Method for screening compounds inhibiting signal transduction through inflammatory cytokines

Abstract

Revealed are that the actions of inflammatory cytokine and the production of inflammatory cytokines such as IL-1 and TNF induced by an inflammatory stimulus as well as the production of other inflammatory cytokines such as IL-6 induced by the former class of inflammatory cytokines are all suppressed by inhibiting the signal transduction through TAK1.

1 - 40 . (canceled)  
     
     
         41 . A method of screening for compounds that inhibit production of an inflammatory cytokine, comprising: 
 (a) providing a sample comprising a TAK1 and a TAB1;    (b) contacting a test compound with the TAK1 and the TAB 1;    (c) detecting binding between the TAK1 and the TAB 1; and    (d) selecting a compound that inhibits the binding;    wherein the TAK1 of (a) is selected from the group consisting of 
 (i) a protein comprising amino acids 76 to 303 of SEQ ID NO:2; and  
 (ii) a protein that binds to amino acids 437 to 504 of SEQ ID NO:4 and comprises an amino acid sequence encoded by a DNA sequence that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO: 1 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide;  
   and wherein the TAB 1 of (a) is selected from the group consisting of 
 (iii) a protein comprising amino acids 437 to 504 of SEQ ID NO:4; and  
 (iv) a protein that binds to amino acids 76 to 303 of SEQ ID NO:2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO:3 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide;  
   wherein the compound inhibits production of an inflammatory cytokine produced in response to binding between TAK1 and TAB 1.    
     
     
         42 . The method of  claim 41 , wherein the TAK1 and/or the TAB1 is fused with a peptide.  
     
     
         43 . The method of  claim 41 , wherein the TAK1 or the TAB1 is linked to a support.  
     
     
         44 . The method of  claim 41 , wherein a label is attached to the TAK1 or the TAB1 and the binding is detected by detecting or measuring the label.  
     
     
         45 . The method of  claim 41 , wherein the binding is detected by detecting or measuring the TAB 1 bound to the TAK1 with a primary antibody against TAB1 or a primary antibody against a peptide fused with the TAB1.  
     
     
         46 . The method of  claim 41 , wherein the binding is detected by detecting or measuring the TAK1 bound to the TAB1 with a primary antibody against TAK1 or a primary antibody against a peptide fused with the TAK1.  
     
     
         47 . The method of  claim 41 , wherein the binding is detected by detecting or measuring the TAB 1 bound to the TAK1 with a primary antibody against the TAB 1 or a primary antibody against a peptide fused with TAB1, and a secondary antibody against the primary antibody.  
     
     
         48 . The method of  claim 41 , wherein the binding is detected by detecting or measuring the TAK1 bound to the TAB1 with a primary antibody against TAK1 or a primary antibody against a peptide fused with the TAK1, and a secondary antibody against the primary antibody.  
     
     
         49 . The method of  claim 47 , wherein the primary antibody or the secondary antibody is labeled with a radioisotope, enzyme, or fluorescent substance.  
     
     
         50 . The method of  claim 41 , wherein the TAK1 of (a) comprises amino acids 76 to 303 of SEQ ID NO:2.  
     
     
         51 . The method of  claim 41 , wherein the TAK1 of (a) is a protein that binds to amino acids 437 to 504 of SEQ ID NO:4 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO: 1 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide.  
     
     
         52 . The method of  claim 41 , wherein the TAK1 of (a) is a protein that binds to amino acids 437 to 504 of SEQ ID NO:4 and comprises an amino acid sequence that is encoded by a DNA that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO: 1 under washing conditions of 60° C., 0.1×SSC, and 0.1% sodium dodecyl sulfate.  
     
     
         53 . The method of  claim 41 , wherein the TAB1 of (a) comprises amino acids 437 to 504 of SEQ ID NO:4.  
     
     
         54 . The method of  claim 41 , wherein the TAB1 of (a) is a protein that binds to amino acids 76-303 of SEQ ID NO:2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO:3 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide.  
     
     
         55 . The method of  claim 41 , wherein the TAB1 of (a) is a protein that binds to amino acids 76-303 of SEQ ID NO:2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO:3 under washing conditions of 60° C., 0.1×SSC, and 0.1% sodium dodecyl sulfate.  
     
     
         56 . The method of  claim 41 , wherein the inflammatory cytokine is IL-1.  
     
     
         57 . The method of  claim 41 , wherein the inflammatory cytokine is TNF.  
     
     
         58 . The method of  claim 41 , wherein the inflammatory cytokine is IL-6.  
     
     
         59 . The method of  claim 41 , wherein the inflammatory cytokine is IL-10.  
     
     
         60 . A method of screening for compounds that inhibit production of an inflammatory cytokine, comprising: 
 (a) providing a sample comprising a TAK1 and a TAB1;    (b) contacting a test compound with the TAK1 and the TAB1;    (c) detecting binding between the TAK1 and the TAB1; and    (d) selecting a compound that inhibits the binding;    wherein the TAK1 of (a) is selected from the group consisting of 
 (i) a protein comprising amino acids 76 to 303 of SEQ ID NO:2; and  
 (ii) a protein that binds to amino acids 437 to 504 of SEQ ID NO:4 and comprises an amino acid sequence encoded by a DNA sequence that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO:1 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide;  
   and wherein the TAB 1 of (a) is selected from the group consisting of 
 (iii) a protein comprising amino acids 437 to 504 of SEQ ID NO:4; and  
 (iv) a protein that binds to amino acids 76 to 303 of SEQ ID NO:2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO:3 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide;  
   wherein the compound inhibits production of an inflammatory cytokine produced in response to IL-1, TNF or IL-6.    
     
     
         61 . The method of  claim 60 , wherein the TAK1 and/or the TAB1 is fused with a peptide.  
     
     
         62 . The method of  claim 60 , wherein the TAK1 or the TAB 1 is linked to a support.  
     
     
         63 . The method of  claim 60 , wherein a label is attached to the TAK1 or the TAB1 and the binding is detected by detecting or measuring the label.  
     
     
         64 . The method of  claim 60 , wherein the binding is detected by detecting or measuring the TAB1 bound to the TAK1 with a primary antibody against TAB1 or a primary antibody against a peptide fused with the TAB1.  
     
     
         65 . The method of  claim 60 , wherein the binding is detected by detecting or measuring the TAK1 bound to the TAB1 with a primary antibody against TAK1 or a primary antibody against a peptide fused with the TAK1.  
     
     
         66 . The method of  claim 60 , wherein the binding is detected by detecting or measuring the TAB1 bound to the TAK1 with a primary antibody against the TAB1 or a primary antibody against a peptide fused with TAB1, and a secondary antibody against the primary antibody.  
     
     
         67 . The method of  claim 60 , wherein the binding is detected by detecting or measuring the TAK1 bound to the TAB1 with a primary antibody against TAK1 or a primary antibody against a peptide fused with the TAK1, and a secondary antibody against the primary antibody.  
     
     
         68 . The method of  claim 67 , wherein the primary antibody or the secondary antibody is labeled with a radioisotope, enzyme, or fluorescent substance.  
     
     
         69 . The method of  claim 60 , wherein the TAK1 of (a) comprises amino acids 76 to 303 of SEQ ID NO:2.  
     
     
         70 . The method of  claim 60 , wherein the TAK1 of (a) is a protein that binds to amino acids 437 to 504 of SEQ ID NO:4 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO:1 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide.  
     
     
         71 . The method of  claim 60 , wherein the TAK1 of (a) is a protein that binds to amino acids 437 to 504 of SEQ ID NO:4 and comprises an amino acid sequence that is encoded by a DNA that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO:1 under washing conditions of 60° C., 0.1×SSC, and 0.1% sodium dodecyl sulfate.  
     
     
         72 . The method of  claim 60 , wherein the TAB1 of (a) comprises amino acids 437 to 504 of SEQ ID NO:4.  
     
     
         73 . The method of  claim 60 , wherein the TAB 1 of (a) is a protein that binds to amino acids 76-303 of SEQ ID NO:2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO:3 under washing conditions of 42° C., 5×SSC, 0.1% sodium dodecyl sulfate, and 50% formamide.  
     
     
         74 . The method of  claim 60 , wherein the TAB1 of (a) is a protein that binds to amino acids 76-303 of SEQ ID NO:2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO:3 under washing conditions of 60° C., 0.1×SSC, and 0.1% sodium dodecyl sulfate.  
     
     
         75 . The method of  claim 60 , wherein the inflammatory cytokine is IL-1.  
     
     
         76 . The method of  claim 60 , wherein the inflammatory cytokine is TNF.  
     
     
         77 . The method of  claim 60 , wherein the inflammatory cytokine is IL-6.  
     
     
         78 . The method of  claim 60 , wherein the inflammatory cytokine is IL-10.  
     
     
         79 . The method of  claim 60 , wherein the compound inhibits production of an inflammatory cytokine produced in response to IL-1.  
     
     
         80 . The method of  claim 60 , wherein the compound inhibits production of an inflammatory cytokine produced in response to TNF.  
     
     
         81 . The method of  claim 60 , wherein the compound inhibits production of an inflammatory cytokine produced in response to IL-6.