Cysteine-containing peptides having antioxidant properties

Abstract

Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I Milano (R173C) and apoA-I Paris , (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

1 . A peptide comprising a sequence selected from the following group 
       
         
           
                 
                 
               
                   SDEL   RQRLAA   RLEA   L   KN; 
                   (SEQ ID NO: 2) 
                 
                     
                 
                   GEEM   RDRAR   A   H   VDALRTH; 
                   (SEQ ID NO: 10) 
                 
                     
                 
                   PVL   ESFKVSFLSA   L   EEY   T; 
                   (SEQ ID NO: 17) 
                 
                     
                 
                   L   KLLDN   WDSVTSTFS   K   LR; 
                   (SEQ ID NO: 33) 
                 
                   and 
                     
                 
                     
                 
                   PALEDL   R   QGLLPVL   ESFKVSFL SA L   EEY   TKKLN; 
                   (SEQ ID NO: 31) 
                 
                     
                 
                   PALEDL   R   QGLLPL   KLLDN   WDSVTSTFS   K   LR, 
                   (SEQ ID NO: 40) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       wherein
 said peptide has one to four cysteine residues substituted for the amino acids shown in bold and underlined. 
 
     
     
         2 . The peptide of  claim 1 , having from 18 to 100 amino acids, wherein the amino acid sequence is at least 80% homologous with the corresponding native Apolipoprotein A-I human protein sequence as set forth in SEQ ID NO: 85. 
     
     
         3 . A peptide comprising the sequence of GADMED VRGR LVQYRGEV (SEQ ID NO: 48), wherein said peptide has one to four cysteine residues substituted for the amino acids shown in bold and underlined. 
     
     
         4 . The peptide of  claim 3 , having from 18 to 100 amino acids, wherein the amino acid sequence is at least 80% homologous with the corresponding native Apolipoprotein E3 human protein sequence as set forth in SEQ ID NO: 86. 
     
     
         5 . A peptide comprising a sequence selected from the following group 
       
         
           
                 
               
                   (SEQ ID NO: 53) 
                 
                 
                 
               
                     
                   ARL   SRGV   QVLSRK   L   TLKA; 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 59) 
                 
                 
                 
               
                     
                   ARL   SRGV   QVLSRK   L   TLKAKALHARIQQNLDQLREEL; 
                 
                     
                   and 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 65) 
                 
                 
                 
               
                     
                   ATL   K   D   SLEQD   LNNMNKFLEKLR, 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
       wherein said peptide has one to four cysteine residues substituted for the amino acids shown in bold and underlined. 
     
     
         6 . The peptide of  claim 5 , having from 18 to 100 amino acids, wherein the amino acid sequence is at least 80% homologous with the corresponding native Apolipoprotein A-V human protein sequence as set forth in SEQ ID NO: 87. 
     
     
         7 . A peptide comprising a sequence selected from the following group 
       
         
           
                 
               
                   (SEQ ID NO: 71) 
                 
                 
                 
               
                     
                   ETGDLWVGCHP; 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 72) 
                 
                 
                 
               
                     
                   ETGDLWVGCHPNGMKIFFYDSEN; 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 73) 
                 
                 
                 
               
                     
                   LKSLDFNTLVDNISVDP ETGDLWVGCHPNGMKIFFYDSEN. 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         8 . The peptide of  claim 7 , having from 18 to 100 amino acids, wherein the amino acid sequence is at least 80% homologous with the corresponding native Human Serum Paraoxonase as set forth in SEQ ID NO: 88. 
     
     
         9 . A peptide comprising the sequence selected from the following group 
       
         
           
                 
               
                   (SEQ ID NO: 75) 
                 
                 
                 
               
                     
                   D   WLKAF   YDK   VA   EKLKEAF; 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 82) 
                 
                 
                 
               
                     
                   LEKLNSCLRDRLSALTDTPLEELRD   S   LRSRLDALRST; 
                 
                     
                   and 
                 
                     
                     
                 
                 
               
                   (SEQ ID NO: 84) 
                 
                 
                 
               
                     
                   LEKLNS   S   LRDRLSALTDT; 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
       wherein said peptide has one to four cysteine residues substituted for the amino acids shown in bold and underlined. 
     
     
         10 . The peptide of  claim 9 , having from 18 to 100 amino acids extended with the sequence of Apolipoprotein A-I, wherein the extended amino acid sequence is at least 80% homologous with the corresponding native Apolipoprotein A-I human protein sequence as set forth in SEQ ID NO: 85. 
     
     
         11 . A method of making an anti-oxidant peptide, comprising the steps of:
 (a) identifying an amphipathic helix in a Human HDL-associated protein, said helix having between 10 and 100 amino acids and further having a hydrophobic side and a hydrophilic side when viewed axially through the helix;   (b) modifying at least one residue near the amphipathic interface from the naturally occurring amino acid to a cysteine residue to create a modified helix peptide;   (c) selecting a modified helix peptide that has at least twice the antioxidant activity as the unmodified peptide; and   (d) synthesizing said peptide in sufficient quantity for antioxidant use.   
     
     
         12 . A method for preventing oxidation of a phospholipid, comprising contacting the phospholipid with a peptide of  claim 1 . 
     
     
         13 . The method of  claim 12  further comprising the addition of an antioxidant. 
     
     
         14 . The method of  claim 13  where the antioxidant is selected from the group consisting of GSH, vitamin C, vitamin E and N-acetyl cysteine. 
     
     
         15 . A method for preventing oxidation of a phospholipid, comprising contacting the phospholipid with a peptide of  claim 3 . 
     
     
         16 . The method of  claim 15  further comprising the addition of an antioxidant. 
     
     
         17 . A method for preventing oxidation of a phospholipid, comprising contacting the phospholipid with a peptide of  claim 5 . 
     
     
         18 . The method of  claim 17  further comprising the addition of an antioxidant. 
     
     
         19 . A method for preventing oxidation of a phospholipid, comprising contacting the phospholipid with a peptide of  claim 9 . 
     
     
         20 . The method of  claim 19  further comprising the addition of an antioxidant. 
     
     
         21 . The method of  claim 11  wherein the peptide is selected from helix 1 (amino acids 44-65), helix 6 (amino acids 145-162) and helix 10 (amino acids 209-238) of apoAI, helix 7 (amino acids 167-184) of apoAI, the helix spanning amino acids 105-122 of apoE3, and amino acids 219-236 of apo AV.