Novel interleukin-1 receptor intracellular ligand proteins and inhibitors of ligand binding

Abstract

Novel IL-1-R intracellular ligand proteins are disclosed. Polynucleotides encoding the IL-1-R intracellular ligand protein are also disclosed, along with vectors, host cells, and methods of making the IL-1-R intracellular ligand protein. Pharmaceutical compositions containing the IL-1-R intracellular ligand protein, methods of treating inflammatory conditions, and methods of inhibiting IL-1-R intracellular domain binding are also disclosed. Methods of identifying inhibitors of IL-1-R intracellular domain binding and inhibitors identified by such methods are also disclosed.

1 . A composition comprising an isolated polynucleotide encoding a protein having IL-1-R intracellular ligand protein activity.  
     
     
         2 . The composition of  claim 1  wherein said polynucleotide is selected from the group consisting of: 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 529;    (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1, which encodes a protein having IL-1-R intracellular ligand protein activity;    (c) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:2;    (d) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 and having IL-1-R intracellular ligand protein activity; and    (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the poly nucleotides specified in (a)-(d), which encodes a protein having IL-1-R intracellular ligand protein activity.    
     
     
         3 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of: 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 961;    (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3, which encodes a protein having IL-1-R intracellular ligand protein activity;    (c) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:4;    (d) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 and having IL-1-R intracellular ligand protein activity; and    (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having IL-1-R intracellular ligand protein activity.    
     
     
         4 . The composition of  claim 1  wherein said polynucleotide is selected from the group consisting of: 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 2 to nucleotide 754;    (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5, which encodes a protein having IL-1-R intracellular ligand protein activity;    (c) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:6;    (d) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 and having IL-1-R intracellular ligand protein activity; and    (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having IL-1-R intracellular ligand protein activity.    
     
     
         5 . A composition comprising a protein having IL-1-R intracellular ligand protein activity.  
     
     
         6 . The composition of  claim 5  wherein said protein comprises an amino acid sequence selected from the group consisting of: 
 (a) the amino acid sequence of SEQ ID NO:2and    (b) fragments of the amino acid sequence of SEQ ID NO:2; said protein being substantially free from other mammalian proteins.    
     
     
         7 . The composition of  claim 5  wherein said protein comprises an amino acid sequence selected from the group consisting of: 
 (a) the amino acid sequence of SEQ ID NO:4; and    (b) fragments of the amino acid sequence of SEQ ID NO:4; said protein being substantially free from other mammalian proteins.    
     
     
         8 . The composition of  claim 5  wherein said protein comprises an amino acid sequence selected from the group consisting of: 
 (a) the amino acid sequence of SEQ ID NO:6; and    (b) fragments of the amino acid sequence of SEQ ID NO:6; said protein being substantially free from other mammalian proteins.    
     
     
         9 . A composition of  claim 1  wherein said polynucleotide is operably linked to an expression control sequence.  
     
     
         10 . A host cell transformed with a composition of  claim 9 .  
     
     
         11 . The host cell of  claim 10 , wherein said cell is a mammalian cell.  
     
     
         12 . A process for producing an IL-1-R intracellular ligand protein, which comprises: 
 (a) growing a culture of the host cell of  claim 10  in a suitable culture medium; and    (b) purifying the IL-1-R intracellular ligand protein from the culture.    
     
     
         13 . A method of identifying an inhibitor of IL-1-R intracellular domain binding which comprises: 
 (a) combining an IL-1-R intracellular domain protein with a composition of  claim 5 , said combination forming a first binding mixture;    (b) measuring the amount of binding between the IL-1-R intracellular domain protein and the IL-1-R intracellular ligand protein in the first binding mixture;    (c) combining a compound with the IL-1-R intracellular domain protein and an IL-1-R intracellular ligand protein to form a second binding mixture;    (d) measuring the amount of binding in the second binding mixture; and    (e) comparing the amount of binding in the first binding mixture    with the amount of binding in the second binding mixture; wherein the compound is capable of inhibiting IL-1-R intracellular domain binding when a decrease in the amount of binding of the second binding mixture occurs.    
     
     
         14 . The method of  claim 13  wherein said IL-1-R intracellular ligand protein comprises an amino acid sequence selected from the group consisting of: 
 (a) the amino acid sequence of SEQ ID NO:2;    (b) fragments of the amino acid sequence of SEQ ID NO:2;    (c) the amino acid sequence of SEQ ID NO:4;    (d) fragments of the amino acid sequence of SEQ ID NO:4    (e) the amino acid sequence of SEQ ID NO6;    (f) fragments of the amino acid sequence of SEQ ID NO:6:    (g) the amino acid sequence of SEQ ID NO:7: and    (h) fragments of the amino acid sequence of SEQ ID NO:7.    
     
     
         15 . A method of identifying an inhibitor of IL-1-R intracellular domain binding which comprises: 
 (a) transforming a cell with a first polynucleotide encoding an IL-1-R intracellular domain protein, a second polynucleotide encoding an IL-1-R intracellular ligand protein, and at least one reporter gene herein the expression of the reporter gene is regulated by the binding of the IL-1-R intracellular ligand protein encoded by the second polynucleotide to the IL-1-R intracellular domain protein encoded by the first polynucleotide:    (b) growing the cell in the presence of and in the absence of a compound; and    (c) comparing the degree of expression of the reporter gene in the presence of and in the absence of the compound;    wherein the compound is capable of inhibiting IL-1-R intracellular domain binding when a decrease in the degree of expression of the reporter gene occurs.    
     
     
         16 . The method of  claim 15  wherein the second polynucleotide is selected from the group consisting of: 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 529;    (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1, which encodes a protein having IL-1-R intracellular ligand protein activity;    (c) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:2;    (d) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 and having IL-1-R intracellular ligand protein activity;    (e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 961.    (f) a polynucleotide comprising, a fragment of the nucleotide sequence of SEQ ID NO:3, which encodes a protein having IL-1-R intracellular ligand protein activity;    (g) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:4;    (h) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 and having IL-1-R intracellular ligand protein activity;    (i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 2 to nucleotide 754;    (j) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5, which encodes a protein having IL-1-R intracellular ligand protein activity;    (k) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:6;    (l) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 and having IL-1-R intracellular ligand protein activity;    (m) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising the amino acid sequence of SEQ ID NO:7;    (n) a polynucleotide encoding an IL-1-R intracellular ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:7 and having IL-1-R intracellular ligand protein activity; and    (o) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(n), which encodes a protein having IL-1-R intracellular ligand protein activity.    
     
     
         17 . A composition comprising an antibody which specifically reacts with the IL-1-R intracellular ligand protein of  claim 5 .  
     
     
         18 . The composition of  claim 5 , further comprising a pharmaceutically acceptable carrier.  
     
     
         19 . A method of preventing or ameliorating an inflammatory condition which comprises administering a therapeutically effective amount of a composition of  claim 18 .  
     
     
         20 . A method of inhibiting IL-1-R intracellular domain binding comprising administering a therapeutically effective amount of a composition of  claim 18 .  
     
     
         21 . IL-1-R intracellular ligand protein produced according to the method of  claim 12 .  
     
     
         22 . A composition comprising an inhibitor identified according to the method of  claim 15 .  
     
     
         23 . The composition of  claim 22  further comprising a pharmaceutically acceptable carrier.  
     
     
         24 . A method of preventing or ameliorating an inflammatory condition comprising administering to a mammalian subject a therapeutically effective amount of the composition of  claim 23 .  
     
     
         25 . A method of inhibiting IL-1-R intracellular domain binding comprising administering to a mammalian subject a therapeutically effective amount of the composition of  claim 23.