Title: Novel peptides

Field of the invention

The present invention relates to the field of peptide drugs for treatment of diseases involving the formation of amyloid deposits, especially Alzheimer's disease.

Background of the invention

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterised by cerebral extracellular amyloid deposits containing amyloid-β peptide (Aβ) [I]. Aβ consists of 40-42 amino acids and is cleaved out from the amyloid precursor protein (APP) by β- and γ- secretase [2]. In the more aggressive forms of AD the highly aggregation-prone Aβi_42 [3] is the dominant species and it is believed that the peptide and its aggregation into amyloid fibrils plays a major role in the disease. The polymerisation process of Aβ in vivo is not fully understood and it is unclear which forms of Aβ that are toxic to neurons. Early studies focused on mature fibrils and their neurotoxicity (see for example ref [4, 5]) but recent findings point to soluble intermediates, such as protofibrils and Aβ-derived diffusible ligands (ADDLs) as being responsible for neuronal death and for blocking long-term potentiation [6- 8].

In order to understand the assembly process of Aβ, especially the early stages, in vitro aggregation studies have been performed by a number of groups and several models have been presented. These models of Aβ self-assembly include a large number of oligomers differing in size and morphology such as protofibrils [9, 10], ADDLs [6], paranuclei [11] and amylospheroids [12] and points to the complexity of Aβ aggregation. It remains to be determined which of these in vitro intermediates are formed in vivo and to what extent specific intermediates are responsible for the neuropathogenic events in AD.

The ability to form amyloid fibrils has been observed for a number of proteins. Besides AD, about 20 diseases, such as prion diseases, familial amyloid polyneuropathy and Parkinson's disease, are associated with amyloid fibrils formed from specific proteins (reviewed in ref [13]). Amyloid fibrils are highly insoluble aggregates that are composed of β-strands running perpendicular to the axis of the fibril [14, 15]. The fact that fibrils formed from different

proteins show similar morphology indicates that this might be an important structure that all proteins can adopt under certain conditions [16]. Given that several diseases are associated with amyloid formation, finding a therapeutic strategy to modulate this phenomenon is important.

A number of strategies to prevent amyloid fibril formation in AD have been suggested and several inhibitors of Aβ fibril formation have been reported [17]. Many of these compounds are so-called β-sheet blockers, i.e. they prevent fibril elongation [18]. One recurrent approach for the design of β-sheet blockers is to design short TV-methylated analogues of the peptide target in question. This strategy relies on binding of the TV- methylated peptide to the complementary part of the target peptide or protein, which is in a β- sheet conformation, and thus prevents further growth of the β-sheet as the other side of the TV- methylated peptide is blocked by the methyl groups. This general idea is illustrated in Figure 1.

The use of TV-methyl amino acids in peptides to block Aβ-structure assembly has already been applied to the design of amyloid-disrupting peptides. Thus, Doig et al. prepared single TV-methyl derivatives of the Aβ-amyloid peptide fragment Aβ25-35 to prevent both aggregation and toxicity of the same Aβ25-35 model peptide [19]. However, no data was provided regarding inhibition of cytotoxicity of natural Aβ-peptides such as Aβi _-42 or Aβi _-40 , which is known to be different from that of shorter derivatives [20]. In parallel, Meredith and colleagues described the inhibition of Aβi.40 fibrillogenesis and disassembly of Aβi.40 fibrils by TV-methylated peptides corresponding to the central hydrophobic core sequence, residues 16-22 [21] and residues 16-20 [22], of the protein. These studies showed that TV-methylation at every second amino acid in the sequence efficiently inhibited the formation of amyloid fibrils, while a peptide containing consecutive TV-methylation did not show such activity. The potential of this approach was further established by Giralt et al, who demonstrated that peptide analogues of Aβ (residues 16-20) containing single TV-methyl amino acids reduced the cytotoxicity of natural Aβi _-4 2 in PC12 cell cultures [23].

The structures of amyloid fibrils composed of Aβi _-40 [24] and Aβi _-42 [25] was recently established. These structures include a β-strand-turn-β-strands motif where the β-

strands form intermolecular, parallel β-sheets. The residues making up the two β-strands differ slightly between the two structures but both indicate that the first strand comprises residues 18-24 and that residues 31-40 make up the second. The first β-strand region has been extensively studied and contains a part that has been shown to form fibrils on its own, residues 14-23, whereas the properties of the second β-strand region is less well known.

Summary of the invention

The present invention is based on the discovery that part of the second β-strand region, comprising residues 31-40, can be used as a target for blocking of β-sheet formation. In a general aspect, the invention relates to peptides and peptide-like compounds capable of binding to the β-strand region of residues 31-40 of Aβ peptides and inhibiting further assembly of Aβ-fibrils.

In a first aspect, the present invention relates to a peptide having the amino acid sequence He- Ile-Gly-Leu-Met-Val-Gly-Gly- VaI- VaI (SEQ ID NO: 1), or a fragment thereof, capable of binding to amyloid-β peptide, wherein the N _α -atom of at least one amino acid residue is substituted with a substituent sterically hindering further binding of amyloid-β peptide, and optionally modified to reduce the polarity of the N- and/or C-terminus.

In a preferred embodiment, the peptide according to the first aspect has the amino acid sequence Ile-Gly-Leu-Met-Val-Gly (SEQ ID NO: 2). In a further preferred embodiment, the Nα-atom of at least five amino acid residues are independently substituted with substituents sterically hindering binding of amyloid-β peptide, e.g. all amino acid residues except Met may be substituted. One or more of the amino acid residues may be the D-enantiomer of the amino acid.

The N _α -substituent should be bulky enough to sterically hinder binding of amyloid-β peptide, yet small enough to allow the peptide to pass cell membranes and the blood-brain barrier. The substituent is preferably selected from the group consisting of Ci _-4 alkyl, Ci _-4 alkoxy, Ci _-4 alkylamino, phenyl, benzyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl. A presently preferred substituent is methyl.

To enhance binding of the peptide to Aβ, the peptide may be modified at the N-terminus, the C-terminus or both the N-terminus and the C-terminus in order to reduce the polarity of the terminal end or ends of the peptide. Such modifications are routine for a person skilled in the art and usually comprise N-terminal acetylation and C-terminal amidation.

In a second aspect, the invention relates to a peptide according to the first aspect for use in medicine.

In a third aspect, the invention relates to the use of the peptide according to the first aspect, or pharmaceutically active salts and esters thereof, for the manufacture of a pharmaceutical composition for the treatment or prevention of diseases involving the formation of amyloid deposits, especially of Alzheimer's disease and to such a composition, optionally further comprising other pharmaceutically acceptable ingredients such as excipients and/or carriers.

In one embodiment, such a composition may further include other therapeutic agents such as other peptides inhibiting β-strand formation. Such peptides are i.a. peptides of the sequence

KLVFFA (SEQ ID NO: 3) capable of binding to residues 16-22 of amyloid- β peptide and inhibiting β-sheet formation as disclosed in WO 01/07473 and WO 01/07474. This aspect also includes a method for treating a patient having, or suspected of having, Alzheimer's disease comprising administering a therapeutically effective amount of such a peptide or composition to said patient.

In a fourth aspect, the invention relates to the use of the peptide according to the first aspect, further comprising a reporter group, for diagnosing Alzheimer's disease, or for the production of a composition for use in diagnosis of Alzheimer's disease. The reporter group may be biotin, a fluorescent group, a chromophore or a radioactive atom or group, or any other group that may be incorporated into the peptide and detected in a relevant assay. A radioactive atom may be introduced in a number of ways known to the person skilled in the art. Generally speaking, atoms may be substituted with radioactive isotopes of the same element or isotopes of other elements having similar chemical properties, e.g. amino acid residues used in the production of the peptide may comprise a certain percentage of radioactive hydrogen or sulphur, a percentage of sulphur atoms may be exchanged to selenium atoms etc.

Selenomethionine is commercially available and may be used to incorporate selenium as a reporter group into the peptide.

This aspect also includes a method for diagnosing Alzheimer's disease using the peptide or the composition according to this aspect. Such a method may comprise the steps of bringing the peptide comprising a reporter group into contact with a sample from a subject suspected of having Alzheimer's disease and detecting binding of the peptide to Aβ peptides in the sample.

The method may also be performed in vivo by administering the peptide to a subject suspected of having Alzheimer's disease and detecting binding of the peptide to Aβ peptides in the central nervous system of the subject.

Brief description of the drawings

Figure 1: A: a schematic beta-sheet with interstrand hydrogen bonds between carbonyl oxygens and amide hydrogens. B: illustration of how an N-methylated peptide blocks further sheet formation by steric hindrance and loss of hydrogen bonding capabilities.

Figure 2: Electron micrographs of fibrils formed from Aβ 1-40 with and without addition of methylated peptides. A) 25 μM Aβl-40 alone, B) 25 μM Aβ 1-40 with 125 μM methylated peptide 1, C) 25 μM Aβ 1-40 with 125 μM methylated peptide 8, D) 25 μM Aβ 1-40 with 250 μM methylated peptide 1 and 250 μM methylated peptide 8. All samples were incubated at 37°C with moderate shaking in 10 mM phosphate buffer pH for 48 hours.

Detailed description of the invention

The invention relates to TV-substituted peptides that targets the second sheet forming part of Aβ, i.e. residues 31-40. The following experimental section illustrates one embodiment of the invention and should not be considered to limit the invention, which is defined by the scope of the appended claims.

Peptides M2, M4, M6 and M8, corresponding to the 32-37 region of Ap ₁ ^ ₀ , amino acid sequence IGLMVG, containing varying number of N-methyl amino acids were designed.

N-methylated peptides Ml, M3, M5 and M7 based on the 17-21 region were also synthesized.

Peptides M1-M8 were all synthesized on solid-phase using Fmoc-protected amino acids, as described in the experimental section. In short, all TV-methylated peptides were synthesized on a hyper-acid labile Sieber resin, due to the known instability of poly-N-methylated peptides under strongly acidic conditions [26], with HATU as coupling reagent. The Fmoc-protected TV-methyl amino acids used were synthesized from the corresponding Fmoc-amino acid as previously described [27]. All peptides were obtained in high purity, but were nevertheless purified by preparative reversed phase HPLC.

To study the effect of the different methylated peptides on Aβl-40 fibrillogenesis, transmission electron microscopy was used. As seen in Table 1 and Fig. 2, the methylated peptides studied here had different effects depending on the number of methylations as well as what region they were directed against. Fibrils formed in the presence of methylated peptides based on the 17-21 region were fewer, shorter and more crystal-like than normal fibrils (Fig. 2B). However, the effect decreased with increasing number of methylations. Methylated peptides directed towards the C-terminal region of Aβ 1-40 were more effective with increasing number of methylations. Here, the morphology was almost unchanged but the amount of fibrils formed decreased (Fig.2C). When co-incubating Aβ 1-40 with methylated peptide Ml and M8 complete inhibition of fibril formation was observed (Fig.2D). Oxidising the methionine in the C-terminally directed methylated peptide 8, reduced its potency as seen in Table 1.

Table 1

Fibril appearance and amount in presence of 1, 5 or 10 times excess of methylated peptide, as determined from transmission electron microscopy. Electron microscopy grids were examined under low magnification, a representative part was selected, magnified and photographed in order to be used for the assessment below. (++++ = 75-100% of Aβi _-40 alone, +++ = 50-75%, ++ = 25-50% of, + = 0-25%, - = no fibrils). See "Aggregation assay" for further details.

Materials and methods

High pressure liquid chromatography (HPLC) coupled to MS and universal evaporative light- scattering detection (ELSD) was done on a Gilson system consisting of a Gilson 322 pump, Gilson 233 XL autosampler, and a Gilson UV/VIS 152 detector, coupled in series with a Finnigan AQA mass spectrometer (electrospray in the positive mode) and an ELSD (Sedex 85 CC) from Sedere. The reverse phase HPLC analyses were done using a Phenomenex Gemini C18 (3 μm, 3.0*150 mm) column (for purity determination) with acetonitrile-water (both containing 0.1 % formic acid) as mobile phase (Gradient: 5-95 % acetonitrile in 6 minutes + 6 minutes at 95%, flow 1.0 ml/min). Preparative HPLC was done using a Grace Vydac Cl 8 (5 μm, 22* 150mm) column with acetonitrile-water (both containing 0.1 % TFA) as mobile phase (Gradient: 10 - 60 % acetonitrile in 10 min + 6 minutes at 95 %, flow 18.0 ml/min). The Sieber resin was purchased from GL Biochem. All standard Fmoc-amino acids were purchased from Senn Chemicals, and all N-methylated Fmoc-amino acids were synthesized as previously described [27]. All solvents were from Fischer Scientific and all other chemicals from Aldrich, and used as supplied.

Synthesis of peptides corresponding to the Ab region 32-37

General procedure IA: Attachment of first amino acid to resin.

Sieber resin, 0.300 g, (0.63 mmol/g) was swelled in DMF for 20 min in a reaction vessel equipped with a sintered glass bottom. A solution of 20% piperidine in DMF was then added for 10 min and then the same step was repeated for another 20 min with fresh solution. The resin was washed with 5 x DMF, 5 x MeOH, 5 x DCM and 2 x DMF (4 ml).

General procedure IB: Coupling of Fmoc-AA-OH to primary amine.

Fmoc-AA-OH 3 eq (0.57 mmol), HATU 2.9 eq (0.55 mmol) were dissolved in 4 ml DMF until a clear solution was obtained. DIPEA 6 eq (1.13 mmol) was then added and the mixture was added to the resin and stirred with aid of nitrogen gas until a negative TNBS test was obtained. The resin was washed with 5 x DMF, 5 x MeOH, 5 x DCM and 2 x DMF (4ml). A solution of 20 % piperidine was then added for 10 min and then the same step was repeated for another 20 min with fresh solution. The resin was washed with 5 x DMF, 5 x MeOH, 5 x DCM and 2 x DMF (4 ml).

General procedure 1C: Coupling of Fmoc-NMe-AA-OH to secondary amine.

Fmoc-NMe-AA-OH 3 eq (0,57 mmol), HATU 2.9 eq (0.55 mmol) were dissolved in 4 ml

DMF until a clear solution was obtained. DIPEA 6 eq (1.13 mmol) was then added and the mixture was added to the resin and stirred with aid of nitrogen gas until a negative chloranile test was obtained. The resin was washed with 5 x DMF, 5 x MeOH, 5 x DCM and 2 x DMF

(4ml). A solution of 20 % piperidine was then added for 10 min and then the same step was repeated for another 20 min with fresh solution. The resin was washed with 5 x DMF, 5 x

MeOH, 5 x DCM and 2 x DMF (4 ml).

General procedure ID: Capping ofN-terminal amino acid A solution of acetic anhydride 50 eq (0.89 ml, 9.45 mmol) and DIPEA 50 eq (1.65 ml, 9.45 mmol) was dissolved in 3 ml of DMF and added to the washed resin. After 40 min the resin was washed with 5x DMF, 5 x MeOH and 5 x DCM.

General procedure IE: Cleavage of peptide from resin

The resin was washed with 1O x DCM before cleavage to wash away residual DMF. The peptide was cleaved from the resin using a cleavage mix 97.9: 2: 0.1 (DCM:TFA:EDT) (5 ml) for 15 min. The cleavage solution was collected into a flask containing 25 ml toluene. Then the resin was washed with 5 x DCM, 5 x MeOH, 5 x DCM and 5x MeOH to wash out

residual cleaved peptide from the resin. The cleavage solution was then evaporated and gave a slightly yellow oil.

General procedure IF: Precipitation of the crude peptide

A white solid precipitated upon addition of water to the yellow oil, the solution was sonicated and acetonitrile was added until a clear solution was obtained. The solution was then lyophilized. The obtained white material was then purified using preparative HPLC.

Purification of peptides M1-M8 on preparative HPLC:

Peptides M1-M8 were purified by reversed phase HPLC using a binary gradient composed of acetonitrile (containing 0.1% TFA) and water (containing 0.1 TFA), and the solvent was removed by lyophilization.

Peptide identification

The purity and identity of the peptides were established by running analytical HPLC-MS of the purified peptides.

Table 2.

Reversed phase HPLC-MS data for peptides M1-M8

Peptides

Aβi.40 was purchased from Bachem GmbH (Germany) and stored at -20°C until use. To ensure a monomeric starting solution, the peptide was dissolved, sonicated and vortexed at 1 mg/ml in DMSO (Merck, Germany) and diluted in 10 mM sodium phosphate buffer, pH 7, to

the working concentration immediately before use. For the aggregation assay, all methylated peptides were dissolved at 5 mM in DMSO.

Aggregation assay Aβi.40, 25 μM, was incubated with or without methylated peptides in 1, 5 or 10 times excess in 10 mM sodium phosphate, pH 7, and 15% DMSO at 37°C with agitation. After 48 hours aliquots of 2 μl were adsorbed for 1 min on 200-mesh cooper grids. The grids were then stained with 2% uranyl acetate for 30 s and examined and photographed using a Hitachi H7100 microscope operated at 75 kV. The importance of sequence homology for inhibition was demonstrated by the lack of activity of pentameric poly-N-methylated alanine. The results are given in Table 1.

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