D-enantiomeric peptides derived from D3 and use thereof

Abstract

The present invention relates to novel D-enantiomeric A-beta-oligomer-binding peptides, homologs, fragments, parts and polymers thereof and use thereof.

Claims

1. A peptide comprising at least 50% D-enantiomeric amino acids and comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2 and homologs thereof having at least 83% sequence identity to SEQ ID NO: 2.

2. The peptide of claim 1, wherein the at least one amino acid sequence is SEQ ID NO: 2.

3. The peptide of claim 1, wherein the at least one amino acid sequence is a homolog of SEQ ID NO: 2 having at least 83% sequence identity to SEQ ID NO: 2.

4. The peptide of claim 1, wherein the at least one amino acid sequence is a homolog of SEQ ID NO: 2 having at least 91% sequence identity to SEQ ID NO: 2.

5. The peptide of claim 1, wherein the peptide is composed of D-amino acids.

6. The peptide of claim 1, wherein the peptide comprises at least 80% D-enantiomeric amino acids.

7. The peptide of claim 1, wherein the peptide comprises at least 90% D-enantiomeric amino acids.

8. The peptide of claim 1, wherein the peptide comprises at least 95% D-enantiomeric amino acids.

9. The peptide of claim 1, wherein the peptide is capable of inhibiting formation of fibrils of amyloid beta peptides.

10. The peptide of claim 1, wherein the peptide is capable of binding to aggregated amyloid beta peptides.

11. The peptide of claim 1, wherein the peptide is capable of being used as a therapeutic agent in medicine.

12. The peptide of claim 1, wherein the peptide is capable of treating Alzheimer's disease.

13. A pharmaceutical composition, wherein the composition comprises as one of the components thereof the peptide of claim 1.

14. A probe for the identification and quantitative and/or qualitative determination of amyloid beta fibrils and/or amyloid beta oligomers, wherein the probe comprises the peptide of claim 1.

15. A kit, wherein the kit comprises the peptide of claim 1.

16. The peptide of claim 1, wherein the peptide is linked to a further substance.

17. A method for preparing the peptide of claim 1, wherein the method involves peptide synthesis or mutagenesis.

18. A method of reducing the formation of amyloid beta oligomers and/or amyloid beta peptide aggregates in a patient in need thereof, wherein the method comprises administering to the patient the peptide of claim 1.

19. A method for the detoxification of toxic amyloid beta oligomers and/or aggregates, wherein the method comprises contacting the oligomers and/or aggregates with the peptide of claim 1.

20. The method of claim 19, wherein the amyloid beta oligomers and/or aggregates form amorphous, non-toxic aggregates with the peptide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the accompanying drawings,

(2) FIG. 1 graphically represents the modulation of the A-beta1-42 aggregation behavior by RD2, analyzed by an iodoxanol gradient as set forth in Example below;

(3) FIG. 2 graphically represents the results of an ELISA assay for the relative quantification of the binding of peptides to various A-beta1-42 conformers as set forth in Example 2 below;

(4) FIG. 3 graphically represents the results of a ThT aggregation test for A-beta1-42 for the quantification of the relative fibril content in the presence of various peptides as set forth in Example 3 below; and

(5) FIG. 4 graphically represents the results of a PepChip experiment as set forth in Example 5 below.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

EXAMPLES

(6) 1.

(7) The D3 variants listed in Table 1 were chemically synthesized.

(8) TABLE-US-00001 TABLE 1 Listing of the D3 derivatives investigated Description of the peptide Amino acid sequence (with explanation) (D-enantiomer) D3 (SEQ ID NO: 11) rprtrlhthrnr NT-D3 (D3-N-Terminus) (SEQ ID NO: 6) rprtrl RD1 (rational D3) (SEQ ID NO: 3) pnhhrrrrrttl RD2 (rational D3) (SEQ ID NO: 2) ptlhthnrrrrr RD3 (rational D3) (SEQ ID NO: 4) rrptlrhthnrr D3hth (D3 with deletion hth) (SEQ ID NO: 1) rprtrlrnr

(9) The effects of the peptides described in the table on A-beta aggregation were investigated by means of density gradient centrifugation. Thus, A-beta was mixed with the respective peptide and the particles formed are separated according to size. The sample to be investigated was placed on the surface of the centrifuge tube in which a density gradient was charged, which consisted of layers of different iodixanol concentrations in this example. During the several hours of separation, the molecules sediment at different rates in the solvent, namely, more rapidly the larger the particles. The centrifugation is terminated at a suitable time point and different constituents of the sample are obtained in the various layers which are analyzed by SDS-PAGE. A-beta without addition of peptide served as control. For example, the evaluation of runs with A-beta and A-beta with RD2 is shown in FIG. 1.

(10) As can be seen in FIG. 1, A-beta could be detected in all fractions in the A-beta sample. This changes on addition of RD2. Here, fewer A-beta oligomers could be detected in the fractions 3-9 (A-beta oligomer fraction). Large aggregates are formed which are detectable in fractions 10-15. D3 showed no effect when the concentration of peptide used was 20 M. In earlier studies, it was found that D3 in higher concentrations precipitates A-beta oligomers and converts them into large, amorphous and non-toxic aggregates (Funke at al., ACS Chem. Neurosci, 2010). All D-enantiomeric peptides which were characterized with A-beta using the method mentioned above were labelled with FITC. The amount of dye detectable in fraction 12 was used to estimate the binding strength of the respective D3 derivative to A-beta oligomers or the oligomer precipitating effects thereof in vitro. The results for all peptides are shown in Table 2. It was shown that the peptide RD2 especially has a significant effect on A-beta aggregation compared to D3.

(11) TABLE-US-00002 TABLE 2 Summary of results of the modulation of the A-beta1-42 aggregation behavior of various D3 derivatives Peptide Peptide bound in fraction 12 [%] A-beta control without peptide 0 D3 4 NT-D3 0 RD1 3 RD2 20 RD3 4 D3hth 2
2.

(12) The peptides were also tested with respect to their in vitro binding to various A-beta conformers (A-beta monomers, oligomers, fibrils) using ELISA. All peptides showed weak binding to A-beta monomers but relatively high affinity to oligomers and fibrils (FIG. 2). Interestingly, biotinylated monomers were not detected at the amino terminus, whereas the seedless monomers (aggregation seed-free preparation) were weakly bound to the carboxy terminus biotinylation. This may be interpreted as a potential indication of an epitope binding site of the D3 derivatives localized to the amino terminus.

(13) 3.

(14) Some peptides were used in the thioflavin T (ThT) aggregation test. ThT is a fluorescent dye which binds to -sheet-rich structures of various amyloid proteins and fluoresces at 440 nm on excitation. The emission may be correlated in this manner with the relative fibril content in the sample. ThT tests are used for measuring the fibrillation of A-beta and are used especially in ligands to detect potential inhibitory effects of these on A-beta aggregation. The peptides were used in a ratio of 1:10 (A-beta: peptide) with a 10 M A-beta solution. Fifteen hours later, after reaching the saturation phase in the A-beta control with no addition of ligand, the ThT fluorescence of the co-incubation of D-peptides and A-beta was evaluated and presented as a percentage of the A-beta control incubation. This shows that all peptides used significantly reduce formation of A-beta fibrils (FIG. 3).

(15) 4.

(16) Furthermore, D3 variants were identified which have an increased binding strength for A-beta, particularly for A-beta oligomers, with at least the same effect on the aggregation thereof, compared to D3. Stronger binding may suggest a more efficient therapeutic effect.

(17) By means of a PepSpot analysis, using a membrane from JPT (Berlin) on which more than 300 D3 variants had been immobilized, variants were investigated in order to identify which bind with more affinity to A-beta (1-42). D3, D3 variants with varying amino acid sequences (so-called saturation mutagenesiseach amino acid was exchanged with their D-enantiomeric form from all 19 other naturally occurring amino acids) and controls were immobilized on a trioxa membrane carboxy terminal. After the incubation with 5 M A-Beta(1-42) oligomers for 5 min and washing steps, the signals from bound A-Beta were detected via an anti-A-Beta antibody (6E10). The binding signals were evaluated as signal intensity/area. For the analysis, the signal intensity of the original D3 (mean value) was compared with that of the derivatives. The amino acid exchanges which resulted in stronger binding of A-beta oligomers are summarized in Table 3.

(18) TABLE-US-00003 TABLE 3 The D-amino acid sequence of D3 is shown in the first row in bold. In the rows below are listed the type and location of the amino acid exchanges which resulted in an increase in binding strength to A-beta oligomers. R P R T R L H T H R N R I T P D Q Q Q E D R S
5.

(19) In order to confirm the results and to obtain novel variants with even stronger affinity for A-beta by combination with the amino acid exchanges described in table 4, further D3 variants were coupled to PepChip arrays from Pepscan and their A-beta binding strength was investigated. The selection of the variants were based on the results of the Pepspot membrane evaluation already described. In six independent experiments, the A-beta binding to the D3 derivatives was detected via coupled FITC labeling. In this case, 5 M A-Beta FITC was used. The detection was performed in a FLA8000 microarray scanner from Fujifilm and the binding signals were measured with the AIDA Array Metrix Software and evaluated with the aid of Mathlab Version 7.10.0.449. FIG. 4 shows the binding signals of a selected PepChip as example.

(20) Five of the peptides immobilized on the PepChips exceeded the limit in at least four of six experiments and thus showed considerably higher binding affinity to A-beta compared to D3. These peptide sequences are summarized in Tab. 4. Among these, sequences with combined exchanges can be found from Tab. 3.

(21) TABLE-US-00004 TABLE4 SequencesoftheD-peptideshavingstronger A-betamonomerbindingstrengthcomparedtoD3 Sequence Name RPITRLHTDRNR(SEQIDNO:7) DB1 RPITTLQTHQNR(SEQIDNO:8) DB2 RPITRLRTHQNR(SEQIDNO:5) DB3 RPRTRLRTHQNR(SEQIDNO:9) DB4 RPITRLQTHEQR(SEQIDNO:10) DB5

DESCRIPTION OF THE FIGURES

(22) FIG. 1: Modulation of the A-beta1-42 aggregation behavior by RD2, analyzed by an iodixanol density gradient. The iodixanol gradient was overlayed with 100 l of a 80 M A-beta1-42 solution and 100 l of a 80 M A-beta and 20 M RD2 mixture. The mixture was then centrifuged at 259 000g for 3 h at 4 C. The 15 fractions (fraction 15 is the pellet boiled with the loading buffer) were harvested manually immediately after the centrifugation and analyzed by Tris-Tricin-SDS-PAGE followed by silver staining. If the run was carried out under identical conditions with 20 M D3, no change was apparent compared to the run with only A-beta.

(23) FIG. 2: ELISA for the relative quantification of the binding of the peptides to various A-beta1-42 conformers. Seedless monomers of carboxy terminal biotinylated A-beta (light grey bars) and oligomers (dark grey bars) and fibrils (black bars) of amino terminal biotinylated A-beta monomers were each immobilized at 5 g/ml and the D-peptide applied at a concentration of 10 g/ml. The relative quantification of the binding of the peptides in a duplicate determination is shown as absorption at 450 nm after subtraction of the background absorption.

(24) FIG. 3: ThT aggregation test of A-beta1-42 for the quantification of the relative fibril content in the presence of various peptides. The concentration of A-beta was 10 M and the peptides were added in a ratio of 1:10 (A-beta: peptide). The fluorescence of 10 M A-beta was set to 100% and the values and standard deviations of the other incubations are given as percentages of this maximum value. None of the peptides showed a significant ThT fluorescence with no A-beta.

(25) FIG. 4: Results of a PepChip experiment. Different D3 variants were immobilized on the PepChip. The PepChip was incubated with FITC labeled A-beta (5 M). The FITC fluorescence intensity was read as a measure of the binding strength of the respective D3 derivatives. Values of the mean and standard deviation were calculated from the 11 values obtained for the D3 controls likewise applied at 11 different positions on the chip. Mean and standard deviation were added and the result was defined as the limit which a D3 derivative must attain in order to clearly have higher affinity for A-beta compared to D3. The integrals of the binding signals of all peptides are shown which exceed this limit. The individual bars are based on the means of three exactly identical peptide spots. a.u.: arbitrary units, relative fluorescence units. Due to the observed variance in the results, this entire experiment was carried out six times.