Protein binding miniature proteins

Abstract

The present invention provides a protein scaffold, such as an avian pancreatic polypeptide, that can be modified by substitution of two or more amino acid residues that are exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.

1 . A modified avian pancreatic polypeptide (aPP) comprising substitution of at least one amino acid residue, said at least one residue being exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form, wherein the modified polypeptide binds to a target protein. 
     
     
         2 . The modified polypeptide of  claim 1 , wherein at least six residues are substituted. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The modified polypeptide of  claim 1 , wherein the site is a protein-binding site. 
     
     
         7 . The modified polypeptide of  claim 1 , wherein said at least one substituted residues are from any site of a known protein through which the known protein interacts with its binding partner. 
     
     
         8 . The modified polypeptide of  claim 7 , wherein the target protein is a binding partner of the known protein. 
     
     
         9 . The modified polypeptide of  claim 7 , wherein the known protein is selected from the group consisting of a Bcl2 protein, p53, a protein kinase inhibitor (PKI), and CREB. 
     
     
         10 . The modified polypeptide of  claim 7 , wherein the binding partner is selected from the group consisting of a Bcl2 protein, MDM2, protein kinase A, and CBP. 
     
     
         11 . The modified polypeptide of  claim 7 , wherein the modified polypeptide inhibits interaction between the known protein and the binding partner. 
     
     
         12 . The modified polypeptide of  claim 7 , wherein the modified polypeptide binds to a deep groove of the target protein. 
     
     
         13 . The modified polypeptide of  claim 12 , wherein the groove is more than 6 Å at deepest point. 
     
     
         14 . The modified polypeptide of  claim 7 , wherein the modified polypeptide binds to a shallow groove of the target protein. 
     
     
         15 . The modified polypeptide of  claim 14 , wherein the groove is less than 6 Å at deepest point. 
     
     
         16 . The modified polypeptide of  claim 1 , wherein the modified polypeptide binds to the target protein with a Kd of less than 1 micromolar. 
     
     
         17 . The modified polypeptide of  claim 1 , wherein the modified polypeptide binds to the target protein with high specificity. 
     
     
         18 . (canceled) 
     
     
         19 . A stabilized miniature protein comprising a miniature protein and a second portion comprising a stabilizing domain, wherein the miniature protein is a modified avian pancreatic polypeptide (aPP) comprising substitution of at least one amino acid residue, said at least one residue being exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form, wherein the modified polypeptide binds to a target protein. 
     
     
         20 . The stabilized miniature protein of  claim 19 , wherein the second portion is a polypeptide covalently fused to the miniature protein. 
     
     
         21 . The stabilized miniature protein of  claim 20 , wherein the second portion is selected from the group consisting of serum albumin and an IgG Fc domain. 
     
     
         22 . The stabilized miniature protein of  claim 19 , wherein the second portion is a non-amino acid moiety. 
     
     
         23 . The stabilized miniature protein of  claim 19 , wherein said miniature protein includes one or more modified amino acid residues selected from the group consisting of a phosphorylated amino acid, a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent. 
     
     
         24 - 38 . (canceled) 
     
     
         39 . A method of preparing a miniature protein that modulates the interaction between a known protein and another molecule, comprising the steps of:
 (a) identifying at least one amino acid residue that contributes to the binding between a known protein and another molecule; and   (b) modifying an avian pancreatic polypeptide by substitution of said at least one amino acid residue, such that it is exposed on the alpha helix domain of the polypeptide when the polypeptide is in a tertiary form.   
     
     
         40 - 63 . (canceled)