Screening assays for agents that alter inhibitor of apoptosis (iap) protein regulation of caspase activity

Abstract

The present invention relates to an action between an inhibitor of apoptosis (IAP) protein and members of the caspase family of cell death proteases, for example, an interaction of the X chromosome linked IAP (XIAP) and caspase-3, caspase-7 or caspase-9, wherein the IAP regulates the activity of the caspases. The invention provides screening assays for identifying agents that alter the specific association of an IAP such as XIAP, c-IAP-1 or c-IAP-2 and a caspase such as caspase-3 or caspase-7 or a pro-caspase such as pro-caspase-9, or that modulate the activity of a caspase in the presence of an IAP and that regulate the activation of a pro-caspase by an IAP. The invention further provides methods of reducing the severity of a pathologic condition in an individual by administering to the individual an agent that alters the caspase inhibitory activity of an IAP.

1 - 9 . (canceled) 
     
     
         10 . A method of identifying an agent that modulates the activation of a pro-caspase by an inhibitor of apoptosis (IAP) protein, comprising the steps of:
 a) contacting in vitro the pro-caspase and the IAP, wherein the IAP inhibits activation of the pro-caspase, under conditions that allow activation of the pro-caspase in the absence of the IAP, and an agent suspected of being able to modulate caspase inhibitory activity of the IAP; and   b) detecting caspase activity, wherein caspase activity identifies an agent that modulates the regulation of activation of the pro-caspase by the IAP.   
     
     
         11 . The method of  claim 10 , wherein said IAP is a eukaryotic IAP. 
     
     
         12 . The method of  claim 11 , wherein said eukaryotic IAP is an X chromosome linked IAP. 
     
     
         13 . The method of  claim 11 , wherein said eukaryotic IAP is selected from the group consisting of c-IAP-1 and c-IAP-2. 
     
     
         14 . The method of  claim 10 , wherein said pro-caspase is selected from the group consisting of pro-caspase-3, pro-caspase-7 and pro-caspase-9. 
     
     
         15 . The method of  claim 10 , wherein said conditions that allow activation of the pro-caspase in the absence of the IAP are incubation of the pro-caspase in a cytosolic extract containing cytochrome c. 
     
     
         16 . The method of  claim 10 , wherein said conditions that allow activation of the pro-caspase in the absence of the IAP are incubation of the pro-caspase in a cytosolic extract containing caspase-8. 
     
     
         17 - 25 . (canceled) 
     
     
         26 . A method of identifying an agent that alters the specific association of a pro-caspase and an inhibitor of apoptosis (IAP) protein, comprising the steps of:
 a) contacting the pro-caspase and the IAP, under conditions that allow the pro-caspase and the IAP to specifically associate, with an agent suspected of being able to alter the association of the pro-caspase and the IAP; and   b) detecting an altered association of the pro-caspase and the IAP, thereby identifying an agent that alters the association of the pro-caspase and the IAP.   
     
     
         27 . The method of  claim 26 , wherein said contacting is performed in vitro. 
     
     
         28 . The method of  claim 26 , wherein said contacting occurs in a cell. 
     
     
         29 . The method of  claim 26 , wherein said IAP is a eukaryotic IAP. 
     
     
         30 . The method of  claim 29 , wherein said eukaryotic IAP is an X chromosome linked IAP. 
     
     
         31 . The method of  claim 29 , wherein said eukaryotic IAP is selected from the group consisting of c-IAP-1 and c-IAP-2. 
     
     
         32 . The method of  claim 26 , wherein said pro-caspase is pro-caspase-9. 
     
     
         33 . A method of reducing the severity of a pathologic condition in an individual by altering the level of apoptosis of a cell population, comprising administering to the individual an agent that alters the caspase inhibitory activity of an IAP. 
     
     
         34 . The method of  claim 33 , wherein said pathologic condition is characterized by a pathologically elevated level of apoptosis and wherein said agent increases the caspase inhibitory activity of an IAP. 
     
     
         35 . The method of  claim 34 , wherein said pathologic condition is a neurodegenerative disease. 
     
     
         36 . The method of  claim 33 , wherein the severity of the pathologic condition is reduced by increasing the level of apoptosis of the cell population and wherein said agent decreases the caspase inhibitory activity of an IAP. 
     
     
         37 . The method of  claim 36 , wherein said pathologic condition is cancer. 
     
     
         38 . The method of  claim 36 , wherein said pathologic condition is selected from the group consisting of psoriasis, an autoimmune disease and restenosis. 
     
     
         39 . (canceled)