METHODS OF SCREENING OF PP1-INTERACTING POLYPEPTIDES OR PROTEINS, PEPTIDES INHIBITING PP1c BINDING TO Bcl-2 PROTEINS, BCL-XL AND BCL-W, AND USES THEREOF

Abstract

The invention relates to methods for identifying novel PP1-interacting polypeptides and proteins, compounds which are able to inhibit the binding of PP1c to certain factors naturally interacting with it, especially proteins of the Bcl-2 family (such as Bcl-x L and Bcl-w).

1 - 23 . (canceled) 
     
     
         24 . A set of polypeptides having both the motifs M1 and M2 on the same polypeptide or M1 on one polypeptide and M2 on the other polypeptide wherein M1 and M2 are mammalian PP1 binding sequences wherein M1 is RRNFVNKLKPL (SEQ ID NO:93) and M2 is NQAKKRWFADSTVKVKNVSFEKK (SEQ ID NO:94) or M1 is LDFRNRLQTN (SEQ ID NO:95) and M2 is KKVKKRVSFAND (SEQ ID NO:96) or M1 is RHFVNKLKPLKS (SEQ ID NO:97) and M2 is NQAKKRWFADS (SEQ ID NO:98) or M1 is TCFRPRLRGS (SEQ ID NO:99) and M2 is SQKKKRWFADM (SEQ ID NO:100) or M1 is GEVFINKGKGF (SEQ ID NO:101) and M2 is RGRQLRVRFATH (SEQ ID NO:102) or M1 is QVKFRRRREG (SEQ ID NO:103) and M2 is YFKRYQVKFRRR (SEQ ID NO:104) or M1 is EDFKAKKKEL (SEQ ID NO:105) and M2 is RITPSYVAFTPE (SEQ ID NO:106) or M1 is SVFMQRLKTNILQ (SEQ ID NO:107) and M2 is IDEVKNVYFKNFVLKVSWITFLLA (SEQ ID NO:108-109) or M1 is LQFELRYRPV (SEQ ID NO:110) and M2 is TTKAVMFAK (SEQ ID NO:111) or M1 is DLFENRKKKN (SEQ ID NO:112) and M2 is VRRVFIM (SEQ ID NO:113) or M1 is FFKNEKMLY (SEQ ID NO:114) and M2 is RRGSPRVRFEDG (SEQ ID NO:115). 
     
     
         25 . The set of polypeptides according to  claim 24 , wherein said peptides are glycosylated, acetylated, phosphorylated or amidated. 
     
     
         26 . A set of isolated nucleic acids encoding a set of peptides according to  claim 24  or a nucleic acid sequence that hybridizes under stringent conditions to one of said nucleic acids in said set of peptides. 
     
     
         27 . A vector comprising the nucleic acids according to  claim 26 . 
     
     
         28 . A method of identifying a PP1-interacting polypeptide or protein, comprising:
 detecting in the sequence said polypeptide or protein, the presence of two PP1-binding motifs M1 and M2 on the same polypeptide, wherein the motif M1 and M2 is chosen from one of the motif pairs in  claim 24 ; and confirming said identified PP-1 interacting polypeptide or protein using a biochemical test.   
     
     
         29 . The method of  claim 28 , wherein said biochemical test consists in coimmunoprecipitating said identified PP-1 interacting polypeptide or protein and PP1. 
     
     
         30 . A method of screening compounds that inhibit or enhance PP1 activity or change its localization by interaction or that interact with PP1 regulators, comprising;
 (a) immobilizing peptides according to  claim 24  on a support; and   (b) testing the interaction of said compounds with the peptides of step (a).   
     
     
         31 . A method of screening compounds that interact with PP1, comprising:
 i. obtaining antibodies to peptides according to 24 1 and   ii. testing the interaction of said compound with said antibodies.   
     
     
         32 . A pharmaceutical compound comprising the set of peptides according to  claim 24 . 
     
     
         33 . A pharmaceutical composition comprising a nucleic acid encoding the set of peptides according to  claim 24 . 
     
     
         34 . A pharmaceutical composition comprising the vector of  claim 27 . 
     
     
         35 . A method for inhibiting in vitro the interaction between PP1 c and Bc1XL, comprising a step of adding a set of peptides having both the motifs M1 and M2 on the same peptide, wherein the set of peptides comprises one of the M1 and M2 motif pairs in  claim 24 . 
     
     
         36 . A kit comprising antibodies to the set of peptides according to  claim 24  and reagents for interpreting the interaction. 
     
     
         37 . A method of treating or preventing diabetes, high blood pressure, a neurological disorder, a viral disease or a microbial infection associated with PP1 said method comprising administering to a patient in need of such treatment the set of polypeptides according to  claim 24 . 
     
     
         38 . The method according to  claim 37 , wherein the neurological disorder is Parkinson's disease or Alzheimers disease. 
     
     
         39 . The method according to  claim 37 , wherein the viral disease is HIV-1. 
     
     
         40 . Antibodies to the peptides according to  claim 24 . 
     
     
         41 . Antibodies according to  claim 40 , wherein said antibodies are monoclonal antibodies. 
     
     
         42 . The set of polypeptides according to  claim 24 , wherein there are two or more M1 and M2 motifs on the same polypeptide. 
     
     
         43 . The set of polypeptides according to  claim 42 , wherein there are 2 to 5, M1 and M2 motifs on the same polypeptide. 
     
     
         44 . The set of polypeptides according to  claim 42 , wherein there are 2 to 10, M1 and M2 motifs on the same polypeptide. 
     
     
         45 . The set of polypeptides according to any one of  claim 42 , wherein said M1 and M2 motifs have a spacer between them. 
     
     
         46 . The set of polypeptides according to  claim 45 , wherein the spacer is selected from a sequence of amino acids, a hydrocarbon chain or a chemical entity which connects the at least two motifs. 
     
     
         47 . The set of polypeptide according to  claim 46 , wherein said amino acid spacer has between 1 to 50 amino acids. 
     
     
         48 . The set of polypeptide according to  claim 46 , wherein amino acid spacer has 36 amino acids. 
     
     
         49 . The set of polypeptide according to  claim 46 , wherein said spacer contains regulatory sequences between the motifs.