DE102022101090A1 - Use of D-enantiomeric peptide ligands from monomeric polyQ-containing proteins for the therapy of various polyglutamine diseases - Google Patents

Abstract

Describes 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and homologues, fragments and parts thereof.

Description

The 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, homologues, fragments and parts thereof, and such a peptide for use in the treatment or diagnosis of polyglutamine misfolding -Diseases.

One of the aims of the present invention was to develop an etiological therapy of polyglutamine diseases by preventing the formation of toxic oligomers or aggregates of various polyglutamine-containing proteins or by refolding misfolded polyglutamine-containing proteins into their native functional conformation.

Millions of people worldwide are affected by neurodegenerative diseases. These include a group of different clinical pictures that are characterized by progressive loss of function and neuronal death and can occur at different ages. Associated symptoms are often dementia and/or movement disorders.

Neurodegenerative diseases are associated with amyloid or non-amyloid deposits of certain proteins. Depending on the symptoms and protein deposits, a distinction is made between prion diseases, taupathies, motor neuron diseases, trinucleotide diseases, synucleinopathies, TDP-43 proteinopathies, FUSopathies, neuroaxonal dystrophies and unclassified cases.

Trinucleotide repeat diseases are caused by intragenic expansions of base triplets, which can be coding or non-coding. These often have an unstable, i. H. expanding inheritance pattern. Our working group deals with polyglutamine diseases, which are caused by expansion of the base triplet CAG (encoding for glutamine) in various genes and thus belong to the coding trinucleotide diseases. Depending on the gene affected, different pathologies are caused, such as Huntington's disease (Huntingtin) or spinobulbar muscular atrophy of the Kennedy type (androgen receptor). It is assumed that affected proteins partially lose their structural integrity as a result of the elongation of the PolyQ stretch, tend to aggregate and thus contribute to the development of the disease. A prion-like propagation of the misfolding of PolyQ-containing proteins between cells has been demonstrated in various studies.

Nine hereditary diseases are known to be caused by unphysiologically elongated polyglutamine tracts in human proteins, leading to misfolding, aggregation and neurodegeneration. Current therapeutic strategies include attempting to inhibit the expression of the particular gene encoding the polyglutamine-containing proteins. However, there is a concern that this could affect the physiological function of the protein in question. An alternative approach is to stabilize the native conformation of the protein by D-enantiomeric peptide ligands to prevent misfolding and aggregation, to shift the equilibrium between aggregates and monomers towards monomers, and to disintegrate pre-existing aggregates into non-toxic and functional monomers .

The folding and aggregation of proteins are usually considered to be competing mechanisms. Aggregating proteins are often intrinsically disordered proteins (IDP). During the aggregation process, the monomers of the aggregating protein form small, soluble oligomers that can assemble into larger aggregates. The most common aggregate structure is the cross-beta amyloid fibril, which has been described for several examples with atomic resolution.

Aggregates formed by misfolded proteins can have cytotoxic effects. Several neurodegenerative diseases are caused by misfolded proteins, such as B. Alzheimer's disease (AD), Parkinson's disease (PD), prion encephalopathies, amyotrophic lateral sclerosis (ALS) or the hereditary polyglutamine diseases, which belong to the triplet repeat diseases. Several proteins contain stretches of consecutive glutamines called "polyglutamine proteins" or "polyQ proteins." Polyglutamine proteins form amyloids in a nuclear growth mechanism, with the critical nuclear size depending on the length of the PolyQ chain. As observed for other amyloid proteins, the fibrils formed by polyglutamine proteins are able to replicate, even between different polyQ lengths.

Today, nine hereditary polyglutamine misfolding diseases are known, namely Huntington's disease, Spinal and Bulbar Muscular Atrophy (SBMA) or Kennedy's disease, Spinocerebellar Muscular Atrophy 1 (SCA1), SCA2, SCA3 or Machado-Joseph disease, SCA6, SCA7, SCA12, SCA17 and dentatorubral pallidoluynary atrophy (DRPLA) or Haw River syndrome. There are still no known effective therapies. Similar to other protein misfolding diseases, the oligomers are believed to be the main toxic species in polyglutamine misfolding diseases.

Since misfolding and aggregation of proteins with an elongated polyglutamine tract is the cause of polyglutamine diseases, inhibition of the aggregation process by stabilizing the native conformation of the polyglutamine protein with a therapeutic agent that binds to natively folded polyglutamine tracts is a promising therapeutic approach.

According to the National Institute of Neurological Disorders and Strake (https://www.ninds.nih.gov/), there are currently no substances that slow or halt the progression of HD. Tetrabenazine, deuterabenazine and antipsychotics can be used to treat the symptoms that arise.

According to the National Institute of Neurological Disorders and Stroke, there is also no curative therapy for hereditary ataxias such as SBMA.

In conclusion, a causal and significantly life-prolonging therapy for most, if not all, polyglutamine misfolding diseases does not exist and is badly needed.

The object of the present invention was therefore the development of new chemical units which can disassemble already existing, toxic aggregates of PolyQ-containing proteins into native functional units and/or inhibit the formation of new PolyQ-containing aggregates. Ideally, the effectiveness of these substances is not limited to a single PolyQ pathology, so that their therapeutic use in different PolyQ diseases is possible.

The chemical unit to be used in therapy or its variants should bind as affinely and specifically as possible to the native, endogenous, monomeric PolyQ-containing protein and thus stabilize it. The balance of misfolded and natively folded conformation is thus shifted in favor of the latter. Ideally, existing PolyQ protein oligomers and fibrils can be broken down into their monomer building blocks and thus eliminated.

This object is achieved 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and homologues, fragments and parts thereof.

Further preferred embodiments are defined in the dependent claims.

In the following, the term “comprise” should also include “consisting of”.

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, SEQ ID NO: 7, SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 were found using optimized mirror image phage display selection.

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

In mirror-image phage display, e.g. B. a recombinant library of randomized peptide sequences displayed on the gp3 protein of M13 phage and encoded in its genome, selected against the exact mirror image (D-enantiomer) of an L-enantiomeric target molecule (particularly a polyglutamine target molecule). The gp3 molecule, also known as gene product 3, is a protein that sits in the phage coat and is required for contact with the host cell.

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

The DNA sequence of the p3 gene of a selected phage is linked to the DNA sequence containing the genetic information about 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 L-enantiomeric form of the target molecule. The totality of the phages which presenting different peptides as a fusion protein with gp3 on their surface is referred to as a phage library in the following. The corresponding peptides represent the biomolecules to be selected in the experiment.

So-called panning rounds (selection rounds) can be carried out, e.g. B. three rounds. The phage library is brought into contact with a fixed target molecule, also known as a bait, and binding phages are isolated from the billions of background other, non-binding phages.

For example, the amount of phages that preferentially bind to oligomeric or fibrillar species of the polyglutamine target molecule is reduced by not offering these species as bait. In this way, phages that show an increased affinity for oligomers and fibrils of the polyglutamine target molecule can be removed from the phage pool.

In order to reduce accumulation of phages with an affinity for plastic, BSA or streptavidin, according to the invention different substrate surfaces are preferably used in all panning rounds. In this case, a combination of the biotinylated substrate used (polystyrene or polypropylene surface which has been derivatized with streptavidin) and the blocking or quenching agents used is defined as the substrate surface. In the successive selection rounds, the selection pressure is successively increased. For this purpose, while the concentration of the target molecule remains stable, the number of washing steps after the phage incubation is continuously increased from the 2nd round of selection in order to remove phages that do not have an affinity for the monomer.

Furthermore, in each selection round of phage display, a different substrate surface is chosen by using different agents to block the surface (e.g., BSA, milk powder, no blocking) after the target molecule has been immobilized on the substrate.

For example, you can switch between a BSA-blocked polystyrene surface in round 1, a milk powder-blocked polypropylene surface in round 2, and a BSA-blocked polystyrene surface in round 3.

The change between different substrate surfaces increases the specificity for the target molecule or the bait compared to the surface. In addition, there is a reduction in the number of ligands that bind non-specifically to plastic surfaces, BSA, or other components of the substrate surface other than the bait molecule.

Parallel to the actual phage display selection, control selections can be carried out, for example, which are identical to the main selection in terms of implementation - with the important difference that no bait is used here. A data analysis of the sequences resulting from the control selections enables the identification of peptides that accumulate during the selection even without decoy and are therefore irrelevant for all subsequent steps.

1. a) Bereitstellen eines immobilisierten Köders auf einer Substratoberfläche.
2. b) In Kontakt bringen des als Köder fungierenden immobilisierten Moleküls mit einer Lösung, die eine Bibliothek von zu selektierenden Molekülen enthält.
3. c) In Kontakt bringen der mit den Molekülen besetzten immobilisierten Köder mit einer Waschlösung.
4. d) Trennung und Vermehrung der nach dem in Kontakt bringen der mit den Molekülen besetzten immobilisierten Köder mit Waschlösung weiterhin an den Köder gebundenen Moleküle.
5. e) Wiederholung der genannten Schritte, wobei bei jeder Wiederholung ein unterschiedliches Substrat verwendet wird,
6. f) Identifizierung der Sequenz der nach der Wiederholung auf den Ködern verbleibenden Moleküle.

The procedure is characterized by the following steps:

1. a) providing an immobilized bait on a substrate surface.
2. b) contacting the baited immobilized molecule with a solution containing a library of molecules to be selected.
3. c) Bringing the immobilized bait, which has been occupied with the molecules, into contact with a washing solution.
4. d) Separation and multiplication of the molecules still bound to the bait after bringing the immobilized bait occupied with the molecules into contact with washing solution.
5. e) repetition of said steps, using a different substrate for each repetition,
6. f) Identification of the sequence of molecules remaining on the baits after repetition.

A different substrate is z. B. by changing the type of substrate and / or its blocking or non-blocking by means of reagents.

A molecule from the group consisting of proteins, peptides, RNA, DNA, m-RNA and chemical compounds is used as a decoy. In particular, the monomeric D-polyglutamine peptide D-K2Q23K2 (23 D-glutamine residues, N- and C-terminal flanked by two D-lysines each, sequence: kkqqqqqqqqqqqqqqqqqqqqqqqqqkk) and/or the monomeric D-polyglutamine peptide D-AR(51- 96)Q23 ("AR" for androgen receptor, residues 51 to 96, sequence: Gasllllqqqqqqqqq q qqqqqqqqqqqqqetsprqqqqqqGedGs) was used.

As a surface to which the bait is immobilized, z. B. used a component from the group consisting of microtiter plates, magnetic particles, agarose or sepharose beads.

The bait according to 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. Proteins, peptides, RNA or DNA molecules can be mentioned as baits by way of example but not limitation. Microtiter plates, magnetic particles, agarose or sepharose beads can be used as possible surfaces, for example. The surface with the immobilized bait can then be quenched, thereby inactivating the functional groups of the substrate. In addition, the hydrophobic free areas remaining on the substrate can be blocked with suitable agents.

In the second step b), the immobilized bait is brought into contact with a randomized library of molecules, specifically biomolecules. These biomolecules compete to bind to the bait. The randomized library is a mixture of very many, for example 10 to the power of 10, but also 1000 or only 100 different molecules in a mixture. Such a library can consist, for example, of peptides, proteins, DNA, RNA or mRNA, which are bound to specific vehicles and which can bind to baits. Examples of suitable vehicles are phages, polysomes or bacterial surfaces. The library can consist of artificial components or components isolated from nature, or a mixture of both. For the purposes of the invention, artificially means, for example, compounds produced from oligonucleotide synthesis.

The immobilized bait covered with biomolecules can be brought into contact with a washing substance in step c). For this purpose, a washing step is carried out in step c), in which a buffer solution is brought into contact with or rinsed with the immobilized baits. This means that the solution with the library of biomolecules is preferably repeatedly exchanged for a possibly similar or identical solution. Thus, those library molecules are removed which dissociate less quickly from the immobilized decoys than other library molecules. The speed of the detachment reaction of the binding library molecules is mainly determined by the different dissociation constants (in particular the koff values) of the individual molecules. Statistically, those with a low koff value remain 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 specificity wash step can be salts, detergents or reducing agents.

After the specificity washing step in step c), the bound biomolecules are separated from the bait and multiplied in step d). The separation or elution can take place, for example, by changing the pH, heating or other changes, in particular increasing the salt concentration. The separated or eluted biomolecules are then multiplied using known methods. For this purpose, for example, phage particles obtained and eluted according to steps a) to c), which carry the biomolecules on their surface, can be introduced into cells and multiplied.

In step e), the concentration of the selected biomolecules in the solution that is fed to the bait after step a) is increased. Preferably 3 to 6 rounds of selection containing steps a) to e) are carried out. However, 1 to 10 or 1 to 20 repetitions can also be carried out. The increase in the competitor concentration in step c), which is preferably carried out here, with an increasing number of cycles also leads to improved selection.

An increase in the washing steps in step c), which is preferably carried out, with an increasing number of cycles leads to improved selection.

• SEQ ID 1: QF2D-1 gnprmteqhqsypphmrrr
• SEQ ID 2: QF2D-2 sqsqwstpqgkwshwprrr
• SEQ ID 3: QF2D-3 hnipqklgvwpwpeerrrr
• SEQ ID 4: QF2D-4 rsfdenswqqflgpgerrr
• SEQ ID 5: QF2D-5 gyptypyntqsisswlrrr
• SEQ ID 6: QF2D-6 sstlmaypnysmqgnerrr
• SEQ ID 7: QF2D-7 hhwntawdpfhsvrrr
• SEQ ID 8: QF2D-8 hqrdpswvlygesrivrrr
• SEQ ID 9: QF2D-9 eyeqhvkwpwinnqqhrrr
• SEQ ID 10: QP1D-13 spgtyitkawpeemnw
• SEQ ID 11: QF1D-14 mpyeirwtpnhvgdmn
• SEQ ID 12: QF1D-15 eheherwgyswhdmqt
• SEQ ID 13: QF1D-16 sqhnvffsqpwdrwht
• SEQ ID 14: QF1D-17 ikaysqqqnwldhphk
• SEQ ID 15: QF1D-18 shlqghyrqvydpgfs
• SEQ ID 16: QF1D-19 hkshgvhpsqspymmi
• SEQ ID 17: QF1D-20 ehswvypysqyhwqrg
• SEQ ID 18: QF1D-21 dsvlppfilsttnqrs
• SEQ ID 19: QF1D-22 hvwkkndhhwepkywp

In this way, 19 D-enantiomeric peptides that specifically bind to polyglutamine protein monomer were designed.

• SEQ ID 1: QF2D-1 gnprmteqhqsypphmrrr
• SEQ ID 2: QF2D-2 sqsqwstpqgkwshwprrr
• SEQ ID 3: QF2D-3 hnipqklgvwpwpeerrrr
• SEQ ID 4: QF2D-4 rsfdenswqqflgpgerrr
• SEQ ID 5: QF2D-5 gyptypyntqsisswlrrr
• SEQ ID 6: QF2D-6 sstlmaypnysmqgnerrr
• SEQ ID 7: QF2D-7 hhwntawdpfhsvrrr
• SEQ ID 8: QF2D-8 hqrdpswvlygesrivrrr
• SEQ ID 9: QF2D-9 eyeqhvkwpwinnqqhrrr
• SEQ ID 10: QP1D-13 spgtyitkawpeemnw
• SEQ ID 11: QF1D-14 mpyeirwtpnhvgdmn
• SEQ ID 12: QF1D-15 Eheherwgyswhdmqt
• SEQ ID 13: QF1D-16 sqhnvffsqpwdrwht
• SEQ ID 14: QF1D-17ikaysqqqnwldhphk
• SEQ ID 15: QF1D-18 shlqghyrqvydpgfs
• SEQ ID 16: QF1D-19 hkshgvhpsqspymmi
• SEQ ID 17: QF1D-20 ehswvypysqyhwqrg
• SEQ ID 18: QF1D-21 dsvlppfilsttnqrs
• SEQ ID 19: QF1D-22 hvwkkndhhwepkywp

Through the sequence processing of QF2D-1 to -19 (e.g. sequence variation), there is the possibility of developing further substances that can be used therapeutically.

The present invention can also relate to other peptides that can be identified using the method disclosed above.

The peptides according to SEQ ID NO: 1 to 19 can be used as a possible drug against polyglutamine protein misfolding diseases due to the specific binding to polyglutamine protein monomers.

The object according to the invention is also achieved by a peptide containing homologues, fragments and parts of the amino acid sequence according to SEQ ID NO: 1 to 19.

In the context of the invention, "homologous sequences" or "homologs" means that an amino acid sequence has an identity with one of the above-mentioned amino acid sequences of the monomers of 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, 100% . 80% and 90% are preferred here. Instead of the term “identity”, the terms “homologous” or “homology” are used interchangeably in the present description. The identity between two nucleic acid sequences or polypeptide sequences is calculated by comparison using the BESTFIT program based on the algorithm of Smith, TF and Waterman, MS (Adv. Appl. Math. 2: 482-489 (1981)) using the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3. The identity between two nucleic acid sequences or polypeptide sequences is preferably defined by the identity of the nucleic acid sequence/polypeptide sequence over the entire sequence length in each case, as determined by comparison using the program GAP based on the algorithm of Needleman, S.B. and Wunsch, C.D. (J. Mol. Biol. 48:443-453) setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids gap creation penalty: 50 and gap extension penalty: 3.

Two amino acid sequences are identical for the purposes of the present invention if they have the same amino acid sequence.

In a further variant, the peptides according to the invention have sequences which differ from the specified sequences by up to two or three amino acids.

Furthermore, sequences which contain the above-mentioned sequences can also be used as peptides.

The peptide according to the invention is further preferably characterized in that at the free C-terminus instead of the carboxyl group there is an acid amide group (CONH ₂ group) or a COH group, COCl group, COBr group, CONH-alkyl radical or a CONH- alkyl-amine residue is present, or the peptide is present in cyclized form.

This also particularly advantageously solves the problem of providing a peptide without a negative charge at the C-terminus. This has the advantageous effect that it can bind to the target molecule with higher affinity than a peptide which has a carboxyl group at the free C-terminus. In the physiological state, peptides with a free, unmodified carboxyl group have a negative charge at this end.

In one embodiment of the invention, the peptide according to the invention is in the physiological state, in particular at pH 6-8, in particular 6.5-7.5, in particular 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 modified in such a way that the C-terminus does not carry a negative charge, but instead is neutral or has one or more positive charges.

In one embodiment, the peptide is characterized in that an acid amide group is present at the free C-terminus instead of the carboxyl group. Instead of the carboxyl group (-COOH group), an acid amide group (-CONH ₂ group) is arranged at the C-terminus.

Accordingly, the peptide is particularly advantageously amidated at the free C-terminus.

Accordingly, the peptide is particularly advantageously amidated at the free C-terminus and not modified at the free N-terminus.

This solves the further problem that there is a peptide without a negative charge excess, which has a higher affinity to the Can bind target molecule and is easily available.

In a further embodiment of the disclosure, the following additional groups are present instead of the carboxyl group: COH, COCl, COBr, CONH-alkyl radical, CONH-alkyl-amine radical (positive net charge), etc., without being limited to this, if the technical teaching of the main claim is followed.

In a further preferred embodiment of the invention, the affinity of the binding of the peptides modified according to the invention without a negative charge at the C-terminus is therefore higher 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, 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, 1 26, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 1 51, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 1 76, 177, 178, 179, 180, 181, 182, 184, 184, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198, 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, 2 25, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 2 50, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 2 75, 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, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 317, 318, 31, 320, 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, 3 49, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 3 74, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 3 99, In particular, 400%, 401, 403, 404, 405, 407, 408, 409, 410, 411, 412, 414, 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, 4 48, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 4 73, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 4 98, 499, advantageous even 500%, 502, 503, 504, 506, 507, 508, 509, 511, 512, 513, 515, 516, 517, 518, 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, 6 45, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 6 70, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 6 95, 696, 697, 698, 699, particularly advantageous 700%, 702, 703, 704, 706, 707, 708, 710, 711, 712, 713, 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 increased by 1000%, or even by 10000%, or even by up to 100000% or 1000000%, where any intermediate value can be accepted.

This is indicated by a correspondingly reduced K _D value. The K _D value as a measure of the affinity of binding of a modified peptide 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, especially 99.1, 99.2, 99.3, 99, 4, 99.5%, 99.6, 99.7, 99.8, 99.9 down to 99.99 or even 99.999%, any intermediate value can be accepted.

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 the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

Variants are also conceivable, with the peptide 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides with the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

Dimers of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7, SEQ SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO : 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, wherein the two monomers are peptides having the same SEQ ID or having a different SEQ ID.

The peptide according to the invention is further preferably characterized in that the peptide essentially consists of D-amino acids.

For the purposes of the present invention, the term “essentially consisting of D-enantiomeric amino acids” means that the monomers to be used according to the invention contain at least 50%, 55%, 60%, 65%, 70%, preferably 75%, 80%, particularly preferably 85%, 90%, 95%, in particular 96%, 97%, 98%, 99%, 100% are made up of D-enantiomeric amino acids.

The peptide according to the invention is further preferably characterized in that it consists of 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO : 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

The peptide according to the invention is also preferably characterized in that the peptide is linked to another substance.

In the context of the invention, the link is a chemical bond as defined in Rompp Chemie Lexikon, 9th edition, volume 1, page 650 et seq., Georg Thieme Verlag Stuttgart, preferably a main valence bond, in particular a covalent bond.

In one variant, the substances are drugs or active ingredients, defined in accordance with the Drugs Act §2 or. §4 (19), as of September 2012. In an alternative, active substances are therapeutically active substances that are used as medicinally active substances. Anti-inflammatory drugs are preferred.

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

Another alternative involves compounds that improve the solubility of the peptides and/or the passage of the blood-brain barrier.

In an alternative, the peptides according to the invention have any combination of at least two or more features of the variants, embodiments and/or alternatives described above.

The peptide according to the invention is further preferably characterized in that several 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 are covalently or non-covalently linked to one another.

A covalent connection or linkage of the peptide units is present 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.

The peptide according to the invention is also 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, SEQ SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 without a linker, ie linked directly to one another, or are linked to one another with a linker group.

A non-covalent linkage within the meaning of the invention is present if the peptides are linked to one another, for example via biotin and streptavidin, in particular streptavidin tetramer.

The peptide according to the invention is also 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, SEQ SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 are linked to one another in a linear or branched manner.

In a variant of the present invention, the peptides can be linked to one another in a linear manner, in particular as described above. In another variant, the peptides are linked to one another in a branched manner to form the peptide according to the invention.

According to the invention, a branched peptide can be a dendrimer in which the monomers are linked to one another covalently or non-covalently.

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

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 (K _D value).

The dissociation constant (K _D value) of a peptide according to the invention is advantageously reduced in an advantageous embodiment of the invention. Associated with this are improved properties of the peptides according to the invention, such as higher binding affinity and higher effectiveness of degradation and/or prevention of the formation of toxic oligomers or aggregates.

In a variant of the invention, such peptides are used that are attached to polyglutamine peptide monomer, in particular to the polyglutamine peptide K2Q23K2 and/or the polyglutamine peptide AR(51-96)Q23 and/or to the polyglutamine peptide ARQ46 with a dissociation constant (K _D value) of at most 500 μM, preferably 250, 100, 50 μM, particularly preferably 25, 10, 1 μM, particularly preferably having a dissociation constant (K _D 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, with any intermediate value.

The peptides according to the invention are also preferably characterized in that they bind to the polyglutamine peptide, in particular to the polyglutamine peptide K2Q23K2 and/or the polyglutamine peptide AR(51-96)Q23 and/or to the polyglutamine peptide ARQ46, with a K _D of less than 25 μM .

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

Another variant is a peptide according to the invention for inhibiting or preventing the formation of PolyQ peptide oligomers and/or PolyQ peptide aggregates.

Another variant is a peptide according to the invention for the detoxification of PolyQ peptide oligomers and/or PolyQ peptide aggregates.

The peptides according to the invention detoxify the PolyQ peptide oligomers and/or PolyQ peptide aggregates or polymers formed therefrom, as well as fibrils, preferably by not binding to them but to PolyQ monomer and by shifting the equilibrium leading to the reduction of the PolyQ oligomers and these thus converted into non-toxic compounds. Accordingly, the subject matter of the present invention is also a method for detoxification of the PolyQ oligomers, aggregates or fibrils formed therefrom.

The inhibition or prevention of the formation of PolyQ peptide oligomers and/or PolyQ peptide aggregates, or the detoxification of the PolyQ peptide oligomers and/or PolyQ peptide aggregates can take place in vitro or in vivo.

The present invention also relates to the peptides of the invention for preventing polyglutamine-induced androgen receptor aggregation.

Furthermore, the present invention relates to the peptides according to the invention for the detoxification of androgen receptor oligomers and/or androgen receptor aggregates.

The present invention further relates to the peptides of the invention for use in the treatment of polyglutamine disorders.

Furthermore, the present invention also relates to the peptides according to the invention for use in removing PolyQ fibrils from blood, blood products, body fluids and/or organs, e.g. B. during dialysis.

In addition, the present invention also relates to the use of peptides according to the invention as probes, in particular as sFIDA probes and/or as ELISA probes. Such diagnostic sFIDA and ELISA methods using the peptides of the invention as probes preferably take place outside the body.

The invention is explained in more detail below in non-limiting examples.

examples

Thioflavin T tests

Thioflavin T (ThT) assays were performed to monitor the time-dependent formation of amyloidogenic aggregates. Assays were performed at 37°C in TBS pH 7.5 (15 µM ThT) with double orbital shaking at 300 rpm. All buffers have been sterile filtered beforehand. Polyglutamine proteins were digested as previously described. The peptides were prediluted in TBS pH 7.5. The disaggregated polyglutamine protein in buffer was mixed with 15 µM ThT and the prediluted peptides in a 96-well flat-bottom microplate (Corning, New York, NY, USA). During the experiment, all wells contained 100 µL of ThT solution. The plate was sealed with foil (Thermo Fisher Scientific, Waltham, MA, USA) to prevent evaporation. The course of the fluorescence intensity was followed with a microplate reader (BMG Labtech, Ortenberg, Germany).

The lag time was estimated as follows: The mean deviation of the curve was determined using the last ten steady state values. Since the signal usually dropped at the beginning of the measurement, these values were not used as a starting point. As soon as the stationary signal initially exceeded the average deviation, the follow-up time was considered to have ended.

For experiments with "seeds" (pre-existing elongating amyloid aggregates), the slope of the curve was considered instead of the lag time, since the lag time was eliminated by the addition of the seeds. The slope was determined by plotting the first values and fitting with a linear fit. The time period considered (2 to 3.5 h) was similar for the curves compared to FIG.

Example 1: Impact on the aggregation of ARQ46

In this experiment it should be investigated whether the selected peptides QF2D-1, QF2D-2, QF2D-3, QF2D-4, QF2D-5, QF2D-6, QF2D-7, QF2D-8 and QF2D-9 inhibit the aggregation of the androgen receptor fragment 51-96 with 46 glutamines (GASLLLLQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQETSPRQQ QQQQGEDGS) (hereinafter referred to as ARQ46) because the aim of the work is to find PolyQ-binding peptides that inhibit aggregation. For this purpose, the ARQ46 was disaggregated into 1:1 TFA/HFIP. The solvents were evaporated under a nitrogen sparge and the ARQ46 redissolved in TBS buffer pH 7.5. The samples were incubated in a plate reader at 37° C., using 15 μM ARQ46 and 15 μM peptide each, as well as 15 μM ThT. Before each measurement, the plate was shaken at 300 rpm for 30 s. The measurement was carried out in triplicate, in 1 the means and standard deviation are shown.

Analogously to this experiment, lower concentrations of QF2D-1 and QF2D-2 were also tested, the results are in 4 shown.

The influence of QF2D-2 on the aggregation of a universal polyglutamine peptide with flanking lysines (K2Q46K2) was also investigated according to the described protocol, the results are in 7 shown.

Example 2: Aggregation of ARQ46 and K2Q46K2 in the presence of soluble aggregate fragments

The purpose of this experiment was to investigate whether the selected peptides affect the aggregation of the androgen receptor fragment 51-96 with 46 glutamines (hereinafter referred to as ARQ46) or of a pure polyglutamine peptide with flanking lysines (K2Q46K2) when soluble aggregate fragments are present. To produce these, the polyglutamine peptides (ARQ46 and K2Q46K2) were mixed for 3 days at 37°C and 300 rpm in a Concentration of 300 µM in TBS pH 7.5 aggregates. The aggregates were centrifuged off for 30 min at 100,000 g and 4° C. and then taken up again in TBS pH 7.5. After the aggregates had been sonicated (3x 15s; amplitude 60%), the insoluble components were centrifuged off for 1 h at 100,000 g and 4°C. The soluble aggregate fragments of ARQ46 obtained in this way were treated with the peptides QF2D-1, QF2D-2, QF2D-3, QF2D-4, QF2D-5, QF2D-6, QF2D-7, QF2D-8 and QF2D-9 for 23 h 37°C and 300 rpm shaking in TBS pH 7.5 preincubated the soluble aggregate fragments of the K2Q46K2 with QF1D-13, QF1D14, QF1D-15, QF1D-16, QF1D18, QF1D-19, QF1D-20, QF1D21 and QF1D-22 ,. Then the respective monomer (ARQ46 or K2Q46K2) was added. For this purpose, the ARQ46 was disaggregated into 1:1 TFA/HFIP and the K2Q46K2 into 100% TFA. The solvents were evaporated under a nitrogen sparge and the ARQ46 or K2Q46K2 redissolved in TBS buffer pH 7.5. The samples were incubated in a plate reader at 37°C using 15 µM ARQ46 or 30 µM K2Q46K2 and peptides in equimolar and substoichiometric ratios, and 15 µM ThT. Before each measurement, the plate was shaken at 300 rpm for 30 s. The measurement was carried out twice in 2 and 3 mean values and standard deviation are shown (except for 15 µM QF1D-13, where one well of the duplicate determination could not be evaluated). Peptide P8, which was selected on SOD1, and QBP1, which, according to the literature, binds to polyglutamine proteins and inhibits their aggregation, served as controls.

Example 3: Binding of QF2D-2 to ARQ46

The binding affinity of peptide QF2D-2 to ARQ46 was examined using SPR technology in the T200. The ARQ46 was immobilized on a CM5 chip by means of amino coupling, with 1400 RU immobilized on the chip after quenching. TBS-T with 0.05% Tween served as running buffer. Concentrations between 0.625 µM and 20 µM QF2D-2 were dissolved in running buffer. The binding curve and affinity fit are in 5 shown.

Example 4: Influence of QF2D-2 on aggregation in the presence of preformed aggregates

In this experiment, it should be investigated whether QF2D-2 affects the aggregation of ARQ46 when preformed, also insoluble, aggregates are present, since the peptides should still inhibit aggregation under these conditions. For this purpose, the ARQ46 was disaggregated into 1:1 TFA/HFIP. The solvents were evaporated under a nitrogen sparge and the ARQ46 redissolved in TBS buffer pH 7.5. The samples were incubated in the plate reader at 37°C using 15 µM ARQ46, 5% monomer equivalent of preformed aggregates and 15 µM QF2D2, and 15 µM ThT. Before each measurement, the plate was shaken at 300 rpm for 30 s. The measurement was carried out in triplicate, in 6 the means and standard deviation are shown.

character description

Figure 1: QF2D-1, QF2D-2, QF2D-3, QF2D-4, QF2D-5, QF2D-6, QF2D-7, QF2D-8, and QF2D-9 influence the formation of amyloid ARQ46 aggregates.

The ThT assay with ARQ46 was performed at 37°C in TBS pH 7.5. The evolution of fluorescence intensity was monitored every 30 min at λ _ex = 410 nm and λ _em = 482 nm with 30 s shaking before each measurement. A ThT assay of ARQ46 (15 µM monomer; red) and QF2D-1 (blue) in triplicate. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-1. B ThT assay of ARQ46 (15 µM monomer; red) and QF2D-2 (blue) in triplicate. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-2. C ThT assay of ARQ46 (15 µM monomer; red) and QF2D-3 (blue) in triplicate. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-3. D ThT assay of ARQ46 (15 µM monomer; red) and QF2D-4 (blue) in triplicate determination. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-4. E ThT assay of ARQ46 (15 µM monomer; red) and QF2D-5 (blue) in triplicate determination. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-5. F ThT assay of ARQ46 (15 µM monomer; red) and QF2D-6 (blue) in triplicate. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-6. G ThT assay of ARQ46 (15 µM monomer; red) and QF2D-7 (blue) in triplicate. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-7. H ThT assay of ARQ46 (15 µM monomer; red) and QF2D-8 (blue) in triplicate determination. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-8. I ThT assay of ARQ46 (15 µM monomer; red) and QF2D-9 (blue) in triplicate determination. Grey: TBS with 15 µM peptide without ARQ46. ThT fluorescence intensity of the ARQ46 probe was reduced in the presence of QF2D-9.

The selected peptides inhibited ARQ46 aggregation and may therefore have therapeutic potential for polyglutamine diseases.

Figure 2: QF2D-1, QF2D-2 and QF2D-6 affect the aggregation of ARQ46 seeded by soluble aggregate fragments

Soluble aggregates were prepared by incubating 300 µM ARQ46 for 3 days at 37°C with 300 rpm shaking. The aggregates were sonicated (amplitude 60%, 3x, 15 s) and the insoluble components were removed from the solution by ultracentrifugation (1 h, 100,000 g, 4°C). The supernatant was diluted down to 600 nM and pre-incubated with the appropriate peptides for 23 h at 37° C. and 300 rpm. Thereafter, ARQ46 monomer was added. Fluorescence intensity evolution was monitored every 30 min at λ _ex = 410 nm and λ _em = 482 with 30 s shaking (300 rpm) before each measurement. P8 served as a control peptide that was not selected on a PolyQ protein. QBP1 is a published PolyQ binder designed to inhibit aggregation. In this experiment, QBP1 slightly reduced the fluorescence intensity of the plateau, but did not delay aggregation. A ThT assay of 15 µM ARQ46 monomer (15 µM P8; grey), QBP1 at two concentrations (7.5 µM and 15 µM; lilac and purple) and with 15 µM QF2D-1 (dark blue) and 7.5 µM QF2D- 1 (light blue) in duplicate. ThT fluorescence intensity of the ARQ46 and QF2D-1 sample is reduced and delayed compared to the control peptides. B ThT assay of 15 µM ARQ46 monomer (15 µM P8; grey), QBP1 at two concentrations (7.5 µM and 15 µM; lilac and purple) and with 15 µM QF2D-2 (dark blue) and 7.5 µM QF2D- 2 (light blue) in duplicate. ThT fluorescence intensity of the ARQ46 and QF2D-2 sample is reduced and delayed compared to the control peptides. C ThT assay of 15 µM ARQ46 monomer (15 µM P8; grey), QBP1 at two concentrations (7.5 µM and 15 µM; lilac and purple) and with 15 µM QF2D-3 (dark blue) and 7.5 µM QF2D- 3 (light blue) in duplicate. QF2D-3 had an effect similar to QBP1. D ThT assay of 15 µM ARQ46 monomer (15 µM P8; grey), QBP1 at two concentrations (7.5 µM and 15 µM; lilac and purple) and with 15 µM QF2D-4 (dark blue) and 7.5 µM QF2D- 4 (light blue) in duplicate. QF2D-4 had an effect similar to QP8. E ThT assay of 15 µM ARQ46 monomer (15 µM P8; grey), QBP1 in two concentrations (7.5 µM and 15 µM; lilac and purple) and with 15 µM QF2D-5 (dark blue) in duplicate. QF2D-5 had an effect similar to QBP1. F ThT assay of 15 µM ARQ46 monomer (15 µM P8; grey), QBP1 in two concentrations (7.5 µM and 15 µM; lilac and purple) and with 15 µM QF2D-6 (dark blue) in duplicate. ThT fluorescence intensity of the ARQ46 and QF2D-6 sample is reduced and delayed compared to the control peptides.

It can be assumed that the patients to be treated will already have aggregates in the affected nerve cells when the therapy is started. Therefore, the peptides that still have an inhibiting effect on aggregation even in the presence of preformed aggregates are particularly promising. Of the peptides tested, QF2D-1, QF2D-2 and QF2D-6 met this requirement.

Figure 3: QF1D-13 and QF1D-14 affect aggregation of ARQ46 seeded by soluble aggregate fragments

Soluble aggregates were prepared by incubating 300 µM K2Q46K2 for 3 days at 37°C with 300 rpm shaking. The aggregates were sonicated (amplitude 60%, 3x, 15 s) and the insoluble components were removed from the solution by ultracentrifugation (1 h, 100,000 g, 4°C). The supernatant was diluted down to 600 nM and pre-incubated with the appropriate peptides for 23 h at 37° C. and 300 rpm. Thereafter, K2Q46K2 monomer was added. Fluorescence intensity evolution was monitored every 30 min at λ _ex = 410 nm and λ _em = 482 with 30 s shaking (300 rpm) before each measurement. P8 served as a control peptide that was not selected on a PolyQ protein. QBP1 is a published PolyQ binder designed to inhibit aggregation. In this experiment, QBP1 accelerated aggregation. A ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 in two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-13 (dark green) in duplicate and 15 µM QF1D-13 ( light green). ThT fluorescence intensity of the sample with K2Q46K2 and QF1D-13 is reduced and delayed compared to the control peptides. B ThT assay of 30 µM K2Q46K2 monomer (red) (30 µM P8; grey), QBP1 at two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-14 (dark green) in duplicate. ThT fluorescence intensity of the sample with K2Q46K2 and QF1D-14 is reduced and delayed compared to the control peptides. C ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 at two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-15 (dark green) and 15 µM QF1D-15 (light green) in double determination. The effect of QF1D-15 was comparable to that of P8. D ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 in two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-16 (dark green) and 15 µM QF1D-16 (light green) in double determination. The effect of QF1D-16 was comparable to that of P8. E ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 in two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-18 (dark green) and 15 µM QF1D-18 (light green) in duplicate. The effect of QF1D-18 was comparable to that of P8. F ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 at two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-19 (dark green) and 15 µM QF1D-19 (light green) in double determination. The effect of QF1D-19 was comparable to that of P8. G ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 at two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-20 (dark green) and 15 µM QF1D-20 (light green) in double determination. The effect of QF1D-15 was comparable to that of QBP1. H ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 in two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-21 (dark green) and 15 µM QF1D-21 (light green) in double determination. The effect of QF1D-21 was comparable to that of QBP1. I ThT assay of 30 µM K2Q46K2 monomer (30 µM P8; grey), QBP1 in two concentrations (15 µM and 30 µM; lilac and purple) and with 30 µM QF1D-22 (dark green) and 15 µM QF1D-22 (light green) in double determination. The effect of QF1D-15 was comparable to that of QBP1.

It can be assumed that the patients to be treated will already have aggregates in the affected nerve cells when the therapy is started. Therefore, the peptides that still have an inhibiting effect on aggregation even in the presence of preformed aggregates are particularly promising. Of the peptides tested, QF1D-13 and QF1D-14 met this requirement.

Figure 4: QF2D-2 has a concentration-dependent effect on ARQ46 aggregation

The ThT assay with ARQ46 was performed at 37°C in TBS pH 7.5. The evolution of fluorescence intensity was monitored every 30 min at λ _ex = 410 nm and λ _em = 482 nm with 30 s shaking before each measurement. A ThT assay with ARQ46 (15 µM monomer; red) and three concentrations of QF2D-1 (green) in triplicate determination. The mean values and standard deviations are plotted in the figure. Fluorescence intensity was reduced by QF2D-1 and aggregation was delayed. Due to the large standard deviation, no clear concentration dependency could be identified. B ThT assay with ARQ46 (15 µM monomer; red) and three concentrations of QF2D-2 (blue) in triplicate determination. The mean values and standard deviations are plotted in the figure. Fluorescence intensity was reduced by QF2D-2 in a concentration-dependent manner and aggregation was delayed.

Figure 5: QF2D-2 binds to ARQ46 with a K _D in the micromolar range

The binding properties of QF2D-2 to ARQ46 were studied using SPR technology in the T200. 1400 RU of ARQ46 was immobilized on a CM5 chip by means of amino coupling. QF2D-2 served as the analyte, multiple injections were performed at concentrations between 0.625 µM and 20 µM. The running buffer was TBS-T with 0.05% Tween. QF2D-2 bound to ARQ46 with a K _D of 11.2 µM.

Figure 6: QF2D-2 affects aggregation in the presence of 5% soluble and insoluble ARQ46 aggregates

The ThT assay with ARQ46 was performed at 37°C in TBS pH 7.5. For this purpose, the ARQ46 was disaggregated into 1:1 TFA/HFIP. The solvents were evaporated under a nitrogen sparge and the ARQ46 redissolved in TBS buffer pH 7.5. The samples were incubated in the plate reader at 37°C using 15 µM ARQ46, 5% monomer equivalent of preformed aggregates and 15 µM QF2D2, and 15 µM ThT. Before each measurement, the plate was shaken at 300 rpm for 30 s. The measurement was carried out in triplicate, the mean values and standard deviation are shown. The evolution of fluorescence intensity was monitored every 30 min at λ _ex = 448 nm and λ _em = 482 nm with 30 s shaking before each measurement. As reported in the literature, 5% aggregates eliminated lag phase in the sample without peptide. In the presence of QF2D-2, aggregation was delayed and fluorescence intensity reduced.

Figure 7: QF2D-2 affects the aggregation of K2Q46K2

The K2Q46K2 was disaggregated into 100% TFA. This was evaporated under nitrogen gas and the K2Q46K2 redissolved in TBS buffer pH 7.5. The samples were incubated in a plate reader at 37°C, using 30 µM K2Q46K2 and 30 µM peptide each (P8, QBP1 or QF2D-2), as well as 15 µM ThT. Before each measurement, the plate was shaken at 300 rpm for 30 s. The measurement was carried out in triplicate; the mean values and standard deviation are shown in the figure. The evolution of fluorescence intensity was monitored every 15 min at λ _ex = 450 nm and λ _em = 480 nm with 30 s shaking before each measurement. P8 and QBP1 served as controls. QF2D-2 inhibited K2Q46K2 aggregation more than P8, but not as effectively as QBP1. It could be shown that the peptide selected on the D-ARQ23 reacts to other PolyQ peptides is transmissible and thus most likely binds the polyglutamine tract.

A sequence listing follows as an electronic document. This can be accessed both in DEPATISnet and in the DPMAregister.

Claims (17)

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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and homologues, fragments and parts thereof. peptide according to claim 1 , characterized in that the peptide has 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19 or homologs with at least 80% identity thereof. Peptide according to any one of the preceding claims, characterized in that at the free C-terminus instead of the carboxyl group there is an acid amide group (CONH ₂ group) or a COH group, COCl group, COBr group, CONH-alkyl radical or a CONH -alkyl-amine residue is present or the peptide is present in cyclized form. Peptide according to any one of the preceding claims, 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, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19 contains. Peptide according to any one of the preceding claims, characterized in that the peptide consists essentially of D-amino acids. Peptide according to any one of the preceding claims, characterized in that the peptide consists of 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, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19. Peptide according to any one of the preceding claims, characterized in that the peptide is linked to another substance. Peptide according to any one of the preceding claims, characterized in that several 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, SEQ SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19 are covalently or non-covalently linked to one another. Peptide according to any one of the preceding claims, characterized in that several 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, SEQ SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and/or SEQ ID NO: 19 without a linker, ie linked directly to one another, or are linked to one another with a linker group. Peptide according to any one of the preceding claims, characterized in that several 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, SEQ SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19 are linked to one another in a linear or branched manner. 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, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and/or SEQ ID NO: 19 are linked to a platform molecule. Peptide according to any one of the preceding claims, characterized in that it has a K _D of less than 500 µM, in particular less than 100 µM, preferably less than 25 µM, more preferably less than 10 µM to the polyglutamine peptide K2Q23K2 and/or the polyglutamine peptide AR(51-96)Q23 and/or to the polyglutamine peptide ARQ46. Peptides according to at least one of the preceding claims for preventing polyglutamine-induced androgen receptor aggregation. Peptide according to any one of the preceding claims for detoxification of androgen receptor oligomers and/or androgen receptor aggregates. A peptide according to any one of the preceding claims for use in the treatment of polyglutamine misfolding diseases. Peptide according to any one of the preceding claims for use in removing PolyQ fibrils from blood, blood products, body fluids and/or organs, in particular during dialysis. Use of the peptide according to at least one of the preceding claims as a probe in a test method, in particular in an sFIDA method and/or in an ELISA method.