Stabilized bioactive peptides and methods of identification, synthesis, and use

Abstract

An intracellular selection system allows screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at the N-terminus, the C-terminus, or both. The stabilizing group can be a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, an α-helical moiety, or one or more proline residues.

What is claimed is:  
     
         1 . A polypeptide comprising a bioactive peptide and a first stabilizing group coupled to a terminus of said bioactive peptide, wherein said first stabilizing group is heterologous to the bioactive peptide and lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide.  
     
     
         2 . The polypeptide of  claim 1 , wherein said polypeptide further comprises a second stabilizing group coupled to the other terminus of said bioactive peptide, wherein the second stabilizing group is heterologous to the bioactive peptide.  
     
     
         3 . The polypeptide of  claim 1 , wherein said first stabilizing group is coupled to the N-terminus of said bioactive peptide.  
     
     
         4 . The polypeptide of  claim 1 , wherein said first stabilizing group is coupled to the C-terminus of said bioactive peptide.  
     
     
         5 . The polypeptide of  claim 2 , wherein said first and said second stabilizing groups are the same.  
     
     
         6 . The polypeptide of  claim 1 , wherein said first stabilizing group is coupled to said bioactive peptide via a peptide bond.  
     
     
         7 . The polypeptide of  claim 2 , wherein said first and said second stabilizing groups are coupled to said bioactive peptide via peptide bonds.  
     
     
         8 . The polypeptide of  claim 1 , wherein said bioactive peptide is selected from the group consisting of insulin, glucagon, calcitonin, somatostatin, gonadotrophin, and secretin.  
     
     
         9 . The polypeptide of  claim 1 , wherein said first stabilizing group is a single α-helix.  
     
     
         10 . The polypeptide of  claim 1 , wherein said first stabilizing group is a two-helix bundle.  
     
     
         11 . The polypeptide of  claim 1 , wherein said first stabilizing group is a three-helix bundle.  
     
     
         12 . The polypeptide of  claim 1 , wherein said first stabilizing group is a four-helix bundle.  
     
     
         13 . The polypeptide of  claim 1 , wherein said first stabilizing group is a five-helix bundle.  
     
     
         14 . The polypeptide of  claim 12 , wherein said four-helix bundle is a Rop polypeptide.  
     
     
         15 . The polypeptide of  claim 1 , wherein said first stabilizing group is Xaa-Pro-Pro- or -Pro-Pro-Xaa, wherein Xaa is any amino acid.  
     
     
         16 . The polypeptide of  claim 15 , wherein Xaa is Ala.  
     
     
         17 . The polypeptide of  claim 1 , wherein said bioactive peptide is 5 to 20 amino acids in length.  
     
     
         18 . The polypeptide of  claim 2 , wherein said second stabilizing group is a single α-helix.  
     
     
         19 . The polypeptide of  claim 2 , wherein said second stabilizing group is a two-helix bundle.  
     
     
         20 . The polypeptide of  claim 2 , wherein said second stabilizing group is a three-helix bundle.  
     
     
         21 . The polypeptide of  claim 2 , wherein said second stabilizing group is a four-helix bundle.  
     
     
         22 . The polypeptide of  claim 2 , wherein said second stabilizing group is a five-helix bundle.  
     
     
         23 . The polypeptide of  claim 21 , wherein said four-helix bundle is a Rop polypeptide.  
     
     
         24 . The polypeptide of  claim 2 , wherein said second stabilizing group is Xaa-Pro-Pro- or -Pro-Pro-Xaa, wherein Xaa is any amino acid.  
     
     
         25 . The polypeptide of  claim 24 , wherein Xaa is Ala.  
     
     
         26 . A polypeptide comprising a bioactive peptide and a stabilizing group coupled to a terminus of said bioactive peptide, wherein said stabilizing group is not a thioredoxin polypeptide.  
     
     
         27 . A polypeptide comprising a bioactive peptide, a first stabilizing group coupled to the N-terminus of said bioactive peptide and a second stabilizing group coupled to the C-terminus of said bioactive peptide, wherein said first and second stabilizing groups are heterologous to the bioactive peptide and to each other.  
     
     
         28 . A polypeptide comprising a bioactive peptide, a first stabilizing group coupled to the N-terminus of said bioactive peptide and a second stabilizing group coupled to the C-terminus of said bioactive peptide, wherein said first and second stabilizing groups are heterologous to the bioactive peptide and do not interact to form a naturally occurring secondary or tertiary structure.  
     
     
         29 . A polypeptide comprising a bioactive peptide, a first stabilizing group coupled to the N-terminus of said bioactive peptide and a second stabilizing group coupled to the C-terminus of said bioactive peptide, wherein said first stabilizing group and second groups are heterologous to the bioactive peptide and do not confine the N-terminus and the C-terminus of the bioactive peptide in close proximity.  
     
     
         30 . A method of making a stabilized polypeptide, said method comprising coupling a stabilizing group to at least one terminus of a bioactive peptide to produce said stabilized polypeptide, wherein said first stabilizing group is heterologous to the bioactive peptide and lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide.  
     
     
         31 . The method of  claim 30 , wherein said coupling step comprises chemically synthesizing said stabilized polypeptide.  
     
     
         32 . The method of  claim 30 , further comprising expressing said stabilized polypeptide in a host cell transformed with a vector, said vector comprising an expression control sequence operably linked to a nucleic acid sequence encoding said stabilized polypeptide, wherein said expression control sequence is tightly regulable in said host cell.  
     
     
         33 . The method of  claim 32 , wherein said method further comprises determining stability of said stabilized polypeptide relative to said bioactive peptide.  
     
     
         34 . A method of making a stabilized polypeptide, said method comprising coupling a heterologous stabilizing group to at least one terminus of a bioactive peptide to produce said stabilized polypeptide; and determining stability of said stabilized polypeptide relative to said bioactive peptide.  
     
     
         35 . An isolated nucleic acid encoding a stabilized polypeptide, wherein said stabilized polypeptide comprises a bioactive peptide and a first stabilizing group coupled to one of said bioactive peptide's termini, wherein said first stabilizing group is heterologous to the bioactive peptide and lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide.  
     
     
         36 . The isolated nucleic acid of  claim 35 , wherein said stabilized polypeptide further comprises a second stabilizing group coupled to the other terminus of said bioactive peptide, wherein the second stabilizing group is heterologous to the bioactive peptide.  
     
     
         37 . The isolated nucleic acid of  claim 35 , wherein said first and said second stabilizing groups are the same.  
     
     
         38 . A method of making a stabilized polypeptide, said method comprising: 
 a) providing host cells transformed with an exogenous nucleic acid encoding said stabilized polypeptide, said stabilized polypeptide comprising a bioactive peptide and a first stabilizing group coupled to one of said bioactive peptide's termini, wherein said first stabilizing group is heterologous to the bioactive peptide and lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide;    b) expressing said stabilized polypeptide; and    c) recovering said stabilized polypeptide.    
     
     
         39 . The method of  claim 38 , wherein said host cells are bacteria.  
     
     
         40 . The method of  claim 38 , wherein said host cells are eukaryotic host cells.  
     
     
         41 . The method of  claim 38 , wherein said stabilized polypeptide further comprises a second stabilizing group coupled to the other terminus of said bioactive peptide, wherein the second stabilizing group is heterologous to the bioactive peptide.  
     
     
         42 . The method of  claim 41 , wherein said first and said second heterologous stabilizing groups are the same.  
     
     
         43 . A method of making a polypeptide comprising: 
 providing a bacteriophage that comprises an exogenous nucleic acid encoding a polypeptide comprising a bioactive peptide, a bacteriophage protein coupled to one terminus of said bioactive peptide, and a stabilizing group coupled to the other terminus of said bioactive peptide; and    culturing said bacteriophage under conditions to cause the bacteriophage to express said polypeptide and display it on the surface of the bacteriophage.    
     
     
         44 . The method of  claim 43  wherein the stabilizing group is coupled to the N-terminus of the bioactive peptide.  
     
     
         45 . The method of  claim 43  wherein the stabilizing group is coupled to the C-terminus of the bioactive peptide.  
     
     
         46 . The method of  claim 43  further comprising cleaving the polypeptide from the host cell surface to yield a stabilized bioactive peptide comprising the bioactive peptide and the stabilizing group.  
     
     
         47 . A method of making a stabilized polypeptide comprising coupling a stabilizing group to at a terminus of a bioactive peptide to produce the stabilized polypeptide, said bioactive peptide having been identified using a phage display process that produces a bacteriophage protein coupled to one terminus of said bioactive peptide, wherein the stabilizing group takes the place of the bacteriophage protein.  
     
     
         48 . The method of  claim 47 , wherein said coupling step comprises chemically synthesizing said stabilized bioactive peptide.  
     
     
         49 . The method of  claim 47 , further comprising expressing said stabilized polypeptide in a host cell transformed with a vector, said vector comprising an expression control sequence operably linked to a nucleic acid sequence encoding said stabilized polypeptide, wherein said expression control sequence is tightly regulable in said host cell.  
     
     
         50 . The method of  claim 47 , further comprising identifying said bioactive peptide using a phage display process.  
     
     
         51 . A polypeptide comprising a bioactive peptide and a first stabilizing group coupled to a terminus of said bioactive peptide, said bioactive peptide having been identified using a phage display process that produces a bacteriophage protein coupled to one terminus of said bioactive peptide, wherein the stabilizing group takes the place of the bacteriophage protein.  
     
     
         52 . A vector comprising an expression control sequence operably linked to a nucleic acid sequence encoding a stabilized polypeptide, wherein said stabilized polypeptide comprises a bioactive peptide and a first stabilizing group coupled to one of said bioactive peptide's termini, wherein said first stabilizing group is heterologous to the bioactive peptide and lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide.  
     
     
         53 . The vector of  claim 52 , wherein said stabilized polypeptide further comprises a second stabilizing group coupled to the other terminus of said bioactive peptide, wherein the second stabilizing group is heterologous to the bioactive peptide.  
     
     
         54 . The vector of  claim 52 , wherein said vector further comprises a tightly regulable expression control sequence operably linked to said nucleic acid sequence encoding said stabilized polypeptide.  
     
     
         55 . The vector of  claim 54 , wherein said tightly regulable expression control sequence is from a wild-type  E. coli  lac promoter/operator region.  
     
     
         56 . The vector of  claim 54 , wherein said expression control sequence contains the auxiliary operator O3, the CAP binding region, the −35 promoter site, the −10 promoter site, the operator O1, lacZ Shine-Dalgarno sequence, and a spacer region.  
     
     
         57 . The vector of  claim 56 , wherein said spacer region is 5 to 10 nucleotides in length.  
     
     
         58 . The vector of  claim 52 , wherein said vector is pLAC11, represented by ATCC Accession No. 207108.  
     
     
         59 . A plurality of vectors, wherein each said vector comprises a nucleic acid sequence encoding a polypeptide, said polypeptide comprising a randomized peptide and a first stabilizing group coupled to one of said randomized peptide's termini, wherein said first stabilizing group lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide, and wherein said plurality of vectors comprises at least two different vectors, each of said at least two vectors encoding a different polypeptide.  
     
     
         60 . The plurality of vectors of  claim 59 , wherein said polypeptide further comprises a second stabilizing group coupled to the other terminus of said randomized peptide.  
     
     
         61 . The plurality of vectors of  claim 59 , wherein each said vector further comprises a tightly regulable expression control sequence operably linked to said nucleic acid sequence encoding said polypeptide.  
     
     
         62 . The plurality of vectors of  claim 59 , wherein said plurality comprises at least 50 different vectors, each of said at least 50 vectors encoding a different polypeptide.  
     
     
         63 . The plurality of vectors of  claim 59 , wherein said plurality comprises at least 1×10 6  different vectors, each of said at least 1×10 6  vectors encoding a different polypeptide.  
     
     
         64 . A plurality of host cells collectively containing the plurality of vectors of  claim 48 .  
     
     
         65 . The host cells of  claim 64 , wherein said host cells are prokaryotic.  
     
     
         66 . The host cells of  claim 64 , wherein said host cells are eukaryotic.  
     
     
         67 . A method for selecting bioactive peptides, said method comprising: 
 a) expressing a plurality of stabilized polypeptides, wherein each said stabilized polypeptide is encoded by a different vector, and wherein each said stabilized polypeptide comprises a randomized peptide and a first stabilizing group coupled to one of said randomized peptide's termini, wherein said first stabilizing group lacks the capacity to participate in the formation of an intramolecular disulfide bond within the polypeptide, and wherein said plurality of stabilized polypeptide comprises at least two different polypeptides; and    b) screening each said stabilized polypeptide of said plurality for a bioactivity; and    c) selecting said bioactive peptides with said bioactivity.    
     
     
         68 . The method of  claim 67 , wherein said stabilized polypeptide further comprises a second stabilizing group coupled to the other terminus of said randomized peptide.  
     
     
         69 . The method of  claim 67 , wherein said plurality of stabilized polypeptides is expressed in eukaryotic cells.  
     
     
         70 . The method of  claim 69 , wherein said eukaryotic cells are cancer cells and said bioactivity is a complete or partial inhibition of cell division, induction of apoptosis, or cell toxicity.  
     
     
         71 . The method of  claim 70 , said method further comprising testing said selected bioactive peptides for bioactivity in normal cells compared with said cancer cells.  
     
     
         72 . The method of  claim 69 , wherein said eukaryotic cells are stem cells or cord blood cells and said bioactivity is an increase in cell growth rate.  
     
     
         73 . A polypeptide comprising a bioactive peptide and a stabilizing group coupled to one or both of said bioactive peptide's termini, wherein said stabilizing group is heterologous to said bioactive peptide and consists of Xaa n -Pro-Pro- or -Pro-Pro-Xaa n , wherein Xaa is any amino acid and n=1 or 2.  
     
     
         74 . The polypeptide of  claim 73 , wherein a heterologous stabilizing group is coupled to both of said bioactive peptide's termini.  
     
     
         75 . The polypeptide of  claim 73 , wherein the Xaa residue of said heterologous stabilizing group is different on each end of said bioactive peptide.  
     
     
         76 . The polypeptide of  claim 73 , wherein said bioactive peptide is selected from the group consisting of insulin, glucagon, calcitonin, somatostatin, gonadotrophin, and secretin.  
     
     
         77 . A polypeptide comprising a bioactive peptide and a first stabilizing group coupled to one terminus of said bioactive peptide, wherein said bioactive peptide is 50 or fewer amino acids in length, and wherein said first stabilizing group is human serum albumin or a fragment thereof.  
     
     
         78 . The polypeptide of  claim 77 , wherein said polypeptide further comprises a second stabilizing group coupled to the other terminus of said bioactive peptide.