TRAF inhibitors

Abstract

The invention concerns novel inhibitors of tumor necrosis factor receptor associated factor-(TRAF) mediated signal transduction. The invention encompasses the novel inhibitor proteins (I-TRAFs), nucleic acid encoding them, methods for their recombinant production, and their use in screening assays and as pharmaceuticals.

1 . An isolated I-TRAF polypeptide comprising the amino acid sequence of a polypeptide selected from the group consisting of: 
 (a) a native sequence I-TRAF polypeptide,    (b) a polypeptide having at least about 70% amino acid sequence identity with the TRAF-binding domain of a native sequence I-TRAF polypeptide and capable of inhibiting a TRAF-mediated signaling event, and    (c) a fragment of a polypeptide of (a) or (b) capable of inhibiting a TRAF-mediated signaling event.    
     
     
         2 . The polypeptide of  claim 1  which has an at least about 60% overall amino acid sequence identity with a native sequence I-TRAF polypeptide.  
     
     
         3 . The polypeptide of  claim 1  which has an at least about 70% overall amino acid sequence identity with a native sequence I-TRAF polypeptide.  
     
     
         4 . The polypeptide of  claim 1  which has an at least about 80% overall amino acid sequence identity with a native sequence I-TRAF polypeptide.  
     
     
         5 . The polypeptide of  claim 1  which has an at least about 90% overall amino acid sequence identity with a native sequence I-TRAF polypeptide.  
     
     
         6 . The polypeptide of  claim 1 , which is a native mammalian I-TRAF or a fragment thereof.  
     
     
         7 . The polypeptide of  claim 6  which is a native murine I-TRAF or a fragment thereof.  
     
     
         8 . The polypeptide of  claim 7  comprising amino acids 35-236 of SEQ. ID. NO 1.  
     
     
         9 . The polypeptide of  claim 6  which is a native human I-TRAF or a fragment thereof.  
     
     
         10 . The polypeptide of  claim 9  comprising amino acids 1 through 202 of SEQ. ID. NO: 5.  
     
     
         11 . The polypeptide of  claim 9  represented by SEQ. ID. NO: 5.  
     
     
         12 . The polypeptide of  claim 9  encoded by a nucleic acid capable of hybridizing under stringent conditions to the complement of SEQ. ID. NO: 6.  
     
     
         13 . An isolated nucleic acid molecule encoding a polypeptide of  claim 1 .  
     
     
         14 . An isolated nucleic acid molecule encoding a polypeptide of  claim 9 .  
     
     
         15 . A vector comprising and capable of expressing, in a suitable host cell, the nucleic acid molecule of  claim 13 .  
     
     
         16 . A vector comprising and capable of expressing, in a suitable host cell, the nucleic acid molecule of  claim 14 .  
     
     
         17 . A host cell transformed with the nucleic acid molecule of  claim 13 .  
     
     
         18 . A host cell transformed with the nucleic acid molecule of  claim 14 .  
     
     
         19 . An antibody capable of specific binding to an I-TRAF polypeptide of  claim 1 .  
     
     
         20 . A hybridoma cell line producing an antibody of  claim 19 .  
     
     
         21 . A method of using a nucleic acid molecule of  claim 13  comprising expressing such nucleic acid molecule in a cultured host cell transformed therewith, and recovering the polypeptide encoded by said nucleic acid molecule from the host cell.  
     
     
         22 . A method of producing an I-TRAF polypeptide of  claim 1  comprising inserting into the DNA of a cell containing nucleic acid encoding said polypeptide a transcription modulatory element in sufficient proximity and orientation to the nucleic acid molecule to influence the transcription thereof.  
     
     
         23 . An assay for identifying a molecule capable of modulating the association of an I-TRAF and a TRAF comprising (a) incubating a mixture comprising said I-TRAF and TRAF with a candidate molecule and (b) detecting of the ability of the candidate molecule to modulate I-TRAF/TRAF association.  
     
     
         24 . The assay of  claim 23  wherein said candidate molecule prevents or inhibits the dissociation of an I-TRAF/TRAF complex.  
     
     
         25 . The assay of  claim 23  wherein said candidate molecule prevents the association of I-TRAF and TRAF.  
     
     
         26 . The assay of  claim 23  wherein said candidate molecule is a small molecule.  
     
     
         27 . An assay for identifying a molecule the signal transduction of which is mediated by the association of a TRAF with a cellular protein, comprising (a) incubating a mixture comprising said TRAF and an I-TRAF with a candidate molecule, and (b) detecting the ability of said candidate molecule to release TRAF from a complex formed with I-TRAF.  
     
     
         28 . The assay of  claim 27  wherein said mixture further comprises said cellular protein, and the ability of said candidate molecule to initiate TRAF-mediated signaling through said cellular protein is detected.  
     
     
         29 . The assay of  claim 23  or  claim 28  wherein said TRAF comprises the TRAF domain of a native TRAF1, TRAF2 or TRAF3 polypeptide.  
     
     
         30 . The assay of  claim 23  or  claim 28  wherein said TRAF, I-TRAF and cellular protein are human.  
     
     
         31 . The assay of  claim 30  wherein said TRAF comprises amino acids 264-501 of a native human TRAF2 polypeptide.  
     
     
         32 . The assay of  claim 30  wherein said I-TRAF comprises the N-terminal portion of a native human I-TRAF polypeptide.  
     
     
         33 . The assay of  claim 28  wherein said cellular protein is a member of the TNF receptor superfamily.  
     
     
         34 . The assay of  claim 33  wherein said cellular protein is selected from the group consisting of TNF-R2, CD40 and CD30.  
     
     
         35 . A method of treating a tumor associated with Epstein-Barr virus comprising administering to a patient having developed or at risk of developing such tumor a therapeutically effective amount of an I-TRAF.  
     
     
         36 . The method of  claim 35  wherein said tumor is selected from the group of Burkitt's lymphoma and nasopharyngal carcinoma.  
     
     
         37 . A pharmaceutical formulation comprising a therapeutically effective amount of an I-TRAF in association with a pharmaceutically acceptable carrier.  
     
     
         38 . A method of modulating TRAF-mediated signal transduction in a cell comprising introducing into said cell a nucleic acid according to  claim 13  whereby said nucleic acid is expressed in said cell and the resultant gene product modulates TRAF-mediated signal transduction in said cell.