WO2009128772A1 - Stable amyloid beta monomers and oligomers - Google Patents
Stable amyloid beta monomers and oligomers Download PDFInfo
- Publication number
- WO2009128772A1 WO2009128772A1 PCT/SE2009/050378 SE2009050378W WO2009128772A1 WO 2009128772 A1 WO2009128772 A1 WO 2009128772A1 SE 2009050378 W SE2009050378 W SE 2009050378W WO 2009128772 A1 WO2009128772 A1 WO 2009128772A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- seq
- amino acids
- disease
- alzheimer
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the invention relates to monomelic and oligomeric amyloid beta peptides that are resistant towards fibrillogenesis for use as antigens or screening reagents for the treatment of Alzheimer's disease and other conditions related to protein misfolding.
- AD Alzheimer's disease
- a ⁇ amyloid- ⁇
- APP amyloid precursor protein
- oligomers are soluble structures containing a plurality of monomers prior to proceeding into the inert fibrillar state.
- This oligomeric state has recently been implicated as a neurotoxic agent and, therefore, as the toxic species involved in AD (Haass, C, and Selkoe, D. J., Nature Reviews MoI. Cell. Biol. 8: 101-112 (2007)).
- the prevalent method for producing these toxic structures at physiological conditions involve incubating peptide solutions at 20-100 ⁇ M and 4 0 C in cell culture medium (F- 12) or in buffered salt solutions for several days (Lambert, M.
- Oligomer preparations therefore have a very limited and unpredictable (aggregation nucleation is a spontaneous event) lifespan once they are formed.
- dilute solutions (20 ⁇ M to 25 ⁇ M) at 4 0 C oligomer preparations of A ⁇ (42) are typically stable only for 24 h, and those of A ⁇ (40) for maybe a week.
- >100 ⁇ M the insolubility of the A ⁇ protein decreases dramatically. The fibrillation process is thus a serious drawback when, as in screening assays for medicaments, stable proteins are required at relatively high concentration.
- the aim of the present invention is to overcome these problems by providing engineered A ⁇ peptides that form stabile oligomers.
- Oxidation of these A ⁇ isomers produces a mixture of 15 possible isoforms with different intramolecular disulphide bonds where only three of the 15 possible isoforms will contain an A21C/A30C disulphide bond, but always in combination with additional disulphide bonds.
- Previous A ⁇ peptide disulfide mutants reported in the literature are L17C/L34C, L17C/M35C, and L17C/V36C (Shivaprasad, S. and Wetzel, R., Biochemistry 43: 153-15317 (2004)). These mutations are all non-conservative replacements. Furthermore, these mutants were made specifically to investigate the proximity of Leul7 to Leu34, Met35, and Val36 in the fibril structure.
- a ⁇ oligomer preparations are described in WO 2007/005358, WO 2007/005359, WO 2007/142320 and WO 2004/067561. These described oligomer preparations have been obtained by using intermolecular crosslmkers (WO 2007/005358), non-physiological pH (pH 9 in WO 2007/005359) and/or additives (40% glycerol or TFE were claimed to stabilize oligomers at 37 0 C in WO 2007/005359; GMl ganglioside in WO 2007/142320; and SDS in WO 2004/067561).
- the present invention provides covalently constrained monomelic and oligomeric A ⁇ A21C/A30C peptides that are resistant towards fibrillogenesis.
- the peptides according to the invention retain most of its wild-type like properties as probed by size exclusion chromatography (SEC), far-UV CD (indicative of coil structure), and NMR (also coil- like).
- the A ⁇ peptides according to the invention populate several low molecular-weight oligomers with coil-structure (eluting from SEC as up to approximately 42 kDa proteins), as well as high molecular-weight oligomers with ⁇ -structure (eluting from SEC as approximately 75-85 kDa and 170 kDa proteins) upon concentration. Furthermore, wild-type-like proto fibrillar structures with dimensions of 6 nm width and typically 30 nm in length are obtained by heating concentrated protein solutions. The high molecular-weight oligomers with ⁇ -structure are recognized by the Al 1 polyclonal antibody.
- the present invention provides monomeric and oligomeric A ⁇ peptides with an unprecedented resistance towards fibrillogenesis.
- the A ⁇ peptides according to the invention comprise amino acids replacements at positions 21 and 30 in the wild-type A ⁇ (40) (SEQ ID NO:1) or A ⁇ (42) (SEQ ID NO:2) peptides with cysteines, resulting in intra-molecularly disulphide bonded A ⁇ (40) A21C/A30C (SEQ ID NO:3) and A ⁇ (42) A21C/A30C (SEQ ID NO:4) peptides ( Figure IA).
- the non-oligomeric coil- like state of the A ⁇ peptides according to the invention also remain wild-type-like as probed by size SEC, far-UV CD spectroscopy, and 15 N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy.
- HSQC heteronuclear single quantum coherence
- NMR nuclear magnetic resonance
- the A ⁇ peptides according to the invention are in fact the first examples of A ⁇ peptides forming stable oligomers under physiological conditions, this being obtained by arresting the fibrillation step. Furthermore, long incubation times and unpredictable outcomes of oligomeric preparations are avoided, as the A ⁇ peptides according to the invention can be purified and rapidly concentrated into the oligomeric states.
- the present invention provides peptides comprising the amino acid sequence LVFFC corresponding to amino acids 17 to 21 of SEQ ID NO:4, and the amino acid sequence CIIGLMV corresponding to amino acids 30 to 36 of SEQ ID NO:4.
- the peptide according to the invention further comprises a disulphide bond between the amino acids corresponding to Cys21 and Cys30 in SEQ ID NO:4.
- said peptide is obtained by linking the peptide comprising the amino acid sequence LVFFC to the peptide comprising the amino acid sequence CIIGLMV by an inter-molecular disulphide bond.
- said two peptides are constituents of a single peptide chain with an intra-molecular disulphide bond between Cys21 and Cys30.
- the amino acid sequence LVFFC is fused to the amino acid sequence CIIGLMV by a peptide comprising 4 to 20 amino acids, such as 6 to 16, preferably 7 to 12, or even more preferably 8 to 10 amino acids, such as 9 amino acids.
- the present invention further provides variants of said peptides wherein one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been replaced by conservative substitution, one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been deleted, or one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been inserted.
- the fusion peptide comprises the amino acid sequence EDVGSNKG corresponding to amino acids 22 to 29 of SEQ ID NO:4.
- the peptides according to the invention preferably comprise the amino acid sequence LVFFCED VGSNKGCIIGLMV corresponding to amino acids 17 to 36 of SEQ ID NO:4, and variants of said peptides wherein one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been replaced by conservative substitution, one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been deleted, or one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been inserted.
- the peptides according to the invention comprise the amino acid sequence 1 to 40 of SEQ ID NO:4, the full amino acid sequence SEQ ID NO:4, and variants of said peptides wherein one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been replaced by conservative substitution, one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been deleted, or one or more, such as one, two, three, four, five, or preferably one or two, amino acids have been inserted.
- the peptides according to the invention can further comprise a glycine substitution at the position corresponding to amino acid 22 (SEQ ID NO:5), or a glutamine substitution at the position corresponding to amino acid 22 (SEQ ID NO:6), or a lysine substitution at the position corresponding to amino acid 22 (SEQ ID NO:8).
- the peptides according to the invention can also comprise an asparagine substitution at the position corresponding to amino acid 23 (SEQ ID NO:5), or a glutamine substitution at the position corresponding to amino acid 22 (SEQ ID NO:6), or a lysine substitution at the position corresponding to amino acid 22 (SEQ ID NO:8).
- the peptides according to the invention can also comprise an asparagine substitution at the position corresponding to amino acid 23 (SEQ ID NO:5)
- the peptides according to the invention can also comprise a methionine sulphoxide residue at the position corresponding to amino acid 35 in SEQ ID NO:4.
- the peptides according to the invention can comprise (a) one or two terminal amino acids being maleimidated; (b) one or two terminal amino acids being cysteinylated; (c), the carboxy terminal end of the peptide has been amidated, i.e. the free COOH at the carboxy terminal has been transformed into CONH 2 ,.; and/or the amino terminal end of the peptide has been acetylated, i.e. the free NH 2 group at the amino terminal has been transformed into the amide CH 3 CONH- (AcNH-).
- the peptides according to the invention can be prepared by chemical synthesis or be prepared by recombinant DNA technology.
- Conservative substitution is meant substitution of one amino acids with an amino acid having similar properties with regard to polarity and hydrophobicity. Examples of groups of amino acids having such similar properties are provided in Table 1. Conservative substitution can also include substitution with a non-standard amino acid. Non-standard amino acids include, but are not limited to, the examples within brackets in Table 1 , where PL is pyrro lysine, DA is dehydroalanine, NL is norleucin, SC is selenocysteine, HC is homocysteine, CL is citrulline, and OR is ornithine. Table 1.
- conservative substitutions and amino acid insertions according to the invention do not allow for insertion of further cysteines in addition to the cysteines corresponding to Cys21 and Cys30 in SEQ ID NO:4.
- conservative substitutions and amino acid deletions according to the invention do not allow for replacement or deletion of the cysteines corresponding to Cys21 and Cys30 in SEQ ID NO:4.
- the A ⁇ peptides according to the invention always comprise exactly two cysteines, i.e. the cysteines corresponding to Cys21 and Cys30 in SEQ ID NO:4.
- the peptides according to the invention are substantially resistant towards fibrillogenesis. By being resistant to fibrillogenesis is meant that said peptides do not form fibrils under near- physiological conditions where wild type A ⁇ peptide fibrillate, as demonstrated by several of the examples presented herein.
- the present invention provides soluble oligomers consisting of peptides according to the invention.
- the present invention provides an antibody specifically reacting with a peptide according to the invention. This antibody can be a monoclonal antibody.
- the present invention provides an antibody fragment specifically reacting with a peptide according to the invention.
- the antibody fragment can be a Fab fragment, a (Fab) 2 fragment, a single chain Fab fragment, a single chain Fv fragment, or a single chain Fv dimer.
- the present invention provides a protein binder specifically reacting with a peptide according to the invention.
- Said protein binders include, but are not limited to, derivatives of the anticalin, Affibody, FNfhlO, neocarzinostatin, ankyrin repeat protein, PDH finger, CDR3 grafted green fluorescent protein, and E. coli periplasmic binding protein scaffolds.
- the present invention provides methods for the identification of a compound suitable for the treatment of a disease caused by, or related to, the deposition of protein fibrils or amyloids, said method comprising the use of a peptide according to the invention.
- the disease can be an amyloidosis, such as an amyloid neuropathy or cerebral amyloid angiopathy, a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie, Parkinson's disease, or Alzheimer's disease.
- the disease is Alzheimer's disease.
- the method for the identification of a compound suitable for the treatment of a disease caused or related to the deposition of protein fibrils or amyloids can comprise: a) providing a test compound, b) contacting said test compound with a peptide according to the invention, c) determining if the test compound binds to the peptide and/or inhibits the formation of peptide oligomers, d) identifying said compound as suitable for the treatment of a disease caused or related to the deposition of protein fibrils or amyloids disease.
- the disease can be an amyloidosis, such as an amyloid neuropathy or cerebral amyloid angiopathy, a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie, Parkinson's disease, or Alzheimer's disease.
- an amyloidosis such as an amyloid neuropathy or cerebral amyloid angiopathy
- a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie
- Parkinson's disease or Alzheimer's disease.
- the disease is Alzheimer's disease.
- the present invention provides methods for identifying and/or selecting binding proteins, said method comprising the use of a peptide according to the invention.
- the binding molecule can be an antibody fragment, such as a Fab fragment, a (Fab) 2 fragment, a single chain Fab fragment, a single chain Fv fragment, or a single chain Fv dimer.
- the binding molecule can also be engineered non-natural receptor derivatives, such as derivatives of the anticalin, Affibody, FNfnlO, neocarzinostatin, ankyrin repeat protein, PDH finger, CDR3 grafted green fluorescent protein, and E. coli periplasmic binding protein scaffolds.
- the present invention provides use of a peptide according to the invention in the preparation of pharmaceutical compositions intended for immunization for prophylactic or therapeutic treatment of a disease caused by, or related to, the deposition of protein fibrils or amyloids, optionally in combination with an adjuvant.
- the disease can be an amyloidosis, such as an amyloid neuropathy or cerebral amyloid angiopathy, a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie, Parkinson's disease, or Alzheimer's disease.
- the disease is Alzheimer's disease.
- the present invention provides pharmaceutical preparations comprising a therapeutically effective amount of a peptide according to the invention, optionally in combination with an adjuvant.
- the present invention provides vaccines for immunization of mammals, including humans, against a disease caused by, or related to, the deposition of protein fibrils or amyloids, comprising a peptide according to the invention, optionally in combination with an adjuvant.
- the disease can be an amyloidosis, such as an amyloid neuropathy or cerebral amyloid angiopathy, a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie, Parkinson's disease, or Alzheimer's disease.
- the disease is Alzheimer's disease.
- the present invention provides vaccines for immunization of mammals, including humans, against a disease caused by, or related to, the deposition of protein fibrils or amyloids, comprising a therapeutically effective amount of an antibody according to the invention.
- the disease can be an amyloidosis, such as an amyloid neuropathy or cerebral amyloid angiopathy, a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie, Parkinson's disease, or Alzheimer's disease.
- the disease is Alzheimer's disease.
- the present invention provides methods for prophylactic or therapeutic treatment of a mammal, including a human being, suffering from a disease caused by, or related to, the deposition of protein fibrils or amyloids or facing the risk of developing a disease caused by, or related to, the deposition of protein fibrils or amyloids, whereby a therapeutically effective amount of a peptide according to the invention is administered to said mammal.
- the disease can be an amyloidosis, such as an amyloid neuropathy or cerebral amyloid angiopathy, a prion disease such as Creuztfeldt- Jakob disease, bovine spongiform encephalopathy, or scrapie, Parkinson's disease, or Alzheimer's disease.
- the disease is Alzheimer's disease.
- the present invention provides methods for prophylactic or therapeutic treatment of a mammal, including a human being, suffering from a disease caused by, or related to, the deposition of protein fibrils or amyloids or facing the risk of developing a disease caused by, or related to, the deposition of protein fibrils or amyloids, whereby a therapeutically effective amount of an antibody according to the invention is administered to said mammal.
- Figure IA represents a drawing of the hairpin structure of the A ⁇ -peptide (Protein Data Bank accession no. 2OTK) with the introduced cysteines at positions 21 and 30 and the disulphide bond linking these two positions (SEQ ID NO: 4).
- Leul7, Phel9, Cys21, Cys30, Ile32, Leu34, and Val36 comprising the upwards facing side of the structure; and Vall8, Phe20, Ile31, and Met35 comprising the downward facing side of the structure, this side also contains Gly33.
- Figure IB represents a schematic depiction of a hypothetical aggregation scheme.
- amfiphilic A ⁇ peptide spontaneously self-associates into micellar-like oligomeric structures of low molecular weight that are weakly stabilized by hydrophobic forces (coil-like oligomers).
- the ⁇ - hairpin is only transiently occupied, but is stable in the high molecular-weight oligomeric structures that form by hydrophobic stacking.
- the color coding of the hairpin is the same as in Figure IA.
- Fibrillogenesis occurs as a result of a conformational switch whereby the hydrogen bonds and hydrophobic van der Waals bonds in the ⁇ -structured oligomer are broken and reformed in a much more stable fibril structure, wherein the two ⁇ -strands colored in orange are now facing each other in the fibril core.
- This conformational step is arrested by the disulphide bond connecting positions 21 and 30, thus allowing for the other states to become populated.
- Figure 2 represents fibrillation assays of the A ⁇ (40) peptide with and without the A21C/A30C replacements, as well as the A ⁇ (40) A21C/A30C peptide with a broken disulphide bond (in 5 mM tris(2-carboxyethyl) phosphine (TCEP)). Fibrillation was monitored by Thioflavin T (TFT) binding (10 ⁇ M) and the assays were carried out at 37 0 C with shaking in phosphate buffer at pH 7.2.
- TFT Thioflavin T
- Figure 3 represents fibrillation assays of the A ⁇ (40) A21C/A30C peptide with formed and broken (by 10 mM TCEP) disulphide bonds at 30 ⁇ M, 50 ⁇ M, 150 ⁇ M and 250 ⁇ M peptide.
- Figure 3E represents the baselines without peptide. Fibrillation was monitored by TFT binding (10 ⁇ M) and the assays were carried out at 37 0 C with shaking in phosphate buffer at pH 7.2.
- Figure 4A represents the SEC elution profile of the A ⁇ (40) A21C/A30C peptide at low concentrations (approximately 25 ⁇ M), whereby said peptide elutes as approximately an 8 kDa protein.
- Figure 4B represents a far-UV CD spectrum of the A ⁇ (40) A21C/A30C peptide as a coil- like protein. This spectrum is virtually identical for all coil- like preparations of said peptide.
- Figure 4C represents an SDS polyacrylamide gel electrophoresis (PAGE) analysis of the A ⁇ (40) A21C/A30C peptide with formed (-TCEP) and broken (+TCEP) disulphide bonds.
- Figure 4D represents a 15 N-HSQC NMR spectrum of 50 ⁇ M A ⁇ (40) A21C/A30C peptide.
- Figure 5 represents an SEC elution profile of a concentrated solution (at approximately 1.8 mM) of A ⁇ (40) A21C/A30C peptide that was denatured in 6M guanidinium chloride (GdmCl) prior to SEC.
- GdmCl guanidinium chloride
- Figure 6A represents an SEC elution profile of a concentrated solution (at approximately 1.5- 1.8 mM) of A ⁇ (40) A21C/A30C peptide that was denatured in 6M GdmCl prior to SEC.
- Figure 6B the same solution was pre-treated with ⁇ -mercaptoethanol prior to SEC on the same column and in the same buffer supplemented with 5 mM TCEP to keep the cysteines reduced. The peptide elutes in the void under these conditions (as >100 kDa proteins).
- Figure 6C represents the SEC elution profile of the top fraction in Figure 6A after 4 days at 4 0 C and a concentration of 200 ⁇ M.
- Figure 6D represents the SEC elution profile of the peak centered around 10.8 ml in Figure 6A concentrated to approximately 1 mM.
- Figure 7A represents an SEC elution profile of a concentrated solution (at approximately 1.7 mM) of A ⁇ (40) A21C/A30C peptide that was denatured in 6M GdmCl prior to SEC.
- the peptide elutes as high molecular-weight oligomers with ⁇ structure, and as low molecular-weight oligomers and monomeric protein with coil structure.
- Figure 7B represents a far-UV CD spectrum of 250 ⁇ M A ⁇ (40) A21C/A30C peptide as a ⁇ -structured peptide oligomer. This spectrum is virtually identical for all preparations of said peptide with ⁇ structure, including protofibrillar states.
- Figure 8 represents a fibrillation assay of the A ⁇ (40) A21C/A30C peptide as ⁇ -structured oligomer with and without (in 10 mM TCEP) the constraining disulphide bond. Fibrillation was monitored by TFT binding (10 ⁇ M) and the assay was carried out at 37 0 C with shaking in phosphate buffer at pH 7.2.
- Figure 9 represents an SEC elution profile of approximately a 168 ⁇ M solution of A ⁇ (40) A21C/A30C peptide as high molecular-weight ⁇ -structured oligomer. The insets below the two peaks represent dot blots of the fractions (evenly distributed throughout the peaks) assayed for Al 1 antibody binding capability.
- Figure 1OA represents an SEC elution profile of a concentrated solution (at approximately
- Figure 1OB represents an SEC elution profile of the first fraction in Figure 1OA that had the highest Al 1 affinity.
- the inset below the profile represents a dot blot of the fractions (evenly distributed throughout the peaks) assayed for Al 1 antibody binding capability.
- Figure HA represents far-UV CD spectra of the A ⁇ (40) A21C/A30C peptide as a coil-like protein at high concentration (1.1 mM), and its transformation to ⁇ -structured protein after thermal denaturation from 20 0 C to 80 0 C and back to 20 0 C with a temperature slope of 2 0 C min "1 .
- Figure 1 IB shows the thermal denaturation profile described in Figure 1 IA as monitored at 220 nm. There was no amorphous or fibrillar aggregation present in this sample neither before nor after thermal denaturation.
- Figure 12A represents transmission electron microscope (TEM) images of a negatively stained (uranyl acetate) solution of coil-like oligomers of A ⁇ (40) A21C/A30C peptide, which was approximately 500 ⁇ M at the time of grid application.
- the spherical structures are 15-35 nm in diameter.
- Figure 12B represents TEM images of the same solution as in Figure 12A incubated at 60 0 C for 20 min prior to grid application.
- the protofibrillar-like structures are 6-7 nm in width and on average approximately 36 nm in length.
- Figure 12C represents TEM images of a similarly negatively stained solution of approximately 150 ⁇ M A ⁇ (40) A21C/A30C as a ⁇ -structured high molecular- weight oligomer.
- the spherical structures are similar to those in Figure 12A, having diameters of 15-35 nm.
- the scale bar is 200 nm in these figures.
- Figure 13 represents a fibrillation assay of the A ⁇ (42) A21C/A30C peptide with formed and broken (by 10 mM TCEP) disulphide bonds at 117 ⁇ M. Fibrillation was monitored by TFT binding (10 ⁇ M) and the assays were carried out at 37 0 C with shaking in phosphate buffer at pH 7.2.
- Figure 14 represents an SEC elution profile of a concentrated solution of A ⁇ (42) A21C/A30C peptide that was denatured in 6M GdmCl prior to SEC.
- Figure 15A represents a time-dependent assay of a 80 kDa A ⁇ (40) A21C/A30C SEC fraction that was concentrated to 20 ⁇ M and incubated at 37 0 C. Time points were taken and assayed for the presence of the Al l epitope.
- Figure 15B represents a time-dependent assay of a 10 kDa A ⁇ (42) A21C/A30C SEC fraction that was concentrated to 50 ⁇ M and incubated at 37 0 C. Time points were taken and assayed for the presence of the Al l epitope.
- the invention provides covalently constrained A ⁇ peptides having A21C/A30C disulphide replacements that are resistant towards fibrillogenesis, and their use in the screening for therapeutic agents against toxic oligomeric species involved in Alzheimer's and other protein misfolding diseases.
- the peptides according to the invention can also be used directly as immunogens to raise an immune response against toxic forms of the A ⁇ peptide.
- the invention is an example of the use of rational peptide engineering to modify peptide properties to suit new needs; the need here being the desire to arrest A ⁇ peptide fibrillogenesis at the toxic oligomeric level so that these species can be isolated and utilized.
- the examples presented below demonstrate that the peptides according to the invention share many properties of the wild-type A ⁇ peptide except the high tendency of wild- type A ⁇ peptide to fibrillate.
- oligomer is used as a collective term for soluble aggregates containing a plurality of A ⁇ -peptide monomers.
- These monomers are peptide fragments derived from the APP (Swiss-Prot entry P05067), and are commonly comprised of 39 to 43 amino acids.
- the full length sequence is described in Kang, J., et ah, Nature 325: 773-776 (1987).
- the invention provides stabilized peptides corresponding to all of the 39 to 43 residue long derivatives of the APP including the two most frequently encountered derivatives, A ⁇ (40) and A ⁇ (42), for which the sequences are:
- one preferred aspect of the invention provides peptides comprising the following sequences:
- the peptides according to the invention was specifically designed in order to prevent fibrillation of the A ⁇ peptides derived from the APP.
- Design of the peptides according to the invention (Figure IA) is based on the recently determined NMR structure (Protein Data Bank accession no. 2OTK) of the A ⁇ peptide (Hoyer, W., et al., Proc. Natl. Acad. Sci. USA. 105: 5099-5104 (2008)).
- the constraining disulfide bond present in the peptides of the invention connecting residues 21 and 30 stabilizes this ⁇ -hairpin structure once it is formed, and further inhibits the conformational switch that triggers amyloidosis, (one hypothesis of such a mechanism is schematically depicted in Figure IB.
- the A ⁇ peptides according to the invention are very resistant towards fibrillogenesis even at high concentrations unless the disulphide bond is reduced (disrupted) to form thiols (RSH) with e.g. dithiothreitol (DTT), ⁇ -mercaptoethanol, or TCEP, under physiological conditions, under which the intramolecular covalent link between Cys21 and Cys30 is stable.
- RSH thiols
- DTT dithiothreitol
- TCEP TCEP
- Positions 21 and 30 of the A ⁇ peptide are the only two residues that can be covalently linked by a disulphide bond without disrupting said ⁇ -hairpin structure.
- the invention is not limited to A21C/A30C A ⁇ peptides corresponding to the 39 to 43 residue long A ⁇ peptides with wild-type sequences, but also includes A21C/A30C A ⁇ peptides corresponding to naturally occurring A ⁇ -peptide mutants that lead to an increased incidence of familial AD. These mutations can be found in any of the 39-43 long A ⁇ - peptide derivatives of the APP.
- the invention thus includes, but is not limited to A21C/A30C A ⁇ peptides carrying, the E22G arctic mutation (SEQ ID N0:5), the E22Q Dutch mutation (SEQ ID N0:6), the D23N Iowa mutation (SEQ ID N0:7), and the Italian E22K mutation (SEQ ID NO: 8), and other familial mutations that are likely to appear.
- the hairpin structure in said sequences containing a methionine sulphoxide or sulphone at position 35 can be stabilized by the invention. This methionine is believed by many to be essential for toxicity (see e.g. Butterfield, D. A., and Kanski, I, Peptides 23: 1299-1309 (2002)).
- A21C/A30C A ⁇ peptides comprising one or more conservative mutations or otherwise made conservative substitutions, alterations, insertions or truncations (e.g. the 9-42 truncated derivative; SEQ ID NO:9) of the above mentioned sequences, including non-standard amino acids and end terminal modifications, such as cysteinylation, maleimidation, acetylation, and amidation, are also part of the invention, and are fibrillization-resistant. Amino acids outside of the ⁇ -hairpin structure, i.e.
- A21C/A30C A ⁇ peptides that are part of the invention include, but is not limited to, peptides wherein one or more consecutive stretches of A ⁇ -peptide sequence have been inverted, such as the 1-8 N-terminal inverted derivative (SEQ ID NO: 10).
- the A ⁇ peptides according to the invention can be produced by chemical synthesis followed by separation of oxidized peptides (sulphides) from reduced peptides (thiols) by reverse-phase high-performance liquid chromatography (HPLC) as previously described for other disulfide derivatives of this peptide (Shivaprasad, S. and Wetzel, R., Biochemistry 43: 153-15317 (2004)). Chemical synthesis is the most common way to produce the A ⁇ peptides.
- a ⁇ peptides are usually lyophilized, it may first be necessary to pretreat the peptides with 100% 1,1, 1,3,3, 3-hexafluoro-2- propanol (HFIP) to remove pre-existing aggregates prior to characterization as discussed previously (Stine, W. B. et al. J. Biol. Chem. 278: 11612-11622 (2003)).
- HFIP 1,1, 1,3,3, 3-hexafluoro-2- propanol
- Heterologous expression of the A ⁇ peptides according to the invention in Escherichia coli is an alternative to chemical synthesis, but requires for the peptides to be expressed as fusion proteins to aid its solubility (see e.g. Lee, E. K., et al, Prot. Expr. Purif. 40:183-189 (2005)).
- a method of choice for cost-efficient production of A ⁇ (40) and A ⁇ (42) is to co-express these peptides with an affinity binding protein (Gr ⁇ nwall, C, et al, J. Biotechnol. 128: 162-183 (2007)) in E. coli.
- the peptides are expressed concomitantly with the binding protein, thus forming a soluble complex that protects the A ⁇ peptide against aggregation and proteolysis.
- the binding protein contains a hexa-histidine tag to aid in the purification of the complex using immobilized metal affinity chromatography (IMAC).
- IMAC immobilized metal affinity chromatography
- the complex can be broken using GdmCl, and the released A ⁇ peptide can be separated from the affinity binding protein again using IMAC under denaturing conditions.
- the GdmCl can then be dialyzed away or removed using SEC.
- This co- expression protocol does not involve lyophilization, and enables for direct selection of the peptides during expression. As a result from expression in E.
- the A ⁇ (40) A21C/A30C peptide resembles the wild-type peptide.
- CD and NMR spectra of the low molecular- weight fraction (eluting at approximately 8 kDa) obtained from SEC demonstrate that said peptide forms coil-like structures that are very similar to wild-type peptide (Example 3).
- oligomeric species containing a plurality of monomers elute from the SEC runs. These frequently elute within the range of >8 to ⁇ 42 kDa (Example 4), and they all have coil- like structures with CD spectra identical to the peptide obtained in the low molecular-weight fraction.
- coil-like oligomers are denoted low molecular- weight oligomers.
- the peptide is also capable of forming larger oligomers with ⁇ structures. These structures elute from the SEC columns as proteins of sizes of approximately 75-85 kDa and 170 kDa (Example 5).
- Some of these high molecular-weight oligomers also bind the Al 1 antibody (Example 6), which is a polyclonal antibody specifically raised against A ⁇ peptide oligomers and is suggested to be generic for disease-related oligomeric species (Kayed, R. et al. Science 300: 486-489 (2003)).
- Low molecular-weight oligomers of the peptides of the invention at high concentration can be made to form protofibril- like structures rapidly by the application of heat (60 0 C) (Example 7). This step has not been optimized, and other elevated temperatures (approximately >20 0 C) will have the same effect, although a temperature below 80 0 C is desirable to prevent irreversible covalent modifications of the peptide from occurring (including deamidation of Asn27 and Glnl5, as well as oxidation of the Cys21-Cys30 disulphide bond and Met35).
- these protofibrils have an average dimension of approximately 6.7 nm in width, and lengths in the order of approximately 36 nm (although the lengths vary) (Example 8).
- the transition is accompanied by the formation of ⁇ structure, for which the CD spectrum is indistinguishable from the high molecular-weight oligomers above.
- These structures also bind TFT. In aggregation assays, this structural transition therefore results in a rapid initial increase in fluorescence, which is followed by a gradual decrease when the protofibrils dissociate as a new equilibrium is established.
- the longer peptides according to the invention i.e. the A ⁇ (42) derivatives (Example 9) are also protected from fibrillization, and the oligomers thus obtained during SEC of A ⁇ (42) A21C/A30C have a similar size distribution as the A ⁇ (40) A21C/A30C oligomers (Example 10).
- the Al 1 epitope is present in the largest of the high molecular- weight oligomers with ⁇ structure that elute from the SEC column (Example 10). This Al 1 -binding epitope is very stable once formed and only decreases with 0.1% per day when incubated at 37 0 C over a two-month period (Example 11).
- the peptides according to the invention when used for the production of covalently stabilized oligomers, provides advantages over previous methods in that these peptide's resistance towards fibrillogenesis enables such oligomers to be prepared under physiological conditions. These oligomers can then be studied at higher concentrations and/or for longer times than previously possible, thus aiding considerably in drug screening assays and immunization trials.
- High molecular-weight oligomers of the A ⁇ peptides according to the invention can be produced ⁇ e.g. as in example 5), concentrated, and then used in methods for the identification of compounds potentially suitable for the treatment of AD. Such a compound should bind to already formed toxic oligomers and dissolve them, and/or prevent their formation.
- the covalently stabilized monomers or oligomers may be used for the preparation of antigen formulations as immunogens for the generation of antibodies or other protein binders. Both active and passive immunization against the hairpin structure here shown to be present in the potentially toxic oligomeric forms of AD may come into question.
- the present invention provides non-human transgenic animals expressing a nucleotide sequence encoding an amyloid precursor protein (APP) comprising the A21C/A30C mutations.
- APP amyloid precursor protein
- the APP is a mutated human APP comprising the A21C/A30C mutations, most preferably the APP is the protein SEQ ID NO:11.
- the nucleotide sequence is a mutated human APP gene comprising the A21C/A30C mutations.
- the non-human transgenic animal is preferably selected from the phylum Chordata (and Hemichordata), such as a vertebrate animal (which include cyclostomes, bony fish, sharks and rays, amphibians, reptiles, mammals, and birds).
- the non-human transgenic animal is a mammal, such as a mouse, rat, pig, rabbit, or guinea pig. Most preferably the non-human transgenic mammal is a mouse.
- non-human transgenic animals can be used as model systems for AD pathogenesis by overexpressing only an A ⁇ peptide according to the invention comprising the A21C/A30C replacements.
- non-human transgenic animals may not only be selected from within the chordates (and hemichordates), but may also include invertebrates which lack endogenous APP comprising the A ⁇ peptide (e.g. selected from any of the phyla Annelida, Arthropoda, Cnidaria, Echinodermata, Mollusca, Nematoda, Nematomorpha,
- Invertebrate models of AD pathogenesis have the advantage of short life spans, low costs, small sizes, and highly characterized genetics. Examples of such invertebrate model systems include Drosophila melanogaster (Crowther, D. C. et al. (2005) Neuroscience 132: 123-135) and Caenorhabditis elegans (Link, CD. (1995) Proc. Natl. Acad. Sci. USA 92: 9368-9372). Accordingly, in yet another aspect the present invention provides non-human transgenic animals expressing a nucleotide sequence encoding an A ⁇ peptide according to the invention comprising the A21C/A30C replacements.
- the heterologously expressed A ⁇ peptide in such an invertebrate model system is the peptide corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 SEQ ID NO:9 and/or SEQ ID NO: 10
- the present invention provides a nucleotide sequence encoding an APP comprising the A21C/A30C mutations.
- the APP is a mutated human APP comprising the A21C/A30C mutations, most preferably the APP is the protein SEQ ID NO:11.
- the nucleotide sequence is a mutated human APP gene comprising the A21C/A30C mutations.
- Another aspect of the present invention is an expression system comprising a nucleotide sequence encoding APP comprising the A21C/A30C mutations.
- Yet another aspect of the present invention is a polypeptide which is an APP comprising the A21C/A30C mutations, preferably a mutated human APP comprising the A21C/A30C mutations, most preferably the polypeptide SEQ ID NO:11.
- the amino acid position 21 in the human A ⁇ peptide corresponds to the amino acid position
- amino acid position 30 in the human A ⁇ peptide corresponds to the amino acid position 701 in human APP.
- the sequence of human APP can be found in Swiss-Prot entry P05067 (SEQ ID NO:12). Consequently, by an APP comprising the A21C/A30C mutations is meant an APP wherein z) the amino acid alanine in position 692, as defined by the sequence of the human APP (SEQ ID NO: 12) or the corresponding position in a homologous or heterologous APP, is replaced with a cysteine, and U) the amino acid alanine in position 701, as defined by the sequence of the human APP (SEQ ID NO: 12) or the corresponding position in a homologous or heterologous APP, is replaced with a cysteine.
- a homologous or heterologous APP is meant an APP derived from an animal, such as a vertebrate, as well as a synthetic, mutated, and/or non naturally occurring APP, which has a sequence identity of at least 80%, such as at least 90%, or at least 95% as compared to human APP (SEQ ID NO: 12).
- the percent identity between two amino acid sequences is determined as follows. First, an amino acid sequence is compared to, for example, SEQ ID NO: 12 using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from the U.S.
- B12seq performs a comparison between two amino acid sequences using the BLASTP algorithm.
- B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C: ⁇ seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C: ⁇ seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C: ⁇ output.txt); and all other options are left at their default setting.
- -i is set to a file containing the first amino acid sequence to be compared (e.g., C: ⁇ seql.txt)
- -j is set to a file containing the second amino acid sequence to be compared (e.g., C: ⁇ seq2.txt)
- -p is set to blastp
- -o is set to any desired file name (e.g., C: ⁇ output.txt); and all other options are left
- the following command can be used to generate an output file containing a comparison between two amino acid sequences: C: ⁇ B12seq -i c: ⁇ seql.txt -j c: ⁇ seq2.txt -p blastp -o c: ⁇ output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
- generation of the transgenic mouse may optionally involve disruption of the murine APP genes and introduction of one or more copies of the gene encoding a mutated human APP into the murine genome, preferably at the same location as the endogenous murine APP gene.
- the transgenic non-human animals of the invention are preferably produced by introducing transgenes into the germline of the animal.
- Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell.
- the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
- strains such as C57BL/6 or C57BL/6 x DBA/2 Fl, or FVB lines are often used (obtained commercially from Charles River Labs, Boston, Mass., The Jackson Laboratory, Bar Harbor, ME, or Taconic Labs.).
- transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
- the transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s).
- the egg may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both.
- One common method is to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
- Retroviral infection can also be used to introduce transgenes into non-human animals.
- the developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci. USA 73:1260-1264).
- Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82:6148- 6152).
- Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
- transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
- a third type of target cell for transgene introduction is the embryonic stem (ES) cell.
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus- mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- an endogenous APP gene in a nonhuman host is functionally disrupted by homologous integration of a heterologous APP gene comprising the A21C/A30C mutations, such that the heterologous APP gene substantially replaces the endogenous APP gene, and preferably completely replaces the coding sequences of the endogenous APP gene.
- the heterologous APP gene is linked, as a consequence of homologous integration, to regulatory sequences (e.g., an enhancer/promoter) of the endogenous APP gene, respectively, so that the heterologous gene is expressed under the transcriptional control of regulatory elements from the endogenous APP gene locus.
- regulatory sequences e.g., an enhancer/promoter
- Nonhuman hosts which are homozygous for such replacement alleles may be produced according to methods described herein. Such homozygous nonhuman hosts generally will express a heterologous APP but do not express the endogenous APP protein.
- the expression pattern of the heterologous humanized APP gene will substantially mimic the expression pattern of the endogenous APP gene, in the naturally-occurring (non- transgenic) nonhuman host.
- a transgenic mouse can be generated that has APP gene sequences comprising the A21C/A30C mutations in place of endogenous murine APP gene sequences and which are transcriptionally controlled by endogenous murine regulatory sequences.
- the APP gene sequences comprising the A21C/A30C mutations generally will be expressed similarly to the murine APP in naturally occurring non-transgenic mice.
- a replacement-type targeting construct is employed for homologous gene replacement. Double-crossover homologous recombination between endogenous APP gene sequences of the targeting construct result in targeted integration of the heterologous APP gene segments.
- the homology targeting regions of the transgene comprise sequences which flank the endogenous APP gene segments, so that homologous recombination results in concomitant deletion of the endogenous APP, and homologous integration of the heterologous APP gene segments.
- Substantially an entire endogenous APP gene may be replaced with a heterologous APP by a single targeting event or by multiple targeting events (e.g., sequential replacement of individual exons).
- One or more selectable markers usually in the form of positive or negative selection expression cassettes, may be positioned in the targeting construct. It is usually preferred that selectable markers are located in intron regions of the heterologous replacement region.
- Transgenic animals comprising transgene APP gene sequences comprising the A21C/A30C mutations can be crossed with other transgenic animals, generating transgenic animals comprising multiple transgenes.
- a manner of preparation is to generate a series of mammals, each containing one of the desired knockout constructs or transgenes. Such mammals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single mammal containing all desired knockout constructs and/or transgenes, where the mammal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout(s) constructs and/or transgene(s).
- crossing and backcrossing is accomplished by mating siblings or a parental strain with an offspring, depending on the goal of each particular step in the breeding process. In certain cases, it may be necessary to generate a large number of offspring in order to generate a single offspring that contains each of the knockout constructs and/or transgenes in the proper chromosomal location. In addition, it may be necessary to cross or backcross over several generations to ultimately obtain the desired genotype.
- Example 1 Fibrillation assay of wild-type A ⁇ (40) and A ⁇ (40) A21C/A30C.
- concentration determinations were carried out spectrophotometrically using an extinction coefficient of 1424 cm “1 M "1 for the difference in absorption at 280 nm and 300 nm.
- the buffered solution used in all examples was 50 mM K + phosphate, 50 mM NaCl, pH 7.2, unless otherwise stated. This buffer was also the storage buffer for the A ⁇ peptides. All peptide samples were prepared fresh, kept at 4 0 C, and used within 3-4 days.
- Fibrillation assays were carried out by monitoring the enhanced fluorescence of the dye TFT upon binding to the fibrils (Levine, H., Methods Enzymol. 309: 274-284 (1999)). Fluorescence was recorded in 96-well plates (Nunc) using a FLUOstar Optima reader (BMG) equipped with 440 nm excitation and 480 run emission filters. The peptide samples were kept at 30 ⁇ M and were supplemented with 10 ⁇ M TFT. In addition, 5 mM TCEP was added to one reference sample of the invention to assay the effect of the formed disulphide bond. TCEP is a more stable reductant than the more commonly used DTT at basic pH.
- the fibrillation assay was carried out as in example 1, except that the concentration of peptide was also assayed at 50, 150, and 250 ⁇ M, and the TCEP concentration was increased to 10 mM TCEP in the reference samples. Also, data points were recorded every 15 min with 5 min of orbital shaking (width 5 mm) preceding each measurement. Dual samples were analyzed at each concentration, and the result is presented in Figure 3.
- Example 7 The result is similar to the result obtained in Example 1. However, there is a rapid increase in fluorescence for the samples with an intact disulphide bond at the 150 and 250 ⁇ M concentrations, which then decline to similar levels with much slower rates.
- the coil-like oligomers can be made to form protofibrillar-like structures when subjected to elevated temperatures, and these do bind TFT weakly thus giving rise to enhanced fluorescence. As time progresses, a new equilibrium is reached in these samples. Observations made by Walsh et al for the wild-type A ⁇ peptide are applicable to the data in Figure 3 (Walsh, D. M., et al, J. Biol. Chem. 274: 25945-25952 (1999)).
- Another feature of the increased peptide concentration is the increase in lag times preceding nucleation for the A ⁇ A21C/A30C peptide with broken disulphide bonds. It is probable that this is the combined result of (z) that the coil- like oligomers present in these samples (see Example 4) have more or less protected disulphide bonds, and (U) that more peptide requires more time to be reduced with TCEP.
- SEC was carried out on a Superdex 75 10/300 column (GE Healthcare) equilibrated with 50 mM K + phosphate, 50 mM NaCl, pH 7.2. The flow rate was 0.8 ml min "1 , and the column was operated by an AKTA Explorer unit (GE Healthcare). All column runs were carried out at room temperature (21 0 C). The column was calibrated with a low molecular- weight gel filtration calibration kit (Amersham Biosciences) under similar conditions. Typically, 500 ⁇ l of peptide was injected into this column during each run.
- the A ⁇ (40) A21C/A30C peptide typically elutes as an approximately 8-10 kDa protein. Even though the A ⁇ peptide is approximately a 4.5 kDa protein, we do not interpret this as this peptide is eluting as a dimer because of the potentially extended structures of unfolded coil- like proteins compared to the well- folded proteins used in the calibration kit (ribonucelase A, chymotrypsinogen A, ovalbumin, and albumin).
- a far-UV CD spectrum was collected on a Jasco J-810 spectropolarimeter (Jasco Corporation) at 20 0 C using a scan rate of 50 nm min "1 .
- 20 ⁇ M of A ⁇ (40) A21C/A30C peptide was scanned between 190-260 nm in a 1.0 mm cuvette.
- the spectrum exhibits a minimum around 200 nm that is typical of coil- like structures, and is very similar to CD spectra of non-aggregated A ⁇ (40) and A ⁇ (42) wild-type peptide (not shown; for examples see e.g. Walsh, D. M., et al, J. Biol. Chem. 274: 25945-25952 (1999)).
- this example thus demonstrates that the peptides according to the invention have structural properties very similar to wild type A ⁇ peptide despite the presence of an intramolecular disulphide bond. That the disulphide bond is formed is demonstrated in Examples 1 and 2.
- Example 4 SEC analysis of the oligomeric tendencies of the A ⁇ (40) A21C/A30C . SEC on the Superdex 75 10/300 column was carried out as in Example 3. The larger
- Superdex 75 16/60 column (GE Healthcare) was operated similarly to the smaller 10/300 column, except that it was run at 1.0 ml min "1 and that 1 ml was loaded.
- Figure 5 shows one experiment to assay the distribution of oligomers under higher concentration of peptide than that used in Figure 4A.
- the A ⁇ (40) A21C/A30C peptide was denatured in 6 M guanidinium chloride (GdmCl), 50 mM K + phosphate, 50 mM NaCl (pH 7.2), which destroys pre-existing oligomers.
- GdmCl is a compound routinely used as a protein denaturant.
- a solution of approximately 1.8 mM denatured peptide was injected into the Superdex 75 16/60 column. On the column, the peptide molecules become separated from the GdmCl and are free to oligomerize.
- the A ⁇ (40) A21C/A30C peptide elutes as a distribution of soluble structures.
- the dominating peak elutes at a position which, according to the protein calibration kit (same as in Example 3), corresponds to approximately a 42 kDa globular protein.
- CD spectroscopy demonstrated that the oligomers in this peak has coil-like structure (not shown; the far-UV CD spectrum is virtually identical to Figure 4B).
- the distribution of these coil structures ranges down to species eluting as approximately 8 kDa proteins.
- the larger oligomeric species that elutes as an 85 kDa species has a far-UV CD spectrum consistent with ⁇ structure (not shown; the far-UV CD spectrum of this fraction is virtually identical to Figure 7B).
- Example 3 SEC was carried out as in Example 3, with a Sephacryl S-300 26/60 column (GE Healthcare) where 1 ml was injected into the column, which was run at 1.3 ml min "1 .
- the column had been calibrated previously as in Example 3.
- a far-UV CD spectrum of A ⁇ (40) A21C/A30C peptide was collected as in Example 3.
- 250 ⁇ M of A ⁇ (40) A21C/A30C peptide that eluted from a SEC run at volumes corresponding to 75-85 kDa was scanned between 185-260 nm in a 0.1 mm cuvette.
- the spectrum exhibits a minimum around 210 nm and positive ellipticity around 190 nm that is typical of ⁇ -like structures of the A ⁇ (40) and A ⁇ (42) peptides as they oligomerize and aggregate (e.g. Walsh, D. M., et al, J. Biol. Chem.
- Figure 9 shows a SEC run of a 1 ml A ⁇ (40) A21C/A30C peptide solution of isolated and concentrated (to approximately 168 ⁇ M) oligomer obtained as in Example 5.
- SEC was carried out as in Example 3, using a Superdex 75 16/60 column (GE Healthcare) run at 1 ml min "1 . Under these conditions the sample elutes partly in the void volume (approximately 42 ml for this column, corresponding to > 100 kDa proteins) and as high molecular- weight oligomers (corresponding to approximately 87 kDa proteins). A smaller fraction also elutes as a 7-8 kDa protein.
- the membrane was then washed three times with TBST buffer for five minutes, and the Al 1 primary antibody (Biosource) at 0.5-0.8 ⁇ g ml "1 in 5% non-fat dry milk dissolved in TBST buffer was applied to the membrane for 1 h at room temperature (21 0 C).
- the secondary antibody a donkey ECLTM anti-rabbit IgG horseradish peroxidase conjugate from GE Healthcare
- the secondary antibody a donkey ECLTM anti-rabbit IgG horseradish peroxidase conjugate from GE Healthcare
- Example 7 Coil- to ⁇ -transition in A ⁇ (40) A21C/A30C.
- Figure 1 IA shows the two CD spectra of a 1.1 mM A ⁇ (40) A21C/A30C peptide before (coil structure) and after ( ⁇ structure) being denatured by a 2 0 C min "1 gradient of increasing temperature from 20 to 80 0 C and back again. The transition is shown in Figure 1 IB as monitored at 220 nm.
- Example 8 it is demonstrated that the ⁇ structures thus formed are protofibrillar-like. Protofibrils are believed by many to be precursors of fibrils (e.g. Walsh, D. M., et al, J. Biol. Chem. 274: 25945-25952 (1999)).
- Example 8 TEM analysis of A ⁇ (40) A21C/A30C coil- and ⁇ -structured oligomers, and protofibrillar-like species.
- TEM images were obtained by a LEO 912 AB OMEGA electron microscope (Carl Zeiss SMT AG) equipped with a MegaView CCD camera (Olympus). Negative staining with uranyl acetate was used in all samples. Formvar/carbon coated nickel grids were activated with UV-light for 5 minutes, after which 5-10 ⁇ l of protein sample was applied to each grid for 2 minutes. Two steps of washing with approximately 10 ⁇ l of filtered deionized H 2 O preceded the staining. A two- minute treatment with 2% uranyl acetate solution in filtered deionized H 2 O completed the preparation of the grids, which were allowed to air dry for a few minutes before storage or immediate analysis.
- the scale bar is 200 nm in Figure 12.
- the spherical structures observed in Figure 12A and Figure 12C are similar in morphology and dimension to each other, having diameters of 15 nm to 35 nm. They are also similar to previously published TEM images that have used the same staining technique. These spherical structures of wild-type A ⁇ (40) peptide were recently observed for oligomers of A ⁇ (40) having ⁇ structure (Chimon, S., et al, Nature Struct. MoI. Biol. 14: 1157- 1164 (2007)). In this publication, Chimon et al. claim that these structures are the toxic species.
- FIG. 12B The structures observed in Figure 12B are protofibrillar-like, with an average dimension of 6- 7 nm in width and approximately 36 nm in length (although the lengths vary). They are very similar in morphology and dimension to the wild-type A ⁇ (40) protofibrils, which have average widths of 6-10 nm and lengths of 5-160 nm (Walsh, D. M., et al, J. Biol. Chem. 272: 22364-22372 (1997)).
- Example 9 Fibrillation assay of A ⁇ (42) A21C/A30C.
- the fibrillation assay was carried out as in example 2 with the exception that data points were recorded every 10 min.
- the concentration of A ⁇ (42) A21C/A30C was approximately 117 ⁇ M. The result is presented in Figure 13.
- Example 11 Stability of the Al 1 -binding epitope.
- a 80-kDa fraction of A ⁇ (40) A21C/A30C peptide obtained as in example 5 was concentrated to 20 ⁇ M and incubated at 37 0 C in 50 mM K + phosphate buffer, 50 mM NaCl, and 0.05% azide. Samples were removed at regular intervals during a two-month period and stored at -24 0 C until analysis. The dot blot was carried out as in example 6, and the result is presented in Figure 15A.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Neurology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Psychology (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Hospice & Palliative Care (AREA)
- Diabetes (AREA)
- Psychiatry (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
Claims
Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK09732855.3T DK2262526T3 (en) | 2008-04-14 | 2009-04-14 | STABLE amyloid-beta MONOMERS monomers and oligomers |
JP2011503942A JP5817060B2 (en) | 2008-04-14 | 2009-04-14 | Purified peptides, soluble oligomers containing purified peptides, methods for identifying compounds suitable for disease treatment using purified peptides or soluble oligomers, purified using purified peptides or soluble oligomers Methods for selecting binding proteins that react with peptides or soluble oligomers, use of purified peptides or soluble oligomers in the production of pharmaceutical compositions, pharmaceutical preparations containing purified peptides or soluble oligomers, purified peptides or soluble oligomers A vaccine comprising a purified peptide or a soluble oligomer, a method for preventing or treating a mammal other than a human, a non-human transgenic gene wherein APP expresses a nucleotide encoding APP containing two cysteines Animals, non-human transgenic animals expressing nucleotides encoding purified peptides, polypeptides where APP is an APP containing two cysteines, purified peptides or nucleotides encoding polypeptides, purified peptides Or an expression system comprising a nucleotide encoding a polypeptide |
US12/937,379 US9688734B2 (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
PL09732855T PL2262526T3 (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
CA2721156A CA2721156C (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
ES09732855.3T ES2562224T3 (en) | 2008-04-14 | 2009-04-14 | Stable beta amyloid monomers and oligomers |
CN200980122412.XA CN102065881B (en) | 2008-04-14 | 2009-04-14 | Stable amyloid BETA monomers and oligomers |
AU2009236699A AU2009236699B2 (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
EP09732855.3A EP2262526B1 (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
HRP20160177T HRP20160177T1 (en) | 2008-04-14 | 2016-02-18 | Stable amyloid beta monomers and oligomers |
US15/599,926 US10138281B2 (en) | 2008-04-14 | 2017-05-19 | Stable amyloid beta monomers and oligomers |
US15/599,906 US10023622B2 (en) | 2008-04-14 | 2017-05-19 | Method of immunization for reducing toxic damaged caused by oligomeric form of the amyloid-beta protein with stable amyloid beta monomers and oligomers |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE0800842-7 | 2008-04-14 | ||
SE0800842 | 2008-04-14 | ||
SE0802433-3 | 2008-11-20 | ||
SE0802433 | 2008-11-20 |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/937,379 A-371-Of-International US9688734B2 (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
US15/599,926 Division US10138281B2 (en) | 2008-04-14 | 2017-05-19 | Stable amyloid beta monomers and oligomers |
US15/599,906 Division US10023622B2 (en) | 2008-04-14 | 2017-05-19 | Method of immunization for reducing toxic damaged caused by oligomeric form of the amyloid-beta protein with stable amyloid beta monomers and oligomers |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009128772A1 true WO2009128772A1 (en) | 2009-10-22 |
Family
ID=41199330
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE2009/050378 WO2009128772A1 (en) | 2008-04-14 | 2009-04-14 | Stable amyloid beta monomers and oligomers |
Country Status (12)
Country | Link |
---|---|
US (3) | US9688734B2 (en) |
EP (1) | EP2262526B1 (en) |
JP (2) | JP5817060B2 (en) |
CN (1) | CN102065881B (en) |
AU (1) | AU2009236699B2 (en) |
CA (1) | CA2721156C (en) |
CY (1) | CY1117283T1 (en) |
DK (1) | DK2262526T3 (en) |
ES (1) | ES2562224T3 (en) |
HR (1) | HRP20160177T1 (en) |
PL (1) | PL2262526T3 (en) |
WO (1) | WO2009128772A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2497782A1 (en) | 2011-03-08 | 2012-09-12 | Alzinova AB | Anti oligomer antibodies and uses thereof |
CN109836470A (en) * | 2019-03-22 | 2019-06-04 | 河北大学 | A method of being automatically synthesized disulfide bond in peptide molecule |
WO2022248650A3 (en) * | 2021-05-26 | 2023-01-05 | Regain Therapeutics Sweden Ab | Compositions and methods for treatment and/or prophylaxis of proteinopathies |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9688734B2 (en) * | 2008-04-14 | 2017-06-27 | Alzinova Ab | Stable amyloid beta monomers and oligomers |
JP7357354B2 (en) * | 2020-02-04 | 2023-10-06 | 国立大学法人京都大学 | Amyloid β42 cross-linked analog peptide |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005089539A1 (en) * | 2004-03-22 | 2005-09-29 | Bioarctic Neuroscience Ab | Transgenic model for alzheimer’s disease |
US20060018918A1 (en) * | 2000-12-27 | 2006-01-26 | Board Of Regents, The University Of Texas System | Production of stabilized conformational isomers of disease associated proteins |
US20070021345A1 (en) * | 2003-06-30 | 2007-01-25 | Ehud Gazit | Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases |
US20070213512A1 (en) * | 2002-10-01 | 2007-09-13 | Krafft Grant A | Amyloid beta peptide analogs and assemblies thereof |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH029933A (en) | 1988-02-29 | 1990-01-12 | Keihin Seiki Mfg Co Ltd | Starting device for carburetor |
JPH0659933A (en) | 1992-08-11 | 1994-03-04 | Toshiba Corp | Code converter |
US6699671B1 (en) * | 1998-09-24 | 2004-03-02 | Pharmacia & Upjohn Company | Alzheimer's disease secretase, APP substrates therefor, and uses therefor |
EP1424892A4 (en) * | 2001-08-10 | 2006-08-23 | Univ South Florida | Modulation of angiogenesis by a-beta peptides |
DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
US20040250302A1 (en) * | 2003-10-21 | 2004-12-09 | Envivo Pharmaceuticals, Inc. | Transgenic flies expressing Abeta42-dutch |
US20060079447A1 (en) * | 2004-10-08 | 2006-04-13 | Wetzel Ronald B | Stabilized A-beta protofibrillar aggregates |
TW200726482A (en) | 2005-06-30 | 2007-07-16 | Merck & Co Inc | Method for preparing a covalently cross linked oligomer of amyloid beta peptides |
TW200726774A (en) | 2005-06-30 | 2007-07-16 | Merck & Co Inc | Composition and method for producing stable amyloid beta oligomers |
JP4876224B2 (en) | 2006-06-05 | 2012-02-15 | 財団法人ヒューマンサイエンス振興財団 | Amyloid β oligomer and method for producing and using the same |
US9688734B2 (en) * | 2008-04-14 | 2017-06-27 | Alzinova Ab | Stable amyloid beta monomers and oligomers |
JP2011529084A (en) * | 2008-07-25 | 2011-12-01 | アボット・ラボラトリーズ | Amyloid β peptide analogues, oligomers thereof, processes for preparing said analogues or oligomers and compositions comprising said analogues or oligomers and their use |
-
2009
- 2009-04-14 US US12/937,379 patent/US9688734B2/en active Active
- 2009-04-14 JP JP2011503942A patent/JP5817060B2/en active Active
- 2009-04-14 AU AU2009236699A patent/AU2009236699B2/en active Active
- 2009-04-14 WO PCT/SE2009/050378 patent/WO2009128772A1/en active Application Filing
- 2009-04-14 CN CN200980122412.XA patent/CN102065881B/en active Active
- 2009-04-14 PL PL09732855T patent/PL2262526T3/en unknown
- 2009-04-14 CA CA2721156A patent/CA2721156C/en active Active
- 2009-04-14 EP EP09732855.3A patent/EP2262526B1/en active Active
- 2009-04-14 DK DK09732855.3T patent/DK2262526T3/en active
- 2009-04-14 ES ES09732855.3T patent/ES2562224T3/en active Active
-
2015
- 2015-04-20 JP JP2015086140A patent/JP6128450B2/en active Active
-
2016
- 2016-02-17 CY CY20161100139T patent/CY1117283T1/en unknown
- 2016-02-18 HR HRP20160177T patent/HRP20160177T1/en unknown
-
2017
- 2017-05-19 US US15/599,906 patent/US10023622B2/en active Active
- 2017-05-19 US US15/599,926 patent/US10138281B2/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060018918A1 (en) * | 2000-12-27 | 2006-01-26 | Board Of Regents, The University Of Texas System | Production of stabilized conformational isomers of disease associated proteins |
US20070213512A1 (en) * | 2002-10-01 | 2007-09-13 | Krafft Grant A | Amyloid beta peptide analogs and assemblies thereof |
US20070021345A1 (en) * | 2003-06-30 | 2007-01-25 | Ehud Gazit | Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases |
WO2005089539A1 (en) * | 2004-03-22 | 2005-09-29 | Bioarctic Neuroscience Ab | Transgenic model for alzheimer’s disease |
Non-Patent Citations (1)
Title |
---|
DATABASE GENESEQ [online] 6 April 2006 (2006-04-06), accession no. EBI Database accession no. AEF68007 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2497782A1 (en) | 2011-03-08 | 2012-09-12 | Alzinova AB | Anti oligomer antibodies and uses thereof |
WO2012120035A1 (en) | 2011-03-08 | 2012-09-13 | Alzinova Ab | Anti oligomer antibodies and uses thereof |
US9062102B2 (en) | 2011-03-08 | 2015-06-23 | Alzinova Ag | Anti oligomer antibodies and uses thereof |
CN109836470A (en) * | 2019-03-22 | 2019-06-04 | 河北大学 | A method of being automatically synthesized disulfide bond in peptide molecule |
CN109836470B (en) * | 2019-03-22 | 2022-03-22 | 河北大学 | Method for automatically synthesizing polypeptide intramolecular disulfide bond |
WO2022248650A3 (en) * | 2021-05-26 | 2023-01-05 | Regain Therapeutics Sweden Ab | Compositions and methods for treatment and/or prophylaxis of proteinopathies |
Also Published As
Publication number | Publication date |
---|---|
AU2009236699B2 (en) | 2013-09-05 |
JP5817060B2 (en) | 2015-11-18 |
US20110064741A1 (en) | 2011-03-17 |
US9688734B2 (en) | 2017-06-27 |
US10023622B2 (en) | 2018-07-17 |
US20170355737A1 (en) | 2017-12-14 |
CN102065881B (en) | 2014-03-12 |
DK2262526T3 (en) | 2016-02-08 |
EP2262526B1 (en) | 2015-12-02 |
JP6128450B2 (en) | 2017-05-17 |
CA2721156C (en) | 2017-09-19 |
PL2262526T3 (en) | 2016-04-29 |
JP2011520423A (en) | 2011-07-21 |
US20180030103A1 (en) | 2018-02-01 |
EP2262526A1 (en) | 2010-12-22 |
CY1117283T1 (en) | 2017-04-26 |
CN102065881A (en) | 2011-05-18 |
HRP20160177T1 (en) | 2016-03-25 |
ES2562224T3 (en) | 2016-03-03 |
US10138281B2 (en) | 2018-11-27 |
JP2015145414A (en) | 2015-08-13 |
AU2009236699A1 (en) | 2009-10-22 |
CA2721156A1 (en) | 2009-10-22 |
EP2262526A4 (en) | 2012-04-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10023622B2 (en) | Method of immunization for reducing toxic damaged caused by oligomeric form of the amyloid-beta protein with stable amyloid beta monomers and oligomers | |
Hanakam et al. | Myristoylated and non‐myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein. | |
Swank et al. | Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila | |
JP6940636B2 (en) | Protoxin-II mutant and how to use it | |
WO1997027296A1 (en) | Nucleic acids and proteins related to alzheimer's disease, and uses therefor | |
JP2003511071A (en) | Cell penetrating peptide inhibitor of JNK signal transduction pathway | |
CA2599792A1 (en) | Screening method, process for purifying of non-diffusible a-beta oligomers, selective antibodies against said non-diffusible a-beta oligomers and a process for manufacturing of said antibodies | |
JPH0767650A (en) | Recombination type app small gene for indication in gene exchangeable mouse as model for alzheimer's disease | |
JP6985151B2 (en) | Protoxin-II mutant and how to use it | |
US20110010785A1 (en) | Products and their use for the diagnosis, prevention and/or care of human and/or animal pathologies characterised by the anomalous deposition of b-amyloid and/or amyloid-like substance in human and/or animal organs and tissues, and screening method for determining the risk of such pathologies | |
JP2015518836A (en) | Fuentoxin IV mutant and method of use thereof | |
WO2000071671A2 (en) | New mutant genes in familial british dementia and familial danish dementia | |
Wakefield et al. | The Drosophila reticulon, Rtnl-1, has multiple differentially expressed isoforms that are associated with a sub-compartment of the endoplasmic reticulum | |
JP4937143B2 (en) | Method for detecting autoantibodies having specificity for non-diffusible globular Aβ (X-38..43) oligomeric structural epitopes | |
EP1027433B1 (en) | Human app or a4ct sequences encoding the mutation i45f | |
WO1998003643A2 (en) | Transgenic animals with mutant human app or a4ct sequences | |
Akter | Accelerating Amyloid Formation by Islet Amyloid Polypeptide | |
US7118873B2 (en) | Polynucleotides encoding cellular transporters and methods of use thereof | |
Scarafone | Creation of model proteins to investigate the mechanism of aggregation of expanded-polyglutamine proteins. Insertion of polyglutamine tracts into the ß-lactamase BlaP | |
EP2328918A1 (en) | Mutant alpha-synuclein, and methods using same | |
Starck | The human myostatin precursor protein: structure, function and amyloid formation: implications for the muscle wastage disease sporadic inclusion body myositis: a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand | |
JP2006020540A (en) | New tfiih subunit |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200980122412.X Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09732855 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2009732855 Country of ref document: EP Ref document number: 2011503942 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2721156 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2009236699 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 6524/CHENP/2010 Country of ref document: IN |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2009236699 Country of ref document: AU Date of ref document: 20090414 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12937379 Country of ref document: US |