Novel ubiquitin lifases as therapeutic tragets

Abstract

The present invention relates to the discovery, identification and characterization of nucleotides 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 present invention. The present invention relates to screening assays that use the novel substrate-targeting subunits to identify potential therapeutic agents such as small molecules, compounds or derivatives and analogues of the novel ubiquitin ligases which modulate activity of the novel 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 disorders.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence which encodes a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60. 
     
     
         2 . An isolated nucleic acid molecule which encodes an F-box protein, or a fragment thereof, having a nucleotide sequence that:
 a) hybridizes under highly stringent conditions to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and   b) does not encompass the nucleotide sequences which encode the following known F-box proteins: Cdc4, Grr1, Met30, Skp2, Cyclin F, Elongin A or mouse Md6.   
     
     
         3 . An isolated nucleic acid sequence derived from a mammalian genome that:
 a) hybridizes under highly stringent conditions to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and   b) encodes a gene product which contains an F-box motif and binds to Skp1.   
     
     
         4 . An isolated nucleic acid molecule which encodes an F-box protein, said nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or 59. 
     
     
         5 . A nucleotide vector containing the nucleotide sequence of  claim 1 ,  2 ,  3 , or  4 . 
     
     
         6 . An expression vector containing the nucleotide sequence of  claim 1 ,  2 ,  3 , or  4  in operative association with a nucleotide regulatory sequence that controls expression of the nucleotide sequence in a host cell. 
     
     
         7 . A genetically engineered host cell that contains the nucleotide sequence of  claim 1 ,  2 ,  3 , or  4  in operative association with a nucleotide regulatory sequence that controls expression of the nucleotide sequence in the host cell. 
     
     
         8 . A transgenic animal having cells which harbor a transgene comprising the nucleic acid of  claim 1 ,  2 ,  3 , or  4 . 
     
     
         9 . An animal inactivated in the loci comprising the nucleotide sequence of  claim 1 ,  2 ,  3 , or  4 . 
     
     
         10 . An isolated F-box protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60. 
     
     
         11 . An antibody that immunospecifically binds the polypeptide of  claim 10 . 
     
     
         12 . A method of diagnosing proliferative and differentiative related disorders comprising measuring FBP gene expression in a patient sample. 
     
     
         13 . A method for screening compounds useful for the treatment of proliferative and differentiative disorders comprising contacting a compound with a cell expressing an F-box protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60, or a fragment thereof, and its substrate, and detecting a change in the F-box protein activity. 
     
     
         14 . The method of  claim 13  wherein the change in the F-box protein activity is detected by detecting a change in the interaction of the F-box protein with one or more proteins. 
     
     
         15 . The method of  claim 14  in which one of the one or more proteins is the substrate of the F-box protein. 
     
     
         16 . The method of  claim 13  in which at least one of the one or more proteins is a component of the ubiquitin pathway. 
     
     
         17 . The method of  claim 13  in which one of the one or more proteins is Skp1. 
     
     
         18 . The method of  claim 13  in which the F-box protein is Fbp1 and the substrate is β-catenin or IKBα. 
     
     
         19 . The method of  claim 13  wherein the change in the F-box protein activity is detected by detecting a change in the ubiquitination or degradation of the substrate. 
     
     
         20 . A method for screening compounds useful for the treatment of proliferative and differentiative disorders comprising contacting a compound with a cell or a cell extract expressing Skp2 and one or both of p27 and E2F, and detecting a change in the activity of Skp2. 
     
     
         21 . The method of  claim 20  wherein the change in the activity of Skp2 is detected by detecting a change in the interaction of Skp2 with either p27 or E2F-1. 
     
     
         22 . The method of  claim 20  wherein the change in the activity of Skp2 is detected by detecting a change in the ubiquitination or degradation of p27 or E2F-1. 
     
     
         23 . A method for treating a proliferative or differentiative disorder in a mammal comprising administering to the mammal a compound to the mammal that modulates the synthesis, expression or activity of an FBP gene or gene product so that symptoms of the disorder are ameliorated. 
     
     
         24 . The method of  claim 23  in which the disorder is breast cancer. 
     
     
         25 . The method of  claim 23  in which the disorder is ovarian cancer. 
     
     
         26 . The method of  claim 23  in which the disorder is prostate cancer. 
     
     
         27 . The method of  claim 23  in which the disorder is small cell lung carcinoma.