Peptide substrates of a proteolytic ADAM33 polypeptide and assays using the same

Abstract

The present invention discloses peptide substrates of a proteolytic ADAM33 polypeptide. The present invention also discloses methods of identifying additional substrates of a proteolytic ADAM33 polypeptide. Furthermore, the present invention discloses methods of identifying compounds that inhibit the proteolytic activity of a proteolytic ADAM33 polypeptide.

1 . A peptide comprising the sequence designated as: P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′, wherein: 
 P5 is Trp, Tyr, Phe, Ile, Leu, Met, Val, or an analog thereof;    P4 is Ala, Cys, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Tyr, an analog thereof, or an amino acid derivative;    P3 is Val, Ile, or an analog thereof;    P2 is Ala, Met, Leu, Lys, Arg, His, or an analog thereof;    P1 is Phe, Tyr, Met, His, Ala, Arg, Glu, Gin, Gly, Leu, Pro, Ser, Trp, or an analog thereof;    P1′ is Gin, Leu, Met, His, or an analog thereof;    P2′ is Ile, Val, Lys, Arg, Leu, Met, Phe, Tyr, Trp, or an analog thereof;    P3′ is Leu, Ile, Met, His, Trp, or an analog thereof;    P4′ is Trp, Ala, Ile, Leu, Met, Val, Phe, Tyr, Arg, His, Lys, or an analog thereof;    and the peptide is a substrate of a proteolytic ADAM33 polypeptide.    
     
     
         2 . The peptide of  claim 1  wherein: 
 P5 is Trp, Tyr, Phe, or an analog thereof;    P2 is Ala or an analog thereof;    P1 is Phe, Tyr, Met, His, or an analog thereof;    P1′ is Gin or an analog thereof;    P2′ is Ile, Val, Lys, or an analog thereof;    P3′ is Leu or an analog thereof; or    P4′ is Trp, Ala, or an analog thereof.    
     
     
         3 . The peptide of  claim 1  comprising 9 to 16 amino acid residues.  
     
     
         4 . The peptide of  claim 1  comprising the amino acid sequence set forth in SEQ ID NO: 1.  
     
     
         5 . The peptide of  claim 4  comprising the amino acid sequence set forth in SEQ ID NO: 2.  
     
     
         6 . The peptide of  claim 5  comprising the amino acid sequence set forth in: 
 a) SEQ ID NO: 3;    b) SEQ ID NO: 4;    c) SEQ ID NO: 5; or    d) SEQ ID NO: 6.    
     
     
         7 . The peptide of  claim 1  comprising the amino acid sequence set forth in: 
 a) SEQ ID NO: 3;    b) SEQ ID NO: 4;    c) SEQ ID NO: 5;    d) SEQ ID NO: 6;    e) SEQ ID NO: 7;    f) SEQ ID NO: 8;    g) SEQ ID NO: 9;    h) SEQ ID NO: 10;    i) SEQ ID NO: 11; or    j) SEQ ID NO: 12.    
     
     
         8 . The peptide of  claim 7  wherein the peptide has one conservative amino acid substitution.  
     
     
         9 . The peptide of  claim 1  comprising the amino acid sequence set forth in SEQ ID NO: 13.  
     
     
         10 . The peptide of  claim 1  further comprising a detectable moiety.  
     
     
         11 . The peptide of  claim 10  wherein the detectable moiety is: 
 a) a fluorescent donor;    b) an acceptor;    c) a fluorophore; and    d) a protein marker.    
     
     
         12 . The peptide of  claim 11  wherein: 
 a) the fluorescent donor is selected from the group consisting of Edans, Mca, Cy3B, and Alexa Fluor 546;    b) the acceptor is selected from the group consisting of Alexa Fluor 647, Cy5, Dabcyl, Dnp, and Cy5Q;    c) the fluorophore is selected from the group consisting of fluorescein, rhodamine, Texas red, BODIPY derivatives, Alexa™ Fluor, and Cy™ dyes; or    d) the protein marker is selected from the group consisting of biotin, digoxin, and phosphotyrosine.    
     
     
         13 . The peptide of  claim 1  further comprising P5′ wherein: 
 a) P4 is an amino acid derivative that comprises a fluorescent donor and P5′ comprises an acceptor;    or    b) P4 is an amino acid derivative that comprises an acceptor and P5′ comprises a fluorescent donor.    
     
     
         14 . The peptide of  claim 1  further comprising P6 and P5′ wherein: 
 a) P6 comprises a fluorescent donor and P5′ comprises an acceptor; or    b) P6 comprises an acceptor and P5′ comprises a fluorescent donor.    
     
     
         15 . The peptide of  claim 14  selected from the group consisting of: 
 a) SEQ ID NO: 14;    b) SEQ ID NO: 15;    c) SEQ ID NO: 16;    d) SEQ ID NO: 17; and    e) SEQ ID NO: 18.    
     
     
         16 . The peptide of  claim 1  further comprising P6 and P5′ wherein: 
 a) P6 comprises a protein marker and P5′ comprises a fluorophore;    or    b) P6 comprises a fluorophore and P5′ comprises a protein marker.    
     
     
         17 . The peptide of  claim 16  selected from the group consisting of: 
 a) SEQ ID NO: 19;    b) SEQ ID NO: 20; and    c) SEQ ID NO: 21.    
     
     
         18 . A method for identifying whether a test compound is a substrate of a proteolytic ADAM33 polypeptide comprising: 
 a) contacting the proteolytic ADAM33 polypeptide with the test compound; and    b) measuring cleavage of the test compound;    wherein cleavage of the test compound indicates that the test compound is a substrate of the proteolytic ADAM33 polypeptide.    
     
     
         19 . The method of  claim 18  wherein the measuring uses a high throughput fluorescence device or a high performance liquid chromatography device.  
     
     
         20 . A method for identifying whether a test compound inhibits proteolytic activity of a proteolytic ADAM33 polypeptide comprising: 
 a) contacting the proteolytic ADAM33 polypeptide with a substrate of the proteolytic ADAM33 polypeptide;    b) measuring cleavage of the substrate;    c) adding the test compound;    d) measuring cleavage of the substrate in the presence of the test compound;    wherein a decrease in cleavage of the substrate in the presence of the test compound indicates that the test compound inhibits the proteolytic activity of the proteolytic ADAM33 polypeptide.    
     
     
         21 . The method of  claim 20  wherein the measuring uses a high throughput fluorescence device or a high performance liquid chromatography device.