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 . An isolated peptide comprising a sequence of at least four amino acids defined by formula P 2 P 1 -P 1 P 2 ′ wherein
 P 2  is a charged amino acid, a polar amino acid, or an aliphatic amino acid but is not an aromatic amino acid;   P 1  is an aromatic amino acid or an aliphatic amino acid but not a polar amino acid or a charged amino acid;   P 1 ′ is a charged amino acid, or aliphatic amino acid, or a polar amino acid but is not an aromatic amino acid;   P 2 ′ is an uncharged aliphatic polar amino acid or an aromatic amino acid; and   
       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. 
     
     
         2 - 13 . (canceled) 
     
     
         14 . The isolated peptide of  claim 1  comprising a first label. 
     
     
         15 . The isolated peptide of  claim 14  further comprising a second label. 
     
     
         16 . An isolated peptide according to  claim 1 , further comprising a detectable label and a quenching moiety, wherein cleavage of the peptide between P 1  and P 1 ′ separate the quenching moiety from the label to permit detection of the label. 
     
     
         17 - 19 . (canceled) 
     
     
         20 . The isolated peptide of  claim 1 , wherein said peptide comprises an amino acid selected from the group consisting of SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:120; SEQ ID NO:133; SEQ ID NO:134; SEQ ID NO:135; SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:144; SEQ ID NO:145; SEQ ID NO:147; SEQ ID NO:148; SEQ ID NO:149; SEQ ID NO:150; SEQ ID NO:151; SEQ ID NO:152; SEQ ID NO:153; SEQ ID NO:154; SEQ ID NO:155; SEQ ID NO:156; SEQ ID NO:157; SEQ ID NO:158; SEQ ID NO:159; SEQ ID NO:160; SEQ ID NO:161; SEQ ID NO:162; SEQ ID NO:163; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; SEQ ID NO:167; SEQ ID NO:168; SEQ ID NO:169; SEQ ID NO:190; SEQ ID NO:191; SEQ ID NO:192 and SEQ ID NO:193. 
     
     
         21 . An isolated peptide comprising a sequence of at least four 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 ′;   with the proviso that if P 1 ′P 2 ′ comprise the sequence DA, P 2 P 1  do not comprise the sequence NL or NNle.   
     
     
         22 - 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 . A polypeptide comprising a peptide according to  claim 1  and further comprising a transmembrane domain amino acid sequence. 
     
     
         29 - 31 . (canceled) 
     
     
         32 . The polypeptide of  claim 28 , wherein said transmembrane domain anchors said polypeptide to an intracellular membrane selected from the group consisting of the Golgi or the endoplasmic reticulum. 
     
     
         33 - 35 . (canceled) 
     
     
         36 . A polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to  claim 20 . 
     
     
         37 . A polynucleotide comprising a nucleotide sequence that encodes a peptide according to  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 37 . 
     
     
         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 peptide according  claim 1 ;   (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 - 49 . (canceled) 
     
     
         50 . A β-secretase modulator identified according to the method of  claim 43 . 
     
     
         51 . (canceled) 
     
     
         52 . A method of producing a substrate for a α-secretase assay comprising:
 growing a host cell transformed or transfected with a vector of  claim 38  in a manner allowing expression of said polypeptide.   
     
     
         53 - 54 . (canceled) 
     
     
         55 . A pharmaceutical composition comprising a modulator of  claim 50  and a pharmaceutically acceptable carrier. 
     
     
         56 - 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 1  and a composition comprising an Hu-Asp2 activity in the presence and absence of a test agent;   (b) determining the cleavage of said peptide 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 - 63 . (canceled) 
     
     
         64 . A method for identifying agents that modulate the activity of Asp2 aspartyl protease, comprising the steps of:
 (a) contacting an Asp2 aspartyl protease and a peptide of  claim 1  in the presence and absence of a test agent, wherein the Asp2 aspartyl protease is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a Hu-Asp2-encoding polynucleotide selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3;   (b) determining the cleavage of said peptide between said P 1  and said P 1 ′ site by said Asp2 in the presence and absence of the test agent; and   (c) comparing the cleavage activity of said Asp2 in the presence of the test agent to the cleavage activity in the absence of the agent to identify agents that modulate the activity of the polypeptide, wherein a modulator that is an Asp2 inhibitor reduces said cleavage and a modulator that is an Asp2 agonist increases said cleavage.   
     
     
         65 - 82 . (canceled)