}VTAVAE.

8. Use of the inhibitory peptide of claim 1 in the preparation of a composition for reducing or inhibiting the aggregation of α-synuclein (SEQ ID NO: 1), wherein the composition is used in a method comprising contacting α-synuclein amyloid fibrils with the inhibitory peptide of claim 1 in an amount sufficient to reduce or inhibit α-synuclein aggregation.

9. The use as claimed in claim 8, wherein the α-synuclein amyloid protofibrils are in vivo.

10. Use of the inhibitory peptide of claim 1 in the preparation of a composition for regulating the size or growth rate of α-synuclein amyloid fibrils, or the diffusion rate of α-synuclein amyloid fibrils, wherein the composition is used in a method comprising: In an environment where the inhibitory peptide is in contact with residues 68-78 or 47-56 of α-synuclein, the protofibril is contacted with a certain amount of the inhibitory peptide of claim 1. This causes the contacted α-synuclein amyloid fibrils to exhibit regulated size or growth rate.

11. Use of the inhibitory peptide of claim 1 in the preparation of a composition for observing the presence or absence of α-synuclein amyloid fibrils in a biological sample, wherein the composition is used in a method comprising: (a) Combining a biological sample with the inhibitory peptide of claim 1; (b) Binding the peptide to α-synuclein amyloid fibrils that may be present in the biological sample; and Monitoring combination (b) indicates the presence of a complex formed between α-synuclein amyloid protoplasts and the peptide; wherein the presence of the complex indicates the presence of α-synuclein amyloid protoplasts in the biological sample by means of MRI or PET or other imaging methods.

12. The use as claimed in claim 11, wherein the presence of a complex formed between the α-synuclein amyloid protoplast and the peptide is monitored using a detectable label conjugated to the peptide.

13. The use as described in claim 12, wherein the detectable tag includes a heteropeptide tag.

14. The use as claimed in claim 11, wherein the method is performed on a biological sample obtained from an individual suspected of having Parkinson's disease.

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