Assays for detecting .beta.-secretase
AssigneeElan Pharmaceuticals, Inc.
InventorsJohn P. Anderson, Kirsten L. Jacobson-Croak, Sukanto Sinha
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Abstract
Compositions comprising a novel protease capable of cleaving .beta.-amyloid precursor protein (APP) on the amino-terminal side of the .beta.-amyloid peptide therein are provided. The protease is designated .beta.-secretase. Reaction systems comprising .beta.-secretase may be used in screening assays to monitor .beta.-secretase modulated cleavage of APP and to identify .beta.-secretase inhibitors, wherein the .beta.-secretase is in the presence of a suitable polypeptide substrate and cleavage of the substrate determined in the presence and absence of the test substance. Antibodies are raised against peptides of .beta.-secretase. Pharmaceutical compositions and methods comprise compounds identified by screening assays.
Claims5
A method for detecting human .beta.-secretase cleavage of a polypeptide substrate, said method comprising:
- providing a reaction system including human .beta.-secretase, which has been purified to have an activity which is at least five-fold greater than that of a solubilized but unenriched membrane fraction from human 293 cells and which has an apparent molecular weight when measured by gel exclusion chromatography in the range from 260 kD to 300 kD, and the polypeptide substrate present in initial amounts, wherein the polypeptide substrate comprises a .beta.-secretase cleavage site of .beta.-amyloid precursor protein (APP) and at least five amino acids upstream and downstream of the cleavage site;
- maintaining the reaction system under conditions which permit .beta.-secretase cleavage of the polypeptide substrate into .beta.-secretase cleavage products;
- and
- detecting the amount of at least one of the .beta.-secretase cleavage products produced as a result of .beta.-secretase cleavage of the substrate relative to a control by binding at least one of an amino terminal end of a carboxyl terminal fragment and a carboxy terminal end of an amino terminal fragment of the polypeptide substrate with an antibody specific for said end.
Dependencies are inferred from the claim text.