Isozyme-specific antagonists of protein kinase C

Abstract

A method of changing or otherwise converting the biological activity of a PKC peptide agonist to a peptide antagonist is described. The method involves substituting one or more amino acid residues so as to effect a change in charge in the peptide and/or to otherwise make the sequence similar to a sequence derived from the PKC binding site on the RACK protein for the respective PKC enzyme. Methods of inhibiting the activity of a PKC enzyme, and various peptide antagonists of εPKC are also disclosed.

1 . A method of converting a protein kinase C (PKC) agonist peptide or peptidomimetic to a PKC antagonist peptide or peptidomimetic, comprising substituting at least one amino acid in said agonist peptide or peptidomimetic with an amino acid that converts the PKC agonist peptide or peptidomimetic into a PKC antagonist peptide or peptidomimetic.  
     
     
         2 . The method of  claim 1 , wherein said at least one amino acid is a charged amino acid which is substituted with an uncharged amino acid.  
     
     
         3 . The method of  claim 2 , wherein said charged amino acid is aspartic acid and said uncharged amino acid is asparagine.  
     
     
         4 . The method of  claim 1 , wherein said PKC agonist peptide is a classical PKC agonist, a novel PKC agonist or an atypical PKC agonist.  
     
     
         5 . The method of  claim 1 , wherein said PKC agonist peptide is an εPKC agonist peptide.  
     
     
         6 . The method of  claim 5 , wherein said εPKC agonist peptide is a ψεRACK peptide having an amino acid sequence set forth in SEQ ID NO:3.  
     
     
         7 . The method of  claim 5 , wherein said εPKC agonist peptide has an amino acid sequence set forth in SEQ ID NO:12; SEQ ID NO:20; SEQ ID NO:16; or SEQ ID NO:21.  
     
     
         8 . The method of  claim 1 , wherein said PKC antagonist peptide is an εPKC antagonist peptide having an amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57.  
     
     
         9 . The method of  claim 1 , wherein said PKC agonist peptide is an εPKC agonist peptide and wherein said at least one amino acid is a charged amino acid which is substituted with an uncharged amino acid.  
     
     
         10 . A method of inhibiting the activity of a protein kinase C (PKC) enzyme, comprising contacting the enzyme with a PKC inhibitor peptide or peptidomimetic, said peptide derived from a PKC agonist peptide or peptidomimetic wherein at least one amino acid in the agonist peptide or peptidomimetic is substituted with another amino acid sufficient to convert the agonist peptide or peptidomimetic into an antagonist peptide or peptidomimetic.  
     
     
         11 . The method of  claim 10 , wherein said PKC agonist peptide is derived from the ψ-RACK sequence of said PKC enzyme.  
     
     
         12 . The method of  claim 10 , wherein said ψ-RACK sequence is a ψεRACK sequence.  
     
     
         13 . The method of  claim 10 , wherein said protein kinase C is εPKC and said PKC agonist peptide is a derivative of a ψRACK sequence of said PKC enzyme.  
     
     
         14 . The method of  claim 13 , wherein said PKC agonist peptide is a ψεRACK agonist peptide having an amino acid sequence set forth in SEQ ID NO:3.  
     
     
         15 . The method of  claim 13 , wherein said PKC agonist peptide has an amino acid sequence set forth in SEQ ID NO:12; SEQ ID NO:16; SEQ ID NO:20 or SEQ ID NO:21.  
     
     
         16 . The method of  claim 13 , wherein said PKC agonist peptide is a ψεRACK peptide having an amino acid sequence set forth in SEQ ID NO:3 and said PKC inhibitor peptide has an amino acid sequence that differs from SEQ ID NO:3 in that said at least one amino acid residue is substituted with another amino acid that has a decreased electrical charge compared to said at least one amino acid.  
     
     
         17 . The method of  claim 16 , wherein said at least one amino acid is negatively charged and said another amino acid is uncharged.  
     
     
         18 . The method of  claim 17 , wherein said at least one amino acid is aspartic acid and said another amino acid is asparagine.  
     
     
         19 . The method of  claim 16 , wherein said PKC inhibitor peptide has the amino acid sequence set forth in SEQ ID NO:55.  
     
     
         20 . The method of  claim 10 , wherein said PKC inhibitor peptide has an amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57.  
     
     
         21 . A peptide, comprising a peptide having PKC antagonistic activity, said peptide derived from a PKC agonist peptide wherein at least one amino acid in the agonist peptide is substituted with another amino acid sufficient to convert the agonist peptide into an antagonist peptide.  
     
     
         22 . The peptide of  claim 21 , wherein said PKC is εPKC and wherein said PKC agonist peptide is an εPKC agonist peptide.  
     
     
         23 . The peptide of  claim 22 , wherein said peptide has an amino acid sequence set forth in SEQ ID NO:55.  
     
     
         24 . The peptide of  claim 21 , wherein said antagonist peptide has an amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57.  
     
     
         25 . The peptide of  claim 21 , wherein said at least one amino acid is substituted with an amino acid that provides a change of electrical charge at the substituted position.  
     
     
         26 . A peptide having PKC antagonistic activity, comprising a peptide having an amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57.  
     
     
         27 . A treatment method, comprising administering to a patient in need thereof a therapeutically effective amount of an εPKC antagonist peptide having the amino acid sequence set forth in SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; or SEQ ID NO:57, or a combination thereof.  
     
     
         28 . The method of  claim 27 , wherein said treatment is for a disease or condition modulated by εPKC.  
     
     
         29 . The method of  claim 28 , wherein said disease or condition is a fibrotic disease or an inflammatory disease.  
     
     
         30 . The method of  claim 29 , wherein said fibrotic disease is scleroderma, liver fibrosis or lung fibrosis.  
     
     
         31 . The method of  claim 29 , wherein said inflammatory disease is an autoimmune disease or a lung disease.  
     
     
         32 . The method of  claim 31 , wherein said autoimmune disease is multiple sclerosis, Guillain-Barre syndrome, psoriasis, Grave's disease, rheumatoid arthritis and Type 1 diabetes mellitus.  
     
     
         33 . The method of  claim 31 , wherein said lung disease is chronic obstructive pulmonary disease or asthma.  
     
     
         34 . The method of  claim 29 , wherein said inflammatory disease is septic shock or inflammatory bowel disease.