Gamma-Secretase Substrates and Methods of Use

Abstract

Polypeptide substrates based on modifications or fragments of the various APP isoforms, assay methods based on the use of these substrates, and screening methods directed toward identifying inhibitors of γ-secretase activity. The assay methods and the screening methods are adapted for use in high throughput multi-well plate assay apparatuses. In many embodiments the substrate polypeptides are labeled for ease of detection, and/or may bind specific ligands that themselves are labeled. Generally the labels promote high specificity as well as high sensitivity of detection. These features render the assay and screening methods that employ the labeled substrates especially suited for use in high throughput assay formats. This disclosure further identifies small polypeptides based on a subsequence motif of Aβ that are shown herein to be potent inhibitors of the activity of γ-secretase.

1 . A method of assaying the activity of γ-secretase comprising the steps of:
 a) providing a container comprising a composition suspected of containing γ-secretase activity; 
 b) adding to the container a polypeptide substrate for γ-secretase comprising at least a portion of an isoform of an APP polypeptide bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a detectably labeled product; 
 c) contacting the labeled product with
 1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and 
 2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and 
 
 d) determining the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand. 
 
     
     
         2 . The method described in  claim 1  wherein the APP isoform is APP695 (SEQ ID NO:1), APP751 (SEQ ID NO:2), or APP770 (SEQ ID NO:3). 
     
     
         3 . The method described in  claim 1  wherein the portion comprises amino acid residues 620-695 of APP695 (SEQ ID NO:1). 
     
     
         4 . The method described in  claim 1  wherein the label comprises biotin. 
     
     
         5 . The method described in  claim 1  wherein the first ligand comprises an avidin and the first tag comprises a detectable fluorescence acceptor. 
     
     
         6 . The method described in  claim 5  wherein the second ligand comprises a first antibody that specifically binds a C-terminus of the product, the first antibody being bound to a second antibody bearing a fluorescence donor that excites the fluorescence acceptor tag bound to the first ligand. 
     
     
         7 . The method described in  claim 1  wherein the method is a high throughput assay method conducted in a plurality of containers. 
     
     
         8 . The method described in  claim 7  wherein each container is a well in a multi-well assay plate, thereby providing a high throughput method of assaying. 
     
     
         9 . The method described in  claim 8  wherein the plate contains at least 96 wells, or at least 384 wells, or at least 1536 wells. 
     
     
         10 - 59 . (canceled)