Amyloid inhibitory peptides

Abstract

The present invention relates to peptides, in particular of amyloid inhibitory peptides, and to pharmaceutical compositions comprising such peptides, for use in methods of treating or preventing or delaying the onset of synucleinopathies, in particular of Parkinson's disease (PD) or dementia with Lewy bodies, and their comorbidities, in particular PD / type 2 diabetes (T2D) and PD / Alzheimer's disease (AD). Furthermore, the present invention relates to such peptides, in particular such amyloid inhibitory peptides, for use in methods of diagnosing such synucleinopathies and related comorbidities. Furthermore, the present invention also relates to a kit for the in-vitro or in-vivo detection and, optionally, quantification of amyloidogenic polypeptides, amyloid fibrils or amyloid aggregates, and / or for the diagnosis of synucleinopathies and related comorbidities, in particular PD / type 2 diabetes (T2D) and PD / Alzheimer's disease (AD), in a patient.

Description

[0001] Amyloid inhibitory peptides

[0002] The present invention relates to peptides, in particular of amyloid inhibitory peptides, and to pharmaceutical compositions comprising such peptides, for use in methods of treating or preventing or delaying the onset of synucleinopathies, in particular of Parkinson’s disease (PD) or dementia with Lewy bodies. Furthermore, the present invention relates to such peptides, in particular such amyloid inhibitory peptides, for use in methods of diagnosing such synucleinopathies. Furthermore, the present invention also relates to a kit for the in-vitro or in-vivo detection and, optionally, quantification of amyloidogenic polypeptides, amyloid fibrils or amyloid aggregates, and / or for the diagnosis of synucleinopathies in a patient.

[0003] Amyloid self-assembly is linked to devastating cell-degenerative diseases including Alzheimer’s disease (AD), type 2 diabetes (T2D), Parkinson’s disease (PD) and dementia with Lewy bodies, amongst others. Molecules blocking amyloidogenesis of the key amyloid polypeptides of AD and T2D, i.e. amyloid-P peptide (APqo(42)) (AD) and islet amyloid polypeptide (JAPP) (T2D), or of synucleinopathies, such as Parkinson’s disease (PD) or dementia with Lewy bodies, i.e. o-synuclein (aSyn), could thus become drug candidates. However, the rational design of amyloid inhibitors is a difficult task. Major reasons are the high conformational flexibility of most amyloidogenic polypeptides and several amyloidogenic proteins, high affinity interactions of amyloid self-assembly, and the large size of involved interfaces. Additional challenges include a low blood-brain- barrier (BBB) permeability, high production costs, and potential immunogenicity of antibodies, low proteolytic stability and usually no BBB crossing of linear peptides, while small molecules often lack high affinity and specificity and cannot block interactions involving large interfaces. Importantly, none of the reported inhibitors of amyloid selfassembly of o-synuclein (ctSyn), Afi4o(42) or JAPP has yet advanced to the clinic.

[0004] Synucleinopathies, also sometimes termed “a-synucleinopathies” are neurodegenerative diseases characterized by the abnormal accumulation of aggregates of a pre-synaptic protein of 140 residues, termed “a-synuclein” (aSyn) and of several mutants thereof. Parkinson’s disease (PD) is the most prominent synucleinopathy and is the second most common neurodegenerative disease (after Alzheimer’s disease) and affects more than 10 million people worldwide. Patients with synucleinopathies have features of parkinsonism, impaired cognition, sleep disorders and visual hallucinations. Parkinsonism is characterized by tremor, bradykinesia, rigidity and postural instability.

[0005] Previously, it was surmised that type 2 diabetes (T2D) is a risk factor for Parkinson’s disease. IAPP fibrils had previously been shown to be able to act as cross-seeding nuclei for amyloid self-assembly of o-synuclein (aSyn). However, both a-synuclein (aSyn) and IAPP are intrinsically disordered proteins, and possible cross-interaction sites and structures of possible hetero-assemblies are unknown, making the design of amyloid inhibitors extremely difficult.

[0006] US 7,745,490 discloses substituted N-aiyl benzamides as potential inhibitors of fibril formation of a-synuclein (aSyn), AB peptide(s), IAPP and others. It is not clear however, whether these inhibitors actually do inhibit such fibril formation, and whether they might also be able to interfere with cross-seeding caused by interactions of pre-existing minute amounts of fibrillar aggregates. Of note, there are no reports so far about molecules which are able to block lAPP / aSyn cross-seeding interactions.

[0007] Furthermore, none of the reported a-synuclein (aSyn) amyloid inhibitors or pipeline therapeutics for Parkinson’s disease, including antibodies, peptides and small molecules, have yet advanced into the clinic or have been shown to suppress cross-seeding of a- synuclein (aSyn). Parkinson’s disease and any other synucleinopathy are still incurable diseases.

[0008] Accordingly, there is a need for new inhibitors of amyloid self-assembly of a-synuclein (aSyn) and / or inhibitors that would interfere with a possible interaction between a- synuclein (aSyn) and IAPP. There is furthermore a need to provide amyloid inhibitors that inhibit amyloid self-assembly of a-synuclein (aSyn). There is also a need to provide amyloid inhibitors that inhibit a cross-seeding of a-synuclein (aSyn) assembly by IAPP.

[0009] In a first aspect, the present invention relates to a peptide, preferably an amyloid inhibitory peptide, having an amino acid sequence according to formula o

[0010] (Formula o) wherein

[0011] Zi and Z2 are selected from the following pairs a) cysteine and cysteine, b) aspartic acid and lysine, or lysine and aspartic acid, c) aspartic acid and ornithine, or ornithine and aspartic acid, d) aspartic acid and 2,4-diaminobutyric acid, or 2,4-diaminobutyric acid and aspartic acid, e) aspartic acid and 2,3-diaminopropionic acid, or 2,3-diaminopropionic acid and aspartic acid, f) glutamic acid and lysine, or lysine and glutamic acid, g) glutamic acid and ornithine, or ornithine and glutamic acid, h) glutamic acid and 2,4-diaminobutyric acid, or 2,4-diaminobutyric acid and glutamic acid, i) glutamic acid and 2,3-diaminopropionic acid, or 2,3-diaminopropionic acid and glutamic acid; with denoting a covalent bond between Zi and Z2, thus providing for a cyclization of the peptide;

[0012] Xi, X2, X3, X4, X5, X6, and X7 are, independently at each occurrence, selected from glycine, asparagine, valine, histidine, leucine, serine, alanine, and threonine;

[0013] F is, independently at each occurrence, phenylalanine;

[0014] L is leucine;

[0015] U is, independently at each occurrence, selected from arginine, homoarginine, citrulline, ornithine, lysine, and norleucine; G is glycine;

[0016] I is isoleucine; wherein Zl, Z2, X1-X7, F, L, U, G and I are L-amino acid residues or D-amino acid residues, or some of Zi, Z2, X1-X7, F, L, U, G and I are L-amino acid residues and others are D-amino acid residues; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof; wherein preferably said peptide has an amino acid sequence according to formula oa

[0017] (Formula oa) wherein

[0018] Zi, Z2, X1-X7, F, L, U, G, I are as defined above, and -methyl, for use in a method of treating, or preventing, or delaying the onset and / or pathogenesis of, a synucleinopathy. In one embodiment, the peptide according to the present invention, preferably the amyloid inhibitory peptide according to the present invention, has an amino acid sequence according to formula i

[0019] (Formula 1) or an amino acid sequence according to formula i

[0020] (Formula 1*) wherein

[0021] C is cysteine;

[0022] Xi, X2, X3, X4, X5, X6, and X7 are, independently at each occurrence, selected from glycine, asparagine, valine, histidine, leucine, serine, alanine, and threonine;

[0023] F is, independently at each occurrence, phenylalanine;

[0024] L is leucine;

[0025] R is arginine;

[0026] G is glycine;

[0027] I is isoleucine; is a disulfide bond; -methyl; C, X1-X7, F, L, R, G and I are L-amino acid residues or D-amino acid residues, or some of C, X1-X7, F, L, R, G and I are L-amino acid residues and others are D-amino acid residues; and pharmaceutically acceptable salts, esters, solvates, polymorphs and other modified forms thereof.

[0028] In an embodiment of the peptide, either a) Xi and X4 are asparagine, X2 is valine, X3 is histidine, X4 is glycine, X5 is glycine, X6 and X7 are glycine; b) X1-X7 are glycine, alanine or serine; c) X1-X7 are glycine; d) X1-X3 are glycine, X4 is asparagine, X5 is alanine, and X6-X7 are glycine; or e) X1-X3 are glycine, X4 is asparagine, X5 is alanine, X6 is leucine, X7 is serine.

[0029] In one embodiment, Zi, Z2, C, X1-X7, F, L, U, R, G, and I are L-amino acid residues.

[0030] In one embodiment, R is, at each occurrence, D-arginine, and / or

[0031] F is, at each occurrence, D-phenylalanine, and / or

[0032] L is D-leucine, and / or

[0033] Zi, Z2 and C are D-amino acid residues, and / or

[0034] I is D-isoleucine or N-methyl-D-isoleucine.

[0035] In one embodiment, the peptide according to the present invention has a sequence according to a formula selected from the following formulae 2a - 2e, 2a* - 2e*: C-GFLGG-RRR-GFGGIGG-C

[0036] (Formula 2b*)

[0037] C-GFLGG- r r r -GFGGIGG-C

[0038] (Formula 2c*)

[0039] (Formula 2e*) wherein upper case letters represent L-amino acid residues or D-amino acid residues, preferably L-amino acid residues, and lower case letters represent D-amino acid residues.

[0040] In a particularly preferred embodiment, the peptide(s) according to the present invention have a sequence according to a formula selected from 2b, 2e, 2b* and 2e*

[0041] Me Me

[0042] C-GFLGG-RRR-GFGGIGG-C

[0043] (Formula 2b) Me Me c-GflGG- r r r -GfGGIGG-c

[0044] (Formula 2e)

[0045] C-GFLGG-RRR-GFGGIGG-C

[0046] (Formula 2b*) c-GIIGG- r r r -GfGGIGG-c

[0047] (Formula 2e*), wherein upper case letters represent L-amino acid residues or D-amino acid residues, preferably L-amino acid residues, and lower case letters represent D-amino acid residues.

[0048] In one embodiment, said peptide consists of a sequence according to any of formulae o, oa, i, i*, 2a - 2e, 2a* - 2e*, as defined above, respectively, preferably of a sequence according to any of formulae 2b, 2e, 2b* and 2e*, as defined herein.

[0049] In one embodiment, said synucleinopathy is selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy-related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD). In one embodiment, said peptide is an amyloid inhibitory peptide that preferably binds to a-synuclein (aSyn), preferably to monomers and / or oligomers and / or fibrils thereof.

[0050] In a particularly preferred embodiment, which may be combined with any other embodiment herein, said peptide binds to o-synuclein (aSyn), preferably to monomers and / or oligomers and / or fibrils thereof, with nanomolar affinity. In a particularly preferred embodiment, said peptide binds to monomers of o-synuclein (aSyn) with nanomolar affinity.

[0051] In one embodiment, which may also be combined with any other embodiment herein, said peptide binds to IAPP, preferably to monomers and / or oligomers and / or fibrils thereof. In a particularly preferred embodiment, said peptide binds to IAPP with nanomolar affinity.

[0052] In one embodiment, which may also be combined with any other embodiment herein, said peptide binds to Abeta4o(42), preferably to monomers and / or oligomers and / or fibrils thereof. In a particularly preferred embodiment, said peptide binds to Abeta4O(42) with nanomolar affinity.

[0053] It should be noted that in preferred embodiments, where reference is made to a “peptide” in general, such peptide may also be referred to as an “amyloid inhibitory peptide”. An “amyloid inhibitory peptide” is a peptide that functions as, or can be used as, an “amyloid inhibitor”.

[0054] Without wishing to be bound by any theory, the present inventors believe that the amyloid inhibitory effect of the amyloid inhibitory peptides described herein is mainly mediated by their binding to key amyloid polypeptides, in particular to o-synuclein (aSyn) and / or to islet amyloid polypeptide (IAPP), more specifically to specific sites of a-synuclein (aSyn). The present inventors furthermore believe, again without wishing to be bound by any theory, that these specific sites mediate both self-assembly of a- synuclein (aSyn) and its possible interactions with islet amyloid polypeptide (IAPP), thus interfering with self-seeding interactions of a-synuclein (aSyn) with itself, but also its cross-interactions and cross-seeding by IAPP. Their potent (because of nanomolar IC50 values (Table 1)) amyloid inhibitory effects are also related to their ability to bind with nanomolar affinity to both a-synuclein (aSyn) and IAPP, in their various conformations (monomeric, oligomeric, or fibrils) and to interfere with their interactions and / or selfassembly.

[0055] Hence, the term "amyloid inhibitory" as used herein in the context of a peptide, refers to the capability of such peptide to block or inhibit amyloid self-assembly or amyloidogenesis or aggregation or amyloid formation, preferably of the key amyloid polypeptide(s) believed to be responsible for synucleinopathies, in particular of a- synuclein (aSyn), but also of islet amyloid polypeptide (IAPP), if / when such IAPP interacts with o-synuclein (aSyn). By binding with high affinity specific aSyn segments or “sites” which are crucial for aSyn self-assembly and its interactions with IAPP, the amyloid inhibitory peptides of the invention are thus capable of suppressing or blocking both self-seeded and cross-seeded amyloid self-assembly of aSyn while their ability to interact with or block amyloid self-assembly of IAPP likely contributes to their potent amyloid inhibitor function.

[0056] In one embodiment, the peptide(s) according to the present invention binds(bind) to at least one of the following stretches of the amino acid sequence of o-synuclein (aSyn): aSyn(i-i4), aSyn(34-52), and aSyn(87-iO5), preferably to at least two of these stretches, more preferably all three of them.

[0057] The terminology “aSyn(x-y)”, as used in this context is meant to refer to a stretch of the amino acid sequence of a-synuclein (aSyn), as shown in Example 1.16 herein, with “x” denoting the first (N-terminal) residue and “y” denoting the last (C-terminal) residue of such stretch.

[0058] Amyloid inhibitory peptides in accordance with the present invention are useful as amyloid inhibitors, and may thus be used for therapeutic and / or diagnostic purposes, i.e. they may be used for treatment and / or diagnosis of diseases involving amyloid selfassembly or amyloidogenesis, in particular of synucleinopathies. Preferably such synucleionpathies are selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy-related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD). In a further aspect, the present invention also relates to a composition comprising a peptide, preferably an amyloid inhibitory peptide, according to the present invention, and a suitable solvent, such as water, and a buffer.

[0059] In a further aspect, the present invention also relates to a pharmaceutical composition comprising a peptide, preferably an amyloid inhibitory peptide, according to the present invention and a pharmaceutically acceptable excipient, for use in a method of treating or preventing or delaying the onset of a synucleinopathy selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy-related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD).

[0060] In such pharmaceutical composition, the peptide may occur as such, or it maybe linked to other entities / molecules that endow the peptide with a specific functionality. For example, there may be a tag attached to increase blood-brain-barrier permeability, or it maybe attached to a specific reporter molecule, such as a dye or a quantum dot, allowing the detection in diagnostic methods (preferably whilst retaining the therapeutic functionality of the peptide - “theranostic applications”). The use of quantum dots may be particularly useful in various imaging technologies, such as MRI, PET, PET-MRI, or specifically quantum-dot-based brain imaging methodologies. In certain embodiments, the peptide may be attached to a nanoparticle, to a suitable carrier molecule, to a targeting entity or other functional molecule.

[0061] Because the peptides according to the present invention, however, are indeed capable of passing the blood-brain-barrier, the present invention also relates to the use of a peptide according to the present invention, as defined above, as a carrier for molecules, substances or compounds to pass the blood-brain-barrier. According to this aspect, in certain embodiments, the molecule to pass the blood-brain-barrier is linked, preferably covalently linked, to a peptide according to the present invention as defined above.

[0062] In one embodiment of such peptide or composition for use, said method comprises administering an effective amount of said peptide or of said composition to a patient in need thereof. In a further aspect, the present invention also relates to a peptide, in particular an amyloid inhibitory peptide, according to the present invention, or the pharmaceutical composition according to the present invention as defined above, for use in a method of diagnosing a synucleinopathy which is selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy-related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD).

[0063] In one embodiment of such peptide or composition for use, said method comprises administering an effective amount of said peptide or of said composition to a subject to be tested for a synucleinopathy.

[0064] In one embodiment of such peptide or composition for use, said peptide, in particular said amyloid inhibitory peptide, is linked to or administered together with a suitable reporter molecule that allows detection of o-synuclein (aSyn) and / or amyloid and / or non-amyloid aggregates and / or co-aggregates (e.g. aSyn / IAPP or aSyn / Abeta) and / or fibrils thereof, by a suitable detection methodology, such as positron emission tomography (PET), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), and PET-MRI and said subject, after administration of said peptide, is subjected to PET, NMR, MRI, PET-MRI.

[0065] In a yet a further aspect, the present invention relates to a kit for the in-vitro or in-vivo detection and / or quantification of o-synuclein (aSyn) and / or amyloid and / or nonamyloid aggregates and / or co-aggregates (e.g. aSyn / IAPP or aSyn / Abeta) and / or fibrils thereof , or for the diagnosis of a synucleinopathy selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy and mitochondrial membrane protein associated neurodegeneration, wherein preferably, said synucleinopathy is Parkinson’s disease (PD), in a patient. Preferably, said kit comprises a peptide, in particular an amyloid inhibitory peptide according to the present invention as defined herein, in a freeze-dried form in a suitable container, a buffered solvent in a separate container for reconstitution of said peptide in solution, and, optionally, means to dispense said peptide once reconstituted in solution, such as a syringe or pipette. Alternatively, the kit may contain the peptide, in particular the amyloid inhibitory peptide according to the present invention as defined above, in an already reconstituted, ready-to-use form. In a yet a further aspect, the present invention also relates to the use of the peptide according to the present invention, in an in-vitro assay, such as an enzyme linked immunosorbent assay (ELISA) or a radioimmuno assay (RIA), for the detection of o- synuclein (aSyn) monomeric or oligomeric aggregates and / or amyloid and / or nonamyloid aggregates and / or co-aggregates (e.g. aSyn / IAPP or aSyn / Abeta) and / or fibrils thereof.

[0066] Such use may, in certain embodiments, involve the analysis of body fluids, including but not limited to blood, cerebrospinal fluid, lymph, urine, and other body fluids or it may involve the analysis of brain biopsies, and may also further involve the use of suitable reporter molecules to which the peptide may be attached or with which the peptide may be used together.

[0067] In a further aspect, the present invention relates to the use of a peptide according to the present invention, as defined above, for the manufacture of a medicament for the treatment, prevention, delay of onset of, or diagnosis of a synucleinopathy selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy and mitochondrial membrane protein associated neurodegeneration, wherein preferably, said synucleinopathy is Parkinson’s disease (PD).

[0068] In yet a further aspect, the present invention also relates to a method of treatment, prevention, delay of onset of, or diagnosis of a synucleinopathy selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy and mitochondrial membrane protein associated neurodegeneration, wherein preferably, said synucleinopathy is Parkinson’s disease (PD), wherein said method comprises administering an effective amount of said peptide or of said composition according to the present invention as defined herein, to a patient in need thereof or to a subject to be tested.

[0069] The present inventors have provided cyclic peptides which function as nanomolar inhibitors of amyloid self-assembly of o-synuclein (aSyn) and which therefore have manifold applications.

[0070] Moreover, these peptides bind, with high (i.e. nanomolar) affinity, to o-synuclein (aSyn) monomers and / or amyloid aggregates, in particular to monomers thereof. Moreover, the peptides “detoxify” oligomers of a-synuclein (aSyn) (as for example shown in the ex-vivo assay of figure 4 as well as in figure 6.) In the present application, use is made of the one-letter-code for amino acid residues and the three-letter-code for amino acid residues. Hence, amino acid residues are designated herein by reference to their respective one-letter-code or three-letter-code. Accordingly, alanine is A or Ala; arginine is R or Arg; asparagine is N or Asn; aspartic acid is D or Asp; cysteine is C or Cys; glutamine is Q or Gin; glutamate is E or Glu; glycine is G or Gly; histidine is H or His; isoleucine is I or He; leucine is L or Leu; lysine is K or Lys; methionine is M or Met; phenylalanine is F or Phe; proline is P or Pro; serine is S or Ser; threonine is T or Thr; tryptophan is W or Trp; tyrosine is Y or Tyr; valine is V or Vai.

[0071] Sometimes, in this application, reference to amino acid sequences is made by reciting the individual residues. Where such amino acid sequence is indicated by using upper case letters only, this means that these amino acids are unspecified in terms of their chirality, i.e. the residues may be L-amino acids or D- amino acids or a mixture of the two possibilities, i.e. some of the residues in the sequence may be L-amino acids and others may be D- amino acids. In one embodiment, the upper case amino acids may be all L- amino acids.

[0072] In those instances, where such amino acid sequence is indicated by using upper case letters and lower case letters together in one sequence, this means that the upper case amino acids maybe L-amino acids or D- amino acids, preferably L-amino acids, and the lower case amino acids are, in any case, D- amino acids.

[0073] Moreover, sometimes, in this application, reference to amino acid sequences is made by reciting the individual residues as free amino acids, such as “glycine”, “glutamic acid”, etc., notwithstanding the fact that these residues appear in the respective amino acid sequence in their respective covalently linked form, i.e. with the individual residues linked by appropriate peptide bonds, i.e. amide bonds, between them.

[0074] The term “N-methyl” or “NMe” or “ I ”, as used herein, refers to a methyl group that is attached to the nitrogen in the amide bond between two amino acid residues.

[0075] For example where a sequence is indicated as this means that a methyl group is attached to the amide nitrogen forming the amide bond between F and G, and a further methyl group is attached to the amide nitrogen forming the amide bond between X and I. This may also be referred to as “N-methylated glycine” and “N-methylated isoleucine” respectively, or “methylated glycine” and a “methylated isoleucine”, respectively, because the respective amide nitrogen belongs to glycine and isoleucine respectively in these cases.

[0076] In some preferred embodiments of the peptide according to the present invention, there is a methyl group attached to the amide bond between Fit and G12 (i.e. a “methylated glycine 12”), and a further methyl group attached to the amide bond between X13 and I14 (i.e. a “methylated isoleucine 14”), if one uses a numbering which is based on a 17- peptide according to any of formulae o, oa, 1, 1*, 2, 2a-2e, 2a*-2e*. By reference to the lAPP-numbering, these positions correspond to the amide bond between F23 and G24 and the amide bond between A25 and I26.

[0077] The symbol refers to a covalent bond between two residues thus linked. For example, if the two residues thus linked are two cysteines, it means a disulfide bond.

[0078] Alternatively, it may mean a lactam bridge involving the sidechains of the respective two amino acids. For example, the sidechains of aspartic acid and lysine, or of glutamic acid and lysine may form such a lactam bridge. Pairs that may be involved in such lactam bridge formation are Asp-Lys, Asp-Orn, Asp-Dab (Dab meaning 2,4 diaminobutyric acid), Asp-Dap (Dap meaning 2,3-diaminopropionic acid), Glu-Lys, Glu-Orn, Glu-Dab, Glu-Dap, or pairs with an inverse arrangement of the aforementioned residues. In one embodiment, it also possible to obtain a cyclisation via the generation of 1,2,3-triazole rings, generated by click reactions between specific amino acids, instead of cysteines.

[0079] In one embodiment, the N-terminus and / or the C-terminus of the peptides according to the present invention are protected. Suitable protecting groups are manifold and are known to a person skilled in the art. For example, the N-terminus may be acetylated or formylated, or there may be an even longer chain attached such as palmitoyl. The C- terminus could be protected via formation of an amide or carbonic acid ester. In one embodiment, the C-termini of the peptide(s) according to the present invention, in particular of the amyloid inhibitory peptides according to the present invention, more particularly of the peptides according to formulae o, oa, i, 1*, 2a-2e, 2a*-2e* according to the present invention, are protected by an amide. In other embodiments, the C- terminus is in its free carboxy-form; in yet other embodiments, it is in esterified form. In particularly preferred embodiments, the C-termini of the peptide(s) according to the present invention, in particular of the amyloid inhibitory peptides according to the present invention, more particularly of the peptides according to formulae o, oa, 1, 1*, 2a-2e, 2a*-2e* according to the present invention, are protected by an amide, and the respective N-termini of the peptides are unprotected, i.e. in their NH2-form (or NH3+- form).

[0080] The term “treatment” or “treating”, as used herein, encompasses both prophylactic treatment and therapeutic treatment. In a preferred embodiment, it specifically refers to therapeutic treatment.

[0081] The present inventors have managed to design peptidic inhibitors of amyloid selfassembly of o-synuclein (aSyn) and of amyloid fibril formation of o-synuclein (aSyn) which is cross-seeded by IAPP fibrils. Without wishing to be bound by any theory, the present inventors believe that the peptidic inhibitors according to the present invention (which mimic IAPP surfaces that interact with IAPP, A or o-synuclein (aSyn)) target interaction surfaces of a-synuclein (aSyn) that are responsible for self-aggregation and / or for interaction and / or for co-aggregation with other surfaces or entities, such as IAPP, while maintaining only minimal lAPP-derived self / cross-recognition elements. In preferred embodiments, the peptides reported herein also exhibit both strongly improved proteolytic stability in human plasma and blood-brain-barrier crossing ability in a cell model. Peptides according to formulae 2a-2e are herein also sometimes simply referred to as “2a”, “2b”, “2c”, “2d”, and “2e”, or sometime also as “2A”, “2B”, “2C”, “2D”, and “2E”.

[0082] Furthermore, reference is made to the figures, wherein

[0083] Figure 1 shows the inventors’ strategy for identification of IAPP regions that interact with a-synuclein (aSyn) by using peptide arrays (a) and determination of the binding affinities of interactions of a-synuclein (aSyn) with IAPP and IAPP(8-28) by fluorescence spectroscopic titrations (b,c). a) Synthetic peptide arrays containing IAPP decamers (bold & underlined) were incubated with Biotin-o-synuclein (aSyn) (0.5 pM); decamers which bound Biotin o-synuclein (aSyn) are in dashed rectangles. Array representative of two arrays synthesized in parallel and two independent incubations with Biotin-o-synuclein (aSyn). b,c) Fluorescence emission spectra of Fluos-IAPP (=fluorescein-labelled JAPP) (b) and Fluos-IAPP(8-28) (c) (5 nM) alone or their mixtures with various molar ratios of a-synuclein (aSyn) (Fluos-peptide / a-synuclein (aSyn)) as indicated; data from 1 representative assay out of 3. Insets show binding curves; data means (±SD) of 3 titration assays.

[0084] Figure 2 shows the effects of 2b, 2e, and 4Ala-2b on non-seeded (a-c), seeded with preformed fibrillary aSyn (faSyn) (d-f), and fibrillary LAPP (flAPP)-cross-seeded aSyn amyloid self-assembly and related cell-damaging effects (g-i). a-c) Fibrillogenesis of aSyn (3 pM) alone or in the presence of 2b and 2e (1 / 1) or 4Ala-2b (1 / 50) determined by ThT binding (means (±SD), 3 assays (3 wells each)) (a); TEM images of solutions (7 day-aged) from (a) as indicated (color code as in (a)) (scale bars, too nm) (b); PC12 cell viability after treatment with solutions from (a) (7 day-aged) determined by the MTT reduction assay (means (±SD), 3 assays (3 wells each)) (c). d-f) Fibril formation of aSyn alone (3 pM) or seeded by preformed faSyn (10%) alone or with 2b and 2e (1 / 1) or 4Ala- 2b (1 / 50) as determined by the ThT binding assay and ThT binding of faSyn seeds (0.3 pM) (means (±SD), 3 assays (3 wells each) (d); TEM images of solutions from (d) aged for 7 days (seeded aSyn / 2b(2e) mixtures) or for 24 h (seeded aSyn alone or with 4Ala- 2b) and from faSyn seeds (scale bars, too nm) (e); PC12 cell viability after treatment solutions from (d) (7 day-aged) determined by the MTT reduction assay (means (±SD), 3 assays (3 wells each)) (f). g-i) Fibrillogenesis of aSyn (3 pM) alone or cross-seeded by flAPP (10%) alone or with 2b and 2e (1 / 1) or 4Ala-2b (1 / 50) determined by ThT binding and ThT binding of flAPP seeds (0.3 pM) (means (±SD), 3 assays (3 wells each)) (g); TEM images of solutions from (g) aged for 7 days (cross-seeded aSyn / 2b(2e)) or for 24 hours (cross-seeded aSyn alone or with 4Ala-2b) as indicated (scale bars, too nm) (h); PC12 cell viability after treatment with solutions from (g) (7 day-aged) determined by the MTT reduction assay (means (±SD), 3 assays (3 wells each).

[0085] Figure 3 shows studies on interactions, hetero-complexes, and mechanism of inhibitory effects of 2e and 2b on aSyn amyloid self-assembly. a,b) Left, app. Kas of interactions of Fluos-2b (a) and Fluos-2e (b) with aSyn determined by fluorescence spectroscopic titrations. Fluorescence emission spectra of Fluos-2b or Fluos-2e (1 nM) and their mixtures with various molar ratios of aSyn are shown as indicated; spectra from 1 representative binding assay out of 3. Right side, binding curves; data means (±SD) of 3 titration assays; app. Kas in Table 2. c,d) Far-UV CD spectra of aSyn (1 pM) alone and its mixtures with 2b (c) or 2e (d) (10 pM) measured at o h and after 48 h of incubation, e) Characterization of aSyn / MCIP hetero- and aSyn homo-oligomers by cross-linking with glutaraldehyde, SDS-PAGE, and Western blot with anti- aSyn (left) or anti-2e(2b) (right) antibodies (aSyn, 10 pM; MCIPs, 50 pM); representative results from 3 assays, f) Characterization of aSyn / 2e hetero-complexes in comparison to aSyn and 2e alone by size exclusion chromatography (SEC). Chromatograms of aSyn (3 pM), 2e (30 pM), and the aSyn / 2e mixture (1 / 10) are shown. Inset, ESI-MS spectrum (deconvoluted) of the 21 min peak from SEC of the aSyn / 2e mixture. Determined MWs as indicated; calculated mass (average) 14460.27 Da (aSyn) and 1695.04 Da (2e). Representative results from 3 SEC analyses and ESI-MS. g) Kinetics of aSyn self-assembly into An-reactive toxic oligomers alone or in the presence of 2e followed by slot blot analysis using the An antibody. Solutions aSyn (3 pM) alone, aSyn / 2e (1 / 1), and 2e (3 pM) alone were analyzed at indicated incubation time points. Representative results from 4 assays, h) Binding of 2b, 2e, and 4Ala-2b to faSyn and flAPP determined by dot blot analysis. Fluos-2b, Fluos-2e, and Fluos-4Ala-2b (1.5 pM) and the buffer alone control were incubated with membranes containing spotted faSyn or flAPP; binding visualized by fluorescence. Representative results from 3 assays.

[0086] Figure 4 shows the suppression of aSyn oligomer-induced LTP impairment in murine hippocampal slices ex-vivo by 2b and 2e. a,b) Time course of synaptic transmission (fEPSP, field excitatory postsynaptic potential) after treatment with a) aCSF medium (buffer control), aSyn oligomers (175 nM), 2b alone (1.75 pM), and aSyn oligomers / 2b mixture (1 / 10) or b) aCSF medium, aSyn oligomers (175 nM), 2e (1.75 pM), and aSyn oligomers / 2e mixture (1 / 10); data means (±SD), from n=io samples / treatments each, c) LTP values: bars show the averages from the last 10 min of recording; data means (±SD), n=io for each group; p-values as indicated; calculated using non-parametric testing with Mann-Whitney U-tests or a Kruskal -Wallis test.

[0087] Figure 5 shows the identification of aSyn segments mediating its interactions with MCIPs and LAPP by synthetic peptide arrays (a,e), determination of binding affinities by fluorescence spectroscopic titrations (b-d), and overview of key aSyn interaction sites and related functions (f). a) Identification of key aSyn regions interacting with 2e using peptide arrays. Top, aSyn sequence; identified 2e-binding regions highlighted in orange; colored arrows indicate P-strands Pi- P8 in the faSyn fold. Bottom, peptide array containing aSyn decamers following incubation with Fluos-2e (1 pM) and bound peptide visualization by fluorescence; identified Fluos-2e-binding segments aSyn(i-i4), aSyn(34-52), and aSyn(87-iO5) in orange rectangles. Array representative of 2 arrays synthesized in parallel and 2 independent incubations with Fluos-2e (data not shown), b-d) Determination of app. Kas of Fluos-2e interactions with identified 2e-binding aSyn segments by fluorescence spectroscopic titrations. Left, fluorescence emission spectra of Fluos-2e alone (5 nM) or with various molar ratios of aSyn(i-i4) (b), aSyn(34-52) (c), and aSyn(87-iO5) (d) (Fluos-2e / peptide as indicated). Spectra from 1 representative assay out of 3. Right, binding curves; data means (±SD) of 3 assays (see Table 2). e) Identification of key aSyn regions interacting with IAPP using peptide arrays. Top, aSyn sequence; identified lAPP-binding regions highlighted in pink; colored arrows as under (a). Bottom, peptide array containing aSyn decamers (as in (a)) following incubation with Fluos-IAPP (1 pM) and visualization. The identified 3 major Fluos-IAPP-binding segments aSyn(i-i3), aSyn(34-46), and aSy(87-iO4) are in pink rectangles made by solid lines; the weaker binding aSyn(68-8o) is in a pink rectangle made by dashed lines. Array representative of two arrays synthesized in parallel and two independent incubations with Fluos-IAPP (data not shown), f) Schematic overview of the 3 identified key aSyn segments mediating its interactions with 2e, 2b, and IAPP and previously reported interactions & functions of related aSyn sequence parts. White arrows indicate P-strands Pi- P8 in the faSyn fold.

[0088] Figure 6 demonstrates a confirmation of the presence of cell-damaging aSyn oligomers in solutions used for studying effects of MCIPs on hippocampal synaptic LTP impairment mediated by aSyn oligomers (see Fig. 4). aSyn oligomers were prepared according to a previously described protocol (M. J. Diogenes, et al., Journal of Neuroscience 2012, 32, 11750-11762.). Following a 5-day aging of aSyn (138 pM) at 37°C, aSyn oligomers were separated from fibrils by centrifugation and supernatants were used for the LTP impairment studies. The presence of cytotoxic oligomers in the supernatant fraction was confirmed by the ThT binding assay (a), TEM (b), dot blot with the anti-oligomer antibody An (c), and the MTT reduction assay (d); for comparison, freshly dissolved aSyn (aSyn monomers) or buffer alone and pellet fractions (fibrils) were studied as well, a) ThT binding properties of aSyn monomers and of the supernatant (aSyn oligomers) and pellet (aSyn fibrils) fractions (aSyn 1.7 pM) as indicated. Data is means (±SD) from 3 aSyn oligomer preparations, b) TEM micrographs of supernatant (aSyn oligomers) and pellet (aSyn fibrils) fractions; scale bars 1 pm and 100 nm, respectively, c) Dot blot analysis of An antibody binding of supernatant (aSyn oligomers) and pellet (aSyn fibrils) fractions as compared to aSyn monomers (5 pg each); a representative dot blot (n=2) is shown, d) Effects of buffer, aSyn monomers, and the supernatant fraction (aSyn oligomers) (aSyn, 300 nM) on PC12 cell viability determined by MTT reduction assay. Data means (±SD) of 4 assays (n=3 wells each); statistical analysis by one-way ANOVA with post-hoc Tukey test.

[0089] Figure 7 shows that the peptides in accordance with the present invention (sometimes herein also referred to as “macrocyclic inhibitory peptides” or “MCIPs”) suppress aSyn cytotoxicity.

[0090] More specifically, MCIPs are shown to be able to (Fig. 7a) suppress aSyn cytotoxicity in aSyn overexpressing human postmitotic dopaminergic neurons (see example 1) and (Fig. 7b-d) block cross-seeding of aSyn amyloid self-assembly by AP42fibrils (fAP42): (a) Suppression of aSyn-mediated toxicity in aSyn overexpressing human postmitotic dopaminergic LUHMES neurons by 2b and 2e (10 nM) measured by lactate dehydrogenase (LDH) release. Grey column (control): untransduced cells; dark yellow column (GFP): cells transduced with GFP (control for virus); black column (aSyn): aSyn overexpressing cells without treatment; red column (aSyn + 2b): aSyn overexpressing cells treated with 2b; blue column (aSyn + 2e): aSyn overexpressing cells treated with 2e. Data means (±SEM) from 3 assays (n=3 each). Statistical significance was determined using one-way ANOVA, followed by Dunnett's multiple comparisons test; p- values as indicated, (b-d) MCIPs block cross-seeding of aSyn amyloid self-assembly by fAP42: (b) Fibrillogenesis of aSyn (3 pM) alone or cross-seeded by fAP42 (20%) alone or with 2b and 2e (1 / 1) determined by ThT binding; data of IAP42 seeds (0.6 mM) is also shown for comparison; data means (±SD), 3 assays (3 wells each); (c) TEM images of 7 day-aged solutions from (e) (scale bars, too nm); (d) PC12 cell viability after treatment with 7 day-aged solutions from (e) determined by the MTT reduction assay (means (±SD), 3 assays (3 wells each)) (g). P values <0.05 were considered significant.

[0091] Figure 8 shows in vitro evidence for remodeling of AP4o(42) fibrils and cytotoxic oligomers into non-toxic and non-fibrillar assemblies by the peptides according to the present invention, as exemplified by peptide 2E. A. Addition of 2E to preformed fAP42 diminishes ThT binding of fAP42. ThT fluorescence of fAP42 (5 pM) (o h) before or following addition of 2E (50-fold molar excess) was measured at various incubation time points (3 independent assays, 3 technical replicates each). B. Bar diagram showing ThT fluorescence from Figure 8A at o h or 6 days of incubation. Data from n=3 independent assays. C. 2E converts IAP42 into amorphous aggregates. Representative TEM images of solutions of Figure 8A. Scale bars: 100 nm. D. 2E addition to IAP42 reduces fAP42- mediated cell damage. Preformed fAP42 (o h), fAP42 (6 day-aged) or fAP42+2E (1 / 50) (6 day-aged) (from Figure 8A) were added to PC12 cells; cell damage was determined by the MIT reduction assay (3 independent assays, 3 technical replicates each). E. Bar diagram showing MIT reduction values from Figure 8D (fAP42, 1 pM) (3 assays). F. Addition of 2E to pre-aggregated AP42, i.e. a mixture of preformed fAP42 and cytotoxic AP42 oligomers (2 h-aged AP42) (see also Figures 8G-H), diminishes ThT binding of fAP42 consistent with fAP42 remodeling. ThT fluorescence measured in pre-aggregated AP42 (5 pM) alone and before or following addition of 2E (20-fold molar excess) as indicated (three assays, 3 technical replicates each). G. Addition of 2E (20-fold molar excess) to pre-aggregated AP42 (2 h-aged) significantly reduces AP42-mediated cell damage. Solutions from Figure 8F were added to PC12 cells and cell damage was determined by the MTT reduction assay. Bar diagram shows MTT reduction values of AP42 (1 pM) alone before (o h- or 2 h-aged) and at different incubation time points after 2E addition. Data from three independent assays, 3 technical replicates each. H. Slot blot analysis of AP42 solutions (5 pM) of Figures 8F-G at different incubation time points using the anti-oligomer An antibody indicates significant amounts of cytotoxic (An reactive) AP42 oligomers in 2-6 h aged solutions. Results representative of 4 assays. I. Addition of 2E (20-fold molar excess) to pre-aggregated (2 h-aged) AP42 containing significant amounts of fibrils and cytotoxic oligomers (see Figures 8F-H) blocks amyloid self-assembly and remodels this species into amorphous aggregates. TEM images of solutions of Figure 8F reveals as major species thin fibrils and amorphous aggregates in

[0092] 2 h-aged AP42, fibrillar assemblies in 6-day aged AP42, and amorphous aggregates in 6- day aged AP42(2 h) / 2E mixtures. Scale bars: 100 nm. J. Addition of 2E to preformed fAP4O markedly reduces ThT binding. ThT fluorescence of preformed fAP4O (5 pM) alone and before or following addition of 2E (50-fold molar excess) versus buffer alone was measured at various incubation time points as indicated (three independent assays,

[0093] 3 technical replicates each). K-L. Bar diagrams showing ThT fluorescence from solutions of Figure 8 J at o h (Figure 8K) and 7 days (Figure 8L) of incubation (3 assays). M. 2E remodels fAP4O into amorphous aggregates. Representative TEM images of solutions of Figure 8J reveal mostly fibrils in fAP4O (o h) and 7 day-aged fAP4O and mainly amorphous aggregates in 7 day-aged mixture of IAP40-1-2E. Scale bars: 100 nm. N. 2E addition to fAP4O significantly reduces its cell damaging effects. Solutions from Figure 8J were added to PC12 cells and cell damage was determined by MTT reduction (three independent assays, 3 technical replicates each). O. Bar diagram showing MTT reduction values from Figure 8N at 7 days of incubation (fAP4O, 1 pM) (three independent assays, 3 technical replicates each), d, days; h, hours. Data are means ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001. Statistical significance was assessed using oneway ANOVA with Tukey’s post hoc test.

[0094] Figure 9 shows that the control peptide 4Ala-2b does not affect fAP42 and toxic AP42 oligomers in vitro. A. Addition of 4Ala-2b to preformed IAP42 does not affect ThT binding of IAP42. ThT fluorescence of fAP42 (5 pM) (o h) alone and before or following addition of 4Ala-2b (50-fold molar excess) was measured at various incubation time points (representative data from one out of 3 independent assays, n=3 technical replicates). B. 4Ala-2b addition to fAP42 does not affect fAP42-mediated cell damage. Solutions of fAP42 (6 day-aged) or fAP42+4Ala-2b (6 day-aged) (from Figure 9A) were added to PC12 cells; cell damage was determined by the MTT reduction assay (representative data from one out of 3 independent assays, n=3 technical replicates). C. 4Ala-2b addition to IAP42 does not affect IAP42 morphology. Representative TEM images of solutions of Figure 9A. Scale bars: 100 nm. D. Addition of 4Ala-2b to preaggregated AP42, i.e. a mixture of preformed fAP42 and cytotoxic AP42 oligomers (2 h- aged AP42), does not affect ThT binding of fAP42 and AP42 aggregation kinetics. ThT fluorescence measured in pre-aggregated AP42 (5 pM) alone and before or following addition of 4Ala-2b (20-fold molar excess) as indicated (data from one assay in 3 technical replicates). E. Addition of 4Ala-2b to pre-aggregated AP42 (2 h-aged; see Figure 9D) does not affect AP42-mediated cell damage. Solutions from Figure 9D were added to PC12 cells and cell damage was determined by the MTT reduction assay (data from one assay in 3 technical replicates). F. Addition of 4Ala-2b to pre-aggregated AP42 (2 h-aged; see Figure 9D) does not intervene with AP42 amyloid self-assembly. Representative TEM images of solutions of Figure 9D reveal fibrillar assemblies as main species in both AP42 alone and its mixtures with 4Ala-2b (both 6 day-aged). Scale bars: too nm. d, days; h, hours. Data are means ± standard error of the mean.

[0095] Figure 10 shows that the peptides in accordance with the present invention as exemplified by peptide 2e, not only block cytotoxic amyloid self-assembly, but are also able to remodel or disassemble preformed aSyn fibrils (faSyn) and cytotoxic assemblies into non-toxic and non-fibrillar species. More specifically, for the experiments in Figure 10, incubations of aSyn amyloid fibrils (faSyn) were prepared by incubating monomeric aSyn (3 pM) for 7 days as for the assays shown in Fig. 2 and as described in Example 1. After 7 days of incubation, ThT, cell viability, and TEM studies (Fig. 2a-c) showed that faSyn and cytotoxic assemblies were major species. These 7-day incubated (“aged”) solutions were then mixed with 2e at the indicated fold excess and kept at 37°C for additional 7 days; ThT binding (Fig. 10a) was measured at the indicated time points. Cell viability studies (MTT reduction studies) (Fig. 10b) were performed by adding dilutions of faSyn / 2e mixtures aged for 7 days to cultured PC12 cells as described in Fig. 2b and Example 1. Fig. 10c shows representative TEM images (scale bars: 100 nm) of solutions used in Fig. 10a from faSyn alone after 7 days of incubation which was found to consist mostly of amyloid fibrils (upper panel) versus its 7-day aged mixture with 2e which was found to consist mostly of amorphous aggregates (lower panel).

[0096] Moreover, reference is made to the following examples which are given to illustrate and not to limit the present invention.

[0097] Examples

[0098] Example 1 - Materials and Methods

[0099] 1. Peptides, peptide synthesis, and proteins

[0100] All peptides, i.e. macrocyclic inhibitory peptides (MCIPs), 4Ala-2b, ISMs, JAPP, and JAPP or aSyn segments, and their Na-terminal fluorescein-labeled analogs (Fluos- peptide) (all C-terminal amides) were synthesized using Fmoc-based solid phase synthesis (SPPS) on Rink resin and purified via RP-HPLC. Briefly, double or triple couplings were performed using Fmoc-protected amino acids (3 -fold molar excess) and as coupling reagents N,N,N\N'-tetramethyl-O-(iH-benzotriazol-i-yl)uronium hexafluorophosphate (HBTU) or 2-(7-aza-iH-benzotriazole-i-yl)-i, 1,3,3- tetramethyluronium hexafluorophosphate (HATU) (3-fold molar excess) for selected couplings and N,N-diisopropyl ethylamine (DIEA) (4.5-fold molar excess) in N,N- dimethylformamide (DMF). Cysteines were coupled using 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC). Na-terminal fluorescein-labels were introduced by coupling 5(6)-carboxyfluorescein (3-fold molar excess) using (HATU (3- fold molar excess) and DIEA (4.5-fold molar excess) in DMF (double coupling). In the case of the SPPS of LAPP, pseudoproline dipeptides (3-fold molar excess) were used at specific sequence positions and coupled using HATU (3-fold excess) and DIEA (4.5 fold excess); SPPS, cleavage of protecting groups / from the resin, disulfide bridge formation, and RP-HPLC purification were performed in accordance with standard protocols. Disulfide bridge formation of the MCIPs and 4Ala-2b was performed in aq. 0.1 M NH4HCO3 solution containing DMSO (40%) (stirring for -1.5 h). SPPS was performed manually for sequence parts containing N-methyl amino acids and either manually or with a CS336X peptide synthesizer (CSBio) for the rest of the sequence parts or peptides containing no N-methyl amino acids. RP-HPLC purifications were preformed using Nucleosil 100 C18 (Grace) or Reprosil Gold 200 C18 columns (Dr. Maisch) according to previously developed standard protocols. Peptide purity was verified by MALDI-TOF mass spectrometry (MS).

[0101] Peptide stock solutions were freshly made in i,i,3,3,3,3-hexafluoro-2-isopropanol (HFIP) on ice; concentrations were determined by peptide weight and / or the BCA assay and by UV spectroscopy in the case of IAPP and fluorescein-labeled peptides in accordance with standard protocols. Stock solutions of IAPP and fluorescein-labeled peptides in HFIP were filtered over 0.2 mm filters just before the determination of their concentration. Peptides were applied from their HFIP stocks following HFIP evaporation with N2and reconstitution with assay buffer. Recombinant human aSyn was produced as described in (S. T. Kumar, S. Donzelli, A. Chiki, M. M. K. Syed, H. A. Lashuel, Journal of Neurochemistry 2020, 153, 103-119.) or was purchased from Eurogentech (Anaspec) (catalog number AS-55555 with an additional Gly residue at the N-terminus) or from AlexoTech (Catalog Number AS-600-100). ctSyn aliquots were prepared in ddH20, frozen immediately, lyophilized, and freshly reconstituted with assay buffer for each experiment. Of note, freshly made aSyn solutions at the low pM concentration range used in the present inventors’ studies consisted predominantly of non-toxic monomers based on CD spectroscopy, SEC, ThT binding, the MIT reduction assay, and TEM (Fig. 2a, 3c, 3f, 3g, 2a).

[0102] 2. Thioflavin T (ThT) binding assays

[0103] Effects of peptides on aSyn self- and cross-seeded amyloid self-assembly were studied by a combination of the ThT binding assay with TEM and the MTT reduction assay according to previously developed protocols (K. Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem,

[0104] L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commun 2022, 13, 5004).

[0105] The ThT binding assays were performed based on a previously published protocol (Shaykhalishahi, A. Gauhar, M. M. Wordehoff, C. S. Gruning, A. N. Klein, O. Bannach,

[0106] M. Stoldt, D. Willbold, T. Hard, W. Hoyer, Angew Chem Int Ed Engl 2015, 54, 8837- 8840; bM. M. Wordehoff, W. Hoyer, Bio-protocol 2018, 8).

[0107] Solutions used for the assays of amyloid self-assembly of aSyn alone (3 pM) or its mixtures with various peptide amounts (as indicated) with or w / o preformed (cross- )seeds (10% or 1% as indicated) or related controls were made in 20 mM sodium phosphate buffer (pH 6.0) containing 50 mM NaCl (abbreviated “ThT assay buffer”) and ThT (40 pM) in 96-well black MTPs (FluoroNunc / Thermo Fisher Scientific) and incubated (“aged”) for up to 7 days at 37°C with shaking at 800 rpm in a MTP shaker (Thermoshaker TPS-4H; 4 More Labor). Of note, each MTP well contained one glass bead (2 mm). ThT fluorescence was measured at 486 nm (excitation at 450 nm) using the Multilabel reader VictorX3 (Perkin Elmer Life Sciences) at the indicated time points with short shaking (1 min) before measuring. In each assay, solutions were prepared in 3 technical replicates (n=3 wells) and the ThT fluorescence signal of the buffer was subtracted from all samples. Data are means (±SD) of 3 independent assays (n=3 wells each) if not stated otherwise.

[0108] Preformed seeds of IAPP fibrils (flAPP) were prepared by incubating IAPP (128 pM) in ThT assay buffer for 24 h (RT; no shaking); fibril formation was verified by TEM (Fig. 2h (inset)). aSyn fibrils (faSyn) were prepared by incubating aSyn (69 pM in ThT assay buffer in a MTP well containing a glass bead (2 mm) for 7 days (at 37°C & 800 rpm); their formation was verified by TEM (Fig. 2e (inset)). Preformed seeds were sonicated for 1 min prior to their use.

[0109] Solutions used for the incubations were made as follows: (1) To study effects of the peptides on (unseeded) aSyn amyloid assembly, freshly made aSyn (3 pM) or aSyn / peptide mixtures (aSyn / peptide molar ratios as indicated) or control solutions in ThT assay buffer containing 40 pM ThT were incubated for 7 days as mentioned above (37°C & 800 rpm). Of note, peptides were preincubated in ThT assay buffer for 6 h prior mixing with aSyn and ThT to ensure complete dissolution. (2) To study effects of the peptides on aSyn amyloid self-assembly (cross-)seeded with preformed faSyn or flAPP seeds, freshly made aSyn (3 pM) and the corresponding aSyn / peptide mixtures with or w / o faSyn or flAPP (10% (0.3 pM) or 1% (0.03 pM) as indicated; aSyn / peptide molar ratios as indicated), or control solutions (e.g. seeds alone) were incubated in ThT assay buffer containing 40 pM ThT for 7 days as mentioned above (at 37°C & 800 rpm). Of note, peptides with or w / o faSyn or flAPP were preincubated in ThT assay buffer (for 6 h, RT) prior to mixing with aSyn and ThT. To investigate the (cross-)seedability of the aSyn / peptide hetero-complexes by faSyn or flAPP seeds, peptides (aSyn / peptide molar ratios as indicated) were preincubated (6 h, RT) with aSyn (3 pM) in ThT assay buffer prior to mixing with fibril seeds to allow for hetero-complex formation. Following addition of faSyn or flAPP seeds (10% (0.3 pM), solutions were incubated in ThT assay buffer containing 40 pM ThT for 7 days as mentioned above (at 37°C & 800 rpm). 3. Assessment of cell damage by the MTT reduction assay

[0110] PC12 cells were obtained from DSMZ - German Collection of Microorganisms and Cell Cultures GmbH (DSMZ number: ACC 159) and were cultured and plated as described in (M. Yan, A. Velkova, M. Tatarek-Nossol, E. Andreetto, A. Kapurniotu, Angew Chem Int Ed Engl 2007, 46, 1246-1252).

[0111] Effects of MCIPs and controls on formation of cell-damaging aSyn assemblies were studied by the MTT reduction assay using solutions from the ThT binding assays aged for o h, 24 h, 48 h, or 7 days (at 37°C & 800 rpm as under “ThT binding assays”) according to previously developed protocols (K. Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commun 2022, 13, 5004). Briefly, aliquots of ThT binding assay solutions were diluted with cell medium at various incubation time points and added to the PC12 cells at the indicated final concentrations of aSyn and peptides (IC50values at 100 nM aSyn; 7 day-aged solutions). Following incubation with the cells for ~20 h (37°C, humidified atmosphere with 5% C02), cells were incubated with MTT (0.92 mg / ml) for ~2 h at 37°C. Following addition of 10% SDS in 20 mM HC1 (pH 4.5) and overnight incubation, generated formazan was quantified by its absorbance at 570 nm with a Multilabel reader VictorXs (Perkin Elmer Life Sciences); for 100% MTT reduction the absorbance of untreated cells and for 0% MTT reduction the absorbance of Triton X-100- treated cells was used.

[0112] 4. Transmission electron microscopy (TEM)

[0113] Aliquots (10 pl) of solutions to be examined, including solutions used for ThT binding and MTT reduction assays, were applied at the indicated incubation time points on formvar / carbon-coated grids (3 min). Grids were washed with ddH20 and stained with aqueous 2% (w / v) uranyl acetate solution for 1 min. Examination of the grids was performed using a JEOL 1400 Plus electron microscope at 120 kV.

[0114] 5. Far-UV CD spectroscopy

[0115] CD measurements were performed using a Jasco 715 spectropolarimeter and spectra (average of 3 spectra) were recorded between 200 and 250 nm, at 0.1 nm intervals, with a response time of 1 sec as previously described in (A. Spanopoulou, L. Heidrich, H. R. Chen, C. Frost, D. Hrle, E. Malideli, K. Hille, A. Grammatikopoulos, J. Bernhagen, M. Zacharias, G. Rammes, A. Kapurniotu, Angew Chem Int Ed Engl 2018, 57, 14503- 14508). For the CD studies on structures and effects of aSyn / MCIP hetero-complexes on aSyn self-assembly (Fig. 3c, d), freshly made solutions of aSyn alone (1 pM), MCIP alone (10 jiM), and aSyn / MCIP mixtures (1 / 10) in ThT assay buffer were made and incubated for 48 h at 370C to mimic the experimental conditions of the ThT binding assays. CD spectra were recorded at the o h incubation time point (after a 20 min preincubation at 37° C) and at the 48 h incubation time point. Of note, the spectra of the MCIPs alone are not shown in Fig. 3c, d as they were the same as reported previously in WO2O19 / 234157. The spectrum of the buffer alone was subtracted from all CD spectra.

[0116] 6. Fluorescence spectroscopic titrations

[0117] Fluorescence spectroscopic titration studies were performed using a Jasco FP-6500 fluorescence spectrophotometer and previously established protocols (Andreetto, E. Malideli, L. M. Yan, M. Kracklauer, K. Farbiarz, M. Tatarek-Nossol, G. Rammes, E. Prade, T. Neumuller, A. Caporale, A. Spanopoulou, M. Bakou, B. Reif, A. Kapurniotu, Angew Chem IntEd Engl 2015, 54, 13095-13100). Briefly, excitation was at 492 nm and emission spectra were recorded between 500 and 600 nm. For all experiments, freshly prepared stocks of peptides and their fluorescently labeled analogs in HFIP were used while aSyn stocks were in aqueous 10 mM sodium phosphate buffer (pH 7.4). Measurements were performed in freshly made solutions containing synthetic Na- terminal fluorescein-labeled JAPP (Fluos-IAPP), IAPP(8-28) (Fluos-IAPP(8-28)), MCIPs (Fluos-2e & Fluos-2b), or 4Ala-2b (Fluos-4Ala-2b) (1 or 5 nM as indicated) alone or with various amounts of the binding partner (aSyn, aSyn segments, or JAPP) in aqueous 10 mM sodium phosphate buffer (pH 7.4) containing 0.5% HFIP (for binding to aSyn or its segments) or 1% HFIP (for binding to LAPP or IAPP(8-28)). Measurements were performed within 2-5 min after solution preparation. Under these experimental conditions, Fluos-peptides were mostly monomeric. Apparent (app.) Kd values were calculated as described using 1 / 1 binding models which fitted the 1 / 1 aSyn / peptide ratio required for full inhibition of amyloid self-assembly and cytotoxicity. However, due to the self-assembly propensities of the examined peptides / proteins and their multi-site binding features more complex models may also apply. App. Kas are means (±SD) of three binding curves derived from 3 titration assays.

[0118] 7. Cross-linking, NuPAGE, and Western blot (WB) analysis

[0119] Cross-linking studies were performed in combination with NuPAGE and WB based on a previously developed assay system (Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commun 2022, 13, 5004). Briefly, for characterizing aSyn homo- and aSyn / MCIP hetero-assemblies, aSyn alone (10 |nM), aSyn / MCIP mixtures (1 / 5), and MCIPs alone (50 pM) were prepared in ThT assay buffer. Following incubation for 30 min, solutions were cross-linked by adding 25% aqueous glutaraldehyde (Sigma-Aldrich) (2 min). Following treatment with 2 M NaBH4(in 0.1 M NaOH, 20 min), cross-linked complexes were precipitated with 10% aqueous trichloroacetic acid (4°C), centrifuged (10 min, 12000 g), and pellets were dissolved in NuPAGE LDS-sample buffer (w / o reducing agent) boiled for 5 min, and subjected to gel electrophoresis (10-20% Tricine gels) with Tricine SDS running buffer according to the manufacturer’s (Invitrogen) recommendations. Equal amounts of aSyn (4.3 pg) and MCIPs (2.5 pg for 2b and 2e and 2.2 pg for 4Ala-2b) were loaded in the different lanes and prestained protein size markers (from 3.5 to 260 kDa (Invitrogen)) were run in the same gels.

[0120] Gels were blotted onto nitrocellulose membranes using an XCell II Blot Module blotting system (Invitrogen) and membranes were blocked by overnight incubation (io°C) with 2% BSA in TBS-T (20 mM Tris, 150 mM NaCl, 0.05% Tween-20). To detect aSyn containing bands, membranes were incubated with a rabbit anti-human aSyn antibody (Sigma -Aldrich, SAB4502829, Lot: 210582) (1:1000) in 0.5% BSA in TBS-T (2 h at RT or overnight at io°C) and thereafter with donkey anti-rabbit-HRP antibody (GE Healthcare, NA934, Lot: 16836138) (1:5000) in 0.5% BSA in TBS-T (2 h at RT or overnight at io°C). Western blots were developed using Super Signal West Dura Extended Duration Substrate (Thermo Scientific, Cat. No. 34075) and imaged using a LAS-4000 mini imager (Fujifilm). To detect 2e- or 2b-containing bands, membranes were stripped by incubating with stripping buffer consisting of 2% SDS and 100 mM - mercaptoethanol in 50 mM Tris (pH 6.8) (40 min at 6o°C and 60 min at RT). After washing with TBS-T, membranes were blocked with 2% BSA in TBS-T (3 h, RT). Detection of 2b and 2e was done by incubation (2 h at RT or overnight at io°C) with mouse anti-2e antibody (Clone MCP2E 26C3), which was produced at Helmholtz Center Munich as described below, and with goat anti-mouse-HRP antibody (Abeam, ab6 89, Lot: GR3257574-1) (1:10000) in 0.5% BSA in TBS-T as secondary antibody (2 h at RT or overnight at io°C). Of note, the anti-2e antibody recognized 2b as well and did not bind with aSyn (Fig. 3e); the anti-aSyn antibody did not bind to 2b and 2e (data not shown).

[0121] 8. Generation of monoclonal antibodies against 2e

[0122] Monoclonal antibodies were developed in cooperation with the core facility monoclonal antibodies of the Helmholtz Center Munich as follows: Thirteen week old female Balb / c wild type mice (Charles River) were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 50 pg ovalbumin-coupled 2e dissolved in a mixture of 200 pl PBS containing 5 nmol CpG2006 (TIB MOLBIOL) and 200 pL incomplete Freund's adjuvant (Sigma -Aldrich). Animal procedures were approved by the local Animal Use and Care Committee with approval by the local authorities of Upper Bavaria, Germany (reference number ROB-55.2Vet-2532.Vet_O3-22-25) in accordance with European and German animal welfare regulations. After 12 weeks, a boost without Freund’s adjuvant was given i.p. and s.c. 3 days before fusion. Fusion of the myeloma cell line P3X63-Ag8.653 with the mouse immune spleen cells was performed using polyethylene glycol 1500 according to standard procedure. After fusion, the cells were plated in 96-well plates with medium consisting of RPMI 1640 with stable glutamine (RPMI-STA, Capricorn) supplemented with 20% fetal calf serum, imM pyruvate, lx non- essential amino acids and HAT media supplement (Hybri-Max, Sigma-Aldrich). Hybridoma supernatants were screened 10 days later in a flow cytometry assay (iQue, Intellicyt; Sartorius) using N-terminal biotinylated 2e captured on streptavidin beads (PolyAN) and incubated for 90 min with hybridoma supernatant and Atto-488-coupled isotype-specific monoclonal rat-anti-mouse IgG secondary antibodies. Antibody binding was analyzed using ForeCyt software (Sartorius). Positive supernatants were further validated by dot blot and Western blot. Hybridoma cells from selected supernatants were cloned by limiting dilution to obtain stable monoclonal cell lines. The present inventors’ assays (Fig. 3e) were performed with hybridoma supernatant of clone MCP2E 26C3 (mouse IgG2a / k).

[0123] 9. Size exclusion chromatography (SEC) and ESI-MS of collected peaks

[0124] For the SEC studies a Superdex 75 10 / 300 GL column (GE Healthcare) and a Dionex UltiMate 3000 device (Thermo Fisher Scientific) were used (Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commurt 2.02.2, 13, 5004). Elution buffer was the ThT assay buffer, the flow rate was 0.5 ml / min, and protein / peptide detection was at 214 nm. The column was calibrated with a gel filtration protein standard (Bio-Rad, Cat. No. 151-1901) (Supporting Fig. S10). The retention time (IR) of ctSyn monomers was determined by injecting aSyn (3 pM) freshly dissolved in 6 M GdnHCl in 0.1 M NH4HCO3(pH 8.4). Of note, aSyn monomers eluted earlier than expected based on the applied globular protein standard likely due to their disordered structure consistent with previous reports. For the analysis of aSyn / MCIP hetero-complexes, freshly made solutions (400 pl) of aSyn alone (3 pM), 2e alone (30 pM), and aSyn / 2e mixtures (1 / 10) in ThT assay buffer were loaded onto the column. Peaks were collected, lyophilized, and analyzed by liquid-chromatography electrospray ionization mass spectrometry (LC-ESI-MS). For sample analysis, a Dionex UltiMate 3000 HPLC system (column: MSPac DS-10 (10 x 2.1 mm, 5 pm) (Thermo Scientific)) coupled to a LCQ Fleet mass spectrometer (Thermo Scientific) with a heated ESI source was used. The mass spectrometer was run in positive mode collecting iontrap scans at scan rate “normal” from 300 to 2000 m / z. Spectra were visualized using XCalibur 2.2 SP1.48 (Thermo Scientific) and deconvoluted using MagTran 1.02 (Amgen Inc.) implementing the ZSCORE algorithm (Z. Zhang, A. G. Marshall, Journal of the American Society for Mass Spectrometry 1998, 9, 225-233).

[0125] 10. Slot blot analysis

[0126] Effects of MCIPs on kinetics of formation of cytotoxic (An-reactive) aSyn oligomers (Fig. 3g) were studied by Slot blot analysis in combination with ThT binding, MTT reduction assays, and TEM (see Fig. 2a-c). Slot blot analysis was performed using a PR648 Slot blot blotting manifold (Hoefer) with nitrocellulose membrane based on a previously described protocol (C. Kontos, O. El Bounkari, C. Krammer, D. Sinitski, K. Hille, C. Zan, G. Yan, S. Wang, Y. Gao, M. Brandhofer, R. T. A. Meg ens, A. Hoffmann, J. Pauli, Y. Asare, S. Gerra, P. Bourilhon, L. Leng, H. H. Eckstein, W. E. Kempf, J. Pelisek, O. Gokee, L. Maegdefessel, R. Bucala, M. Dichgans, C. Weber, A. Kapurniotu, J. Bernhagen, Nat Commun 2020, 11, 5981). Briefly, solutions containing aSyn (3 pM) alone, 2e (3 pM) alone, and their mixture (1 / 1) in ThT assay buffer were incubated for 7 days (37°C, 800 rpm) in MTPs as for the ThT binding assay and added (200 pl each) at the indicated incubation time points to the slots which had been pre-wetted with ThT assay buffer. Solutions were left in the slots for 2 h (RT) to allow for binding with the membrane; thereafter, they were removed with vacuum and slots were washed with ThT assay buffer (1x100 pL). The membrane was washed 3x5 min with TBS-T and blocked with 2% BSA in TBS-T overnight at io°C. Then, the membrane was incubated with a rabbit An antibody (Invitrogen, AHB0052, Lot: WH329538) (1:1000 in 0.5% BSA in TBS-T) (overnight at io°C) and, following washing with TBS-T (3x5 min), incubated with donkey anti-rabbit-HRP antibody (GE Healthcare, NA934, Lot: 16836138) (1:5000 in 0.5% BSA in TBS-T) 2h at RT or overnight at io°C. Following washing with TBS-T (3x5 min), the membrane was developed using Super Signal West Dura Extended Duration Substrate (Thermo Scientific, Cat. No. 34075) and imaged with a LAS-4000 mini imager (Fujifilm).

[0127] 11. Dot blot assays

[0128] Binding of 2b, 2e and 4Ala-2b to flAPP or faSyn (Fig. 3I1) was studied by dot blot analysis performed according to a previously described protocol (Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commurt 2.02.2, 13, 5004). Briefly, flAPP was prepared by incubating an JAPP solution (128 pM) in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl and 0.5% HFIP for 24 h at RT (flAPP confirmed by ThT and TEM) (Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commurt 2022, 13, 5004). faSyn were prepared by incubating aSyn (69 pM) in ThT assay buffer at 37°C & 800 rpm for 7 days as for the ThT binding assay (confirmed by ThT and TEM; data not shown). flAPP (20 pg) or faSyn (10 pg) were spotted on nitrocellulose membranes. Membranes were washed with TBS-T, blocked with 5% milk in TBS-T overnight at io°C, and washed again with TBS-T. Then, membranes were incubated with solutions of N-terminal fluorescein-labeled peptides (Fluos-peptide) (1.5 pM) in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl and 0.5% HFIP (for flAPP binding studies) or in ThT assay buffer (for faSyn binding studies) overnight at io°C. To control for fibril autofluorescence, membranes containing spotted flAPP or faSyn were incubated in parallel with buffer alone (Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commurt 2022, 13, 5004). Following washing with TBS-T, bound peptides were visualized with a LAS- 4000 mini imager equipped with a suitable fluorescence filter (Fujifilm).

[0129] 12. Studies using peptide arrays

[0130] To identify the aSyn binding sites for its interactions with MCIPs and JAPP, the present inventors used synthetic peptide arrays. Peptide arrays containing JAPP or aSyn decamers covering full length JAPP or aSyn and positionally shifted by one residue were synthesized on a modified cellulose membrane support using stepwise SPOT synthesis protocols and a MultiPep RSi (Intavis) peptide synthesizer as previously described (Andreetto, L. M. Yan, M. Tatarek-Nossol, A. Velkova, R. Frank, A. Kapurniotu, Angew Chem Int Ed Engl 2010, 49, 3081-3085). Thereafter, arrays were immobilized on glass slides according to the manufacturer’s instructions and processed and developed using previously described protocols (Andreetto, L. M. et al., 2010, ibid.). Briefly, glass slides blocked for 4 h at RT with 1% BSA in TBS-T and incubated with synthetic Na-terminal fluorescein-labeled 2e (Fluos-2e) or JAPP (Fluos-IAPP) (1 pM in 1% BSA in TBS-T) or with recombinant Biotin-labeled MiC-aSyn (Biotin-aSyn; Cysi(side-chain)-labeled MiC-aSyn via thiol-maleimide-chemistry) (0.5 pM in 1% BSA in TBS-T) overnight (at io°C) followed by washing with TBS-T. Bound peptides were visualized by using a LAS- 4000 mini instrument as follows (Fujifilm): In the case of fluorescein-labeled peptides, visualization was based on their fluorescence readout. In the case of Biotin-aSyn, visualization was performed via enhanced chemiluminescence (ECL) following incubation (2h at RT) of the glass slides with streptavidin-POD antibody (Roche Diagnostics, 11089153001, Lot: 56790500) (1:1000 in 1% BSA in TBS-T) and development by the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Cat. No. 34075) as described (Andreetto, L. M. et al., 2010, ibid.). For quantification, the optical density of each spot was determined using ImageJ software. For each array, the highest optical density value was defined as 100%, while the optical density value of the negative control spot was defined as 0% and relative intensity of each spot was determined by using the following formula: optical density (spot) — optical density (neg. control, 0%)

[0131] Rel. intensity = - - - r - highest optical density (100%) — optical density (neg. control, 0%)

[0132] Binding regions corresponded to clusters of at least 4 strong binding decamers defined as indicated in the legends of Supporting Fig. S16 and S18. Of note, results of densitometric analyses were consistent with the results of the visual inspection of the arrays.

[0133] 13. Fluorescence polarization assays

[0134] Fluorescence polarization (FP) assays were performed using a Jasco FP-8550 fluorescence spectrophotometer. Excitation was at 492 nm and emission was recorded at 522 nm. Bandwidth for excitation and emission was set at 5 nm and time response at 0.5 s. Measurements were performed within 2-5 min upon solution preparation. To study interactions of the three identified aSyn key segments aSyn(i-i4), aSyn(34-52), and aSyn(87-iO5) with flAPP, the present inventors measured FP of freshly made Fluos- flAPP alone (too nM) in aqueous 10 mM sodium phosphate buffer (pH 7.4) containing 0.5% HFIP and its mixtures with each of the 3 segments (1.5 pM). Fluos-flAPP was made by incubating Fluos-IAPP (20 pM) in aqueous 10 mM sodium phosphate buffer (pH 7.4) containing 0.5% HFIP for 48 h; solutions were sonicated for 30 seconds prior to their use for FP assays; the presence of fibrils was verified by TEM (not shown).

[0135] 14. Preparation and characterization of aSyn oligomers for the ex-vivo hippocampal LTP measurements aSyn oligomers for the hippocampal LTP measurements were prepared based on a previously reported protocol (M. J. Diogenes, R. B. Dias, D. M. Rombo, H. Vicente Miranda, F. Maiolino, P. Guerreiro, T. Nasstrom, H. G. Franquelim, L. M. Oliveira, M. A. Castanho, L. Lannfelt, J. Bergstrom, M. Ingelsson, A. Quintas, A. M. Sebastiao, L. V. Lopes, T. F. Outeiro, Journal of Neuroscience 2.012, 32, 11750-11762). Briefly, lyophilized aSyn was suspended at 2 mg / ml (138 pM) in ddH20 and incubated under continuous shaking for 5 days at 37°C in a Thermomixer (Eppendorf) at 1400 rpm. Solutions were centrifuged at 22000g for 15 min, the resulting supernatant (oligomers) was lyophilized. The concentration of ctSyn oligomer solutions (supernatants) was determined using the BCA assay (Pierce). Supernatant fractions containing the oligomers were lyophilized, kept at -6o°C, and reconstituted with aCSFjust prior to the LTP measurements. In addition, assemblies present in supernatants and pellets of the aSyn oligomer preparations were characterized by TEM, the ThT binding assay, the MTT reduction assay, and a Dot blot assay to assess An-reactivity using above protocols (). Briefly, TEM grids were prepared and imaged as described under TEM. ThT binding was determined in aliquots of solutions containing aSyn (1.7 pM) monomers (freshly made and supernatant (oligomer) or pellet (fibril) fractions in ThT assay buffer containing 20 pM ThT as described under “ThT binding assays”. For determination of All-reactivity, solutions containing mostly aSyn monomers and the supernatants or re-suspended pellets (69 pM in ddH20) were spotted on a nitrocellulose membrane (as described under dot blot assays) and the membrane was blocked with 2% BSA in TBS-T overnight at io°C and incubated with rabbit An antibody and developed as described under “Slot blot assays”. For the assessment of the cell-damaging effects of aSyn oligomer-containing fractions, lyophilized supernatant fractions were dissolved in ThT assay buffer to a concentration of 3 pM (as for the ThT binding assays). Following dilution with cell medium, aSyn oligomers were added to the cultured PC12 cells plated in MTPs (aSyn, 300 nM) and incubated with the PC12 cells for ~20 h as described under “MIT reduction assays”. MTT reduction was assessed as described under “MTT reduction assays”. Data is means (±SD) of four assays (n=3 wells each) performed using three independent aSyn oligomer preparations.

[0136] 15. Hippocampal long-term potentiation (LTP) measurements (ex vivo)

[0137] LTP measurements were performed as previously described (K. Tas, B. D. Volta, C. Lindner, O. El Bounkari, K. Hille, Y. Tian, X. Puig-Bosch, M. Ballmann, S. Hornung, M. Ortner, S. Prem, L. Meier, G. Rammes, M. Haslbeck, C. Weber, R. T. A. Megens, J. Bernhagen, A. Kapurniotu, Nat Commun 2022, 13, 5004). Briefly, sagittal hippocampal slices (350 pm) were obtained from C57BL / 6N mice (6-8 weeks of age, male) (Charles River Laboratories) in ice-cold Ringer solution bubbled with a mixture of 95% 02and 5% C02according to protocols approved by the ethical committee on animal care and use of the government of Bavaria Germany (according to §11 TierschG and §4 TierschG; no accreditation number). Ethics oversight by ethical committee on animal care and use of the government of Bavaria (Regierung von Oberbayern, ROB). Extracellular recordings were performed using artificial cerebrospinal fluid (aCSF)-filled glass microelectrodes (2-3 Mil) at RT. aCSF consisted of 125 mM NaCl, 2.5 mM KC1, 25 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, 25 mM D-glucose, and 1.25 mM NaH2PO4(pH 7.3) and was bubbled with 95% 02and 5% C02. Field excitatory postsynaptic potentials (fEPSPs) were evoked in the hippocampal CA1 dendritic region via two independent inputs by stimulating the Schaffer collateral commissural pathway (Seep). For LTP induction, high-frequency stimulation (HFS; 100Hz / too pulses) conditioning pulses were delivered to the same Seep inputs. Both stimulating electrodes were used to utilize the input specificity of LTP, thus allowing for the measurement of internal control within the same slice. aSyn oligomers (175 nM) (made as described in the previous chapter), their mixtures with MCIPs (1 / 10), or MCIPs alone (1.75 pM) were freshly dissolved in aCSF and applied to the slices 60-90 min before HFS. Responses were measured for 60 min after HFS. fEPSP slope measurements (20-80% of peak amplitude) are presented as %fEPSP slope of baseline (the 20 min control period before tetanic stimulation was set to 100%). For statistical comparisons, the present inventors performed non-parametric testing with Mann-Whitney U tests or a Kruskal-Wallis test. A correction for multiple comparisons was not performed, which is acceptable as for instance stated by Rothman (K. J. Rothman, Epidemiology 1990, 1, 43-46) and uncorrected p-values are presented in Fig. 4-

[0138] 16. Effects of the peptides on aSyn toxicity in a-Syn overexpressing postmitotic dopaminergic LUHMES neurons

[0139] An earlier developed cell model of aSyn toxicity in aSyn overexpressing dopaminergic postmitotic Lund human mesencephalic (LUHMES) neurons was used (as described under (a) M. Hollerhage, J. N. Goebel, A. de Andrade, T. Hildebrandt, A. Dolga, C. Culmsee, W. H. Oertel, B. Hengerer, G. U. Hoglinger, Neurobiol Aging 2014, 35, 1700-1711 and (b) M. Hollerhage, C. Moebius, J. Melms, W. H. Chiu, J. N. Goebel, T. Chakroun, T. Koeglsperger, W. H. Oertel, T. W. Rosier, M. Bickle, G. U. Hoglinger, Sci Rep 2017, 7, 11469.). Briefly, LUHMES cells (ATCC, catalogue number: CRL-2927) were cultivated (~ioo.ooo cells / cm2), differentiated into a postmitotic dopaminergic phenotype, and transduced to overexpress aSyn by adding adenoviral vectors encoding wild type aSyn (on day 1 after the begin of differentiation). The virus-containing medium was removed, and after a cellwash step (PBS), peptides (10 nM in cell medium) were added to the cells (on day 2 after the begin of differentiation) and cell death in peptide-treated or untreated transduced cells and in untransduced control cells and GFP-transduced cells (control for adenovirus effects) was quantified by the lactate dehydrogenase (LDH) release assay which was performed 6 days after transduction. Data in Fig. 7a are presented as “relative LDH release” (% of LDH release of untreated cells overexpressing aSyn which was set to 100%); background values were measured in wells containing medium but no cells and their averages were subtracted from all data.

[0140] 17. Sequences

[0141] The sequences of the peptides according to the present invention are mentioned and disclosed elsewhere herein and in the appended claims as formulae o, oa, 1, 1*, 2a - 2e, 2a* - 2e*.

[0142] The key amyloid protein in Parkinson’s disease (PD) is “o-synuclein” (aSyn) which has an amino acid sequence of 140 residues as follows:

[0143] MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH

[0144] GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL

[0145] GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0146] The sequence of the key amyloid protein in diabetes type 2 is islet amyloid polypeptide (IAPP) which has the following 37 residue amino acid sequence:

[0147] KCNTATCATQ RLANFLVHSSN NFGAILSSTN VGSNTY

[0148] The key amyloid peptide in Alzheimer’s disease (AD) is the amyloid B-peptide (“AB” or “Abeta”) two forms of which (“AB40” and “AB42” - jointly herein also sometimes referred to as “AB4o(42)”) play an important role in AD. These two forms are derived from amyloid precursor protein (APP) by enzymatic cleavage and differ from each other in their respective lengths by 2 amino acids at the C-terminus. AB40 has 40 amino acid residues and is shorter by two amino acids at the C-terminus than AB42 which has 42 amino acid residues. The respective sequences are (with the two additional amino acids of AB42 shown in brackets at the C-terminus):

[0149] AB4O(42): DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA I IGLMVGGVV ( IA)

[0150] R3-GI, an IAPP interaction interaction surface mimic (or “ISM”) is a linear peptide which has the sequence:

[0151] ATQRLANFLV HRRRNFGAIL S in which G17 and I19 (= G24 and I26, when using an lAPP-based numbering) are N- methylated (Andreetto, Kapurniotu et al. ACIE (2015)).

[0152] The negative control “4Ala-2b” (Spanopoulou, Kapurniotu et al. ACIE (2018)) in which all four lAPP-derived key residues (for IAPP self / cross-interactions) of 2b and 2e (F15, L16, F23 and I26 of the lAPP-sequence) were replaced by Ala, has a cyclized sequence of

[0153] CGAAGGRRRG AGGAGGC disulfide cyclization occurring via the terminal cysteines, with G12 and A14 additionally being N-methylated.

[0154] Example 2

[0155] Nanomolar Affinity lAPP / ctSyn Cross-Interactions Mediated by LAPP Amyloid Core Region IAPP(8-28)

[0156] The inventors first determined the IAPP regions that mediate its cross-interactions with aSyn. Synthetic peptide arrays containing IAPP decamers covering full-length IAPP and positionally shifted by one residue were incubated with biotin-labeled aSyn (Biotin- aSyn) and Biotin-aSyn-bound decamers were visualized by chemiluminescence. The inventors found a major cluster of 4 consecutive decamers within IAPP(8-2o), while a second weaker cluster localized in IAPP(i3-27) (Fig. la). The inventors then titrated synthetic Na-terminal fluorescein-labeled IAPP (Fluos-IAPP) and IAPP(8-28) (Fluos- IAPP(8-28)) with aSyn. Determined app. K<is were 26.7 (±6.0) nM for the Fluos- lAPP / aSyn interaction and 8.2 (±2.3) nM for the Fluos-IAPP(8-28) / aSyn interaction (Figure ib,c). These data revealed that IAPP binds aSyn with low nanomolar affinity and that the IAPP amyloid core IAPP(8-28) contains the key recognition elements for the lAPP / aSyn interaction as earlier found (WO2O19 / 234157A1) for the IAPP / IAPP and the IAPP / AP4O(42) interactions. Macrocyclic peptides 2b and 2e are Nanomolar Inhibitors of Self- and IAPP- Cross-Seeded Amyloid Self-Assembly of aSyn

[0157] Based on the above, the inventors hypothesized that the IAPP(8-28)-derived macrocyclic peptides as described earlier in WO2O19 / 234157 of formulae o, oa, 1, 1*, 2a - 2e, 2a* - 2e* might mimic putative ZAPP / aSyn cross-interaction surfaces and interfere with aSyn amyloid self-assembly and its cross-seeding by flAPP. To test this they used peptides of formulae 2b and 2e as examples. Notably, initial studies showed that both IAPP(8-28), which is intrinsically amyloidogenic, and its non-amyloidogenic analogs IAPP(8-28)-GI and R3-GI, which are linear MCIP precursors, were unable to inhibit (data not shown). Hence, in the following, peptides 2b and 2e were used as exemplary test peptides. However, the present inventors believe that all the other peptides of formulae o, oa, 1, 1*, 2a - 2e, 2a* - 2e*, as described herein, behave similarly and show the same inhibitory effects, as demonstrated herein.

[0158] The effects of 2b and 2e on aSyn amyloid self-assembly were then studied (Fig. 2, Table 1). In parallel, the inventors also studied the effects of the negative control peptide 4Ala- 2b. According to the amyloid specific ThT binding assay and transmission electron microscopy (TEM), aSyn fibrillogenesis started after a lag-time of ~24 h and was accomplished after -48-72 h (Fig. 2a, b). However, in the presence of 2b or 2e (aSyn / peptide 1 / 1) a full suppression of aSyn fibrillogenesis was observed (Figures 2a, b). In addition, 2b and 2e strongly suppressed formation of cell-damaging aSyn assemblies according to the results of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay in cultured rat pheochromocytoma (PC12) cells (Fig. 2c). In fact, aSyn titrations with 2b and 2e revealed nanomolar IC50values, i.e. 62.3 (±33.9) nM (2b) and 66.0 (±23.3) nM (2e) (Table 1). No attenuating effects were found for the negative control 4Ala-2b up to a 50-fold higher molar excess than 2b or 2e (Fig. 2a-c).

[0159] Table 1. IC50of inhibitory effects of 2b, 2e, and 4Ala-2b on cell-damaging effects of unseeded, faSyn-seeded, and flAPP-cross-seeded aSyn amyloid self-assembly.

[0160] IC5o (±SD) (nM) IC5o (±SD) (nM) IC5O(±SD) (nM)

[0161] Peptide Inhibition of Inhibition of Inhibition of

[0162] 2b 62.3 (±33-9) 51-8 (±4-8) 75-2 (±21.1)

[0163] 2e 66.0 (±23.3) 54.7 (±4.4) 42.0 (±18.5) 4Ala-2b > 5000 > 5000 > 5000

[0164] [a] IC5o values, means (±SD) from 3 titration assays (n=3 wells each); aSyn, too nM w / o or with preformed faSyn or flAPP seeds (10%).

[0165] The inventors next asked whether 2b and 2e might also suppress seeding of aSyn fibrillogenesis by preformed aSyn fibrils (faSyn). Addition of faSyn seeds (10%) to aSyn strongly accelerated formation of aSyn fibrils and cell-damaging aggregates as expected (Fig. 2d-f). However, in the presence of 2b and 2e (1 / 1), aSyn fibrillogenesis and cell toxicity were fully suppressed whereas again 4Ala-2b (aSyn / 4Ala-2b, 1 / 50) did not inhibit (Fig. 2d-f). Titrations with the two inhibitors yielded IC50values of 51.8 (±4.8) nM (2b) and 54.7 (±4.4) nM (2e) which were nearly identical to the IC50values of effects on unseeded aSyn fibrillogenesis (Table 1). Notably, 2b and 2e inhibited aSyn fibrillogenesis and cytotoxicity when seeding was performed both with 10% and 1% faSyn seeds, indicative of effects on secondary nucleation and fibril elongation events (Fig. 2d- f).

[0166] The inventors then asked whether the two peptides might also interfere with the crossseeding effect of LAPP fibrils (flAPP) on aSyn fibrillogenesis. Addition of seed amounts (10%) of preformed JAPP fibrils (flAPP) to aSyn strongly accelerated its fibrillogenesis consistent with previous findings (Fig. 2g, h). In parallel, a strong acceleration of formation of cell-damaging aSyn species was also observed (data not shown). Importantly, in the presence of 2b or 2e (1 / 1) a full suppression of cross-seeding of aSyn fibrillogenesis and cytotoxicity was observed and titrations yielded nanomolar IC50values for both peptides, i.e. 75.2 (±21.1) nM (2b) and 42.0 (±18.5) nM (2e) (Table 1). As expected, 4Ala-2b did not inhibit (aSyn / peptide, 1 / 50).

[0167] Taken together, the above studies identified 2b and 2e as nanomolar inhibitors of both self- and flAPP-cross-seeded amyloid self-assembly of aSyn.

[0168] MCIPs Bind aSyn with Nanomolar Affinity and Sequester it into Non- Fibrillar and Non-Cytotoxic Co-Assemblies

[0169] To learn more about the inhibition mechanism, aSyn / peptide interactions and coassemblies were studied by various biophysical and biochemical methods. First, the affinities of aSyn / peptide interactions were determined by titrating synthetic N -terminal fluorescein-labeled 2b (Fluos-2b) and 2e (Fluos-2e) with aSyn (Fig. 3a, b). Low nanomolar apparent (app.) Kd values were obtained for both peptides (Fluos-2b, app. Kd=i7-2 (±2.6) nM; Fluos-2e, 22.0 (±5.1) nM) (Table 2) in good agreement with their IC5o values (Table 1). Notably, the aSyn binding affinities of 2b and 2e were very similar to their IAPP binding affinities while 4Ala-2b did not bind either IAPP or aSyn (Table 2).

[0170] The far-UV CD spectrum of freshly dissolved aSyn exhibited a pronounced minimum at ~200 nm indicative of mainly disordered structure consistent with previous reports (Fig. 3c,d). Following aging for 48 h, a marked reduction of the CD magnitude was observed indicative of aSyn oligomerization. In the presence of 2b or 2e, however, no / slower reduction of the CD magnitude was observed in line with their inhibitory activity on aSyn amyloid self-assembly (Fig. 3c, d). Of note, in addition to the minimum at ~200 nm, the CD spectra of aSyn / inhibitor mixtures exhibited a weaker but clear minimum between 220-230 nm. Their shapes and magnitudes suggested that hetero-complexes were more ordered than aSyn (Fig. 3c, d).

[0171] Next, aSyn / inhibitor hetero-complexes were cross-linked with glutaraldehyde and following separation by NuPAGE visualized by Western blot (WB) with anti-aSyn and anti-2e(2b) antibodies (Fig. 3e). In freshly made aSyn solutions, monomers and dimers were major species; trimers and other medium-to-high MW aggregates were less abundant consistent with previous findings. In aSyn / 2b(2e) mixtures, a similar pattern as in aSyn alone was observed with the difference that the bands stained with both the anti-aSyn and a monoclonal anti-2e(2b) antibody; in addition, bands corresponding to aSyn mono-, di-, and trimers were slightly shifted upwards (Fig. 3e). These data indicated that 2b and 2e co-assemble with aSyn monomers and low MW oligomers into hetero-dimers and low MW hetero-oligomers (Fig. 3e).

[0172] Hetero-complexes formed at early steps of aSyn / 2e co-assembly were then studied by size exclusion chromatography (SEC) (Fig. 3f)- aSyn monomers (-15 kDa) present in freshly made aSyn alone solutions eluted at a retention time (tn) of ~21 min corresponding to a globular protein of ~44 kDa; this was due to its natively unfolded nature resulting in a higher hydrodynamic radius (Fig. 3f). In 2e alone (~2 kDa) solutions, the major fraction eluted at ~38 min and corresponded to 2e monomers while a smaller fraction corresponding to 2e oligomers eluted at ~32 min. Importantly, in the aSyn / 2e mixtures, the 21 min peak found in aSyn alone was still present but the 2e alone peaks were strongly diminished (Fig. 3O- These findings were consistent with formation of aSyn / 2e hetero-complexes which eluted at ~2i min and were confirmed by electrospray ionization mass spectrometry (ESI-MS) (Fig. 3f)- The observed lack of a shift of the aSyn peak to higher MWs in the aSyn / 2e mixture was most likely due to the low MW of 2e and the resolution limit of the column. Together, the above studies suggested aSyn / 2e hetero-dimers and low MW hetero-oligomers as early species in the aSyn / 2e co-assembly pathway. Formation of cytotoxic aSyn oligomers is associated with neurodegeneration and PD pathogenesis. The present inventors’ ThT binding and MTT reduction assays suggested that in the presence of the MCIPs formation of cytotoxic assemblies of aSyn was strongly suppressed (Fig. 2a-c). To characterize the effects of MCIPs on formation of aSyn oligomers more directly, kinetics of cytotoxic oligomer formation in aSyn alone and its mixtures with 2e were followed. The inventors used slot blot analysis and the antibody An reported to recognize toxic oligomers of various different proteins including aSyn (Fig. 3g). Formation of cytotoxic aSyn oligomers was confirmed by MTT reduction and TEM (data not shown). In aSyn alone, large amounts of cytotoxic An-reactive oligomers were present in ~48 h-aged solutions (Fig. 3g). By contrast, no An-reactive oligomers and no cytotoxic effects were observed in the aSyn / 2e mixtures (1 / 1) (Fig. 3g, Fig. 2c). The potent inhibitory activity of the MCIPs could also be mediated by binding to faSyn and / or flAPP resulting in suppression of secondary nucleation and / or fibril elongation. In fact, dot blot (DB) assays showed that Fluos-2b and Fluos-2e are able to bind both faSyn and flAPP (Fig. 3I1). However, the non-inhibitor Fluos-4Ala-2b also bound -most likely non-specifically- (Fig. 3I1). In addition, sub-stoichiometric amounts of 2b and 2e did not markedly affect self- / cross-seeded aSyn fibrillogenesis (data not shown). Furthermore, aSyn / 2e(2b) hetero-complexes were unable to become (cross-) seeded by faSyn or flAPP consistent with a key role in MCIPs’ anti-amyloid function (data not shown).

[0173] Collectively, the findings suggested that the inhibitory effects of 2b and 2e are mainly mediated by nanomolar affinity binding to aSyn monomers and / or prefibrillar species and their sequestration into amorphous, non-cytotoxic, and non-(cross-)seedable aSyn / MCIP co-assemblies.

[0174] Table 2. App. Kas of interactions of Fluos-2b, -2e, and -LAPP with LAPP, aSyn, and the three identified aSyn key segments determined by fluorescence spectroscopic titrations.[a]

[0175] Binding app. Ka (±SD) app. Ka (±SD) pp. Ka (±SD) partner (11M) (2b) (nM) (2e) )1M) (LAPP)

[0176] IAPP 29.6 (±19.3) 46.9 (±33-4) 9-7 (±0.9) ctSyn 17.2 (±2.6) 22.0 (±5.1) 26.7 (±6.0) aSyn(i-i4) 366.2 (±115.8) 461.3 (±47.5) 886.4 (±552.0) aSyn(34-52) 662.8 (±9.4) 504.7 (±171.2) 347.5 (±103.2) aSyn(87-iO5) 72.1 (±20.5) 122.2 (±17.1) 31.9 (±0.6) [a] App. KdS, means (±SD) from 3 binding curves using Na-terminal fluorescein-labeled 2b (Fluos-2b), 2e (Fluos-2e), and IAPP (Fluos-IAPP) (pH 7.4). Fluos-peptides 5 nM except for titrations with aSyn and of Fluos-IAPP with IAPP (data from Yan et al. PNAS (2006)) (Fluos-peptides 1 nM).

[0177] MCIPs Ameliorate aSyn Oligomer-Mediated Synaptic Damage in Mouse Brains ex vivo

[0178] The impairment of hippocampal synaptic long term potentiation (LTP) by aSyn oligomers is believed to be directly linked to neuronal dysfunction in PD. Therefore, to obtain first information about the potential physiological relevance of the in vitro findings, the inventors investigated the effects of the two MCIPs on aSyn oligomer- mediated impairment of hippocampal synaptic LTP in mouse brains ex vivo (Fig. 4). In fact, the electrophysiological studies showed that synaptic LTP damage caused by preformed cytotoxic aSyn oligomers was significantly reduced in the presence of 2b or 2e (Fig. 4).

[0179] Three aSyn Key Regions Mediate its High Affinity Interactions with both the MCIPs and LAPP: Multi-Site Binding Underlies MCIP Anti-Amyloid Function

[0180] To identify the aSyn regions mediating its high affinity interactions with the MCIPs, the inventors incubated synthetic peptide arrays containing aSyn decamers covering its entire sequence and positionally shifted by one residue with Fluos-2e (Fig. 5a). The inventors identified 3 clusters of strong binding decamers: one localized within the N- terminal segment aSyn(i-i4), a 2ndone within aSyn(34-52), and a 3rdone within aSyn(87-iO5) (Fig. 5a). The results of the peptide array studies were confirmed and quantified by fluorescence spectroscopic titrations which revealed nanomolar app. Kas for the interactions of 2e and 2b with all 3 aSyn segments (Fig.5b-d Table 2). These data showed that the high affinity binding of MCIPs to aSyn is mediated via the 3 aSyn regions aSyn(i-i4), aSyn(34-52), and aSyn(87-iO5).

[0181] Because MCIPs might mimic IAPP sites mediating its cross-interactions with aSyn, the inventors hypothesized that they might interact with the same / similar aSyn regions as IAPP which could underlie their potent inhibitory activity on lAPP-mediated crossseeding. To address this, the aSyn peptide array was incubated with Fluos-IAPP. The inventors identified 3 major binding clusters corresponding to aSyn(i-i3), aSyn(34-46), and aSyn(87-iO4) and a weaker one within the NAC region corresponding to aSyn(68- 8o) (Fig. 5e). Importantly, the 3 major lAPP-binding aSyn regions were nearly identical to the MCIP-binding ones which was consistent with the inventors’ hypothesis. Furthermore, fluorescence spectroscopic titrations confirmed that the 3 major MCIP- binding aSyn segments bind (f)IAPP as well and revealed that the affinities of their interactions with IAPP were very similar to the affinities of their interactions with 2b and 2e (Table 2). Together, the above studies identified segments aSyn(i-i4), aSyn(34- 52), and aSyn(87-iO5) as key sites of the high affinity interactions of aSyn with both the MCIPs and IAPP.

[0182] The present inventors’ findings suggest that MCIPs’ multi-site binding to aSyn blocks interactions underlying aSyn misfolding cytotoxic di- / oligomerization, (self-)seeding, and flAPP-mediated mediated cross-seeding and support the suggestion that multi-site targeting of aSyn could be a key requirement for effective anti-amyloid function. The present inventors’ results also suggest that MCIPs’ ability to mimic IAPP sites mediating lAPP / aSyn cross-interactions accounts for multi-site targeting of aSyn and support the notion that common molecular recognition features of AP, IAPP, and aSyn exist which can be exploited to develop multi-functional anti-amyloid molecules.

[0183] Example 3

[0184] Suppression of aSyn toxicity

[0185] The data of Fig. 7 provide additional support that MCIPs are applicable as anti-amyloid treatments in PD, AD, and their comorbidities. In particular, as shown in Fig. 7a the effects of MCIPs 2b and 2e on aSyn toxicity were also studied using aSyn overexpressing postmitotic dopaminergic Lund human mesencephalic (LUHMES) neurons, a previously developed disease-relevant cell model for the screening of putative modulators of aSyn toxicity. These studies were performed as described under Example 1. aSyn cytotoxicity was quantified by the lactate dehydrogenase (LDH) release assay which measures the release of LDH from the cells as an indicator of cell damage and was performed as under Example 1. Importantly, significant protection of the neurons was found for both peptides providing additional support for the anti-amyloid function of the MCIPs (Fig. 7a). In addition, as AP-mediated cross-seeding of aSyn may play an important role in AD / PD co-pathology the inventors also asked whether 2b and 2e, found to also inhibit A amyloid self-assembly (Spanopoulou et al. ACIE (2018)), may affect this process as well. In fact, ThT binding, TEM, and cell viability studies revealed a full suppression of fAP42-cross-seeding of aSyn in their presence (1 / 1) (Fig. 7b-d). Example 4

[0186] 2E-mediated remodeling of A04o(42) fibrils and cytotoxic oligomers into non-toxic and non-fibrillar assemblies

[0187] The peptides according to the present invention (“MCIPs”) (here exemplarily represented by “2e” which is herein also sometimes referred to as “2E”) are useful for anti-amyloid treatments in PD / AD comorbidities since the data (Fig. 8) provide evidence that 2E remodels or disassembles preformed A04o(42) fibrils into non-toxic and non- fibrillar species as detailed below. The fAP-remodeling function of 2E is of great importance for the observed suppressing effect of 2E on fAP-mediated cross-seeding of aSyn amyloid self-assembly which could be involved in PD pathogenesis.

[0188] It was previously shown that 2E binds AP4o(42) with nanomolar affinity and inhibits its amyloid self-assembly and formation of cell damaging aggregates in vitro (Spanopoulou et al. ACIE (2018)). To investigate the effects of 2E on already formed AP42 fibrils (fAP42), 2E was added to preformed AP42 fibrils. A dramatic time-dependent reduction of ThT binding of fAP42 within 48 h following 2E addition was observed (Figure 8A-B). TEM confirmed the absence of fibrils in the 6-day aged AP42 / 2E mixture and showed that amorphous aggregates were major species (Figure 8C). fAP42 and aged fAP42 / 2E mixtures were then incubated with cultured rat pheochromocytoma cells (PC12). According to the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction assay, 2E addition to IAP42 resulted in a significant reduction of fAP42- mediated cell damage (Figure 8D-E). In addition, to more directly address the effects of 2E on cytotoxic AP42 oligomers, 2E was added to pre-aggregated AP42, i.e. at an early time point of its amyloid self-assembly process (2 h) at which both fibrils and a large amount of cytotoxic (An reactive) AP42 oligomers were already present (Figure 8F-H). The results of the ThT binding assay (Figure 8F), the MTT reduction assay (Figure 8G- H), and TEM (Figure 81) were very similar to the results found following 2E addition to preformed fAP42. Of note, the macrocyclic control peptide 4Ala-2b, in which all 4 IAPP- derived hot spot residues were substituted for Ala, was unable to interfere with AP42 amyloidogenesis (Figure 9A-F). Furthermore, the effects of 2E on preformed AP40 fibrils (fAP4o) were also studied and similar effects to the ones on fAP42 were found (Figure 8J-O). The above results were consistent with 2E being able to remodel or disassemble AP4O(42) fibrils and cytotoxic oligomers into amorphous and non-toxic species. These results provide evidence that the effective anti-amyloid function of the peptides on AP4o(42)-related amyloid self-assembly events according to the present invention, as exemplified by 2E, are mediated at least in part by their ability (a) to slow down ongoing AP 40(42) amyloid self-assembly and (b) to remodel or disassemble already formed fAP4o(42) fibrils and cytotoxic assemblies into less / non-toxic ones.

[0189] Example 5

[0190] A negative control peptide (”4Ala-2b”) is unable to affect or remodel preformed fAB.

[0191] In contrast to peptide 2E, its mutant 4Ala-2b (designed as a negative control peptide) (as described herein). Results are shown in Figure 9: As shown in Figure 9, 4Ala-2b is unable to affect or remodel performed fAp.

[0192] Example 6

[0193] Remodelling of preformed ctSyn fibrils (faSyn) into non-toxic and non- fibrillar species

[0194] The peptides in accordance with the present invention (here represented by “2e”) are useful for anti-amyloid treatments in PD since the data shown in Fig. 10 provide evidence that in addition to slowing down the amyloid self-assembly process of aSyn, 2e is also able to remodel (or disassemble) preformed aSyn fibrils (faSyn) into non-toxic and non- fibrillar species as shown in Fig. 10.

[0195] Conclusion

[0196] The present inventors surprisingly show that macrocyclic peptides of formulae o, oa, 1, 1*, 2a - 2e, 2a* - 2e*, designed to mimic JAPP self- / cross-interaction sites and previously found to be potent inhibitors of amyloid self-assembly of LAPP and / or the amyloid-P peptide (AP) of Alzheimer’s disease (AD), are also nanomolar inhibitors of both self- and lAPP-cross-seeded amyloid self-assembly of aSyn, as exemplified by, in particular, peptides 2b and 2e. The inventors’ results suggest that their anti-amyloid function is mediated by nanomolar affinity interactions with aSyn via three aSyn segments which are identified as key sites of both aSyn self- and its cross-interactions with LAPP. Based on their broad spectrum amyloid inhibitor activity and additional drug-like properties, these macrocyclic peptides are now promising leads for multifunctional anti-amyloid drugs in synucleinopathies, such as PD and PD-related synucleinopathies, as well as T2D, AD, and their comorbidities. In addition, the identified key aSyn segments shall serve as valuable targets for the design of further novel, multi-site targeting molecules as effective anti-amyloids in PD, related synucleinopathies and their comorbidities.

Claims

Claims1. A peptide having an amino acid sequence according to formula o(Formula o) whereinZi and Z2 are selected from the following pairs a) cysteine and cysteine, b) aspartic acid and lysine, or lysine and aspartic acid, c) aspartic acid and ornithine, or ornithine and aspartic acid, d) aspartic acid and 2,4-diaminobutyric acid, or 2,4-diaminobutyric acid and aspartic acid, e) aspartic acid and 2,3-diaminopropionic acid, or 2,3-diaminopropionic acid and aspartic acid, f) glutamic acid and lysine, or lysine and glutamic acid, g) glutamic acid and ornithine, or ornithine and glutamic acid, h) glutamic acid and 2,4-diaminobutyric acid, or 2,4-diaminobutyric acid and glutamic acid, i) glutamic acid and 2,3-diaminopropionic acid, or 2,3-diaminopropionic acid and glutamic acid; with denoting a covalent bond between Zi and Z2, thus providing for a cyclization of the peptide;Xi, X2, X3, X4, X5, X6, and X7 are, independently at each occurrence, selected from glycine, asparagine, valine, histidine, leucine, serine, alanine, and threonine;F is, independently at each occurrence, phenylalanine;L is leucine;U is, independently at each occurrence, selected from arginine, homoarginine, citrulline, ornithine, lysine, and norleucine;G is glycine;I is isoleucine; wherein Zi, Z2, X1-X7, F, L, U, G and I are L-amino acid residues or D-amino acid residues, or some of Zi, Z2, X1-X7, F, L, U, G and I are L-amino acid residues and others are D-amino acid residues; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof; wherein preferably said peptide has an amino acid sequence according to formula oa(Formula oa) whereinZi, Z2, X1-X7, F, L, U, G, I are as defined above, and-methyl,for use in a method of treating, or preventing, or delaying the onset and / or pathogenesis of, a synucleinopathy.

2. The peptide for use according to claim i, having an amino acid sequence according to formula 1(Formula 1) or an amino acid sequence according to formula 1*(Formula 1*) whereinC is cysteine;Xi, X2, X3, X4, X5, X6, and X7 are, independently at each occurrence, selected from glycine, asparagine, valine, histidine, leucine, serine, alanine, and threonine;F is, independently at each occurrence, phenylalanine;L is leucine;R is arginine;G is glycine;I is isoleucine; is a disulfide bond;-methyl;wherein C, X1-X7, F, L, R, G and I are L-amino acid residues or D-amino acid residues, or some of C, X1-X7, F, L, R, G and I are L-amino acid residues and others are D-amino acid residues; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof.

3. The peptide for use according to any of claims 1 and 2, wherein either a) Xi and X4 are asparagine, X2 is valine, X3 is histidine, X4 is glycine, X5 is glycine, X6 and X7 are glycine; b) X1-X7 are glycine, alanine or serine; c) X1-X7 are glycine; d) X1-X3 are glycine, X4 is asparagine, X5 is alanine, X6-X7 are glycine; or e) X1-X3 are glycine, X4 is asparagine, X5 is alanine, X6 is leucine, X7 is serine.

4. The peptide for use according to any of claims 1 and 2, whereinZi, Z2, C, X1-X7, F, L, U, R, G, and I are L-amino acid residues.

5. The peptide for use according to any of claims 1 - 3, wherein R is, at each occurrence, D-arginine, and / or wherein F is, at each occurrence, D-phenylalanine, and / or wherein L is D-leucine, and / or wherein Zi, Z2 and C are D-amino acid residues, and / or wherein I is D-isoleucine or N-methyl-D-isoleucine.

6. The peptide for use according to any of claims 1 -2, having a sequence according to a formula selected from the following formulae 2a - 2e, 2a* - 2e*:(Formula 2c)(Formula 2d)wherein upper case letters represent L-amino acid residues or D-amino acid residues, preferably L-amino acid residues, and lower case letters represent D-amino acid residues.

7. The peptide for use according to claim 6, having a sequence according to a formula selected from 2b, 2e, 2b* and 2e*8. The peptide for use according to any of the foregoing claims, wherein said peptide consists of a sequence according to any of formulae o, oa, 1, 1*, 2a - 2e, 2a* - 2e*, as defined in any of claims 1, 2,6, and 7, respectively, preferably of a sequence according to any of formulae 2b, 2e, 2b* and 2e*, as defined in any of claims 1, 2, 6 and 7.

9. The peptide for us according to any of the foregoing claims, wherein said synucleinopathy is selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy-related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD).

10. The peptide for use according to any of the foregoing claims, wherein said peptide is an amyloid inhibitory peptide that binds to o-synuclein (aSyn), preferably to monomers and / or oligomers and / or fibrils thereof, more preferably with nanomolar affinity.

11. A pharmaceutical composition comprising a peptide according to any of the foregoing claims and a pharmaceutically acceptable excipient, for use in a method of treating, or preventing, or delaying the onset and / or pathogenesis of, a synucleinopathy selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy- related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD).

12. The peptide for use according to any of claims 1 - 10 or the pharmaceutical composition for use according to claim 11, wherein said method comprises administering an effective amount of said peptide or of said composition to a patient in need thereof.

13. The peptide as defined in any of claims 1 - 10 or the pharmaceutical composition as defined in claim 11, for use in a method of diagnosing a synucleinopathy which is selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, mitochondrial membrane protein associated neurodegeneration, and synucleinopathy-related comorbidities, including Parkinson’s disease (PD) / Alzheimer’s disease (AD), and Parkinson’s disease (PD) / type 2 diabetes (T2D), wherein preferably, said synucleinopathy is Parkinson’s disease (PD).14- The peptide or pharmaceutical composition for use according to claim 13, wherein said method comprises administering an effective amount of said peptide or of said composition to a subject to be tested for a synucleinopathy.

15. The peptide or pharmaceutical composition for use according to any of claims 13 - 14, wherein said peptide is linked to or administered together with a suitable reporter molecule that allows detection of o-synuclein (aSyn) and / or amyloid and / or nonamyloid aggregates and / or co-aggregates (e.g. aSyn / IAPP or aSyn / Abeta) and / or fibrils thereof by a suitable detection methodology, such as positron emission tomography (PET), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), and PET-MRI and wherein said subject, after administration of said peptide, is subjected to said suitable detection methodology, such as PET, NMR, MRI, PET-MRI.

16. A kit for the in-vitro or in-vivo detection and / or quantification of o-synuclein (aSyn) and / or amyloid and / or non-amyloid aggregates and / or co-aggregates (e.g. aSyn / IAPP or aSyn / Abeta) and / or fibrils thereof, or for the diagnosis of a synucleinopathy selected from Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy and mitochondrial membrane protein associated neurodegeneration, wherein preferably, said synucleinopathy is Parkinson’s disease (PD), in a patient, said kit comprising the peptide as defined in any of claims 1 - 10, in a freeze-dried form in a suitable container, a buffered solvent in a separate container for reconstitution of said peptide in solution, and, optionally, means to dispense said peptide once reconstituted in solution, such as a syringe or pipette.

17. Use of the peptide as defined in any of claims 1 - 10, in an in-vitro assay, such as an enzyme linked immunosorbent assay (ELISA) or a radioimmuno assay (RIA), for the detection of o-synuclein (aSyn) monomeric or oligomeric aggregates and / or amyloid and / or non-amyloid aggregates and / or co-aggregates (e.g. aSyn / IAPP or aSyn / Abeta) and / or fibrils thereof.

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