Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
1999-07-16
2001-06-12
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S087000, C436S089000, C436S095000, C435S105000, C435S106000
Reexamination Certificate
active
06245575
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns screening agents for treatment of Alzheimer's disease, and concerns a method of screening an agent for potential use in the treatment of Alzheimer's disease.
BACKGROUND TO THE INVENTION
Alzheimer's disease is the leading cause of dementia in the elderly. The brains of patients with Alzheimer's disease contain characteristic structural lesions known as plaques and tangles. Plaques will not be considered here. Neurofibrillary tangles represent intracellular accumulations of paired helical filaments (PHFs), which are quite unlike any of the normal structural elements of the neuronal cytoskeleton. Tangles become extracellular on the death of the affected cell. PHFs are also found in abnormal neurites associated with neuritic plaques and as an extensive distribution of fine neurophil threads throughout affected regions of the brain. The extent of neurofibrillary degeneration found post mortem appears to provide the most reliable pathological correlate of the degree of dementia observed in life. These topics are discussed further in references 1 and 2.
PHFs are made of microtubule-associated protein tau in a hyperphosphorylated state (3-8). Hyperphosphorylation of tau is known to result in its inability to bind to microtubules (9, 10) and is believed to precede PHF assembly (11). However, it is unclear whether hyperphosphorylation of tau is either necessary or sufficient for PHF formation. A major reason for this lack of understanding is that it has not hitherto been possible to form paired helical-like filaments for full-length tau protein either in vitro or in vivo.
Tau protein consists in adult human brain of six isoforms, produced from a single gene by alternative mRNA splicing. The isoforms, which range in size from 352 to 441 amino acids, contain towards the carboxyl terminus a tandem repeat region with three or four homologous stretches of 31 or 32 amino acids. The other isoforms are generated by the presence of a 29- or 58- amino-acid insertion in the amino-terminal half of the protein.
The repeat region of tau represents the microtubule-binding domain, and the amino-terminal half of the protein appears to form an arm-like projection from the surface of the microtubule.
When tau protein is in hyperphosphorylated state (PHF-tau) the protein is unable to bind to microtubules and assembles into PHFs or structural variants known as straight filaments (SFs). The repeat region of tau constitutes the core of the resulting filaments.
See references 31 and 32 for further discussion of tau protein.
The present invention is based on the discovery that suitable sulphated carbohydrates can induce in vitro assembly of tau protein into filaments like those in Alzheimer's disease under physiological conditions in an essentially quantitative manner. This discovery forms the basis of an in vitro assay or screen for agents that inhibit the assembly and so may have potential use in the treatment of Alzheimer's disease.
SUMMARY OF THE INVENTION
The present invention thus provides a method of screening an a gent for potential use in the treatment of Alzheimer's disease, comprising reacting, in the presence of the agent, tau protein with a suitable sulphated carbohydrate under appropriate conditions to form filaments, and monitoring for the presence of filaments.
Tau protein and sulphated carbohydrate, ea sulphated glycosaminoglycan, will react under appropriate conditions to form filaments, either paired helical filaments or straight filaments. If filament formation is affected when the reaction is carried out in the presence of an agent being screened, this is possibly due to an interfering, inhibiting or blocking effect of the agent. An agent which inhibits assembly of PHFs in vitro may also have an inhibiting effect in vivo and thus have potential therapeutic value in delaying the dementing effects of Alzheimer's disease. The invention can thus provide a screen to identify agents worthy of further investigation for use in the treatment of Alzheimer's disease.
The tau protein may be the full length protein or a fragment thereof which forms filaments. Filament-forming fragments of tau generally include at least the portion thereof including three microtubule-binding repeats. Any of the 6 isoforms which occur in the adult human brain, as discussed above, may be used. The full length 3 repeat 381 amino acid isoform, which forms paired helical filaments very similar to the paired helical filaments found in the brain of patients with Alzheimer's disease as explained above, works very well and so is currently favoured. The tau protein may be modified in known manner by amino acid additions, substitutions and/or deletions that do not significantly affect filament formation. The term “tau protein” is used to cover all such proteins and fragments that form filaments under appropriate conditions.
The currently preferred sulphated carbohydrate is sulphated glycosaminoglycan, conveniently heparin or heparan sulphate.
The reaction may be carried out under physiological conditions, eg. by incubating the reagents in MOPS and AEBSF as described below, and provides quantitative results. Good results have been obtained by incubating with 30 mM MOPS. 1 mM AEBSF, pH 7.4 at 30° C., with filament formation starting after 5 h and reaching a maximum after 48 h. Tau concentration is suitably at least 40 &mgr;M, with a tau/sulphated glycosaminoglycan molar ratio of approximately 4:1 giving good results.
The presence (or otherwise) of filaments may be monitored by using electron microscopy or other appropriate methods.
REFERENCES:
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patent: 5643562 (1997-07-01), Kisilevsky et al.
patent: 5728375 (1998-03-01), Kisilevsky et al.
patent: 5840294 (1998-11-01), Kisilevsky et al.
patent: 93 10459 (1993-05-01), None
patent: 93 11231 (1993-06-01), None
Su et al. “Localization of heparan sulfate glycosaminoglycan and proteoglycan core protein in aged brain and Alzheimer's disease”, abstract 1992.*
Yang et al. “Protein kinase Fa/glycogen synthase kinase-3alpha after heparin potentiation phosphorylates tau on sites abnormally phosphorylated in Alzheimer's Disease brain”, J. Neurochem. (1994), 63 (4), 1416-25.*
Song et al. “Tau protein kinase I/GSK-3 beta/kinase FA in heparin phosphorylatesd tau on Ser199, Thr231, Ser235, Ser262, Ser369 and Ser400 sites phosphorylated in Alshemer disease brain”, abstract, 1995.*
Goedert et al. “Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease”, Neuron (1989), 3(4), 519-26.*
Moreno et al: “Glysogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin)” FEBS LETTERS., vol. 372, No. 1, Sep. 18, 1995, pp. 65-68, XP002041922.
Chemical Abstract, vol. 121, No. 17, Oct. 24, 1994, abstract No. 202384, Yang et al: “Protein kinase Fa/glycogen synthase kinases-3-alpha after heparin potentiation phosphorylates tau on sites abnormally phosphorylated in Alzheimer's disease brain”, p. 879, col 2, XPOOSO41923.
Crowther Richard Anthony
Goedert Michel
Hasegawa Masato
Jakes Ross
Smith Michael John
Gakh Yelena
Medical Research Council
Pillsbury & Winthrop LLP
Warden Jill
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