Inducible Nitric Oxide Synthase Binds, S-Nitrosylates, and Activates Cyclooxygenase-2

Abstract

Cyclooxygenase (COX2) and inducible nitric oxide synthase (iNOS) are two major inflammatory mediators. Inducible NOS specifically binds to COX2 and S-nitrosylates it, enhancing COX2 catalytic activity. Selectively disrupting iNOS-COX2 binding prevents NO-mediated activation of COX2. The synergistic molecular interaction between two inflammatory systems permits assays for developing anti-inflammatory drugs.

1 . A method of screening for substances useful for relieving inflammation, comprising:
 contacting a test substance with a first protein and a second protein under conditions in which the first protein and the second protein bind to each other, wherein the first protein is selected from the group consisting of mammalian iNOS, a fragment of mammalian iNOS from the N-terminal 10% of iNOS sufficient to bind COX2, and a fusion protein comprising said fragment of mammalian iNOS, wherein the second protein is selected from the group consisting of mammalian COX2, a fragment of mammalian COX2 from the C-terminal 20% of COX2 sufficient to bind iNOS, and a fusion protein comprising said fragment of COX2;   determining amount of free or bound of said first or said second protein;   identifying a test substance which increases the amount of free first or second protein or decreases the amount of bound first or second protein as a candidate drug for relieving inflammation.   
     
     
         2 . The method of  claim 1  wherein the step of contacting is done in vitro. 
     
     
         3 . The method of  claim 1  wherein the step of contacting is done in yeast cells containing recombinant forms of the first and second proteins. 
     
     
         4 . The method of  claim 3  wherein the first and second recombinant proteins are each fused to a first and second yeast protein, wherein the first and second yeast proteins reconstitute a functional transcriptional activator when brought into physical proximity by binding of the first recombinant protein to the second recombinant protein. 
     
     
         5 . The method of  claim 1  further comprising the step of:
 testing an identified candidate drug in an animal to determine if the candidate drug relieves inflammation in the animal.   
     
     
         6 . The method of  claim 1  wherein the test substance is contacted with iNOS and COX2. 
     
     
         7 . The method of  claim 1  wherein the test substance is contacted with said fragment of mammalian iNOS and said fragment of mammalian COX2. 
     
     
         8 . The method of  claim 1  wherein the test substance is contacted with said fusion protein comprising mammalian iNOS and said fusion protein comprising mammalian COX2. 
     
     
         9 . The method of  claim 1  wherein the test substance is contacted with one of said mammalian proteins and one of said fragments. 
     
     
         10 . The method of  claim 1  wherein the test substance is contacted with one of said fusion proteins and one of said fragments. 
     
     
         11 . The method of  claim 1  wherein the test substance is contacted with one of said mammalian proteins and one of said fusion proteins. 
     
     
         12 . The method of  claim 1  wherein antibodies are used to determine said amount. 
     
     
         13 . The method of  claim 1  wherein bound protein is determined by co-immunoprecipitation. 
     
     
         14 . The method of  claim 1  wherein said step of contacting a test substance with a first protein and a second protein is accomplished by contacting the test substance with a cell which comprises the first protein and the second protein. 
     
     
         15 . The method of  claim 1  wherein the mammalian iNOS fragment comprises amino acids 1-114 of human iNOS. 
     
     
         16 . The method of  claim 1  wherein the mammalian COX2 fragment comprises amino acids 488-604 of human COX2. 
     
     
         17 . The method of  claim 1  wherein the mammalian COX2 fragment comprises amino acids 484-604 of murine COX2.