USE OF D-ENANTIOMER PEPTIDI DIGANDS OF MONOMEREM TAU FOR THE THERAPY OF VARIOUS TAUOPATHIES

Description

[0001] The invention relates to a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: and SEQ ID NO: 5, and to such a peptide for use in the treatment of tauopathies.

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide, affecting an estimated two-thirds of the population—approximately 27 million people. It belongs to a heterogeneous group of tauopathies, all characterized by progressive dementia. The intraneuronal deposits found in the brain, which contain amyloid structures of hyperphosphorylated tau protein, generally correlate with the degree of cognitive decline over the course of the disease.

[0003] Tau, as a neuronal microtubule-associated protein, is significantly involved in the assembly and stabilization of microtubules.

[0004] Hyperphosphorylation of tau triggers its misfolding and aggregation, leading to the formation of neurofibrillary tangles, which are a neuropathological hallmark of most tauopathies. In the brains of adults, six tau isoforms are expressed through alternative splicing, ranging in length from 352 to 441 amino acids. Depending on the composition of isoforms present, different malformations can develop, which are specific to the respective tau pathology.

[0005] Toxic, self-replicating, and propagating tau aggregates are a hallmark of various tauopathies. These pathologies are characterized by a prion-like spread of misfolded tau aggregates throughout the central nervous system (CNS), accompanied by neurodegeneration of the affected brain regions. Several studies have demonstrated the uptake of tau aggregates by cells, seeding effects (aggregates that act as templates, inducing the conversion of monomeric to misfolded, oligomeric, or fibrillar tau), and tau aggregate transfer between cell cultures. The pathways of propagation in tau-associated pathologies reflect neuroanatomically connected brain regions.

[0006] Currently, there is no active ingredient or medication that addresses the underlying causes of Alzheimer's disease (AD). The medications used and approved so far alleviate some of the symptoms of Alzheimer's dementia. However, they are not able to slow the progression of the disease or cure it. Some substances have shown promise in preventing AD in animal studies, but not necessarily in treating it.

[0007] However, five medications (Aricept, Exelon, Razadyne, Donepezil and Namenda) are available that alleviate the symptoms of the disease and make everyday life easier for those affected.

[0008] Frontotemporal dementia is also not treatable at its root cause, meaning that the progression of the disease cannot be slowed or stopped. The primary treatment involves antidepressants, which can alleviate symptoms.

[0009] For progressive supranuclear palsy, there is currently no way to halt or slow the progression of the condition. Some symptoms do not respond to any medication. Antidepressants (e.g., Prozac, Elavil, and Tofranil) are used to alleviate other symptoms, while special glasses and walking aids help with daily life.

[0010] Subacute sclerosing panencephalitis (SSP), caused by measles infection, is currently incurable. Antiviral drugs (isoprinosine and ribavirin) and immunomodulating agents (interferon alpha) can halt disease progression and increase patient life expectancy. However, the side effects of long-term treatment are still unknown.

[0011] Currently, there is no medication available that slows or halts the progression of corticobasal degeneration. The symptoms of corticobasal degeneration are often resistant to treatment. Clonazepam is used to treat myoclonus. Physiotherapy and speech therapy can help with daily living.

[0012] ZHANG XIANCHENG ET AL., ACS CHEMICAL NEUROSCIENCE, Vol. 11, No. 24, December 7, 2020, pages 4240-4253, describe D-peptides that are directed against tau and can be used for the treatment of tauopathies.

[0013] In summary, a causal and significantly life-extending therapy is currently unavailable for most, if not all, tauopathies and is urgently needed.

[0014] The object of the present invention was therefore the development of new chemical units which minimize the formation of new aggregates and can disassemble existing toxic tau aggregates into native functional tau monomers, thus enabling their therapeutic use in various tauopathies.

[0015] The chemical unit or its variants used in therapy should bind as affinely and specifically as possible to the native, endogenous, monomeric tau protein, thereby stabilizing it. The equilibrium between misfolded and natively folded tau conformations is thus shifted in favor of the latter. Ideally, this allows existing tau oligomers and fibrils to be broken down into their monomeric building blocks and thus eliminated.

[0016] This problem was solved by a peptide according to claim 1.

[0017] Further preferred embodiments are defined in the dependent claims.

[0018] In the following, the term "comprise" should also include "consist of".

[0019] The peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ

[0020] The peptides ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 were found using an optimized mirror-image phage display selection. The peptides of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7 are not claimed.

[0021] It should be noted that, in addition to the selection of specific monomer binders mentioned here, the mirror-image phage display method can of course also be used to find specific oligomer binders or even to find ligands for other species occurring in protein misfolding disease.

[0022] In mirror-image phage display, for example, a recombinant library of randomized peptide sequences, presented on the gp3 protein of the M13 phage and encoded in its genome, is selected against the exact mirror image (D-enantiomer) of a naturally occurring L-enantiomeric target molecule (e.g., Tau).

[0023] The peptide sequence is advantageously presented at the N-terminus of the gp3 protein of the M13 phage and is encoded in its genome.

[0024] The gp3 molecule, also called gene product 3, is a protein located in the coat of the phage and is required for contact with the host cell.

[0025] The DNA sequence of the p3 gene of a selected phage is linked to the DNA sequence containing the genetic information for the corresponding peptide sequence on the gp3 molecule, allowing it to be sequenced. After sequencing, the genomic sequence can be transcribed into an amino acid sequence and synthesized as a D-enantiomeric peptide that binds to the physiological L-enantiomeric form of the target molecule (e.g., tau). The entirety of phages that present different peptides as fusion proteins with gp3 on their surface is referred to as the phage library. The corresponding peptides represent the biomolecules to be selected in the experiment.

[0026] So-called panning rounds can be performed, e.g., three rounds. In this process, the phage library is brought into contact with a fixed target molecule, also called a bait, and binding phages are isolated from the billions of other, non-binding phages.

[0027] For example, the number of phages that preferentially bind to oligomeric or fibrillar species of tau is reduced by not offering these species as bait. Phages exhibiting an increased affinity for tau oligomers and fibrils can thus be removed from the phage pool, allowing, for instance, tau monomer-specific phages to accumulate. This method can, of course, be adapted analogously to identify ligands and peptides that specifically bind to tau oligomers.

[0028] To reduce the accumulation of plastic-, BSA-, or streptavidin-affine phages, according to the invention, different substrate surfaces are used in preferably all panning rounds. In this case, the substrate surface is defined as a combination of the substrate used (plastic plate, in this case polystyrene with a streptavidin matrix) and the blocking or quenching agents used. The selection pressure is successively increased in the successive selection rounds.

[0029] To achieve this, while the concentration of the target molecule (e.g., monomeric tau) remains stable, the number of washing steps after phage incubation is continuously increased from the 2nd selection round onwards in order to remove phages that are not affine to tau monomer.

[0030] Furthermore, a different substrate surface is selected in each selection round of the phage display by using different agents to block the surface after immobilizing the target molecule on the substrate (e.g., BSA, milk powder, no blocking). For example, the surface can be changed between a milk powder-blocked surface in addition to a biotin-treated one in round 1, a BSA-blocked surface in round 2, and a milk powder-treated surface in round 3.

[0031] Switching between different substrate surfaces increases the specificity for the target molecule or bait relative to the surface. Furthermore, it reduces the number of ligands that bind non-specifically to plastic surfaces or the blocking agent.

[0032] Parallel to the actual phage display selection, control selections can be performed, which are identical to the main selection in terms of procedure – with the important difference that no decoy is used. Data analysis of the sequences resulting from the control selections allows the identification of peptides that accumulate even without decoy during the selection and are therefore irrelevant for all subsequent steps.

[0033] The procedure is thus characterized by the following steps: a) Deploying an immobilized bait on a substrate surface. b) Contacting the immobilized molecule acting as bait with a solution containing a library of molecules. c) Contacting the immobilized bait, now bound to the molecules, with a washing solution. d) Separating and amplifying the molecules that remain bound to the bait after contact with the washing solution. e) Repeating these steps, using a different substrate each time. f) Identifying the sequence of molecules remaining on the bait after repetition.

[0034] A different substrate is used, for example, by changing the type of substrate and / or blocking or not blocking it using reagents.

[0035] The bait used is a molecule from the group consisting of proteins, peptides, RNA, DNA, mRNA, and chemical compounds. In this particular case, monomeric tau protein is used as the bait.

[0036] The surface or substrate to which the bait is immobilized can be, for example, a component from the group consisting of microtiter plates, magnetic particles, agarose or sepharose beads.

[0037] The bait described in point a) is therefore a compound to which the biomolecule to be selected is to be bound. It is fixed to a first surface using methods known in the art. Examples of suitable baits include proteins, peptides, RNA, or DNA molecules, especially tau monomers. Possible surfaces include, for example, microtiter plates, magnetic particles, agarose or Sepharose beads.

[0038] The surface with the immobilized bait can then be quenched, inactivating the functional groups of the substrate. Additionally, the remaining free areas on the substrate can be blocked with suitable reagents.

[0039] In the second step b), the immobilized bait is brought into contact with a randomized library of molecules—specifically biomolecules. These biomolecules compete for binding to the bait. The randomized library is a mixture of a very large number of molecules, for example, 1012, 104, or even as few as 100 different molecules. Such a library can consist, for example, of peptides, proteins, DNA, RNA, or mRNA, each bound to specific vehicles capable of binding to the bait. Suitable vehicles include, for example, phages, polysomes, or bacterial surfaces. The library can consist of artificial components, components isolated from nature, or a mixture of both. For the purposes of this invention, "artificial" refers, for example, to compounds produced by oligonucleotide synthesis.

[0040] In step c), the immobilized bait coated with 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 solution is brought into contact with, or rinsed over, the immobilized baits. This means that the solution containing the library of biomolecules is preferably repeatedly replaced with a similar or identical solution. This removes those library molecules that dissociate less rapidly from the immobilized baits than other library molecules. The rate of the dissociation reaction of the binding library molecules is primarily determined by the different dissociation constants (especially the koff values) of the individual molecules.Those with a small k-off value remain statistically bound to the immobilized bait the longest and therefore have a lower statistical probability of being washed away by the wash buffer. The liquid containing the wash buffer is preferably aqueous and may contain a pH buffer. Optional components of the solution for the wash step may be salts, detergents, or reducing agents.

[0041] After the specificity washing step in step c), the bound biomolecules are separated from the bait and multiplied in step d).

[0042] The separation in step d) is achieved, for example, by elution of phages from the bait. This separation can be accomplished, for instance, by changing the pH, heating, or altering, particularly increasing, the salt concentration. During the subsequent amplification of the library molecules remaining on the bait, the phage particles obtained, for example, after steps a) to c) can be introduced into cells and multiplied.

[0043] In step e), the concentration of the selected biomolecules in the solution added to the bait after step a) is increased. Preferably, 3 to 6 selection rounds, comprising steps a) to e), are performed. However, 1 to 10 or 1 to 20 repetitions can also be carried out. The preferably increased competitor concentration in step c) with increasing number of cycles also leads to improved selection.

[0044] A particularly relevant mirror-image phage display involves N-terminally biotinylated D-enantiomeric tau monomer in step a), a recombinant phage library in step b), and a buffer solution in step c) alongside tau monomer as bait. Elution as a separation step is achieved, for example, by lowering the pH and by phage amplification in bacterial cells.

[0045] In this way, seven D-enantiomeric peptides that bind specifically against tau monomer were selected. SEQ ID NO 1: TF2D-1 (free N-terminus, amidated C-terminus): nemylwhwqypqhlrvg SEQ ID NO 2: TF2D-5 (free N-terminus, amidated C-terminus): dggyqilfkipgghih SEQ ID NO 3: TF2D-1a (free N-terminus, amidated C-terminus): nemylwhwqypqhlrvrrrrr SEQ ID NO 4: TF2D-1b (free N-terminus; amidated C-terminus): nelylwhwqypqhlrvrrrrr SEQ ID NO 5: TF2D-5a (free N-terminus, amidated C-terminus): dggyqilfkipgghihrrrrr SEQ ID NO 6: TF2D-5b (free N-terminus, amidated C-terminus): rssyqilfripsshihrrrrr SEQ ID NO 7: TF2D-5c (free N-terminus, amidated C-terminus): yqilfripsshihrrrrr

[0046] The peptides according to SEQ ID NO: 1 to 7 can be used as a possible drug against tauopathies, such as Alzheimer's dementia, through specific binding to tau monomers.

[0047] Here, the terms tau peptide and tau protein preferably refer to the human tau peptide and tau protein, respectively.

[0048] The peptides according to the invention are further preferably characterized in that an amide group (CONH 2 group) or a COH group, COCI group, COBr group, CONH alkyl residue or a CONH alkyl amine residue is present at the free C-terminus instead of the carboxyl group, or the peptide is cyclized.

[0049] This also advantageously solves the additional problem of providing a peptide without a negative charge at the C-terminus. This allows it to bind to the target molecule with a higher affinity than a peptide with a carboxyl group at its free C-terminus. Peptides with a free, unmodified carboxyl group exhibit a negative charge at this end in their physiological state.

[0050] In one embodiment of the invention, the peptides according to the invention are modified in the physiological state, 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, such that the C-terminus does not carry a negative charge, but is instead neutral or has one or more positive charges.

[0051] In one embodiment, the peptide is characterized in that an amide group is present at the free C-terminus instead of the carboxyl group. Thus, instead of the carboxyl group (-COOH group), an amide group (-CONH₂ group) is arranged at the C-terminus.

[0052] The peptide is therefore particularly advantageously amidated at the free C-terminus and unmodified at the free N-terminus.

[0053] The peptide is therefore particularly advantageously amidated at the free C-terminus and unmodified at the free N-terminus.

[0054] This particularly advantageously solves the further problem of having a peptide without negative charge excess, which can bind more affinely to the target molecule and is easily obtainable.

[0055] In a further embodiment of the invention, the following additional groups are present instead of the carboxyl group: COH, COCl, COBr, CONH alkyl residue, CONH alkyl amine residue (net positive charge), etc., and this is not the limit, provided that the technical teaching of the main claim is followed.

[0056] In a further preferred embodiment of the invention, the binding affinity of the peptides modified according to the invention without a negative charge at the C-terminus, compared to linear peptides with a negative charge at the C-terminus but otherwise the same amino acid sequence, is therefore reduced by 1%, 2, 3, 4, 5, 6, 7, 8, 9, in particular 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, especially 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, 187, 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, in particular 300%, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 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, 403, 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, 434, 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, advantageously even 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, 588, 589, 590, 591, 592, 593, 594 595, 596, 597, 598, 599, particularly advantageous 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, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691 692, 693, 694, 695, 696, 697, 698, 699, particularly advantageous 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, 772, 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, also particularly advantageous 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, 832, 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, 863, 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, also particularly advantageous 900%, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923,924, 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, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or even by 1000%, or even by 10000%, or even by up to 100000% or 1000000%, with any intermediate value being acceptable.

[0057] This is indicated by a correspondingly lower KD value. The KD value, as a measure of the affinity of the binding of a modified peptide to the tau monomer, is, compared to a linear, binding peptide with a negative charge at the free C-terminus, by 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, in particular 99.1, 99.2, 99.3, 99.4, 99.5%, 99.6, 99.7, 99.8, reduced by 99.9% to 99.99% or even 99.999%, with any intermediate value being acceptable.

[0058] The peptide according to the invention is further preferably characterized in that it contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 5.

[0059] Variants are also conceivable in which the peptide contains 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides with SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 5.

[0060] Particularly preferred are dimers of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 5, wherein the two monomers of the dimer are peptides with the same SEQ ID or with different SEQ IDs.

[0061] The peptides according to the invention are characterized in that they consist of D-amino acids.

[0062] The peptide according to the invention is further preferably characterized in that the peptide is linked to another substance.

[0063] The linkage, as defined in the invention, is a chemical bond as defined in Römpp Chemie Lexikon, 9th edition, volume 1, page 650 ff, Georg Thieme Verlag Stuttgart, preferably a primary valence bond, in particular a covalent bond.

[0064] In one sense, these substances are medicinal products or active ingredients, as defined in the German Medicines Act, Sections 2 and 4 (19), as amended in September 2012. Alternatively, active ingredients are therapeutically active substances used as medicinal agents. Anti-inflammatories are the preferred choice.

[0065] In another variant, the substances are compounds that enhance the effect of the peptides.

[0066] Another alternative involves compounds that improve the solubility of the peptides and / or their passage across the blood-brain barrier.

[0067] Alternatively, according to the invention, the peptides have any combination of at least two or more features of the variants, embodiments and / or alternatives described above.

[0068] The peptide according to the invention is further preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5 are linked together covalently or non-covalently.

[0069] A covalent connection or linkage of the peptide units exists within the meaning of the invention if the peptides are linearly linked head to head, tail to tail or head to tail, with or without linkers or linker groups inserted in between.

[0070] The peptide according to the invention is further preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5 are linked together without linkers, i.e. directly, or with a linker group.

[0071] A non-covalent linkage within the meaning of the invention exists if the peptides are linked to each other, for example, via biotin and streptavidin, in particular streptavidin tetramers.

[0072] The peptide according to the invention is further preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 are linked together linearly or branched.

[0073] In one embodiment of the present invention, the peptides can be linked linearly to one another, in particular as described above. In another embodiment, the peptides are linked to one another in a branched manner to form the peptide according to the invention.

[0074] According to the invention, a branched peptide can be a dendrimer in which the monomers are linked together covalently or non-covalently.

[0075] Alternatively, the peptides can also be linked to a platform molecule (such as PEG or sugar) to form a branched peptide.

[0076] Alternatively, combinations of these options are also possible. In one embodiment of the invention, the affinity of the binding of the peptides is defined via the dissociation constant (KD value).

[0077] In an advantageous embodiment of the invention, the dissociation constant (KD value) of a peptide according to the invention is advantageously reduced. This results in improved properties of the peptides according to the invention, such as higher binding affinity and higher effectiveness in degradation and / or prevention of the formation of toxic tau oligomers or aggregates.

[0078] In one embodiment of the invention, peptides are used which bind to a tau monomer with a dissociation constant (KD value) of at most 500 µM, preferably 250, 100, 50 µM, particularly preferably 25, 10, 1 µM, particularly preferably with a dissociation constant (KD value) of at most 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 sub-pM, wherein any intermediate value can be assumed.

[0079] The peptides according to the invention are further preferably characterized in that they bind to the tau protein, preferably the natively folded tau protein, with a KD of less than 1 µM.

[0080] The peptides can be produced, for example, via chemical synthesis or peptide synthesis.

[0081] In another variant, the invention relates to a peptide for inhibiting or preventing the formation of tau peptide oligomers and / or tau peptide aggregates.

[0082] In another variant, the invention relates to a peptide for the detoxification of tau peptide oligomers and / or tau peptide aggregates.

[0083] The peptides according to the invention detoxify the tau peptide oligomers and / or tau peptide aggregates or polymers formed therefrom, as well as fibrils, preferably by binding not to them but to tau monomers and, by shifting the equilibrium, leading to the reduction of the tau oligomers and thus converting them into non-toxic compounds. Accordingly, the present invention also relates to a method for detoxifying the tau oligomers, aggregates formed therefrom, or fibrils.

[0084] The inhibition or prevention of the formation of tau peptide oligomers and / or tau peptide aggregates, or the detoxification of the tau peptide oligomers and / or tau peptide aggregates, can be carried out in vitro or in vivo.

[0085] The present invention also relates to a peptide according to any of the preceding claims for use in the treatment of tauopathies.

[0086] The present invention also relates to a peptide according to any of the preceding claims, in particular for use in the treatment of Alzheimer's dementia (Alzheimer's).

[0087] The invention is explained in more detail below using non-limiting examples. Examples

[0088] The peptides TF2D-1, TF2D-5 and TF2D-5a were investigated in more detail.

[0089] The results are in the Figure 1 , 2a , 2b , 3 , 4 , 5 and 6summarized. Figure 1: TF2D-1 and TF2D-5 inhibit tau aggregation, analyzed by ThT analysis

[0090] The effects of TF2D-1 and TF2D-5 on tau aggregation are shown. The ThT assay was performed by incubating 15 µM tau with 8 µM heparin, 20 µM THT, and 15 µM of either the two peptides TF2D-1 or TF2D-5 at 37°C in PBS pH 7.4. The sample without the addition of the peptide is shown in red. The samples containing TF2D-1 or TF2D-5 are shown in green (TF2D-1) and blue (TF2D-5), respectively. Figure 2a: TF2D-5 binds with high affinity to tau protein

[0091] SPR measurements of TF2D-5 with the analyte tau. Seven tau concentrations were prepared for the measurement, with the highest concentration being 100 nM and the lowest 1.56 nM. The running buffer contained TBS with 0.1% tween. The Koff was determined to be approximately 6 x 10⁻⁵ s⁻¹. Figure 2b: TF2D-5 and TF2D-5a bind to the tau protein with high affinity.

[0092] SPR measurement of TF2D-5 and TF2D-5a with full-length tau 441 A) The binding curves and the calculated fit for the affinity of TF2D-5 to monomeric full-length tau 441 yielded a KD of 1.3 nM. The tau concentration during the measurement varied from 3.125 nM to 25 nM. B) The binding affinity of TF2D-5a to full-length tau was determined to be 6.1 nM. Tau was used in concentrations from 31.25 nM to 500 nM. The SPR measurements of A and B were recorded on a T200 Biacore instrument, with the respective peptide immobilized on a Cytiva CM5 chip, while monomeric tau served as the analyte. The tests were performed using TBS pH 7.4, 0.05% Tween 20, and 10 µM DTT as running buffer. The fit calculation was performed using the internal evaluation software of the T200 Biacore. A 1:1 model was used for both fits. Figure 3: TF2D-5 inhibits tau aggregation, analyzed by ThT and AFM analysis

[0093] The graph shows the effects of TF2D-5 on tau aggregation. The ThT assay was performed by incubating 15 µM tau with 8 µM heparin, 20 µM ThT, 10% DMSO, and different concentrations of TF2D-5 at 37°C, pH 7.0. The images next to the graph show the respective AFM measurements of the individual samples, taken after 5 days. Image A shows the sample without TF2D-5. Image 6 shows sample A with a 1:10 dilution. Image C corresponds to the blue ThT curve without dilution, to which TF2D-5 was added after 24 hours. All AFM measurements were performed by drying 1 µl on a mica, followed by two washing steps with 100 µl of water and measuring on JPK Nanowizard3 with intermittent contact air and an OMCL AC160TS as a cantilever. Figure 4: TF2D-5 and TF2D-5a affinity for Tau is sequence-specific

[0094] MST measurements of TF2D-5, TF2D-5a, and CP1 with full-length tau. A) Boltzmann fit of TF2D-5: MST measurements with full-length tau. TF2D-5 was labeled with CF633. The concentration of TF2D-5 was 500 nM, while the concentration of full-length tau ranged from 2.5 µM to 0.2 pM. The measurement was performed in 1x PBS pH 7.4 with 150 mM NaCl. The calculated KD is 308 pM ± 148 pM. B) Boltzmann fit of TF2D-5a: MST measurements with full-length tau. TF2D-5a was labeled with CF633. The concentration of TF2D-5a was 62.5 nM, while the concentration of full-length tau varied between 300 nM and 0.5 nM. The measurement was performed in 0.1 M sodium phosphate buffer pH 7.4 with 75 mM sodium sulfate and 0.05% Tween 20. The calculated KD is 11.7 nM ± 1.3 nM. C) Average of the CP1-MST measurements with full-length tau. CP1 consists of the same amino acids as TF2D-5, but with a different amino acid distribution. CP1 was labeled with CF633.The concentration of CP1 was 62.5 nM, while the concentration of full-length tau varied between 2.5 µM and 0.2 pM. The measurement was performed in 0.1 M sodium phosphate buffer pH 7.4 with 75 mM sodium sulfate and 0.05% Tween 20. No Boltzmann fit or KD could be calculated for the tested concentration range. D) Overview of the calculated KD values ​​of the individual peptides to full-length tau. All experiments were repeated four times, and the average of all measurements was calculated, which was then used to calculate the fits (Boltzmann). Figure 5: TF2D-5a inhibits the aggregation of tau in cells.

[0095] A) TF2D-5a and the D-peptide CP-1, as a negative control, were tested for their ability to inhibit tau aggregation in tauK18(LM)-YFP cells. The scale bar represents 50 µm and applies to bd. Significance was calculated using a one-way ANOVA followed by Dunnett's test for multiple comparisons. Three asterisks indicate a p-value of less than 0.001, and four asterisks indicate a p-value of less than 0.0001. B) In contrast to unseeded cells, which showed no tau aggregates on day three post-implantation, seeding with sonified tau fibrils resulted in aggregation in approximately 40% of the cells after three days of incubation. C) Three days of treatment with the D-peptide CP-1 as a negative control did not inhibit tau aggregation.D) In ​​contrast, treatment with increasing concentrations of TF2D-5a led to a concentration-dependent reduction in the number of cells with tau aggregates, which was significant at 12.5 µM and was further reduced with 25 µM and 50 µM TF2D-5a. Figure 6: TF2D-5 and TF2D-5a are very stable in various body fluids.

[0096] Stability study of TF2D-5 and TF2D-5a in various simulated human body fluids. A) Stability of the peptides in simulated gastric fluid (SGF). The simulated gastric fluid consisted of 2 mg / ml sodium chloride, 3.2 mg / ml pepsin, and 80 mM HCl; pH 1. B) Stability of TF2D-5 and TF2D-5a in simulated intestinal fluid (SIF). The simulated intestinal fluid consisted of 6.8 mg / ml potassium dihydrogen phosphate, 10 mg / ml pancreatic powder, and 15.4 mM NaOH; pH 6.8. C) Stability of TF2D-5 and TF2D-5a in human blood plasma. Human K3EDTA blood plasma was obtained from BioTrend. D) Stability of TF2D-5 and TF2D-5a in human liver microsomes. Human liver microsomes consisted of 6 mg / ml protein (containing cytochrome P450 (CYP) enzymes), 75 mM sucrose, 1.3 mM NADPH, 3.3 mM D-glucose-6-phosphate, 0.2 u / ml D-glucose-6-phosphate dehydrogenase, 3.3 mM magnesium chloride and 100 mM potassium phosphate pH 7.4.

Claims

1. A peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5, characterized in that the peptide consists of D-amino acids.

2. The peptide according to claim 1, characterized in that an acid amide group (CONH2 group) or a COH group, COCI group, COBr group, CONH alkyl residue, or a CONH alkyl amine residue is present at the free C-terminus in place of the carboxyl group, or the peptide is present in a cyclized form.

3. The peptide according to claim 1 or claim 2, characterized in that it contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5.

4. The peptide according to any one of the preceding claims, characterized in that the peptide is linked to a further substance.

5. The peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5 are covalently or non-covalently linked to each other.

6. The peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5 are linked without a linker, i.e., directly to each other, or are linked to each other with a linker group.

7. The peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5 are linked to each other in a linear or branched manner.

8. The peptide according to any one of the preceding claims, characterized in that it is a dendrimer, wherein peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5 are linked to a platform molecule.

9. The peptide according to any one of the preceding claims, characterized in that it binds to the tau peptide with a KD of less than 1 µM.

10. The peptide according to any one of the preceding claims for use in a method for preventing the formation of tau peptide oligomers and / or tau peptide aggregates.

11. The peptide according to any one of the preceding claims for use in a method for detoxifying tau peptide oligomers and / or tau peptide aggregates.

12. The peptide according to any one of the preceding claims for use in a method for treating tauopathies.