Use of hepatitis B X-interacting protein (HBXIP) in modulation of apoptosis

Abstract

Novel methods of regulating cellular apoptosis by affecting the interaction of hepatitis B X-interacting protein (HBXIP) with Survivin are described. More specifically, these novel methods of enhancing apoptosis of neoplastic cells comprises inhibiting interaction of hepatitis B X-interacting protein (HBXIP) with Survivin.

What is claimed is:  
     
         1 . A method for enhancing apoptosis of neoplastic cells comprising inhibiting interaction of hepatitis B X-interacting protein (HBXIP) with Survivin.  
     
     
         2 . The method of  claim 1 , further comprising using siRNA or antisense to downregulate expression of HBXIP.  
     
     
         3 . The method of  claim 1 , further comprising using HBXIP- or Survivin-specific antibodies to inhibit the interaction of HBXIP with Survivin.  
     
     
         4 . The method of  claim 1 , further comprising using specific inhibitors, molecular decoys, or the like to downregulate HBXIP.  
     
     
         5 . A compound that inhibits HBXIP in the presence of Survivin.  
     
     
         6 . A pharmaceutical composition comprising a compound that inhibits HBXIP in the presence of Survivin.  
     
     
         7 . A method of treating neoplastic disease comprising administration of an inhibitor of HBXIP in the presence of Survivin.  
     
     
         8 . A method for treating human liver disease associated with HBV, comprising administration of an inhibitor of HBXIP in the presence of Survivin.  
     
     
         9 . A compound that inhibits Survivin in the presence of HBXIP.  
     
     
         10 . A pharmaceutical composition comprising a compound that inhibits Survivin in the presence of HBXIP.  
     
     
         11 . A method for identifying an effective agent that alters the association of Survivin and HBXIP comprising: 
 contacting Survivin and HBXIP under conditions that allow Survivin and HBXIP to associate in the presence or absence of a compound; and    detecting an altered association between Survivin and HBXIP, thereby determining whether said compound is an effective agent for altering association of Survivin with HBXIP.    
     
     
         12 . The method of  claim 11 , wherein Survivin and HBXIP associate in the presence of a compound and pro-Caspase-9.  
     
     
         13 . The method of  claim 11 , wherein the altered association between Survivin and HBXIP is detected by measuring the activation of pro-Caspase-9.  
     
     
         14 . A method for identifying an agent that effectively inhibits the activity of Survivin, comprising: 
 contacting Survivin with a cell extract containing HBXIP and pro-Caspase-9 in the presence of a compound;    inducing activation of pro-Caspase-9 using cytochrome C; and    measuring the activation of pro-Caspase-9, thereby determining whether said compound is an agent that effectively inhibits the activity of Survivin.    
     
     
         15 . A method for identifying an agent that effectively inhibits the activity of HBXIP, comprising: 
 contacting HBXIP with a cell extract containing Survivin and pro-Caspase-9 in the presence of a compound;    inducing activation of pro-Caspase-9 using cytochrome C; and    measuring the activation of pro-Caspase-9, thereby determining whether said compound is an agent that effectively inhibits the activity of HBXIP.    
     
     
         16 . A method for identifying an agent that effectively inhibits the activity of HBX, comprising: 
 contacting HBX with a cell extract containing Survivin and pro-Caspase-9 in the presence of a compound;    inducing activation of pro-Caspase-9; and    measuring the activation of pro-Caspase-9, thereby determining whether said compound is an agent that effectively inhibits the activity of HBX.    
     
     
         17 . A method for identifying an agent that effectively inhibits the activity of Survivin, comprising: 
 contacting Survivin with purified pro-Caspase-9 and Apaf1 in the presence of HBXIP and a compound;    inducing activation of pro-Caspase-9; and    measuring the activation of pro-Caspase-9, thereby determining whether said compound is an agent that effectively inhibits the activity of Survivin.    
     
     
         18 . A method for identifying an agent that effectively inhibits the activity of HBXIP, comprising: 
 contacting HBXIP with purified pro-Caspase-9 and Apaf1 in the presence of Survivin and a compound;    inducing activation of pro-Caspase-9; and    measuring the activation of pro-Caspase-9, thereby determining whether said compound is an agent that effectively inhibits the activity of HBXIP.    
     
     
         19 . A method for identifying an effective agent that alters the association of Survivin and HBXIP comprising: 
 contacting Survivin and HBXIP under conditions that allow Survivin and HBXIP to associate in the presence a compound, pro-Caspase-3, pro-Caspase-9, Apaf1 and cytochrome C; and    detecting an altered association between Survivin and HBXIP, thereby determining whether said compound is an effective agent for altering association of Survivin with HBXIP.    
     
     
         20 . The method of  claim 19 , wherein the altered association between Survivin and HBXIP is detected by measuring the activation of pro-Caspase-3.  
     
     
         21 . The method of  claim 20 , wherein the activation of pro-Caspase-3 is measured by monitoring the cleavage of a caspase 3 substrate selected from the group consisting of DEVD-AFC, DEVD-pNA and DEVD-AMC.  
     
     
         22 . A method for identifying an effective agent that alters the association of Survivin and HBXIP comprising: 
 contacting Survivin and HBXIP under conditions that allow Survivin and HBXIP to associate in the presence a cell extract and a compound; and    detecting an altered association between Survivin and HBXIP, thereby determining whether said compound is an effective agent for altering association of Survivin with HBXIP.    
     
     
         23 . The method of  claim 22 , wherein said cell extract comprises pro-Caspase-9 and pro-Caspase-3.  
     
     
         24 . The method of  claim 22 , wherein the altered association between Survivin and HBXIP is detected by measuring the activation of pro-Caspase-3.  
     
     
         25 . The method of  claim 24 , wherein the activation of pro-Caspase-3 is measured by monitoring the cleavage of a caspase 3 substrate selected from the group consisting of DEVD-AFC, DEVD-pNA and DEVD-AMC.  
     
     
         26 . A method of identifying an agent that potentially alters the association of Survivin and HBXIP comprising: 
 identifying a site of interaction between Survivin and HBXIP; and    determining whether a compound binds to said interaction site, thereby determining whether said compound is an agent that potentially alters the association of Survivin with HBXIP.    
     
     
         27 . The method of  claim 26 , further comprising confirming that said compound alters the association of Survivin with HBXIP by detecting an altered association between Survivin and HBXIP in the presence said compound.  
     
     
         28 . The method of  claim 26 , wherein said interaction site is a portion of Survivin that binds HBXIP.  
     
     
         29 . The method of  claim 26 , wherein said interaction site is a portion of HBXIP that binds Survivin.  
     
     
         30 . The method of  claim 26 , wherein said interaction site is identified using transverse relaxation-optimized spectroscopy.  
     
     
         31 . The method of  claim 26 , wherein said interaction site is identified using cross relaxation-enhanced polarization transfer.  
     
     
         32 . The method of  claim 26 , wherein nuclear magnetic resonance spectroscopy (NMR) is used to determine whether said compound binds to said interaction site.  
     
     
         33 . The method of  claim 26 , wherein fluorescence polarization assay (FPA) is used to determine whether said compound binds to said interaction site.  
     
     
         34 . The method of  claim 26 , wherein computational-docking studies are used to determine whether said compound binds to said interaction site.