Use of D-enantiomerized peptide ligands of monomer α-synuclein for the treatment of various synuclein diseases

Abstract

The present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, and such peptide for use in the treatment of a synucleinopathic disease.

Description

Technical Field

[0001] The present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, their homologs, fragments and moieties, and such peptides for use in the treatment of synucleinopathies.

Background Art

[0002] Synucleinopathies encompass a heterogeneous group of neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are associated with misfolding and aggregation of the protein α-synuclein in specific cells.

[0003] Insoluble inclusions found in neurons affected by PD contain the amyloid form of α-synuclein, suggesting a causal relationship between amyloid formation and disease pathology. α-Synuclein aggregates in various synucleinopathies are presumed to correspond to different conformational states of α-synuclein that propagate in a prion-like manner and transfer from cell to cell.

[0004] According to data from the National Institute of Neurological Disorders and Stroke (https: / / www.ninds.nih.gov / ), there are currently no substances that can cure or effectively halt PD, DLB and MSA. Nevertheless, there are many active ingredients that can be used to treat the respective symptoms.

[0005] In summary, therapies that significantly extend lifespan based on causal relationships are currently not available, or at least not for most synucleinopathies, and are urgently needed.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, the objective of the present invention was to develop a novel chemical substance that can decompose existing toxic α-synuclein aggregates into naturally occurring functional α-synuclein monomers, thereby enabling therapeutic use in various synuclein diseases.

[0007] The chemical substances or their variants used in treatment are intended to bind to the native endogenous α-synuclein protein with the highest possible affinity and specificity, thereby stabilizing it. As a result, the balance between the misfolded α-synuclein structure and the naturally folded α-synuclein structure shifts in favor of the latter. Thus, ideally, already present α-synuclein oligomers and protofibrils can be broken down into their monomeric components and thus eliminated. [Means for solving the problem]

[0008] This objective is achieved by the peptide described in claim 1, in particular by a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and their homologs, fragments, and parts.

[0009] Further preferred embodiments are provided in the dependent claims. [Brief explanation of the drawing]

[0010] [Figure 1] Thioflavin-T assay using recombinant full-length L-enantiomer α-syn and D-enantiomer 16-mer peptide. [Figure 2] MST measurement of SVD-1 and α-syn. [Figure 3] Multi-cycle SPR experiments with wild-type α-synuclein and SVD-1. [Figure 4] Elimination of α-syn oligomers by SVD-1. [Figure 5]Quasi-stoichiometric inhibition of α-syn aggregation in ThT assays. [Figure 6] Gradient centrifugation of α-syn aggregated samples in the presence and absence of SVD-1. [Figure 7] Thioflavin-T assay using recombinant full-length L-enantiomer α-syn and D-enantiomer SVD-1a. [Figure 8] Single-cycle SPR experiments using wild-type α-syn and SVD-1a, SVD-6a, or SVD-10a. [Figure 9] De novo ThT assay and seeded ThT assay using SVD-1 and SVD-1_scrambled. [Figure 10] MST measurement of α-syn using SVD-1 and SVD-1a. MST measurement of the interaction between SVD-1 and SVD-1a and full-length α-syn at 25°C in PBS, pH 7.4. [Figure 11] A single-cycle kinetic assay using α-syn and immobilized SVD-1 and SVD-1a. [Figure 12] NMR analysis of 15N-labeled α-syn in interaction with SVD-1a (unlabeled). [Figure 13] Size exclusion chromatography of monomer α-syn after incubation with gradually increasing concentrations of SVD-1a. [Figure 14] Thioflavin-T de novo aggregation of monomer α-syn with SVD-1a and SVD-1_scrambled+5r. [Figure 15] De novo aggregation analysis of α-syn in the presence and absence of SVD-1a using ThT, DGC, CD, and AFM. [Figure 16] A ThT assay using SVD-1a and SVD-1_scrambled+5r as seeds. [Figure 17] SVD-1a decomposes PFF oligomers into α-syn monomers. [Figure 18] Analysis of agglutination assays in the presence and absence of SVD-1a using ThT assay and CD spectroscopy. [Figure 19] SVD-1a inhibits the aggregation of seeded α-syn in cells. [Figure 20] SVD-1 and SVD-1a increase cell viability in MTT assays using PFF oligomer seeds. [Modes for carrying out the invention]

[0011] Hereafter, "comprise" shall also include "consist of".

[0012] The peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 were found using optimized enantiomer phage display selection.

[0013] It should be noted that the enantiomer phage display method, along with the selection of specific monomer binders described herein, can be used to identify specific oligomer binders, or even ligands for other species that form in protein misfolding diseases.

[0014] In enantiomer phage display, for example, a recombinant library of randomized peptide sequences displayed on the gp3 protein of the M13 phage and encoded in its genome is selected for the exact mirror image (D-enantiomer) of a naturally occurring L-enantiomer target molecule (e.g., α-synuclein). The gp3 molecule, also known as gene product 3, is a protein in the phage capsid required for contact with the host cell.

[0015] The peptide sequence is advantageously presented at the N-terminus of the gp3 protein of the M13 phage and encoded within its genome.

[0016] The DNA sequence of the p3 gene of a selected phage is ligated to a DNA sequence containing genetic information about the corresponding peptide sequence on the gp3 molecule, which can then be sequenced. After sequencing, the genome sequence can be translated into an amino acid sequence and synthesized as a D-enantiomer peptide that binds to a target molecule in a physiological L-enantiomer form (e.g., α-synuclein). All phages that present different peptides on their surface as fusion proteins with gp3 are hereafter referred to as a phage library. The corresponding peptides represent biomolecules selected in the experiment.

[0017] Three rounds, called "biopanning" rounds (or selection rounds), can be performed. In this process, the phage library is brought into contact with immobilized target molecules, also known as bait, and bound phages are isolated from millions of other unbound phages that are similarly present.

[0018] For example, the number of phages that preferentially bind to α-synuclein oligomer or fibril species is reduced by not providing these very species as bait. This allows for the removal of phages with increased affinity for α-synuclein oligomers and fibrils from the phage pool, which means, for example, the accumulation of α-synuclein monomer-specific phages. This method can, of course, be similarly adapted to specifically determine ligands and peptides that bind to α-synuclein oligomers.

[0019] According to the present invention, in order to reduce any accumulation of phages having an affinity for plastic, BSA, or streptavidin, different substrate surfaces are preferably used in all biopanning rounds. In this case, the substrate surface is defined as a combination of the biotinylated substrate used (a surface of polystyrene or polypropylene derivatized with streptavidin) and the blocking or quenching agent used. In successive selection rounds, the selective pressure is gradually increased. For this purpose, the concentration of the target molecule (e.g., monomer α-synuclein) remains stable, but the number of washing steps after phage incubation is increased starting from the second selection round so as not to remove phages having an affinity for the α-synuclein monomer.

[0020] Furthermore, by using other agents (e.g., BSA, milk powder, or no blocking) to block (block) the surface after the target molecule has been immobilized on the substrate, different substrate surfaces can be selected in every selection round of phage display.

[0021] For example, there may be a switch between the BSA block polystyrene surface in Round 1, the milk powder block polypropylene surface in Round 2, and the BSA block polystyrene surface in Round 3.

[0022] Switching between different substrate surfaces increases the specificity of the target molecule or bait to the surface. Furthermore, this results in a reduction of ligands that nonspecifically bind to other components of the substrate surface other than the plastic surface, BSA, or bait molecule.

[0023] In parallel with the actual phage display selection, a control selection may be performed, for example, which is identical to the main selection but differs in that it does not use bait. Data analysis of sequences obtained from the control selection makes it possible to identify peptides that accumulate during selection even in the absence of bait and are therefore irrelevant to all subsequent steps.

[0024] Therefore, the method in question is as follows: a) A step of providing immobilized bait on the surface of a substrate, b) A step of contacting an immobilized molecule that acts as a bait with a solution containing a library of molecules to be selected, c) A step of bringing the immobilized bait surrounded by the above molecules into contact with a washing solution, d) A step of separating and propagating (Vermehrung) the molecules still bound to the bait after the immobilized bait surrounded by the above molecules has been brought into contact with a washing solution, e) A step of repeating the above process using a different substrate in each iteration, f) A step to identify the sequence of molecules remaining on the bait after iteration. It is characterized by the following.

[0025] For example, different substrates are used by changing the type of substrate and / or by blocking or not blocking the substrate with a reagent.

[0026] Molecules from the group consisting of proteins, peptides, RNA, DNA, mRNA, and chemical compounds are used as bait. The monomer α-synuclein is particularly used as bait.

[0027] For example, materials (components) from the group consisting of a microtitration plate, magnetic particles, agarose beads, or sepharose beads are used as the surface to which the bait is immobilized.

[0028] Therefore, the bait relating to point a) is a compound to which the biomolecule to be selected is bound. According to methods known from the prior art, the compound is immobilized on a first surface. Non-limiting examples of baits include proteins, peptides, RNA or DNA molecules, particularly α-synuclein monomers. Possible surfaces include, for example, microtitration plates, magnetic particles, agarose beads or Sepharose beads. Subsequently, the surface having the immobilized bait can be quenched, and the functional groups of the substrate are inactivated in this process. Furthermore, any remaining hydrophobic free surfaces on the substrate can be blocked with appropriate agents.

[0029] In the second step (b), the immobilized bait is brought into contact with a randomized library of molecules, particularly biomolecules. These biomolecules compete for binding to the bait. The randomized library contains a very large number of molecules in the mixture, e.g., 10 12 Different types of molecules, or 10 4 It is a mixture of one or as many as 100 different molecules. Such a library may consist, for example, of peptides, proteins, DNA, RNA, or mRNA that can bind to a specific vehicle and, in each case, to a bait. Examples of vehicles include phages, polysomes, or bacterial surfaces. The library may consist of artificial components, components isolated from nature, or a mixture of both. In the context of this invention, "artificial" means, for example, compounds produced from oligonucleotide synthesis.

[0030] In step c), the immobilized bait surrounded by biomolecules can be brought into contact with a washing substance. For this purpose, a washing step is performed in step c), in which a buffer is brought into contact with the immobilized bait or used to rinse the immobilized bait. This means that the solution containing the biomolecule library is repeatedly replaced, preferably with a similar or identical solution. This removes library molecules from the immobilized bait that do not dissociate faster than other library molecules. The rate of dissociation reactions of the bound library molecules is mainly determined by the different dissociation constants (especially k) of the individual molecules. off It is determined by the value. Therefore, statistically, a small k off Molecules with a value remain bound to the immobilized bait the longest and are therefore statistically less likely to be washed away by the washing buffer. The liquid containing the washing buffer is preferably aqueous and may contain a pH buffer. Optional components of the solution for the specific washing step may be salts, surfactants, or reducing agents.

[0031] Following the specificity washing step in step c), in step d), the bound biomolecules are separated from the bait and grown. Separation or elution may be performed, for example, by changing the pH, heating, or other changes, particularly by increasing the salt concentration. The separated or eluted biomolecules are then grown using known methods. For this purpose, for example, phage particles obtained and eluted after steps a) to c), with biomolecules supported on their surface, can be introduced into cells and grown.

[0032] In step e), the concentration of selected biomolecules in the solution supplied to the bait after step a) is increased. Preferably, 3 to 6 selection rounds including steps a) to e) are performed. However, 1 to 10 or 1 to 20 iterations may also be performed. Preferably, increasing the concentration of competing substances performed in step c) also results in improved selection with increasing number of cycles. Preferably, increasing the number of washing steps performed in step c) also results in improved selection with increasing number of cycles.

[0033] In particular, the relevant enantiomer phage display provides the N-terminally biotinylated D-enantiomer α-synuclein monomer in step a), the recombinant phage library in step b), and the buffer in step c), along with the α-synuclein monomer as bait. Elution as a separation step is performed, for example, by lowering the pH as a separation step, and phage amplification is performed as proliferation in step d).

[0034] In this way, seven D-enantiomer peptides that specifically bind to α-synuclein monomers were developed.

[0035] Sequence ID 1: SVD-1 (free N-terminus, amidated C-terminus): kmpthetywqehiwha

[0036] Sequence ID 2: SVD-6 (free N-terminus, amidated C-terminus): ydwkqpmsasrflapw

[0037] Sequence ID 3: SVD-10 (free N-terminus, amidated C-terminus): sshwqqwnppywntds

[0038] Sequence ID 4: SVD-14 (free N-terminus, amidated C-terminus): ydwkqpmsasrflapwr

[0039] Sequence ID 5: SVD-1a (free N-terminus, amidated C-terminus): rlpthetywqehiwharrrrr

[0040] Sequence ID 6: SVD-6a (free N-terminus, amidated C-terminus): ydwrqplsasrflapwrrrrr

[0041] Sequence ID 7: SVD-10a (free N-terminus, amidated C-terminus): sshwqqwnppywntdsrrrrr

[0042] Sequence processing (e.g., sequence mutations) of SVD-1, SVD-6, SVD-10, SVD-14, SVD-1a, SVD-6a, or SVD-10a offers the potential to develop other substances that can be used therapeutically in synuclein disease.

[0043] The present invention may also relate to other peptides that may have been identified using the methods disclosed above.

[0044] The peptides related to Sequence IDs 1-7 can be used as potential pharmaceuticals for synuclein diseases as a result of their specific binding to α-synuclein monomers.

[0045] The objectives of the present invention can also be achieved by peptides containing homologs, fragments, and portions of the amino acid sequences relating to SEQ ID NOs: 1 to 7.

[0046] In this specification, α-synuclein peptide or α-synuclein protein preferably means human α-synuclein peptide or α-synuclein protein.

[0047] In the context of the present invention, homologous sequence or “homogen” means that an amino acid sequence has at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with one of the above-mentioned amino acid sequences of the above-mentioned monomer. Of these, 80% and 90% are preferred. In this specification, the terms “homogen” and “homogeny” are used synonymously with “identity”. Identity between two nucleic acid sequences or polypeptide sequences is calculated by comparison using the BESTFIT program based on the algorithm by Smith, TF and Waterman, MS (Adv. Appl. Math. 2:482-489 (1981)), by adjusting the parameters for amino acids: Gap creation penalty: 8 and Gap extension penalty: 2, and for nucleic acids: Gap creation penalty: 50 and Gap extension penalty: 3. Preferably, identity between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid / polypeptide sequences over the same sequence length in each case, such that identity is calculated by comparison using the GAP program based on the algorithm by Needleman, SB and Wunsch, CD (J. Mol. Biol. 48:443-453), by adjusting the parameters for amino acids: Gap creation penalty: 8 and Gap extension penalty: 2, and for nucleic acids: Gap creation penalty: 50 and Gap extension penalty: 3.

[0048] In the context of this invention, two amino acid sequences are considered identical if they have the same amino acid sequence.

[0049] In further modified forms, the peptide according to the present invention has a sequence different from the sequence described for only two or three amino acids.

[0050] Furthermore, sequences containing the above sequence can also be used as peptides.

[0051] The peptide according to the present invention is more preferably characterized in that, at the free C-terminus, instead of a carboxyl group, there is an acid amide group (CONH2 group), a COH group, a COCl group, a COBr group, a CONH-alkyl group, or a CONH-alkylamine group, or the peptide is in a cyclized form.

[0052] This also achieves the objective of providing peptides that do not have a negative charge at the C-terminus, which is particularly advantageous. This means that the peptides can bind to target molecules with higher affinity than peptides that have a carboxyl group at the free C-terminus. Under physiological conditions, peptides with a free, unmodified carboxyl group have a negative charge at this terminus.

[0053] In one embodiment of the present invention, the peptide according to the present invention is modified such that, under physiological conditions, particularly at pH 6-8, particularly at pH 6.5-7.5, particularly at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, or pH 8.0, the C-terminus does not have a negative charge, but is instead neutral or has one or more positive charges.

[0054] In one embodiment, the peptide is characterized by the presence of an acid amide group instead of a carboxyl group at its free C-terminus. Therefore, an acid amide group (-CONH2 group) is positioned at the C-terminus instead of a carboxyl group (-COOH group).

[0055] Therefore, the peptide is particularly advantageously amidated at its free C-terminus.

[0056] Therefore, the peptide is particularly advantageous in that it is amidated at the free C-terminus and unmodified at the free N-terminus.

[0057] This also achieves the objective of having a peptide without excess negative charge that can bind to the target molecule with affinity and can be obtained in a simple manner, which is particularly advantageous.

[0058] In further embodiments of the present disclosure, the following additional groups may be present instead of the carboxyl group, such as COH, COCl, COBr, ONH-alkyl groups, CONH-alkylamine groups (with a net positive charge), but this is a non-limiting list as long as it follows the technical teachings of the main claims.

[0059] Therefore, in a further preferred embodiment of the present invention, the binding affinity of peptides modified according to the present invention that do not have a negative charge at the C-terminus is 1%, 2, 3, 4, 5, 6, 7, 8, 9, especially 10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 ,93,94,95,96,97,98,99,100%,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 1 87, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, especially 200%, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 ,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, especially 300%, 301, 302, 303, 304, 305, 306, 307, 308, 309, 31 0, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 ,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, especially 400%, 401, 402, 4 03, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 43 4, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465 ,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497, 498, 499, and even better, 500%, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 ,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 5 88, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, especially advantageous at 600%, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617 ,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 6 80, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, especially advantageous at 700%, 701, 702, 703, 704, 705, 706, 707, 708, 709 ,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 7 72, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, and similarly, particularly advantageous at 800%, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 8 32, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 86 3, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894 ,895,896,897,898,899, similarly particularly advantageous at 900%,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918,919,920,921,922,92 3, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954 ,955,956,957,958,959,960,961,962,963,964,965,966,967,968,969,970,971,972,973,974,975,976,977,978,979,980,981,982,983,984,985,It increases to 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or even 1000%, or even 10000%, or even 100000%, or 1000000% (any intermediate value can be used).

[0060] This corresponds to a decrease in K. D The value is indicated by K as a measure of the binding affinity of the modified peptide to the α-synuclein monomer. D The values ​​are compared to linearly bound peptides with a negative charge at the free C-terminus: 1%, 2, 3, 4, 5, 6, 7, 8, 9, especially 10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , decreases to 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, especially 99.1, 99.2, 99.3, 99.4, 99.5%, 99.6, 99.7, 99.8, 99.9~99.99 or even 99.999% (any intermediate value can be used).

[0061] The peptide according to the present invention is more preferably characterized in that the peptide contains two, three, four, five, six, seven, eight, nine, ten or more copies of the sequence having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0062] It is also possible to recall variants in which the peptide contains 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0063] Dimers of sequences having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7 are particularly preferred, wherein both monomers are peptides having the same SEQ ID NO: or different SEQ ID NOs.

[0064] The peptide according to the present invention is more preferably characterized in that the peptide consists substantially of D-amino acids.

[0065] In the context of the present invention, the term “substantially consisting of D-enantiomer amino acids” means that the monomers used in accordance with the present invention are formed from D-enantiomer amino acids in a proportion of at least 50%, 55%, 60%, 65%, 70%, preferably 75%, 80%, particularly preferably 85%, 90%, 95%, and especially up to 96%, 97%, 98%, 99%, and 100%.

[0066] The peptide according to the present invention is more preferably characterized in that the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0067] The peptide according to the present invention is more preferably characterized in that the peptide is linked to another substance.

[0068] In the context of the present invention, a bond is a chemical bond, preferably a principal valence bond, and more particularly a covalent bond, as defined in Roempp Chemie Lexikon, 9th edition, Vol. 1, pp. 650 onwards, by Georg Thieme Verlag, Stuttgart.

[0069] The above-mentioned substances are medicinal products or variants of active ingredients as defined in accordance with Article 2 or 4(19) of the German Medicines Act, September 2012 edition. Alternatively, the active ingredient is a therapeutic active substance used as a pharmacoactive substance. Preferably, anti-inflammatory agents are used.

[0070] In a further modified form, the substance is a compound that enhances the effect of the peptide.

[0071] In a further alternative form, the substance is a compound that improves the solubility and / or crossing of the peptide across the blood-brain barrier.

[0072] Alternatively, according to the present invention, the peptide has any desired combination of at least two or more features of the above-described modified forms, embodiments, and / or alternative forms.

[0073] The peptide according to the present invention is more preferably characterized in that a plurality of peptides, such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, are linked to each other by covalent or non-covalent bonds.

[0074] In the context of the present invention, covalent bonding or linking of peptide units exists when two peptides are linearly linked to each other either head-to-head, tail-to-tail, or head-to-tail, with or without a linker group inserted between them.

[0075] The peptide according to the present invention is more preferably characterized in that a plurality of peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 are linked to each other without a linker, that is, they are directly linked to each other or linked to each other using a linker group.

[0076] In the context of the present invention, non-covalent linkage exists when peptides are linked to each other, for example, via biotin and streptavidin, particularly streptavidin tetramer.

[0077] The peptide according to the present invention is more preferably characterized in that a plurality of peptides, such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, are linked to each other in a linear or branched manner.

[0078] In a modified version of the present invention, the peptides may be linked linearly to one another, as described above. In another modified version, the peptides are linked to one another in a branched manner to give the peptide according to the present invention.

[0079] According to the present invention, the branched peptide may be a dendrimer in which the above monomers are linked to each other by covalent or non-covalent bonds.

[0080] Alternatively, the peptide can be linked to a platform molecule (e.g., PEG or sugar) to form a branched peptide.

[0081] Alternatively, a combination of these options is also possible.

[0082] In one configuration of the present invention, the binding affinity of the peptide is determined by the dissociation constant (K D Defined by the value.

[0083] In one advantageous configuration of the present invention, the dissociation constant (K) of the peptide according to the present invention is D The value is advantageously reduced. This is related to the improved properties of the peptide according to the present invention, such as higher binding affinity and higher degradation efficiency, and / or prevention of the formation of toxic α-synuclein oligomers or aggregates.

[0084] In a modified version of the present invention, the dissociation constant (K) is up to 500 μM, preferably 250, 100, 50 μM, and particularly preferably 25, 10, 1 μM.D value), particularly preferably up to 500 nM, 250, 100, 50, particularly preferably 25, 10, 1 nM, 500 pM, 100, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 pM to less than 1 pM (any intermediate value can be adopted) of the dissociation constant (K D value), a peptide that binds to the α-synuclein monomer is used.

[0085] The peptides according to the present invention are more preferably those having a K of less than 50 μM D and bind to an α-synuclein peptide, preferably an unfolded α-synuclein peptide.

[0086] The peptide can be produced, for example, by chemical synthesis or peptide synthesis.

[0087] A peptide according to any one of the preceding claims for preventing the formation of α-synuclein peptide oligomers and / or α-synuclein peptide aggregates.

[0088] In a further variant, the peptide is a peptide for inhibiting or preventing the formation of α-synuclein peptide oligomers and / or α-synuclein peptide aggregates.

[0089] In a further variant, the peptide is a peptide for detoxifying α-synuclein peptide oligomers and / or α-synuclein peptide aggregates.

[0090] The peptides according to the present invention detoxify α-synuclein peptide oligomers and / or α-synuclein peptide aggregates or polymers formed therefrom, such as protofibrils, by, preferably, binding to α-synuclein monomers rather than to them, and by shifting the equilibrium, thereby reducing the α-synuclein oligomers and preventing them from being converted into toxic compounds. Accordingly, another subject of the present invention is a method for detoxifying α-synuclein oligomers and aggregates or protofibrils formed therefrom.

[0091] Inhibition or prevention of the formation of α-synuclein peptide oligomers and / or α-synuclein peptide aggregates, and detoxification of α-synuclein peptide oligomers and / or α-synuclein peptide aggregates can be performed in vitro or in vivo.

[0092] The present invention also relates to the above-mentioned peptides for use in the treatment of synuclein disease.

[0093] The present invention also relates to the above peptides for use in the treatment of Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA), particularly those related to the misfolding and aggregation of the protein α-synuclein in specific cells.

[0094] The present invention also relates in particular to the above-mentioned peptides for use in the treatment of Parkinson's disease (PD).

[0095] The present invention will be described in more detail below using non-limiting embodiments. [Examples]

[0096] Peptides SVD-1, SVD-6, SVD-10, and SVD-14, SVD-1a, SVD-6a, and SVD-10a were examined in more detail.

[0097] Materials and methods: Recombinant expression and cleaning of wild-type (WT) α-syn and α-syn A140C monomers:

[0098] N-terminally acetylated wild-type α-syn (hereinafter referred to as α-syn) and acetylated α-syn-A140C were expressed together with N-terminal acetyltransferases from Schizosaccharomyces pombe in E. coli BL21(DE3) vectors containing codon-optimized α-syn in pT7 and pNatB vectors. Expression was performed using LB containing 1 mM IPTG or 15 In N-saturated M9 minimum medium, 1.2 OD 600 Elution occurred after reaching the target level and subsequently incubation at 37°C for 4 hours (h). A pellet from 1 liter of expression was resuspended in 25 ml of 20 mM Tris, pH 8.0, and boiled at 95–100°C for 2 × 15 minutes (min). After centrifugation at 20,000 × g for 30 minutes at 4°C, the supernatant was precipitated at a final concentration of 0.45 g / ml ammonium sulfate. This protein was pelletized at 20,000 × g for 30 minutes and resuspended in 50 ml of 20 mM Tris-HCl, pH 8.0. After sterile filtration, the sample was loaded onto a HiPrep QFF 16 / 10 (Cytiva, USA, CV=20 ml) anion exchange column. Gradient elution was performed over 20 CVs with the target concentration of 800 mM NaCl. Recombinant α-syn eluted with an electrical conductivity of 28–32 mS / cm. The fraction containing recombinant α-syn was pooled and precipitated with ammonium sulfate as described above. The pellet was resuspended in 5 ml of 50 mM Tris-HCl, pH 7.4, 50 mM NaCl and loaded onto a HiLoad Superdex 60 / 75 pg gel filtration column (Cytiva, USA, CV=120 ml). Expression was measured at 5600 M. -1 cm -1 The concentration was determined using A275, which has an extinction coefficient, to be 20-30 mg / l. This protein aliquot was frozen in liquid nitrogen and stored at -80°C.

[0099] D-enantiomer peptide: D-enantiomer peptides having a C-terminal amidation were obtained from CASLO (Denmark) as lyophilized chloride salt powder with a purity of >95%. The peptides, e.g., SVD-1 and SVD-1a, were tested under different buffers including PBS, pH 7.4, and under various conditions. UV-vis absorbance measurements after incubation at 37°C for 1 hour and centrifugation at 20,800 × g showed that both compounds remained completely in the supernatant up to an initial concentration of at least 1 mM.

[0100] Thioflavin T assay: The thioflavin T (ThT) assay is conventionally used to visualize α-syn fibrillation. This is because the dye ThT can bind to the amyloid-forming cross-β-pleated sheet portion of the fibril structure. Recombinant α-syn was thawed on ice and centrifuged at 21,000 × g and 4°C for 30 minutes. The concentration of the supernatant was measured. Lyophilized D-peptide was thawed at room temperature (RT) for 1 hour and dissolved in 500 μl of PBS, pH 7.4. After centrifuging at 21,000 × g for 30 minutes, A 280 The supernatant concentration was determined by UV-vis using the corresponding extinction coefficients. All ThT assay experiments were performed at 37°C using 15 μM ThT and 0.05% sodium azide (w / v) in PBS, pH 7.4, unless explicitly stated otherwise. ThT fluorescence was measured orbitally averaging up to 3 mm from the center using a fluorescence plate reader (Clariostar or Polarstar Optima, BMG Labbiotech, Germany) with λ ex =448nm and λ emMonitoring was performed with a baseline optical configuration at 482 nm. Before starting measurements, 120 μl of sample solution was added to an unbound 96-well half-area plate (Corning, USA) with a clear, flat base. For all de novo agglutination assays, borosilicate glass beads (d=3.0 mm, Hilgenberg, Germany) were added to each well. For inoculated agglutination assays, beads were not used, and the samples were incubated under static conditions. The wells surrounding the sample wells were filled with the same volume of buffer to improve heat distribution. Unless explicitly stated otherwise, experiments were performed in 5 replicates (n=5).

[0101] The ThT assay was used for different purposes. Firstly, the de novo ThT agglutination assay served as a screening platform for agglutination delay by synthetic D-peptides. In this case, 50 μM recombinant α-syn was incubated with each D-peptide in a 3-fold molar excess. Samples were shaken at 300 rpm for 30 seconds (s) in orbital shaking mode before each cycle. Peptides insoluble in aqueous buffer were dissolved in 2.5 μl of DMSO (0.5 mg peptide) and gradually mixed with PBS, pH 7.4 until a final concentration of 2.5% (v / v) DMSO was reached. Reference agglutination of α-syn alone in PBS, pH 7.4 containing 2.5% (v / v) DMSO was also performed for these samples. The agglutination curves for each repeat were individually adjusted using a symmetric Boltzmann sigmoid fit (OriginPro 2020, OriginLab, USA) with the following formula.

number

[0102] (A1 = starting value, A2 = ending value, x0 = inflection point [s], dx = time constant [1 / s]).

[0103] The inflection point of the adjustment is the aggregation half-life t. 1 / 2 Determine the delay time, and the formula is t 1 / 2 -2·dt 1 / 2Approximate using, in the formula, dt 1 / 2 is, x=t 1 / 2 It is defined as the gradient of the adjustment (unit: 1 / s). Half-life and delay time were calculated as the average value of each adjustment. To determine the concentration dependence of aggregation delay, different concentrations of the above compound were applied to 50 μM recombinant α-syn. Delay time and t 1 / 2 The calculations were performed as described above. For graphical representation, the adjusted steady state was normalized to 1 for all conditions, and the mean error was calculated based on the adjustments. Statistical tests for the significance of time shifts were performed using Welch's t-test on two random samples (random samples) with p<0.05 (OriginPro 2020, OriginLab, USA). 10 μM α-syn was used for the de novo ThT assay at quasi-stoichiometric concentrations. In contrast to the screening agglutination assay, to shorten the agglutination time, the α-syn sample was continuously shaken at 300 rpm in orbital shaking mode, and measurements were taken every 5 minutes. t-tests were performed for delayed time shifts and steady state reductions in the presence of inhibitors and corresponding control peptides at the same concentrations. 1 / 2 Statistical evaluation was performed as described above by comparing the significance of the results. For the inoculated ThT assay, 50 nM monomer equivalents of PFF oligomers were incubated in a fluorescence plate reader at static conditions of 37°C with or without the above compounds at different concentrations. After 20 hours, 20 μM monomer α-syn was added to the sample mixture to initiate seeding. Measurements were taken every 5 minutes (n=3).

[0104] PFF oligomer generation: PFF oligomers were produced as follows: First, insoluble PFF was produced by incubating 300 μM recombinant α-syn in 20 mM NaPI, pH 7.0, and 150 mM 0.05% NaCl (w / v) sodium azide in a LoBind reaction tube (Eppendorf GmbH, Germany) containing borosilicate glass beads (d=3.0 mM; Hilgenberg, Germany) at 37°C for 1 week. This insoluble PFF was recovered by ultracentrifugation at 100,000 × g for 30 minutes at 4°C, and the pellet was washed several times with 20 mM NaPI, pH 7.0, and 150 mM NaCl. The monomer equivalent concentration was determined by measuring the α-syn concentration in the supernatant after the initial centrifugation and subtracting this from the starting concentration for fibrillation. The insoluble PFF was resuspended in buffer and frozen at -80°C with liquid N2. Oligomer PFF was obtained by vigorously sonicating 200 μl of insoluble PFF with a monomer equivalent concentration of 300 μM using a tip sonicator (MS72 microtip, Sonopolus, Brandelin, Germany) at 3 × 15 seconds (1 second start / end) and 60% amplitude. The insoluble PFF was separated by centrifugation at 100,000 × g for 1 hour at 4°C. The supernatant containing the PFF oligomer was separated, aliquoted, and frozen in liquid N2 at -80°C.

[0105] Fluorescent labeling: Peptides were fluorescently labeled by cysteine-maleimide coupling. SVD-1_Cys and SVD1a_Cys were fluorescently labeled at the C-terminus with 5-fold or 10-fold molar excess TCEP and CF633-PEG2-maleimide (Sigma-Aldrich, USA). This labeling reaction was carried out at room temperature for 2 hours in 25 mM NaPI, pH 7.0. The labeled peptides were purified at 25°C using an Agilent 1260 Infinity II System and a C-18 RP-HPLC column (Zorbax 300 SB-C8 / SB-C 18, Agilent, USA). The mobile phase consisted of A: water + 0.1% TFA and B: acetonitrile + 0.1% TFA. The fluorescently labeled peptides and proteins were eluted over 30 minutes with a gradient of 5–40% (v / v) B. The fluorescently labeled products were fractionated using the absorption wavelength of the fluorophore (CF633: 633 nm), and the pooled samples were subsequently lyophilized and redissolved in appropriate buffer. Protein concentrations were determined by UV-vis, and the fluorescently labeled samples were stored at -20°C.

[0106] Microscale thermophoresis Microscale thermophoresis (MST) experiments were performed using a Monolith NT.115 instrument (NanoTemper Technologies GmbH, Germany). Fluorescently labeled peptides were diluted to a final concentration of 100 nM in PBS, pH 7.4. Recombinant α-syn was diluted in a series of sequential dilutions in PBS, pH 7.4 to final concentrations ranging from 50 μM to 3 pM, and then homogeneously mixed with the peptide solution. These samples were loaded into standard surface area glass capillaries for SVD1 and premium surface area capillaries (NanoTemper Technologies GmbH, Germany) for SVD-1a, and measurements were performed at 25°C with 20% LED output and 60% MST output. Manufacturer-recommended standard parameters were used (30-second heating period and 5-second re-equilibrium delay period). The data were evaluated for thermophoresis effect using manufacturer-supplied NT analysis software (version 1.5.41).

[0107] Surface plasmon resonance dynamics experiments: Measurements were performed using an 8K-Biacore instrument (Cytiva, USA). Interactions were measured using single-cycle kinetic experiments. In all assays, peptide compounds were immobilized on the sensor surface as ligands, and recombinant α-syn was injected into the flow as the analyte. Peptides, particularly SVD-1 and SVD-1a, were immobilized via primary amino groups onto a CMD200M carboxyledextran matrix chip (Xantec, Germany). Immobilization was performed using 50 μg / ml peptide in 10 mM NaAc at pH 5.0 for SVD-1 and pH 7.0 for SVD-1a, after 7 minutes of EDC / NHS activation at 10 μl / min until a saturation signal (SVD-1: 400 RU, SVD-1a: 500 RU) was reached. Surface quenching was performed with 1 M ethanolamine at pH 8.3. Unless otherwise explicitly stated, kinetic experiments were performed at a flow rate of 30 μl / min in PBS, pH 7.4. Surfaces were regenerated between cycles using 30 μl / min injections of 2 M Gua-HCl for 30 seconds. Data evaluation was performed using the evaluation software Biacore insight v3.0 (Cytiva, USA).

[0108] Generation of spin-labeled SVD-1a_Cys_MTSL: A nitroxyl radical-spin-labeled analog of SVD-1a was generated by reacting SVD-1a_Cys with (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)methanethiosulfonate (MTSL) (Toronto Research Chemicals, USA), resulting in covalent bonding of the nitroxyl radical-spin-labeled MTSL to the C-terminal D-cysteine ​​residue. A 5-fold molar excess of MTSL relative to the peptide, i.e., approximately 4 mg of MTSL (approximately 15 μmol), was dissolved in 90 μl of N,N-dimethylformamide (DMF) and mixed with 810 μl of 200 mM HEPES / NaOH buffer, pH 7.6. This solution was then added to approximately 5 mg (approximately 3 μmol) of lyophilized SVD-1a_Cys. After incubation at room temperature for 1-2 hours, the reaction mixture was used for semi-preparative RP-HPLC on a C8 column (Zorbax-300 SB, Agilent, Germany) with a connected HPLC system (Agilent 1260, Agilent, Germany). Spin-labeled peptide SVD-1a_Cys_MTSL was divided into 4 ml portions at 25°C using an aqueous acetonitrile (ACN) gradient (from 8% ACN, 0.1% trifluoroacetic acid (TFA) in Milli-Q water to 60% ACN, 0.1% TFA within 40 minutes). -1 The sample was purified by throughput and detection at 214 nm. The purified spin-labeled sample was collected, aliquoted, rapidly frozen in liquid N2, and lyophilized under vacuum (LT-105, Martin Christ, Germany). This described method enables complete spin labeling of SVD-1a_Cys_MTSL with a purity of 98% or higher.

[0109] NMR spectroscopy: The sample was measured in PBS buffer with 5% D2O added as an internal standard, pH 7.4, and measured to a full length of 25 μM. 15 The final concentration of N-labeled acetyl-α-syn was used to prepare the samples in the absence (reference) and in the presence of equimolar amounts of SVD-1a (unlabeled and therefore invisible to NMR). 2D- 1 H- 15N-HSQC spectra were recorded continuously (Ruecken an Ruecken) at a proton Larmor frequency of 1200 MHz using a Bruker AVANCE NEO spectrometer (Bruker, USA). The experimental temperature was 10°C. The spectral dimension was 16.02 ppm. 1 H) × 30 ppm ( 15 N) and 1 2048 points in H dimension. 15 In N dimensions, this is an increment of 256. 1 53ms in H dimension, 15 A recording time of 35 ms was given in N dimensions. 32 scans were acquired for each increment using a 1-second recovery delay between scans, resulting in a total test period of 4.8 hours per spectrum.

[0110] NMR paramagnetic relaxation enhancement data were obtained in the presence of 25 μM of paramagnetically labeled (but not isotope-enriched) SVD-1a_Cys_MTSL (using MTSL spin labeling covalently bonded to the C-terminus of SVD-1a) that gives a 1:1 α-syn:SVD-1a ratio, and 25 μM of full-length NMR. 15 The intensity (Ipara) was obtained using N-labeled acetyl-α-syn. 2D- 1 H- 15 The data was extracted from the N-Best-TROSY-NMR spectrum. The measurement involved 128 scans for each increment, with a total test period of 16 hours. A 20-fold molar excess of ascorbic acid was added to the same sample, resulting in a quench of the spin-labeled paramagnetic effect and obtaining a diamagnetic reference sample, thus yielding reference data. The diamagnetic reference spectrum and intensity (Idia) were continuously recorded under the same conditions as the paramagnetic sample.

[0111] The NMR dataset was processed using Bruker TopSpin software (version 4.1.1) and visualized using CcpNmr Analysis (v2.4.2)(50). Peak positions were extracted using CcpNmr Analysis software to evaluate the change in chemical shift of α-syn resonances in the presence of SVD-1a compared to a reference spectrum (without SVD1a). 1 H and 15 The absolute change in chemical shift was calculated according to the following formula based on specific changes in the residues of the chemical shift in the N dimension.

number

[0112] To analyze the PRE data, the resonance intensity of the paramagnetic sample and the peak intensity of the diamagnetic reference sample were extracted using CcpNmr Analysis.

[0113] Size exclusion chromatography (SEC) The exclusion of PFF oligomers was evaluated by SEC and subsequent detection of peaks for PFF oligomers and monomers. PFF oligomers were incubated in low-retention Eppendorf tubes (Eppendorf, Germany) in PBS, pH 7.4, with or without SVD-1a, under static conditions at 37°C for 3 days (d). Before injection, the samples were centrifuged at 20,800 × g for 2 minutes, and 100 μl of the sample was injected onto an SEC-HPLC column (Bio SEC-3 300 Å, Agilent, USA) using an Agilent 1260 Infinity II system (Agilent, USA) at a flow rate of 1 ml / min with PBS, pH 7.4 as the mobile phase. Proteins were detected with A214.

[0114] Dynamic light scattering (DLS) Measurements were performed using a SpectroSize 300 131 (XtalConcepts, Germany) device and a 1 ml sample volume in a sealed quartz cuvette (Hellma Group, Germany). Samples were incubated under static conditions at 37°C. Prior to measurement, all samples were centrifuged at 4°C and 21,000 × g for 30 minutes to remove any possible impurities from the solution. Data points were recorded every 30 seconds for time-dependent DLS measurements. The diffusion coefficient was obtained from an analysis of the decay of the autocorrelation function of the scattering intensity and used to determine the apparent hydrodynamic radius using the Stokes-Einstein equation.

[0115] Atomic force microscopy (AFM): Samples were prepared by diluting them to an α-syn monomer concentration of 1 μM. 5 μl was incubated on a freshly cleaved mica surface and dried. The surface was then washed three times with 200 μl of ddH2O and dried using a gentle N2 stream. Measurements were performed using a Nanowizard 3 system (JPK BioAFM - Bruker Nano GmbH, Germany) in intermittent contact mode, with line scanning speeds of 0.5-2 Hz at 2×2 and 5×5 μm lines, under ambient conditions, using a silicon cantilever and tip (Olympus OMCL-AC160TS-R3) with a nominal spring constant of 26 N / m, an average tip radius of 9 ± 2 nm, and a resonant frequency of approximately 300 kHz. Images were processed using JPK data processing software (version spm-5.0.84). For the height profiles shown, polynomial adjustments were first subtracted from each scan line independently, and then using a limited data range.

[0116] Circular dichroism (CD) spectroscopy: Far-UV circular dichroism (CD) data were acquired using a Jasco J-1100 spectropolarizer (Jasco, Germany). 350 μl of sample replicas from the inoculated assay were pooled and packed into high-precision quartz cuvettes (Hellma-Gruppe, Germany) with a layer thickness of 1 mm. Five integrations were performed per sample at a scanning speed of 20 nm / min and far-UV wavelengths from 260 nm to 190 nm. The baseline was corrected by subtracting only the buffer measurement.

[0117] Cellular assay of α-synuclein aggregation: A construct encoding the full-length A53T mutant human α-syn, fused with YFP at the C-terminus, was synthesized and introduced into the pMK-RQ expression vector (GeneArt; Thermo Fisher Scientific, USA). The α-synA53T-YFP construct was subcloned using the restriction sites NheI(5') and NotI(3') in the vector pIRESpuro3 (Clontech; Takara Bio, Japan). HEK293T cells (American Type Culture Collection) were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, USA), 50 units / ml penicillin, and 50 μg / ml streptomycin (Sigma-Aldrich, USA). These cells were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0118] Cells plated in DMEM were transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, USA). Stable cells were selected in DMEM containing 1 μg / ml puromycin (EMD Millipore, Merck Millipore, USA). Monoclonal cell lines were generated by fluorescence-activated cell sorting of polyclonal cell populations in 96-well plates using a MoFlo XDP cell sorter (Beckman Coulter, USA). Clonal cell line B5 was ultimately selected from 24 clonal cell lines. This was named αSynA53T-YFP cells.

[0119] The peptide was incubated with 1.5% Lipofectamine 2000 in OptiMEM at room temperature for 2 hours. α-synA53T-YFP cells were plated at a density of 1000 cells per well using 0.1 μg / ml Hoechst 33342 (Thermo Fisher Scientific, USA) in a 384-well plate with poly-D-lysine coating (Greiner, Austria), and the pre-prepared transfection mixture was directly added to the cells in the wells.

[0120] To seed α-syn cell aggregation into α-synA53T-YFP cells, 30 nM soluble α-syn PFF oligomers were incubated with 1.5% Lipofectamine in OptiMEM at room temperature for 2 hours and added to each well 3 hours after the initial transfection.

[0121] Next, the plates were incubated at 37°C in a humidified atmosphere of 5% CO2. On day 3, cells were imaged using blue and green fluorescence channels on an IN Cell Analyzer 6500HS system (Cytiva, USA) and analyzed using IN Carta Image Analysis software (Cytiva, USA), which was used to establish an algorithm for identifying intracellular aggregates in living cells. Four wells were used for each condition, and 16 images were recorded per well to prevent distortion, and the data were analyzed using a fully automated algorithm. Statistical analysis was performed using one-way ANOVA and Dunnett's test for subsequent multiple comparisons (GraphPad Prism 9, GraphPad Software, USA). Error bars represent the standard deviation.

[0122] Cell viability assay; CellGlo trial: The number of viable cells in the culture was determined based on the quantification of ATP present, an indicator of metabolically active cells, using the CellTiter-Glo Luminescent Cell Viability Assay (Promega GmbH, Germany).

[0123] After culturing cells in a 384-well plate for 3 days, 35 μl of culture medium was removed from the wells, and 40 μl of CellTiter-Glo reagent was added directly to each well. After mixing, luminescence was measured 10 minutes later using a Fluostar (BMG labtech, Germany).

[0124] Immunofluorescence cytostaining After 3 days of cell culture on a 384-well plate, the cells were fixed in 4% formaldehyde (Sigma-Aldrich, USA) in PBS (pH 7.4) for 15 minutes. After washing three times with PBS (each time lasting 5 minutes), the cells were permeabilized in 0.25% Triton X-100 (Sigma-Aldrich, USA) in PBS for 10 minutes.

[0125] After washing with PBS for 5 minutes three times, the cells were blocked for 30 minutes with 1% bovine serum albumin (Sigma-Aldrich, USA) in PBS containing 0.1% Tween-20 (Sigma-Aldrich, USA). These cells were stained with CF633 (Biotium, USA) fluorescently labeled antibody at a concentration of 8 μg / ml in 1% bovine serum albumin in PBS containing 0.1% Tween-20 for 1–3 hours at room temperature in the dark. The entire α-syn was detected using the anti-α-syn antibody syn211 (Abcam, UK).

[0126] Oligomeral α-syn and fibrillary α-syn were detected using the anti-aggregating α-syn antibody 5G4 (Sigma-Aldrich, USA). Serine-129 phosphorylated α-syn was detected using the recombinant anti-α-syn (phospho S129) antibody EP1536Y (Abcam, UK). After three subsequent washes in PBS, each lasting 5 minutes, cells in PBS were imaged at 40x magnification using an IN-Cell Analyzer 6500HS system (Cytiva, USA).

[0127] Cell viability assay; MTT assay: The potential to save PC12 cells (Leibniz-Institut DSMZ, Germany) from α-syn toxicity by adding SVD-1, SVD-1a, or SVD-1_scrambled was measured in an MTT (3(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay.

[0128] PC12 cells (Leibniz-Institut DSMZ, Germany) were cultured at 37°C in collagen A-coated (Biochrom GmbH, Germany) tissue culture flasks in RPMI1640 medium supplemented with 5% fetal bovine serum and 10% equine serum, in a 95% humidified atmosphere containing 5% CO2. 10,000 cells per 100 μl well were seeded into collagen A-coated 96-well plates (Thermo Fisher Scientific, USA) and incubated in a thermocycler at 37°C and 300 rpm for 24 hours.

[0129] Subsequently, a final concentration of 30 nM α-syn was added to the cells either without prior incubation or after prior incubation with 15 μM SVD-1, SVD-1_scrambled, or 0.5 μM SVD-1a. Furthermore, 15 μM peptide alone, cell medium, peptide-free buffer, and 0.1% Triton X-100 (cytotoxic compound) were used as controls. After further incubation at 37°C for 24 hours in a 95% humidified atmosphere containing 5% CO2, cell viability was measured using Cell Proliferation Kit I (MTT) (Roche Applied Science, Switzerland) according to the manufacturer's protocol.

[0130] MTT-formazan products were quantified by measuring the absorbance at 570 nm, corrected by subtracting the absorbance at 660 nm, using a FluoroStar Optima plate reader (BMG labtech, Germany). All results were normalized to untreated cells grown only in culture medium. Significance was tested by one-way ANOVA and Bonferroni post-hoc analysis (OriginPro 2020, OriginLab, USA; n=4).

[0131] The results are shown in Figures 1 to 20.

[0132] Figure 1: Thioflavin-T assay using recombinant full-length L-enantiomer α-syn and D-enantiomer 16-mer peptide.

[0133] 50 μM α-synuclein (α-syn) was incubated in PBS, pH 7.4 with or without 50, 150, or 250 μM D-peptide. Measurements were performed in unbound 96-well plates at 37°C with shaking at 300 rpm for 30 seconds every 20 minutes. One borosilicate glass bead (d=3 mm) was added to each well. Five replicas for each condition were individually fitted to a Boltzmann fit, normalized, and the mean was calculated along with the standard deviation (error bars). X1 / 2 and delay time shifts were determined by calculating the difference between samples containing and not containing the peptide. Significance of the time shift was determined using Welch's t-test, where two random samples showed a significance of 0.05 (significant: * The following tests were conducted to determine if the following were true: (A) SVD-1, (B) SVD-6, (C) SVD-10, (D) SVD-14.

[0134] Figure 2: MST measurements of SVD-1 and α-syn.

[0135] SVD-1 was incubated with 140C-C2-Alexa 647 at concentrations of 159.5 μM to 19 nM and 30 nM. 70% LED output; 60% MST output. K D It was determined to be 55 nM.

[0136] Figure 3: Multi-cycle SPR experiments with wild-type α-synuclein and SVD-1.

[0137] 400RU of SVD-1 was immobilized on the surface of a 2D CMD sensor chip. Wild-type α-syn was injected in PBS, pH 7.4, at concentrations ranging from 0.39 to 100 (0.1%). Regeneration: 2M Gua-HCl and 0.1% SDS. D This is determined by heterologous ligand adjustment, K D 1 is 12.5 nM, and K D The concentration of 2 was 2.0 μM.

[0138] Figure 4: Elimination of α-syn oligomers by SVD-1.

[0139] 5 μM monomer equivalents of oligomers were incubated with or without SVD-1 at 900 rpm and 37°C for 3 days. The samples were centrifuged at 21,000 × g, and the supernatant was loaded onto an HPLC-SEC column. α-syn oligomers could not be detected in the supernatant at SVD-1 concentrations of 150 and 250 μM.

[0140] Figure 5: Quasi-stoichiometric inhibition of α-syn aggregation in the ThT assay.

[0141] 25 μM monomer α-syn was incubated with quasi-stoichiometric concentrations of SVD-1 at 2.5 and 12.5 μM. Measurements were performed in 96 unbound coated wells with continuous shaking at 37°C. One borosilicate bead (d=3 mm) was added per well. Each replica was adjusted using a Boltzmann sigmoid fit, and the mean value and delay time were calculated for each condition.

[0142] Figure 6: Gradient centrifugation of α-syn aggregated samples in the presence and absence of SVD-1.

[0143] Monomer α-syn was incubated in a highly bonded surface-coated 96-well plate (CORNING) with or without equimolar concentrations of SVD-1. No shaking, no glass beads, 37°C. After 9 days, the three samples were combined, and 100 μl was added to an iodixanol gradient (5–50% (w / v)). Fifteen fractions of 140 μl each were analyzed by RP-HPLC using a Zorbax 300 SB-C8 column and integrated to the α-syn peak. No fibrillation (F6–F12) was observed in the presence of SVD-1.

[0144] Figure 7: Thioflavin-T assay using recombinant full-length L-enantiomer α-syn and D-enantiomer SVD-1a.

[0145] 20 μM α-syn was incubated in PBS, pH 7.4, with or without quasi-stoichiometric concentrations of SVD-1a. Measurements were performed in unbound coated 96-well plates with continuous shaking at 400 rpm and 37°C. One borosilicate bead (d=3 mm) was added per well. The mean and standard error of five replicas were calculated for each condition.

[0146] Figure 8: Single-cycle SPR experiment with wild-type α-syn and SVD-1a, SVD-6a, or SVD-10a.

[0147] Each peptide was immobilized on the surface of the CM5 sensor chip. Wild-type α-syn was injected in PBS, pH 7.4, at a concentration range of 30-500 nM in 0.005% (v / v) Tween-20. Regeneration was performed after each cycle with 2M Gua-HCl for 2 × 30 seconds. D The following values ​​were determined using a 1:1 coupling model fit: SVD-1a: 15.7 nM; SVD-6a: 14.0 nM; SVD-10a: 12.3 nM.

[0148] Figure 9: De novo ThT assay and seeded ThT assay using SVD-1 and SVD-1_scrambled.

[0149] Both assays were performed in a PBS buffer system at pH 7.4. The course of ThT fluorescence was recorded using a Fluorostar plate reader (BMG Labtech, Germany) at λ. ex =448nm and λ em Measurements were taken at 482 nm in a 96-well half-area plate (Corning, USA). Peptide sequences are shown in gray as single-letter amino acid codes.

[0150] (A) De novo agglutination assay using SVD-1 and SVD 1_scrambled. 10 μM α-syn monomer was added to each well along with 5, 10, and 20 μM peptides, with one borosilicate glass bead (d=3.0 mm, Hilgenberg, Germany) per well. The mixture was continuously shaken at 300 rpm between measurements and incubated at 37°C. Mean data ± SD (n=5) are reported.

[0151] (B) For seeded aggregation, 50 nM monomer equivalents of PFF oligomers were pre-incubated as seeds at 37°C for 20 hours under static conditions, with or without the peptide. Only then was 20 μM of α-syn monomer added to induce seeding. Mean data ± SD (n=3) are reported.

[0152] Figure 10: MST measurement of α-syn using SVD-1 and SVD-1a. MST measurement of the interaction between SVD-1 and SVD-1a and full-length α-syn at 25°C in PBS, pH 7.4.

[0153] α-syn was diluted in a serial dilution series from 50 μM to 1 pM and mixed with a fluorophore-labeled peptide (Alexa 647 C2) at a final concentration of 100 nM. The experiment was performed as individual measurements. K D The values ​​were determined to be 170 nM ± 100 nM for SVD-1 and 390 nM ± 150 nM for SVD-1a.

[0154] Figure 11: A single-cycle kinetic assay using α-syn and immobilized SVD-1 and SVD-1a.

[0155] SVD-1 and SVD-1a were immobilized on a carboxyl dextran matrix (CMD200M, Xantec, Germany) by amino coupling to saturation. α-syn was injected into PBS 7.4 at a rate of 30 μl / min for 100 seconds in serial dilutions of 30–500 nM, followed by a dissociation time of 60 or 30 minutes. Experiments were performed as individual measurements. Interaction kinetics were adjusted using a kinetic 1:1 interaction model:SVD-1:K D :880pM, K on : 6.56×104M -1 s -1 , K off :5.78s -1 ×10 -5 SVD-1a:K D :100pM, K on :3.13×105M -1 s -1 , K off :3.13×10 -5 s -1 .

[0156] Figure 12: In interactions with SVD-1a (unlabeled isotope) 15 NMR analysis of N-labeled α-syn.

[0157] (A) 25 μM in the presence (red) and absence (black) of equimolar amounts of SVD-1a 15 N-α-syn two-dimensional 1 H- 15 Superposition of N-HSQC spectra.

[0158] (B) Expansion of numerous resonances in the spectrum shown in (A) showing small chemical changes in residues in the presence (red) and absence (black) of SVD-1a (130E, 129S, 126E, 122N, 119D, 65N). For comparison, the 77V resonance, which does not show a change in chemical shift, is shown.

[0159] (C) Compared to the absence of SVD-1a, in the presence of SVD-1a 15Changes in residue-specific absolute NMR chemical shifts in the spectrum of N α-syn. The standard deviation σ and double standard deviation 2σ of the distribution of observed chemical shift changes are shown by dashed lines.

[0160] (D) In ​​the presence of paramagnetically labeled SVD-1a 15 NMR PRE intensity ratio of N α-syn. This is a two-dimensional comparison of a paramagnetic sample with a diamagnetic sample. 1 H- 15 The residue-specific intensity ratio Ipara / Idia of the cross-peak intensity in the N NMR spectrum is shown. A lower Ipara / Idia intensity ratio indicates that the paramagnetically labeled SVD-1a is closer to the target α-syn residue. An intensity of 1 indicates a lack of interaction. The data suggest a somewhat more pronounced (transient) binding interaction of SVD-1a with the C-terminal residue of α-syn compared to the average effect on the remaining N-terminal residues.

[0161] Figure 13: Size exclusion chromatography of monomer α-syn after incubation with gradually increasing concentrations of SVD-1a.

[0162] 10 μM α-syn monomer was incubated with 0, 5, 10, 20, 40, and 80 μM SVD-1a in PBS, pH 7.4 for 2 hours at 37°C and 300 rpm. SEC was performed using a Bio SEC-3 column (150 Å, Agilent, USA) at 1 ml / min with PBS, pH 7.4 as the mobile phase. After centrifugation of the samples at 20,800 × g for 5 minutes, the supernatant was injected. Proteins were detected with A214. α-syn monomer eluted after a retention time of 6.91 minutes. For the 10 μM α-syn sample, the peak area of ​​α-syn monomer was integrated and set to 100%. The concentration of monomer α-syn did not decrease in the presence of gradually increasing concentrations of SVD-1a. SVD-1a itself was eluted only during the SEC purification and regeneration procedure, which is likely due to its high net positive charge.

[0163] Figure 14: Thioflavin-T de novo aggregation of monomer α-syn with SVD-1a and SVD-1_scrambled+5r.

[0164] The experiment was conducted in a PBS buffer system at pH 7.4. The progression of ThT fluorescence was recorded using a Fluorostar plate reader (BMG Labtech, Germany) with a λ-scale. ex =448nm and λ em Measurements were performed at 482 nm in a 96-well half-area plate (Corning, USA). 10 μM of α-syn monomer was added along with 5, 10, and 20 μM of peptides, one borosilicate glass bead (d=3.0 mm, Hilgenberg, Germany) per well. The plate was continuously shaken at 300 rpm between measurements and incubated at 37°C. Data are reported as mean ± SD (n=5). The amino acid sequences of the peptides used are shown in gray text.

[0165] Figure 15: De novo aggregation analysis of α-syn in the presence and absence of SVD-1a using ThT, DGC, CD, and AFM.

[0166] (A) De novo ThT assay with or without 20 μM SVD-1a and 10 μM α-syn. ThT fluorescence progression was recorded using a Fluorostar plate reader (BMG Labtech, Germany) with λ ex =448nm and λ em Measurements were taken at 482 nm on a 96-well half-area plate (Corning, USA), with the plate continuously shaken at 300 rpm between measurements. Data are reported as mean ± SD (n=5).

[0167] (B) CD secondary structure analysis of de novo aggregate samples. Samples were incubated as described in (A) (n=3) without the addition of any ThT, and then pooled for CD analysis. The far-UV ellipticity of these samples was measured in a quartz cuvette (l=10 mm) using a J-1100 CD spectrometer (Jasco, Germany). In addition to (A), a sample containing only 20 μM SVD-1a was incubated under the same conditions and subsequently used as a reference for the sample containing α-syn and SVD-1a. The CD spectra of these samples (α-syn + SVD-1a (after incubation)) subtracted from the SVD-1a reference are shown.

[0168] (C) The sample from (A) was isolated immediately after incubation and diluted in PBS, pH 7.4 to a final concentration of 1 μM α-syn monomer equivalent. 5 μl of the diluted sample was incubated, dried on a freshly cleaved mica surface, washed with ddH2O, and dried using a gentle N2 stream. Analysis was performed using a NanoWizard 3 system (J-1100, JPK BioAFM, USA), and multiple surface sections were recorded. The section shown in (C) is representative of the types identified and observed in all surface sections.

[0169] Figure 16: A ThT assay using SVD-1a and SVD-1_scrambled+5r as seeds.

[0170] The progression of ThT fluorescence was measured using a Fluorostar plate reader (BMG Labtech, Germany) with a λ ex =448nm and λ emMeasurements were taken at 482 nm in 96-well half-area plates (Corning, USA). D-peptide sequences are shown in gray as single-letter amino acid codes. 50 nM monomer equivalents of PFF oligomers were pre-incubated as seeds for 20 hours at 37°C under static conditions, with or without the peptide. Only then was 20 μM of α-syn monomer added to induce seeding and initiate incubation with the α-syn monomer. Mean data ± SD (n=3) are reported.

[0171] Figure 17: SVD-1a decomposes PFF oligomers into α-syn monomers.

[0172] PFF oligomers were produced as described above. 100 nM monomer equivalents of PFF oligomers were incubated in PBS, pH 7.4 at 37°C for 3 days, with or without 400 nM and 1600 nM SVD-1a.

[0173] (A) The HPLC-SEC sample was injected into a Bio SEC-3 column (300 Å, Agilent, USA). The PFF oligomer eluted after approximately 5 minutes, and the α-syn monomer was detected after approximately 8.6 minutes.

[0174] (B) Time-dependent DLS measurements using 100 nM PFF oligomers in the presence (red) and absence (black) of 400 nM SVD-1a. Using a SpectroSize 300 Instrument (XtalConcepts, Germany), 1 ml of sample was continuously measured under static conditions at 37°C every 30 seconds for 24 hours in a sealed quartz cuvette. The data are shown as radius diagrams, with the signal amplitude of each particle represented by the diameter of the data point.

[0175] (C) For AFM analysis, 5 μl of the sample described in (A) was removed before centrifugation, incubated, dried on a freshly cleaved mica surface, subsequently washed with ddH2O, and dried using a gentle N2 stream. Analysis was performed using a NanoWizard 3 system (J-1100, JPK BioAFM, USA), and multiple surface sections were recorded. The sections shown in C are representative of the observed types and particle densities identified in all surface sections.

[0176] Figure 18: Analysis of agglutination assays in the presence and absence of SVD-1a using ThT assay and CD spectroscopy.

[0177] 500 nM α-syn PFF was incubated in PBS, pH 7.4, with or without 15 μM α-syn monomer and / or 30 μM SVD-1a, under static conditions at 37°C for 24 hours.

[0178] (A) The progression of ThT fluorescence was measured using a Fluorostar plate reader (BMG Labtech, Germany) and λ ex =448nm and λ em Measurements were taken at 482 nm on a 96-well unbonded half-area plate (Corning, USA). Data are reported as mean ± SD (n=3). Arrows indicate time points analyzed by CD spectroscopy.

[0179] (B) The samples shown in (A) were incubated under the same conditions without the addition of ThT. Three replicas (120 μl each) for each condition were pooled and measured by CD spectroscopy at time points of 0 hours (h), 2 hours, 8 hours, and 24 hours. Far-UV ellipticity was measured in a quartz cuvette (l=10 mm) using a J-1100 CD spectrometer (Jasco, Germany). The data shown for (d) were subtracted from (c) for the corresponding time point.

[0180] Figure 19: SVD-1a inhibits the aggregation of seeded α-syn in cells.

[0181] To verify the inhibitory effect of SVD-1a in cells, we used an α-synA53T-YFP cell line that stably expresses human α-syn fused with YFP and carrying the familial A53T mutation in HEK293T cells. Fluorescence-based detection of aggregates allowed for clear indication of strong intracellular fluorescence spots against the intracellular background after PFF oligomer seeding. To prevent the peptide from interfering with PFF oligomer assimilation, a two-step transfection was performed, starting with a first transfection with the peptide and a second transfection with the PFF oligomer. SVD-1a inhibited α-syn aggregation in αSynA53T-YFP cells in a dose-dependent manner, but did not inhibit a peptide derived from a negative control (kgvgnleyqlwalegk-NH2) (A). A fully automated algorithm for image analysis was used to quantify the number of cells containing aggregates in our images and to prevent experimental distortion. Significance was calculated using one-way ANOVA and Dunnett's test for subsequent multiple comparisons. One asterisk represents a p-value less than 0.05, and three asterisks represent a p-value less than 0.001. Error bars represent the standard deviation. In contrast to unseeded cells (B-E) that had no α-syn aggregates at 3 days post-seeding, seeding with soluble α-syn-PFF oligomers resulted in aggregation in 85% of cells treated with a negative control peptide (25 μM) that does not bind to α-syn (F-I). Treatment with gradually increasing concentrations of SVD-1a, in this case 25 μM, resulted in a concentration-dependent decrease in the number of cells with aggregates (J-M). Panels B, E, and H show nuclei stained with Hoechst 33342. Panels C, F, and I show αSynA53T-YFP fluorescence. Panels D, G, and J show composite images. The L scale bar corresponds to 100 μm and applies to panels B-D, F-H, and J-L. Panels E, I, and M show magnified areas of the enclosed regions of D, H, and L. The M scale bar applies to panels E, I, and M and corresponds to 25 μm.

[0182] Figure 20: SVD-1 and SVD-1a increase cell viability in MTT assays using PFF oligomer seeds.

[0183] SVD-1, SVD1a, and SVD-1_scrambled were incubated overnight at 37°C and 300 rpm, with or without 30 nM PFF oligomers, and subsequently incubated with PC12 cells at final concentrations of 15 μM (SVD-1 and SVD-1_scrambled) or 0.5 μM (SVD-1a). Cell viability decreased to 50% with only 30 nM PFF oligomers, but 89% or 82% of cells could be saved in the presence of SVD-1 or SVD-1a. A slight increase in cell viability salvage was also observed with 15 μM of the control peptide SVD-1_scrambled. Significance testing was performed by one-way ANOVA and Bonferroni post-hoc analysis. * p ≤ 0.5, *** p ≤ 0.01. The mean value is listed along with ±SD (n=4).

Claims

1. A peptide comprising an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 5, which binds to the α-synuclein peptide at a K / D ratio of less than 50 μM.

2. At the free C-terminus, instead of a carboxyl group, an acid amide group (CONH ２ The peptide according to claim 1, characterized in that it contains a group, or a COH group, a COCl group, a COBr group, a CONH-alkyl group, or a CONH-alkylamine group, or the peptide is in a cyclized form.

3. The peptide according to claim 1 or 2, characterized in that it contains two, three, four, five, six, seven, eight, nine, ten or more copies of the sequence having SEQ ID NO: 1 or SEQ ID NO:

5.

4. The peptide according to any one of claims 1 to 3, characterized in that the peptide consists substantially of D-amino acids.

5. The peptide according to any one of claims 1 to 4, characterized in that it is linked to another substance.

6. The peptide according to any one of claims 1 to 5, characterized in that a plurality of peptides of sequence number 1 or sequence number 5 are linked to each other by covalent or non-covalent bonds.

7. The peptide according to any one of claims 1 to 6, characterized in that a plurality of peptides of SEQ ID NO: 1 or SEQ ID NO: 5 are linked to each other without a linker, that is, directly linked to each other or linked to each other using a linker group.

8. The peptide according to any one of claims 1 to 7, characterized in that a plurality of peptides of SEQ ID NO: 1 or SEQ ID NO: 5 are linked to each other in a linear or branched manner.

9. The peptide according to any one of claims 1 to 8, characterized in that the peptide is a dendrimer in which the peptide of SEQ ID NO: 1 or SEQ ID NO: 5 is linked using a platform molecule.

10. The peptide according to any one of claims 1 to 9 for preventing the formation of α-synuclein peptide oligomers and / or α-synuclein peptide aggregates.

11. The peptide according to any one of claims 1 to 10 for detoxification of α-synuclein peptide oligomers and / or α-synuclein peptide aggregates.

12. A peptide according to any one of claims 1 to 11 for use in the treatment of synuclein disease.

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