Methods of regulating apoptosis

Abstract

Compounds that modulate the function of anti-apoptotic proteins such as Bcl-2 and related Bcl-2 family members are identified. These compounds have the ability to convert the activity of Bcl-2-family member proteins from anti-apoptotic to pro-apoptotic. Methods for inducing or preventing apoptosis are described, together with methods for identifying molecules that induce or prevent apoptosis through interaction with Bcl-2-family members. Methods for treatment of proliferative diseases and neurodegenerative diseases using the modulators of Bcl-2 and related family members are also disclosed.

1 . A method of inducing apoptosis in a mammalian cell, comprising:
 providing a compound comprising:
 Nur 77 or a fragment thereof; and 
 a cell-penetrating peptide; and 
   contacting said mammalian cell with an effective amount of said compound, wherein said contacting results in apoptosis of said mammalian cell.   
     
     
         2 . The method of  claim 1 , wherein contacting said mammalian cell is performed in vitro. 
     
     
         3 . The method of  claim 1 , wherein contacting said mammalian cell is performed in vivo. 
     
     
         4 . The method of  claim 1 , wherein said mammalian cell is a breast cancer cell. 
     
     
         5 . The method of  claim 1 , wherein said compound comprises D-amino acids. 
     
     
         6 . The method of  claim 1 , wherein said compound comprises at least one non-naturally occurring amino acid. 
     
     
         7 . The method of  claim 1 , wherein said compound is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 60. 
     
     
         8 . The method of  claim 1 , wherein said compound is a Nur77-derived Bcl-2-converting peptide (NuBCP). 
     
     
         9 . The method of  claim 8 , wherein said NuBCP peptide is D-NuBCP-9-r8. 
     
     
         10 . The method of  claim 1 , wherein said compound is a DC-3 peptide. 
     
     
         11 . The method of  claim 1 , wherein said compound is 4-20 amino acids in length. 
     
     
         12 . A method of inducing apoptosis in a mammalian cell, comprising:
 providing a compound consisting of:
 peptide having an amino acid sequence of SEQ ID NO:60 or mutation or fragment thereof, wherein the peptide is pro-apoptotic; and 
 a cell-penetrating-peptide, wherein said compound induces apoptosis of mammalian cells expressing Bcl-2 or Bcl-2 related proteins; and 
   contacting said mammalian cell with an effective amount of said compound, wherein said contacting results in apoptosis of said mammalian cell.   
     
     
         13 . The method of  claim 12 , wherein said cell-penetrating peptide consists of transportan10 or penetratin (SEQ ID NO: 65). 
     
     
         14 . The method of  claim 12 , wherein said pro-apoptotic fragment consists of the amino acid sequence Phe Gly Asp Trp Ile Asp Ser Ile Leu (SEQ ID NO:16). 
     
     
         15 . The method of  claim 12 , wherein said pro-apoptotic fragment consists of the amino acid sequence Phe Ser Arg Ser Leu His Ser Leu Leu (SEQ ID NO:9). 
     
     
         16 . The method of  claim 12 , wherein said pro-apoptotic fragment consists of the amino acid sequence Phe Ala Cys Leu Ser Ala Leu Val Leu (amino acid residues 37-45 of SEQ ID NO:60). 
     
     
         17 . The method of  claim 12 , wherein said pro-apoptotic fragment consists of the amino acid sequence Phe Tyr Leu Lys Leu Glu Asp Leu Val (SEQ ID NO:18). 
     
     
         18 . The method of  claim 12 , wherein said mutation is selected from the group consisting of an amino acid deletion, an amino acid insertion, a conservative amino acid substitution, an amino acid substitution with alanine, and an amino acid substitution with a non-naturally occurring amino acid. 
     
     
         19 . The method of  claim 12 , wherein said compound comprises one or more D-amino acids. 
     
     
         20 . A method of inducing apoptosis in a breast cancer cell, comprising:
 providing a compound comprising:
 Nur 77 or a fragment thereof; and 
 a cell-penetrating peptide; and 
   contacting said breast cancer cell with an effective amount of said compound, wherein said contacting results in apoptosis of said breast cancer cell.   
     
     
         21 . The method of  claim 20 , wherein contacting said breast cancer cell is performed in vitro. 
     
     
         22 . The method of  claim 20 , wherein contacting said breast cancer cell is performed in vivo. 
     
     
         23 . The method of  claim 20 , wherein said compound is a NuBCP. 
     
     
         24 . The method of  claim 21 , wherein said NuBCP is L-NuBCP-9-r8 or D-NuBCP-9-r8. 
     
     
         25 . The method of  claim 20 , wherein said cell-penetrating peptide is Lyp-1 or F3.