Novel beta-secretase and modulation of beta-secretase activity

Abstract

The invention concerns a novel β-secretase, a method of partially purifying this novel β-secretase, and its use in assays to screen for potential drug candidates against Alzheimer's disease and other neurological diseases. The novel β-secretase has an estimated molecular weight of about 32-39 kDa or 22-26 kDa in HEK293 cell membrane extracts and human brain samples, respectively, as calculated from radiation inactivation analysis, and has a pH optimum at about pH 6.5-7.0.

1 . A method for reducing β-amyloid protein (Aβ) formation, comprising: 
 providing to a test mixture comprising an amyloid precursor protein (APP) and a β-secretase enzyme, a β-secretase inhibitor in an amount sufficient to reduce β-secretase activity from the β-secretase enzyme, wherein the enzyme is obtainable from HEK293 cells or human brain, having a pH optimum at about pH 6.5-7.0, and an estimated molecular weight of about 32-39 kDa as calculated from radiation inactivation analysis of HEK293 cell membrane extract, or about 20-26 kDa as calculated from radiation inactivation analysis of human brain samples; and    whereby the reduced β-secretase activity results in a reduction of Aβ formation relative to a control mixture comprising APP and the β-secretase in the absence of the inhibitor.    
   
   
       2 . The method of  claim 1 , wherein the reduction of Aβ formation results from decreased levels of N-terminal proteolysis of APP.  
   
   
       3 . The method of  claim 1 , wherein the β-secretase activity is between 3% and 80% below normal.  
   
   
       4 . The method of  claim 3 , wherein the β-amyloid release levels are 20-80% of normal.  
   
   
       5 . The method of  claim 1 , wherein the β-secretase inhibitor non-specifically inhibits the β-secretase enzyme.  
   
   
       6 . The method of  claim 5 , wherein the β-secretase inhibitor is 4-(2-Aminoethyl) benzenesulphonyl fluoride (AEBSF).  
   
   
       7 . The method of  claim 1 , wherein the β-secretase inhibitor preferentially inhibits the activity of the β-secretase enzyme.  
   
   
       8 . The method of  claim 1 , wherein the test mixture and the control mixture further comprise a β-secretase enzyme having a pH optimum at about pH 4.5-5.0 and an estimated molecular weight of about 50-60 kDa as calculated from radiation inactivation analysis of HEK293 cell membrane extracts or human brain samples.  
   
   
       9 . The method of  claim 8 , wherein the β-secretase inhibitor preferentially inhibits the activity of a β-secretase enzyme having a pH optimum at about pH 4.5-5.0.  
   
   
       10 . The method of  claim 1 , wherein the test mixture and the control mixture further comprise cardiolipin.  
   
   
       11 . A method for reducing β-amyloid plaque formation, comprising: 
 providing a candidate inhibitory compound determined to inhibit β-secretase activity in an amount sufficient to reduce β-secretase activity, wherein the compound is identified by contacting an isolated and purified human β-secretase enzyme characterized by    (i) having a pH optimum at about pH 6-6.5,    (ii) an estimated molecular weight of about 32-39 kDa as calculated from radiation inactivation analysis of human brain samples, and    (iii) insensitive to statine substrate analog inhibitor (StatVal),    with a candidate inhibitory compound, in the presence of a β-amyloid precursor protein (APP) substrate of said β-secretase enzyme to perform enzymatic cleavage of said substrate, and monitoring the effect of the candidate compound on the activity of said β-secretase enzyme, thereby identifying the compound.    
   
   
       12 . The method of  claim 11 , wherein said inhibition results in reduced release of β-amyloid peptide in brain tissue.  
   
   
       13 . The method of  claim 11 , wherein said inhibition results in decreased levels of N-terminal proteolysis of APP.  
   
   
       14 . The method of  claim 11 , wherein the β-secretase activity is between 3% and 80% below normal.  
   
   
       15 . The method of  claim 12 , wherein the β-amyloid release levels are 20-80% of normal.  
   
   
       16 . The method of  claim 11 , wherein said compound preferentially inhibits the activity of a β-secretase enzyme having a pH optimum at about pH 6.5-7.0.  
   
   
       17 . The method of  claim 11 , wherein said candidate compound preferentially inhibits the activity of a β-secretase enzyme having a pH optimum at about pH 4.5-5.0.