Methods to identify compounds useful for the treatment of proliferative and differentiative disorders

Abstract

The present invention relates to the discovery, identification and characterization of nucleotide sequences that encode novel substrate-targeting subunits of ubiquitin ligases. The invention encompasses nucleotides encoding novel substrate-targeting subunits of ubiquitin ligases: FBP1, FBP2, FBP3, FBP4, FBP5, FBP6, FBP7, FBP8, FBP9, FBP10, FBP11, FBP12, FBP13, FBP14, FBP15, FBP16, FBP17, FBP18, FBP19, FBP20, FBP21, FBP22, FBP23, FBP24, and FBP25, transgenic mice, knock-out mice, host-cell expression systems and proteins encoded by the nucleotides of the novel substrate-targeting subunits. The present invention relates to screening assays that use novel and known substrate-targeting subunits of ubiquitin ligases to identify potential therapeutic agents such as small molecules, compounds or derivatives and analogues of the novel and known ubiquitin ligases which modulate activity of the novel and known ubiquitin ligases for the treatment of proliferative and differentiative disorders, such as cancer, major opportunistic infections, immune disorders, certain cardiovascular diseases, and inflammatory disorders. The invention further encompasses therapeutic protocols and pharmaceutical compositions designed to target ubiquitin ligases and their substrates for the treatment of proliferative and differentiative disorders.

1 . A method for identifying a compound useful for the treatment of a proliferative or differentiative disorder comprising 
 a) incubating components comprising the compound, an F-box protein (FBP), or a fragment thereof, and an FBP-associated protein, wherein the incubating is carried out under conditions sufficient to allow the components to interact; and    b) measuring the effect of the compound on the activity of the FBP.    
     
     
         2 . The method of  claim 1  wherein the FPB is Fbx9.  
     
     
         3 . The method of  claim 1  wherein the FPB is Fbl10.  
     
     
         4 . The method of  claim 1  wherein the FPB is Fbl11.  
     
     
         5 . The method of  claim 1  wherein the FPB is Fbl12.  
     
     
         6 . The method of any one of claims  1 - 5  wherein the FBP-associated protein is an FPB target substrate.  
     
     
         7 . The method of  claim 6  wherein the FBP target substrate is a component of the ubiquitin pathway.  
     
     
         8 . The method of any one of claims  1 - 5  wherein the FBP-associated protein is an FPB pseudosubstrate.  
     
     
         9 . The method of any one of claims  1 - 5  wherein the FBP-associated protein is a protein which regulates the binding of the FPB to the FBP target substrate.  
     
     
         10 . The method of  claim 1  or  2  wherein the FBP-associated protein is Elongation Factor 2.  
     
     
         11 . The method of  claim 1  or  2  wherein the FBP-associated protein is endostatin-like protein.  
     
     
         12 . The method of  claim 1  or  2  wherein the FBP-associated protein is BAF57.  
     
     
         13 . The method of  claim 1  or  2  wherein the FBP-associated protein is splice isoform SP100-HMG of P23497 nuclear autoantigen SP100.  
     
     
         14 . The method of  claim 1  or  2  wherein the FBP-associated protein is H326 protein.  
     
     
         15 . The method of  claim 1  or  4  wherein the FBP-associated protein is Kap1.  
     
     
         16 . The method of  claim 1  or  4  wherein the FBP-associated protein is Kap1.  
     
     
         17 . The method of any one of claims  1 - 16  wherein the FBP activity measured is the interaction of FBP with other components of the ubiquitin ligase complex, the binding of FPB to target substrates or to other FBP-associated proteins, the ubiquitination of an FBP substrate, or the degradation of an FBP substrate.  
     
     
         18 . The method of any one of claims  1 - 16  wherein the FBP activity measured is the binding of FBP to DNA.  
     
     
         19 . A method for treating a proliferative or differentiative disorder in a mammal comprising administering to a mammal in need thereof a compound identified by the method of any one of claims  1 - 18  so that the symptoms of the disorder are ameliorated.  
     
     
         20 . A method for one of claims  1 - 19  wherein the compound is selected from the group consisting of a small molecule, a peptide, an antibody, an antisense molecule, or a ribozyme.