Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-06-27
2003-04-08
Borin, Michael (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C702S019000, C530S300000, C536S023100
Reexamination Certificate
active
06545127
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is in the area of the expression of catalytically active Memapsin 2 (beta-secretase) and use thereof in the design and screening of specific inhibitors which are useful in the treatment and/or prevention of Alzheimer's Disease.
Alzheimer's disease (AD) is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia (
The early story of Alzheimer's Disease,
edited by Bick et al. (Raven Press, N.Y. 1987)). It is the leading cause of dementia in elderly persons. AD patients have increased problems with memory loss and intellectual functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibit personality changes, socially inappropriate actions and schizophrenia (
A Guide to the Understanding of Alzheimer's Disease and Related Disorders,
edited by Jorm (New York University Press, New York 1987). AD is devastating for both victims and their families, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration.
AD is associated with neuritic plaques measuring up to 200 &mgr;m in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the principal constituents of neuritic plaques is amyloid, which is stained by Congo Red (Fisher (1983); Kelly Microbiol. Sci. 1(9):214-219 (1984)). Amyloid plaques stained by Congo Red are extracellular, pink or rust-colored in bright field, and birefringent in polarized light. The plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mechanisms of AD neuropathy. Available evidence strongly indicates that there are distinct types of genetic predispositions for AD. First, molecular analysis has provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate et al.
Nature
349:704-706 (1991); Murrell et al.
Science
254:97-99 (1991); Chartier-Harlin et al.
Nature
353:844-846 (1991); Mullan et al.,
Nature Genet.
1:345-347 (1992)). Additional genes for dominant forms of early onset AD reside on chromosome
14
and chromosome
1
(Rogaev et al.,
Nature
376:775-778 (1995); Levy-Lahad et al.,
Science
269:973-977 (1995); Sherrington et al.,
Nature
375:754-760 (1995)). Another loci associated with AD resides on chromosome
19
and encodes a variant form of apolipoprotein E (Corder,
Science
261:921-923 (1993)).
Amyloid plaques are abundantly present in AD patients and in Down's Syndrome individuals surviving to the age of 40. The overexpression of APP in Down's Syndrome is recognized as a possible cause of the development of AD in Down's patients over thirty years of age (Rumble et al.,
New England J. Med.
320:1446-1452 (1989); Mann et al.,
Neurobiol. Aging
10:397-399 (1989)). The plaques are also present in the normal aging brain, although at a lower number. These plaques are made up primarily of the amyloid &bgr; peptide (A&bgr;; sometimes also referred to in the literature as &bgr;-amyloid peptide or &bgr; peptide) (Glenner and Wong,
Biochem. Biophys. Res. Comm.
120:885-890 (1984)), which is also the primary protein constituent in cerebrovascular amyloid deposits. The amyloid is a filamentous material that is arranged in beta-pleated sheets. A&bgr; is a hydrophobic peptide comprising up to 43 amino acids.
The determination of its amino acid sequence led to the cloning of the APP cDNA (Kang et al.,
Nature
325:733-735 (1987); Goldgaber et al.,
Science
235:877-880 (1987); Robakis et al.,
Proc. Natl. Acad. Sci.
84:4190-4194 (1987); Tanzi et al.,
Nature
331:528-530 (1988)) and genomic APP DNA (Lemaire et al.,
Nucl. Acids Res.
17:517-522 (1989); Yoshikai et al.,
Gene
87, 257-263 (1990)). A number of forms of APP cDNA have been identified, including the three most abundant forms, APP695, APP751, and APP770. These forms arise from a single precursor RNA by alternate splicing. The gene spans more than 175 kb with 18 exons (Yoshikai et al. (1990)). APP contains an extracellular domain, a transmembrane region and a cytoplasmic domain. A&bgr; consists of up to 28 amino acids just outside the hydrophobic transmembrane domain and up to 15 residues of this transmembrane domain. A&bgr; is normally found in brain and other tissues such as heart, kidney and spleen. However, A&bgr; deposits are usually found in abundance only in the brain.
Van Broeckhaven et al.,
Science
248:1120-1122 (1990), have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This was confirmed by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al.,
Science
248:1124-1128 (1990)). The mutation substituted a glutamine for glutamic acid at position 22 of the A&bgr; (position 618 of APP695, or position 693 of APP770). In addition, certain families are genetically predisposed to Alzheimer's disease, a condition referred to as familial Alzheimer's disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full length protein (Goate et al. (1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the families and are absent in families with late-onset AD. This mutation at amino acid 717 increases the production of the A&bgr;
1-42
form of A&bgr; from APP (Suzuki et al.,
Science
264:1336-1340 (1994)). Another mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al. (1992)). This mutation to amino acids 670 and 671 increases the production of total A&bgr; from APP (Citron et al.,
Nature
360:622-674 (1992)).
APP is processed in vivo at three sites. The evidence suggests that cleavage at the &bgr;-secretase site by a membrane associated metalloprotease is a physiological event. This site is located in APP 12 residues away from the lumenal surface of the plasma membrane. Cleavage of the &bgr;-secretase site (28 residues from the plasma membrane's lumenal surface) and the &bgr;-secretase site (in the transmembrane region) results in the 40/42-residue &bgr;-amyloid peptide (A &bgr;), whose elevated production and accumulation in the brain are the central events in the pathogenesis of Alzheimer's disease (for review, see Selkoe, D. J.
Nature
399:23-31 (1999)). Presenilin 1, another membrane protein found in human brain, controls the hydrolysis at the APP (&bgr;-secretase site and has been postulated to be itself the responsible protease (Wolfe, M. S. et al.,
Nature
398:513-517 (1999)). Presenilin 1 is expressed as a single chain molecule and its processing by a protease, presenilinase, is required to prevent it from rapid degradation (Thinakaran, G. et al.,
Neuron
17:181-190 (1996) and Podlisny, M. B., et al.,
Neurobiol. Dis.
3:325-37 (1997)). The identity of presenilinase is unknown. The in vivo processing of the &bgr;-secretase site is thought to be the rate-limiting step in A &bgr; production (Sinha, S. & Lieberburg, I.,
Proc. Natl. Acad. Sci., USA,
96:11049-11053 (1999)), and is therefore a strong therapeutic target.
The design of inhibitors effective in decreasing amyeloid plaque formation is dependent on the identification of the critical enzyme(s) in the cleavage of APP to yield the 42 amino acid peptide, the A&bgr;
1-42
form of A&bgr;. Although several enzymes have been identified, it has not been possible to produce active enzyme. Without active enzyme, one cannot confirm the substrate specificity, determine the subsite specificity, nor determine the kinetics or critical active site residues, all of which are essential for the design of inhib
Hong Lin
Koelsch Gerald
Lin Xinli
Tang Jordan J. N.
Borin Michael
Hamilton Brook Smith & Reynolds P.C.
Oklahoma Medical Research Foundation
Zhou Shuba
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