Assays using amyloid precursor proteins with modified beta-secretase cleavage sites to monitor beta-secretase activity

Abstract

Provided are methods of identifying inhibitors of β-secretase that employ modified β-secretase substrates. The modified β-secretase substrates have β-secretase cleavage sites that are altered from wild type. The amino acid sequences of the altered β-secretase cleavage sites contain different amino acids in at least one of the positions P2-P1-P1′-P2′ of the β-secretase cleavage site. Many of the modified β-secretase substrates are more efficient substrates for β-secretase than are corresponding substrates having wild-type sequences, that is, these modified substrates are more susceptible to enzymatic breakdown by β-secretase. Recombinant polynucleotide molecules encoding the modified β-secretase substrates are provided. Antibodies that recognize cleavage products of the modified β-secretase substrates are provided. Stable cell lines expressing the modified β-secretase substrates are provided. Transgenic animals expressing the modified β-secretase substrates are provided.

1 - 46 . (canceled)  
     
     
         47 . An APP molecule or an APP biologically active fragment thereof, selected from the group consisting of APP695, APP751 and APP770, having a modified β-secretase cleavage site comprising a sequence designated P2-P1-P1′-P2′, where 
 P2 is selected from the group consisting of N, K and F;    P1 is selected from the group consisting of F, L, Y and M;    P1′ is selected from the group consisting of E, D and A; and    P2′ is selected from the group consisting of V and A;    with the proviso that P2, P1, P1′ and P2′ are not selected such that P2-P1-P1′-P2′ is KMDA (SEQ ID NO:1) or NLDA (SEQ ID NO:2); and    where said modified β-secretase cleavage site, when cleaved by a human β-secretase, is cleaved by β-secretase at a rate higher than a substrate having a wild type (SEQ ID NO: 1) or Swedish mutation (SEQ ID NO: 2) β-secretase cleavage site.    
     
     
         48 . An APP molecule of  claim 47  wherein the modified β-secretase cleavage site comprises an amino acid sequence selected from the group consisting of SEQ. ID. NO.:3 to SEQ. ID. NO.:23.  
     
     
         49 . An APP molecule of  claim 48  wherein the modified β-secretase cleavage site is located at positions corresponding to positions 595-596-597-598 of APP695, positions 651-652-653-654 of APP751 and positions 670-671-672-673 APP770.  
     
     
         50 . An antibody that specifically recognizes an epitope formed by the cleavage by β-secretase of an APP molecule or an APP biologically active fragment thereof, selected from the group consisting of APP695, APP751 and APP770, having a modified β-secretase cleavage site.  
     
     
         51 . An antibody of  claim 50  wherein the β-secretase cleavage site comprises an amino acid sequence selected from the group consisting of SEQ. ID. NO.:3 to SEQ. ID. NO.:23.  
     
     
         52 . An antibody of  claim 51  wherein the β-secretase cleavage site comprises an amino acid sequence of NFEV (SEQ. ID. NO.:3).  
     
     
         53 . An antibody of  claim 50  wherein said antibody is a monoclonal antibody.  
     
     
         54 . An antibody of  claim 50  wherein said antibody is a humanized antibody.  
     
     
         55 . An antibody of  claim 50  that specifically recognizes the free carboxy-terminal end of a modified sAPPβ fragment, wherein said modified sAPPβ fragment results from the cleavage by β-secretase of an APP molecule or an APP biologically active fragment thereof having a modified β-secretase cleavage site.  
     
     
         56 . An antibody of  claim 55  wherein said APP molecule or an APP biologically active fragment thereof comprises a modified β-secretase site selected from the group consisting of NFEV (SEQ. ID. NO.:3), NFDA (SEQ. ID. NO.:4, NFEA (SEQ. ID. NO.:5), NFDV (SEQ. ID. NO.:8), NFTV (SEQ. ID. NO.:9) and NFAA (SEQ. ID. NO.:16).  
     
     
         57 . An antibody of  claim 56  wherein the free carboxy-terminal end has the sequence SEVNF (SEQ. ID. NO.:57).  
     
     
         58 . An antibody of  claim 50  that specifically recognizes the free amino-terminal end of a modified CTFβ fragment, wherein said modified CTFβ fragment results from the cleavage by β-secretase of an APP molecule or an APP biologically active fragment thereof having a modified β-secretase cleavage site.  
     
     
         59 . An antibody of  claim 58  wherein said APP molecule or an APP biologically active fragment thereof comprises a modified β-secretase site selected from the group consisting of NFEV (SEQ. ID. NO.:3), NFDA (SEQ. ID. NO.:4, NFEA (SEQ. ID. NO.:5), NFDV (SEQ. ID. NO.:8), NFTV (SEQ. ID. NO.:9) and NFAA (SEQ. ID. NO.:16).  
     
     
         60 . An antibody of  claim 59  wherein the free amino-terminal end has the sequence EVEFR (SEQ. ID. NO.:58).