Identification of compounds that regulate apoptosis

Abstract

An assay for identifying compounds that affect conversion of Bcl-2 family protein from an antiapoptotic to a proapoptotic form or inhibit activity of Bcl-2 family protein is described.

What is claimed is: 
     
         1 . A method of screening for compounds capable of converting a Bcl-2 family protein from an antiapoptotic form to a proapoptotic form, comprising:
 providing a Bcl-2 family protein;   providing a fluorescently labeled compound known to bind to and convert said Bcl-2 family protein to a proapoptotic form;   contacting said Bcl-2 family protein and said fluorescently labeled compound in the presence or absence of a test compound; and   determining fluorescence of said Bcl-2 family protein, wherein a decrease in fluorescence indicates that said test compound is capable of converting said Bcl-2 family protein from an antiapoptotic form to a proapoptotic form.   
     
     
         2 . The method of  claim 1 , wherein said test compound is a natural product or natural product derivative. 
     
     
         3 . The method of  claim 1 , wherein said test compound is a peptidomimetic. 
     
     
         4 . The method of  claim 1 , wherein said test compound is a small molecule. 
     
     
         5 . The method of  claim 1 , wherein said test compound is a Nur77 peptide. 
     
     
         6 . The method of  claim 5 , wherein said Nur77 peptide is NuBCP-9. 
     
     
         7 . The method of  claim 5 , wherein said Nur77 peptide is D-NuBCP-9. 
     
     
         8 . The method of  claim 1 , wherein said fluorescent label is Fluorescein. 
     
     
         9 . The method of  claim 1 , wherein said fluorescent label is Green Fluorescent Protein (GFP). 
     
     
         10 . The method of  claim 1 , wherein said fluorescently labeled compound is a Nur77 peptide. 
     
     
         11 . The method of  claim 10 , wherein said Nur77 peptide is NuBCP-9-r8 peptide. 
     
     
         12 . The method of  claim 1 , wherein said Bcl-2 family protein is Bcl-2. 
     
     
         13 . The method of  claim 1 , wherein said Bcl-2 family protein is Bcl-B. 
     
     
         14 . The method of  claim 1 , wherein said Bcl-2 family protein is Bfl-1. 
     
     
         15 . The method of  claim 1 , further comprising at least one secondary screen to confirm that said test compound converts said Bcl-2 family protein from an antiapoptotic to a proapoptotic form. 
     
     
         16 . The method of  claim 15 , wherein said secondary screen is an apoptosis assay. 
     
     
         17 . The method of  claim 16 , wherein said apoptosis assay is selected from the group consisting of staining annexin-v, detecting caspase activity, and detecting DNA fragmentation. 
     
     
         18 . The method of  claim 1 , wherein said fluorescence is measured by fluorescence polarization. 
     
     
         19 . A method of screening for compounds capable of inhibiting a Bcl-2 family protein, comprising:
 providing a Bcl-2 family protein;   providing a fluorescently labeled compound known to bind to said Bcl-2 family protein;   contacting said Bcl-2 family protein and said fluorescently labeled compound in the presence or absence of a test compound; and   determining fluorescence of said Bcl-2 family protein, wherein a decrease in fluorescence indicates that said test compound is capable of inhibiting said Bcl-2 family protein.   
     
     
         20 . The method of  claim 19 , wherein said test compound is a Nur77 peptide. 
     
     
         21 . The method of  claim 20 , wherein said Nur77 peptide is NuBCP-9. 
     
     
         22 . The method of  claim 20 , wherein said Nur77 peptide is D-NuBCP-9. 
     
     
         23 . The method of  claim 19 , wherein said fluorescent label is Fluorescein or Green Fluorescent Protein (GFP). 
     
     
         24 . The method of  claim 19 , wherein said fluorescently labeled compound is a Nur77 peptide. 
     
     
         25 . The method of  claim 24 , wherein said Nur77 peptide is NuBCP-9-r8 peptide. 
     
     
         26 . The method of  claim 19 , wherein said Bcl-2 family protein is Bcl-2, Bcl-B, or Bfl-1.