Substrates and Assays for Beta Secretase Activity

Abstract

The present invention is directed to novel substrates for Hu-Asp. More particularly, the invention provides peptide substrates and fusion polypeptide substrates comprising a β-secretase cleavage site. Methods and compositions for making and using the peptides are disclosed.

1 - 20 . (canceled)  
     
     
         21 . An isolated peptide comprising an amino acid sequence of at least six amino acids defined by formula P 2 P 1 -P 1 ′P 2 ′, wherein: 
 P 2  comprises an amino acid selected from the group consisting of N, S, and D;    P 1  comprises an amino acid selected from the group consisting of Y, L, and Nle;    P 1 ′ comprises an amino acid selected from the group consisting of E, A, and D;    P 2 ′ comprises an amino acid selected from the group consisting of A and V; and    wherein a human Aspartyl protease encoded by the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 3 (Hu-Asp2) cleaves said peptide between P 1  and P 1 ′;    wherein said peptide is cleaved between P 1  and P 1 ′ by a human aspartyl protease encoded by the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 and said peptide does not comprise the corresponding P 2 P 1 -P 1 ′P 2 ′ portion of amino acid sequences depicted in SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39; or SEQ ID NO:40.    
     
     
         22 . An isolated peptide according to  claim 21 , wherein the peptide amino acid sequence consists of 6-50 amino acids.  
     
     
         23 . An isolated peptide according to  claim 21 , wherein the Hu-Asp2 cleaves the peptide at a rate greater than the Hu-Asp2 cleaves a corresponding peptide having the P 2 P 1 -P 1 ′P 2 ′ amino acid sequence KMDA.  
     
     
         24 - 26 . (canceled)  
     
     
         27 . A polypeptide comprising a peptide sequence according to  claim 21 , and further comprising a transmembrane domain to localize the polypeptide to a cellular membrane when the polypeptide is expressed in a eukaryotic cell.  
     
     
         28 - 35 . (canceled)  
     
     
         36 . A polynucleotide comprising a nucleotide sequence that encodes a polypeptide of  claim 27 .  
     
     
         37 . A polynucleotide comprising a nucleotide sequence that encodes a peptide of claim  1 .  
     
     
         38 . A vector comprising a polynucleotide according to  claim 36 .  
     
     
         39 . A vector comprising a polynucleotide according to  claim 37 .  
     
     
         40 . (canceled)  
     
     
         41 . A host cell transformed or transfected with a polynucleotide according to  claim 36 .  
     
     
         42 . A host cell transformed or transfected with a vector according to  claim 39 .  
     
     
         43 . A method for assaying for modulators of β-secretase activity, comprising the steps of: 
 (a) contacting a first composition with a second composition both in the presence and in the absence of a putative modulator compound, wherein the first composition comprises a mammalian β-secretase polypeptide or biologically active fragment thereof, and wherein the second composition comprises a substrate, wherein said substrate comprises a peptide according to  claim 21;     (b) measuring cleavage of the substrate peptide in the presence and in the absence of the putative modulator compound; and    (c) identifying modulators of β-secretase activity from a difference in cleavage in the presence versus in the absence of the putative modulator compound, wherein a modulator that is a β-secretase antagonist reduces such cleavage and a modulator that is a β-secretase agonist increases such cleavage.    
     
     
         44 . The method of  claim 43 , wherein said first composition comprises a purified human Asp2 polypeptide.  
     
     
         45 . The method of  claim 43 , wherein said first composition comprises a soluble fragment of a human Asp2 polypeptide that retains Asp2 β-secretase activity.  
     
     
         46 . The method of  claim 45 , wherein said soluble fragment is a fragment lacking an Asp2 transmembrane domain.  
     
     
         47 - 51 . (canceled)  
     
     
         52 . A method of producing a substrate for a β-secretase assay comprising: 
 growing a host cell transformed or transfected with a vector of  claim 39  in a manner allowing expression of said polypeptide.    
     
     
         53 - 57 . (canceled)  
     
     
         58 . A method for identifying agents that inhibit the activity of human Asp2 aspartyl protease (Hu-Asp2), comprising the steps of: 
 (a) contacting a peptide of  claim 21  and a composition comprising an Hu-Asp2 activity in the presence and absence of a test agent;    (b) determining the cleavage of said peptide or polypeptide between said P 1  and P 1 ′ by said Hu-Asp2 in the presence and absence of the test agent; and    (c) comparing said cleavage activity of the Hu-Asp2 in the presence of the test agent to the activity in the absence of the test agent to identify an agent that inhibits said cleavage by the Hu-Asp2, wherein reduced activity in the presence of the test agent identifies an agent that inhibits Hu-Asp2 activity.    
     
     
         59 . A method according to  claim 58 , wherein the Hu-Asp2 is a recombinant Hu-Asp2 purified and isolated from a cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes Hu-Asp2.  
     
     
         60 - 69 . (canceled)  
     
     
         70 . A kit for performing a β-secretase assay comprising a β-secretase substrate comprising a peptide according to  claim 21  and a β-secretase enzyme.  
     
     
         71 - 72 . (canceled)  
     
     
         73 . An isolated peptide comprising a sequence of at least 10 amino acids having the sequence SEISY-EVEFR (SEQ ID NO:152).  
     
     
         74 - 82 . (canceled)