Compositions and methods for screening pro-apoptotic compounds

Abstract

The present invention is directed to compositions of matter useful for the enhancement of apoptosis in mammals and to methods of using those compositions of matter for the same.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identitiy to the nucleotide sequence shown in  FIG. 1  (SEQ ID NO: 1).  
     
     
         2 . An isolated nucleic acid molecule comprising the nucleotide sequence shown in  FIG. 1  (SEQ ID NO:1).  
     
     
         3 . The nucleic acid molecule of  claim 2  which is a variant nucleic acid molecule.  
     
     
         4 . An isolated nucleic acid molecule that hybridizes under stringent conditions to the compliment of the nucleotide sequence shown in  FIG. 1  (SEQ ID NO:1).  
     
     
         5 . An expression vector comprising the nucleic acid of claim  1 - 4 .  
     
     
         6 . The expression vector of  claim 5 , wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.  
     
     
         7 . A host cell comprising the expression vector of  claim 6 .  
     
     
         8 . The host cell of  claim 7 , which is a mammalian cell, an  E.coli  cell or a yeast cell.  
     
     
         9 . A process for producing a polypeptide comprising culturing the host cell of  claim 8 , under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.  
     
     
         10 . An isolated ML-IAP chimera polypeptide comprising at least 90% amino acid sequence identity to a polypeptide encoded by the nuclotide sequence shown in  FIG. 1  (SEQ ID NO:1).  
     
     
         11 . An isolated polypeptide having at least 90% amino acid sequence identity to: 
 (a) the polypeptide shown in  FIG. 2  (SEQ ID NO:2);    (b) the polypeptide shown in  FIG. 2  (SEQ ID NO:2); with a C-terminal truncation;    (c) the polypeptide shown in  FIG. 2  (SEQ ID NO:2); with a N-terminal truncation;    (d) the polypeptide shown in  FIG. 2  (SEQ ID NO:2), with an alternative BIR domain at residues 160-179;    (e) the polypeptide shown in  FIG. 2  (SEQ ID NO:2) with an amino acid substituion for Serine 150; and    (f) the polypeptide shown in  FIG. 2  (SEQ ID NO:2); with an amino acid substituion for Histadine 167.    
     
     
         12 . An isolated polypeptide comprising the ML-IAP chimera amino acid sequence shown in  FIG. 2  (SEQ ID NO:2).  
     
     
         13 . A chimeric polypeptide comprising the polypeptide of  claim 10  or  11  fused to a heterologous polypeptide.  
     
     
         14 . A method of screening for a compound that modulates the caspase inhibition of an IAP polypeptide comprising: 
 (a) contacting a crystal structure of the molecule found in  FIG. 2  (SEQ ID NO:2) in spacegroup P41212, with unit cell dimentions of about 87× 87 × 74  Å, with a known IAP inhibitor;    (b) soaking a potential inhibitor with the complex found in (a); and    (c) determining if the potential inhibitor displaces the known IAP inhibitor.    
     
     
         15 . A method of screening for a compound that modulates the caspase inhibition of an IAP polypeptide comprising; 
 b. co-crystallizing the IAP polypeptide with a compound;    c. resolving the structure of the bound molecules by x-ray crystallography;    d. using the data generated from step (b) to make a computer model of the bound molecules;    e. applying an iterative process using the computer generated model to identify potential agonists or antagonists of the IAP polypeptide;    f. synthesizing the agonist or antagonist;    g. contacting the agonist or antagonist with the IAP polypeptide to determine its ability to interact with said IAP polypeptide.    
     
     
         16 . A method of screening for a compound that modulates the caspase inhibition of an ML-IAP chimera comprising; 
 h. contacting a caspase polypeptide and an IAP polypeptide in the presence of said compound;    i. detecting the amount of caspase activity when compared to the amount of caspase activity without the presence of the modulating compound.    
     
     
         17 . The method of  claim 16 , wherein said caspase is selected from the group consisting of caspase-3, and caspase-9.  
     
     
         18 . The method of  claim 16 , wherein caspase activity is detected using an antibody.  
     
     
         19 . The method of  claim 16 , wherein caspase activity is detected by cleavage of a protein substrate.  
     
     
         20 . The method of  claim 16 , wherein the IAP polypeptide is shown in  FIG. 2  (SEQ ID NO:2).  
     
     
         21 . A method of screening antagonists of IAP polypeptides comprising: 
 (a) transfecting cells with the IAP chimera found in  FIG. 1  (SEQ ID NO:1);    (b) contacting the transfected cells with a potential IAP antagonist;    (c) determining whether said antagonist increases apoptosis in cells expressing an IAP relative to antagonist untreated cells.    
     
     
         22 . A method of increasing the survival of a cell, comprising contacting the cell with a compound that alters the caspase inhibitory activity of an ML-IAP chimera polypeptide in the cells.  
     
     
         23 . A method of identifying a compound that alters the specific binding of a caspase and an ML-IAP chimera comprising: 
 (a) contacting the caspase and the ML-IAP chimera under conditions that allow the caspase and the ML-IAP chimera to specifically bind, with a compound potentially being able to alter the association of the caspase and the ML-IAP chimera; and    (b) detecting the altered association of the caspase and the ML-IAP chimera, thus identifying the compound that alters the association of the caspase and the ML-IAP chimera.    
     
     
         24 . The method of  claim 23 , wherein the contacting is performed in vitro.  
     
     
         25 . The method of  claim 23 , wherein the contacting occurs in a cell.  
     
     
         26 . The method of  claim 23 , wherein the caspase is selected from the group consisting of caspase 3 and caspase 9.  
     
     
         27 . The method of  claim 23  comprising contacting a ML-IAP chimera and a compound wherein the binding of the molecules can be measured by fluorescence polarization.