Synuclein-binding peptides and uses thereof

Abstract

The present invention relates to peptides binding to monomeric a-synuclein which are capable of stabilizing the monomeric form of a-synuclein, preventing the aggregation of a-synuclein and / or promoting the disaggregation of a-synuclein aggregates and their use in the diagnosis and treatment of synucleinopathies.

Description

[0001] Synuclein-binding peptides and uses thereof

[0002] Field of the Invention

[0003] The present invention relates to peptides binding to monomeric a-synuclein which are capable of stabilizing the monomeric form of a-synuclein, preventing the aggregation of a-synuclein and / or promoting the disaggregation of a-synuclein aggregates and their use in the diagnosis and treatment of synucleinopathies.

[0004] Introduction

[0005] Pathological aggregation of a-synuclein monomers into oligomers and fibrils is toxic to neurons and leads to cell death, resulting in progressively worsening symptoms of Parkinson’s disease or other synucleinopathies. Parkinson’s disease (PD) is an incurable and progressive neurodegenerative disease characterized by motor impairment and a variety of non-motor symptoms. PD is rare under the age of 50 and peaks between 85 years and 89 years (1.7 % for men and 1.2 % for women) with an estimated 6.1 million people affected globally (Collaborators GBDPsD, Lancet Neurol 2018, 17(11):939-53). The neuropathological hallmark of PD is the loss of dopaminergic neurons within the substantia nigra pars compacta of the midbrain that is preceded by an intraneuronal accumulation of a-synuclein (a-syn) deposits in Lewy bodies and Lewy neurites (Spillantini MG, et al. Nature 1997, 388(6645):839-40). The role of a-syn in PD was first suggested when mutations like A53T in the SNCA gene encoding a-syn were linked to familial PD (Polymeropoulos MH, etal. Science 1997, 276(5321 ):2045-7).

[0006] a-Syn is a soluble and intrinsically disordered presynaptic protein of 140 amino acids and a molecular weight of 14.6 kD (Maroteaux L, et al. J Neurosci 1988, 8(8):2804-15). PD pathogenesis is driven by the progressive aggregation of a-syn monomers into insoluble fibrillar aggregates that are toxic to neurons (Forloni G, et al. Ann Neurol 2000, 47(5):632-40). The accumulation of a-syn aggregates disturbs multiple cellular processes, such as the ubiquitin-proteasome system and the autophagy-lysosomal system responsible for protein homeostasis, mitochondrial function, endocytosis and cellular trafficking, membrane integrity, and synaptic transmission, which ultimately result in neuronal death and PD (Morris HR, et al. Lancet 2024, 403(10423):293-304). With time the a-syn pathology spreads within the central nervous system (CNS), primarily by transsynaptic transmission of a-syn aggregates from diseased to healthy neurons, where pathological a-syn can seed further a-syn aggregation by recruiting a-syn monomers into growing a-syn fibrils (Peng C, et al. Nature reviews Neurology 2020). Because of these characteristic propagation properties, a-syn is frequently referred to as a prion or prion-like protein (Woerman AL, Tamguney G, Neurobiol Dis 2022, 164:105625). Extensive research inboth human subjects and animal models support the hypothesis that a-syn pathology can spread systemically over large anatomical distances overtime, including from the brain to the peripheral nervous system and also from the enteric nervous system to the brain, potentially via peripheral nerves or the bloodstream (Arotcarena ML, etal. Brain 2020, 143(5): 1462-75).

[0007] In addition to PD, pathological a-syn aggregates are also associated with a number of other progressive neurological diseases, including PD dementia (PDD), dementia with Lewy body dementia (LDB), and multiple system atrophy (MSA), which are collectively referred to as synucleinopathies (Spillantini MG, etal., supra).

[0008] Currently, there are no approved neuroprotective or neurorestorative therapies for PD and other synucleinopathies. Therapeutic approaches targeting a-syn in PD aim to enhance clearance or modulate post-translational modificationsand aggregation. Targeting a-syn aggregation would be particularly advantageous, as it directly disrupts the downstream cascade of a-syn oligomer and fibril formation. These pathological a-syn fibrils are promising targets for therapeutic interventions aimed at curing or protecting against these diseases.

[0009] The present invention provides peptides that specifically bind and stabilize monomeric a-syn over aggregated a-syn, thereby inhibiting and preventing a-syn aggregation and fibril formation; the peptides are capable of disassembling or disaggregating existing a-syn oligomers, fibrils and / or aggregates. The inventive peptides provide a new therapeutic way to treat and prevent synucleinopathies such as PD.

[0010] Summary of the Invention

[0011] In a first aspect, the present invention relates to a peptide of no more than 42 amino acids, comprising the amino acid sequence X1X2X3X4X5X6X7X8X9VRFLVHRR (SEQ ID NO:1), with no more than one amino acid substitution in the motif VRFLVHRR, wherein each of Xi , X2 , X3 , X4, X5 , Xe , X7 , Xs and X9 is independently from each other absent or is any amino acid, and wherein SEQ ID NO: 1 comprises at least one D-amino acid residue. In particular embodiments, the peptide consists of more than 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of D-amino acids, preferably consists of more than 90% of D-amino acids, more preferably consists entirely of D-amino acids.

[0012] In one preferred embodiment the inventive peptide is as defined above, wherein Xi , X2 , X3 , X4 , X5 , Xa , X7 , Xa and X9 is independently as follows: Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably Y and V; X2 is independently chosen from the group consisting of absent, small amino acids, preferably A or G, polar amino acids, preferably N, and charged amino acids, preferably D and R; X3is independently chosenfrom the group consisting of small amino acids, preferably A, and non-polar amino acids or hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably W and F; X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R and K, and hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably L; X5 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably V, and polar amino acids, preferably Q; X8is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably H, K and R; X7 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R; X8is independently chosen from the group consisting of polar amino acids, preferably T and S, and charged amino acids, preferably positively charged amino acids, more preferably R; and X9 is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y.

[0013] In one preferred embodiment the inventive peptide is as defined above, wherein:

[0014] a) said peptide comprises the sequence XiX2[X3 / W]X4QX8X7X8YVRFLV[H / Xi5]RR (SEQ ID NO:2), wherein Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y, with Y being preferred; X2 is independently chosen from the group consisting of absent, small amino acids, preferably A or G, polar amino acids, preferably N, and charged amino acids, preferably negatively charged amino acids, more preferably D, with A, N or D being most preferred; X3 is absent or is a non-polar amino acid, preferably A; X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K, with R being preferred; X8is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K, with R being preferred; X7 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R, with R being preferred; X8is independently chosen from the group consisting of polar amino acids, preferably T or S, with T being preferred; X15 is a non-polar amino acid, preferably A, wherein when position 15 of SEQ ID NO:2 is H, R at position 11 or R at position 16 may be A; or

[0015] b) said peptide comprises the sequence X1RFLVHRRVRFLVHRR (SEQ ID NO:3), wherein Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably V; orc) said peptide comprises the sequence YDWX4QX6X7X8YVRFLVHRR (SEQ ID NO: 4), wherein X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R and K, with R being preferred; Xe is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R and K, with R being preferred; X7 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R; and Xs is independently chosen from the group consisting of polar amino acids, preferably T or S, with T being preferred; or

[0016] d) said peptide comprises the sequence X1X2WKQKRTYVRFLVHRR (SEQ ID NO: 5), wherein Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y; and X2 is independently chosen from the group consisting of absent, and charged amino acids, preferably negatively charged amino acids, more preferably D; or

[0017] e) said peptide comprises the sequence YDWRQRX7X8YVRFLVHRR (SEQ ID NO: 6), wherein X7 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R; with I being preferred; Xs is independently chosen from the group consisting of polar amino acids, preferably T and S, with T being preferred; or

[0018] f) said peptide comprises the sequence YDWRQRIXsYVRFLVHRR (SEQ ID NO: 7), wherein Xs is independently chosen from the group consisting of polar amino acids, more preferably T or S; or

[0019] g) said peptide comprises the sequence YDWX4QX6RTYVRFLVHRR (SEQ ID NO: 8), wherein X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K; and Xs is independently chosen from the group consisting of charged amino acids, preferably charged amino acids, more preferably R and K; or

[0020] h) said peptide comprises the sequence YX2WKQKRTYVRFLVX15RR (SEQ ID NO: 9), wherein X2 and X15 are independently a non-polar amino acid, preferably a small amino acid, most preferably A; or

[0021] i) said peptide comprises the sequence YX2WKQKRTYVRFLVHRR (SEQ ID NO: 10), wherein X2 is a polar amino acid, preferably N; orj) said peptide comprises the sequence YX2X3X4QX6RTX9VRFLVHRR (SEQ ID NO: 51), wherein X2 is independently chosen from the group consisting of small amino acids, preferably A or G, polar amino acids, preferably N, and charged amino acids, preferably negatively charged amino acids, more preferably D, with A, N or D being most preferred; X3 is independently chosen from the group consisting of a hydrophobic amino acid, preferably W and a small amino acid, preferably A; X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, with R or K being most preferred; Xe is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, with R or K being most preferred; X9 is independently chosen from the group consisting of a non-polar amino acid, with A or Y being most preferred; and / or one amino acid of the motif VRFLVHRR may substituted with a small amino acid, preferably A; most preferably j1) X3 is a small amino acid, preferably A, R at position 16 is substituted, preferably with A and X2is selected from A, N and D, or j2) X3is a small amino acid, preferably A and X9 is a small amino acid, preferably A, or j3) X3 is a small amino acid, preferably A and F at position 12 or V at position 14 is A; or

[0022] k) said peptide comprises the sequence YDWKQKRTYVRFLVHRR (SEQ ID NO: 14), wherein any one amino acid is substituted with a small amino acid, preferably A; preferably the peptide comprises the sequence YDWKQKRTYVRFLVHRR, wherein Y at position 1 is A, or wherein D at position 2 is A, or wherein W at position 3 is A, or wherein K at position 4 is A, or wherein Q at position 5 is A, or wherein K at position 6 is A, or wherein R at position 7 is A, or wherein T at position 8 is A, or wherein Y at position 9 is A, or wherein V at position 10 is A, or wherein R at position 11 is A, or wherein F at position 12 is A, or wherein L at position 13 is A, or wherein V at position 14 is A, or wherein H at position 15 is A, or wherein R at position 16 is A, or wherein R at position 17 is A.

[0023] In one particular aspect, the invention relates to a peptide of no more than 42 amino acids, comprising the amino acid sequence motif YDWKQKRTYVRFLVHRR (SEQ ID NO: 14) with no more than 6, preferably no more than 5, most preferably no more than 4 (i.e. 4, 3, 2 or 1) amino acid substitutions, wherein peptide consists of more than 50% D-amino acids, preferably consists of more than 90% of D-amino acids, more preferably consists entirely of D-amino acids. Preferably, the peptide consists of at least 12, 13, 14, 15, 16 or 17 amino acids and of less than 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19 or 18 amino acids.

[0024] In a preferred embodiment, the inventive peptide as defined hereinabove or -below has a length of 12-24 amino acids while maintaining the motif VRFLVHRR with one optional amino acidsubstitution in that motif, preferably said peptide has a length of 14-18 amino acids, more preferably a length of 15 amino acids, 16 amino acids or 17 amino acids.

[0025] The peptide of any one of the above embodiments has preferably no more than 25 amino acids and comprises an amino acid sequence chosen from the group consisting of SEQ ID NOs: 1-51, preferably SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a).

[0026] More preferably, the peptide according to the invention consists of an amino acid sequence chosen from the group consisting of SEQ ID NOs: 1-51, preferably SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at) , SEQ ID NO: 27 (SVD-14j_a1), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a). Particularly preferred is a peptide of any one of SEQ ID NOs: 11-50 with all D-amino acids.

[0027] The peptide of any one of the above embodiments can comprise one or more (i.e. two, three or all) of the following features (i) to (iv): (i) a C-terminal modification, wherein the C-terminalmodification is selected from the group consisting of an acid amide group (CONH2-group), a CONH-alkyl group, a CONH-alkyl amine group, a COOH-group, preferably wherein the C-terminal modification is an acid amide group (CONH2-group), (ii) an additional cysteine residue at the C-terminus and the N-terminus or within 1-2 amino acid (aa) distance of the C-terminus and the N-terminus, respectively, or an N- and / or C-terminal modification that allows a cyclization of the peptide, (iii) a truncation at the C- and / or N terminus of cumulatively no more than 4 amino acid residues, (iv) the peptide is a cyclic peptide.

[0028] As a particularly preferred embodiment, the peptide consists of the amino acid sequence chosen from the group consisting of SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a), wherein all amino acids of said peptide are D-amino acids, and wherein said peptide comprises a C-terminal modification in the form of an acid amide group (CONH2-group).

[0029] The inventive peptide binds to monomeric a-synuclein and / or inhibits a-synuclein aggregation and / or is capable of disaggregating a-synuclein aggregates.

[0030] The present invention further relates to a peptide which is an enantiomeric retro-inverso peptide of any one of the peptides defined herein, wherein the retro-inverso peptide binds to monomeric a-synuclein and / or inhibits a-synuclein aggregation and / or is capable to disaggregate a-synuclein aggregates.

[0031] In another aspect the invention relates to a multimer of a peptide defined herein. Preferably the multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides as defined herein. A particularly preferred multimer is a dimer, which can be a homodimer of two identical peptides or a heterodimer of two different peptides. Particularly preferred multimers comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies (e.g. two to form a dimer) of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-51, preferably selected from the group consisting ofSEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD- 14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD- May), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a). In the multimer, multiple copies of said peptides may form a continuous amino acid sequence (i.e. via peptide bonds) or are otherwise covalently or non-covalently linked to one another. In a particular embodiment multiple copies of the inventive peptides are linked to one another by a linker, preferably the linker is or comprises any one of Gamma-aminobutyric acid (GABA), 6-aminohexanoic acid, or polyethylene glycol (PEG) or the inventive peptides are linked with a peptide linker of no more than 10 amino acids length.

[0032] The peptide of any one of the above can be a peptide that is linked to a further substance, preferably the substance is selected from any one of a peptide, a protein, a marker, a detectable label, a drug, an antibody, a radioisotope-containing moiety, a half-life-extending moiety, or a nucleic acid such as siRNA, miRNA, shRNA or antisense RNA.

[0033] In a further aspect, the invention provides a pharmaceutical composition comprising the peptide or multimer as defined herein.

[0034] In a further aspect, the invention provides an in vitro or ex vivo use of the peptide or multimer as defined herein for (i) detecting a-synuclein oligomers and / or a-synuclein aggregates and / or (ii) detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates.

[0035] In a further aspect, the invention provides the in vivo use of the peptide or multimer or pharmaceutical composition as defined herein. In particular, the peptide, the multimer or the pharmaceutical composition are provided for use in detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates in a subject. Preferably the subject is a mammalian subject, more preferably a human subject.In a further aspect, the invention provides the peptide or multimer or the pharmaceutical composition as defined herein, for use in diagnosing and / or treating and / or preventing a synucleinopathy in a subject, preferably a mammalian subject, more preferably a human subject. The synucleinopathy is selected from the group consisting of Parkinson' disease (PD), Lewy body dementia (LBD), multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) and a disease exhibiting a a-synuclein co-pathology such as Alzheimer's disease (AD), such as Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB).

[0036] The invention further relates to methods of treatment comprising administering a peptide, a multimer or a pharmaceutical composition as defined herein, to a subject. Preferably, the subject is a subject in need of detoxification from and / or disaggregation of a-synuclein oligomers and / or a-synuclein aggregates in the subject. Preferably the subject is a mammalian subject, more preferably a human subject. The method comprise administering the subject an effective amount of a peptide, a multimer or a pharmaceutical composition as defined herein to treat or prevent a synucleinopathy such as Parkinson's disease (PD), Lewy body dementia (LBD), multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) or a disease exhibiting a a-synuclein co-pathology such as Alzheimer's disease (AD) such as Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB).

[0037] Description of the Figures

[0038] Figure 1: Surface Plasmon Resonance data for SVD-14j (A) and SVD-14u (B) binding to a-synuclein monomer on a streptavidin-coated sensorchip.

[0039] Figure 2: Surface Plasmon Resonance data for SVD-14af (A), SVD-14ag (B) and SVD-14ah (C) to a-synuclein monomer on a streptavidin-coated sensorchip.

[0040] Figure 3: Inhibition of de novo a-synuclein aggregation (ThT assay) for SVD-14j and SVD-14jb (A) and SVD-14u and SVD-14ua (B).

[0041] Figure 4: Inhibition of de novo a-synuclein aggregation (ThT assay) for SVD-14af (A), SVD-14ah (B) and SVD-14ag (C).

[0042] Figure 5: Relative ThT signal on incubation endpoint for different SVD-14 peptides in a seeded aggregation assay with a-synuclein PFF.

[0043] Figure 6: Reduction of seeded aggregation in cell-based assay for different SVD-14 peptides.Figure 7: Rescue from a-synuclein pre-formed fibril induced cell toxicity by SVD-14j and SVD-14u (A), SVD-14ua (B), and SVD-14af, SVD-14ah and SVD14jb (C).

[0044] Figure 8: Determination of the EC50 for SVD-14af and SVD-14j in inhibiting the seeding capacity of a-synuclein pre-formed fibrils in a seeded aggregation assay.

[0045] Figure 9: Incubation with SVD-14j (b) and SVD-14u (c) eliminates small a-syn fibrils in a timedependent manner, (a): dynamic light scattering (DLS) measurement of small a-syn fibrils (100 nM) in the absence of SVD-14j and SVD-14u, (b / c): DLS measurement of a-syn fibrils in the presence of 5 pM SVD-14j and 400 nM SVD-14u, respectively.

[0046] Detailed Description of the Invention

[0047] The pathological aggregation of a-synuclein monomers into oligomers and fibrils is toxic to neurons and leads to cell death, resulting in progressively worsening symptoms of Parkinson’s disease (PD) or other synucleinopathies. The present invention is based on the identification and generation of peptides that exhibit the effect of stabilizing a-synuclein (a-syn) monomers and destabilizing a-synuclein oligomers, fibrils and / or aggregates. Thus, the present invention provides tailored peptide compounds as defined hereinabove and -below that can stabilize a-synuclein monomers in their monomer conformation, can inhibit their seeded aggregation and can disassemble a-synuclein aggregates into physiologically harmless monomers and, thus, protect neurons from aggregate-induced toxicity.

[0048] The inventive peptide compounds bind a-syn monomers with high affinity, inhibit their non-seeded and seeded aggregation, efficiently eliminate a-syn fibrils in vitro and ex vivo. Due to this anti-prionic mode of action of the inventive peptide compounds, they can be used for the treatment of PD and related synucleinopathies.

[0049] In a first aspect, the present invention relates to a peptide of no more than 42 (i.e. 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 or 14) amino acids, comprising the amino acid sequence

[0050] XiX2X3X4X5X6X7X8X9VRFLVHRR (SEQ ID NO:1),

[0051] with no more than one amino acid substitution in the motif VRFLVHRR, wherein each of Xi , X2 , X3 , X4 , X5 , Xe , X7 , Xs and X9 is independently from each other absent or is any amino acid, and wherein SEQ ID NO: 1 comprises at least one D-amino acid residue. D-Amino acids are amino acids where the stereogenic carbon alpha to the amino group has the D-configuration. The inventive peptide may be a peptide which comprises in SEQ ID NO:1 one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen, D-amino acid residues. In particular embodiments, the peptide consists of more than 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of D-amino acids, preferably consists of more than 90% or more than 95% of D-amino acids, and more preferably consists entirely of D-amino acids. D-amino acid-containing peptides are advantageous for their greater stability in the physiological context, i.e. they have a greater serum half-life, are less prone to protease degradation and are less likely to induce adverse immunological reactions. Also included in the term “peptide” are “peptide variants” as further described herein below.

[0052] In one embodiment the inventive peptide is as defined above, wherein Xi , X2 , X3 , X4 , X5 , Xe , X7 , X8and X9 is independently as follows: Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably Y and V; X2 is independently chosen from the group consisting of absent, small amino acids, preferably A or G, polar amino acids, preferably N, and charged amino acids, preferably D and R; X3 is independently chosen from the group consisting of small amino acids, preferably A, and non-polar amino acids or hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably W and F; X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R and K, and hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably L; X5 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably V, and polar amino acids, preferably Q; X6is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably H, K and R; X7 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R; Xs is independently chosen from the group consisting of polar amino acids, preferably T and S, and charged amino acids, preferably positively charged amino acids, more preferably R; and X9 is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y.

[0053] The peptide as defined above can be a peptide that has no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18 or 17 amino acids and comprises the amino acid sequence of any one of (a) to (i) below:

[0054] a) the sequence XIX2[X3 / W]X4QX6X7XBYVRFLV[H / XI5]RR (SEQ ID NO:2), wherein Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y, with Y being preferred; X2 is independently chosen from the group consisting of absent, small amino acids, preferably A or G, polar amino acids, preferably N, and charged amino acids, preferably negatively charged aminoacids, more preferably D, with A, N or D being most preferred; X3 is absent or is a non-polar amino acid, preferably A; X4 is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K, with R being preferred; Xa is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K, with R being preferred; X? is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R, with R being preferred; Xs is independently chosen from the group consisting of polar amino acids, preferably T or S, with T being preferred; X15 is a non-polar amino acid, preferably A, wherein when position 15 of SEQ ID NO:2 is H, R at position 11 or R at position 16 may be A; the above preferred specifically identified amino acid positions give rise to 1 (Xi = A) *4 (X2= A, G, N or D) * 3 (X3= absent, W or A) * 2 (X4 = R or K) * 2 (X6= R or K) * 2 (X7= I or R) * 2 (Xs = T or S) * 1 (X15 = A) = 192 possible individual variants of SEQ ID NO:2, each of which in addition to SEQ ID NO:2 itself, are herewith individually included as preferred embodiments;

[0055] b) the sequence X1RFLVHRRVRFLVHRR (SEQ ID NO:3), wherein Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably V;

[0056] c) the sequence YDWX^XeXyXgYVRFLVHRR (SEQ ID NO: 4), wherein X4is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R and K, with R being preferred; Xa is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R and K, with R being preferred; X7 is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R; and Xs is independently chosen from the group consisting of polar amino acids, preferably T or S, with T being preferred; the above preferred specifically identified amino acid positions give rise to 2 (X4= R or K) * 2 (Xa = R or K) * 2 (X7= I or R) * 2 (Xs = T or S) = 16 possible individual variants of SEQ ID NO:4, each of which in addition to SEQ ID NO:4 itself, are herewith individually included as preferred embodiments;

[0057] d) the sequence X1X2WKQKRTYVRFLVHRR (SEQ ID NO: 5), wherein Xi is independently chosen from the group consisting of absent, and hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y; and X2is independently chosen from the group consisting of absent, and charged amino acids, preferably negatively charged amino acids, more preferably D;e) the sequence YDWRQRX7XBYVRFLVHRR (SEQ ID NO: 6), wherein X7is independently chosen from the group consisting of hydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, and charged amino acids, preferably positively charged amino acids, more preferably R; with I being preferred; X8is independently chosen from the group consisting of polar amino acids, preferably T and S, with T being preferred; the above preferred specifically identified amino acid positions give rise to 2 (X7= I or R) * 2 (X8= T or S) = 4 possible individual variants of SEQ ID NO:6, each of which in addition to SEQ ID NO:6 itself, are herewith individually included as preferred embodiments;

[0058] f) the sequence YDWRQRIX8YVRFLVHRR (SEQ ID NO: 7), wherein X8is independently chosen from the group consisting of polar amino acids, more preferably T or S;

[0059] g) the sequence YDWX4QX6RTYVRFLVHRR (SEQ ID NO: 8), wherein X4is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K; and X8is independently chosen from the group consisting of charged amino acids, preferably charged amino acids, more preferably R and K; the above preferred specifically identified amino acid positions give rise to 2 (X4= R or K) * 2 (X8= R or K) = 4 possible individual variants of SEQ ID NO:8, each of which in addition to SEQ ID NO:8 itself, are herewith individually included as preferred embodiments;

[0060] h) the sequence YX2WKQKRTYVRFLVXI5RR (SEQ ID NO: 9), wherein X2and Xi5are independently a non-polar amino acid, preferably a small amino acid, most preferably A; or

[0061] i) the sequence YX2WKQKRTYVRFLVHRR (SEQ ID NO: 10), wherein X2is a polar amino acid, preferably N; or

[0062] j) said peptide comprises the sequence YX2X3X4QX6RTX9VRFLVHRR (SEQ ID NO: 51), wherein X2is independently chosen from the group consisting of small amino acids, preferably A or G, polar amino acids, preferably N, and charged amino acids, preferably negatively charged amino acids, more preferably D, with A, N or D being most preferred; X8is independently chosen from the group consisting of a hydrophobic amino acid, preferably W and a small amino acid, preferably A; X4is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, with R or K being most preferred; X8is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, with R or K being most preferred; X9 is independently chosen from the group consisting of a non-polar amino acid, with A or Y being most preferred; and / or one amino acid of the motif VRFLVHRR may substituted with a small amino acid, preferably A; most preferably j1) X3 is a small amino acid, preferably A, R at position 16 is substituted, preferably with A and X2is selected from A, N and D, or j2) X3is a small amino acid, preferably A and X9 is a small amino acid,preferably A, or j3) X3 is a small amino acid, preferably A and F at position 12 or V at position 14 is A; or

[0063] k) said peptide comprises the sequence YDWKQKRTYVRFLVHRR (SEQ ID NO: 14), wherein any one amino acid is substituted with a small amino acid, preferably A; preferably the peptide comprises the sequence YDWKQKRTYVRFLVHRR, wherein Y at position 1 is A, or wherein D at position 2 is A, or wherein W at position 3 is A, or wherein K at position 4 is A, or wherein Q at position 5 is A, or wherein K at position 6 is A, or wherein R at position 7 is A, or wherein T at position 8 is A, or wherein Y at position 9 is A, or wherein V at position 10 is A, or wherein R at position 11 is A, or wherein F at position 12 is A, or wherein L at position 13 is A, or wherein V at position 14 is A, or wherein H at position 15 is A, or wherein R at position 16 is A, or wherein R at position 17 is A.

[0064] In one particular aspect, the invention relates to a peptide of no more than 42 amino acids, comprising the amino acid sequence motif YDWKQKRTYVRFLVHRR (SEQ ID NO: 14) with no more than 6 or 5, preferably no more than 4 or 3, most preferably no more than 2 or 1 amino acid substitutions, wherein peptide consists of more than 50% D-amino acids, preferably consists of more than 90% of D-amino acids, more preferably consists entirely of D-amino acids. Preferably, the peptide consists of at least 12, 13, 14, 15, 16 or 17 amino acids and of less than 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19 or 18 amino acids.

[0065] In a preferred embodiment, the inventive peptide as defined hereinabove or -below has a length of 12-24 amino acids while maintaining the motif VRFLVHRR with one optional amino acid substitution in that motif, preferably said peptide has a length of 14-18 amino acids, more preferably a length of 15 amino acids, 16 amino acids or 17 amino acids.

[0066] Preferred embodiments are peptides as defined herein, wherein the peptide has no more than 25 amino acids, comprises at least one D-amino acid and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 11-50 (Table 1).

[0067] Designation Sequence SEQ ID NO:

[0068] SVD-14ag YDWRQRITYVRFLVHRR 11

[0069] SVD-14ah YDWRQRISYVRFLVHRR 12

[0070] SVD-14af YDWRQRRTYVRFLVHRR 13

[0071] SVD-14j YDWKQKRTYVRFLVHRR 14

[0072] SVD-14jb WKQKRTYVRFLVHRR 15

[0073] SVD-14u VRFLVHRRVRFLVHRR 16

[0074] SVD-14ua RFLVHRRVRFLVHRR 17

[0075] SVD-14al YAAKQKRTYVRFLVHRR 18

[0076]

[0077] SVD-14am YAWKQKRTYVAFLVHRR 19Designation Sequence SEQ ID NO:

[0078] SVD-14an YAWKQKRTYVRFLVARR 20

[0079] SVD-14ao YAWKQKRTYVRFLVHAR 21

[0080] SVD-14ap YNWKQKRTYVRFLVHRR 22

[0081] SVD-14aq YNWKQKRTYVRFLVHAR 23

[0082] SVD-14ar YNWKQKRTYVAFLVHRR 24

[0083] SVD-14as YXXKQKRTYVRFLVHAR 25

[0084] SVD-14at YGWKQKRTYVRFLVHAR 26

[0085] SVD-14j_a1 ADWKQKRTYVRFLVHRR 27

[0086] SVD-14j_a2 YAWKQKRTYVRFLVHRR 28

[0087] SVD-14j_a3 YDAKQKRTYVRFLVHRR 29

[0088] SVD-14j_a4 YDWAQKRTYVRFLVHRR 30

[0089] SVD-14j_a5 YDWKAKRTYVRFLVHRR 31

[0090] SVD-14j_a6 YDWKQARTYVRFLVHRR 32

[0091] SVD-14j_a7 YDWKQKATYVRFLVHRR 33

[0092] SVD-14j_a8 YDWKQKRAYVRFLVHRR 34

[0093] SVD-14j_a9 YDWKQKRTAVRFLVHRR 35

[0094] SVD-14j_a10 YDWKQKRTYARFLVHRR 36

[0095] SVD-14j_a11 YDWKQKRTYVAFLVHRR 37

[0096] SVD-14j_a12 YDWKQKRTYVRALVHRR 38

[0097] SVD-14j_a13 YDWKQKRTYVRFAVHRR 39

[0098] SVD-14j_a14 YDWKQKRTYVRFLAHRR 40

[0099] SVD-14j_a15 YDWKQKRTYVRFLVARR 41

[0100] SVD-14j_a16 YDWKQKRTYVRFLVHAR 42

[0101] SVD-14j_a17 YDWKQKRTYVRFLVHRA 43

[0102] SVD-14au YDAKQKRTYVRFLVHAR 44

[0103] SVD-14av YAAKQKRTYVRFLVHAR 45

[0104] SVD-14aw YNAKQKRTYVRFLVHAR 46

[0105] SVD-14ax YDAKQKRTAVRFLVHRR 47

[0106] SVD-14ay YDAKQKRTYVRALVHRR 48

[0107] SVD-14az YDAKQKRTYVRFLAHRR 49

[0108]

[0109] SVD-14af_a WRQRRTYVRFLVHRR 50 Table 1: Exemplary alpha-synuclein monomer stabilizing peptides.

[0110] Particularly preferred peptides are peptides consisting of any one of SEQ ID NOs: 11-50, wherein one or more (i.e. two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen) of the amino acids are replaced by the corresponding D-amino acid(s), preferably all amino acids are replaced by the corresponding D-amino acids. Particularly preferred are peptides consisting of any one of SEQ ID NOs: 11-50, wherein all amino acid residues are D-configured. Further preferred is a peptide as defined herein (and in particular a peptide of any one of SEQ ID NOs: 11-50), which is C-terminally amidated, particularly an all-D peptide which is C-terminally amidated.The peptide of any one of the above embodiments can comprise one or more (i.e. two, three or all) of the following features (i) to (iv) in any combination: (i) a C-terminal modification, wherein the C-terminal modification is selected from the group consisting of an acid amide group (CONH2-group), a CONH-alkyl group, a CONH-alkyl amine group, or a COOH-group, preferably the C-terminal modification is an acid amide group (CONH2-group), (ii) an additional cysteine residue at the C-terminus and the N-terminus or within 1-2 amino acid (aa) distance of the C-terminus and the N-terminus, respectively, or an N- and / or C-terminal modification that allows a cyclization of the peptide, (iii) a truncation at the C- and / or N terminus of cumulatively no more than 4 amino acid residues, (iv) the peptide is a cyclic peptide. The peptide may also comprise an N-terminal modification alone or in conjunction with one or more of the above modifications (i) to (iv). Advantages of such modifications include increased physiological stability, better bioavailability, improved cell permeation, in particular neuronal cell permeation, higher serum half-life and increased resistance to proteolytic degradation. Such modifications also improve the peptides’ ability to cross of the blood-brain barrier. Particularly preferred is a peptide with a C-terminal amidation, which has the advantage of increasing the cellular uptake of the peptide into cells by potentially reducing overall negative peptide charge.

[0111] As a particularly preferred embodiment, the peptide consists of the amino acid sequence chosen from the group consisting of SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a), wherein all amino acids of said peptide are D-amino acids, and wherein said peptide comprises a C-terminal modification in the form of an acid amide group (CONH2-group).

[0112] The peptide of any one of the above can be a peptide that is linked to a further substance e.g. to form a peptide conjugate as further described in detail below (under the heading “Attachment of peptides to other molecules (peptide conjugates)”. Preferably the substance is selected from any one of a peptide, a protein, a marker, a detectable label, a drug, an antibody, a radioisotopecontaining moiety, a half-life extending moiety, or a nucleic acid such as siRNA, miRNA, shRNA or antisense RNA as further defined below.

[0113] The inventive peptide binds to monomeric a-synuclein and / or inhibits a-synuclein aggregation and / or is capable of disaggregating a-synuclein aggregates. The peptide of the present invention stabilizes the monomeric conformation of a-synuclein and thereby prevents a-synuclein aggregation. The stabilization of the monomeric form over the aggregated form leads to an overall degradation of the aggregates. This mechanism, which is purely driven by thermodynamics, destabilizes aggregates in the presence of the compounds and disassembles them into harmless and potentially functional native monomers. Without being bound to any theory, it is believed that the inventive peptide may also act as specific chaperone (protein folding helper) for a-synuclein, in that they, upon interaction with “misfolded” a-synuclein (a beta sheet structures-containing form that has the tendency to form aggregates), force the misfolded protein into its native form (an unordered and eventually alpha helix structure containing form which does not have a tendency to form aggregates). This active interaction of the inventive peptides with monomeric subunits of “misfolded” alpha synuclein is responsible for their capacity to actively degrade existing a-synuclein aggregates, by actively converting misfolded molecules to molecules with a native conformation. After conversion of the misfolded a-synuclein molecule into the native form, the inventive peptides are released from the molecule and are able to continue converting misfolded a-synuclein molecules.

[0114] The present invention further relates to a peptide which is an enantiomeric retro-inverso peptide of any one of the peptides defined herein, wherein the retro-inverso peptide binds to monomeric a-synuclein and / or inhibits a-synuclein aggregation and / or is capable to disaggregate a-synuclein aggregates. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules, while maintaining an identical array of side chains and similar structure. For example, a retro-inverso peptide of an all-D peptide will be an all-L peptide in which the particular amino acid positions are changed in order to substantially maintain the three dimensional space occupied by the D-peptide as far as possible. Retro-inverso peptides can be designed and produced as described in more detail further below.

[0115] In another aspect the invention relates to a multimer of a peptide defined herein. The term “multimer” refers to a plurality of monomers or units (a peptide of the invention), i.e. e.g. to dimers, trimers, 4mers, 5mers, ... etc.. The inventive peptides that form a multimer may have the same or different sequence, i.e. form homomultimers or heteromultimers. The number of different inventive peptide sequences (peptide monomers or peptide units) that may be linked to form a multimer is not limited. For example, a multimer that is a 3mer can comprise three copies of oneparticular peptide sequence as defined herein or may comprise the sequence of 2 or 3 different inventive peptides in any order.

[0116] Preferably the multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides as defined herein. Particularly preferred multimers comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-10 and 51 or the further possible preferred peptides of SEQ ID NOs: 11-50 described above. Particularly preferred are multimers with 2, 3, 4 or 5 peptide units. In the multimer, multiple copies of said peptides may form a continuous amino acid sequence (i.e. via peptide bonds) or be otherwise covalently or non-covalently linked to one another. Preferably, the monomers of the multimer are covalently linked to each other, preferably via peptide bonds. The multimers may be linear peptides or branched peptides. The peptide units that form a multimer can also be linked to a platform molecule (such as PEG or sugar) to form a branched peptide or dendrimer. Particularly preferred multimers of the inventive peptides herein are dimers and trimers.

[0117] In a particular embodiment, multiple copies of the inventive peptides are linked to one another by a branched or linear linker, preferably the linker is or comprises any one of Gamma-aminobutyric acid (GABA), 6-aminohexanoic acid, or polyethylene glycol (PEG) or the inventive peptides are linked with a peptide linker of no more than 10 amino acids length. Further linker moieties that can be used are described below under the heading “Linkers”.

[0118] In a further aspect, the invention provides a pharmaceutical composition comprising the peptide or multimer as defined herein. Specific embodiments of pharmaceutical compositions are described below under the heading “Pharmaceutical compositions”.

[0119] In a further aspect, the invention provides an in vitro or ex vivo use of the peptide or multimer as defined herein for (i) detecting a-synuclein oligomers and / or a-synuclein aggregates and / or (ii) detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates. Particular embodiments of in vitro uses of the inventive peptides are described below under the heading “In vitro uses”. A further in vitro use of the inventive peptides is the use for diagnostic purposes as described in more detail below under the heading “Diagnostics”.

[0120] In a further aspect, the invention provides the peptide or multimer or pharmaceutical composition as defined herein for various in vivo therapeutic uses. In particular, the peptide, the multimer or the pharmaceutical composition are for use in detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates in a subject. Preferably, the subject is a mammalian subject, more preferably a human subject. In a further aspect, the invention provides a peptide or multimer or a pharmaceutical composition as defined herein, for use in diagnosing and / or treating and / or preventing a synucleinopathy in a subject, preferably a mammalian subject, morepreferably a human subject. The synucleinopathy is selected from the group consisting of Parkinson's disease (PD), Lewy body dementia (LBD), multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) and a disease exhibiting a a-synuclein co-pathology such as Alzheimer's disease (AD) e.g. Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB).

[0121] The invention further relates to methods of treatment comprising administering a peptide, a multimer or a pharmaceutical composition as defined herein, to a subject. Preferably, the subject is a subject in need of detoxification from, and / or disaggregation of, a-synuclein oligomers and / or a-synuclein aggregates. Preferably the subject is a mammalian subject, more preferably a human subject suffering from a synucleinopathy such as Parkinson disease (PD), Lewy body dementia (LBD) or dementia with Lewy bodies (DLB), multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) or a disease exhibiting a a-synuclein copathology such as Alzheimer's disease (AD) such as Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB). The method comprises administering the subject an effective amount of a peptide, a multimer or a pharmaceutical composition as defined herein, thereby treating or preventing the synucleinopathy.

[0122] Further embodiments of the above in vivo aspects (medical uses and methods of treatment) of the invention are described below under the heading “Medical uses and methods of treatment”.

[0123] In the following further modifications and embodiments are described that universally apply to the peptides, multimers or pharmaceutical compositions of the present invention. The headings used are not to be construed to limit the corresponding section to one particular aspect of the invention, but may apply to each and every aspect of the present invention as far as technically applicable.

[0124] Structural features of peptide compounds

[0125] Synthetic peptides

[0126] Peptides comprising or consisting of D-amino acids can be chemically synthesized by solid phase peptide synthesis techniques known in the art. Said peptides can also be synthesized by chemical ligation such as native chemical ligation (NCL) techniques. Peptides comprising one or more of D-amino acids within a sequence of L-amino acids may also be produced using engineered ribosomes, post-translational modification systems (PLP-dependent enzyme) or non-ribosomal peptide synthetases (NPRS).

[0127] Synthetic peptides can also be obtained from various commercial sources, e.g. as lyophilized chloride salts. The peptides can be synthesized using all-D amino acids, optionally with amidatedC-terminus. Synthetic peptides preferably exhibit a purity >95 % as verified by high-performance liquid chromatography.

[0128] Length

[0129] In accordance with the invention, the peptide has a length of 12 to 42 amino acids. Accordingly, peptides in accordance with the present invention may have a length of 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 or 42 amino acids. The length range of the peptide can also be from 12-40, 13-35, 14-30, 15-21 or 16- 19. A preferred length range for the peptide is from 14 to 22 amino acids, more preferably 15 to 19 amino acids or 14 to 18 amino acids. Preferred specific lengths are 14, 15, 16, 17, 18, 19, 20 and 21 amino acids, most preferably 15, 16 or 17.

[0130] In the context of conjugates of the peptides, or multimers of the peptides, the above length definitions apply to the peptide unit that is part of said conjugate, or to the peptide unit that is a monomer of said multimer.

[0131] Position

[0132] The term “position” or “amino acid position” refers to the numerical position of a given amino acid within a peptide sequence. E.g. for SEQ ID NO:3 (XiRFLVHRRVRFLVHRR), position 1 is Xi, position 2 is R, position 3 is F and so forth. The same also applies where Xi is defined as absent; also in that case it would still occupy “position 1”, with position 1 simply being in that case absent.

[0133] The expression “within 1 -2 amino acids distance to the terminus” indicates that that the so-defined modification occurs at the second or third position from the N-terminus, or the penultimate and second-to-penultimate position from the C-terminus.

[0134] The terms “identical” and “identity” in the context of two or more peptide or polypeptide sequences in question, refer to two or more sequences or subsequences that are the same. Two amino acid sequences are identical in the sense of the present invention if they have the same amino acid sequence. In the context of the present invention, a D-amino acid residue will not be considered “identical” to the same amino acid in L-configuration with respect to the calculation of the Coidentity of two peptides.

[0135] The term “percent identity” or“%-identity” of two biological, e.g. peptide, sequences is used herein to describe the percentage of identical (not similar) amino acids or nucleotides of two sequences. “Percent identity” or “%-identity” means the percent of identical residues between the amino acidsor nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared.

[0136] In calculating percent identity, the sequences being compared are aligned in a way that gives the largest match between the sequences. Percent identity may be determined with the GCG program package, which includes GAP (Devereux et al., (1984) NucL Acid Res. 12:387) using the default parameters. The computer algorithm GAP is used to align the two sequences for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm (Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-52 for the PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10915-9 for the BLOSUM 62 comparison matrix).

[0137] A further common method to determine the %-identity of two sequences is the BLAST algorithm (Needleman S.B. and Wunsch C.D. (1970), J. Mol. Biol. 48(3): 443-53) such as implemented in EMBOSS needle (Rice P., Longden I. and Bleasby A. (2000), EMBOSS: The European Molecular Biology Open Software Suite, Trends Genet. 16(6): 276-7) using the default configuration.

[0138] Amino acids

[0139] As used herein, the terms “amino acid” and “amino acid residue” are interchangeable and, when used in the context of a peptide or polypeptide, refer to both naturally occurring and synthetic amino acids, as well as amino acid analogs and non-naturally occurring amino acids that are chemically similar to the naturally occurring amino acids.

[0140] In the present disclosure all generic references to an amino acid or amino acid residue refers to both the D- and the L-amino acid or residue. Preferably, the amino acid residues of the peptides are D-amino acids. “D-Amino acids” as used herein are amino acids in which the stereogenic alpha carbon has the D-configuration. Especially preferred are the D-configurated proteinogenic amino acids.

[0141] A “naturally occurring amino acid” or “proteinogenic amino acid” is an amino acid that is encoded by the genetic code, as well as those amino acids that are encoded by the genetic code that aremodified by a living cell after synthesis, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.

[0142] A “non-naturally occurring amino acid” or “non-proteinogenic amino acid,” or “amino acid analog” which terms can be used interchangeably herein, includes amino acids that occur by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids) which are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. A non-limiting list of examples of non-naturally amino acids that can be inserted into a peptide sequence include p-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit), Na-methylcitrulline (NMeCit), Na-methyl-homocitrulline (Na-MeHoCit), ornithine (Orn), Na-Methylornithine (Na-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Na-methylarginine (NMeR), Na-methylleucine (Na-MeL or NMeL), N-methylhomolysine (NMeHoK), Na-methylglutamine (NMeQ), norleucine (Nle), homoserine, norvaline (Nva), 1 ,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pl-Phe), para-aminophenylalanine (4AmP or4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (“K(N£-glycyl)” or “K(glycyl)” or “K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), y-carboxyglutamic acid (y-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), a-aminoadipic acid (Aad), Na-methyl valine (NMeVal), N-a-methyl leucine (NMeLeu), Na-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), a, p-diaminopropionoic acid (Dpr), a, y-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), p,P-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), a-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N -ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methyl-glycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), y-carboxyglutamate, s-N,N,N-trimethyllysine, s-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, w-methylarginine, 4-amino-O-phthalic acid (4APA), methionine sulfoxide, methionine methyl sulfonium and derivatized forms of any of those specifically listed. Further non-natural amino acids are those listed in Table S2, pages S9 to S16 of the supporting information of Kruger, D.M. et al. (J. Med. Chem. 2017, 60, 21, 8982-8988, supporting information at DOI: 10.1021 / acs.jmedchem.7b01221), the contents of which are herewith specifically incorporated by reference).The peptide or peptide variant of the present invention comprises at least one D-amino acid residue, preferably the peptide or peptide variant consists of more than 50% of D-amino acids, or consists essentially of D-amino acids. Most preferably, the peptide or peptide variant consist of all-D amino acids (i.e. every amino acid of the peptide is a D-amino acid). A peptide in accordance with the present invention may also comprise one or more non-proteinogenic or non-naturally occurring amino acids or amino acid analogs such as those defined herein.

[0143] For the purposes of the present invention, the term "consists essentially of D-amino acids" means that the peptide monomers are composed of at least 55%, 60%, 65%, 70% preferably 75%, 80%, particularly preferably 85%, 90%, 95%, especially 96%, 97%, 98%, 99%, 100% of D-amino acids. It is especially preferred that a peptide of the invention is composed of 100% D-amino acids.

[0144] Polar or hydrophilic amino acids: Herein, the following proteinogenic amino acids are considered polar / hydrophilic amino acid residues: S, T, H, N, Q, E, D, K, R. Regarding the 'charge' of the amino acid residues, the 'polar' class includes positive charged amino acids are: R, H, K and negative charged amino acids are: D, E.

[0145] Non-polar and hydrophobic amino acids: Herein, the following proteinogenic amino acids are considered non-polar amino acid residues A, C, G, I, L, M, F, P, W, V, Y. Regarding the non-polar group of amino acids, the group includes hydrophobic amino acids V, I, L, F, W, Y, M (with F, W, and Y being aromatic amino acids and V, I, L, M being aliphatic amino acids) and the small amino acids P, G, A.

[0146] Peptide variants

[0147] The term “variant” in the context of the a-syn binding peptides of the invention encompasses a peptide in which an amino acid residue of the peptide has been modified, thus generating a “peptide variant”. Such modifications include, but are not limited to, one or more (up to four) amino acid substitutions, including substitutions with non-naturally-occurring amino acids and amino acid analogs defined herein. Peptide variants of the invention retain the ability to bind to monomeric a-synuclein and / or to inhibit a-synuclein aggregation and / or are capable of disaggregating a-synuclein aggregates.

[0148] In various embodiments, a peptide or peptide variant comprises an amino acid sequence that is at least about 70 or 80 percent identical to the herein disclosed sequences. In other embodiments, a peptide comprises an amino acid sequence that is at least about 85 or 90 percent, or about 95, percent identical to the herein disclosed sequences.

[0149] The invention further relates to a peptide which is an enantiomeric retro-inverso peptide of the peptide of the invention, wherein the retro-inverso peptide binds to monomeric a-synuclein and / orinhibits a-synuclein aggregation and / or is capable to disaggregate a-synuclein aggregates. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and a similar structure. A retro-inverso peptide is topologically very similar to the original peptide since it includes all three elements of transformation: reversal of peptide-bond direction, inversion of chirality in each center, at least each alpha carbon, and repositioning of end groups that reproduce the original disposition relative to the side chains. Methods for generating retro-inverso generation based on existing peptides are described in Al Musaimi, O. ..Unlocking the Potential of Retro-inverso (Rl) Peptides as Future Drug Candidates". Int J Pept Res Ther 30, 56 (2024); the sections ..Linear Peptides", „End-group problems", ..Cyclic peptides" and ..Synthesis of Gem-Diamino Alkyl Derivatives" are hereby incorporated by reference.

[0150] Terminally modified peptides:

[0151] The C-terminus of the inventive peptide, peptide variant or multimer may be free or protected. The peptide or peptide variant or multimer of the present invention may be modified at the C-terminus, the N-terminus, or both. In one embodiment the peptide or variant or multimer comprises a C-terminal modification, wherein the C-terminal modification is selected from the group consisting of an acid amide group (CONH2-group), a CONH-alkyl group, a CONH-alkyl amine group, a COOH-group, preferably the C-terminal modification is an acid amide group (CONH2-group). In a further embodiment the peptide or peptide variant or multimer comprises an additional cysteine residue at the C-terminus and the N-terminus or within the peptide sequence, for example in 1-2 aa distance of the C-terminus and the N-terminus, respectively, or comprises an N- and / or C-terminal modification that allows a cyclization of the peptide. In a further embodiment the peptide or peptide variant or multimer comprises a truncation at the C- and / or N terminus of no more than 4 amino acid residues, preferably no more than 3, 2 or 1 amino acid residue.

[0152] The peptide, peptide variant or multimer of the present invention may be cyclic. Cyclization may be achieved by terminal (or near terminal, i.e. within 1-2 amino acids distance to the respective terminus) cysteine residues by way of a disulfide bond as described above, or may be achieved by other means (e.g. by a bispecific linker). The peptide of the invention may also be head-to-tail cyclized. Peptides with a free, unmodified carboxyl group have a negative charge at this end in the physiological state. Cyclization provides a peptide without a negative charge at the C-terminus. This has the advantage that the peptide has a potentially better cell membrane permeability as compared with a peptide which has a carboxyl group at the free C-terminus.

[0153] In another embodiment the N-terminus, the C-terminus and / or the side-chain N of K (Lys) may linked to natural or synthetic polymers like PEG, HES and / or PAS or may be linked to a carrierprotein like serum albumin, an antibody or an antibody fragment. These modifications increase the half-life in the patient’s body. In fact, any art-established measure for half-life extension may be used. In that regard, it is of note that some loss of activity or efficacy is tolerated, especially if the half-life can be significantly increased at the same time. One preferred modification of the peptide is modification with polyethylene glycol (PEG), or PEGylation (Fee & Domadaran, Eur. Pharm. Rev., Issue 1 (2010)). Modification with HES, or HESylation, is a covalent modification of a peptide by the attachment of hydroxyethyl starch (Liebner et. al. Eur J Pharm Biopharm., 87(2):378-85 (2014)). Modification with PAS, or PASylation, refers to the attachment of a conformationally disordered polypeptide chain comprising or consisting of Pro, Ala and Ser residues to a peptide of the present invention (Ahmadpour & Hosseinimehr, Curr Drug Deliv.

[0154] 15(3):331-41 (2018)).

[0155] In a further aspect, the invention relates to peptide constructs where the N-terminus is biotinylated via a linker of two molecules of y-aminobutyric acid (GABA) in 1stand 2ndposition (GABA2). All remaining amino acids (except for GABA) may be in D-configuration as defined herein. Such modified peptides are particularly useful for surface plasmon resonance (SPR) experiments. For quenching in SPR experiments, a C-terminal amidated and N-terminally biotinylated GABA2 spacer construct (biotin_GABA2-NH2) of the inventive peptides can be used.

[0156] In a particularly preferred embodiment the peptide, peptide variant or multi mer of the invention is amidated at the free C-terminus. In another particularly preferred embodiment, the peptide is amidated at the free C-terminus and unmodified at the free N-terminus.

[0157] Linkers

[0158] Any suitable linker can be employed to generate peptide conjugates or a multimeric form of the inventive peptides or peptide variants to link two of the inventive peptides, whether the same or different, with each other or to link the inventive peptide with a further entity to form a conjugate. Linker molecules can be branched or unbranched and can be attached to a peptide using various known chemistries. The chemical structure of a linker is not critical, since it serves primarily as a spacer. The linker can be independently the same or different from any other linker, or linkers, that may be present in a multimer. In one embodiment, a linker can be made up of amino acids linked together by peptide bonds (peptidyl linker). Some of these linker amino acids can be glycosylated. For example, a useful linker sequence constituting a sialylation site is X1X2NX3X4G, wherein Xi, X2, X4 and X5are each independently any amino acid residue.

[0159] In embodiments in which a peptidyl linker is present (J.e., made up of amino acids linked together by peptide bonds) the linker has a length, preferably, of from 1 up to about 10 amino acid residues, more preferably, of from 1 up to about 20 amino acid residues, and most preferably of from 1 toabout 10 amino acid residues. In one embodiment, the amino acid residues in the linker are selected from any the twenty canonical (naturally occurring) amino acids. In another embodiment the amino acid residues in the linker are selected from cysteine, glycine, alanine, proline, asparagine, glutamine, and / or serine. In yet another embodiment, a peptidyl linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, serine, and alanine linked by a peptide bond. It is often desirable that, if present, a peptidyl linker be selected that avoids rapid proteolytic turnover in circulation in vivo. Thus, preferred peptidyl linkers include polyglycines, particularly (Gly)4 (SEQ ID NO:52); (Gly)s (SEQ ID NO:53); poly(Gly-Ala); and polyalanines. Other preferred peptidyl linkers include (GGGGS)n(SEQ ID NO:54), wherein n is selected from 1, 2 or 3. Further suitable linkers include all D- or partly D-configured linkers, including basic and / or hydrophobic residues.

[0160] In embodiments of a multimer that comprise a peptide linker moiety, acidic residues, for example, glutamate or aspartate residues, are placed in the amino acid sequence of the linker moiety. Examples include the following peptide linker sequences, preferably in all-D or partly D-configured form: GGEGGG (SEQ ID NO:55); GGEEEGGG (SEQ ID NO:56); GEEEG (SEQ ID NO:57); GEEE (SEQ ID NO:58); GGDGGG (SEQ ID NO:59); GGDDDGG (SEQ ID NO:60); GDDDG (SEQ ID NO: 61); GDDD (SEQ ID NO: 62); GGGGSDDSDEGSDGEDGGGGS (SEQ ID NO: 63); WEWEW (SEQ ID NO: 64); FEFEF (SEQ ID NO: 65); EEEWWW (SEQ ID NO: 66); EEEFFF (SEQ ID NO: 67); WWEEEWW (SEQ ID NO: 68); or FFEEEFF (SEQ ID NO: 69).

[0161] In other embodiments, a peptidyl linker constitutes a phosphorylation site, e.g., X1X2YX3X4G, X1X2SX3X4G, or X1X2TX3X4G, wherein in each case Xi, X2,X3and X4are each independently any amino acid residue.

[0162] Non-peptide linkers can also be used in a multimer. For example, alkyl linkers such as -NH-(CH2)S-C(O)-, wherein s = 2 to 20 may be used. These alkyl linkers can further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-Ce), acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc..

[0163] In a further embodiment, a linker can be a PEG molecule, which can have a molecular weight of 1 to 100 kDa, preferably 10 to 50 kDa (e.g., 10, 20, 30 or 40 kDa) and more preferably 20 kDa. When a linker is a PEG molecule, attachment can be achieved by employing standard chemistry and a free sulfhydryl or amine group, such as those found on cysteine residues (which can be introduced into the peptide or variant peptide sequence by substitution or addition or can be naturally occurring) or on lysine (which can be introduced into the peptide or variant peptide sequence by substitution or addition or can be naturally occurring) or N-terminal amino groups. Other examples of useful linkers include aminoethyloxyethyloxy-acetyl linkers.In a further exemplary embodiment, the linker used to produce a multimer described herein is a homobifunctional bis-maleimide PEG molecule with the general structure: X-(CH2CH2O)nCH2CH2-X, where X is a maleimide group and n is an integer from 4-50000. In other embodiments, X can be an orthopyridyl-disulphide, an iodoacetamide, a vinylsulfone or any other reactive moiety known to the art to be specific for thiol groups. In yet another embodiment X can be an aminospecific reactive moiety used to tether two peptides or peptide variants through either the N-terminus or an engineered lysyl group. In still another embodiment, a linker can have the general structure: X-(CH2CH2O)nCH2CH2-Y, where X and Y are different reactive moieties selected from the groups in the present paragraph. Such a linker allows the conjugation of different peptides to generate heteromultimers.

[0164] Functional activity

[0165] The peptides, peptide variants and multimers of the present invention bind to monomeric a-synuclein and / or inhibit a-synuclein aggregation and / or are capable of disaggregating a-synuclein aggregates. The inventive peptides can be used for preventing the formation of a-synuclein oligomers and / or a-synuclein aggregates.

[0166] The term “a-synuclein” or “a-syn” (or “alpha-synuclein” or “a-syn”) refers to human a-synuclein as well as mammalian homologs thereof. Most preferably a-synuclein refers to human a-synuclein with the UniProtKB / Swiss-Prot. accession number P37840.1 (database entry version of 27. Nov.

[0167] 2024, which is hereby incorporated by reference in its entirety) or any reported isoform thereof.

[0168] Without being bound to any theory, the inventive peptides particularly stabilize the unfolded and / or the alpha-helical form of a-synuclein and / or destabilize the aggregated form of a-synuclein, such as fibrils or oligomers. Thereby, the formation and growth of a-synuclein fibrils and oligomers is inhibited and existing a-synuclein fibrils or oligomers are destabilized and disintegrated.

[0169] In the context of the present invention, the term “disintegration of a-synuclein aggregates” or “disaggregating of a-synuclein” includes “detoxification” or grammatically related terms such as “detoxify” of a-synuclein. The peptides, peptide variants and multimers of the present invention can therefore be used for the detoxification of a-synuclein oligomers and / or a-synuclein aggregates.

[0170] The terms “a-synuclein aggregate” and “a-synuclein oligomers” are used interchangeably herein and refer to a plurality of a-synuclein monomers which are non-covalently associated with each other. Aggregation of a-syn usually involves the formation of beta-sheet-like assemblies (dimers to oligomers), which precede the formation of more complex fibrillated forms of the protein foundin Lewy Bodies. Therefore, the expression “a-synuclein aggregate” refers to oligomeric a-syn as well as fibrillary forms of a-syn, (also referred to as fibrillary a-synuclein or a-synuclein fibrils).

[0171] The peptides according to the invention de-toxify the a-synuclein oligomers and / or a-synuclein aggregates or polymers formed therefrom, as well as fibrils, preferably not by specifically binding to these aggregate forms but binding to a-synuclein monomers and leading to the reduction of the a-synuclein oligomers by shifting the equilibrium and thus converting them into non-toxic compounds (monomeric a-synuclein).

[0172] The inhibition or prevention of the formation of a-synuclein oligomers and / or a-synuclein aggregates, or the detoxification of the a-synuclein oligomers and / or a-synuclein aggregates can be carried out in vitro or in vivo.

[0173] Binding to a-synuclein

[0174] The peptides according to the invention are further preferably characterized in that they specifically bind to a-synuclein, preferably monomeric a-synuclein with a dissociation constant (KD value) of at most 5 nM, preferably 2.5 nM, 1 nM, 0.5 nM, particularly preferably 250 pM, 100 pM, 10 pM, particularly preferably with a dissociation constant (KD value) of at most 5 pM, 2.5 pM, 1 pM, 500 nM, particularly preferably 250 nM, 100 nM, 10 nM, 5 nM, 1 nM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM to sub-pM whereby any intermediate value can be assumed.

[0175] The term “specifically binds” in the context of the peptides of the present invention relates to peptides that bind to a-synuclein, preferably monomeric a-synuclein, but do not bind to other proteins of the same subject. Alternatively, the inventive peptide, peptide variant or multimer may bind to other proteins of the same subject with an affinity that is one order of magnitude, or two, three, four, five or six or more orders of magnitude lower than the binding affinity of the peptide, peptide variant or multimer to a-synuclein.

[0176] Binding affinity of the peptides, peptide variants or multimers can be performed by techniques known in the art, such as quantitative ELISA, Surface plasmon resonance (SPR), Biolayer interferometry (BLI), Grating-coupled interferometry (GCI), Microscale thermophoresis (MST) or Isothermal titration calorimetry (ITC). SPR is an optical sensing technique that detects molecular interactions as refractive index changes within a localized evanescent field of the surface plasmon. BLI uses an optical biosensor to measure changes in the interference pattern of light reflected from the biosensor tip. The biosensor tip is typically coated with a capture molecule (monomeric or aggregated a-syn) and dipped in a sample solution containing the peptide, peptide variant or multimer of interest. GCI is another optical sensing technique that involves coupling asample and reference beam into a fiber optic waveguide; the binding affinity and kinetics are analyzed by measuring the time-dependent phase-shift signals that result from the interaction. ITC measures the heat change associated with the binding interaction and determines its binding stoichiometry and thermodynamics.

[0177] Preferably, the affinity of the peptides is determined by surface plasmon resonance (SPR).

[0178] Surface plasmon resonance (SPR)

[0179] SPR measurements can be performed using an S200 Biacore instrument (Cytiva), preferably at room temperature (RT). Interactions of inventive peptides and a-syn monomer can be measured using a single cycle kinetics experiment in PBS with 0.05 % (v / v) Tween 20 (pH 7.4). Biotin-labeled peptides are immobilized as ligand on a streptavidin derivatized SAD200M chips until a response of 1000 RU is reached (Xantec). Surface quenching can be performed by injection of 10 pM Biotin-labeled linker for 300 s on reference and active surface. A single cycle experiment can be performed using five concentrations of recombinant monomeric a-syn as analyte in range of 58 to 500 nM with association times of 200 s and dissociation time of 3600 s. Data evaluation can be performed using a 1:1 kinetic model in Biacore S200 evaluation software (Cytiva).

[0180] SPR measurements can also be performed using an 8K Biacore device (Cytiva, USA), peptides may be immobilized via primary amino groups on a CMD200M carboxyldextran matrix chip (Xantec, GE). Immobilization can be performed after 7 min EDC / NHS activation at 10 pl / min with 50 pg / ml peptide in 10 mM NaAc pH 5.0 to pH 7.0 until a saturation signal is reached. Surface quenching can also be performed with 1 M ethanolamine pH 8.3. Kinetic experiments may be performed at a flow rate of 30 pl / min in PBS pH 7.4. The surface can be regenerated between cycles with 30 s injections of 2 M Gua-HCI at 30 pl / min. Data analysis may be performed with the analysis software Biacore insight v3.0 (Cytiva, USA).

[0181] Mirror image phage display for identifying a-syn binding peptides

[0182] In the 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 construct (a protein composed fully of D-amino acids and glycine) of the naturally occurring target molecule (e.g. a-synuclein). The gp3 molecule, also known as gene product 3, is a protein that is located in the phage's envelope and is required for contact with the host cell.

[0183] The peptide sequence is advantageously presented at the N-terminus of the gp3 protein of the 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 that contains 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 peptide in D-enantiomeric configuration that binds to the physiological target molecule, which is in L-enantiomeric configuration (e.g. a-synuclein). The entirety of phages that present different peptides as fusion proteins with gp3 on their surface is referred to below as the phage library. The corresponding peptides represent the biomolecules to be selected in the experiment.

[0184] So-called panning rounds (selection rounds) can be carried out, e.g. three rounds. The phage library is brought into contact with a fixed target molecule, also known as bait, and binding phages are isolated from the billion-fold background of other, non-binding phages.

[0185] For example, the amount of phages that preferentially bind to oligomers or fibrillar species of a-synuclein is reduced by not offering these species as bait. Phages that show an increased affinity for a-synuclein oligomers and fibrils can be removed from the phage pool in this way so that, for example, a-synuclein monomer-specific phages accumulate. The method can of course be adapted in an analogous manner to identify specific a-synuclein oligomer binding ligands and peptides.

[0186] In order to reduce the accumulation of plastic-, BSA- or streptavidin-affine phages, different substrate surfaces may also be used according to the invention, preferably in all panning rounds. In this case, the substrate surface is defined as a combination of the biotinylated substrate used (e.g. polystyrene or polypropylene surface derivatized with streptavidin) and the blocking or quenching agents used. In the successive selection rounds, the selection pressure is successively increased. For this purpose, while the concentration of the target molecule (e.g. monomeric a-synuclein) remains stable, the number of washing steps after phage incubation is continuously increased from the 2ndselection round onwards in order to remove phages with no affinity for a-synuclein monomers.

[0187] Furthermore, a different substrate surface is selected in each selection round of the phage display by using different agents to block the surface after immobilization of the target molecule on the substrate (e.g. BSA, milk powder, no blocking). For example, it is possible to 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. Switching between different substrate surfaces increases the specificity for the target molecule or the bait in relation to the surface. In addition, there is a reduction in ligands that bind non-specifically to plastic surfaces, BSA or other components of the substrate surface apart from the bait molecule.Parallel to the actual phage display selection, control selections can be carried out as an example, which are identical to the main selection in terms of execution - 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 bait and are therefore irrelevant for all subsequent steps. The process is thus characterized by the following steps:

[0188] a) Providing an immobilized bait on a substrate surface.

[0189] b) Bringing the immobilized molecule acting as bait into contact with a solution containing a library of molecules to be selected.

[0190] c) Bringing the immobilized bait containing the molecules into contact with a washing solution.

[0191] d) Separation and multiplication of the molecules still bound to the bait after the immobilized bait occupied with the molecules has been brought into contact with the washing solution.

[0192] e) Repetition of the above steps, using a different substrate for each repetition,

[0193] f) Identification of the sequence of molecules remaining on the baits after repetition.

[0194] A different substrate is used, for example, by changing the type of substrate and / or blocking or not blocking it using reagents.

[0195] The surface to which the bait is immobilized is, for example, a component from the group consisting of microtiter plates, magnetic particles, agarose or Sepharose beads.

[0196] 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. By way of example but not limitation, proteins, peptides, RNA or DNA molecules, in particular a-synuclein monomers, can be mentioned as baits. Most preferably, in the context of the present invention, monomeric a-synuclein is used as bait.

[0197] Possible surfaces include microtiter plates, magnetic particles, agarose or Sepharose beads. The surface with the immobilized bait can then be quenched, whereby the functional groups of the substrate are inactivated. 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 for binding to the bait. The randomized library is a mixture of very many, for example 1012, but also 104or only 100 different molecules in a mixture. Such a library can, for example, consist of peptides, proteins, DNA, RNA or m-RNA, which are bound to specific vehicles and can bind to baits. Possible vehicles include phages, polysomes or bacterial surfaces. The library can consist of artificial components or components isolated from nature or a mixture of both. Artificial in the sense of the invention means, for example, compounds produced from oligonucleotide synthesis.

[0198] The immobilized bait containing 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 replaced with a similar or identical solution. In this way, those library molecules are removed which dissociate less quickly from the immobilized baits 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. Those with a low kOfr value statistically remain bound to the immobilized bait the longest and thus 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 may be salts, detergents or reducing agents.

[0199] 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 be carried out, for example, by changing the pH value, heating or other changes, in particular increasing the salt concentration.

[0200] The separated or eluted biomolecules are then multiplied using known methods. 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.

[0201] 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 containing steps a) to e) are carried out. However, 1 to 10 or 1 to 20 repetitions can also be carried out. Increasing the competitor concentration in step c) as the number of cycles increases also preferably leads to improved selection.

[0202] Preferably increasing the number of washing steps in step c) as the number of cycles increases leads to improved selection.A particularly relevant mirror image phage display provides N-terminally biotinylated D-enantiomeric a-synuclein monomer in step a), a recombinant phage library in step b) and a buffer solution in step c) in addition to a-synuclein monomer as bait. Elution as a separation step is carried out, for example, by lowering the pH value as a separation step and phage amplification as propagation in step d).

[0203] Aqqreqation / disaqqreqation of g-svnuclein

[0204] Preparation of a-syn monomers and fibrils: Expression of a-syn can be performed by any expression system known in the art. Exemplarily, and not by way of limitation, N-terminally acetylated human wild-type a-syn can be expressed in E. coli BL21(DE3) carrying the pT7 vector for codon-optimized a-syn and the pNatB vector for the N-terminal acetyltransferase B complex from Schizosaccharomyces pombe.

[0205] Bacteria can be cultured in 120 mL lysogeny broth (LB) medium with 100 pg / mL ampicillin and 34 pg / mL chloramphenicol at 37 °C and 120 rpm overnight. The next day, the optical density is measured at 600 nm and the culture is diluted to an optical density of 0.1 in 1 L of LB medium. The culture can then be incubated with 100 pg / mL ampicillin and 34 pg / mL chloramphenicol at 37 °C until the optical density reaches 1.0-1.2. Expression can be induced with 1 mM of isopropyl P-d-1 -thiogalactopyranoside (IPTG). After 4.5 h, the cells can be pelleted at e.g. 5000 x g and 4 °C. The pellets may be re-suspended in e.g. 20 mM Tris (pH 8.0) containing a protease inhibitor (Roche) and can be boiled for e.g. 2 x 15 min, then centrifuged at e.g. 20,000 x g and 4 °C for 30 min. Ammonium precipitation can be performed with e.g. 0.45 g / mL of (NH^S crystals, which are added to the supernatant over about 5 min and stirred for about 15 min. The centrifugation may be repeated and the pellet can re-suspended in e.g. 50 mL of 20 mM Tris-HCI (pH 8.0). a-Syn can purified using the HiPrep QFF 16 / 10 anion exchange chromatography column and a linear gradient from 20 mM Tris-HCI (pH 8.0) binding buffer to 1 M NaCI in 20 mM Tris-HCI (pH 8.0) elution buffer on an AKTA pure chromatography system (GE Healthcare). The ammonium precipitation may be repeated and the pellet may be re-suspended in e.g. 50 mM Tris-HCI (pH 7.2) and purified using a HiLoad 16 / 60 Superdex 75 pg size exclusion column (Cytiva) over 1.5 column volumes. NaCI can be added to obtain 50 mM Tris-HCI and 150 mM NaCI. The protein can be concentrated to e.g. 5 mg / mL using a vivaspin concentrator (Sartorius).

[0206] Preparation of small a-syn fibrils for in vitro experiments: Fibrillar a-syn (also called pre-formed fibrils (PFF)) can be prepared by any method known in the art. Exemplarily, insoluble fibrils are prepared by incubating 300 pM recombinant a-syn monomer in a LoBind reaction tube (Eppendorf GmbH) with a borosilicate glass bead (d = 3.0 mm; Hilgenberg, DE) in PBS (pH 7.4) with 0.05 % (w / v) sodium azide for one week at 37 °C. The insoluble fibrils can be harvested by ultracentrifugation at 100,000 x g for 30 min at 4 °C, and the pellet is washed several times withPBS (pH 7.4). The monomer equivalent concentration can be determined by measuring the a-syn concentration in the supernatant after the first centrifugation and subtracting it from the starting concentration for fibrillation. Insoluble fibrils can be resuspended in buffer and frozen at -80 °C with liquid N2. Soluble fibrils can be generated by harsh sonication of 300 pl of insoluble fibrils with 300 pM monomer equivalent concentration for 3 x 15 s (1 s on / off) and 60 % amplitude using a tip sonicator (MS 1.5 microtip, Sonoplus HD 5020). Insoluble fibrils can be separated by centrifugation at 100,000 x g for 1 h at 4 °C. The supernatant containing the soluble small a-syn fibrils can be separated, aliquoted and frozen at -80 °C with liquid N2.

[0207] Spontaneous aggregation assay: The effect of the inventive peptides to prevent a-syn aggregation can be exemplarily determined with a spontaneous aggregation assay. Recombinant a-syn monomer is thawed on ice and centrifuged at 21.000 x g for 30 min. The concentration of the supernatant is determined by A275 with an extinction coefficient of 5600 cm-1M"1. Thioflavin (ThT) fluorescence can be measured in a sealed 96-well non-binding half-area plate (Corning) with a FLUOstar Omega plate reader (BMG Labtech) at Aex = 448 nm and Aem= 482 nm. Ten pM a-syn monomer can be incubated with e.g. 2, 5, 10, 20 and 50 pM peptide at 37 °C in PBS (pH 7.4), adding one borosilicate glass bead per well (d = 3.0 mm, Hilgenberg) with continuous orbital shaking at 300 rpm in between reads (n = 5).

[0208] In addition to ThT fluorescence, aggregate formation can be followed by OD measurement or by dynamic light scattering. Alternatively, the aggregation can be monitored by a cell assay for a-syn aggregation as described in Schulz, Celina M. et al. (Cell Reports Physical Science, Volume 5, Issue 9, 102180).

[0209] A further alternative to produce a-syn aggregates and to monitor the capacity of peptides to prevent the formation of a-syn aggregates is a seeded aggregation assay or ThT assay (seeded aggregation assay).

[0210] Seeded aggregation assay: The ThT test is commonly used to visualize a-syn fibrillation, as the dye ThT is able to bind to the amyloidogenic cross-B-sheet portions of the fibril structures.

[0211] The course of Thioflavin T (ThT) fluorescence can be measured in a 96-well half plate (Corning) with a FLUOstar Omega plate reader (BMG Labtech) at Aex = 448 nm and Aem= 482 nm. Small a-syn fibrils (50 nM monomer equivalent) as seeds can be pre-incubated for 20 h with or without peptide at 37 °C under quiescent conditions in PBS (pH 7.4). Only then is 20 pM a-syn monomer is added to induce aggregation (corresponds to x = 0 h). For evaluation, ThT fluorescence is set to zero for t = 0 and the area under the curve (AUC) can be determined using the corresponding function in GraphPad Prism 10 (GraphPad Software Inc.). AUC values are normalized to 100 %for the control samples without compound (no peptide + monomer + seeds). The EC50 value can be determined using the Absolute IC50 function in GraphPad Prism 10 (GraphPad Software Inc.).

[0212] Dynamic light scattering measurements: The aggregation and disaggregation of a-syn can also be monitored by dynamic light scattering (DLS).

[0213] DLS can be measured with a SpectroSize 300 device (XtalConcepts) and a sample volume of 1 mL in a sealed quartz cuvette (Hellma Group). The samples can be incubated at 37 °C under quiescent conditions in PBS at pH 7.4. Prior to measurements, all buffers are sterile filtered (pore size 0.22 pm) and diluted samples can be centrifuged at 21,000 x g for 30 minutes at RT to remove possible impurities from the solution. Data points may be recorded every 60 s for the time-dependent DLS measurements. Diffusion coefficients can be obtained from analyzing the decay of the autocorrelation function of the scattered intensity and can used to determine the apparent hydrodynamic radii via the Stokes-Einstein equation. Kinetic fitting for the degradation of small a-syn fibrils can be performed with an inventive peptide + small a-syn fibril sample using the single-phase exponential decay function of GraphPad Prism 10 (GraphPad Software Inc.), considering only particle sizes in the range of 1 to 100 nm with an amplitude > 0.2. Outliers can be identified with a Q value of 1 % and excluded from the fitting. Protein quantification after centrifugation may be performed by incubating identical samples as in the DLS measurements with additional compound and buffer controls in low binding reaction tubes at 37 °C under quiescent conditions (n = 3). After e.g. 0, 20, and 72 h of incubation the samples can be centrifuged at 21,000 x g for 30 min at RT and the supernatant can be carefully transferred to a fresh reaction tube, while potential pellets are resuspended in 1 mL PBS pH 7.4. Protein quantification of pellet and supernatant can be performed using a Micro-BCA kit following the manufacturer's instructions (Thermo Fisher Scientific).

[0214] Attachment of peptides to other molecules (peptide conjugates)

[0215] The invention further relates to a peptide or multimer as defined herein, wherein said peptide or multimer is linked to a further substance, preferably the substance is selected from any one of a peptide, a protein, a marker, a detectable label, a drug, an antibody, a radioisotope containing moiety, or a nucleic acid such as siRNA, miRNA, shRNA or antisense RNA. The combination of a peptide or peptide variant of the present invention with another entity is also referred to as peptide conjugate.

[0216] As used herein, the term “peptide” refers to a compound comprised of amino acid residues covalently linked by peptide bonds. A peptide must contain at least two amino acids and, in the context of the present invention, the maximum number of amino acids that may constitute a peptide’s sequence is preferably 42.As used herein, the terms “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein must contain at least 50, preferably at least 60 amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein’s sequence. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or a combination thereof.

[0217] A "detectable label" or “marker” is a molecule or atom which can be conjugated to a peptide moiety to e.g. produce a molecule useful for diagnosis. Examples of detectable labels or markers include chelators, photoactive agents, radioisotopes or radioisotope containing moieties, fluorescent agents, paramagnetic ions or other marker moieties. In a preferred embodiment, the peptide, peptide variant or multimer is fluorescently labelled.

[0218] A fluorescent label may be a fluorescent protein (such as any kind of Green fluorescent protein (GFP), Yellow fluorescent protein (YFP) or Red fluorescent protein (RFP)) or another fluorescent dye. Fluorescent dyes that can be used to fluorescently label peptides include amine-reactive dyes that include active esters, carboxylates, isothiocyanates, and sulfonyl halides which react with primary amines within the peptides. Further suitable fluorescent dyes include thiol -reactive dyes that include iodoacetamides, maleimides, benzylic halides, and bromomethylketones which react with cysteines within the protein or peptide.

[0219] Exemplary fluorescent dyes are amine-reactive dyes such as fluorescein, fluorescein derivatives (such as 5-DTAF, Oregon Green 488 and Oregon Green 514), rhodamine 6G, tetramethyl-rhodamine, Texas Red fluorophores, CF633, CF488, Alexa fluor dyes (ThermoFisher Scientific) such as Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790, BIODYP fluorophores such as BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR (ThermoFisher Scientific), lissamine rhodamine B and rhodamine Red-X yyes, X-rhodamine, Texas Red and Texas Red-X Dyes, naphthofluorescein, carboxyrhodamine 6G, QSY dyes (fluorescence quenchers), coumarins, pyrenes, Pacific orange dye, cascade blue, cascade yellow and other pyridyloxazole derivatives, naphthalenes (including dansyl chloride), dapoxyl dye, bimane. Exemplary thiol-reactive dyes include Alexa Fluor maleimides (such as the C5-maleimide of Alexa Fluor 350 Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568 , Alexa Fluor 594, Alexa Fluor 633 or Alexa Fluor 750 or the C2-maleimide of Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680 or AlexaFluor 555 (ThermoFisher Scientific)), BODIPY derivatives, fluorescein derivatives (such as thiolreactive Oregon green dyes), rhodamine derivatives, PyMPO maleimide, benzoxadiazole derivatives, lucifer yellow iodoacetamide, coumarin derivatives, pacific orange maleimide, pyrene derivatives, and naphthalene derivatives. Particularly preferred fluorescent markers are CF488 and CF633.

[0220] In an exemplary embodiment of fluorescent labeling of the peptides, peptide variants and multimers of the invention, the peptides can be fluorescently labeled using cysteine-maleimide coupling. C-terminally cysteine containing variants of the inventive peptides can be fluorescently labeled C-terminally with a fivefold and tenfold molar excess of TCEP and CF633-PEG2-maleimide (Sigma-Aldrich, USA). The labeling reaction can be carried out in 25 mM NaOH pH 7.0 for 2 h at RT. The labeled peptides can be purified using an Agilent 1260 Infinity II system and a C-18 RP-HPLC column (Zorbax 300 SB-C8 / SB-C 18, Agilent, USA) at 25 °C. The mobile phases can consist of A: water + 0.1 % TFA and B: acetonitrile + 0.1 % TFA. Elution of the fluorescently labeled peptides and multimers can be performed with a gradient of 5 - 40 % (v / v) B in 30 min. The fluorescently labeled products can be analyzed using the absorption wavelength of the fluorophore (CF633: 633 nm), and the pooled samples can be subsequently lyophilized and re-dissolved in the desired buffer. Protein concentrations can be determined by UV-VIS and the fluorescently labeled peptides can be stored at -20 °C.

[0221] The term “drug” relates to any substance other than a nutrient or an essential dietary ingredient, which, when administered to a living organism, produces a physiological effect. The term “drug” includes “pharmaceutical drug”, also called “medicament” or “medicine”, which is used to treat, cure, prevent, or diagnose a disease or to promote well-being.

[0222] The term “antibody” or “Ab” as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”) and humanized antibodies. The term “antibody” also includes antibody fragments.

[0223] In a preferred embodiment the peptide (or variant or multimer thereof) is linked to an antibody (or antibody fragment) that binds to a membrane receptor of a cell (for example a cell in which a-syn aggregation needs to be prevented or a cell in need of a-syn disaggregation). Preferably, the membrane receptor is a receptor that enables to shuttle the peptide across a cellular barrier, e.g. the blood-brain-barrier (BBB).The term “antibody fragment” refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, F(ab)2, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), VHH domains, and multispecific antibodies formed from antibody fragments.

[0224] “Radioisotope-containing moiety” refers to a molecule that comprises a radioactive label, such as the isotopes3H,11C,14C,18F,32P,35S,36C1,51Cr,52Co,57Co,59Fe,67Cu,90Y,99MTc,111ln,117Lu, 121 |, 124| 125| 131 | 198A(J211^ 213^ 225Ac and186Re

[0225] The term “nucleic acid” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acid molecules can be composed of monomers present in naturally-occurring nucleotides (such as in DNA and RNA), or analogs of naturally-occurring nucleotides, or a combination of both. Nucleic acids can be either single stranded or double stranded. Preferred nucleic acids for conjugation or linkage with the inventive peptides are ribonucleotides such as siRNA, miRNA, shRNA or antisense RNA.

[0226] A “half-life extending moiety” relates to an entity that extends the serum half-life of the molecule to which it is attached as compared to said molecule without the half-life extending entity. Exemplary half-life extending moieties include or comprise polymers such as PEG, polysachosine, polysaccharides (sialic acid, hydroxyethyl starch (HES)), or moieties such as glycosaminoglycans (chondroitin sulfate, heparin, hyaluronic acid), lipids or polypeptides. The serum half-life of the peptides may exemplarily be extended by lipidation, PEGylation, fusion to an Fc moiety or serum albumin, or by N-glycosylation and / or O-glycosylation and any of the aforementioned entities represent half-life extending moieties after attachment to the inventive peptides.

[0227] In one embodiment, peptides of the present invention can be modified by covalent attachment of one or more polymers. For example, the polymer selected is typically water-soluble so that the peptide to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Included within the scope of suitable polymers is a mixture of polymers. Preferably, for therapeutic use of the end-product preparation, the polymer is pharmaceutically acceptable. Exemplary polymers each can be of any molecular weight and can be branched or unbranched. The polymers each typically have an average molecular weight of between about 2 kDa to about 100 kDa. The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably between about 20 kDa and about 35 kDa.Suitable water-soluble polymers include, but are not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(Ci-Cio), alkoxy-, or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polysialic acid and polyvinyl alcohol.

[0228] In embodiments of the instant disclosure wherein the polymer is PEG, the PEG group can be of any convenient molecular weight, and can be linear or branched. The average molecular weight of the PEG group will preferably range from about 2 kD to about 100 kDa, and more preferably from about 5 kDa to about 50 kDa, e.g., 10, 20, 30, 40, or 50 kDa. The PEG groups will generally be attached to the peptide or multimer via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the peptide or multimer (e.g., an aldehyde, amino, or ester group).

[0229] Pharmaceutical compositions

[0230] In a further aspect, the present invention relates to pharmaceutical compositions comprising a peptide, peptide variant or multimer of the invention. Such peptide, -peptide variants or -multimer containing pharmaceutical compositions can comprise a therapeutically effective amount of said peptide, peptide variant or multimer together with a pharmaceutically or physiologically acceptable carrier or formulation agent.

[0231] The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation agents suitable for accomplishing or enhancing the delivery of a peptide or peptide variant or multimer into the body of a subject such as a human or non-human subject, and for use in the methods disclosed herein. The term includes any and all solvents, dispersion media, isotonic and absorption delaying agents and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, as well as combinations thereof.

[0232] Further, a pharmaceutical composition for use in the applications and methods disclosed herein can contain one or more formulation agent(s) for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, betacyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, saltforming counterions (such as sodium or potassium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides - preferably sodium or potassium chloride - or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (see, e.g., Remington's PHARMACEUTICAL SCIENCES, 16th-20thand subsequent editions). Such formulation agents can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the peptide, peptide variant or multimer. The optimal pharmaceutical composition can be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage (see, e.g., Remington's PHARMACEUTICAL SCIENCES, supra). The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers can be used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[0233] The primary vehicle or carrier in a pharmaceutical composition for use in the methods disclosed herein is preferably aqueous in nature. For example, a suitable primary vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, optionally supplemented with other agents described herein. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary vehicles comprise Tris buffer, or acetate buffer.

[0234] In one embodiment of the present invention, peptide, peptide variant or multimer compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional further formulation agents (Remington’s PHARMACEUTICAL SCIENCES, supra) followed by lyophilization to generate a lyophilized cake.The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, subcutaneous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by inhalation (which may use an inhalation or spraying device); by intranasal application (which may include the use of application devices such as nasal spray or drops); by sustained release systems (which may also be injected); or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion.

[0235] A particularly preferred mode of administration is oral administration. Also preferred modes are subcutaneous administration and nasal administration.

[0236] Alternatively or additionally, the peptide composition can be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired peptide, peptide variant or multimer has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0237] For parenteral administration, the pharmaceutical compositions for use in the disclosed methods can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired peptide, peptide variant or multimer in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a peptide, peptide variant or multimer is formulated as a sterile, isotonic solution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the peptides, peptide variants or multimers include implantable drug delivery devices.

[0238] It is also contemplated that certain formulations can be administered orally. In one embodiment a peptide, peptide variant or multimer that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and presystemic degradation is minimized. Additional agents can be included to facilitate absorption ofthe peptide, peptide variant or multimer. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed. Suitable excipients of tables or capsules include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0239] Additional peptide, peptide variant or multimer containing pharmaceutical compositions include formulations involving said peptide, peptide variant or multimer in sustained- orcontrolled-delivery formulations. Techniques for formulating such sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles, hydrogels, porous beads, depot injections, or semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules are known to those skilled in the art. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by methods known in the art (see, e.g., Epstein etal. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3688-92).

[0240] A pharmaceutical composition comprising an inventive peptide, peptide variant or multimer to be used for in vivo administration typically should be sterile. This can for example be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.

[0241] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0242] In a specific embodiment, the present invention is directed to kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried peptide, peptide variant or multimer of the invention and a second container having an aqueous formulation of a primary pharmaceutically acceptable carrier and one or more excipients as described above. Kits can also contain single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0243] The term "effective amount" refers to an amount of peptide, peptide variant or multimer, or a pharmaceutical composition comprising the peptide, peptide variant or multimer (i.e. the “drug”) that is sufficient to effect one or more beneficial or desired outcomes. An "effective amount" can be provided by administering the drug once, twice, three times, four times, multiple times orintermittently. The duration between the administrations may be a few hours, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours. The duration between the administrations may be a few days, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more days. The administration may also be about monthly or bi-monthly, in particular if sustained release formulations are used.

[0244] The effective amount of a pharmaceutical composition comprising an inventive peptide, peptide variant or multimer to be employed therapeutically in the applications and methods disclosed herein will depend, for example, upon the therapeutic context and objectives. The appropriate dosage levels for treatment can vary depending, in part, upon the molecule delivered, the indication for which an inventive peptide, peptide variant or multimer is being used, the route of administration, and the size (body weight, body surface, or organ size), the gender and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 0.1 pg / kg to up to about 100 mg / kg or more, such as, e.g., from about 1 mg to 600 mg / patient, depending on the factors mentioned above.

[0245] The frequency of dosing employed in the methods disclosed herein will depend upon the pharmacokinetic parameters of the peptide, peptide variant or multimer in the pharmaceutical formulation used. Typically, the administration is continued until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which mayor may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Appropriate dosages can be ascertained through use of appropriate dose-response data, such as data obtained from a clinical trial involving the treatment of a synucleopathic disorder or condition, including PD, with a peptide, peptide variant or multimer of the present invention.

[0246] In vitro uses

[0247] A further aspect of the present invention is the use of the peptides or peptide variants or multimers for diagnostic purposes, in particular the in vitro use of the peptide or multimer as defined herein for detecting a-synuclein oligomers and / or a-synuclein aggregates in a sample. The sample may be a sample obtained from a subject, such as a mammalian subject, preferably a human subject. Samples obtained from a subject include, but are not limited to, a blood sample, a cerebrospinal fluid sample, a tissue sample which may have been obtained by biopsy or surgical resection, a cell sample, a saliva sample, nose fluid sample or a urine sample.

[0248] Briefly, a peptide, peptide variant or multimer of the present invention that carries a detectable label as defined herein is brought into contact with the sample under conditions suitable forallowing said peptide, peptide variant or multimer to contact a-synuclein aggregates that may be present in the sample, and monitoring / visualizing the detectable label to identify a signal associated with the label. Alternatively, the peptide, peptide variant or multimer may be immobilized and a liquid sample may be brought into contact with the immobilized peptide, peptide variant or multimer for a time sufficient to allow a-synuclein aggregates to bind. Following removal of the liquid sample and optional washing step, a-synuclein aggregates that remain bound to the immobilized peptide, peptide variant or multimer can be detected by any suitable means. A preferred way to detect the presence of a-synuclein aggregates is using BiaCore or surface plasmon resonance as described herein.

[0249] In a further embodiment of this aspect of the invention, the peptide, peptide variant or multimer can be used for detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates in vitro. In this aspect, any non-living object that may contain a-synuclein aggregates is brought into contact with the peptide, peptide variant or multimer of the invention under conditions suitable and for a time sufficient, to allow the inventive the peptide, peptide variant or multimer to disaggregate the a-synuclein oligomers and / or a-synuclein aggregates. The clearance from a-synuclein oligomers and / or a-synuclein aggregates can be monitored by any suitable means, e.g. by a-synuclein- or a-synuclein aggregate-specific antibodies.

[0250] Diagnostics

[0251] In a further aspect of the present invention, the peptide, peptide variant or multimer as defined herein can be used in diagnosing a synucleinopathy in a subject, preferably a mammalian subject, more preferably a human subject.

[0252] “Diagnosing” or “diagnosis” of a synucleinopathy relates to detecting the presence or absence of a synucleinopathy, as well as the detection and monitoring of the severity of the synucleinopathy or the monitoring of the course of a synucleinopathy.

[0253] Using an immobilized sample from a subject or patient the immobilized sample may be incubated with a peptide, peptide variant or multimer of the present invention for under conditions and for a time suitable to allow the peptide, peptide variant or multimer to disaggregate a-synuclein oligomers and / or a-synuclein aggregates that may be present in the sample to release a-synuclein monomers in the supernatant of the reaction vessel or the liquid phase of a column containing the immobilized sample. Accumulation of a-syn monomers in the supernatant, or detection of a-syn monomers in the flow through is an indication that the sample contained or contains a-synuclein oligomers and / or a-synuclein aggregates.Alternatively or additionally, for monitoring the course of a synucleinopathy in a subject, the presence or amount of a-syn aggregates can be monitored at different time points using an immuno-fluorescent (or immune-fluorescent) staining as described below.

[0254] Immuno-fluorescent cell staining

[0255] A sample comprising cells from a subject to be diagnosed may be obtained by any suitable means. The isolated cells may be cultivated in any suitable means appropriate for the cell type. After three days of cell cultivation, the cells may be fixed in 4 % formaldehyde (Sigma-Aldrich, USA) in PBS (pH 7.4) for 15 minutes. After washing three times with PBS for 5 minutes each time, the cells can be permeabilized with 0.25 % Triton X-100 (Sigma-Aldrich, USA) in PBS for 10 minutes.

[0256] After three further 5-minute washes with PBS, the cells can be blocked for 30 minutes with 1 % bovine serum albumin (Sigma-Aldrich, USA) in PBS containing 0.1 % Tween 20 (Sig ma -Aid rich, USA). Cells can be stained with CF633 (Biotium, USA) fluorescently labeled antibody at a concentration of e.g. 8 pg / ml in 1 % bovine serum albumin in PBS containing 0.1 % Tween-20 for 1-3 h at room temperature in the dark. To detect total a-syn, the anti-a-syn antibody syn211 (Abeam, UK) may be used.

[0257] For the detection of oligomeric and fibrillar a-syn the anti-aggregation a-syn antibody, clone 5G4 (Sigma-Aldrich, USA) can be used. For the detection of a-syn phosphorylated at serine 129, the recombinant anti-a-syn (phospho S129) antibody EP1536Y (Abeam, UK) can be used. After a final three washes in PBS for 5 minutes each, the cells can be imaged in PBS using an IN-Cell Analyzer 6500HS system and 40x magnification (Cytiva, USA).

[0258] In order to monitor the synucleinopathy in the subject, samples from the subject are obtained at different times. The subject may undergo an actual treatment with the peptides, peptide variants, multimers or the pharmaceutical composition of the present invention. Quantification of a-syn aggregates at different time points provides an indication whether the treatment with the peptides, peptide variants, multimers or the pharmaceutical composition of the present invention leads to a reduction in the presence or amount of a-syn aggregates in the samples from the patient over the course of time. Likewise, an increase in the presence of a-syn aggregates over time indicates a progression of the disease.

[0259] Medical uses and methods of treatment

[0260] In a further aspect of the present invention, the peptide, peptide variant or multimer as defined herein or the pharmaceutical composition of the invention is used in detoxifying and / ordisaggregating a-synuclein oligomers and / or a-synuclein aggregates in a subject, preferably a mammalian subject, more preferably a human subject.

[0261] The present invention also relates to a peptide, peptide variant or multimer as described above for use in the treatment of synucleinopathies. The present invention relates to a peptide, peptide variant or multimer as described above, in particular for use in the treatment of Parkinson's disease (PD), Lewy body dementia (DLB) and multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) and a disease exhibiting a a-synuclein copathology such as Alzheimer's disease (AD), which are diseases that are associated with the misfolding and aggregation of the protein a-synuclein in certain cells.

[0262] In particular, the present invention relates to a peptide, peptide variant or multimer or a pharmaceutical composition as described above for use in the treatment of Parkinson's disease (PD).

[0263] As used herein, a "disease" or “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. In the present invention, the ongoing accumulation of a-synuclein aggregates leads to a continuous progression and aggravation of the a-synucleinopathy. The inventive peptides are capable of stopping the progression of the a-synucleinopathy and are capable of reverting the symptoms of the a-synucleinopathy.

[0264] A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. A prophylactic treatment is performed to prevent the occurrence of a disease, such as a synucleinopathy.

[0265] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

[0266] "Treating," as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject, or reducing the severity with which symptoms are experienced by a patient or subject.

[0267] A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0268] The invention relates to a method of treating a synucleinopathy in a subject in need thereof, the method comprising administering to the subject an effective amount of a peptide, peptide variantor multimer of the present invention, ora pharmaceutical composition of the present invention as defined herein to the subject. Synucleinopathies (also called a-Synucleinopathies) are neurodegenerative diseases characterized by the abnormal accumulation of aggregates of alpha-synuclein protein in e.g. neurons, nerve fibres or glial cells. There are three main types of synucleinopathy: Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Other rare disorders, such as various neuroaxonal dystrophies (pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD)), also have a-synuclein pathologies. Additionally, autopsy studies have shown that around 6% of sporadic Alzheimer's Disease exhibit a-synuclein positive Lewy pathology, and are sub-classed as Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB).

[0269] Parkinson's disease (PD), or simply Parkinson's, is a neurodegenerative disease primarily of the central nervous system, affecting both motor and non-motor systems. Symptoms typically develop gradually, with non-motor issues becoming more prevalent as the disease progresses. Common motor symptoms include tremors, bradykinesia (slowness of movement), rigidity, and balance difficulties, collectively termed parkinsonism. In later stages, Parkinson's disease dementia, falls, and neuropsychiatric problems such as sleep abnormalities, psychosis, mood swings, or behavioral changes arise.

[0270] Dementia with Lewy bodies (DLB) is a type of dementia characterized by changes in sleep, behavior, cognition, movement, and regulation of automatic bodily functions. The disease worsens over time and is usually diagnosed when cognitive impairment interferes with normal daily functioning. Together with Parkinson's disease dementia, DLB is one of the two Lewy body dementias. REM sleep behavior disorder (RBD) — in which people lose the muscle paralysis (atonia) that normally occurs during REM sleep and act out their dreams — is a core feature of DLB. RBD may appear years or decades before other symptoms. Other core features are visual hallucinations, marked fluctuations in attention or alertness, and parkinsonism (slowness of movement, trouble walking, or rigidity).

[0271] Multiple system atrophy (MSA) is a rare neurodegenerative disorder characterized by tremors, slow movement, muscle rigidity, postural instability (collectively known as parkinsonism), autonomic dysfunction and ataxia. This is caused by progressive degeneration of neurons in several parts of the brain including the basal ganglia, inferior olivary nucleus, and cerebellum. MSA was first described in 1960 by Milton Shy and Glen Drager and was then known as Shy-Drager syndrome. Subjects affected by MSA experience dysfunction of the autonomic nervous system, which commonly manifests as orthostatic hypotension, impotence, loss of sweating, dry mouth and urinary retention and incontinence. Palsy of the vocal cords is an important and sometimes initial clinical manifestation of the disorder.Pure autonomic failure (PAF) is an uncommon, sporadic neurodegenerative condition marked by a steadily declining autonomic regulation. Patients usually present with orthostatic hypotension or syncope in midlife or later. In addition, genitourinary, thermoregulatory, and bowel dysfunction can be signs of autonomic failure. Pure autonomic failure originates from peripheral autonomic nervous system lesions. The majority of symptoms that patients with PAF exhibit are associated with neurogenic orthostatic hypotension, or orthostatic hypotension brought on by severe sympathetic failure.

[0272] Idiopathic rapid eye movement (REM) sleep behavior disorder (IRBD) manifests as unpleasant dreams and vigorous behaviors during REM sleep that can result in injuries. Patients with IRBD have no known neurological diseases or motor or cognitive complaints; however, this sleep disorder is not harmless. Idiopathic rapid eye movement (REM) sleep behavioural disorder (RBD) is now recognized as an early marker of a-synucleinopathies. IRBD is the prelude of the synucleinopathies Parkinson's disease, dementia with Lewy bodies or multiple system atrophy.

[0273] Preferred synucleinopathies that are subject to treatment with the inventive peptide, peptide variant or multimer or pharmaceutical compositions comprising these are Parkinson's disease (PD), Lewy body dementia (LDB) or dementia with Lewy bodies (DLB) and multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) and a disease exhibiting a a-synuclein co-pathology such as Alzheimer's disease (AD) such as Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB). A particularly preferred synucleinopathy is PD.

[0274] The therapeutic potential of the inventive peptides, peptide variants or multimers was determined by way of cell-viability assays described below, and by way of established mouse models.

[0275] Cell-viability assay: The potential rescue of PC12 cells (Leibniz Institute DSMZ) from of a-syn aggregate induced toxicity by the inventive peptides can be measured in an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay.

[0276] PC12 cells can be cultivated in collagen A-coated (Biochrom GmbH) tissue culture flasks in RPMI 1640 medium supplemented with 5 % fetal calf serum and 10 % horse serum in a 95 % humidified atmosphere with 5 % CO2 at 37 °C. Ten thousand cells per well in a volume of 100 pL may be seeded on collagen A-coated 96-well plates (Thermo Fisher Scientific) and can be incubated for 20 h at 37 °C and 300 rpm in a thermocycler.

[0277] Then, final concentrations of 30 nM (monomer equivalent) small a-syn fibrils either in the absence or after pre-incubation with increasing concentrations of the peptides of the invention can be added to the cells. Cell medium and 1 % Triton X-100 may serve as controls. After furtherincubation in a 95 % humidified atmosphere with 5 % CO2 at 37 °C for 24 h, cell viability can be measured using the Cell Proliferation Kit I (MTT) (Roche Applied Science) according to manufacturer's protocol.

[0278] The MTT formazan product can be quantified by measuring the absorbance at 570 nm corrected by subtraction of the absorbance at 660 nm in a FLUOstar Omega plate reader (BMG Labtech). Results can be normalized to untreated cells grown in medium only. Significance can be tested by one-way ANOVA with Bonferroni post-hoc analysis using OriginPro 2020 (OriginLab). EC50 values can be determined using the Absolute IC50 function in GraphPad Prism 10 (GraphPad Software Inc.).

[0279] Cell assay for g-synuclein aggregation: A construct encoding full-length A53T-mutated human a-syn fused to YFP at the C-terminus was synthesized and introduced into the pMK-RQ expression vector (GeneArt; Thermo Fisher Scientific, USA). The a-synA53T-YFP construct can be subcloned into the plRESpuro3 vector (Clontech; Takara Bio, JPN) using the Nhel (5') and Notl (3') restriction sites. HEK293T cells (American Type Culture Collection) can be cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM; Sig ma -Aid rich, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, USA) and 50 units / ml penicillin and 50 pg / ml streptomycin (Sigma-Aldrich, USA). The cells can be cultivated in a humidified atmosphere with 5 % CO2 at 37 °C.

[0280] Cells plated in DMEM can be transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, USA). Stable cells can be selected in DMEM with 1 pg / ml puromycin (EMD Millipore, USA). Monoclonal lines can be generated by fluorescence-activated cell sorting of a polyclonal cell population in 96-well plates using a M0FI0 XDP cell sorter (Beckman Coulter, USA). The clonal cell line B5 can finally be selected from 24 clonal cell lines and is referred to as a-synA53T-YFP cells.

[0281] The peptides were incubated with 1.5 % Lipofectamine 2000 in OptiMEM for 2 hours at room temperature. The a-synA53T-YFP cells can be plated in a 384-well plate with poly-D-lysine coating (Greiner, AT) at a density of 1,000 cells per well with 0.1 pg / ml Hoechst 33342 (Thermo Fisher Scientific, USA), and the previously prepared transfection mixture can be added directly to the cells in the wells.

[0282] To seed cellular aggregation of a-syn in a-synA53T YFP cells, 30 nM soluble a-syn PFF oligomers can be incubated with 1.5% Lipofectamine in OptiMEM for 2 hours at room temperature and added to each well 3 hours after the first transfection.The plate can then be incubated in a humidified atmosphere with 5 % CO2 at 37 °C. On the third day, cells can be imaged with an IN Cell Analyzer 6500HS system (Cytiva, USA) using the blue and green fluorescence channels and analyzed with IN Carta Image Analysis Software (Cytiva, USA) using an algorithm to identify intracellular aggregates in living cells. Four wells may be used for each condition and 16 images per well may be acquired and analyzed using a fully automated algorithm to avoid bias. Statistical analysis can be performed using one-way ANOVA followed by Dunnett's test for multiple comparisons (GraphPad Prism 9, GraphPad Software, USA).

[0283] Cell viability assay (CellGlo test): The CellTiter-Glo Luminescent Cell Viability Assay (Promega GmbH, GE) can be used to determine the number of viable cells in the culture based on the quantification of ATP present, an indicator of metabolically active cells.

[0284] After the cells are cultured in 384-well plates for three days, 35 pl of the medium is removed from the wells and 40 pl of CellTiter-Glo reagent is added directly to each well. After mixing, the luminescence can be measured 10 minutes later using a Fluostar (BMG labtech, GE). A higher level of luminescence indicates a higher number of viable cells.

[0285] Microscale thermophoresis (microscale thermophoresis)

[0286] Microscale thermophoresis (MST) for determining the affinity of two interactants, whereby interactants remain in solution can performed using a Monolith NT.115 device (NanoTemper Technologies GmbH, GE). Fluorescently labeled peptides are diluted to a final concentration of 100 nM in PBS pH 7.4. Recombinant a-syn is diluted in a serial dilution series from 50 pM to 3 pM final concentration in PBS pH 7.4 and mixed uniformly with the peptide solutions. Samples are loaded into standard surface glass capillaries or premium surface capillaries (NanoTemper Technologies GmbH, GE) and measurements are performed at 20 % LED power and 60 % MST power at 25 °C. The standard parameters recommended by the manufacturer are used (heating period delay time of 30 s and re-equilibration period of 5 s). The data can be analyzed with the thermophoresis effect using the NT analysis software (version 1.5.41) supplied by the manufacturer.

[0287] Circular dichroism (CD) spectroscopy

[0288] Circular dichroism (CD) spectroscopy can be used to determine the secondary structure of an analyte. For the present invention it can be used to investigate the status of the alpha synuclein monomer in the presence of drug (SVD peptide). Far-UV circular dichroism (CD) data can determined using a Jasco J-1100 spectropolarimeter (Jasco, GE). 350 pl of sample replicates from the inoculated assays are pooled and loaded into a high-precision quartz cuvette with a path length of 1 mm (Hellma Group, GE). A scan speed of 20 nm / min is performed with fiveaccumulations per sample at far UV wavelengths from 260 to 190 nm. The baseline can be corrected by subtracting only the measurements of the buffer.

[0289] General terms

[0290] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, typical methods and materials are described.

[0291] Following convention, as used herein “a” and “an” mean “one or more” unless specifically indicated otherwise.

[0292] The use of the term “comprising” as well as other grammatical forms such as “comprises” and “comprised” is not limiting. The terms “comprising”, “comprises” and “comprised” should be understood as referring to an open-ended description of an embodiment of the present invention that may, but does not have to, include additional technical features in addition to the explicitly stated technical features. The same applies for the term “including” and other grammatical forms such as “includes” and “included”. Further, the terms “comprising”, “involving” and “including”, and any grammatical forms thereof, are not to be interpreted as exclusively referring to embodiments that include features additional to those explicitly recited. These terms equally refer to embodiments that consist of only those features that are explicitly mentioned.

[0293] In contrast the term “consisting” or “consists” indicates that the embodiment does not include additional features to those that are explicitly mentioned. The term “consisting essentially” or “consists essentially” indicates that the embodiment does not include features that change the overall nature of the embodiment, but may contain features that do not alter the overall nature of the embodiment; e.g. a pharmaceutical composition consisting essentially of a peptide of the present invention and a pharmaceutically acceptable excipient, may additionally contain binders or flavorings, but does not contain e.g. a second active ingredient.

[0294] Section headings throughout the description are for organizational purposes only. In particular, they are not intended as limiting for various embodiments described therein, and it is to be understood that embodiments (and features therein) described under one subheading may be freely combined with embodiments (and features therein) described under another subheading.

[0295] As used herein, when a single recitation of a modifier precedes the first element in a list of elements, unless otherwise stated that modifier is to be read as applying to each respective subsequent element in the list. Merely as an example, “at least 65%, 70%, 75% or 80%” should be understood as “at least 65%, at least 70%, at least 75% or at least 80%”.As used herein, the term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, in addition to that measurable value itself, variations of +20% or +10%, in some instances +9%, +8%, +7%, +6%, +5%, +4%, +3%, +2% or +1%, and in some instances +0.9%, +0.8%, +0.7%, +0.6%, +0.5%, +0.4%, +0.3%, +0.2% or +0.1% from the specified value. It is to be understood that the term “about”, in reference to a particular value, includes that exact particular value itself, irrespective of any explicit mention that that exact particular value is included. Thus, the absence of an explicit indication that the term “about” includes the particular exact recited value is not to be understood that the particular recited value is excluded from the range of variations created by the term “about”; even in the absence of an explicit indication that the term “about” includes the particular exact recited value, that exact particular value is still included in said range of variations created by the term “about”. The skilled person will recognize when deviations around a stated value may be either integral or fractional (as for example for a temperature or a pressure), and when such deviations may only be integral (as for example for discrete moieties within a larger molecule).

[0296] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range including the endpoints of that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range or the context in which this range is disclosed.

[0297] All publications, including but not limited to patents, patent applications and scientific publications, cited in this description are herein incorporated by reference for all purposes as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0298] The following examples demonstrate the efficacy of the peptides of the present invention in the prevention of a-syn aggregation, and the disaggregation of a-syn aggregates and thus the treatment and prevention of various synucleinopathies.

[0299] The compositions used in the examples are exemplary compositions and shall not be construed to limit the present invention.Examples

[0300] Various different SVD-14 peptides were used in various in vitro experiments. The previously developed peptide SVD-1a (RLPTHETYWQEHIWHARRRRR-NH2; SEQ ID NO:70) was used as reference (see Table 3 below). All-D peptides were synthesized using D amino acids with amidated C-terminus and a purity >95 % as verified by high-performance liquid chromatography.

[0301] Example 1

[0302] SVD-14 peptides bind to a-syn monomers with high affinity and inhibit de novo aggregation

[0303] SVD-14 was designed to bind monomeric a-syn and stabilize the intrinsically unfolded structure and dissolve pre-existing aggregates. Since a high affinity for the intrinsically disordered protein structure of the a-syn monomer is important for this mode of action, a surface plasmon resonance (SPR) experiment was performed. Biotinylated a-synuclein monomer was immobilized on a streptavidin-coated sensor chip. The peptides were then injected onto the sensor surface at different concentrations in a multicyclic experiment and the affinity (KD) and kinetic rates (ka and kd) were determined via global fitting with a 1:1 kinetic binding model.

[0304] SVD-14 peptide concentrations of 625 nM, 1250 nM, 2500 nM, 5000 nM and 10000nM (SVD-14j, Fig. 1A), 625 nM, 1250 nM, 2500 nM and 5000 nM (SVD-14u, SVD-14af and SVD-14ag; Figs.

[0305] 1B, 2A and 2B, respectively), or 19.53 nM, 39.06 nM, 78.13 nM and 156.25 nM (SVD-14ah, Fig.

[0306] 2C) with 300 s association and 1200 s dissociation. The interactions were fitted using a 1 :1 kinetic fitting model.

[0307] In this way, high affinity binding of SVD-14 to the a-syn monomer was demonstrated with a KD, kaand, kd values as follows:

[0308] Peptide KD ka kd

[0309] SVD-14j 188 nM 1.83E+3 [1 / Ms] 3.44E-4[1 / s] SVD-14U 17 nM 2.48E+3 [1 / Ms] 4.32E-5[1 / s] SVD-14af 63.7 nM 5.9E+3 [1 / Ms] 3.8E-4 [1 / s] SVD-14ag 64.6 nM 3.8E+3 [1 / Ms] 2.4E-4 [1 / s] SVD-14ah 1.67 nM 1.0E+5 [1 / Ms] 1.7E-4 [1 / s]

[0310]

[0311] Table 2: KD, ka and kd of different SVD-14 peptidesThe peptides SVD-14jb and SVD-14ua are N-terminal truncations of the sequences SVD-14j and SVD-14u, respectively. Since similar binding behaviors were identified for these peptides, they are not listed separately in Table 2.

[0312] Dissociation constants in nM could be determined for all investigated peptides. The example shows that all investigated SVD-14 peptides exhibit a significant binding affinity towards monomeric a-syn.

[0313] Next, the influence of SVD-14 peptides on the a-syn aggregation cascade was investigated. The initial process in the aggregation cascade is primary nucleation, which precedes the exponential proliferation of fibril particles through secondary nucleation, fibril elongation, and fragmentation. Since all these steps utilize monomeric a-syn, a compound that stabilizes the a-syn monomer in its native conformation should delay, if not completely inhibit, the whole aggregation process.

[0314] To monitor spontaneous a-syn aggregation, ThT fluorescence was continuously measured for 6 days using 30 pM a-syn monomer alone or in the presence of 30 pM SVD-14 peptide. The aggregation processes for SVD-14j, SVD-14jb, SVD-14u, SVD-14ua, SVD-14af, SVD-14ah and SVD-14ag are shown in Figures 3 and 4 (Fig. 3A: SVD-14j and SVD-14jb, Fig. 3B: SVD-14u and SVD-14ua, Fig. 4A: SVD-14af, Fig. 4B: SVD-14ah and Fig. 4C: SVD-14ag). Mean data shown in Figures 3 and 4 (n = 5).

[0315] All SVD-14 peptides inhibited aggregation in a dose dependent manner (dose escalation data not shown). Spontaneous aggregation of a-syn in the ThT-positive de novo aggregation assay was significantly inhibited by all investigated SVD-14 peptides. A complete inhibition was shown for equimolar concentrations of SVD-14af, SVD-14ah and SVD-14ag.

[0316] Thus, the correlation between monomer stabilization and aggregate formation inhibition was demonstrated for various different SVD-14 peptides. Several SVD-14 peptides were able to completely inhibit the aggregation of a-syn at micromolar concentrations.

[0317] Example 2.

[0318] SVD-14 peptides reduce seeded aggregation of a-syn fibrils

[0319] The SVD-14 peptides are capable of inhibiting the seeded aggregation (starting from already aggregated particles). The seeded aggregation is also a proof of the aggregation of a-synuclein. The inhibition of seeded aggregation was investigated using the ThT assay described hereinabove. In contrast to the de novo aggregation shown above, however, the aggregation does not start with a-synuclein monomer but with preformed fibrillar particles (a-synuclein PFF).50 nM monomer equivalents of the a-synuclein PFF were pre-incubated with different concentrations of the SVD-14 peptides (SVD-14j, SVD-14u, SVD-14af, SVD-14ag, SVD-14ah, SVD-14ua and SVD-14jb) overnight, to allow the peptides to eliminate the seeding ability of the PFF. Subsequently, 20 pM a-synuclein monomer was added to the sample to induce ThT-positive aggregation. For peptides that are able to reduce the seeding capacity of the PFF, a reduced ThT signal is achieved at the incubation endpoint (6 d). The relative end signal at the incubation time endpoint is shown in Figure 5 with the addition of various peptide concentrations. All SVD-14 peptides completely eliminated the seeding capacity of PFF at a peptide concentration of 1.25 pM.

[0320] The efficacy of the SVD-14 peptides to prevent a-synuclein seeded aggregation at a peptide concentration of only 1.25 pM was surprising and demonstrates the potential of the peptides to prevent a-synuclein aggregation and to stabilize monomeric a-synuclein.

[0321] Example 3.

[0322] SVD-14 peptides reduce the seeding capacity of small a-syn fibrils in vitro with EC50 in the nM range

[0323] Aggregation of a-syn was measured by ThT fluorescence. The monomer equivalent of 50 nM a-syn PFF was pre-incubated for 20 hours at 37 °C under quiescent conditions without or with increasing compound concentrations (SVD-14af and SVD-14j). Seeding was then initiated by adding 20 pM a-syn monomer. The area under the curve (AUC) was determined for each concentration after the initiation of seeding and plotted against the concentration of the compound, while the untreated control was set to 100 %. On this basis, a fitting was performed to determine the EC50 values.

[0324] The kinetics of the aggregation reactions showed that increasing SVD-14 peptide concentrations reduced the seeding activity of small a-syn fibrils in a concentration-dependent manner. By plotting the resulting area under the curve (AUC) values against the respective SVD-14 peptide concentration, an effective concentration (EC50) of 264 nM for SVD-14af and of 300 nM for SVD-14] that rendered 50 % of small a-syn fibrils seeding incompetent within a 20 h incubation period was determined (see Figure 8).

[0325] EC50 values in the nanomolar range indicate a surprisingly superior activity of the SVD-14 peptides in the elimination of a-syn seeding capacity and also the disaggregation potential of the peptides. SVD-14 peptides showed higher efficacy in reduction of seeding activity than the previously described peptides SVD-1 and SVD-1a which reached EC50 values in the single digit pmolar range.Example 4.

[0326] SVD-14 peptides reduce intracellular aggregation of a-syn fibrils

[0327] In this example, the ability of the SVD-14 peptides to inhibit intracellular seeded aggregation was investigated. For this purpose, HEK293T cells were generated that stably express a fusion protein from the a-synuclein mutant A53T and the fluorescent protein YFP (yellow fluorescent protein). When a-synuclein PFF is introduced into the cells via transfection with lipofectamine, intracellular aggregation of the a-synuclein fusion protein is induced. As a result of the aggregation, the a-synuclein-YFP fusion protein is accumulated locally. If the YFP fluorescence signal within a cell exceeds a certain limit, this cell was rated as aggregate-positive. Four wells were imaged for each condition by taking 16 images per well with an IN Cell Analyzer 6500HS System (Cytiva) using the blue and green fluorescence channels. Image analysis was performed using IN Carta image analysis software (Cytiva) after establishing an automated algorithm to identify intracellular aggregates in live cells. Statistical analysis and nonlinear fitting to obtain an EC50 value were done with GraphPad Prism 10. 25 pM of each SVD-14 peptide was added to the sample 2 h after PFF transfection.

[0328] For all investigated SVD-14 peptides, a reduction in aggregate-positive cells was observed compared to the positive control as shown in Figure 6. The most significant reduction was observed for the peptides SVD-14ua, SVD-14ah and SVD-14ag.

[0329] The results in Figure 6 show that all SVD-14 peptides are able to enter the cells in a functional form and significantly reduce intracellular a-syn aggregation.

[0330] Example 5.

[0331] SVD-14 peptides reduce cell toxicity of small preformed a-syn fibrils

[0332] This example shows the effect of different SVD-14 peptides on the reduction of the cell toxicity of pre-aggregated a-synuclein particles.

[0333] For this purpose, 50 nM monomer equivalent of a-synuclein PFF was pre-incubated with or without the addition of different peptide concentrations for 20 h at 37 °C and then added to PC12 cells. SVD-14 peptides SVD-14j, SVD-14jb, SVD-14u, SVD-14ua, SVD-14af and SVD-14ah were investigated. The cell viability was then checked using an MTT assay as described hereinabove. A dose-response relationship of small a-syn fibril toxicity and compound concentration has been found for the SVD 14 peptides.If the PFF are incubated with the cells without adding the peptides, the cell viability is reduced. While small a-syn fibrils (30 nM monomer equivalent) reduced the cell viability of PC12 cells by approximately 50 %, pre-incubation of small a-syn fibrils with SVD-14 resulted in a significant concentration-dependent increase in cell viability. For all SVD-14 peptides, a concentrationdependent increase in cell viability was observed as shown in Figure 7 (Fig. 7A: SVD-14j and SVD-14u, Fig. 7B: SVD-14ua, Fig. 7C: SVD-14af, SVD-14ah and SVD-14jb). Mean data shown with + SD (n S 4). The EC50 of the peptides was determined by fitting with a Boltzmann fit.

[0334] The EC50 for the rescue of the cells was 53.64 nM for SVD-14j, 305.73 nM for SVD-14jb, 14.33 nM for SVD-14u, 21.62 nM for SVD-14ua, 8.8 nM for SVD-14af and 12.43 nM SVD-14ah. For comparison, the previously developed peptide SVD-1a exhibited an EC50 of 1880 nM, i.e. the SVD-14 peptides were about 6-fold to about 210-fold more effective in the cell viability assay. Thus, the new SVD-14 peptides provide for vastly superior cell rescue properties as previous peptide compounds.

[0335] Example 6

[0336] SVD-14 peptides disassemble small a-syn fibrils into monomers

[0337] The elimination of small a-syn fibrils in the presence of SVD-14j and SVD-14u was monitored over time by dynamic light scattering (DLS), with a particular focus on changes in particle size distribution (Fig. 9).

[0338] Time-dependent dynamic light scattering (DLS) measurements with 100 nM small a-syn fibrils in the absence (Fig. 9a) or presence (Fig. 9b / c) of 5 pM SVD-14j or 400 nM SVD-14u show that incubation with compound eliminates a-syn fibrils over time. One milliliter samples were measured every 60 s for 72 h under quiescent condition in a sealed quartz cuvette at 37 °C. Data is shown as a radius plot where the signal amplitude of each particle size is represented by the data point diameter.

[0339] In the absence of SVD-14j and SVD-14u, the small a-syn fibrils exhibited a stable particle size distribution within the range of approximately 25 nm over a 3-d period (Fig. 9a). In contrast, the presence of SVD-14j and SVD-14u resulted in a gradual reduction in particle size (Fig. 9b / c).

[0340] Example 7

[0341] Additional variants of SVD-14 peptides reduce the seeding capacity of small preformed a-syn fibrilsA series of further SVD-14 peptide variants (SEQ ID NOs: 27-50) was produced based on an alanine scan of peptide SVD-14j (peptides SVD-14j_a1 to ~a17) and by a rational design approach (peptides SVD-14 au to ~az and SVD-17af_a). These peptides and the peptides SVD-14 af, SVD-14al, SVD-14am, SVD-14an, SVD-14ao, SVD-14ap, SVD-14aq, SVD-14ar, SVD-14as, SVD-14at were subjected to further series of disaggregation assays as follows.

[0342] The reduction in seeding capacity of small a-syn fibrils in vitro of these peptides as well as peptides was determined by seeded ThT assays as essentially described in Example 3. Deviating from this setup, SVD-14j was used as the control sequence, and SVD-14 variants were incubated either at a fixed concentration or as a concentration series to determine the EC50 value. In both cases, the fold change was calculated by dividing the value obtained for the SVD-14 variant by that of the control (SVD-14j). Fold-change values <1 therefore indicate that the variant inhibits aggregation more strongly than SVD-14j and thus exhibits improved activity. Conversely, foldchange values >1 indicate slightly weaker inhibition. However, values lower than 17 are considered to exhibit superior activity compared to the previously described peptide SVD-1a.

[0343] The results of these measurements are given in Table 3.

[0344] SEQ ID NO: Designation average fold-change

[0345] 70 SVD-1a 17.00

[0346] 14 SVD-14j 1.00

[0347] 28 SVD-14j_a2 0.17

[0348] 29 SVD-14j_a3 1.27

[0349] 30 SVD-14j_a4 1.47

[0350] 31 SVD-14j_a5 1.26

[0351] 32 SVD-14j_a6 1.31

[0352] 33 SVD-14j_a7 1.69

[0353] 34 SVD-14j_a8 1.62

[0354] 36 SVD-14j_a10 1.56

[0355] 37 SVD-14j_a11 1.28

[0356] 39 SVD-14j_a13 1.58

[0357] 41 SVD-14j_a15 1.12

[0358] 42 SVD-14j_a16 1.20

[0359] 43 SVD-14j_a17 1.60

[0360] 18 SVD-14al 0.89

[0361] 19 SVD-14am 0.51

[0362] 20 SVD-14an 0.29

[0363] 21 SVD-14ao 0.47

[0364] 22 SVD-14ap 0.31

[0365]

[0366] 23 SVD-14aq 0.4324 SVD-14ar 0.52

[0367] 25 SVD-14as 0.89

[0368] 26 SVD-14at 0.46

[0369] 44 SVD-14au 3.52

[0370] 45 SVD-14av 2.96

[0371] 46 SVD-14aw 3.73

[0372]

[0373] 13 SVD-14af 0.17

[0374] Table 3: Seeded aggregation assay (ThT) comparison of SVD-14 variant peptides with SVD-14j. Fold change was calculated by dividing the aggregate reduction value of the variant (either reduction at a fixed compound concentration or EC50 values) by the value of the reference substance (SVD-14j). A fold change <1 means that the ability to eliminate seeding capacity of PFF is better than that of SVD-14j. However, peptides with a fold change <17 relative to SVD-14] are still at least as effective as SVD-1a.The results show that the efficacy of the peptides was preserved over a large panel of mutational sites covering the entire sequence length of the reference peptide SVD-14j, indicating that the peptide sequence of SVD-14j provides a robust signature sequence. Moreover, the Example shows that it is possible to generate further effective peptides based on the consensus sequences described hereinabove.

[0375] Therefore, it has been shown that SVD-14 peptides of the present invention cover a considerable area of efficacy that is valuable for dealing with additional properties that are of importance for further therapeutic development towards clinical proof -of-concept. The panel of peptides covers a wide therapeutic window for treating synucleinopathies in divergent patient populations.

[0376] Summary of results

[0377] In summary, the results show that SVD-14 peptides not only bind a-syn monomers with considerably high affinity (Figures 1 and 2) but also disassemble small a-syn fibrils into monomers, which reduces their seeding capacity (Figures 3 and 4 and Figure 5) intracellular aggregation (Figure 6) and toxicity (Figure 7) in a concentration-dependent manner. The results obtained for different SVD-14 peptides are summarized in Table 3.

[0378] Parameter Description (unit) Assay Remark SVD-14-peptide SVD-1a SVD-14j: 24

[0379] Delay of 11 / 2 with SVD-14af: >168*

[0380] de novo equimolar incubation ThT higher is

[0381] SVD-14ag: >168* >168 * aggregation of monomer and assay better

[0382] SVD-14ah: >168*

[0383] SVD peptide (h)

[0384]

[0385] SVD-14u: 24SVD-14ua: 57.6

[0386] SVD-14jb: 62.4

[0387] SVD-14j: 300

[0388] SVD-14af: 264

[0389] SVD-14ag: n.d.

[0390] SVD-14ah: n.d.

[0391] EC50 of reduction of

[0392] Seeded ThT lower is SVD-14u: n.d.

[0393] seeding capacity of 5100 aggregation assay better SVD-14ua: n.d.

[0394] 50 nM PFF (nM)

[0395] SVD-14jb: n.d.

[0396] + fold-change of

[0397] further variants (see

[0398] Table 3)

[0399] SVD-14j: 54

[0400] SVD-14af: 9

[0401] EC50 of reduction of SVD-14ag: n.d.

[0402] Reduction in MTT lower is

[0403] cytotoxicity of 30 nM SVD-14ah: 12 1880 cytotoxicity assay better

[0404] PFF (nM) SVD-14u: 14

[0405] SVD-14ua: n.d.

[0406] SVD-14jb: n.d.

[0407] SVD-14j: 188

[0408] SVD-14af: 64

[0409] SVD-14ag: 65

[0410] lower is

[0411] Binding KD with a-syn as SVD-14ah: 2

[0412] SPR higher n.d. affinity ligand (nM) SVD-14u: 17

[0413] affinity

[0414] SVD-14ua: n.d.

[0415] SVD-14jb: n.d.

[0416] SVD-14j: 1.23

[0417] SVD-14af: 1.06

[0418] lower is SVD-14ag: n.d.

[0419] Binding KD with peptide as

[0420] SPR higher SVD-14ah: n.d. 0.1 affinity ligand (nM)

[0421] affinity SVD-14u: n.d.

[0422] SVD-14ua: n.d.

[0423] SVD-14jb: n.d.

[0424] SVD-14j: 40 Reduction of SVD-14af: 40 intracellular SVD-14ag: 48 Intracellular aggregation with 25 Cell higher is SVD-14ah: 49 n.d. aggregation pM peptide (%); assay better

[0425] compounds added SVD-14u: 40 to medium SVD-14ua: 67

[0426]

[0427] SVD-14jb: 21

[0428] Table 4: Summary of results (*: >168 = complete inhibition up to 168 h)

[0429] SVD-14 peptides exhibited a binding affinity to a-syn monomers with a KD between 2 to 188 nM, which is higher than SVD-1a's KD of 100 pM. However, the SVD-14 peptides SVD-14j and SVD-14af inhibited seeded a-syn aggregation in vitro more potently with an EC50 of 0.300 pM and0.264 pM, respectively, as compared to SVD-1a's EC50 of 5.1 pM, despite of the lower binding affinity for a-syn monomers.

[0430] In a cell viability assay, the SVD-14 peptides conferred dose-dependent protection against a-syn fibril induced toxicity. In this assay, the SVD-14 peptides were approximately 35-fold to about 200-fold more potent than SVD-1a with an EC50 of 1880 nM.

[0431] Both the seeded aggregation assay and the cell viability assay examine the inactivation of seed functionality with respect to seeding activity and toxicity in the absence and presence of the peptides of the present invention. Increased efficacy in the reduction of intracellular aggregation by SVD-14 peptides may be because these peptides are taken up into cells more rapidly than SVD-1a, which was not efficient when only added to the medium, or because they act more efficiently in the disassembly of a-syn oligomers and fibrils.

[0432] The property to passively enter cells could also enable SVD-14 to cross the blood-brain barrier more efficiently and to enter diseased neurons in the CNS, where a-syn is aggregating.

[0433] Proof that the a-syn seeds are indeed inactivated by sequential disassembly / disaggregation is provided by monitoring the particle size in presence of the compound using DLS (Figure 9).

[0434] Overall, the results show that SVD-14 peptides are more efficient in disaggregating alpha-synuclein aggregates than the previously developed SVD-1 peptides. This is best recognized by the significantly lower EC50 for inactivating the seeding capacity. This may partially be due to their lower measured affinity to alpha-synuclein aggregates. Said lower KD indicates a higher koff rate of the SVD-14 peptides, which in the present case results in a higher activity of releasing native monomers from alpha-synuclein aggregates. Thus, the novel SVD-14 peptides represent improved compounds for the disaggregation of alpha-synuclein in the treatment of synucleinopathies, such as PD, DLB and MSA.

Claims

PCT Application No.: To be assignedApplicant: Priavoid GmbHOur Ref: PCT153775-SZ163Date: March 16, 2026Claims1. A peptide of no more than 42 amino acids, comprising the amino acid sequence X1X2X3X4X5X6X7X8X9VRFLVHRR (SEQ ID NO:1), with no more than one amino acid substitution in the motif VRFLVHRRwherein each of Xi , X2 , X3 , X4 , X5 , Xe , X7 , Xs and X9 is independently from each other absent or is any amino acid, and wherein SEQ ID NO:1 comprises at least one D-amino acid residue.

2. The peptide according to claim 1 , wherein the peptide consists of more than 50% of D-amino acids, preferably consists of more than 90% of D-amino acids, more preferably consists entirely of D-amino acids.

3. The peptide according to claim 1 or 2, whereinXi is independently chosen from the group consisting ofabsent, andhydrophobic amino acids, preferably Y and V;X2is independently chosen from the group consisting ofabsent,small amino acids, preferably A or G,polar amino acids, preferably N, andcharged amino acids, preferably D and R;X3is independently chosen from the group consisting ofsmall amino acids, preferably A, andnon-polar amino acids or hydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably W and F;X4is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, more preferably R and K, andhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably L;X5is independently chosen from the group consisting ofhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably V, andpolar amino acids, preferably Q;X6is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, more preferably H, K and R;X7is independently chosen from the group consisting ofhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, andcharged amino acids, preferably positively charged amino acids, more preferably R;X8is independently chosen from the group consisting ofpolar amino acids, preferably T and S, andcharged amino acids, preferably positively charged amino acids, more preferably R; andX9is independently chosen from the group consisting ofabsent, andhydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y.

4. The peptide according to any one of claims 1 -3, whereina) said peptide comprises the sequenceXiX2[X3 / W]X4QX6X7X8YVRFLV[H / Xi5]RR (SEQ ID NO:2), wherein Xi is independently chosen from the group consisting ofabsent, andhydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y,with Y being preferred;X2is independently chosen from the group consisting ofabsent,small amino acids, preferably A or G,polar amino acids, preferably N, andcharged amino acids, preferably negatively charged amino acids, more preferably D,with A, N or D being most preferred;X3is absent or is a non-polar amino acid, preferably A;X4is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, more preferably R or K,with R being preferred;X6is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, more preferably R or K,with R being preferred;X7is independently chosen from the group consisting ofhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, andcharged amino acids, preferably positively charged amino acids, more preferably R,with R being preferred;X8is independently chosen from the group consisting ofpolar amino acids, preferably T or S,with T being preferred; andX15 is a non-polar amino acid, preferably A,wherein when position 15 of SEQ ID NO:2 is H, R at position 11 or R at position 16 may be A; orb) said peptide comprises the sequenceXiRFLVHRRVRFLVHRR (SEQ ID NOS), wherein Xi is independently chosen from the group consisting ofabsent, andhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably V; orc) said peptide comprises the sequenceYDWX4QX6X7X8YVRFLVHRR(SEQ ID NO: 4), wherein X4is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, more preferably R and K,with R being preferred;X6is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, more preferably R and K,with R being preferred;X7is independently chosen from the group consisting ofhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, andcharged amino acids, preferably positively charged amino acids, more preferably R; andX8is independently chosen from the group consisting ofpolar amino acids, preferably T or S,with T being preferred; ord) said peptide comprises the sequenceX1X2WKQKRTYVRFLVHRR (SEQ ID NO: 5), wherein Xi is independently chosen from the group consisting ofabsent, andhydrophobic amino acids, preferably aromatic hydrophobic amino acids, more preferably Y; andX2is independently chosen from the group consisting ofabsent, andcharged amino acids, preferably negatively charged amino acids, more preferably D; ore) said peptide comprises the sequenceYDWRQRX7X8YVRFLVHRR (SEQ ID NO: 6), wherein X7is independently chosen from the group consisting ofhydrophobic amino acids, preferably aliphatic hydrophobic amino acids, more preferably I, andcharged amino acids, preferably positively charged amino acids, more preferably R;with I being preferred;X8is independently chosen from the group consisting ofpolar amino acids, preferably T and S,with T being preferred; orf) said peptide comprises the sequenceYDWRQRIXsYVRFLVHRR (SEQ ID NO: 7), wherein X8is independently chosen from the group consisting of polar amino acids, more preferably T orS; org) said peptide comprises the sequenceYDWX4QX6RTYVRFLVHRR (SEQ ID NO: 8), wherein X4is independently chosen from the group consisting of charged amino acids, preferably positively charged amino acids, more preferably R or K; andX6is independently chosen from the group consisting of charged amino acids, preferably charged amino acids, more preferably R and K; orh) said peptide comprises the sequenceYX2WKQKRTYVRFLVX15RR (SEQ ID NO: 9), wherein X2and X15 are independently a non-polar amino acid, preferably a small amino acid, most preferably A; ori) said peptide comprises the sequenceYX2WKQKRTYVRFLVHRR (SEQ ID NO: 10), wherein X2is a polar amino acid, preferably N; orj) said peptide comprises the sequenceYX2X3X4QX6RTX9VRFLVHRR (SEQ ID NO: 51), wherein X2is independently chosen from the group consisting ofsmall amino acids, preferably A or G,polar amino acids, preferably N, andcharged amino acids, preferably negatively charged amino acids, more preferably D,with A, N or D being most preferred;X3is independently chosen from the group consisting ofa hydrophobic amino acid, preferably W anda small amino acid, preferably A;X4is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, with R or K being most preferred;X6is independently chosen from the group consisting ofcharged amino acids, preferably positively charged amino acids, with R or K being most preferred;X9is independently chosen from the group consisting ofa non-polar amino acid,with A or Y being most preferred; and / orone amino acid of the motif VRFLVHRR may substituted with a small amino acid, preferably A, ork) said peptide comprises the sequence YDWKQKRTYVRFLVHRR (SEQ ID NO:14), wherein any one amino acid is substituted with a small amino acid, preferably A.

5. A peptide of no more than 42 amino acids, comprising the amino acid sequence motif YDWKQKRTYVRFLVHRR (SEQ ID NO:14) with no more than 6, preferably no more than 5, most preferably no more than 4 amino acid substitutions, wherein peptide consists of more than 50% D-amino acids, preferably consists of more than 90% of D-amino acids, more preferably consists entirely of D-amino acids.

6. The peptide according to any one of the preceding claims, wherein said peptide has a length of 12-24 amino acids while maintaining the motif VRFLVHRR with one optional amino acid substitution, preferably said peptide has a length of 14-18 amino acids, more preferably a length of 15 amino acids, 16 amino acids or 17 amino acids.

7. The peptide according to any one of the preceding claims, wherein said peptide has no more than 25 amino acids and comprises an amino acid sequence chosen from the group consisting of SEQ ID NOs, 11-50, preferably SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD- 14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1 ), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11 ), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a).

8. The peptide according to any one of the previous claims, wherein said peptide consists of an amino acid sequence chosen from the group consisting of SEQ ID NOs, 11-50, preferably SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13(SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11 ), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a).

9. The peptide according to any one of the previous claims, wherein the peptide (i) comprises a C-terminal modification, wherein the C-terminal modification is selected from the group consisting of an acid amide group (CONH2-group), a CONH-alkyl group, a CONH-alkyl amine group, a COOH-group, preferably wherein the C-terminal modification is an acid amide group (CONH2-group), and / or (ii) comprises an additional cysteine residue at the C-terminus and the N-terminus or within 1 -2 aa distance of the C-terminus and the N-terminus, respectively, or comprises an N- and C-terminal modification that allows a cyclization of the peptide, and / or (iii) comprises a truncation at the C- and / or N terminus of cumulated no more than 4 amino acid residues while maintaining the motif VRFLVHRR with one optional amino acid substitution, and / or (iv) is a cyclic peptide.

10. The peptide according to any one of the previous claims,wherein said peptide consists of an amino acid sequence chosen from the group consisting of SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1 ), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD-14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a),wherein all amino acids of said peptide are D-configu rated amino acids, and wherein said peptide comprises a C-terminal modification in the form of an acid amide group (CONH2-group).

11. A peptide which is an enantiomeric retro-inverso peptide of the peptide of any one of the previous claims, wherein the retro-inverso peptide binds to monomeric alpha-synuclein and / or inhibits alpha-synuclein aggregation and / or is capable of disaggregating alpha-synuclein aggregates.

12. The peptide of any one of the previous claims, wherein the peptide binds to monomeric alpha-synuclein and / or inhibits alpha-synuclein aggregation and / or is capable of disaggregating alpha-synuclein aggregates.

13. A multimer of the peptide of any one of the previous claims, preferably the multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-51 , preferably selected from the group consisting of SEQ ID NO: 11 (SVD-14ag), SEQ ID NO: 12 (SVD-14ah), SEQ ID NO: 13 (SVD-14af), SEQ ID NO: 14 (SVD-14j), SEQ ID NO: 15 (SVD-14jb), SEQ ID NO: 16 (SVD-14u), SEQ ID NO: 17 (SVD-14ua), SEQ ID NO: 18 (SVD-14al), SEQ ID NO: 19 (SVD-14am), SEQ ID NO: 20 (SVD-14an), SEQ ID NO: 21 (SVD-14ao), SEQ ID NO: 22 (SVD-14ap), SEQ ID NO: 23 (SVD-14aq), SEQ ID NO: 24 (SVD-14ar), SEQ ID NO: 25 (SVD-14as), SEQ ID NO: 26 (SVD-14at), SEQ ID NO: 27 (SVD-14j_a1 ), SEQ ID NO: 28 (SVD-14j_a2), SEQ ID NO: 29 (SVD-14j_a3), SEQ ID NO: 30 (SVD-14j_a4), SEQ ID NO: 31 (SVD-14j_a5), SEQ ID NO: 32 (SVD-14j_a6), SEQ ID NO: 33 (SVD-14j_a7), SEQ ID NO: 34 (SVD-14j_a8), SEQ ID NO: 35 (SVD-14j_a9), SEQ ID NO: 36 (SVD-14j_a10), SEQ ID NO: 37 (SVD-14j_a11), SEQ ID NO: 38 (SVD-14j_a12), SEQ ID NO: 39 (SVD-14j_a13), SEQ ID NO: 40 (SVD-14j_a14), SEQ ID NO: 41 (SVD-14j_a15), SEQ ID NO: 42 (SVD-14j_a16), SEQ ID NO: 43 (SVD- 14j_a17), SEQ ID NO: 44 (SVD-14au), SEQ ID NO: 45 (SVD-14av), SEQ ID NO: 46 (SVD-14aw), SEQ ID NO: 47 (SVD-14ax), SEQ ID NO: 48 (SVD-14ay), SEQ ID NO: 49 (SVD-14az), and SEQ ID NO: 50 (SVD-14af_a), wherein optionally the multiple copies of said peptides form a continuous amino acid sequence or are covalently or non-covalently linked to one another.

14. The multimer of claim 13, wherein multiple copies are linked to one another by a branched or linear linker, preferably the linker is or comprises Gamma-aminobutyric acid(GABA), 6-aminohexanoic acid, or polyethylene glycol (PEG) or a peptide linker of no more than 10 amino acids length.

15. The peptide or multimer according to any one of the previous claims, wherein said peptide or multimer is linked to a further substance distinct from said peptide, preferably the substance is selected from any one of a peptide, a protein, a marker, a detectable label, a drug, an antibody, a radioisotope-containing moiety, a half-life-extending moiety, or a nucleic acid such as siRNA, miRNA, shRNA or antisense RNA.

16. A pharmaceutical composition comprising the peptide or multimer of any one of the previous claims.

17. Use in vitro of the peptide or multimer according to any one of claims 1 -15 for(i) detecting a-synuclein oligomers and / or a-synuclein aggregates and / or(ii) detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates.

18. The peptide or multimer according to any one of claims 1 -15, or the pharmaceutical composition according to claim 16, for use in detoxifying and / or disaggregating a-synuclein oligomers and / or a-synuclein aggregates in a subject, preferably a mammalian subject, more preferably a human subject.

19. The peptide or multimer according to any one of claims 1 -15, or the pharmaceutical composition according to claim 16, for use in diagnosing and / or treating and / or preventing a synucleinopathy in a subject, preferably a mammalian subject, more preferably a human subject.

20. The peptide or multimer or pharmaceutical composition for use according to claim 19, wherein the synucleinopathy is selected from the group consisting of Parkinson's disease (PD), Lewy body dementia (LBD), and multisystem atrophy (MSA), pure autonomic failure (PAF), idiopathic REM-sleep behavior disorder (iRBD) and a disease exhibiting a a-synuclein co-pathology such as Alzheimer's disease (AD) such as Alzheimer's Disease with Amygdala Restricted Lewy Bodies (AD / ALB).

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