A Novel Mechanism of Cyclooxygenase-2 Inhibition Involving Interactions with Ser-530 and Tyr-385

Abstract

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in inhibition by and A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in inhibition by and cyclooxygenase (COX) anti-inflammatory anti-inflammatory activity of prostaglandin endoperoxide the of of into arachidonic acid to the prostaglandin G2 the cyclooxygenase active site and binds the active site is to the the to and COX that in mode of and COX-1 is the of the enzyme is in a of COX-2 is to in by a of and of COX by non-steroidal anti-inflammatory drugs anti-inflammatory and of COX-2 inhibitors the and is an of the of and COX-1 and COX-2 have by a of the to a This the and its the cyclooxygenase active cyclooxygenase active site is the the by a of the Arg-120, is in with that of is in with that of Tyr-355, and is with its carboxylic acid to Arg-120, its to the important and its into a that of COX with carboxylic that the inhibitors in a with to Arg-120 and into the cyclooxygenase active site Tyr-385 inhibitors of COX-2 bind in the cyclooxygenase active site Arg-120 and or groups into a side by of a of arachidonic acid with a conformation of in the arachidonic acid was inverted with its carboxylate to Ser-530 and Tyr-385 this the of Arg-120 and that its was to and This orientation was to a or conformation of is evidence inhibition in of arachidonic acid to the the of this conformation of arachidonic its orientation Tyr-385 and Ser-530 that can in the of or that an is a of the of of Ser-530 of Tyr-385 to of Ser-530 by by Tyr-385 is to the of the that Ser-530 and evidence the of Tyr-385 and Ser-530 in the of to groups important the of with Ser-530 is cyclooxygenase the S530A represents an enzyme that is active but to with Tyr-385 in this and its to inhibition by of the of suggest that is inhibition of COX-2 by the and this of that that diclofenac is in an orientation in its carboxylic acid is to Tyr-385 and to the inverted orientation arachidonic acid to the of Tyr-385 and Ser-530 to with COX-2 and a binding mode that to important COX inhibition by of acid and acid and and was inhibitors and was a COX-2 the was into the the and the was the and and the S530A was by of the site in and the site in the and to and and in and described activity of was the described was with a with a and a to in a and of in the described with of and arachidonic with with of in the with acid and by in the described inhibition by the inhibitor and enzyme and to the of acid was to that in a the of the inhibition of inhibitor and of acid to the by of enzyme the a the and in the murine COX-2 was in in a described that the was first was a of to a COX-2 and was a containing in the to in the of the described was described a of and to crystal was and the the an crystal to and the with the crystal to group and with the a and an of structure was by the acid complex the COX-2 in the by and with the and in the of to with of and and and diclofenac have been into the of series of site-directed in murine COX-2 and in and Arg-120 and the the and the active and Tyr-385 and the active hydrogen-bonded to mutants by and of the was by of to mutants to or also the and cyclooxygenase of the of the to the activity with that the in of the of the a in COX a in the conversion of arachidonic acid to COX cyclooxygenase activity this is that of of and COX-2 the cyclooxygenase activity and in the described cyclooxygenase activity of enzyme was arachidonic acid the of of in a tested to inhibition by a series of that COX inhibitors and of was to the of cyclooxygenase inhibitors and inhibitor was with to the of arachidonic and against enzyme and S530A but inhibition of the inhibition was to mutation of Arg-120 acid inhibition was This is with the of and with the crystal structure of a complex in the carboxylic acid with Arg-120 and of and activity against the of inhibition of COX-2 and COX-2 mutants by and the described the of or in the to in a A of was by is a of inhibition of and the S530A enzyme to inhibition by been to of Ser-530 by inhibition by diclofenac was with in that inhibit or but its against S530A was Interestingly, a carboxylic inhibition of the was to diclofenac in that was against S530A but the of the was with diclofenac inhibition of cyclooxygenase activity of and mutants by and S530A in with diclofenac by a to of acid and was described inhibition of cyclooxygenase activity of and the S530A by acid and or in cyclooxygenase was by the of and S530A a of of described Ser-530 to an important binding of the tested in also the of the inhibitors and acid against the S530A mutation a in the of and and a in of acid and mutants in this side important in binding the carboxylate of to inhibition by inhibitors of COX-2 and S530A COX-2 by and the described or with the in the to in a to the of the in of diclofenac by the mutation of Ser-530 to to its to a or inhibitor of COX-2 and is a inhibitor of enzyme and of arachidonic acid its inhibition of S530A was and the of diclofenac S530A was the of of the inhibition a series of of the of by COX-2 a of of the experiments by of enzyme to a containing with to the of of the of that is This is with inhibition or a of the of inhibition with inhibition of COX-2 by is and with the of of arachidonic acid by the inhibition can to the and is of the in the of experiments with the S530A is a inhibitor against this but of the with with mutation of Ser-530 to inhibition by but against enzyme and against of mutation of Ser-530 inhibition by diclofenac suggests that binds to COX-2 in a to with the Ser-530 this of in the of the of diclofenac in the cyclooxygenase active site is in the active is the of a that the of inhibitor is the of the cyclooxygenase active site of COX-2 with in the side of diclofenac to experimental to the of the inhibitor to the of the binding of diclofenac in the COX-2 active carboxylic acid of diclofenac and the side of Tyr-385 and Ser-530 in carboxylate of diclofenac to the side of Tyr-385 and Ser-530 the of the cyclooxygenase active site the inhibitor with the active of and the group that is to the acid of the inhibitor with the of and group is in with group with and Arg-120 the the inhibitor and Arg-120 side is This is with the of the experiments in that the the mutants a inhibition by that Ser-530 and Tyr-385 important the inhibition of COX-2 by aspirin. of Ser-530 to inhibition by diclofenac and important and and inhibition by with the structure of the arachidonic COX-2 diclofenac binds in the COX-2 active site in a inverted to in its carboxylate is by Ser-530 and Tyr-385 of diclofenac to inhibit the and is with this to the in to diclofenac inhibition by the of COX-2 evidence the binding of diclofenac in the of Ser-530 is by of the COX-2 a in diclofenac enzyme been that diclofenac the of COX-1 but the of the enzyme This suggests that diclofenac bind in the active site of the its carboxylate is by Ser-530 and Tyr-385 in the diclofenac into the active site of COX-2 and that in of of of its carboxylate by Tyr-385 and Ser-530. orientation that in the crystal structure of diclofenac to COX-2 to the first conformation by in the group into the of the active is against COX-1 and COX-2 but is active against COX-2 carboxylic have the crystal of and and and acid inhibitors to Interestingly, of the inhibitors in with carboxylic to Arg-120. by the and diclofenac a binding mode with inhibitors to This is in the structure of diclofenac in the active site of COX-2 with of and arachidonic acid a and binds with its carboxylate to Arg-120 and Tyr-355, with the of diclofenac of the by arachidonic acid in the structure and is to Arg-120 and of inhibitor binding to of the of diclofenac and to the and of the of diclofenac and the mode of arachidonic acid acidic groups with the side of Tyr-385 and Ser-530 of is of COX-2 by and inhibition of COX-2 by in and suggest that is important in the binding of and S530A is by and is to inhibition by inhibition of COX by is a carboxylic acid have the first the of Arg-120, Tyr-355, and Ser-530 in the binding of that binds to COX-2 in an conformation with of mutation of of the that with is binding in the COX active is an that is COX-2 and been to bind in the side by that mutation of this in COX-2 to or inhibition and the of this and into the active site with its group in the side by the or in that mutation of Ser-530 to inhibition but inhibition suggests with Ser-530 and Tyr-385 in to and a polar group that with Ser-530 in to a that bind in the side and that to and inhibitor binding in the COX active first site the of Arg-120 and the the of inhibitors and the orientation of is the side by Tyr-355, and and of inhibitors that or in by in this side pocket. diclofenac and arachidonic acid in its orientation with Tyr-385 and Ser-530 the of the active in the binding modes of diclofenac and the orientation of arachidonic acid the of inhibition in is to that COX into the of inhibitors that bind to cyclooxygenase (COX) anti-inflammatory anti-inflammatory activity of prostaglandin endoperoxide the of of into arachidonic acid to the prostaglandin G2 the cyclooxygenase active site and binds the active site is to the the to and COX that in mode of and COX-1 is the of the enzyme is in a of COX-2 is to in by a of and of COX by non-steroidal anti-inflammatory drugs anti-inflammatory and of COX-2 inhibitors the and is an of the of and COX-1 and COX-2 have by a of the to a This the and its the cyclooxygenase active cyclooxygenase active site is the the by a of the Arg-120, is in with that of is in with that of Tyr-355, and is with its carboxylic acid to Arg-120, its to the important and its into a that of COX with carboxylic that the inhibitors in a with to Arg-120 and into the cyclooxygenase active site Tyr-385 inhibitors of COX-2 bind in the cyclooxygenase active site Arg-120 and or groups into a side by of a of arachidonic acid with a conformation of in the arachidonic acid was inverted with its carboxylate to Ser-530 and Tyr-385 this the of Arg-120 and that its was to and This orientation was to a or conformation of is evidence inhibition in of arachidonic acid to the the of this conformation of arachidonic its orientation Tyr-385 and Ser-530 that can in the of or that an is a of the of of Ser-530 of Tyr-385 to of Ser-530 by by Tyr-385 is to the of the that Ser-530 and evidence the of Tyr-385 and Ser-530 in the of to groups important the of with Ser-530 is cyclooxygenase the S530A represents an enzyme that is active but to with Tyr-385 in this and its to inhibition by of the of suggest that is inhibition of COX-2 by the and this of that that diclofenac is in an orientation in its carboxylic acid is to Tyr-385 and to the inverted orientation arachidonic acid to the of Tyr-385 and Ser-530 to with COX-2 and a binding mode that to important COX inhibition by of acid and acid and and was inhibitors and was a COX-2 the was into the the and the was the and and the S530A was by of the site in and the site in the and to and and in and described activity of was the described was with a with a and a to in a and of in the described with of and arachidonic with with of in the with acid and by in the described inhibition by the inhibitor and enzyme and to the of acid was to that in a the of the inhibition of inhibitor and of acid to the by of enzyme the a the and in the murine COX-2 was in in a described that the was first was a of to a COX-2 and was a containing in the to in the of the described was described a of and to crystal was and the the an crystal to and the with the crystal to group and with the a and an of structure was by the acid complex the COX-2 in the by and with the and in the of to with of and and and diclofenac have been into the acid and acid and and was inhibitors and was a COX-2 the was into the the and the was the and and the S530A was by of the site in and the site in the and to and and in and described activity of was the described was with a with a and a to in a and of in the described with of and arachidonic with with of in the with acid and by in the described inhibition by the inhibitor and enzyme and to the of acid was to that in a the of the inhibition of inhibitor and of acid to the by of enzyme the a the and in the murine COX-2 was in in a described that the was first was a of to a COX-2 and was a containing in the to in the of the described was described a of and to crystal was and the the an crystal to and the with the crystal to group and with the a and an of structure was by the acid complex the COX-2 in the by and with the and in the of to with of and and and diclofenac have been into the of series of site-directed in murine COX-2 and in and Arg-120 and the the and the active and Tyr-385 and the active hydrogen-bonded to mutants by and of the was by of to mutants to or also the and cyclooxygenase of the of the to the activity with that the in of the of the a in COX a in the conversion of arachidonic acid to COX cyclooxygenase activity this is that of of and COX-2 the cyclooxygenase activity and in the described cyclooxygenase activity of enzyme was arachidonic acid the of of in a tested to inhibition by a series of that COX inhibitors and of was to the of cyclooxygenase inhibitors and inhibitor was with to the of arachidonic and against enzyme and S530A but inhibition of the inhibition was to mutation of Arg-120 acid inhibition was This is with the of and with the crystal structure of a complex in the carboxylic acid with Arg-120 and of and inhibition of COX-2 and COX-2 mutants by and the described the of or in the to in a A of was by is a of inhibition of and the S530A enzyme to inhibition by been to of Ser-530 by inhibition by diclofenac was with in that inhibit or but its against S530A was Interestingly, a carboxylic inhibition of the was to diclofenac in that was against S530A but the of the was with diclofenac inhibition of cyclooxygenase activity of and mutants by and S530A in with diclofenac by a to of acid and was described inhibition of cyclooxygenase activity of and the S530A by acid and or in cyclooxygenase was by the of and S530A a of of described Ser-530 to an important binding of the tested in also the of the inhibitors and acid against the S530A mutation a in the of and and a in of acid and mutants in this side important in binding the carboxylate of to inhibition by inhibitors of COX-2 and S530A COX-2 by and the described or with the in the to in a to the of the in of diclofenac by the mutation of Ser-530 to to its to a or inhibitor of COX-2 and is a inhibitor of enzyme and of arachidonic acid its inhibition of S530A was and the of diclofenac S530A was the of of the inhibition a series of of the of by COX-2 a of of the experiments by of enzyme to a containing with to the of of the of that is This is with inhibition or a of the of inhibition with inhibition of COX-2 by is and with the of of arachidonic acid by the inhibition can to the and is of the in the of experiments with the S530A is a inhibitor against this but of the with with mutation of Ser-530 to inhibition by but against enzyme and against of mutation of Ser-530 inhibition by diclofenac suggests that binds to COX-2 in a to with the Ser-530 this of in the of the of diclofenac in the cyclooxygenase active site is in the active is the of a that the of inhibitor is the of the cyclooxygenase active site of COX-2 with in the side of diclofenac to experimental to the of the inhibitor to the of the binding of diclofenac in the COX-2 active carboxylic acid of diclofenac and the side of Tyr-385 and Ser-530 in carboxylate of diclofenac to the side of Tyr-385 and Ser-530 the of the cyclooxygenase active site the inhibitor with the active of and the group that is to the acid of the inhibitor with the of and group is in with group with and Arg-120 the the inhibitor and Arg-120 side is This is with the of the experiments in that the the mutants a inhibition by of series of site-directed in murine COX-2 and in and Arg-120 and the the and the active and Tyr-385 and the active hydrogen-bonded to mutants by and of the was by of to mutants to or also the and cyclooxygenase of the of the to the activity with that the in of the of the a in COX a in the conversion of arachidonic acid to COX cyclooxygenase activity this is that tested to inhibition by a series of that COX inhibitors and of was to the of cyclooxygenase inhibitors and inhibitor was with to the of arachidonic and against enzyme and S530A but inhibition of the inhibition was to mutation of Arg-120 acid inhibition was This is with the of and with the crystal structure of a complex in the carboxylic acid with Arg-120 and of and A of was by is a of inhibition of and the S530A enzyme to inhibition by been to of Ser-530 by inhibition by diclofenac was with in that inhibit or but its against S530A was Interestingly, a carboxylic inhibition of the was to diclofenac in that was against S530A but the of the was with diclofenac Ser-530 to an important binding of the tested in also the of the inhibitors and acid against the S530A mutation a in the of and and a in of acid and mutants in this side important in binding the carboxylate of to inhibition by inhibitors to the of the in of diclofenac by the mutation of Ser-530 to to its to a or inhibitor of COX-2 and is a inhibitor of enzyme and of arachidonic acid its inhibition of S530A was and the of diclofenac S530A was the of of the inhibition a series of of the of by COX-2 a of of the experiments by of enzyme to a containing with to the of of the of that is This is with inhibition or a of the of inhibition with inhibition of COX-2 by is and with the of of arachidonic acid by the inhibition can to the and is of the in the of experiments with the S530A is a inhibitor against this but of the with with mutation of Ser-530 to inhibition by but against enzyme and against of mutation of Ser-530 inhibition by diclofenac suggests that binds to COX-2 in a to with the Ser-530 this of in the of the of diclofenac in the cyclooxygenase active site is in the active is the of a that the of inhibitor is the of the cyclooxygenase active site of COX-2 with in the side carboxylate of diclofenac to the side of Tyr-385 and Ser-530 the of the cyclooxygenase active site the inhibitor with the active of and the group that is to the acid of the inhibitor with the of and group is in with group with and Arg-120 the the inhibitor and Arg-120 side is This is with the of the experiments in that the the mutants a inhibition by that Ser-530 and Tyr-385 important the inhibition of COX-2 by aspirin. of Ser-530 to inhibition by diclofenac and important and and inhibition by with the structure of the arachidonic COX-2 diclofenac binds in the COX-2 active site in a inverted to in its carboxylate is by Ser-530 and Tyr-385 of diclofenac to inhibit the and is with this to the in to diclofenac inhibition by the of COX-2 evidence the binding of diclofenac in the of Ser-530 is by of the COX-2 a in diclofenac enzyme been that diclofenac the of COX-1 but the of the enzyme This suggests that diclofenac bind in the active site of the its carboxylate is by Ser-530 and Tyr-385 in the diclofenac into the active site of COX-2 and that in of of of its carboxylate by Tyr-385 and Ser-530. orientation that in the crystal structure of diclofenac to COX-2 to the first conformation by in the group into the of the active is against COX-1 and COX-2 but is active against COX-2 carboxylic have the crystal of and and and acid inhibitors to Interestingly, of the inhibitors in with carboxylic to Arg-120. by the and diclofenac a binding mode with inhibitors to This is in the structure of diclofenac in the active site of COX-2 with of and arachidonic acid a and binds with its carboxylate to Arg-120 and Tyr-355, with the of diclofenac of the by arachidonic acid in the structure and is to Arg-120 and of is of COX-2 by and inhibition of COX-2 by in and suggest that is important in the binding of and S530A is by and is to inhibition by inhibition of COX by is a carboxylic acid have the first the of Arg-120, Tyr-355, and Ser-530 in the binding of that binds to COX-2 in an conformation with of mutation of of the that with is binding in the COX active is an that is COX-2 and been to bind in the side by that mutation of this in COX-2 to or inhibition and the of this and into the active site with its group in the side by the or in that mutation of Ser-530 to inhibition but inhibition suggests with Ser-530 and Tyr-385 in to and a polar group that with Ser-530 in to a that bind in the side and that to and inhibitor binding in the COX active first site the of Arg-120 and the the of inhibitors and the orientation of is the side by Tyr-355, and and of inhibitors that or in by in this side pocket. diclofenac and arachidonic acid in its orientation with Tyr-385 and Ser-530 the of the active in the binding modes of diclofenac and the orientation of arachidonic acid the of inhibition in is to that COX into the of inhibitors that bind to that Ser-530 and Tyr-385 important the inhibition of COX-2 by aspirin. of Ser-530 to inhibition by diclofenac and important and and inhibition by with the structure of the arachidonic COX-2 diclofenac binds in the COX-2 active site in a inverted to in its carboxylate is by Ser-530 and Tyr-385 of diclofenac to inhibit the and is with this to the in to diclofenac inhibition by the of COX-2 evidence the binding of diclofenac in the of Ser-530 is by of the COX-2 a in diclofenac enzyme been that diclofenac the of COX-1 but the of the enzyme This suggests that diclofenac bind in the active site of the its carboxylate is by Ser-530 and Tyr-385 in the diclofenac into the active site of COX-2 and that in of of of its carboxylate by Tyr-385 and Ser-530. orientation that in the crystal structure of diclofenac to COX-2 to the first conformation by in the group into the of the active is against COX-1 and COX-2 but is active against COX-2 carboxylic have the crystal of and and and acid inhibitors to Interestingly, of the inhibitors in with carboxylic to Arg-120. by the and diclofenac a binding mode with inhibitors to This is in the structure of diclofenac in the active site of COX-2 with of and arachidonic acid a and binds with its carboxylate to Arg-120 and Tyr-355, with the of diclofenac of the by arachidonic acid in the structure and is to Arg-120 and of is of COX-2 by and inhibition of COX-2 by in and suggest that is important in the binding of and S530A is by and is to inhibition by inhibition of COX by is a carboxylic acid have the first the of Arg-120, Tyr-355, and Ser-530 in the binding of that binds to COX-2 in an conformation with of mutation of of the that with is binding in the COX active is an that is COX-2 and been to bind in the side by that mutation of this in COX-2 to or inhibition and the of this and into the active site with its group in the side by the or in that mutation of Ser-530 to inhibition but inhibition suggests with Ser-530 and Tyr-385 in to and a polar group that with Ser-530 in to a that bind in the side pocket. and that to and inhibitor binding in the COX active first site the of Arg-120 and the the of inhibitors and the orientation of is the side by Tyr-355, and and of inhibitors that or in by in this side pocket. diclofenac and arachidonic acid in its orientation with Tyr-385 and Ser-530 the of the active in the binding modes of diclofenac and the orientation of arachidonic acid the of inhibition in is to that COX into the of inhibitors that bind to We and the of COX-2