Protease-resistant lipidated glp-1 analogs

Abstract

The present invention provides protease-resistant peptides, methods of making such peptides, as well as compositions comprising protease-resistant peptides and method of treatment utilizing such peptides. A combination of lipidation of certain amino acid residues and substituition of alpha-methyl functionalized amino acids for natural amino acids has been determined to produce protease-resistant peptides.

1 . An isolated polypeptide comprising the amino acid sequence: 
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   HX2EGS X6TSDV X11X12X13LE GEAAX20 EX22IX24X25 
                 
                     
                 
                   VVX28GG; 
                 
             
                
                
                
                
               
            
           
         
         wherein X2 is A or Aib, 
         X6 is F or an alpha-methyl functionalized amino acid, 
         X11 is S or an alpha-methyl functionalized amino acid, 
         X12 is S or a lipid modified K, 
         X13 is Y or an alpha-methyl functionalized amino acid, 
         X20 is a lipid modified K, K, or an alpha-methyl functionalized amino acid, 
         X22 is F or an alpha-methyl functionalized amino acid, 
         X24 is A or a lipid modified K, 
         X25 is W or an alpha-methyl functionalized amino acid, and 
         X28 is K, E, or an alpha-methyl functionalized amino acid; and 
         wherein the polypeptide is lipidated on only one of X12, X20, or X24. 
       
     
     
         2 . The polypeptide of  claim 1 , wherein the peptide comprises a C-terminal amide. 
     
     
         3 . The polypeptide of  claim 1 , wherein X2 is Aib. 
     
     
         4 . The polypeptide of any one of  claim 1 , wherein the lipid modified K is selected from the group consisting of: K(ε-(PEG) 2 -(PEG) 2 -γE-Stearate), K(ε-γE-Palmitoyl), K(ε-(PEG) 2 -(PEG) 2 -γE-Stearate), K(γE-Palmitoyl), K(ε-(PEG) 2 -(PEG) 2 -(PEG) 2 -Stearoyl), K(ε-γE-Lauroyl), K(ε-γE-γE-Lauroyl), K(ε-γE-γE-γE-Lauroyl), K(ε-Ahx-Lauroyl), K(ε-Ahx-Ahx-Lauroyl), K(ε-Ahx-Ahx-Ahx-Lauroyl), K(ε-(PEG) 2 -Lauroyl), K(ε-(PEG) 2 -(PEG) 2 -Lauroyl), K(ε-(PEG) 2 -(PEG) 2 -(PEG) 2 -Lauroyl), K(ε-γE-12-(4-carboxyphenoxy)dodecanoyl), K(ε-γE-γE-12-(4-carboxyphenoxy)dodecanoyl), K(ε-γE-γE-γE-12-(4-carboxyphenoxy)dodecanoyl), K(ε-Ahx-12-(4-carboxyphenoxy)dodecanoyl), K(ε-Ahx-Ahx-12-(4-carboxyphenoxy)dodecanoyl), K(ε-Ahx-Ahx-Ahx-12-(4-carboxyphenoxy)dodecanoyl), K(ε-(PEG) 2 -12-(4-carboxyphenoxy)dodecanoyl), K(ε-(PEG) 2 -(PEG) 2 -12-(4-carboxyphenoxy)dodecanoyl), K(ε-(PEG) 2 -(PEG) 2 -(PEG) 2 -12-(4-carboxyphenoxy)dodecanoyl), K(ε-γE-Stearoyl), K(ε-γE-γE-Stearoyl), K(ε-γE-γE-γE-Stearoyl), K(ε-Ahx-Stearoyl), K(ε-Ahx-Ahx-Stearoyl), K(ε-Ahx-Ahx-Ahx-Stearoyl), K(ε-(PEG) 2 -Stearoyl), K(ε-(PEG) 2 -(PEG) 2 -Stearoyl), K(ε-(PEG) 2 -(PEG) 2 -(PEG) 2 -Stearoyl), K(ε-γE-Stearate), K(ε-γE-γE-Stearate), K(ε-γE-γE-γE-Stearate), K(ε-Ahx-Stearate), K(ε-Ahx-Ahx-Stearate), K(ε-Ahx-Ahx-Ahx-Stearate), K(ε-(PEG) 2 -Stearate), K(ε-(PEG) 2 -(PEG) 2 -Stearate), K(ε-(PEG) 2 -(PEG) 2 -(PEG) 2 -Stearate), and any combination thereof. 
     
     
         5 . The polypeptide of  claim 4 , wherein the lipid modified K is K(ε-(PEG) 2 -(PEG) 2 -γE-Stearate). 
     
     
         6 . The polypeptide of  claim 1 , wherein X6 is α-MeF, X11 is α-MeS, X13 is α-MeF, X22 is α-MeF, X25 is α-MeF, X28 is α-MeK, or any combination thereof. 
     
     
         7 . The polypeptide of  claim 6 , wherein X2 is Aib, X6 is α-MeF, X11 is a-MeS, X13 is α-MeF, X20 is K(ε-(PEG) 2 -(PEG) 2 -γE-Stearate), X22 is α-MeF, X25 is α-MeF, and X28 is α-MeK (SEQ ID NO: 3). 
     
     
         8 . The polypeptide of  claim 1 , wherein the polypeptide is substantially resistant to proteolytic degradation. 
     
     
         9 . The polypeptide of  claim 8 , wherein the polypeptide is substantially resistant to DPP-IV, neprilysin, α-chymotrypsin, plasmin, thrombin, kallikrein, trypsin, elastase and/or pepsin degradation. 
     
     
         10 . The polypeptide of  claim 1 , wherein the polypeptide at least maintains substantially the same receptor potency as a corresponding non-lipidated polypeptide. 
     
     
         11 . The polypeptide of  claim 10 , wherein the polypeptide at least maintains substantially the same receptor selectivity as a corresponding non-lipidated polypeptide. 
     
     
         12 . The polypeptide of  claim 11 , wherein the polypeptide exhibits increased receptor potency over a corresponding non-lipidated polypeptide. 
     
     
         13 . An isolated polypeptide comprising the amino acid sequence: 
       
         
           
                 
               
                   (SEQ ID NO: 4) 
                 
                   HX2EGX5 X6TSDX10 X11X12X13X14E GX17AAX20 
                 
                     
                 
                   EX22IX24X25 X26VX28GX30; 
                 
             
                
                
                
                
               
            
           
         
         wherein X2 is A or Aib, 
         X5 is T or S, 
         X6 is F or an alpha-methyl functionalized amino acid, 
         X10 is V or a lipid modified K, 
         X11 is S or an alpha-methyl functionalized amino acid, 
         X12 is S or a lipid modified K, 
         X13 is Y, F, or a lipid modified K, 
         X14 is L or a lipid modified K, 
         X17 is Q or E, 
         X20 is K, E, or an alpha-methyl functionalized amino acid, 
         X22 is F, norleucine, tyrosine methyl ester, or an alpha-methyl functionalized amino acid, 
         X24 is A or a lipid modified K, 
         X25 is W, F, or a lipid modified K, 
         X26 is L, V, or a lipid modified K, 
         X28 is K or E, and 
         X30 is R or G; 
         wherein the polypeptide comprises two lipid modified K residues, and wherein one of X10, X12, X13, or X14 is a lipid modified K residue and one of X24, X25, or X26 is a lipid modified K residue. 
       
     
     
         14 . The polypeptide of  claim 13 , wherein X6 is F, α-MeF, α-MeS, or α-MeK, X11 is S, α-MeF, α-MeS, or α-MeK, X20 is K, E, α-MeF, α-MeS, or α-MeK, and X22 is F, or α-MeF. 
     
     
         15 . The polypeptide of  claim 13 , wherein the peptide comprises a C-terminal amide. 
     
     
         16 . The polypeptide of  claim 13 , wherein X2 is Aib. 
     
     
         17 . The polypeptide of  claim 13 , wherein the two lipid modified K residues are the same or are different, and are selected from the group consisting of: K(ε-(PEG) 2 -(PEG) 2 -γE-Lauroyl), K(ε-(PEG) 2 -(PEG) 2 -γE-Palmitate), K(ε-(PEG) 2 -(PEG) 2 -γE-Myristoyl), K(ε-(PEG) 2 -(PEG) 2 -γE-Palmitoyl), K(ε-(PEG) 2 -(PEG) 2 -γE-Stearoyl), K(ε-(PEG) 2 -(PEG) 2 -γE-Stearate), and any combination thereof. 
     
     
         18 . The polypeptide of  claim 17 , wherein the two lipid modified K residues are both K(ε-(PEG) 2 -(PEG) 2 -γE-Lauroyl), both K(ε-(PEG) 2 -(PEG) 2 -γE-Palmitate), both K(ε-(PEG) 2 -(PEG) 2 -γE-Myristoyl), both K(ε-(PEG) 2 -(PEG) 2 -γE-Palmitoyl), both K(ε-(PEG) 2 -(PEG) 2 -γE-Stearoyl), or both K(ε-(PEG) 2 -(PEG) 2 -γE-Stearate). 
     
     
         19 . The polypeptide of  claim 13 , wherein X10 is a lipid modified K and any one of X24, X25, or X26 is a lipid modified K. 
     
     
         20 . The polypeptide of  claim 13 , wherein X12 is a lipid modified K and any one of X24, X25, or X26 is a lipid modified K. 
     
     
         21 . The polypeptide of  claim 13 , wherein X13 is a lipid modified K and any one of X24, X25, or X26 is a lipid modified K. 
     
     
         22 . The polypeptide of  claim 13 , wherein X14 is a lipid modified K and any one of X24, X25, or X26 is a lipid modified K. 
     
     
         23 . The polypeptide of  claim 13 , wherein X24 is a lipid modified K and any one of X10, X12, X13, or X14 is a lipid modified K. 
     
     
         24 . The polypeptide of  claim 13 , wherein X25 is a lipid modified K and any one of X10, X12, X13, or X14 is a lipid modified K. 
     
     
         25 . The polypeptide of  claim 13 , wherein X26 is a lipid modified K and any one of X10, X12, X13, or X14 is a lipid modified K. 
     
     
         26 . The polypeptide of  claim 13 , wherein:
 X6 is α-MeF,   X11 is α-MeS,   X20 is α-MeK,   X6 is α-MeF and X11 is α-MeS,   X6 is α-MeF and X20 is α-MeK,   X11 is α-MeS and X20 is α-MeK, or   X6 is α-MeF, X11 is α-MeS, and X20 is α-MeK.   
     
     
         27 . The polypeptide of  claim 26 , wherein X13 is a lipid modified K and one of X24, X25, or X26 is a lipid modified K. 
     
     
         28 . The polypeptide of  claim 26 , wherein X14 is a lipid modified K and one of X24, X25, or X26 is a lipid modified K. 
     
     
         29 . The polypeptide of  claim 13 , wherein X5 is S. 
     
     
         30 . The polypeptide of  claim 13 , wherein X17 is E, X28 is E, and X30 is G. 
     
     
         31 . The polypeptide of  claim 13 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 252, SEQ ID NO: 263, SEQ ID NO: 269, SEQ ID NO: 405, SEQ ID NO: 408, SEQ ID NO: 409, or SEQ ID NO: 410. 
     
     
         32 . The polypeptide of  claim 13 , wherein the polypeptide is substantially resistant to proteolytic degradation. 
     
     
         33 . The polypeptide of  claim 32 , wherein the synthetic peptide is substantially resistant to DPP-IV, neprilysin, α-chymotrypsin, plasmin, thrombin, kallikrein, trypsin, elastase and/or pepsin degradation. 
     
     
         34 . The polypeptide of  claim 33 , wherein the polypeptide, which comprises two lipid modified K residues, at least maintains substantially the same receptor potency as a corresponding non-lipidated peptide. 
     
     
         35 . The polypeptide of  claim 34 , wherein the polypeptide, which comprises two lipid modified K residues, at least maintains substantially the same receptor selectivity as a corresponding non-lipidated peptide. 
     
     
         36 . The polypeptide of  claim 35 , wherein the polypeptide, which comprises two lipid modified K residues, exhibits increased receptor potency over a corresponding non-lipidated polypeptide. 
     
     
         37 - 63 . (canceled)