Stable amyloid beta monomers and oligomers
The invention provides monomeric and oligomeric amyloid beta peptide isomers that are resistant towards fibrillogenesis and their use as screening reagents or antigens in methods and pharmaceutical preparations for the treatment of Alzheimer's disease and other conditions related to protein misfolding. The alanines at positions 21 and 30, in wild type amyloid beta peptide amino acid sequence, are according to the invention replaced by cysteins, which results in an intra molecular disulphide bond. The invention further provides transgenic animals expressing modified amyloid precursor proteins or amyloid beta peptides.
1. A purified peptide comprising the amino acid sequence L-V-F-F-C corresponding to amino acids 17 to 21 of SEQ ID N0:4 and the amino acid sequence C-I-I-G-L-M-V corresponding to amino acids 30 to 36 of SEQ ID NO:4, the peptide further comprising:
a disulfide bond between the cysteine residues corresponding to amino acids 21 and 30 of SEQ ID N0:4;
said peptide comprising exactly two cysteines, said cysteines corresponding to Cys21 and Cys30 in SEQ ID N0:4, and comprising exactly one disulfide bond; and
said peptide being resistant towards fibrillogenesis.
2. The purified peptide according to claim 1 comprising the amino acid sequence L-V-F-F-C-E-D-V-G-S-N-K-G-C-I-I-G-L-M-V corresponding to amino acids 17 to 36 of SEQ ID N0:4.
3. The purified peptide according to claim 2 comprising an amino acid sequence corresponding to amino acids 1 to 40 of SEQ ID N0:4.
4. The purified peptide according to claim 2 comprising the amino acid sequence SEQ ID N0:4.
5. The purified peptide according to claim 2 comprising a glycine substitution at the position corresponding to amino acid 22 in SEQ ID N0:4.
6. The purified peptide according to claim 5 comprising the amino acid sequence SEQ ID N0:5.
7. The purified peptide according to claim 2 comprising a glutamine substitution at the position corresponding to amino acid 22 in SEQ ID N0:4.
8. The purified peptide according to claim 7 comprising the amino acid sequence SEQ ID N0:6.
9. The purified peptide according to claim 2 comprising a lysine substitution at the position corresponding to amino acid 22 in SEQ ID N0:4.
10. The purified peptide according to claim 9 comprising the amino acid sequence SEQ ID N0:8.
11. The purified peptide according to claim 2 comprising an asparagine substitution at the position corresponding to amino acid 23 in SEQ ID N0:4.
12. The purified peptide according to claim 11 comprising the amino acid sequence SEQ ID N0:7.
13. The purified peptide according to claim 1 comprising a methionine sulphoxide residue substitution at the position corresponding to amino acid 35 in SEQ ID N0:4.
14. The purified peptide according to claim 1 wherein,
(a) one or two terminal amino acids have been maleimidated;
(b) the N-terminal amino acid has been acetylated; and/or
(c) the C-terminal amino acid has been amidated.
15. The purified peptide according to claim 1 prepared by chemical synthesis.
16. The purified peptide according to claim 1 prepared by recombinant DNA technology.
17. A pharmaceutical preparation comprising a therapeutically effective amount of a peptide according to claim 1 , optionally in combination with an adjuvant.
18. A vaccine for immunization of mammals, including humans, against a disease selected from the group consisting of an amyloidosis; a prion disease; Parkinson's disease; or Alzheimer's disease comprising a peptide according to claim 1 , optionally in combination with an adjuvant.
19. The vaccine according to claim 18 , wherein said amyloidosis is selected from the group consisting of an amyloid neuropathy or cerebral amyloid angiopathy.
20. The vaccine according to claim 18 , wherein said prion disease is selected from the group consisting of Creutzfeld-Jacob disease, bouvine spongiform encephalopathy or scapie.
21. The peptide according to claim 1 , wherein said peptide is capable of forming oligomers consisting of multiple molecules of said peptide.
22. The peptide according to claim 1 , wherein said peptide forms a beta structure similar to a wild-type amyloid beta peptide as defined by SEQ ID N0:1 or SEQ ID N0:2 as probed by far-UV circular dichroism (CD) spectroscopy.
23. A purified soluble oligomer comprising a plurality of purified peptides according to claim 1 .
24. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:11.
25. A method for the identification of an inhibiting compound for an amyloid-beta protein, said method comprising
a) providing a test compound,
b) contacting said test compound with a peptide according to claim 1 ,
c) determining if said test compound binds to said peptide and inhibits formation of peptide oligomers of said peptide using a fibrillation or aggregation detection assay with size exclusion chromatography (SEC), far-UV circular dichroism LCD) spectroscopy or nuclear magnetic resonance (NMR) spectroscopy,
d) identifying said test compound as an inhibiting compound for said amyloid-beta protein if said test compound binds to said purified peptide and inhibits formation of peptide oligomers as determined in step (c).