WO2019179985A1

WO2019179985A1 - Inhibitors of mint and uses thereof

Info

Publication number: WO2019179985A1
Application number: PCT/EP2019/056783
Authority: WO
Inventors: Kristian STRØMGAARD; Christian Reinhard Otto BARTLING; Linda HAUGAARD-KEDSTRÖM
Original assignee: University Of Copenhagen
Priority date: 2018-03-19
Filing date: 2019-03-19
Publication date: 2019-09-26

Abstract

The present invention relates to compounds with high affinity for the PTB domain of Mint proteins. The compound interacts with Mint, blocking the native protein-protein interactions between Mint and their natural ligands. The invention furthermore relates to the therapeutic use of these compounds in prevention and/or treatment of neurodegenerative diseases, such as cognitive diseases, for example Alzheimer's disease, as well as to the use for diagnosing Alzheimer's disease.

Description

Inhibitors of Mint and uses thereof

Field of invention

The present invention relates to ligands targeting the phosphotyrosine binding (PTB) domain of Munc-18-interacting protein (Mint) and their therapeutic and diagnostic use.

Background

Neurodegenerative diseases are characterized by the loss of neurons in certain regions of the central nervous system. Alzheimer’s disease (AD), the most common form of dementia, is one of these neurodegenerative diseases and distinguished by the formation and accumulation of amyloid plaques (Selkoe, D.J. et al. 2016, Wang, J. et al. 2017). These amyloid plaques are - in part - deposits of the neurotoxic amyloid b (Ab) peptide of which production, aggregation, and accumulation in the brain are believed to be initial steps in the cause of AD (Mullard, A. et al. 2016). Ab is generated through sequential cleavages of the amyloid precursor protein (APP) by b-secretase and y- secretase.

The Mint protein family, Mint1-3, are multidomain scaffolding proteins. Two members, Mintl and Mint2, are predominantly expressed in neurons and are being assigned to key functions in synaptic vesicle exocytosis, protein transport, and synapse formation (Okamoto, M. et al. 1997, Duclos F. et al. 1993, Motodate, R. et al. 2016). Furthermore, Mintl and Mint2 are important for APP processing (McLoughlin, D. M. et al 1996, Miller, C. C. J. et al. 2006). Direct protein-protein interactions (PPIs) of Mintl and Mint2 with APP and the g-secretase subunit presenilin and the hypothesized formation of a trimeric protein complex link these two proteins to Ab formation and AD. PPIs are generally vital for cellular and biochemical processes and hence are promising drug targets.

Multiple studies demonstrate the effect of Mint proteins on Ab formation. Mint proteins are thus potential targets for reducing Ab formation and hence prevention and/or treatment of AD (Ho, A. et al. 2008, Mitchell, C. J. et al. 2009). To date, there is no approved disease-modifying therapy for AD. Summary

This present invention provides compounds which are ligands of Mint proteins. The compounds target the Mint-PTB domain, which is involved in PPI with APP (Figure 1 ) and thus the compounds inhibit the APP-Mint interaction. The Mint-APP complex further interacts with g-secretase, ultimately resulting in Ab formation. Through inhibition of the Mint-APP complexation, Ab formation is inhibited, leading to reduced amyloid plaque formation and subsequent prevention/slowing of the development of AD. Thus, the present invention comprises compounds that target and block the PTB binding sites of Mint proteins and hence further inhibit the formation of APP-Mint protein complex crucial for the formation of Ab.

The present invention is based on a peptide derived from the intracellular binding motif of APP, and developed to significantly increase affinity for Mint2 thus generating a super binding peptide (SBP). The PTB domains of the Mint proteins are highly conserved, and it is therefore highly plausible that the SBP will also bind the PTB domains of Mintl and Mint3.

In one aspect the present invention concerns a compound (Pi) comprising or consisting of at least twelve amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100), wherein:

Xi is selected from the group consisting of L-asparagine (N), D-asparagine (n), L- alanine (A), D-alanine (a), A/-methyl-D-asparagine (N Me-n) and N-methyl-D-alanine (L/Me-a);

X2 is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1 - naphthyl)alanine (1 NAL) and L-phenylalanine (F);

X₃ is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L- lysine (K), L-ornithine (ORN) and L-2,3-diaminopropionic acid (DAP);

X₄ is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W) and L- phenylalanine (F);

X5 is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L- lysine (K), L-ornithine (ORN) and L-2,3-diaminopropionic acid (DAP);

C_Q is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), L-3-(1 -naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL);

X₇ is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (IGL), L-3-(1 -naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and Xe is selected from the group consisting of L-glutamic acid (E) and D-glutamic acid (e); wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), L-3-(1-naphthyl)alanine (1 NAL) and L-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L-phenylalanine (F).

In one aspect, the present invention relates to a compound as described herein for use as a medicament.

In one aspect, the present invention relates to a compound as described herein for use in the prevention and/or the treatment of a neurodegenerative disease, such as for example AD.

In another aspect, the present invention relates to a method of preventing and/or treating a neurodegenerative disease, such as for example AD, comprising administering a therapeutically effective amount of a compound as described herein to an individual in need thereof.

In another aspect, the present invention relates to a method of isolating Mint proteins, the method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with a compound as described herein, thus obtaining a compound:Mint complex; and

c) isolating the Mint protein bound to said compound, thus obtaining pure Mint.

In a further aspect, the present invention relates to a method of diagnosing AD, the method comprising the steps of:

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein, wherein the compound comprises a detectable moiety; and

c) visualizing the localization of compound bound to Mint in b);

wherein the presence and localization of compound bound to Mint in the biological sample is indicative of the presence of amyloid plaques and resulting AD in the individual from which the biological sample is derived. In one embodiment, a method of diagnosing AD is provided, the method comprising the steps of:

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein, wherein the compound comprises a detectable moiety;

c) quantifying the amount of compound bound to Mint in b); and

d) comparing the amount of compound bound to Mint in c), to a control;

wherein an amount of compound bound to Mint in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

Description of Drawings

Figure 1 : Illustration of the proposed mode of action of Mint mediated progression of Ab plaque formation in AD. A. APP is cleaved by g-secretase to liberate the Ab peptide. The g-secretase cleavage is dependent on the formation of a trimeric protein complex of APP, Mint and secretase. The Ab peptide then associates into toxic oligomers and plaques affecting neuron survival. The APP binding site of Mint is located at the PTB domain. B. The strategy of the present invention targets the PTB domain of neuronal scaffold protein Mint, which is involved in the g-secretase cleavage of APP to generate Ab. Binding of a compound of the invention (Mint inhibitor) to the PTB domain, results in inhibition of the protein-protein interaction between Mint and APP, whereby the formation of Ab is blocked and the number of toxic plaques is reduced. The reduction in Ab formation positively affects neuron survival.

Figure 2: Structure of ligand 83 (super binding peptide (SBP), SEQ ID: 83).

Figure 3: Saturation binding affinities ( _d ) measured using isothermal titration calorimetry (ITC) of ligand 83 _d = 30 ± 8 nm (SBP, SEQ ID: 83, A) and of ligand 3 K_d = 2.4 mM (APP^wt peptide, SEQ ID: 3, B). Representative raw heat signatures (top) and integrated molar heat release (bottom) obtained from titrating ligands with recombinant Mint2-PTB.

Figure 4: In vitro plasma stability of ligands 83 (SBP, SEQ ID: 83, A) and ligand 3 (APP^wt peptide, SEQ ID NO: 3, B) compared to control conditions of ligand 3 in PBS (a). Figure 5: In vitro hepatic clearance of ligands 83 (SBP, SEQ ID: 83, A) and ligand 3 (APP^wt peptide, SEQ ID NO: 3, B) compared to control compound Propranolol (a) and control conditions containing no co-factor NADPH (b) and using heat-deactivated ezymes (c).

Figure 6: Representative pull down of Mint2 from neuronal cell lysate using neuronal cells either expressing Mint2 (lanes 1-3) or not expressing Mint2 (lanes 4-5). Pulldown of Mint2 using ligands 85 (C-PEG2-SBP, SEQ ID: 85) and 84 (C-PEG2-APP^wt peptide, SEQ ID: 84) demonstrated that the SBP but not APP^wt peptide was able to pull down Mint2 in lysate from Acre neurons; blotting for GAPDH was performed as loading control. Total experiments n = 3.

Figure 7: Inhibitory effect of ligand 87 (TAT-SBP, SEQ ID: 87) on Ab formation. Ab42 content in neuronal media (MFT neurons, DIV 10) was measured after being exposed to ligands 86 (TAT-APP^wt peptide, SEQ ID: 86) and 87 (TAT-SBP, SEQ ID: 87) for 24 h using an Ab42 ELISA assay kit. Data is normalized to the vehicle (0.5% DMSO). One- way ANOVA was used for statistical analysis (^***P < 0.001 ). Data represent

mean ± S.E.M (n = 4).

Figure 8: Peptide macrocyclization via side-chain to side-chain cyclization. (a)

Schematic representation of a-helix stabilization via H-bonds in a model peptide (b) Side-chain to side-chain cyclization strategy using various chemical linkers between residues R¹ and R⁵ aiming to introduce helicity. (c-g) Structures of lactam-,

all-hydrocarbon-, triazole-, thioether- and perfluoroaryl cyclized peptides.

Figure 9: Cell viability of neuroblastoma cells (SH-SY5Y) incubated with increasing concentrations of compound (a) DMSO control (n = 1 1 ). (b) APPWT peptide (CB407)

(n = 4). (c) Lactam cyclized peptide CB520 (n = 4). (d) RCM cyclized peptide CB499 (n = 4). (e) Triazole cyclized peptide FA017b (n = 4). (f) hCys X-linked peptide FA034 (n = 4). (g) Abi-40 (n = 3). (h) Abi-42 (n = 3). Data obtained after 48 h; presented as % viability relative to 0.25% DMSO (100%; vehicle control = highest DMSO

concentration) and expressed as mean + SD. Figure 10: C-terminal Cys crosslinked cyclic APPWT variants (a) Schematic

representation of cyclic APPWT analogues stapled through thiol-reactive crosslinkers between residues T761 Cys and F765Cys (highlighted in blue). Cyclization site bridging the ⁷⁵⁹NPTY⁷⁶² recognition motif is highlighted in dark gray (b) Structures of the used di-bromo linkers (L1 - L6) and perfluoroaryl linkers (L7 & L8). (c) K_\ values of APPWT analogues FA001 -1 - FA001 -8 measured by FP, best hit FA001 -5 highlighted in blue; data expressed as mean + SEM (n = 3).

Detailed description

Definitions

The term“albumin binding moiety” refers to a moiety capable of binding to albumin, i.e. having albumin binding affinity.

The term “Alzheimer’s disease” (AD) refers to a neurodegenerative disorder characterized by progressive dementia, loss of cognitive abilities, and deposition of fibrillar amyloid proteins as intraneuronal neurofibrillary tangles, extracellular amyloid plaques and vascular amyloid deposits. The major constituents of these plaques are neurotoxic amyloid-beta proteins, that are produced by the proteolysis of the APP protein.

The term“APP” refers to the protein amyloid precursor protein or amyloid-beta A4 protein (Uniprot: P05067).

Proteinogenic“amino acids” (AA) are named herein using either their 1 -letter or 3-letter code according to the recommendations from IUPAC, see for example http://www.chem.qmul.ac.uk/iupac/AminoAcid/. Capital letter abbreviations indicate L- amino acids, whereas lower case letter abbreviations indicate D-amino acids.

The term“amyloid-beta” (amyloid b or Ab) refers to peptides of 36-43 amino acids that are involved in AD as the main component of the amyloid plaques found in brains of AD patients. Amyloid-beta is generated by cleavage of APP by secretases. The term“amyloid plaque” refers to aggregates of peptides or proteins which aggregate due to improper processing or folding to form amyloid fibrils. Amyloid plaques have been linked to the development of various diseases, such as for example Alzheimer’s disease wherein APP has been improperly processed to form Ab peptides which aggregates into amyloid plaques.

The term“cognitive disorder”, also known as neurocognitive disorder (NCD), refers to a mental health disorders that primarily affect cognitive abilities including learning, memory, perception, and problem solving. Cognitive disorders are defined by deficits in cognitive ability that are acquired, and may have an underlying brain pathology.

The term“cell penetrating peptide” (CPP) refers to a peptide characterised by the ability to cross the plasma membrane of mammalian cells, and thereby ability to facilitate the intracellular delivery of cargo molecules, such as peptides, proteins or oligonucleotides to which it is linked.

The term“detectable moiety” refers to a a moiety which can be detected by analytical means. A detectable moiety may be selected from the group consisting of fluorophores, radiocontrasts, MRI contrast agents and radioisotopes.

The term“D-SynB3 peptide” refers to a CPP peptide having the sequence frrrsyslrr (SEQ ID NO: 96).

The term“effective amount”, as used herein, refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.

The term “Kd” refers to a dissociation constant and is a measure of the affinity of a molecule for another molecule. The lower the K_d, the higher the affinity of a peptide for its binding site.

The term“ ” refers to a inhibitory constant of a protein-inhibitor complex. The lower the K, the higher the affinity of a peptide for its binding site. The term“linker” refers to a molecular moiety which is capable of binding two molecules to one another. The linker may be of varying length and structure. The linker may be a non-degradable linkage or a linkage containing an intracellularly degradable bond, e.g. a disulphide bond. Potentially, the degradable linker is designed as a self-immolative linker (SIL) capable of releasing the native compound upon degradation.

The term “mini-AP4 peptide” refers to a CPP peptide having the sequence c[DLATEPAK(DAP)] (SEQ ID NO: 97), wherein c indicates side-chain to side-chain cyclization.

The term“Mint” refers to the protein family Mund 8-interactions protein family. The protein family consist of the proteins Mintl , Mint2 and Mint3.

The term“Mintl” refers to the protein Mund 8-interaction protein 1 /Amyloid-beta A4 precursor protein-binding family A member 1 , and may be human Mintl (Uniprot: Q02410).

The term“Mint2” refers to the protein Mund 8-interaction protein 2/Amyloid-beta A4 precursor protein-binding family A member 2, and may be human Mint2 (Uniprot: Q99767).

The term“Mint3” refers to the protein Mund 8-interaction protein 3/Amyloid-beta A4 precursor protein-binding family A member 3, and may be human Mint3 (Uniprot: 096018).

The term“mixed TAT peptide” refers to a CPP peptide having the sequence rRrGrKkRr (SEQ ID NO: 94).

The term“neurodegenerative disease” refers to a disease caused by progressive loss of structure or function of neurons, including death of neurons. This causes problems with movement (called ataxias), or mental functioning (called dementias). Alzheimer’s disease is an example of a neurodegenerative disease. The term“non-canonical amino acids”, also referred to as non-coded, non-standard, non-cognate, unnatural or non-natural amino acids, are amino acids, as used herein which are not encoded by the genetic code. Examples are 2-indanyl- L-glycine (IGL), L- 3-(1 -naphthyl)alanine (1 NAL), i_-3-(2-naphthyl)alanine (2NAL), L-ornithine (ORN) and L- 2,4-diaminobutyric acid (DAB). A list of non-canonical amino acids including their structures can be found in Example 1 1 .

The term“L/PEG”, is a linker derived from the classical polyethylene glycol (PEG) moiety, but where one or more of the backbone oxygen atoms is replaced with a nitrogen atom.

The term “PARM” refers to a protein fragment of the Mint2 protein consisting of its phosphotyrosine-binding (PTB) domain and the a-helical linker (ARM) domain.

The term “penetratin peptide” refers to a CPP peptide having the sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 98).

The term “polyArg peptide” refers to a CPP peptide having the sequence RRRRRRRRR (SEQ ID NO: 95). The number of arginine residues in the sequence may be varied.

The term“presenilin” refers to the protein g-secretase subunit PEN-1 and/or PEN-2 essential for intermembrane cleavage of integral membrane proteins such as APP (Uniprot: P49768 (PEN-1 ) and Q9NZ42 (PEN-2)).

The term “PTB” refers to phosphotyrosine-binding (PTB) domain, also known as phosphotyrosine-interacting domain (PID or Pi-domain).

The term “reactive group” refers to a chemical entity, which comprises a reactive functional group, which is capable of reacting with and forming a bond with a second chemical entity. The reactive group may be a nucleophilic group or an electrophilic group. As exemplified in the present invention, the reactive group may be a cysteine, which is capable of reacting with an electrophilic second chemical entity, thereby linking the compound to said second chemical entity. The term “retroinverso” refers to peptides that are composed of D-amino acids assembled in the reverse order from that of the parent L-amino acid sequence.

The term “streptavidin binding moiety” refers to a moiety capable of binding to streptavidin, i.e. having streptavidin binding affinity. One example of a streptavidin binding moiety is biotin.

The term “TAT peptide” refers to an 1 1-mer CPP peptide having the sequence YGRKKRRQRRR (SEQ ID NO: 93) derived from the human immunodeficiency virus- type 1 (HIV-1 ) Tat protein, which facilitates permeability across biological membranes, including the blood-brain barrier.

Mint proteins and amyloid precursor protein

Processing of amyloid precursor protein (APP) by secretases result in Ab formation and subsequent amyloid plaque formation. This aggregation of Ab is toxic to cells and leads to neuron degradation and/or death and resulting disease. Amyloid plaque formation is involved in neurodegenerative diseases such as for example cognitive diseases, for example AD.

The Mint protein family, Mint1-3, are multidomain scaffolding proteins. Two members, Mintl and Mint2, are predominantly expressed in neurons and are being assigned to key functions in synaptic vesicle exocytosis, protein transport, and synapse formation. Furthermore, Mintl and Mint2 have been identified as being important for APP processing. The PTB-domains of Mintl and Mint2 binds APP via PPI. The Mintl -APP and Mint2-APP complexes further interact with g-secretase, ultimately resulting in cleavage of APP and Ab formation.

Multiple studies demonstrate the effect of Mint proteins on Ab formation. Mint proteins are thus potential targets for reducing Ab formation and subsequent prevention and/or treatment of AD. Through inhibition of the Mint-APP complexation, Ab formation is inhibited, leading to reduced amyloid plaque formation and thus prevention/slowing of the development of AD. Thus, in one aspect, the present invention provides a compound as described herein which is capable of preventing the PPI of Mint proteins and APP and thereby inhibit Ab formation.

In one embodiment, the present invention provides a compound as described herein which is capable of preventing the PPI of Mintl and/or Mint2 and APP and thereby inhibit Ab formation.

In one embodiment, the present invention provides a compound as described herein which is capable of preventing the PPI of Mint2 and APP and thereby inhibit Ab formation.

The compound of the invention may be used for treatment of neurodegenarative diseases, such as cognitive diseases, for example AD.

Inhibition of Mint proteins

In one embodiment, the present invention provides a compound as described herein which is capable of binding to Mint proteins.

In one embodiment, the present invention provides a compound as described herein which is capable of binding to human Mint proteins.

More specifically, in one embodiment, the present invention provides a compound as described herein which bind to the PTB-domain of Mint. Such binding may result in inhibition of the PPI of Mint and APP.

In one embodiment, the present invention provides a compound as described herein which inhibit the Mint-APP, Mint-y-secretase and/or Mint-APP-y-secretase interaction.

In one embodiment, the present invention provides a compound as described herein which inhibit the Mint-APP interaction.

In one embodiment, the compound as described herein has a K_d for Mint below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

In one embodiment, the compound as described herein has a K, for Mint below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

In one embodiment, the present invention provides a compound as described herein which is capable of binding to Mintl and/or Mint2.

In one embodiment, the present invention provides a compound as described herein which is capable of binding to human Mintl (Uniprot: Q02410) and/or Mint2 (Uniprot: Q99767).

More specifically, in one embodiment, the present invention provides a compound as described herein which bind to the PTB-domain of Mintl and/or Mint2. Such binding may result in inhibition of the PPI of Mintl and/or Mint2 and APP.

In one embodiment, the present invention provides a compound as described herein which inhibit the Mintl -APP, Mintl -g-secretase, Mintl -ARR-g-secretase, Mint2-APP, Mint2-y-secretase and/or Mint2-APP-y-secretase interaction.

In one embodiment, the present invention provides a compound as described herein which inhibit the Mintl -APP and/or Mint2-APP interaction.

In one embodiment, the compound as described herein has a K_d for Mintl and/or Mint2 below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

In one embodiment, the compound as described herein has a K, for Mintl and/or Mint2 below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM. In one embodiment, the present invention provides a compound as described herein which is capable of binding to Mint2.

In one embodiment, the present invention provides a compound as described herein which is capable of binding to human Mint2 (Uniprot: Q99767).

More specifically, in one embodiment, the present invention provides a compound as described herein which bind to the PTB-domain of Mint2. Such binding may result in inhibition of the PPI of Mint2 and APP.

In one embodiment, the present invention provides a compound as described herein which inhibit the Mint2-APP, Mint2-y-secretase and/or Mint2-APP-y-secretase interaction.

In one embodiment, the present invention provides a compound as described herein which inhibit the Mint2-APP interaction.

In one embodiment, the compound as described herein has a K_d for Mint2 below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

In one embodiment, the compound as described herein has a K, for Mint2 below 1 pM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

Mint Inhibitors

The present invention provides a compound as described below.

In one embodiment, the compound as described herein is a peptide or peptide analogue. In one embodiment, compound (Pi) is provided comprising at least twelve amide- bonded proteinogenic or non-proteinogenic amino acid residues of the sequence X1GX2X3NPOX4X5X6X7X8 (SEQ ID NO: 248), wherein:

Xi is selected from the group consisting of L-asparagine (N), D-histidine (h), L-histidine (H), D-asparagine (n), L-alanine (A), D- alanine (a), /V-methyl-L-asparagine (L/Me-N), /V-methyl-d-asparagine (L/Me-n), L-arginine (R), /V-methyl-L-alanine (L/Me-A), and N- methyl-D-alanine (L/Me-a);

X2 is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1 - naphthyl)alanine (1 Nal), 2-indanyl-L-glycine (Igl), (S)-2-amino-3-(3,4- dichlorophenyl)propionic acid (Phe-3,4-Cl2), 2,4-dichloro-L-phenylalanine (Phe-2,4-Cl2), 4-fluoro-L-phenylalanine (Phe-4-F), 3-(3-pyridyl)- L-alanine (3Pal), 3-(4-pyridyl)-L- alanine (4Pal) L-phenylalanine (F), L-homophenylalanine (hPhe), L-3-(2- naphthyl)alanine (2Nal), 2-fluoro-L-phenylalanine (Phe-2-F), 3-fluoro-L-phenylalanine (Phe-3-F), 3,4-difluoro-L-phenylalanine (Phe-3,4-F₂), 2-chloro-L-phenylalanine (Phe-2- Cl), 3-chloro-L-phenylalanine (Phe-3-CI), 3-iodo-L-phenylalanine (Phe-3-l) and 4-iodo- L-phenylalanine (Phe-4-l);

X₃ is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L- lysine (K), L-ornithine (Orn), (S)-2-amino-heptanedioic acid (Api), L-homoglutamine (hGIn), (S)-2-aminoadipic acid (Aad), L-citrulline (Cit), (R)-2-amino-3-(3-carboxy- propylsulfanyl)propionic acid (Cpc), 4-(((R)-2-amino-2-carboxyethyl)-sulfinyl)butanoic acid (Cpc^ox), L-cysteine (C), L-fe/f-leucine (Tie), (S)-2-cyclopentylglycine (Cpg), (S)-2- cyclohexylglycine (Chg), and L-isoleucine (l),L-2,3-diaminopropionic acid (Dap) and L- 2,4-diaminobutyric acid (Dab);

O is selected from the group consisting of: L-threonine (T), L-lysine (K), L-glutamic acid (E), L-ornithine (Orn), L-aspartic acid (D), L-2,4-diaminobutyric acid (Dab), (R)-2-(4- pentenyl)alanine (R5), (S)-2-(4-pentenyl)alanine (S5), (S)-2-(7-octenyl)alanine (Ss), 6- azido-L-lysine (Lys^a), propargylglycine (Pra), 5-azido-L-ornithine (Orn^a), L- homopropargylglycine (hPra), L-cysteine (C), D-cysteine (c), L-2,3-diaminopropionic acid (Dap), and L-homocysteine (hC);

X₄ is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), (S)-2-(4- pentenyl)alanine (Ss), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal) and L-phenylalanine (F);

X₅ is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L- lysine (K), L-ornithine (Orn), L-arginine (R), L-homoglutamine (hGIn), L-homoarginine (hArg), L-homoglutamine (hLys), (S)-2-aminoadipic acid (Aad), (S)-2-amino- heptanedioic acid (Api), L-citrulline (Cit), and L-2,3-diaminopropionic acid (Dap), L-2,4- diaminobutyric acid (Dab), (S)-2-amino-4-guanidinobutanoic acid (Arb), (S)-2-amino-3- guanidinopropanoic acid (Arp) and L-glutamine(Q);

C_Q is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (Igl), L-3-(1-naphthyl)alanine (1 Nal), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L- alanine (4Pal), (S)-2-amino-3-(3-iodophenyl)propanoic acid (Phe-3-l), 2-amino-3-(3- chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L-phenylalanine (Phe-2,4-Cl2), (S)-3-(3-tert-butyl-phenyl)-2-amino-propionic acid (Phe-3-tBu), (S)-3-(4-tert-butyl- phenyl)-2-amino-propionic acid (Phe-4-tBu), (S)-2-(4-pentenyl)alanine (S₅), L-histidine (H), 4-fluoro-L-phenylalanine (Phe-4-F), L-homophenylalanine (hPhe), and L-3-(2- naphthyl)alanine (2Nal);

X₇ is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (Igl), L-3-(1-naphthyl)alanine (1 Nal), L-glutamic acid (E), L-lysine (K), L-aspartic acid (D), L-ornithine (Orn), L-2,4-diaminobutyric acid (Dab), (S)-2-(4-pentenyl)alanine (S₅), (S)-2-(7-octenyl)alanine (Ss), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal), (S)-2-amino-3-(3-iodophenyl)propanoic acid (Phe-3-l), 2-amino-3-(3- chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L-phenylalanine (Phe-2,4-Cl2), (S)-3-(3-tert-butyl-phenyl)-2-amino-propionic acid (Phe-3-tBu), propargylglycine (Pra), 6-azido-L-lysine (Lys^a), L-homopropargylglycine (hPra), 5-azido-L-ornithine (Orn^a), L- cysteine (C), D-cysteine (c), L-homocysteine (hC), L-3-(2-naphthyl)alanine (2Nal), L- homophenylalanine (hPhe), (S)-2-amino-3-(3,4-dichlorophenyl)propionic acid (Phe-3,4- CI₂), 3,4-difluoro-L-phenylalanine (Phe-3,4-F₂) and 4-fluoro-L-phenylalanine (Phe-4-F); and

Xs is selected from the group consisting of L-glutamic acid (E), D-glutamic acid (e), (S)- 2-aminoadipic acid (Aad), (S)-2-amino-heptanedioic acid (Api), (S)-2-(4- pentenyl)alanine (Ss), L-alanine (A), D-glutamine (q), L-homoglutamine (hGIn), L- citrulline (Cit), and L-glutamine (Q); wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), L-3-(1 -naphthyl)alanine (1 Nal) and l-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L-phenylalanine (F).

The compound (Pi) as defined herein comprises at least twelve amide-bonded amino acid residues, which can be referred to by a number from 755 to 766 corresponding to their position in wild type amyloid precursor protein (APP).

In one embodiment, the compound (Pi) is of the sequence X1GX2X3NPOX4X5X6X7X8 (SEQ ID NO: 249), wherein:

Xi is selected from the group consisting of L-asparagine (N), D-histidine (h), L-alanine (A), L-histidine (H), D-alanine (a), /V-methyl-L-asparagine (L/Me-N), /V-methyl-D-alanine (N Me-a), /V-methyl-L-alanine (L/Me-A), and L-arginine (R);

X2 is selected from the group consisting of L-tyrosine (Y), L-3-(1 -naphthyl)alanine (1 Nal), 2-indanyl-L-glycine (Igl), (S)-2-amino-3-(3,4-dichlorophenyl)propionic acid (Phe- 3,4-C ), 2,4-dichloro-L-phenylalanine (Phe-2,4-CI2), 4-fluoro-L-phenylalanine (Phe-4- F), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal), L-phenylalanine (F), L- homophenylalanine (hPhe), L-3-(2-naphthyl)alanine (2Nal), 2-fluoro-L-phenylalanine (Phe-2-F), 3-fluoro-L-phenylalanine (Phe-3-F), 3,4-difluoro-L-phenylalanine (Phe-3,4- F₂), 2-chloro-L-phenylalanine (Phe-2-CI), 3-chloro-L-phenylalanine (Phe-3-CI), 3-iodo- L-phenylalanine (Phe-3-l) and 4-iodo-L-phenylalanine (Phe-4-l);

X3 is selected from the group consisting of L-glutamic acid (E), (S)-2-amino- heptanedioic acid (Api), L-homoglutamine (hGIn), (S)-2-aminoadipic acid (Aad), L- citrulline (Cit), (R)-2-amino-3-(3-carboxy-propylsulfanyl)propionic acid (Cpc),4-(((R)-2- amino-2-carboxyethyl)-sulfinyl)butanoic acid (Cpc^ox), L-cysteine (C), L-fe/f-leucine (Tie), (S)-2-cyclopentylglycine (Cpg), (S)-2-cyclohexylglycine (Chg), L-isoleucine (I), and L-2,4-diaminobutyric acid (Dab);

O is selected from the group consisting of: L-threonine (T), L-lysine (K), L-glutamic acid (E), L-ornithine (Orn), L-aspartic acid (D), L-2,4-diaminobutyric acid (Dab), (R)-2-(4- pentenyl)alanine (Rs), (S)-2-(4-pentenyl)alanine (Ss), (S)-2-(7-octenyl)alanine (Ss), 6- azido-L-lysine (Lys^a), propargylglycine (Pra), 5-azido-L-ornithine (Orn^a), L- homopropargylglycine (hPra), L-cysteine (C), D-cysteine (c), L-2,3-diaminopropionic acid (Dap), and L-homocysteine (hC);

X₄ is selected from the group consisting of: L-tyrosine (Y), (S)-2-(4-pentenyl)alanine (Ss), L-phenylalanine (F), 3-(3-pyridyl)-L-alanine (3Pal), and 3-(4-pyridyl)-L-alanine (4Pal);

X₅ is selected from the group consisting of L-lysine (K), L-arginine (R), L- homoglutamine (hGIn), L-homoarginine (hArg), L-homoglutamine (hLys), (S)-2- aminoadipic acid (Aad), (S)-2-amino-heptanedioic acid (Api), L-citrulline (Cit), L-2,4- diaminobutyric acid (Dab), (S)-2-amino-4-guanidinobutanoic acid (Arb), (S)-2-amino-3- guanidinopropanoic acid (Arp) and L-glutamine(Q);

C_Q is selected from the group consisting of: L-phenylalanine (F), (S)-2-(4- pentenyl)alanine (S₅), L-histidine (H), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L- alanine (4Pal), (S)-2-amino-3-(3-iodophenyl)propanoic acid (Phe-3-l), 2-amino-3-(3- chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L-phenylalanine (Phe-2,4-Cl2), (S)-3-(3-tert-butyl-phenyl)-2-amino-propionic acid (Phe-3-tBu), (S)-3-(4-tert-butyl- phenyl)-2-amino-propionic acid (Phe-4-tBu), (S)-2-(4-pentenyl)alanine (S₅), L-histidine (H), L-3-(1-naphthyl)alanine (1 Nal), L-homophenylalanine (hPhe), and 4-fluoro-L- phenylalanine (Phe-4-F);

X₇ is selected from the group consisting of L-phenylalanine (F), L-glutamic acid (E), L- lysine (K), L-aspartic acid (D), L-ornithine (Orn), L-2,4-diaminobutyric acid (Dab), (S)-2- (4-pentenyl)alanine (S₅), (S)-2-(7-octenyl)alanine (Ss), l-3-(1-naphthyl)alanine (1 Nal), 3- (3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal), (S)-2-amino-3-(3- iodophenyl)propanoic acid (Phe-3-l), 2-amino-3-(3-chlorophenyl)propanoic acid (Phe-3- Cl), 2,4-dichloro-L-phenylalanine (Phe-2,4-Cl2), (S)-3-(3-tert-butyl-phenyl)-2-amino- propionic acid (Phe-3-tBu), propargylglycine (Pra), 6-azido-L-lysine (Lys^a), L- homopropargylglycine (hPra), 5-azido-L-ornithine (Orn^a), L-cysteine (C), D-cysteine (c), L-homophenylalanine (hPhe), (S)-2-amino-3-(3,4-dichlorophenyl)propionic acid (Phe- 3,4-C ), 3,4-difluoro-L-phenylalanine (Phe-3,4-F₂), and 4-fluoro-L-phenylalanine (Phe- 4-F), L-homocysteine (hC); and

Xe is selected from the group consisting of: L-glutamic acid (E), D-glutamic acid (e), (S)-2-aminoadipic acid (Aad), (S)-2-amino-heptanedioic acid (Api), (S)-2-(4- pentenyl)alanine (Ss), L-alanine (A), D-glutamine (q), L-homoglutamine (hGIn), L- citrulline (Cit), and L-glutamine (Q); wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of l-tryptophan (W), l-3-(1-naphthyl)alanine (1 Nal) and l-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L-phenylalanine (F).

In one embodiment, the compound (Pi) according to the present disclosure has the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 247.

In one embodiment, the compound (Pi) is provided, wherein (Pi) is cyclized between at least two of the side chains via crosslinking of thiols using any one of linkers (1 ) to (8).

In one embodiment, the compound (Pi) is cyclized between at least two of the side chains via copper(l)-catalyzed azide alkyne cycloaddition (CuAAC).

In one embodiment, the compound (Pi) is cyclized between at least two of the side chains via an amide bond.

In one embodiment, the compound (Pi) has an acyl or alkyl on the N-terminus. In one embodiment, the acyl is acetyl. In one embodiment, the alkyl is methyl. In one embodiment, the alkyl is selected from the group consisting of methyl, ethyl propyl, butyl, pentyl, iso-propyl, and iso-butyl.

In one embodiment, the proteolytic stability determined as half-life time (T1_/2) using (Trypsin) and/or (Proteinase K) of the compound (Pi) is above 120 min.

In one embodiment, a compound (Pi) comprising at least twelve amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein: Xi is selected from the group consisting of hydrophobic amino acids, b-branched amino acids and N-methylated amino acids;

X₂ is selected from the group consisting of polar amino acids and hydrophobic amino acids;

X₃ is an amino acid comprising a side chain carboxylic acid or amine;

X₄ is selected from the group consisting of polar amino acids and hydrophobic amino acids;

X5 is an amino acid comprising a side chain carboxylic acid or amine;

C_Q is a hydrophobic amino acids;

X₇ is a hydrophobic amino acids; and

Xe is an acidic amino acid;

wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), i_-3-(1-naphthyl)alanine (1 NAL) and L-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L-phenylalanine (F).

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein:

Xi is selected from the group consisting of hydrophobic amino acids, b-branched amino acids and N-methylated amino acids;

X2 is selected from the group consisting of polar amino acids and hydrophobic amino acids;

X₃ is an amino acid comprising a side chain carboxylic acid or amine;

X5 is an amino acid comprising a side chain carboxylic acid or amine;

C_Q is a hydrophobic amino acids;

X₇ is a hydrophobic amino acids; and

Xe is an acidic amino acid;

with the proviso that Pi does not comprise the sequence of YENPTY.

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein: Xi is selected from the group consisting of L-asparagine (N), D-asparagine (n), L- alanine (A), D-alanine (a), A/-methyl-D-asparagine (N Me-n) and A/-methyl-D-alanine (L/Me-a);

X₂ is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1- naphthyl)alanine (1 NAL) and L-phenylalanine (F);

C_Q is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), L-3-(1-naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL);

X₇ is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), L-3-(1-naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and Xe is selected from the group consisting of L-glutamic acid (E) and D-glutamic acid (e);

wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), L-3-(1-naphthyl)alanine (1 NAL) and L-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L-phenylalanine (F).

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein:

Xi is selected from the group consisting of L-asparagine (N), D-asparagine (n), L- alanine (A), D-alanine (a), A/-methyl-D-asparagine (N Me-n) and A/-methyl- D-alanine (L/Me-a);

X2 is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1- naphthyl)alanine (1 NAL) and L-phenylalanine (F);

X5 is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L- lysine (K), L-ornithine (ORN) and L-2,3-diaminopropionic acid (DAP); C_Q is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), i_-3-(1-naphthyl)alanine (1 NAL) and i_-3-(2-naphthyl)alanine (2NAL);

X7 is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), L-3-(1-naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and Xe is selected from the group consisting of L-glutamic acid (E) and D-glutamic acid (e);

with the proviso that Pi does not comprise the sequence of YENPTY.

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein:

X2 is selected from the group consisting of L-tryptophan (W), L-3-(1-naphthyl)alanine (1 NAL) and L-phenylalanine (F);

X₇ is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), L-3-(1-naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and Xe is selected from the group consisting of L-glutamic acid (E) and D-glutamic acid (e).

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein:

Xi is selected from the group consisting of L-asparagine (N), D-asparagine (n), L- alanine (A), D-alanine (a), A/-methyl-D-asparagine (N Me-n) and A/-methyl- D-alanine (L/Me-a); X₂ is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1- naphthyl)alanine (1 NAL) and L-phenylalanine (F);

X₃ is selected from the group consisting of L-aspartic acid (D), L-lysine (K), L- ornithine (ORN) and L-2,3-diaminopropionic acid (DAP);

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein:

Xi is selected from the group consisting of L-asparagine (N), D-asparagine (n), L- alanine (A), D- alanine (a), A/-methyl-D-asparagine (N Me-n) and A/-methyl- D-alanine (L/Me-a);

X4 is selected from the group consisting of L-tryptophan (W) and L-phenylalanine (F); X5 is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L- lysine (K), L-ornithine (ORN) and L-2,3-diaminopropionic acid (DAP);

X₇ is selected from the group consisting of L-phenylalanine (F), 2-indanyl- L-glycine (IGL), L-3-(1-naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and Xe is selected from the group consisting of L-glutamic acid (E) and D-glutamic acid (e). In one embodiment, a compound (Pi) comprising at least twelve amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100) is provided, wherein:

Xi is selected from the group consisting of /V-methyl-D-asparagine (L/Me-n) and N- methyl-D-alanine (L/Me-a);

X2 is selected from the group consisting of L-tyrosine (Y), and L-3-(1 - naphthyl)alanine (1 NAL);

X₃ is selected from the group consisting of L-lysine (K) and L-ornithine (ORN);

X₄ is selected from the group consisting of L-tyrosine (Y) and L-phenylalanine (F);

X5 is selected from the group consisting of L-glutamic acid (E) and L-aspartic acid

(D);

C_Q is selected from the group consisting of L-phenylalanine (F), L-3-(1- naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL);

X₇ is selected from the group consisting of L-phenylalanine (F), L-3-(1- naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and

Xs is D-glutamic acid (e).

In one embodiment, the compound (Pi) is cyclized between the side chains of X₃ and X₅. Said cyclization may be via an amide bond formed between a carboxylic acid of one side chain and an amine of the other side chain. The side chains of X₃ may comprise the carboxylic acid and the side chain of X₅ may comprise the amine. Alternatively, the side chains of X₃ may comprise the amine and the side chain of X₅ may comprise the carboxylic acid.

In some embodiments, the compound comprises between 12 and 40 amino acid residues, for example between 15 and 35 amino acid residues, such as between 20 and 30 amino acid residues, for example between 20 and 25 amino acid residues.

In some embodiments, the compound comprises between 12 and 40 amino acid residues, for example between 12 and 35 amino acid residues, such as between 12 and 30 amino acid residues, for example between 12 and 25 amino acid residues, such as between 12 and 20 amino acid residues, for example between 12 and 15 amino acid residues. In some embodiments, the compound comprises between 12 and 40 amino acid residues, for example between 15 and 40 amino acid residues, such as between 20 and 40 amino acid residues, for example between 25 and 40 amino acid residues, such as between 30 and 40 amino acid residues, for example between 35 and 40 amino acid residues.

In some embodiment, the compound comprises at least 12 amino acids residues, such as at least 13 amino acid residues, such as at least 14 amino acid residues, such as at least 15 amino acid residues, such as at least 16 amino acid residues, such as at least 17 amino acid residues, such as at least 18 amino acid residues, such as at least 19 amino acid residues, such as at least 20 amino acid residues, such as at least 21 amino acid residues, such as at least 22 amino acid residues, such as at least 23 amino acid residues, such as at least 24 amino acid residues, such as at least 25 amino acid residues, such as at least 26 amino acid residues, such as at least 27 amino acid residues, such as at least 28 amino acid residues, such as at least 29 amino acid residues, such as at least 30 amino acid residues, such as at least 31 amino acid residues, such as at least 32 amino acid residues, such as at least 33 amino acid residues, such as at least 34 amino acid residues, such as at least 35 amino acid residues, such as at least 36 amino acid residues, such as at least 37 amino acid residues, such as at least 38 amino acid residues, such as at least 39 amino acid residues, such as at least 40 amino acid residues.

In some embodiments, the compound comprises no more than 40 amino acid residues, such as no more than 39 amino acid residues, such as no more than 38 amino acid residues, such as no more than 37 amino acid residues, such as no more than 36 amino acid residues, such as no more than 35 amino acid residues, such as no more than 34 amino acid residues, such as no more than 33 amino acid residues, such as no more than 32 amino acid residues, such as no more than 31 amino acid residues, such as no more than 30 amino acid residues, such as no more than 29 amino acid residues, such as no more than 28 amino acid residues, such as no more than 27 amino acid residues, such as no more than 26 amino acid residues, such as no more than 25 amino acid residues, such as no more than 24 amino acid residues, such as no more than 23 amino acid residues, such as no more than 22 amino acid residues, such as no more than 21 amino acid residues, such as no more than 20 amino acid residues, such as no more than 19 amino acid residues, such as no more than 18 amino acid residues, such as no more than 17 amino acid residues, such as no more than 16 amino acid residues, such as no more than 15 amino acid residues, such as no more than 14 amino acid residues, such as no more than 13 amino acid residues, such as no more than 12 amino acid residues.

In one embodiment, the compound is (/VMe-a)GYc[(ORN)NPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 101 ).

In one embodiment, the compound is (/VMe-a)GYc[KNPTYE](1 NAL)(1 NAL)e (SEQ ID NO: 102).

In one embodiment, the compound is (/VMe-a)GYc[(ORN)NPTYE](1 NAL)(1 NAL)e (SEQ ID NO: 103).

In one embodiment, the compound is (/VMe-a)GYc[KNPTYD](2NAL)(2NAL)e (SEQ ID NO: 104).

In one embodiment, the compound is (/VMe-n)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 105).

In one embodiment, the compound is (/VMe-a)GYc[KNPTFD](1 NAL)(1 NAL)e (SEQ ID NO: 106).

In one embodiment, the compound is (/VMe-a)G(1 NAL)c[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 107).

In one embodiment, the compound is (/VMe-a)GYc[KNPTYD](1 NAL)Fe (SEQ ID NO: 108).

In one embodiment, the compound is (/VMe-a)GYc[KNPTYD]F(1 NAL)e (SEQ ID NO: 109).

In one embodiment, the compound is (/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 83). This compound is also referred to herein as super binding peptide or SBP. In one embodiment, the compound as described herein, further comprises one or more conjugated moieties.

In one embodiment, the one or more conjugated moieties is selected from the group consisting of a cell penetrating peptide (CPP), an albumin binding moiety, a detectable moiety, a streptavidin binding moiety, a reactive group and/or a linker (L).

In one embodiment, the compound as described herein has the generic structure of Formula I:

Z - L - Pi (I), wherein Z is selected from the group consisting of a CPP, an albumin binding moiety, a detectable moiety, a streptavidin binding moiety and a reactive group and L is an optional linker.

In one embodiment, the compound as described herein has the generic structure of Formula II:

Z - Ri (II),

wherein Z is selected from the group consisting of a CPP, an albumin binding moiety, a detectable moiety, a streptavidin binding moiety and a reactive group.

The one or more conjugated moieties may be attached to the N- or C-terminal end of the compound as described herein. Aternatively, the one or more conjugated moieties may be attached to the compound as described herein via an amino acid side chain.

Attachment of an albumin binding moiety may provide increased circulation time of the compound. Examples of albumin binding moieties include but are not limited to aliphatic chains and peptides.

Attachment of a streptavidin binding moiety may allow for immobilization of the compound on a solid support such as for example a bead or a microarray. An example of a streptavidin binding moiety is biotin.

Attachment of a reactive group may allow for conjugation of the compound to a another entity. The entity may for example be a separate moiety as described above or a solid support such as for example a bead or a microarray. Examples of reactive groups include but are not limited to thiols, N- hydroxy succinimide esters and maleimides.

In one embodiment, the linker (L) comprises or consists of an alkane chain, a peptide, diaminoacetic acid, maleimide, ethylene glycol, PEG, L/PEG or any combination thereof.

Side-chain to side-chain cyclization

In order to introduce structural ohelicity, which is often important for protein-protein interactions, various synthetic strategies are employed in the art. One of such strategies relies on side-chain to side-chain cyclization. In the present disclosure, five different cyclization methods known to introduce helicity are presented, namely:

sidechain to sidechain lactam cyclization, ring closing metathesis (RCM), copper(l)- catalyzed azide alkyne cycloaddition (CuAAC) and the crosslinking of thiols using either dibromo or perfluoroaryl linkers. Crosslinking is also referred to as X-linking throughout. Cysteine alkylation using bifunctional alkyl or aryl linkers such as dibromo- p xylene yielding macrocyclic strucutres has been reported previously. Similarly, perfluoroaryl linkers have been shown to react in a nuclear aromatic substitution (S_NAr) reaction with thiols to form macrocyclic structures. After cyclization, the S-atom of the cysteine or cysteine analogue has displaced the halide (Br or F) of the unattached linker. A wavy line intersecting the bond formed may be used to illustrate the bond formed between the S-atom and a carbon atom of the linker.

In one embodiment, the structure of (Pi) is selected from the group consisting of:

Asn Gly Tyr Glu Asn Pro S₅ Tyr Lys Phe S, Glu

Asn Gly Tyr Glu Asn Pro S₅ (4Pal) Lys Phe S5 Glu

Asn Gly Tyr Glu Asn Pro S₅ Tyr Lys Phe S₅ (d-Glu) and

In one embodiment, the compound (Pi) according to the present disclosure is cyclized between at least two of the side chains of the residues selected from the group consisting of Xi, X₂, X3, O, X₄, X5 CQ X7, and Xs.

In one embodiment, the compound (Pi) according to the present disclosure is cyclized between O and X₇, cyclized between X₄ and Xs, or cyclized between O and C_Q. In one embodiment ,the compound (Pi) is cyclized as represented by the following formula: XIGX₂X3NP(#)[OX₄X₅X₆X7]^XX8,

wherein ^x is ¹ to ⁵ representing the cyclization type, wherein:

¹ is cyclization via an amide bond,

² is cyclization via ring-closing metathesis (RCM),

3 is cyclization via copper(l)-catalyzed azide alkyne cycloaddition (CuAAC),

⁴ is cyclization via crosslinking of thiols through linkers (1 ) to (6),

⁵ is cyclization via crosslinking of thiols through linkers (7) to (8); and

wherein (#) is (1 ) to (8) representing the linker, when ^x is ⁴ or ⁵;

wherein (#) is not included when ^x is ¹ to ³;

; wherein the wavy bond“¾” indicates the bond formed between the sidechain of a thiol and the linker after crosslinking;

and wherein Xi, G, X₂, X3, O, X₄, X5, CQ, X7, and Xs are defined herein.

In one embodiment, the compound (Pi) is cyclized between at least two of the side chains via ring-closing metathesis (RCM).

In one embodiment, the compound (Pi) according to the present disclosure has a double bond formed by RCM that is mostly (E)-configu ration, mostly (Z)-configu ration, or a 1 :1 mixture thereof.

In one embodiment, the compound (Pi) is of the following structure:

In one embodiment, the compound (Pi) according to the present disclosure, has the double bond formed by RCM further derivatized, such as by C-H activation through transition metal catalysis or by addition of an electrophile. In one embodiment, the compound (Pi) according to the present disclosure is provided, wherein X1 is selected from the group consisting of: (N- Me-A) and (L/Me-a);

wherein X₂ is selected from the group consisting of: (Phe-3,4-CI₂) and (Phe-2,4-CI₂) or (Phe-F-4);

wherein X₃ is selected from the group consisting of: (Aad), (hGIn) and (Api);

wherein X₄ is selected from the group consisting of: (3Pal) and (4Pal);

wherein X₅ is selected from the group consisting of: (R), (hArg) and (hLys);

wherein Xe is selected from the group consisting of: (Phe-4-tBu), (Phe-3-CI), (3Pal) and (4Pal); and

wherein Xe is selected from the group consisting of: (e), (Aad) and (Api).

Intracellular Target

Mint proteins are intracellular proteins and any potential ligand must therefore enter the cell to interact with and/or bind any Mint protein. Preferably, a ligand must be able to cross the blood brain barrier (BBB) of an animal, including a human, to reach the intracellular target in the central nervous system (CNS).

Thus, in one embodiment, the compound as described herein has the generic structure of Formula I:

Z - L - Pi (I), wherein Z is a CPP and L is an optional linker. A CPP may provide for the compound to cross the cell membrane and enter the cell for interaction with and/or binding to any Mint protein.

Z - Ri (II),

wherein Z is a CPP.

In one embodiment, the CPP has a polycationic structure.

In one embodiment, the CPP comprises at least 4 amino acid residues individually selected from the group consisting of lysine (K or k) and arginine (R or r).

In one embodiment, the CPP comprises a retroinverso peptide. In one embodiment, the CPP comprises a TAT peptide, a mixed TAT peptide, a PolyArg peptide, a D-SynB3 peptide or a mini-AP4 peptide.

In one embodiment, the CPP comprises a TAT peptide.

In one embodiment, the CPP is selected from the group consisting of SEQ ID NO: 93 to 97.

In one embodiment, the CPP comprises at least 4 amino acids having cationic or basic side chains that are analogous to arginine (R) or lysine (K), such as for example 5- hydroxylysine, ornithine, 2-amino-3 (or-4)-guanidinopropionic acid, and homoarginine.

In one embodiment, the CPP has an amphipathic structure and comprises an alternating pattern of polar/charged amino acids and non-polar/hydrophobic amino acids.

In one embodiment, the CPP is selected from the group consisting of penetratin (SEQ ID NO: 98), retroinverso-penetratin (SEQ ID NO: 99) and amphipathic model peptide (SEQ ID NO: 100).

In one embodiment, the compound is conjugated to the CPP via a linker (L). The linker (L) may comprise or consist of an alkane chain, a peptide, diaminoacetic acid, maleimide, ethylene glycol, PEG, L/PEG or any combination thereof.

In one embodiment, the compound as described herein has a structure according to formula I, wherein formula I is Ac-YGRKKRRQRRR-(/VMe- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 87).

In one embodiment, the compound as described herein has a structure according to formula I, wherein formula I is Ac-rRrGrKkRr-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 88).

In one embodiment, the compound as described herein has a structure according to formula I, wherein formula I is Ac-RRRRRRRRR-(/VMe- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 89). In one embodiment, the compound as described herein has a structure according to formula I, wherein formula I is Ac-frrrsyslrr-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 90).

In one embodiment, the compound as described herein has a structure according to formula I, wherein formula I is c[DLATEPAK(DAP)]-(/VMe- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 91 ).

In one embodiment, the compound as described herein has a structure according to formula II, wherein formula II is Ac-YGRKKRRQRRR-(/VMe- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 87).

In one embodiment, the compound as described herein has a structure according to formula II, wherein formula II is Ac-rRrGrKkRr-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 88).

In one embodiment, the compound as described herein has a structure according to formula II, wherein formula II is Ac-RRRRRRRRR-(/VMe- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 89).

In one embodiment, the compound as described herein has a structure according to formula II, wherein formula II is Ac-frrrsyslrr-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 90).

In one embodiment, the compound as described herein has a structure according to formula II, wherein formula II is c[DLATEPAK(DAP)]-(/VMe- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 91 ).

Diagnostic method

It has previously been demonstrated that Mint proteins are upregulated in patients with AD and that the proteins colocalize with the amyloid plaques found in AD (Jacobs, E. H. et al. 2006, McLoughlin, D. M. et al. 1999). Hence visualization of Mint protein amount and localization in the brain or brain tissue may be usefull for diagnosis of AD. Thus, in one embodiment, the compound as described herein has the generic structure of Formula I:

Z - L - Pi (I), wherein Z is a detectable moiety and L is an optional linker. The detectable moiety may provide for visualization of the compound and hence localization of amyloid plaques in the brain or in brain tissue.

Z - Ri (II),

wherein Z is a detectable moiety.

In one embodiment, the detectable moiety is a fluorophore. The fluorophore may be any fluorophore, such as for example 5,6-carboxyltetramethylrhodamine (TAMRA) or indodicarbocyanine (Cy5).

In one embodiment, the detectable moiety comprises or consists of a radioisotope.

In one embodiment, the radioisotope is selected from the group consisting of ¹²⁵l, ^99mTc, ¹¹¹ In, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ¹²³l, ¹⁸F and ²⁰¹TI.

In one embodiment the detectable moiety is selected from the group consisting of ¹²⁵l- indodicarbocyanine, indodicarbocyanine, ¹²⁵l-indotricarbocyanine and

indotricarbocyanine.

Attachment of a radioisotope to the compound as described herein may allow for visualization of the compound using for example fluorescent microscopy, magnetic resonance imaging (MRI), computed tomography (CT), positron-emission tomography (PET) or single-photon emission computed tomography (SPECT).

In one embodiment, the compound is conjugated to the detectable moiety via a linker (L). The linker (L) may comprise or consist of an alkane chain, a peptide, diaminoacetic acid, maleimide, ethylene glycol, PEG, L/PEG or any combination thereof. In one embodiment, the visualization may be combined with visualization with one or more additional amyloid binding compounds.

In one embodiment, a method of diagnosing AD is provided, the method comprising the steps of:

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein,

wherein the compound comprises a detectable moiety; and c) visualizing the localization of compound bound to Mint in b);

wherein the presence and localization of compound bound to Mint in the biological sample is indicative of the presence of amyloid plaques and resulting Alzheimer’s disease in the individual from which the biological sample is derived.

In one embodiment, a method of diagnosing Alzheimer’s disease is provided, the method comprising the steps of:

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a microchip as described herein; and c) visualizing the localization of compound bound to Mint in b);

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein,

wherein the compound comprises a detectable moiety;

c) quantifying the amout of compound bound to Mint in b); and

d) comparing the amount of compound bound to Mint in c), to a control; wherein an amount of compound bound to Mint in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

In one embodiment, a method of diagnosing Alzheimer’s disease is provided, the method comprising the steps of: a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a microchip as described herein;

c) quantifying the amountel of compound bound to Mint in b); and d) comparing the amount of compound bound to Mint in c), to a control; wherein an amount of compound bound to Mint in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein,

wherein the compound comprises a detectable moiety; and c) visualizing the localization of compound bound to Mint 1 and/or Mint2 in b);

wherein the presence and localization of compound bound to Mintl and/or Mint2 in the biological sample is indicative of the presence of amyloid plaques and resulting Alzheimer’s disease in the individual from which the biological sample is derived.

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a microchip as described herein; and c) visualizing the localization of compound bound to Mintl and/or Mint2 in b);

wherein the presence and localization of compound bound to Mintl and/or Mint2 in the biological sample is indicative of the presence of amyloid plaques and resulting Alzheimer’s disease in the individual from which the biological sample is derived. In one embodiment, a method of diagnosing AD is provided, the method comprising the steps of:

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein,

wherein the compound comprises a detectable moiety; c) quantifying the amount of compound bound to Mintl and/or Mint2 in b); and

d) comparing the amount of compound bound to Mintl and/or Mint2 in c), to a control;

wherein an amount of compound bound to Mintl and/or Mint2 in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a microchip as described herein; and c) quantifying the amount of compound bound to Mintl and/or Mint2 in b);

and

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein,

wherein the compound comprises a detectable moiety; and c) visualizing the localization of compound bound to Mint2 in b);

wherein the presence and localization of compound bound to Mint2 in the biological sample is indicative of the presence of amyloid plaques and resulting Alzheimer’s disease in the individual from which the biological sample is derived.

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a microchip as described herein; and c) visualizing the localization of compound bound to Mint2 in b); wherein the presence and localization of compound bound to Mint2 in the biological sample is indicative of the presence of amyloid plaques and resulting Alzheimer’s disease in the individual from which the biological sample is derived.

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a compound as described herein,

wherein the compound comprises a detectable moiety;

c) quantifying the amount of compound bound to Mint2 in b); and d) comparing the amount of compound bound to Mint2 in c), to a control; wherein an amount of compound bound to Mint2 in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with a microchip as described herein; and c) quantifying the amount of compound bound to Mint2 in b); and d) comparing the amount of compound bound to Mint2 in c), to a control; wherein an amount of compound bound to Mint2 in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

In one embodiment, the sample is a body fluid and/or a tissue sample.

Composition and method of use

In one embodiment, a composition comprising the compound as described herein is provided.

In one embodiment, the composition is a pharmaceutical composition. The

pharmaceutical composition may be any pharmaceutical composition. For example, said pharmaceutical composition may be suitable for administration via enteral or parenteral routes. Parenteral routes may include but are not limited to intraveneous, epidural, intracerebral or intracerebroventricular routes.

In one embodiment, a method of reducing the production of Amyloid b by a cell, the method comprising contacting the cell with a compound as described herein, or a composition as described herein.

In one embodiment, a compound as described herein, or a composition as described herein is provided, for use as a medicament.

In one embodiment, a compound as described herein, or a composition as described herein is provided, for use in the prevention and/or the treatment of a

neurodegenerative disease.

In one embodiment, the neurodegenerative disease is a cognitive disorder.

In one embodiment, the neurodegenerative disease is dementia.

In one embodiment, the neurodegenerative disease is Alzheimer’s disease.

In one embodiment, the neurodegenerative disease is characterized by formation of amyloid plaques comprising Amyloid b.

In one embodiment, a method of preventing and/or treating a neurodegenerative disease is provided, said method comprising administering a therapeutically effective amount of a compound as described herein, or a composition as described herein, to an individual in need thereof.

In one embodiment, the invention is directed to the use of a compound as described herein, or a composition as described herein, for the manufacture of a medicament for prevention and/or treatment of a neurodegenerative disease.

Isolation of Mint proteins

The compound as described herein may be immobilized on a solid support. Examples of solid supports include but are not limited to beads and microarrays. Thus, in one embodiment, the compound as described herein is immobilized on a bead.

In one embodiment, a microchip comprising an array of compounds as described herein is provided.

The immobilized compound, as described herein, may be used for detection of Mint proteins or for purification of Mint proteins.

In one embodiment, the immobilized compound, as described herein, may be used for detection of Mintl and/or Mint2 or for purification of Mintl and/or Mint2.

In one embodiment, the immobilized compound, as described herein, may be used for detection of Mint2 or for purification of Mint2.

Thus, in one embodiment, a method of isolating Mint is provided, said method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with an immobilized compound as described herein, thus obtaining a compound:Mint complex; and c) isolating the Mint protein bound to said immobilized compound, thus obtaining pure Mint.

In one embodiment, a method of isolating Mint is provided, said method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with a microchip as described herein, thus obtaining a compound:Mint complex; and

c) isolating the Mint protein bound to said microchip, thus obtaining pure Mint.

a) providing a composition comprising Mint; b) contacting said composition of a) with an immobilized compound as described herein, thus obtaining a compound:Mint complex; and c) isolating the Mint protein bound to said immobilized compound, thus obtaining pure Mint.

a) providing a composition comprising Mint;

b) contacting said composition of a) with a microchip as described herein, thus obtaining a compound:Mint complex; and

In one embodiment, a method of isolating Mintl and/or Mint2 is provided, said method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with an immobilized compound as described herein, thus obtaining a compound:Mint1 and/or compound:Mint2 complex; and

c) isolating the Mintl and/or Mint2 protein bound to said immobilized compound, thus obtaining pure Mintl and/or Mint2.

In one embodiment, a method of isolating Mint 1 and/or Mint2 is provided, said method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with a microchip as described herein, thus obtaining a compound:Mint1 and/or compound:Mint2 complex; and

c) isolating the Mint 1 and/or Mint2 protein bound to said microchip, thus obtaining pure Mintl and/or Mint2.

a) providing a composition comprising Mintl and/or Mint2; b) contacting said composition of a) with an immobilized compound as described herein, thus obtaining a compound:Mint1 and/or compound:Mint2 complex; and

a) providing a composition comprising Mintl and/or Mint2;

b) contacting said composition of a) with a microchip as described herein, thus obtaining a compound:Mint1 and/or compound:Mint2 complex; and c) isolating the Mintl and/or Mint2 protein bound to said microchip, thus obtaining pure Mintl and/or Mint2.

In one embodiment, a method of isolating Mint2 is provided, said method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with an immobilized compound as described herein, thus obtaining a compound:Mint2 complex; and c) isolating the Mint2 protein bound to said immobilized compound, thus obtaining pure Mint2.

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with a microchip as described herein, thus obtaining a compound:Mint2 complex; and

c) isolating the Mint2 protein bound to said microchip, thus obtaining pure

Mint2.

a) providing a composition comprising Mint2;

b) contacting said composition of a) with an immobilized compound as described herein, thus obtaining a compound:Mint2 complex; and c) isolating the Mint2 protein bound to said immobilized compound, thus obtaining pure Mint2.

a) providing a composition comprising Mint2;

b) contacting said composition of a) with a microchip as described herein, thus obtaining a compound:Mint2 complex; and

c) isolating the Mint2 protein bound to said microchip, thus obtaining pure

Mint2.

Examples

Example 1 : Peptide synthesis

Ligands 1 to 91 (SEQ ID: 1 to 91 ), exemplified by ligand 83 (SEQ ID: 83) in Figure 2, were manually synthesized using Fmoc-based solid phase peptide synthesis on 2- chlorotrityl chloride resin. The resin was first swelled in diemthylformamide (DMF) for 1 h at room temperature after which 4 eq. (relative to the resin) of the first amino acid was dissolved in 8 eq. diisopropylethylamine (DIPEA) in DMF and added to the resin. The reaction was allowed to proceed overnight under agitation. The resin was washed in DMF and capped by treatment with 5 mL DMF/methanol (MeOH)/DIPEA (16:3:1 ) for 5 min. The capping procedure was repeated twice followed by extensive washing with DMF. Fmoc-deprotection was carried out by treating the resin with 20% piperidine in DMF for 2 x 1 min. The resin was washed extensively with DMF before the next Fmoc- protected amino acid were coupled to the resin by dissolving 4 eq. of amino acid in 4 eq. /V,/V,/V',/V'-tetramethyl-0-(1 /-/-benzotriazol-1 -yl)uronium hexafluorophosphate (HBTU), 8 eq DIPEA in DMF or in 4 eq. 1 -[bis(dimethylamino)methylene]-1 /-/-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate (HATU), 8 eq. DIPEA in DMF and adding the solution to the resin. The coupling reaction was allowed to proceed for 30 min - 16 h at room temperature. The steps of Fmoc-deprotection and coupling of amino acids were repeated until the desired peptide sequence was obtained. The desired peptide was treated with 20 eq. PhSiFh and 0.2 eq. Pd(PPh3)₄ in DCM under nitrogen for 2 x 15 min to remove the allyl and alloc protecting groups. The resin was washed with DCM followed by DMF. The cyclication reaction between the side chains of Glu/Lys, and/or Glu/Orn, and/or Glu/Dap, and/or Asp/Lys, and/or Asp/Orn, and/or Asp/Dap was performed by treatment of the peptide with 2 eq. (benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 2 eq. DIPEA in DMF for 2 h - 16 h at room temperature followed by DMF wash. The resin was washed extensively with DMF and DCM and dried. Finally, the Fmoc-group was removed from the N-terminus. For ligands 86 to 90 (SEQ ID: 86 to 90), the N-terminus was capped on resin by treatment with 5 mL

DMF/acetic anhydride/DIPEA (16:3:1 ) for 2 x 15 min. The resin was washed extensively with DMF and DCM and dried.

The peptide was cleaved from the solid support by treatment with trifluoroacetic acid (TFA)/ triisopropylsilane (TIPS)/ water (95:2.5:2.5) for 2 h at room temperature followed by evaporation and ice-cold diethyl ether precipitation. The crude peptide was collected by centrifugation and purified by RP-HPLC followed by lyophilisation. The final peptide ligands were characterised by LC-MS and UPLC (214 nm). In conclusion, example 1 demonstrates that the ligands can be synthesized, purified and obtained in pure form.

Example 2: Method for determining affinity to PARM of Mint2

The binding affinity of the compounds to PTB-ARM (PARM) of Mint2 was measured with fluorescent polarisation (FP). First saturation binding curves for the 5,6- carboxyltetramethylrhodamine (TAMRA) labelled ligands 1 (TAMRA-APP^wt peptide, SEQ ID: 1 ) and 2 (SEQ ID: 2) were obtained by using increasing concentrations of PARM of Mint2 in the presence of 50 nM fluorescent labelled ligand. The fluorescence polarisation of the sample was measured at excitation/emission wavelength of 530/585 nm and the generated FP values were fitted to a one site binding model using the software Prism. The Kd value was determined at 50% of the maximum response, Table 1 .

Table 1 : Affinity of fluorescently labelled peptides to PARM of Mint2, presented as mean ± SEM.

Sequence Ligand SEQ ID NO: FP

Kd (mM)

TAMRA-NNG-QNGYENPTYKFFEQMQN 1 ¹ 10,9 ± 0,5

TAMRA-NNG-NGYENPTYK (INAL) (INAL) E 2 2 8,1 ± 0,4

1 NAL - L-3-(1-naphthyl)alanine; data represented as n ³ 3. Using the saturation binding curves from the two TAMRA labelled ligands, the affinity between the non-fluorescent labelled ligands and PARM of Mint2 was determined by a competitive FP based inhibition assay where increasing concentrations of non- fluorescently labelled ligands in the presence of 50 nM of TAMRA labelled ligand 1 (SEQ ID: 1 ) or 2 (SEQ ID: 2) and 15000 nM or 10000 nM PARM of Mint2, respectively, competed for the binding site. The FP values were fitted to a one site competition with variable slope model in Prism. The generated IC₅₀ value in Prism was converted to the competition inhibition constant (K,), as described (Nikolovska-Coleska, Z. et al. 2004). In conclusion, example 2 describes how to determine the affinity of ligands binding to PARM of Mint2.

Example 3: Affinity to PARM of Mint2 measured with fluorescent polarisation (FP)

The affinity of synthesized ligands towards PARM of Mint2 was determined using FP as described in example 2. Ligands 3 to 82 (SEQ ID: 3 to 82) were diluted in 25 mM HEPES, 150 mM NaCI, 1 mM beta-mercaptoethanol, pH 7.4 and incubated at increasing concentrations with 50 nM of ligand 1 or 2 and 15000 nM or 10000 nM PARM of Mint2, respectively, at room temperature. The FP values were determined and converted to K,- values based on the K_d values (table 1 ) of ligands 1 (SEQ ID: 1 ) and/or 2 (SEQ ID: 2), see Table 2 to Table 8. The affinity of parent ligand 3 (APP^wt peptide, SEQ ID: 3) to PARM of Mint2 was determined to 4 mM. A subset of ligands 16 (SEQ ID: 16), 39 (SEQ ID: 39), 64 (SEQ ID: 64), 65 (SEQ ID: 65), 70 (SEQ ID: 70) and 76 (SEQ ID: 70) show significantly lower affinities towards PARM of Mint2 compared to the parent ligand 3 (SEQ ID: 3). Introducing mutations at position Xi, X₃, X₅, C_Q, X₇ and Xs generated ligand 83 (SBP, SEQ ID: 83), resulted in a synergistic affinity effect measured by ITC.

Table 2: Binding affinities (K,), presented as mean ± SEM, of N-terminal truncated peptides.

Sequence Ligand SEQ ID NO:

NGYENPTYKFFE 3 3 4,0 ± 0,1

QNGYENPTYKFFEQMQN 4 4 2,9 ± 0,3

NGYENPTYKFFEQMQN 5 5 4,2 ± 0,4

GYENPTYKFFEQMQN 6 6 37,7 ± 3,8

YENPTYKFFEQMQN 7 7 >500

Data represented as n ³ 3. Table 3: Binding affinities (Ki), presented as mean ± SEM, of C-terminal truncated peptides.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0, 1 QNGYENPTYKFFEQMQN 8 8 2.9 ± 0,3 QNGYENPTYKFFEQMQ 9 9 4.9 ± 0,9 QNGYENPTYKFFEQM 10 10 5,6 ± 0,5 QNGYENPTYKFFEQ 11 1 1 6,5 ± 0,7 QNGYENPTYKFFE 12 12 7.2 ± 0,8 QNGYENPTYKFF 13 13 30.2 ± 0,9 QNGYENPTYKF 14 14 329 ± 1 1 ,7 QNGYENPTYK_ 15 15 >500

Data represented as n ³ 3. Table 4: Binding affinities (Ki), presented as mean ± SEM, for Ala scan.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,1

AGYENPTYKFFE 16 16 1.6 ± 0.2

NAYENPTYKFFE 17 17 1 1.9 ± 1 .3

NGAENPTYKFFE 18 18 305 ± 16

NGYANPTYKFFE 19 19 12.2 ± 1.2

NGYEAPTYKFFE 20 20 >500

NGYENATYKFFE 21 21 25.4 ± 2.3

NGYENPAYKFFE 22 22 14.2 ± 1 .3

NGYENPTAKFFE 23 23 9.3 ± 1.0

NGYENPTYAFFE 24 24 5.5 ± 0.4

NGYENPTYKAFE 25 25 56.9 ± 4.4

NGYENPTYKFAE 26 26 72.9 ± 7.7

NGYENPTYKFFA 27 27 5.4 ± 0.5

Data represented as n ³ 3.

Table 5: Binding affinities (Ki), presented as mean ± SEM, for D-AA scan.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,1 nGYENPTYKFFE 28 28 5,1 ± 0,2 NGyENPTYKFFE 29 29 266 ± 30,6 NGYeNPTYKFFE 30 30 >500 NGYEnPTYKFFE 31 31 >500 NGYENpTYKFFE 32 32 >500 NGYENPtYKFFE 33 33 170 ± 27,8 NGYENPTyKFFE 34 34 208 ± 1 ,5 NGYENPTYkFFE 35 35 89.4 ± 5,8 NGYENPTYKfFE 36 36 79,7 ± 4, 1 NGYENPTYKFfE 37 37 32.4 ± 2,5 NGYENPTYKFFe 38 38 5,4 ± 0,7

Data represented as n ³ 3.

Table 6: Binding affinities (Ki), presented as mean ± SEM, for A/(Me)-AA scan.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,1 N (Me) GYENPTYKFFE 39 39 2,5 ±0,1 NG(Me) YENPTYKFFE 40 40 4,7 ± 0,3 NGY (Me) ENPTYKFFE 41 41 >500 NGYE (Me) NPTYKFFE 42 42 9,8 ±0,7 NGYEN (Me) PTYKFFE 43 43 38,2 ±8,2 NGYENPT (Me) YKFFE 44 44 44,8 ±6,0 NGYENPTY (Me) KFFE 45 45 51.1 ± 7,2 NGYENPTYK (Me) FFE 46 46 270,2 ± 188,5 NGYENPTYKF (Me) FE 47 47 55.2 ±5,5 NGYENPTYKFF (Me) E 48 48 98.3 ± 9,1 NGYENPTYKFFE (Me) 49 49 7,2 ±0,2

Data represented as n ³ 3.

Table 7: Binding affinities (Ki), presented as mean ± SEM, for side-chain substitutions.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ±0,1

NGYpENPT YKFFE 50 50 6,8 ± 0,8

NGFENPT YKFFE 51 51 5,1 ± 0,5

NG (INAL) ENPTYKFFE 52 52 1,7 ± 0,2

NGYEQPT YKFFE 53 53 >500

NGYEDPT YKFFE 54 54 >500

NGYEEPT YKFFE 55 55 >500

NGYE (DAP) PTYKFFE 56 56 >500

NGYE (DAB) PTYKFFE 57 57 >500

NGYE (ORN) PTYKFFE 58 58 >500

NGYE (2ABU) PTYKFFE 59 59 >500

NGYE (NVA) PTYKFFE 60 60 >500

NGYENPTKKFFE 61 61 422 ± 40,2 NGYENPTWKFFE 62 62 9,8 ± 0,9 NGYENPTFKFFE 63 63 0,9 ± 0,1

NGYENPTYK ( INAL) FE 64 64 1,53 ± 0,4

NGYENPTYKWFE 65 65 5,16 ± 0,3

NGYENPTYKF (INAL) E 66 66 2,63 ± 0,5

NGYENPTYKFWE 67 67 5,37 ± 0,9

NGYENPTYK (IGL) (IGL) E 68 68 5,98 ± 0,3

NGYENPTYK (2NAL) (2NAL) E 69 69 1 ,73 ± 01

NGYENPTYK (INAL) (1NAL)E 70 70 1,12 ± 0,3

1NAL - L-3-(1-naphthyl)alanine; DAP - L-2,3-diaminopropionic acid; DAB - L-2,4-diaminobutyric acid; ORN - L-ornithine; 2ABU - L-2-aminobutyric acid; NVA - L-norvaline; IGL - 2-indanyl-L-glycine; 2NAL - L-3-(2-naphthyl)alanine; data represented as n ³ 3.

Table 8: Binding affinities (Ki), presented as mean ± SEM, for cyclized peptides.

Sequence _ Ligand _ SEQ ID NO: _ Ki (mM)

NGYENPTYKFFE _ 3 _ 3 _ 4,0 ±0,1

NGYc [ENPTYK] FFE 71 71 2,7 ± 0,2

NGY c [KNPTYE] FFE _ 72 _ 72 _ 2,4 ± 0,2

NGYc [DNPTYK] FFE 73 73 4,2 ± 0,4

NGYc [ENPTY (ORN) ] FFE 74 74 4,4 ± 0,2

NGYc [ (ORN) NPTYE] FFE 75 75 2,8 ± 0,4

NGY c [KNPTYD] FFE _ 76 _ 76 _ 2,2 ± 0,3

NGY c [DNPTY (ORN) ] FFE 77 77 13,6 ± 0,9

NGYc [ENPTY (DAB) ] FFE 78 78 19,6 ±1,0 NGY c [ (DAB) NPTYE] FFE 79 79 35,0 ±2,0

NGY c [ (ORN) NPTYD] FFE 80 80 2,2 ± 0,1

NGYc[DNPTY (DAP) ] FFE 81 81 6,4 ± 0,5

NGYc[ (DAP) NPTYD] FFE 82 82 69,3 ± 10,6

DAP - L-2,3-diaminopropionic acid; DAB - L-2,4-diaminobutyric acid; ORN - L-ornithine; c - indicates side-chain to side-chain cyclization; data represented as n ³ 3.

Table 9: Binding affinities (K,), presented as mean ± SEM, for lactam cyclized peptides.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,10

NGYENP [KYKFE] ¾ CB441 1Ϊ0 9,0 ±0,90

NGYENP [EYKFK ] ¾ CB444 111 9,1 ± 0,90

NGYENP [KYKFD ] ¾ CB433 112 1 ,0 ± 0,10

NGYENPKYKFDE CB524 113 292,0 ± 14,8

NGYENP [ (Orn) YKFE] ¾ CB449 114 46.7 ± 2,60

NGYENP (Orn ) YKFEE CB525 115 n.b.

NGYENP [EYKF (Orn ) ] ¾ CB450 116 44,2 ± 0,70

NGYENPEYKF (Orn ) E CB526 117 257 ± 29,0

NGYENP [DYKFK ] ¾ CB451 118 56.8 ± 5,00

NGYENPDYKFKE CB527 119 338,0 ± 5,40

NGYENP [ (Orn) YKFD] ¾ CB521 120 74.2 ±4,80

NGYENP [ (Dab) YKFE ] ¾ CB442 121 102,0 ± 1,00

NGYENP [EYKF (Dab ) ] ¾ CB445 122 79,9 ± 2,30

NGYENP [DYKF (Orn ) ] ¾ CB522 123 38.2 ± 1,40

NGYENP [ (Dab) YKFD ] ¾ CB443 124 92,0 ± 2,40

NGYENP [DYKF (Dab ) ] ¾ CB446 125 214,0 ±7,70

(Dab) - L-2,4-diaminobutyric acid; (Orn) - L-ornithine; [xxxxx]¹ - indicates lactam side-chain to side-chain cyclization; data represented as n ³ 3.

Table 10: Binding affinities (K), presented as mean ± SEM, for all-hydrocarbon cyclized peptides synthesized via RCM.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,10

NGYENP [ (R₅) YK (S₅) ] ²FE CB437a 126 3,4 ± 0,20 NGYENP (R₅) YK (S₅) FE CB437b 127 3.1 ± 0,20

NGYENP [ ( S₅ ) YKF ( S₅ ) ] ²E CB447a 128 0,51 ± 0,05 NGYENP (S₅) YKF (S₅) E CB447b 129 3.3 ± 0,20

NGYENPT [ ( S₅ ) KFF ( S₅ ) ] ² CB448a 130 1.2 ± 0,20 NGYENPT (S₅) KFF (S₅) CB448b 131 5.4 ± 0,40

NGYENP [ ( S₈ ) YKF ( S₅ ) ] ²E FA018b1 132 2.3 ± 0,30 NGYENP [ ( S₈ ) YKF ( S₅ ) ] ²E FA018b2 133 1.3 ± 0,20 NGYENP (S₈) YKF (S₅) E FA018a 134 2,7 ± 0,60

NGYENP [ ( S₅ ) YKF ( S₈ ) ] ²E FA019b1 135 1 1 ± 0, 10 NGYENP [ ( S₅ ) YKF ( S₈ ) ] ²E FA019b2 136 n.d. NGYENP (S₅) YKF (S₈) E FAQ 19a 137 10,6 ± 1 ,20

NGYENP [ (S₈) YKF (S₈) ] ²E FA020b 138 2,1 ± 0,60 NGYENP (S₈) YKF (S₈) E FA020a 139 12,0 ± 1 ,10

NGYENP [ (S₅) YKF (S₅) ] ²E-N¾ CB544 140 1 ,45 ± 0,05 NGYENP (S₅) YKF (S₅) E-N¾ CB545 141 5,16 ± 0,14

Ac-NGYENP [ (S₅) YKF (S₅) ] ²E-N¾ CB548 142 3,48 ± 0,07 Ac-NGYENP (S₅) YKF (S₅) E-N¾ CB549 143 17,3 ± 0,70

(F¾) - (R)-N-Fmoc-a-4-n-pentenylalanine; (S5) - (S)-N-Fmoc-a-4-n-pentenylalanine; (Se) - (S)-N-Fmoc-a- (7-Octenyl)alanine; [xxxxx]² - indicates all-hydrocarbon side-chain to side-chain cyclization using RCM; data represented as n ³ 3.

Table 11 : Binding affinities (Ki), presented as mean ± SEM, for all-hydrocarbon cyclized peptides synthesized via RCM containing single substitutions.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 _ 3 4,0 ± 0,10

NGYENP [ (S₅) YK (INal) (S₅) ] ²E CB535 144 0,31 ± 0,01 NGYENP [ (S₅) YK (3Pal) (S₅) ] ²E CB615 145 0,39 ± 0,01 NGYENP [ (S₅) YK (4 Pal) (S₅) ] ²E CB616 146 0,60 ± 0,01 NGYENP [ (S₅) YK (Phe-3-I) (S₅) ] ²E CB564 147 0,34 ± 0,02 NGYENP [ (S₅) YK(Phe-3-Cl) (S₅) ] ²E CB565 148 0,31 ± 0,01 NGYENP [ (S₅) YK (Phe-2, 4-

CB566 149 0,32 ± 0,04 Cl₂) (S₅) ] ²E

NGYENP [ (S₅) YK (Phe-3-^tBu) (S₅) ] ²E CB567 150 0,27 ± 0,01

NGYENP [ ( S₅ ) YRF ( S₅ ) ] ²E CB536 151 0,25 ± 0,02

NGYENP [ ( S₅ ) FKF ( S₅ ) ] ²E CB537 152 0,40 ± 0,02 NGYENP [ (S₅) (3Pal) KF (S₅) ] ²E CB617 153

NGYENP [ (S₅) (4 Pal) KF (S₅) ] ²E CB618 154

NGYEAP [ ( S₅ ) YKF ( S₅ ) ] ²E CB550 155 122,6 ± 5,80

NGY (Api ) NP [ ( S₅ ) YKF ( S₅ ) ] ²E CB604 156 0,17 ± 0,02 NGY (hGln) NP [ (S₅) YKF (S₅) ] ²E CB605 157 0,15 ± 0,01 NGY (Aad) NP [ (S₅) YKF (S₅) ] ²E CB606 158 0,11 ± 0,01 NGY (Cit) NP [ (S₅) YKF (S₅) ] ²E CB607 159 0,20 ± 0,04 NGY (Cpc) NP [ (S₅) YKF (S₅) ] ²E CB608b 160 0,12 ± 0,02 NGY (Cpc^ox) NP [ (S₅) YKF (S₅) ] ²E CB608a 246 0,21 ± 0,02

NG (INal) ENP [ (S₅) YKF (S₅) ] ²E CB546 161 0,55 ± 0,01 NG (Igl) ENP [ (S₅) YKF (S₅) ] ²E CB594 162 0,58 ± 0,03 NG (Phe-3, 4- 0,23 ± 0,02

CB595 163

Cl₂) ENP [ (S₅) YKF (S₅) ] ²E

NG (3Pal) ENP [ (S₅) YKF (S₅) ] ²E CB619 164

NG (4 Pal) ENP [ (S₅) YKF (S₅) ] ²E CB596 165 0,67 ± 0,01 NGFENP [ ( S₅ ) YKF ( S₅ ) ] ²E CB597 166 0,37 ± 0,02

N ( bA) YENP [ (S₅) YKF (S₅) ] ²E CB551 167 1 ,50 ± 0,03 (NMe-a) GYENP [ (S₅) YKF (S₅) ] ²E CB538 168 0,32 ± 0,03 hGYENP [ ( S₅ ) YKF ( S₅ ) ] ²E CB547 169 0,50 ± 0,06 NNGNGYENP [ ( S₅ ) YKF ( S₅ ) ] ²E CB495 247 0,40 ± 0,01

(R₅) - (R)-N-Fmoc-a-4-n-pentenylalanine; (S5) - (S)-N-Fmoc-a-4-n-pentenylalanine; (Se) - (S)-N-Fmoc-a- (7-Octenyl)alanine; [xxxxx]² - indicates all-hydrocarbon side-chain to side-chain cyclization using RCM; for structures of subsitutions consult Example 1 1 ; data represented as n ³ 3.

Table 12: Binding affinities (K), presented as mean ± SEM, for triazole cyclized peptides synthesized via CuAAc.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0, 10

NGYENP [ (Lys^a) YKF (Pra) ] ³E CB463b 170 1 ,2 ± 0,20 NGYENP (Lys^a) YKF (Pra) E CB463a 171 6,3 ± 0,20 NGYENP [ (Pra) YKF (Lys^a) ] ³E CB464b 172 12,8 ± 0,50 NGYENP (Pra) YKF (Lys^a) E CB464a 173 23,7 ± 0,90

NGYENP [ (Orn^a) YKF (hPra) ] ³E CB496b 174 64.6 ± 1 ,90 NGYENP (Orn^a) YKF (hPra) E CB496a 175 4,7 ± 0,70 NGYENP [ (hPra) YKF (Orn^a) ] ³E CB497b 176 19.7 ± 1 ,40 NGYENP (hPra) YKF (Orn^a) E CB497a 177 3,5 ± 0,60

NGYENP [ (Orn^a) YKF (Pra) ] ³E CB490b 178 39,0 ± 2,90 NGYENP (Orn^a) YKF (Pra) E CB490a 179 8, 1 ± 0,50 NGYENP [ (Pra) YKF (Orn^a) ] ³E CB491 b 180 21 ,4 ± 0,70 NGYENP (Pra) YKF (Orn^a) E CB491 a 181 23,3 ± 0,60

(Lys^a) - (S)-2-amino-6-azido-hexanoic acid; (Orn^a) - (S)-2-amino-5-azido-pentanoic acid; (Pra) - L-propargylglycine; (hPra) - L-homo-propargylglycine; [xxxxx]³ - indicates triazole cyclization using CuAAC; data represented as n ³ 3.

Table 13: Binding affinities (Ki), presented as mean ± SEM, for thiol (Cys/hCys) cyclized peptides synthesized via crosslinking with various linkers.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0, 10

NGYENPCYKFCE FA001 182 21 ,4 ± 0,70

NGYENP (1) [CYKFC] ⁴E FA001J 183 1 ,7 ± 0, 10 NGYENP (2) [CYKFC] ⁴E FA001_2 184 1 ,0 ± 0,30 NGYENP (3) [CYKFC] ⁴E FA001_3 185 1.5 ± 0,60 NGYENP (4) [CYKFC] ⁴E FA001_4 186 6.5 ± 0,70 NGYENP (5) [CYKFC] ⁴E FA001_5 187 0,4 ± 0, 10 NGYENP (6) [CYKFC] ⁴E FA001_6 188 2.5 ± 0,30

NGYENP (7) [CYKFC] ⁵E FA001_7 189 1 ,3 ± 0,20 NGYENP (8) [CYKFC] ⁵E FA001_8 190 0,9 ± 0,30

NGYENP (5) [cYKFC] ⁴E FA013 191 2.6 ± 0,50 NGYENPcYKFCE CB509 192 48,8 ± 1 ,50 NGYENP (5) [CYKFc] ⁴E FA014 193 8,0 ± 0,50 NGYENPCYKFCE CB510 194 9.2 ± 0,30 NGYENP (5) [cYKFc] ⁴E FA015 195 5.2 ± 0,50 NGYENPcYKFCE CB51 1 196 39,2 ± 2,00

NGYENP (5) [ (hC) YKFC] ⁴E FA024 197 1 , 1 ± 0,30 NGYENP (hC) YKFCE FA021 198 4.3 ± 0,20 NGYENP (5) [CYKF(hC) ] ⁴E FA025 199 3,5 ± 0,20 NGYENPCYKF (hC) E FA022 200 1.3 ± 0,20 NGYENP (5) [ (hC) YKF (hC) ] ⁴E FA026 201 n.d.

NGYENP (hC) YKF (hC) E FA023 202 1.7 ± 0,50

(hC) - L-homo-cysteine; (x)[xxxxx]⁴ - indicates cyclization via crosslink between thiols using linkers (1 ) to (6); (x)[xxxxx]⁵ - indicates cyclization via crosslink between thiols using linkers (7) and (8); data represented as n ³ 3. Table 14: Binding affinities (K), presented as mean ± SEM, for thiol (Cys/hCys) cyclized peptides synthesized via X-linking using linker (5) containing single substitutions.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,10

(Mde-N) GYENP (5) [ (hC) YKF (hC) ] ⁴E FA030 203 1 ,7 ± 0,20 (Mde-A) GYENP (5) [ (hC) YKF (hC) ] ⁴E FA040 204 0,5 ± 0,20 HGYENP (5) [ (hC) YKF (hC) ] ⁴E FA031 205 1.5 ± 0,20 hGYENP (5) [ (hC) YKF (hC) ] ⁴E FA041 206 1.6 ± 0,50 (Mde-a) GYENP (5) [ (hC) YKF (hC) ] ⁴E FA055 207 3.4 ± 0,20 NGY (Tie) NP (5) [ (hC) YKF (hC) ] ⁴E FA038 208 1.4 ± 0,20 NGY (^tBug) NP (5) [ (hC) YKF (hC) ] ⁴E FA039 209 1.7 ± 0,50 NGY (Cpg) NP ( 5 ) [ (hC) YKF(hC) ] ⁴E FA065 210 2.5 ± 0,10 NGY (Chg) NP (5) [ (hC) YKF (hC) ] ⁴E FA066 211 2,2 ± 0,10 NGYENP (5) [ (hC) FKF (hC) ] ⁴E FA032 212 1 ,2 ± 0,10 NGYENP (5) [ (hC) YRF (hC) ] ⁴E FA033 213 0,5 ± 0,10 NGYENP (5) [ (hC) Y (hGln) F (hC) ] ⁴E FA050 214 1.5 ± 0,20 NGYENP (5) [ (hC) Y (Aad) F (hC) ] ⁴E FA051 215 2.5 ± 0,30 NGYENP (5) [ (hC) Y (Api) F (hC) ] ⁴E FA052 216 2.8 ± 0,50 NGYENP (5) [ (hC) Y (Cit) F (hC) ] ⁴E FA056 217 2,1 ± 0,10 NGYENP (5) [ (hC) YK (hPhe) (hC) ] ⁴E FA042 218 3,0 ± 0,30 NGYENP (5) [ (hC) YK (Phe-2, 4-Cl₂) (hC) ] ⁴E FA043 219 1.4 ± 0,20 NGYENP (5) [ (hC) YK (INal) (hC) ] ⁴E FA057 220 2.7 ± 0,30 NGYENP (5) [ (hC) YK (Phe-4-F) (hC) ] ⁴E FA059 221 3,0 ± 0,20 NGYENP (5) [ (hC) YKF (hC) ] ⁴A FA047 222 2.6 ± 0, 10 (NMe-A) GYINP (5) [ (hC) YKF (hC) ] ⁴E FA067 223 1.5 ± 0,20 (NMe-A) GYINP (5) [ (hC) FKF (hC) ] ⁴E FA068 224 n.d. (NMe-A) GYINP (5) [ (hC) FRF (hC) ] ⁴E FA069 225 n.d. (Mde-A) GYINP (5) [ (hC) YK (Phe-2, 4-Cl₂) (hC) ] ⁴E FA070 226 n.d.

(NMe-A) GYENP (5) [ (hC) FKF (hC) ] ⁴E FA079 227 1.6 ± 0, 10 (NMe-A) GYENP (5) [ (hC) YRF (hC) ] ⁴E FA080 228 1 ,6 ± 0,20 (Mde-A) GYENP (5) [ (hC) YK (Phe-2, 4-Cl₂) (hC) ] ⁴E FA081 229 2.9 ± 0,30

NGYINP (5) [ (hC) FKF (hC) ] ⁴E FA087 230 1.7 ± 0,10 NGYINP (5) [ (hC) YRF (hC) ] ⁴E FA088 231 2,0 ± 0,30 NGYINP (5) [ (hC) YK (Phe-2, 4-Cl₂) (hC) ] ⁴E FA089 232 3,4 ± 0,20 NGYENP (5) [ (hC) FRF (hC) ] ⁴E FA090 233 1 ,0 ± 0,20 NGYENP (5) [ (hC) FK (Phe-2, 4-Cl₂) (hC) ] ⁴E FA091 234 1 ,7 ± 0,20 NGYENP (5) [ (hC) YR ( Phe-2 , 4-Cl₂) (hC) ] ⁴E FA092 235 2,2 ± 0,10

(hC) - L-homo-cysteine; (5)[xxxxx]⁴ - indicates cyclization via X-link between thiols using linker (5); for structures of subsitutions (...) consult Example 11 ; data represented as n ³ 3.

Table 15: Binding affinities (Ki), presented as mean ± SEM, for N- and C-terminal modified cyclic peptides.

Sequence Ligand SEQ ID NO: Ki (mM)

NGYENPTYKFFE 3 3 4,0 ± 0,10

Ac-NGYENPTYKFFE-NH₂ CB539 236

Ac-NGYENP [KYKFD] ⁴E-NH₂ CB520 237

Ac-NGYENP [ (S₅) YKF (S₅) ] ²E-NH₂ CB499 238 3.48 ± 0.07 Ac-NGYENP [ (Lys^a) YKF (Pra) ] ³E-NH₂ FA017b 239

Ac-NGYENP (5) [ (hC) YKF (hC) ] ⁴E-NH₂ FA034 240 Table 16: Binding affinities (K), presented as mean ± SEM, for N- and C-terminal modified cyclic peptides containing charge optimized substitutions.

Sequence _ Ligand _ SEQ ID NO: _ Ki (mM)

_ NGYENPTYKFFE _ 3 _ 3 _ 4,0 ± 0,10

AC-RGYENPTYRFFQ-NH₂ CB580 24Ϊ 27,2 ± 4,30

Ac-RGYENP [ KYRFD ] ⁴Q-NH₂ CB581 242 9,4 ± 1 ,10

Ac-RGYENP [ ( S₅ ) YRF ( S₅ ) ] ²Q-NH₂ CB582 243 9,4 ± 0,60

Ac-RGYENP [ ( Lys^a) YRF ( Pra ) ] ³Q-NH₂ CB583 244 5,8 ± 0,30

Ac-RGYENP ( 5 ) [ ( hC ) YRF ( hC ) ] ⁴Q-NH₂ CB584 245 2,2 ± 0,20

In conclusion, by introducing specific mutations in positions Xi, X₂, X₃, O, X₄, X₅, C_Q, X_Z and Xe the affinity for PARM of Mint2 can be increased. The combination of selected mutations led to the development if ligand 83 (SEQ ID: 83) for which the affinity was determined using isothermal titration calorimetry (ITC, see example 4). Further, by employing different side chain-to-side chain cyclization modes in combination with specific amino acid substitutions, the affinity can be further enhanced and an optimal balance between affinity and other pharmacologically relevant parameters can be achieved.

Example 4: Affinity to PARM of Mint2 measured with isothermal titration calorimetrie (ITC)

The affinity of ligands 3 (SEQ ID: 3) and 83 (SEQ ID: 83) towards PARM of Mint2 was determined using isothermal titration calorimetrie (ITC) (Table 17). Experiments were conducted at 25 °C and 1 ,000 r.p.m. stirring and designed so that C values were generally within 10-200 (C value = K_A * [protein] ^c N, wherein _A is estimated equilibrium association constant; [protein] is protein concentration in the cell; N is stoichiometry). Ligand-to-buffer titrations were performed to subtract the heat produced by injection, mixing and dilution. The binding enthalpy was directly measured, while dissociation constant ( K_D ) and stoichiometry ( N = 7) were obtained by data analysis (Figure 3).

Table 17: Binding affinities (K_d), presented as mean ± SEM, of ligands 3 and 83 from ITC.

Sequence Ligand SEQ ID NO: ITC

Kd [nM]

NGYENPTYKFFE 3 ^d 2400

D ] ( INAL ) ( INAL ) e 83 83 30 ± 8

CB433 112 5770 ± 370 ) ] ²E CB447a 128 240 ± 50 ( Pra ) ] ³E CB463b 170 1000 ± 110

F ( hC ) ] ⁴E FA026 201 1040 ± 100 c - indicates side-chain to side-chain cyclization.

In conclucsion, example 4 demonstrates a method of assessing the affinity of ligands to PARM of Mint2 characterized by low nM affinity.

Example 5: In vitro plasma stability of ligand 3 (SEQ ID: 3) and 83 (SEQ ID: 83)

The in vitro stability of ligand 3 (SEQ ID: 3) and 83 (SEQ ID: 83) in human plasma was determined by incubating 250 mM ligand in undiluted plasma at 37 °C for 0-120 minutes or for 0-24 h, depending on stability, see Table 18. At selected timepoints during the incubation, the ligands were extracted from 45 mI_ plasma by pretreatment with 50 mI_ 6 M urea for 10 min at 4 °C followed by addition of 50 mI_ 20% trichloroacetic acid (TCA) in water and incubation at 4 °C for 10 min. The samples were centrifuged at 13400 rpm for 15 min. The supernatant was filtered and analysed by UPLC to determine the amount of ligand remaining (Figure 4). Stability of ligand 3 (SEQ ID NO: 3) in PBS was included as a control (Figure 4).

Table 18: In vitro plasma stability, presented as mean ± SEM, of ligand 3 (SEQ ID: 3) and 83 (SEQ ID:

83).

Sequence Compound Plasma Stability

T [min]

NGYENPTYKFFE 3 26 ± 11

( fe-a) GYc [KNPTYD] (INAL) (lNAL)e 83 ³ 1440 c - indicates side-chain to side-chain cyclization; data represented as n ³ 3.

In conclusion, example 5 demonstrates a method of assessing the stability in human plasma in vitro. Ligand 83 (SEQ ID NO: 83) comprising non-canonical AA, D-AA, N- methylated AA and side-chain to side-chain cyclization has increased stability and half- life compared to the natural ligand 3 (SEQ ID: 3).

Example 6: In vitro hepatic clearance of ligands 3 (SEQ ID: 3) and 83 (SEQ ID: 83)

The in vitro stability of ligand 3 (SEQ ID: 3) and 83 (SEQ ID: 83) in mouse microsomes (BALB-C) was determined by incubating 0.5 mg/mL mouse microsomes with 3 mM MgCh, 1 mM NADPH and 5 mM ligand at 37 °C for 0, 5, 15, 30, 45 and 60 minutes, see Table 1 1. At selected timepoints during the incubation, the ligands were extracted from the microsomes by pretreatment with 100 pL acetonitrile (ACN) for 15 min at 4 °C. The samples were centrifuged at 13400 rpm for 15 minutes. The supernatant was filtered and analysed by UPLC to determine the amount ligand remaining (Figure 5). Hepatic clearance of Propranolol (a) and of ligands 3 and 83 at conditions containing no co-factor NADPH (b) or using heat-deactivated ezymes (c) were included as positive and negative controls, respectively (Figure 5).

Table 19: In vitro plasma stability, presented as mean ± SEM, of ligand 3 (SEQ ID: 3) and 83 (SEQ ID:

83).

Sequence Compound Clearance

CL(int)

[pL/(mg*min)]

NGYENPTYKFFE 3 15,4 ± 2,5

( fe-a) GYc [KNPTYD] (INAL) (lNAL)e 83 >60 c - indicates side-chain to side-chain cyclization; data represented as n ³ 3.

In conclusion, example 6 demonstrates a method of assessing the stability and hepatic clearance in mouse liver microsomes in vitro. Ligand 83 (SEQ ID NO: 83) comprising non-canonical AA, D-AA, /V-methylated AA and side-chain to side-chain cyclization has increased stability and half-life compared to the natural ligand 3 (SEQ ID: 3).

Example 7: Pull down of Mint2 from primary neuron cultures using ligands 84 (SEQ ID: 84) and 85 (SEQ ID: 85)

Pull-down experiments were performed using lysate from primary neuron cultures. Neurons were cultured from a mouse line carrying triple floxed Mint1/Mint2/Mint3, allowing for knock out of all Mint isoforms by treatment with cre-virus. Ligands 3 (SEQ ID: 3) and 83 (SEQ ID: 83) were /V-terminally modified by attaching cysteine via a PEG2- linker (Table 12). Ligands 84 (SEQ ID: 84) and 85 (SEQ ID: 85) were loaded to magnetic beads coated with epoxy groups (Dynabeads M-270 Epoxy resin) and exposed to lysate of cultured primary neurons either expressing Mint2 or not expressing Mint2. Protein bound to the ligand-loaded beads was washed off the beads, loaded onto a SDS-PAGE gel followed by Western blotting for Mint2 using anti-Mint2 anitbody (see Figure 6).

Table 20: Sequence of ligand 84 (SEQ ID: 84) and 85 (SEQ ID: 85) used for pull down experiments.

Sequence Ligand SEQ ID NO: Parent

Sequence

c - indicates side-chain to side-chain cyclization. In conclusion, example 7 demonstrates a method of confirming the interaction of ligand 85 (SEQ ID: 85) with Mint2 under complex in vitro conditions using lysate of cultured primary neurons. Noteworthy, ligand 84 (SEQ ID NO: 84), resembling the wild type ligand 3 (SEQ ID: 3), was not able to pull down Mint2 under the same conditions. This highlights the importance of the significantly improved affinity of lead ligand 83 (SEQ ID: 83) to efficiently interact with Mint2. Further, this data strongly suggests that ligands derived from ligand 83 (SEQ ID: 83) are able to inhibit protein-protein interaction in living neurons. Example 8: Inhibition of Ab formation by treatment with ligand 86 (SEQ ID: 86) and 87 (SEQ ID NO: 87)

As Mint2 is an intracellular target, a series of CPP-tagged peptide ligands 86 to 91 (SEQ ID: 86 to 91 ) were synthesised (Table 21 ). Table 21 : Sequence of ligand 86 (SEQ ID: 86) to 91 (SEQ ID: 91 ) used for Ab inhibiton assay.

SEQ ID

Sequence Ligand

Ac-YGRKKRRQRRR-NGYENPTYKFFE 86 86

Ac-YGRKKRRQRRR- (I\ZMe-a) GYc [ KNPTYD] (INAL) (lNAL)e 87 87

Ac-rRrGrKkRr- (I\ZMe-a) GYc [ KNPTYD] (INAL) (lNAL)e 88 88

Ac-RRRRRRRRR- (I\ZMe-a) GYc [ KNPTYD] (INAL) (lNAL)e 89 89

Ac-frrrsyslrr- ( Me-a) GYc [KNPTYD] (INAL) (INAL) e 90 90 c [ DLATEPAK (DAP) ] - (I\ZMe-

91 91 a) GYc[KNPTYD] (INAL) ( INAL) e

c - indicates side-chain to side-chain cyclization.

The effect of ligands 86 (SEQ ID: 86) and 87 (SEQ ID: 87) on Ab₄₂ formation was assessed using an in vitro model of Alzheimer’s disease. Dissociated high-density hippocampal and neocortical neurons (MFT neurons) were prepared from new born mice carrying the APPswe/Ps1 dE9 double mutation resulting in overproduction of human Ab. Neurons were incubated for 24 h with ligand 87 (SEQ ID: 87) (10, 5 and 1 mM), ligand 86 (SEQ ID: 86) (50 mM), DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester; g-secretase inhibitor, 20 mM) and vehicle (0.5% DMSO). The amount of Ab₄₂ in the cell media was analyzed using a commercial Ab ELISA kit from Invitrogen (Human Ab42 Ultrasensitive ELISA Kit, Cat. No. KHB3544).

Incubation of neurons with ligand 87 (SEQ ID: 87) at 10 mM resulted in a 31% reduced extracellular Ab concentration compared to the vehicle treated control. Importantly, incubation with 5-fold higher concentration of wild-type ligand 86 (SEQ ID: 86) (50 mM) has no effect on Ab-formation (Figure 7).

In conclusion, example 8 demonstrates a method of assessing the inhibition of Ab₄₂ formation by treatment of ligands 86 and 87 (SEQ ID NO: 87 and 87). Ligands 87 (SEQ ID NO: 87), but not ligand 86 (SEQ ID NO: 86) was able to inhibit Ab₄₂ formation and is hence a drug lead for treatment of Alzheimer’s disease by targeting the intracellular protein Mint2. Example 9: Diagnostic method

A correlation between the expression of Mint proteins in Ab plaques and AD diagnosis has been reported (Jacobs, E. H. et al. 2006). Accordingly, a ligand capable of binding to Mintl and/or Mint2 can be of use for detection of Ab plaques and therefore AD. Ligand 1 , 2 or 83 (SEQ ID NO: 1 , 2 or 83) labelled with ¹²⁵l-indodicarbocyanine, indodicarbocyanine, ¹²⁵l-indotricarbocyanine or indotricarbocyanine are used to monitor Mintl and/or Mint2 expression and Ab plaque load. The ligands of the invention are injected intravenously in an AD mouse model. The fluorescence and radioactivity signals are monitored over time. The quantification and biodistribution parameters are determined. Cryo-preserved brain tissue is used for histopathology analysis of AD progression, by staining the brain sections with ligand 1 or 2 (SEQ ID NO: 1 or 2). First, the sections are washed in PBS, followed by incubation with ligand 1 or 2 (SEQ ID NO: 1 or 2) e.g. in PBS overnight at 4°C and additional washing with PBS is conducted. The slides are analysed using fluorescent microscopy.

The results from this study will demonstrate if a certain ligand is useful for detecting amyloid plaques and/or for the diagnosis of AD progression.

Example 10: List of compounds

c - indicates side-chain to side-chain cyclization; Ac - acetylated N-terminus; (pY) - O-Phospho-L- tyrosine; 1 NAL - L-3-(1-naphthyl)alanine; DAP - L-2,3-diaminopropionic acid; DAB - L-2,4-diaminobutyric acid; ORN - L-ornithine; 2ABU - L-2-aminobutyric acid; NVA - L-norvaline; IGL - 2-indanyl-L-glycine; 2NAL - L-3-(2-naphthyl)alanine; A/Me-n - /V-methyl-D-asparagine; A/Me-a - A/-methyl-D-alanine; PEG2 - (2-(2-aminoethoxy)ethoxy)acetic acid.

The following table lists a series of cyclized peptides according to the present disclosure.

The structural definitions of the non-canonical amino acids presented in the above table can be found in the following Example 1 1. The abbreviations for the non- canonical amino acids are surrounded by“()” brackets to indicate the prescence of a non-canonical amino acid. The brackets“[ ]^x”- indicate side-chain to side-chain cyclization, wherein the“^x” specifies the cyclization chemistry: x = 1 is lactam cyclization, x = 2 is ring closing metathesis (RCM), x = 3 is copper(l)-catalyzed azide alkyne cycloaddition (CuAAC), x = 4 is cross-linking of thiols based on linkers (1 )-(6), and x = 5 is cross-linking of thiols based on linkers (7)-(8). The type of linker used in the cyclization are specified by the bracketed number (1 )-(8) in front of the cyclization symbols“[ ]”.

Example 11 : List of non-canonical amino acids and linkers

Example 12: In vitro proteolytic stability of ligands

The in vitro proteolytic stability of the ligands was determined by incubating 250 mM ligand in phosphate buffered saline (PBS) supplemented with Trypsin, and/or protease from strep griseus, and/or proteinase K (0.025 mg/ml_) at 37 °C for 0-120 minutes, see Table 18. At selected timepoints during the incubation, the ligands were extracted from 20 mI_ of assay matrix by treatment with 180 mI_ of 50% acetonitrile (ACN). The sample was filtered and analysed by UPLC to determine the amount of ligand remaining. For the ligands a negative control in PBS was performed treated as described above. LCMS analysis was performed to confirm ligend integrity for compounds with T1/2 > 120 min.

Table 22: In vitro proteolytic stability, presented as mean ± SEM.

Trypsin Stability

Sequence Ligand SEQ ID NO:

T 1/2 [min]

NGYENPTYKFFE 3 3 48.6 ± 1 .9

NGYENP [KYKFE] ²E CB441 r >120 NGYENP [EYKFK] ²E CB444 1 1 1 >120

NGYENP [KYKFD] ²E CB433 ΪT2 >120

NGYENPKYKFDE CB524 1 13 >120 NGYENP [ (Orn) YKFE] ²E CB449 1 14 >120

NGYENP (Orn) YKFEE CB525 1 15 >120 NGYENP [EYKF (Orn) ] ²E CB450 1 16 >120

NGYENPEYKF (Orn) E CB526 1 17 >120 NGYENP [DYKFK] ²E CB451 1 18 >120

NGYENPDYKFKE CB527 1 19 >120

NGYENP [ (Orn) YKFD] ²E CB521 120 >120 NGYENP [ (Dab) YKFE] ²E CB442 121 >120 NGYENP [EYKF (Dab) ] ²E CB445 122 >120 NGYENP [DYKF (Orn) ] ²E CB522 123 >120

NGYENP [ (Dab) YKFD] ²E CB443 124 >120 NGYENP [DYKF (Dab) ] ²E CB446 125 >120

(Dab) - L-2,4-diaminobutyric acid; (Orn) - L-ornithine; [ ]¹ - indicates lactam side-chain to side-chain cyclization.

Table 23: In vitro proteolytic stability, presented as mean ± SEM.

Sequence Ligand SEQ ID NO: Trypsin Stability

NGYENPTYKFFE 3 3 48.6 ± 1 .9

NGYENP [ (R₅) YK (S₅) ] ²FE CB437a 126

NGYENP (R₅) YK (S₅) FE CB437b 127

NGYENP [ ( S_{5 )} YKF ( S_{5 )} ] ²E CB447a Ϊ28

NGYENP (S₅) YKF (S₅) E CB447b 129

NGYENPT [ ( S₅ ) KFF ( S₅ ) ] ² CB448a 130

NGYENPT (S₅) KFF (S₅) CB448b 131

NGYENP [ ( S₈ ) YKF ( S₅ ) ] ²E FA018b1 Ϊ32

NGYENP [ ( S₈ ) YKF ( S₅ ) ] ²E FA018b2 133

NGYENP (S₈) YKF (S₅) E FA018a 134

NGYENP [ ( S₅ ) YKF ( S₈ ) ] ²E FA019b1 135

NGYENP [ ( S₅ ) YKF ( S₈ ) ] ²E FA019b2 136

NGYENP (S₅) YKF (S₈) E FA019a 137

NGYENP [ (S₈) YKF (S₈) ] ²E FA020b 138

NGYENP (S₈) YKF (S₈) E FA020a 139

NGYENP [ (S₅) YKF (S₅) ] ²E-N¾ CB544 140

NGYENP (S₅) YKF (S₅) E-NH₂ CB545 141

Ac-NGYENP [ (S₅) YKF (S₅) ] ²E-N¾ CB548 142

Ac-NGYENP (S₅) YKF (S₅) E-NH₂ CB549 143

(R5) - (R)-N-Fmoc-a-4-n-pentenylalanine; (S5) - (S)-N-Fmoc-a-4-n-pentenylalanine; (Se) - (S)-N-Fmoc-a- (7-Octenyl)alanine; [ ]² - indicates all-hydrocarbon side-chain to side-chain cyclization using RCM. Table 24: In vitro proteolytic stability, presented as mean ± SEM.

Trypsin Stability

Sequence Ligand SEQ ID NO:

T 1/2 [min]

NGYENPTYKFFE 3 3 48.6 ± 1.9

NGYENP [ (Lys^a) YKF (Pra) ] ³E CB463b 170 >120

NGYENP (Lys^a) YKF (Pra) E CB463a 171 66

NGYENP [ (Pra) YKF (Lys^a) ] ³E CB464b 172 >120

NGYENP (Pra) YKF (Lys^a) E CB464a 173 22 NGYENP [ (Orn^a) YKF (hPra) ] ³E CB496b 174 >120

NGYENP (Orn^a) YKF (hPra) E CB496a 175 15

NGYENP [ (hPra) YKF (Orn^a) ] ³E CB497b 176 >120

NGYENP (hPra) YKF (Orn^a) E CB497a 177 17 NGYENP [ (Orn^a) YKF (Pra) ] ³E CB490b 178 >120

NGYENP (Orn^a) YKF (Pra) E CB490a 179 71 NGYENP [ (Pra) YKF (Orn^a) ] ³E CB491 b 180 >120

NGYENP (Pra) YKF (Orn^a) E CB491a 181 31

(Lys^a) - (S)-2-amino-6-azido-hexanoic acid; (Orn^a) - (S)-2-amino-5-azido-pentanoic acid; (Pra) - L-propargylglycine; (hPra) - L-homo-propargylglycine; [ ]³ - indicates triazole cyclization using CuAAC.

Table 25: In vitro proteolytic stability, presented as mean ± SEM.

Sequence Ligand SEQ ID NO: Trypsin Stability

NGYENPTYKFFE 3 3 48.6 ± 1.9

NGYENPCYKFCE FA001 182

NGYENP (1) [CYKFC] ⁴E FA001J 183

NGYENP (2) [CYKFC] ⁴E FA001_2 184

NGYENP (3) [CYKFC] ⁴E FA001_3 185

NGYENP (4) [CYKFC] ⁴E FA001_4 186

NGYENP (5) [CYKFC] ⁴E FA001_5 187

NGYENP (6) [CYKFC] ⁴E FA001_6 188

NGYENP (7) [CYKFC] ⁵E FA001_7 189

NGYENP (8) [CYKFC] ⁵E FA001_8 190

NGYENP (5) [cYKFC] ⁴E FA013 191

NGYENPcYKFCE CB509 192

NGYENP (5) [CYKFc] ⁴E FA014 193

NGYENPCYKFCE CB510 194

NGYENP (5) [cYKFc] ⁴E FA015 195

NGYENPcYKFCE CB511 196

NGYENP (5) [ (hC) YKFC] ⁴E FA024 197

NGYENP (hC) YKFCE FA021 198

NGYENP (5) [CYKF(hC) ] ⁴E FA025 199

NGYENPCYKF (hC) E FA022 200

NGYENP (5) [ (hC) YKF (hC) ] ⁴E FA026 201

NGYENP (hC) YKF (hC) E FA023 202

(hC) - L-homo-cysteine; (#)[ ]⁴ - indicates cyclization via X-link between thiols using linkers (1) to (6), wherein (#) is (1 ) to (6); (#)[ ]⁵ - indicates cyclization via X-link between thiols using linkers (7) and (8), wherein (#) is (7) to (8). Table 26: In vitro proteolytic stability determined for protease from strep griseus, presented as mean ± SEM.

Sequence Ligand SEQ ID NO: T1/2 [min]

NGYENPTYKFFE 3 3 19.6 ± 3.4

NGYENP [KYKFD] ⁴E CB433 Ϊ12 27

NGYENP[ (S_{5) YKF(Ss) ]} ²E CB447a 128 >120

NGYENP [ (Lys^a) YKF (Pra) ] ³E CB463b 170 20

NGYENP (5) [ (hC)YKF(hC) ]⁴E FA026 201 28

Table 27: In vitro proteolytic stability determined for proteinase K, presented as mean ± SEM.

Sequence Ligand SEQ ID NO: T1/2 [min]

NGYENPTYKFFE 3 3 17.6 ± 1.8

NGYENP [KYKFD] ⁴E CB433 Ϊ12 >120

NGYENP [ (Ss) YKF(Ss) ] ²E CB447a 128 >120

NGYENP [ (Lys^a) YKF (Pra) ] ³E CB463b 170 >120

NGYENP (5) [ (hC) YKF (hC) ] ⁴E FA026 201 >120

Example 13: General cyclization procedures Synthesis of all-hydrocarbon stapled peptides (Fig. 8d):

Fmoc-S5-OH, Fmoc-S₈-OH and Fmoc-Rs-OH were employed for incorporation of non- canonical amino acids at positions 761 & 764, 761 & 765 and 762 & 766, respectively.

The corresponding linear peptides were synthesized on solid support as described in the procedure of Example 1. The resin was flow-washed with DCM and drained. After swelling of the resin in dichloroethane (DCE) (5 mL, 10 min), it was flow-washed with DCE and drained. Ring-closing metathesis (RCM) was performed on resin by adding Grubb’s catalyst 1st generation (20 mol%) dissolved in DCE (1 mL) under nitrogen atmosphere. After 2 h, the resin was washed with DCE and the RCM reaction repeated. Completion of the cyclization reactions was confirmed by micro test-cleavage in neat TFA. The cyclized peptides were cleaved and purified as described in Example 1 . Synthesis of triazole stapled peptides (Fig. 8e):

Fmoc-(Lys^a)-OH, Fmoc-(Orn^a)-OH, Fmoc-(Pra)-OH and Fmoc-(hPra)-OH were employed for incorporation of non-canonical amino acids at positions 761 & 765, respectively.

The corresponding linear peptides were synthesized on solid support as described in the Example 1. The resin was flow-washed with DMF, DCM and drained. After swelling of the resin in DMF (5 ml_, 10 min), it was flow-washed with DMF and drained. CuAAC was performed on resin by adding DIPEA (10 eq.), 2,5-lutidine (10 eq.), sodium ascorbate (3 eq.) dissolved in degassed DMF (1 ml.) and CuBr (1 eq.) dissolved in degassed ACN (200 mI_) under nitrogen atmosphere. After overnight agitation, the resin was washed with DMF and DCM. Completion of the CuAAC cyclization reactions was confirmed by micro test-cleavage in neat TFA. The cyclized peptides were cleaved and purified as described in Example 1.

Synthesis of thiol-crosslink stapled peptides with linkers L1 -L6 (Fig. 8f):

Fmoc-L-Cys-OH, Fmoc-D-Cys-OH and Fmoc-L-(hCys)-OH were employed for incorporation of non-canonical amino acids at positions 761 & 765, respectively.

The linear peptides were synthesized on solid support as described in Example 1. The resin was flow-washed with DMF, DCM, drained, cleaved and purified as described in Example 1. The stapled peptides were obtained by in solution cyclization using linkers L1-L6 (also referred to as (1 ) - (6)). A linear peptide (1.5 mg/ml_) was dissolved in NH4HCO3 buffer (100 mM, pH 8.0) and pretreated with TCEP (1.5 eq.) for 3 h. A stock solution of the respective crosslinker in DMF (3.0 eq.) was added and the solution agitated for 5 h. After acidification of the reaction mixture (pH 2.0), completion of the cyclization reactions was confirmed by LC-MS analysis. The cyclized peptides were lyophilized and purified as described in Example 1.

Synthesis of thiol-crosslink stapled peptides with linkers L7-L8 (Fig. 8g):

The linear peptides were synthesized on solid support as described in Example 1. The resin was flow-washed with DMF, DCM, drained, cleaved and purified

as described in Example 1. The stapled peptides were obtained by in solution cyclization using linkers L7 and L8. A linear peptide (1.5 mg/ml_) was dissolved in Tris base (50 mM) in DMF. A stock solution of the respective perfluoroarene crosslinker in DMF (10.0 eq.) was added and the solution agitated for 4.5 h. After acidification of the reaction mixture (pH 2.0), completion of the cyclization reactions was confirmed by LC-MS analysis. The cyclized peptides were lyophilized

and purified as described in Example 1.

Example 14: Link between disruption of the APP-Mint interaction and reduction Ab production in vivo

An optimized peptide termed Tat-opAPPc, comprising twelve C-terminal amino acids of APP and optimized towars Mint2 acts by perturbing the protein-protein interactions of Mint2, a neuronal scaffolding protein that links APP and gamma-secretase to the formation of neurotoxic Ab peptides. Modulation of the APP-Mint2 interaction and treatment with Tat-opAPPc is neuroprotective and reduces Ab levels in rodent models of Alzheimer’s disease.

To determine whether the Tat-opAPPc peptide could be delivered into the brain in the intact animal, C57BL/6 mice (25 g) is injected intraperitoneally with a 500 mM dose of Tat-opAPPc peptide, scrambled Tat-opAPPc peptide or Tat-opAPPc (N759A) . Saggital brain sections is taken 1 h after injection and examined by confocal microscopy to detect fluorescent peptide uptake. Preliminary results indicate that brains from animals injected with the peptide exhibited fluorescence in the brain confirming that the Tat-peptides enter the brain upon peripheral administration. An additional quantitative approach to study brain penetration in vivo is the determination of the brain/plasma ratio whereby both plasma and brain tissue are sampled at 3-5 time points up to 24 h after dosing. Plasma is separated by centrifugation and stored at -80°C before analysis. The brain tissue is homogenized and precipitated and the total brain concentration of the compound is determined by HPLC/mass spectrometry and related to its concentration in plasma.

To determine the effect of the Tat-opAPPc peptide in vivo, a dose-response experiment (ranging from 1 -100 mg/kg) is performed 24 h after dosing in APP/PS1 transgenic mice (6 months of age). This time point was deemed most favorable from previous pharmacokinetics of dose related-effects for b-secretase inhibitors. To evaluate whether high enough doses can be achieved in vivo, 60 mg/kg of Tat-opAPPc or scrambled Tat- opAPPc peptide is injected intraperitoneally into APP/PS1 transgenic mice at 8 month of age.

Delivery of Tat-Mint2-o3 mimetic peptide in vivo selectively reduces soluble Ab42 levels by 41 % compared to mice that received scrambled peptide within 24 h. These preliminary results suggest that Tat-opAPPc peptide has profound effects on Ab production in vivo.

Our in vivo studies aim to decrease Ab production in mouse models of AD. To further extend this finding, plasma, CSF and brain samples is collected at specific time points up to 24 h after dosing. Brain concentrations of the Tat-opAPPc peptide is measured by immunoblotting with anti-FITC antibody and by HPLC/mass spectrometry. The plasma and brain concentrations of the Tat-opAPPc peptide increases in a dose-dependent manner over the dose ranged tested. Ab40 and Ab42 levels is measured. As controls, the scrambled Tat-opAPPc peptide or Tat-opAPPc N759A peptide should not have any effect on Ab levels. The results from the dose-response experiment provides the ED₅o values for the effect of the Tat-opAPPc peptide on the inhibition of Ab production in brain. By plotting Ab levels as a function of plasma and brain drug concentrations, the drug concentration required to inhibit brain Ab by 50% (ECso) will be calculated. Once the dose has been determined, we will next examine the half-life of the Tat-opAPPc peptide. The Tat-opAPPc peptide will be administered intravenously and the peptide concentration will be measured up to 24 h post-dose by HPLC/mass spectrometry. If the peptide exhibits a low brain clearance, which translates to a long half-life, this will demonstrate that the peptide is suitable for once-a-day dosing.

To examine the changes of Ab production in vivo upon chronic dosing of the Tat-opAPPc peptide, Ab reduction is studied after a single, acute dose and after chronic (1 week) dosing of once-a-day. Mutant APP/PS1 mice at 6 months of age is injected with saline or Tat-peptide bolus (Tat-opAPPc, scrambled Tat-opAPPc or Tat-opAPPc-N759A ). Mice is culled immediately 24 h after the last dose. One half of the brain is collected for biochemistry and the other half for morphological assessment. For visualization and quantification of Ab deposits, a series of brain sections is systematically collected (i.e., every third saggital section from a complete series with the starting section chosen at random) and stain with Ab42 antibody (U6590) that has been previously characterized. The Ab deposition is quantified based on morphological classifications that include diffuse and compact, the latter of which is frequently associated with neuritic changes. To complement the morphological analysis, we biochemically examine soluble, membrane and insoluble fractions of Ab40 and Ab42 levels in the cortex and hippocampus by ELISA. It is important to quantify the processing of APP by immunoblotting for total APP, secreted APP ectodomain and APP-CTFs, which are substrates for secretase-mediated proteolysis of APP. The control peptides do not have a measurable effect on Ab levels. If we successfully decrease Ab levels within 1 week, we will further examine the effects of Tat-opAPPc peptide in a longer time treatment (2- 4 weeks). We will also evaluate the effect of chronic administration of Tat-opAPPc peptide on Ab levels in aged mutant APP/PS1 mice whereby mice will be dosed from 12 months of age for 3 months.

Pitfalls and alternative approaches: We expect that introducing into cells an exogenous peptide containing the optimized C-terminal amino acids of APP will have profound effects on APP binding and processing events in vivo. However, quantification of Ab levels may be a problem due to different factors such as genetic background, sex or environment. To reduce the genetic and experimental variability, we use a hybrid genetic background and age- and sex-matched littermate pairs that are housed in similar environments to minimize discrepancies. We will also examine whether the Tat- peptide has any off-target effects that are not directly related to the neuropathology of AD by examining histopathology sections of several peripheral tissues such as the ileum, thymus and spleen.

References

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Claims

1. A compound (Pi) comprising at least twelve amide-bonded proteinogenic or non- proteinogenic amino acid residues of the sequence X1GX2X3NPOX4X5X6X7X8 (SEQ ID NO: 248), wherein:

Xi is selected from the group consisting of L-asparagine (N), D-histidine (h), L- histidine (H), D-asparagine (n), L-alanine (A), D- alanine (a), /V-methyl-L- asparagine (L/Me-N), /V-methyl-D-asparagine (L/Me-n), L-arginine (R), /V-methyl-L- alanine (L/Me-A), and /V-methyl-D-alanine (L/Me-a) ;

X2 is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1- naphthyl)alanine (1 Nal), 2-indanyl-L-glycine (Igl), (S)-2-amino-3-(3,4- dichlorophenyl)propionic acid (Phe-3,4-Cl₂), 2,4-dichloro-L-phenylalanine (Phe-2,4- CI2), 4-fluoro-L-phenylalanine (Phe-4-F), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4- pyridyl)-L-alanine (4Pal), L-phenylalanine (F), L-homophenylalanine (hPhe), L-3- (2-naphthyl)alanine (2Nal), 2-fluoro-L-phenylalanine (Phe-2-F), 3-fluoro-L- phenylalanine (Phe-3-F), 3,4-difluoro-L-phenylalanine (Phe-3,4-F₂), 2-chloro-L- phenylalanine (Phe-2-CI), 3-chloro-L-phenylalanine (Phe-3-CI), 3-iodo-L- phenylalanine (Phe-3-l) and 4-iodo-L-phenylalanine (Phe-4-l);

X₃ is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L-lysine (K), L-ornithine (Orn), (S)-2-amino-heptanedioic acid (Api), L- homoglutamine (hGIn), (S)-2-aminoadipic acid (Aad), L-citrulline (Cit), (R)-2-amino- 3-(3-carboxy-propylsulfanyl)propionic acid (Cpc), 4-(((R)-2-amino-2-carboxyethyl)- sulfinyl)butanoic acid (Cpc^ox), L-cysteine (C), L-fe/f-leucine (Tie), (S)-2- cyclopentylglycine (Cpg), (S)-2-cyclohexylglycine (Chg), and L-isoleucine (I), L-2,3- diaminopropionic acid (Dap), and L-2,4-diaminobutyric acid (Dab);

O is selected from the group consisting of: L-threonine (T), L-lysine (K), L-glutamic acid (E), L-ornithine (Orn), L-aspartic acid (D), L-2,4-diaminobutyric acid (Dab), (R)-2-(4-pentenyl)alanine (R₅), (S)-2-(4-pentenyl)alanine (S₅), (S)-2-(7- octenyl)alanine (Ss), 6-azido-L-lysine (Lys^a), propargylglycine (Pra), 5-azido-L- ornithine (Orn^a), L-homopropargylglycine (hPra), L-cysteine (C), D-cysteine (c), L- 2,3-diaminopropionic acid (Dap), and L-homocysteine (hC); X₄ is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), (S)-2- (4-pentenyl)alanine (Ss), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal) and L-phenylalanine (F);

X₅ is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L-lysine (K), L-ornithine (Orn), L-arginine (R), L-homoglutamine (hGIn), L- homoarginine (hArg), L-homoglutamine (hLys), (S)-2-aminoadipic acid (Aad), (S)-

2-amino-heptanedioic acid (Api), L-citrulline (Cit), and L-2,3-diaminopropionic acid (Dap), L-2,4-diaminobutyric acid (Dab), (S)-2-amino-4-guanidinobutanoic acid (Arb), (S)-2-amino-3-guanidinopropanoic acid (Arp) and L-glutamine(Q);

C_Q is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (Igl), L-3-(1 -naphthyl)alanine (1 Nal), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L- alanine (4Pal), (S)-2-amino-3-(3-iodophenyl)propanoic acid (Phe-3-l), 2-amino-3- (3-chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L-phenylalanine (Phe-2,4- C ), (S)-3-(3-tert-butyl-phenyl)-2-amino-propionic acid (Phe-3-tBu), (S)-3-(4-tert- butyl-phenyl)-2-amino-propionic acid (Phe-4-tBu), (S)-2-(4-pentenyl)alanine (S₅), L- histidine (H), 4-fluoro-L-phenylalanine (Phe-4-F), L-homophenylalanine (hPhe), and L-3-(2-naphthyl)alanine (2Nal);

X₇ is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (Igl), L-3-(1 -naphthyl)alanine (1 Nal), L-glutamic acid (E), L-lysine (K), L-aspartic acid (D), L-ornithine (Orn), L-2,4-diaminobutyric acid (Dab), (S)-2-(4- pentenyl)alanine (S₅), (S)-2-(7-octenyl)alanine (Ss), 3-(3-pyridyl)-L-alanine (3Pal),

3-(4-pyridyl)-L-alanine (4Pal), (S)-2-amino-3-(3-iodophenyl)propanoic acid (Phe-3- I), 2-amino-3-(3-chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L- phenylalanine (Phe-2,4-Cl2), (S)-3-(3-tert-butyl-phenyl)-2-amino-propionic acid (Phe-3-tBu), propargylglycine (Pra), 6-azido-L-lysine (Lys^a), L- homopropargylglycine (hPra), 5-azido-L-ornithine (Orn^a), L-cysteine (C), D- cysteine (c), L-homocysteine (hC), L-3-(2-naphthyl)alanine (2Nal), L- homophenylalanine (hPhe), (S)-2-amino-3-(3,4-dichlorophenyl)propionic acid (Phe-3,4-Cl2), 3,4-difluoro-L-phenylalanine (Phe-3,4-F2) and 4-fluoro-L- phenylalanine (Phe-4-F); and

Xs is selected from the group consisting of L-glutamic acid (E), D-glutamic acid (e), (S)-2-aminoadipic acid (Aad), (S)-2-amino-heptanedioic acid (Api), (S)-2-(4- pentenyl)alanine (Ss), L-alanine (A), L-glutamine (Q),-D-glutamine (q); L- homoglutamine (hGIn), and L-citrulline (Cit); wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), L-3-(1-naphthyl)alanine (1 Nal) and L-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L- phenylalanine (F).

2. The compound according to claim 1 , wherein the compound (Pi) is of the

sequence Xi GX₂X₃N POX4X5X6X7X8 (SEQ ID NO: 249), wherein:

Xi is selected from the group consisting of L-asparagine (N), D-histidine (h), L- alanine (A), L-histidine (H), D-alanine (a), /V-methyl-L-asparagine (L/Me-N), N- methyl-D-alanine (L/Me-a), /V-methyl-L-alanine (L/Me-A), and L-arginine (R);

X₂ is selected from the group consisting of L-tyrosine (Y), L-3-(1-naphthyl)alanine (1 Nal), 2-indanyl-L-glycine (Igl), (S)-2-amino-3-(3,4-dichlorophenyl)propionic acid (Phe-3,4-CI₂), 2,4-dichloro-L-phenylalanine (Phe-2,4-CI2), 4-fluoro-L-phenylalanine (Phe-4-F), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal), L- phenylalanine (F), L-homophenylalanine (hPhe), L-3-(2-naphthyl)alanine (2Nal), 2- fluoro-L-phenylalanine (Phe-2-F), 3-fluoro-L-phenylalanine (Phe-3-F), 3,4-difluoro- L-phenylalanine (Phe-3,4-F₂), 2-chloro-L-phenylalanine (Phe-2-CI), 3-chloro-L- phenylalanine (Phe-3-CI), 3-iodo-L-phenylalanine (Phe-3-l) and 4-iodo-L- phenylalanine (Phe-4-l);

X₃ is selected from the group consisting of L-glutamic acid (E), (S)-2-amino- heptanedioic acid (Api), L-homoglutamine (hGIn), (S)-2-aminoadipic acid (Aad), L- citrulline (Cit), (R)-2-amino-3-(3-carboxy-propylsulfanyl)propionic acid (Cpc),4- (((R)-2-amino-2-carboxyethyl)-sulfinyl)butanoic acid (Cpc^ox), L-cysteine (C), L-tert- leucine (Tie), (S)-2-cyclopentylglycine (Cpg), (S)-2-cyclohexylglycine (Chg), L- isoleucine (I), and L-2,4-diaminobutyric acid (Dab);

O is selected from the group consisting of: L-threonine (T), L-lysine (K), L-glutamic acid (E), L-ornithine (Orn), L-aspartic acid (D), L-2,4-diaminobutyric acid (Dab), (R)-2-(4-pentenyl)alanine (Rs), (S)-2-(4-pentenyl)alanine (Ss), (S)-2-(7- octenyl)alanine (Ss), 6-azido-L-lysine (Lys^a), propargylglycine (Pra), 5-azido-L- ornithine (Orn^a), L-homopropargylglycine (hPra), L-cysteine (C), D-cysteine (c), L-

2.3-diaminopropionic acid (Dap), and L-homocysteine (hC);

X₄ is selected from the group consisting of: L-tyrosine (Y), (S)-2-(4- pentenyl)alanine (Ss), L-phenylalanine (F), 3-(3-pyridyl)-L-alanine (3Pal), and 3-(4- pyridyl)-L-alanine (4Pal);

X₅ is selected from the group consisting of L-lysine (K), L-arginine (R), L- homoglutamine (hGIn), L-homoarginine (hArg), L-homoglutamine (hLys), (S)-2- aminoadipic acid (Aad), (S)-2-amino-heptanedioic acid (Api), L-citrulline (Cit), L-

2.4-diaminobutyric acid (Dab), (S)-2-amino-4-guanidinobutanoic acid (Arb), (S)-2- amino-3-guanidinopropanoic acid (Arp) and L-glutamine(Q);

C_Q is selected from the group consisting of: L-phenylalanine (F), (S)-2-(4- pentenyl)alanine (Ss), L-histidine (H), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L- alanine (4Pal), (S)-2-amino-3-(3-iodophenyl)propanoic acid (Phe-3-l), 2-amino-3- (3-chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L-phenylalanine (Phe-2,4- C ), (S)-3-(3-tert-butyl-phenyl)-2-amino-propionic acid (Phe-3-tBu), (S)-3-(4-tert- butyl-phenyl)-2-amino-propionic acid (Phe-4-tBu), (S)-2-(4-pentenyl)alanine (Ss), L- histidine (H), L-3-(1-naphthyl)alanine (1 Nal), L-homophenylalanine (hPhe), and 4- fluoro-L-phenylalanine (Phe-4-F);

X7 is selected from the group consisting of L-phenylalanine (F), L-glutamic acid (E), L-lysine (K), L-aspartic acid (D), L-ornithine (Orn), L-2,4-diaminobutyric acid (Dab),

(S)-2-(4-pentenyl)alanine (Ss), (S)-2-(7-octenyl)alanine (Ss), l-3-(1-naphthyl)alanine (1 Nal), 3-(3-pyridyl)-L-alanine (3Pal), 3-(4-pyridyl)-L-alanine (4Pal), (S)-2-amino-3- (3-iodophenyl)propanoic acid (Phe-3-l), 2-amino-3-(3-chlorophenyl)propanoic acid (Phe-3-CI), 2,4-dichloro-L-phenylalanine (Phe-2,4-Cl₂), (S)-3-(3-tert-butyl-phenyl)- 2-amino-propionic acid (Phe-3-tBu), propargylglycine (Pra), 6-azido-L-lysine (Lys^a), L-homopropargylglycine (hPra), 5-azido-L-ornithine (Orn^a), L-cysteine (C), D- cysteine (c), L-homophenylalanine (hPhe), (S)-2-amino-3-(3,4- dichlorophenyl)propionic acid (Phe-3,4-Cl₂), 3,4-difluoro-L-phenylalanine (Phe-3,4- F2), and 4-fluoro-L-phenylalanine (Phe-4-F), L-homocysteine (hC); and

Xe is selected from the group consisting of: L-glutamic acid (E), D-glutamic acid (e), (S)-2-aminoadipic acid (Aad), (S)-2-amino-heptanedioic acid (Api), (S)-2-(4- pentenyl)alanine (S₅), L-alanine (A), D-glutamine (q), L-homoglutamine (hGIn), L- citrulline (Cit), and L-glutamine (Q); wherein when X₃ is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), L-3-(1-naphthyl)alanine (1 Nal) and L-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L- phenylalanine (F).

3. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized between at least two of the side chains of the residues selected from the group consisting of X1, X₂, X3, O, X₄, X5 CQ X7, and Xs.

4. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized between O and X₇, cyclized between X₄ and Xs, or cyclized between O and CQ.

5. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized as represented by the following formula: XIGX₂X3NP(#)[OX₄X5X6X7]^XX8, wherein ^x is ¹ to ⁵ representing the cyclization type, wherein:

¹ is cyclization via an amide bond,

² is cyclization via ring-closing metathesis (RCM),

³ is cyclization via copper(l)-catalyzed azide alkyne cycloaddition (CuAAC),

⁴ is cyclization via crosslinking of thiols through linkers (1 ) to (6),

⁵ is cyclization via crosslinking of thiols through linkers (7) to (8); and

wherein (#) is (1 ) to (8) representing the linker, when ^x is ⁴ or ⁵;

wherein (#) is not included when ^x is ¹ to ³;

wherein the wavy bond“¾” indicates the bond formed between the sidechain of a thiol and the linker after crosslinking;

and wherein Xi, G, X₂, X3, O, X₄, X5, CQ, X7, and Xs are defined as in any one of the preceding claims.

6. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized between at least two of the side chains via ring-closing metathesis (RCM).

7. The compound (Pi) according to claim 6, wherein the double bond formed by RCM is mostly (E)-configu ration, mostly (Z)-configuration, or a 1 :1 mixture thereof.

8. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is of the following structure:

Asn Gly Tyr Giu Asn Pro S₅ Tyr Lys Phe S5 Glu

wherein Ss is (S)-2-(4-pentenyl)alanine which has been subjected to RCM.

9. The compound (Pi) according to any one of claims 6 to 8, wherein the double bond formed by RCM is further derivatized, such as by C-H activation through transition metal catalysis or by addition of an electrophile.

10. The compound (Pi) according to any one of the preceding claims,

wherein Xi is selected from the group consisting of: (N- Me-A) and (L/Me-a); wherein X₂ is selected from the group consisting of: (Phe-3,4-CI2) and (Phe-2,4- CI2) or (Phe-F-4);

wherein X₃ is selected from the group consisting of: (Aad), (hGIn) and (Api);

wherein X₄ is selected from the group consisting of: (3Pal) and (4Pal);

wherein X₅ is selected from the group consisting of: (R), (hArg) and (hLys);

wherein Xe is selected from the group consisting of: (e), (Aad) and (Api).

1 1. The compound (Pi) according to any one of the preceding claims, wherein (Pi) consists of the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 247.

12. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized between at least two of the side chains via crosslinking of thiols using any one of linkers (1 ) to (8).

13. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized between at least two of the side chains via copper(l)-catalyzed azide alkyne cycloaddition (CuAAC).

14. The compound (Pi) according to any one of the preceding claims, wherein (Pi) is cyclized between at least two of the side chains via an amide bond.

15. The compound (Pi) according to any one of the preceding claims, wherein (Pi) has an acyl or alkyl on the N-terminus.

16. The compound (Pi) according to claim 15, wherein the acyl is acetyl.

17. The compound (Pi) according to claim 15, wherein the alkyl is methyl.

18. The compound according to claim 1 , comprising at least twelve amide-bonded proteinogenic or non-proteinogenic amino acid residues of the sequence

X1GX2X3NPTX4X5X6X7X8 (SEQ ID: 100), wherein:

Xi is selected from the group consisting of L-asparagine (N), D-asparagine (n), L- alanine (A), D- alanine (a), A/-methyl-D-asparagine (N Me-n) and /V-methyl-D-alanine (L/Me-a);

X₂ is selected from the group consisting of L-tyrosine (Y), L-tryptophan (W), L-3-(1 - naphthyl)alanine (1 NAL) and L-phenylalanine (F);

X3 is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L-lysine (K), L-ornithine (ORN) and L-2,3-diaminopropionic acid (DAP);

X5 is selected from the group consisting of L-glutamic acid (E), L-aspartic acid (D), L-lysine (K), L-ornithine (ORN) and L-2,3-diaminopropionic acid (DAP);

C_Q is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (IGL), L-3-(1 -naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL);

X7 is selected from the group consisting of L-phenylalanine (F), 2-indanyl-L-glycine (IGL), L-3-(1 -naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL); and

Xe is selected from the group consisting of L-glutamic acid (E) and D-glutamic acid

(e); wherein when X3 is L-glutamic acid (E) then X₂ is selected from the group consisting of L-tryptophan (W), L-3-(1-naphthyl)alanine (1 NAL) and L-phenylalanine (F), and/or X₄ is selected from the group consisting of L-tryptophan (W) and L- phenylalanine (F).

19. The compound (Pi) according to any one of the preceding claims, wherein:

X2 is selected from the group consisting of L-tyrosine (Y), and L-3-(1 - naphthyl)alanine (1 NAL); X3 is selected from the group consisting of L-lysine (K) and L-ornithine (ORN);

(D);

C_Q is selected from the group consisting of L-phenylalanine (F), L-3-(1

naphthyl)alanine (1 NAL) and L-3-(2-naphthyl)alanine (2NAL);

X7 is selected from the group consisting of L-phenylalanine (F), L-3-(1- naphthyl)alanine (1 NAL) and i_-3-(2-naphthyl)alanine (2NAL); and

Xs is D-glutamic acid (e).

20. The compound according to any one of the preceding claims, wherein (Pi) is cyclized between the side chains of X3 and X5.

21. The compound according to any one of the preceding claims, wherein the

cyclization is via an amide bond.

22. The compound according to any one of the preceding claims, wherein Pi is

capable of binding to Mint.

23. The compound according to any one of the preceding claims, wherein Pi is

capable of binding to Mintl and/or Mint2.

24. The compound according to any one of the preceding claims, wherein Pi is

capable of binding to Mint2.

25. The compound according to any one of the preceding claims wherein Pi is capable of binding to human Mintl (Uniprot: Q02410).

26. The compound according to any one of the preceding claims wherein Pi is capable of binding to human Mint2 (Uniprot: Q99767).

27. The compound according to any one of the preceding claims wherein Pi is capable of binding to human Mint3 (Uniprot: 096018).

28. The compound according to any one of the preceding claims, wherein Pi

comprises between 12 and 40 amino acids.

29. The compound according to any one of the preceding claims, wherein Pi

comprises at least 12 amino acids residues, such as at least 13 amino acid residues, such as at least 14 amino acid residues, such as at least 15 amino acid residues, such as at least 16 amino acid residues, such as at least 17 amino acid residues, such as at least 18 amino acid residues, such as at least 19 amino acid residues, such as at least 20 amino acid residues, such as at least 21 amino acid residues, such as at least 22 amino acid residues, such as at least 23 amino acid residues, such as at least 24 amino acid residues, such as at least 25 amino acid residues, such as at least 26 amino acid residues, such as at least 27 amino acid residues, such as at least 28 amino acid residues, such as at least 29 amino acid residues, such as at least 30 amino acid residues, such as at least 31 amino acid residues, such as at least 32 amino acid residues, such as at least 33 amino acid residues, such as at least 34 amino acid residues, such as at least 35 amino acid residues, such as at least 36 amino acid residues, such as at least 37 amino acid residues, such as at least 38 amino acid residues, such as at least 39 amino acid residues, such as at least 40 amino acid residues.

30. The compound according to any one of the preceding claims, wherein Pi

comprises no more than 40 amino acid residues, such as no more than 39 amino acid residues, such as no more than 38 amino acid residues, such as no more than 37 amino acid residues, such as no more than 36 amino acid residues, such as no more than 35 amino acid residues, such as no more than 34 amino acid residues, such as no more than 33 amino acid residues, such as no more than 32 amino acid residues, such as no more than 31 amino acid residues, such as no more than 30 amino acid residues, such as no more than 29 amino acid residues, such as no more than 28 amino acid residues, such as no more than 27 amino acid residues, such as no more than 26 amino acid residues, such as no more than 25 amino acid residues, such as no more than 24 amino acid residues, such as no more than 23 amino acid residues, such as no more than 22 amino acid residues, such as no more than 21 amino acid residues, such as no more than 20 amino acid residues, such as no more than 19 amino acid residues, such as no more than 18 amino acid residues, such as no more than 17 amino acid residues, such as no more than 16 amino acid residues, such as no more than 15 amino acid residues, such as no more than 14 amino acid residues, such as no more than 13 amino acid residues, such as no more than 12 amino acid residues.

31. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 83).

32. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[(ORN)NPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 101 ).

33. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[KNPTYE](1 NAL)(1 NAL)e (SEQ ID NO: 102).

34. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[(ORN)NPTYE](1 NAL)(1 NAL)e (SEQ ID NO: 103).

35. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[KNPTYD](2NAL)(2NAL)e (SEQ ID NO: 104).

36. The compound according to any one of the preceding claims, wherein Pi is (L/Me- n)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 105).

37. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[KNPTFD](1 NAL)(1 NAL)e (SEQ ID NO: 106).

38. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)G(1 NAL)c[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 107).

39. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[KNPTYD](1 NAL)Fe (SEQ ID NO: 108).

40. The compound according to any one of the preceding claims, wherein Pi is (L/Me- a)GYc[KNPTYD]F(1 NAL)e (SEQ ID NO: 109).

41. The compound according to any one of the preceding claims, further comprising one or more conjugated moieties.

42. The compound according to any one of the preceding claims, wherein the one or more conjugated moieties are selected from the group consisting of a cell penetrating peptide (CPP), an albumin binding moiety, a detectable moiety, a streptavidin binding moiety, a reactive group and/or a linker (L).

43. The compound according to any one of the preceding claims, wherein said

compound has the generic structure of Formula I:

Z - L - Pi (I) wherein Z is selected from the group consisting of a CPP, an albumin binding moiety, a detectable moiety, a streptavidin binding moiety and a reactive group and L is an optional linker.

44. The compound according to any one of the preceding claims, wherein the

conjugated moiety is a CPP.

45. The compound according to any one of the preceding claims, wherein the CPP has a polycationic structure.

46. The compound according to any one of the preceding claims, wherein the CPP comprises at least 4 amino acid residues individually selected from the group consisting of lysine (K or k) and arginine (R or r).

47. The compound according to any one of the preceding claims, wherein the CPP comprises a retroinverso peptide.

48. The compound according to any one of the preceding claims, wherein the CPP comprises a TAT peptide, a mixed TAT peptide, a PolyArg peptide, a D-SynB3 peptide or a mini-AP4 peptide.

49. The compound according to any one of the preceding claims, wherein the CPP is selected from the group consisting of SEQ ID NO: 92 to 96.

50. The compound according to any one of the preceding claims, wherein the CPP comprises at least 4 amino acids having cationic or basic side chains that are analogous to arginine (R) or lysine (K), such as for example 5-hydroxylysine, ornithine, 2-amino-3 (or-4)-guanidinopropionic acid, and homoarginine.

51 . The compound according to any one of the preceding claims, wherein the CPP has an amphipathic structure and comprises an alternating pattern of

polar/charged amino acids and non-polar/hydrophobic amino acids.

52. The compound according to any one of the preceding claims, wherein the CPP is selected from the group consisting of penetratin (SEQ ID NO: 97), retroinverso- penetratin (SEQ ID NO: 98) and amphipathic model peptide (SEQ ID NO: 99).

53. The compound according to any one of the preceding claims, wherein the

conjugated moiety is a detectable moiety.

54. The compound according to any one of the preceding claims, wherein the

detectable moiety is a fluorophore.

55. The compound according to any one of the preceding claims, wherein the

detectable moiety is 5,6-carboxyltetramethylrhodamine ( TAMRA ) or

indodicarbocyanine (Cy5).

56. The compound according to any one of the preceding claims, wherein the detectable moiety comprises or consists of a radioisotope.

57. The compound according to any one of the preceding claims, wherein the radioisotope is selected from the group consisting of ¹²⁵l, ^99mTc, ¹¹¹ In, ⁶⁷Ga, 6⁸Ga, ⁷² As, ⁸⁹Zr, ¹²³l, ¹⁸F and ²⁰¹TI.

58. The compound according to any one of the preceding claims, wherein the radioisotope is selected from the group consisting of ¹²⁵l-indodicarbocyanine, indodicarbocyanine, ¹²⁵l-indotricarbocyanine or indotricarbocyanine.

59. The compound according to any one of the preceding claims, wherein the linker (L) comprises or consists of an alkane chain, a peptide, diaminoacetic acid, maleimide, ethylene glycol, PEG, L/PEG or any combination thereof.

60. The compound according to any one of the preceding claims, wherein formula I is Ac-YGRKKRRQRRR-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 87).

61. The compound according to any one of the preceding claims, wherein formula I is Ac-rRrGrKkRr-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 88).

62. The compound according to any one of the preceding claims, wherein formula I is Ac-RRRRRRRRR-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 89).

63. The compound according to any one of the preceding claims, wherein formula I is Ac-frrrsyslrr-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO: 90).

64. The compound according to any one of the preceding claims, wherein formula I is c[DLATEPAK(DAP)]-(/VMe-a)GYc[KNPTYD](1 NAL)(1 NAL)e (SEQ ID NO:

91 ).

65. The compound according to any one of the preceding claims, wherein said compound inhibits the Mint-APP, Mint-y-secretase and/or Mint-ARR-g- secretase interaction.

66. The compound according to any one of the preceding claims, wherein said compound inhibits the Mint1-APP, Mint1-Y-secretase, Mint1-APP-Y-secretase, Mint2-APP, Mint2-Y-secretase and/or Mint2-APP-Y-secretase interaction.

67. The compound according to any one of the preceding claims, wherein said compound inhibits the Mint2-APP, Mint2-Y-secretase and/or Mίhί2-ARR-g- secretase interaction.

68. The compound according to any one of the preceding claims, wherein said compound has a Kd for Mint below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

69. The compound according to any one of the preceding claims, wherein said compound has a K for Mint below 1 mM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

70. The compound according to any one of the preceding claims, wherein said compound has a K_d for Mintl and/or Mint2 below 1 pM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

71. The compound according to any one of the preceding claims, wherein said compound has a for Mintl and/or Mint2 below 1 pM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

72. The compound according to any one of the preceding claims, wherein said

compound has a K_d for Mint2 below 1 pM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

73. The compound according to any one of the preceding claims, wherein said

compound has a K for Mint2 below 1 pM, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

74. The compound according to any one of the preceding claims, wherein the

compound has been immobilized on a solid support.

75. A microchip comprising an array of compounds according to any one of the

preceding claims.

76. A composition comprising the compound according to any one of the preceding claims.

77. The composition according to any one of the preceding claims, wherein the

composition is a pharmaceutical composition.

78. A method of reducing the production of Amyloid b by a cell, the method comprising contacting the cell with the compound or the composition according to any one of the preceding claims.

79. A compound or the composition according to any one of the preceding claims, for use as a medicament.

80. A compound or the composition according to any one of the preceding claims, for use in the prevention and/or the treatment of a neurodegenerative disease.

81. A compound or the composition according to any one of the preceding claims, for use according to claim 80, wherein the neurodegenerative disease is a cognitive disorder.

82. A compound or the composition according to any one of the preceding claims, for use according to claim 80, wherein the neurodegenerative disease is dementia.

83. A compound or the composition according to any one of the preceding claims, for use according to claim 80, wherein the neurodegenerative disease is Alzheimer’s disease.

84. A compound or the composition according to any one of the preceding claims, for use according to claim 80, wherein the neurodegenerative disease is characterized by formation of amyloid plaques comprising Amyloid b.

85. A method of preventing and/or treating a neurodegenerative disease, comprising administering a therapeutically effective amount of the compound or the composition according to any one of the preceding claims, to an individual in need thereof.

86. Use of the compound or the composition according to any one of the preceding claims, for the manufacture of a medicament for prevention and/or treatment of a neurodegenerative disease.

87. A method of diagnosing Alzheimer’s disease, the method comprising the steps of: a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with the compound or the microchip according to any one of the preceding claims, wherein the compound comprises a detectable moiety; and

c) visualizing the localization of compound bound to Mint in b);

88. A method of diagnosing Alzheimer’s disease, the method comprising the steps of: a) providing a biological sample isolated from an individual;

c) visualizing the localization of compound bound to Mintl and/or Mint2 in b); wherein the presence and localization of compound bound to Mintl and/or Mint2 in the biological sample is indicative of the presence of amyloid plaques and resulting

Alzheimer’s disease in the individual from which the biological sample is derived.

89. A method of diagnosing Alzheimer’s disease, the method comprising the steps of: a) providing a biological sample isolated from an individual;

c) visualizing the localization of compound bound to Mint2 in b);

90. A method of diagnosing Alzheimer’s disease, the method comprising the steps of: a) providing a biological sample isolated from an individual; b) contacting the sample of a) with the compound or the microchip according to any one of the preceding claims, wherein the compound comprises a detectable moiety;

c) quantifying the amount of compound bound to Mint in b); and

d) comparing the amount of compound bound to Mint in c), to a control;

wherein an amonut of compound bound to Mint in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

91. A method of diagnosing Alzheimer’s disease, the method comprising the steps of: a) providing a biological sample isolated from an individual;

b) contacting the sample of a) with the compound or the microchip according to any one of the preceding claims, wherein the compound comprises a detectable moiety;

c) quantifying the amount of compound bound to Mintl and/or Mint2 in b); and d) comparing the amount of compound bound to Mintl and/or Mint2 in c), to a control;

92. A method of diagnosing Alzheimer’s disease, the method comprising the steps of: a) providing a biological sample isolated from an individual;

c) quantifying the amount of compound bound to Mint2 in b); and

d) comparing the amount of compound bound to Mint2 in c), to a control;

wherein an amount of compound bound to Mint2 in the biological sample, higher than the amount in a control indicates that the individual from which the biological sample is derived is afflicted with AD.

93. A method of isolating Mint, the method comprising the steps of:

a) providing a whole brain lysate; b) contacting said whole brain lysate of a) with the compound or the microchip according to any one of the preceding claims, thus obtaining a compound:Mint complex; and

c) isolating the Mint protein bound to said compound or said microchip, thus obtaining pure Mint.

94. A method of isolating Mintl and/or Mint2, the method comprising the steps of: a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with the compound or the microchip according to any one of the preceding claims, thus obtaining a compound Mintl and/or compound:Mint2 complex; and

c) isolating the Mintl and/or Mint2 protein bound to said compound or said

microchip, thus obtaining pure Mintl and/or Mint2.

95. A method of isolating Mint2, the method comprising the steps of:

a) providing a whole brain lysate;

b) contacting said whole brain lysate of a) with the compound or the microchip according to any one of the preceding claims, thus obtaining a

compound:Mint2 complex; and

c) isolating the Mint2 protein bound to said compound or said microchip, thus obtaining pure Mint2.

96. A method of isolating Mint, the method comprising the steps of:

a) providing a composition comprising Mint;

b) contacting said composition of a) with the compound or the microchip

according to any one of the preceding claims, thus obtaining a compound:Mint complex; and

97. A method of isolating Mintl and/or Mint2, the method comprising the steps of: a) providing a composition comprising Mint;

b) contacting said composition of a) with the compound or the microchip

according to any one of the preceding claims, thus obtaining a compound Mintl and/or compound:Mint2 complex; and c) isolating the Mintl and/or Mint2 protein bound to said compound or said microchip, thus obtaining pure Mintl and/or Mint2.

98. A method of isolating Mint2, the method comprising the steps of:

a) providing a composition comprising Mint;

b) contacting said composition of a) with the compound or the microchip according to any one of the preceding claims, thus obtaining a

compound:Mint2 complex; and