Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-04-02
2001-12-11
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S024000, C435S007100, C435S007950, C436S518000
Reexamination Certificate
active
06329163
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the cleavage of &bgr;-amyloid precursor protein to produce &bgr;-amyloid peptide. More particularly, the present invention relates to isolated and purified compositions containing an enzyme responsible for such cleavage (&bgr;-secretase) and assays for identifying inhibitors of &bgr;-secretase.
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of Alzheimer's disease patients, particularly in those regions involved with memory and cognition. &bgr;-amyloid peptide is a major constituent of amyloid plaque which is produced by cleavage of &bgr;-amyloid precursor protein. It is presently believed that a normal (non-pathogenic) processing of the &bgr;-amyloid precursor protein occurs via cleavage by a putative “&agr;-secretase” which cleaves between amino acids 16 and 17 of the &bgr;-amyloid peptide region within the protein. It is further believed that pathogenic processing occurs in part via a putative “&bgr;-secretase” which cleaves at the amino-terminus of the &bgr;-amyloid peptide region within the precursor protein. Heretofore, however, the existence of &bgr;-secretase has not been confirmed.
The identification, isolation, and characterization of novel biological molecules having unique activities is generally useful. For example, novel enzymes can be used to catalyze reactions of a type associated with their class. In particular, novel proteases can be used to cleave proteins for a variety of purposes, and the availability of new proteases provides unique capabilities. In addition to such uses associated with enzymes in general, the identification, isolation, and purification of the putative &bgr;-secretase enzyme would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit &bgr;-secretase activity.
For these reasons, it would be desirable to isolate, purify, and characterize the enzyme responsible for the pathogenic cleavage of &bgr;-amyloid precursor protein at the amino-terminus of the &bgr;-amyloid peptide region. In particular, it would be desirable to utilize such an enzyme (referred to hereinafter as &bgr;-secretase) in methods for screening candidate drugs for the ability to inhibit the activity of &bgr;-secretase in in vitro systems. It would be particularly desirable if such screening assays could be performed in a rapid format which would permit the screening of large numbers of test drugs in automated fashion.
2. Description of the Background Art
&bgr;-amyloid precursor protein is expressed in three differently-spliced forms of 695, 751, and 770 amino acids, and “normal” processing involves proteolytic cleavage at a site between residues Lys
16
and Leu
17
in the &bgr;-amyloid peptide. Kang et al. (1987)
Nature
325:773-776. Soluble &bgr;-amyloid peptide which has been cleaved at the putative &bgr;-secretase site has also been found in the culture medium of non-diseased cells (Haass et al. (1992)
Nature
359:322-325) and in CSF from healthy humans and animals (Seubert et al. (1992)
Nature
359:325-327). The possible existence of the putative &bgr;-secretase is discussed in, for example, Selkoe, “Cell Biology of the Amyloid &bgr;-Protein and the Mechanism of Alzheimer's Disease,” in
Annual Review of Cell Biology,
Spudich et al., eds., Annual Review, Inc., Palo Alto, Calif., vol. 10, 1994.
SUMMARY OF THE INVENTION
The present invention provides an assay which utilizes a novel &bgr;-secretase composition comprising an isolated and purified enzyme which cleaves &bgr;-amyloid precursor protein (APP) at the amino-terminus of &bgr;-amyloid peptide (&bgr;AP) within APP, referred to hereinafter as “&bgr;-secretase activity.” The compositions of the present invention will generally have a &bgr;-secretase activity which is at least 5-fold, preferably 10-fold, and more preferably 100-fold greater than that of a solubilized but unenriched membrane fraction from human 293 cells. The &bgr;-secretase enzyme is characterized by (1) a molecular weight in the range from 260 kD to 300 kD, (2) a gel pattern as shown in
FIG. 1
or
2
(discussed in more detail in the Experimental section hereinafter), (3) a net negative charge at pH 5 and a net negative charge at pH 7.5, and (4) binding to wheat germ agglutinin.
The methods of the present invention allow identification of test substances which inhibit proteolytic cleavage of APP resulting from the novel &bgr;-secretase. The method comprises exposing a polypeptide comprising the &bgr;-secretase site of APP (located at the amino-terminus of the &bgr;AP region within APP) to an at least partially purified &bgr;-secretase in the presence of the test substance under conditions such that the &bgr;-secretase would be expected to cleave the polypeptide into an amino-terminal fragment and a carboxy-terminal fragment in the absence of test substance which inhibits such cleavage. Test substances which inhibit such cleavage are thus identified as having &bgr;-secretase inhibition activity. Such test methods preferably employ the &bgr;-secretase compositions described above. Usually, generation of the amino-terminal fragment and/or the carboxy-terminal fragment is detected by an antibody specific for the carboxy end of the amino-terminal fragment or the amino end of the carboxy-terminal fragment. The polypeptide substrate for the &bgr;-secretase preferably comprises at least the 125 carboxy-terminal amino acids of APP, and may comprise a fusion polypeptide including an amino-terminal portion having a binding epitope. Use of such a fusion polypeptide as the &bgr;-secretase substrate facilitates detection of cleavage by capture of the amino-terminal portion and labelling of the amino-terminal portion.
REFERENCES:
patent: 5200339 (1993-04-01), Abraham et al.
patent: 5424205 (1995-06-01), Dovey
patent: 0 576 152 (1993-05-01), None
patent: WO 91/13904 (1991-09-01), None
patent: WO 92/03542 (1992-03-01), None
patent: WO 92/07068 (1992-04-01), None
Simian et al, J. Biol. Chem, 268(22):16602-16609, 1993.*
Glenner et al Advances in Behavioral Biology vol. 44, Alzheimer's + Parkinson's Diseases, Plenum Press, NY 1993.*
Savage et al Neuroscience 60(3):607-619, 1994.*
Evin et al, Amyloid: Int. J. Exp. Clin. Invest 1: 263-280, 1994.*
Elvin, G., et al., “Alzheimer's disease amyloid precursor protein (Ap p): proteolytic processing, secretases and A4 amyloid production,”Amyloid: Int. J. Exp. Clin. Invest., 1:263-280 (1994).
Matsumo, A., et al., “Ca2+-Dependent 68-Kilodalton Protease in Familial Alzheimer's Disease Cells Cleaves the n-Terminus of -Amyloid,”Biochemestry, 33:3941-3948 (1994).
Citron, M., et al., “Generation of Amyloid Protein from Its Precursor is Sequence Specific,”Neuron., 14:661-670 (1995).
Matsumoto, A., et al., “Molecular cloning of human cDNA with a sequence hihgly similar to that of the dihydrofolate reductase gene in brain librairies derived from Alzheimer'disease patients,”Eur. J. Biochem., 230:337-343 (1995).
Brown, A., et al., “Evaluation of Cathespins d and G and EC 3,4,24,15 as Candidate -Secretase Proteases Using Peptide and Amyloid Precursor Protein Substrates,”J. Neurochem. vol. 66. No. 6, 2436-2445 (1996).
Tagawa, et al., “Alzheimer's disease amyloid -cliiping enzyme (APP secretase): Identification, purification, and characterization of the enzyme,”Biochemical and Biophyusical Research Communication., vol. 177. No. 1,377-387, especially pp. 379-380 (1991).
Sambamurti, et al., “Evidence for intracellular cleavage of the Alzheimer's amyloid precursir in PC12 cells,”Journal of Neuroscience Research., 33:319-328, entire document.
Nelson, et al., “Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid -Protein,”Journal of Neurochemistry., vol. 61, No. 2, 567-577, espec
Anderson John P.
Jacobson-Croak Kirsten L.
Sinha Sukanto
Duffy Patricia A.
Elan Pharmaceuticals Inc.
Stratford Carol A.
Townsend and Townsend / and Crew LLP
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