Beta-secretase enzyme compositions and methods

Abstract

Disclosed are various forms of an active, isolated β-secretase enzyme in purified and recombinant form. This enzyme is implicated in the production of amyloid plaque components which accumulate in the brains of individuals afflicted with Alzheimer's disease. Recombinant cells that produce this enzyme either alone or in combination with some of its natural substrates (β-APPwt and β-APPsw) are also disclosed, as are antibodies directed to such proteins. These compositions are useful for use in methods of selecting compounds that modulate β-secretase. Inhibitors of β-secretase are implicated as therapeutics in the treatment of neurodegenerative diseases, such as Alzheimer's disease.

1 - 107 . (canceled) 
     
     
         108 . A knock-out mouse, characterized by inactivation or deletion of an endogenous β-secretase gene. 
     
     
         109 . The knock-out mouse of  claim 108 , wherein said β-secretase gene encodes a protein having at least 90% sequence identity to the sequence SEQ ID NO: 65. 
     
     
         110 . The knock-out mouse of  claim 108 , wherein said deletion is inducible 
     
     
         111 . The knock-out mouse of  claim 110 , wherein said inducible expression is effected by a Cre-lox expression system inserted into the mouse genome. 
     
     
         112 - 131 . (canceled) 
     
     
         132 . A method for identifying a compound that decreases the activity of a beta-secretase polypeptide comprising steps of: (a) contacting a beta-secretase polypeptide comprising an amino acid sequence at least 95% identical to a fragment of the beta-secretase amino acid sequence of SEQ ID NO: 43, wherein said fragment is a continuous fragment that includes active aspartic acid catalytic sites and exhibits beta-secretase activity, which is an ability to cleave a 695 isotype of APP between amino acids 596 and 597 with a test compound and a substrate cleavable by the beta secretase polypeptide; (b) measuring the beta-secretase activity of the polypeptide on the substrate in the presence and absence of the test compound, and (c) comparing the beta-secretase activity of the polypeptide cell in the presence and absence of the test compound, wherein the beta-secretase activity is determined using an antibody specific for the C-terminus of the N-terminal fragment or the N-terminus of the C-terminal fragment generated by the beta secretase cleavage of the substrate, wherein decreased beta-secretase activity in the presence of the test compound identifies the test compound as a compound that decreases the beta-secretase activity of the beta-secretase polypeptide. 
     
     
         133 . A method according to  claim 132 , wherein the substrate is APP or a fragment thereof cleavable by β-secretase. 
     
     
         134 . A method according to  claim 132 , wherein the substrate comprises a Swedish mutation of APP. 
     
     
         135 . A method according to  claim 132 , wherein the substrate is a fragment of the amyloid precursor protein comprising SEQ ID NO: 54 or its Swedish mutation SEQ ID NO: 51. 
     
     
         136 . The method of  claim 132 , wherein the substrate is selected from the group consisting of MBP-C125st, MBP-C124sw, APPwt, APPsw and beta-secretase cleavable fragments thereof.