Glu-524 is displayed in red, and the ion-pair interaction with Arg-513 is highlighted with a dashed black line. reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations Bioymifi indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings. Cyclooxygenases (COX-1 and COX-2) play important roles in a wide range of physiological and pathophysiological responses and are the molecular targets for nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors.1?3 The two COX isoforms are approximately 60% identical in amino acid sequence and virtually superimposable in three-dimensional structure.4?7 Although their active sites exhibit approximately 85% sequence identity,8 subtle structural differences have enabled the design of isoform-selective inhibitors for both COX-1 and COX-2.9?18 Each COX isoform is a structural homodimer that functions as a heterodimer. One subunit, containing the required heme prosthetic group, acts as the catalytic site, whereas the other serves as an allosteric site.19,20 Prior evidence suggests that inhibitors may act at either or both sites, depending on the inhibitors structure and concentration.19,21?23 Regardless of site, binding requires that a small molecule must first enter through the four-helix membrane-binding domain into an open area termed the lobby.7 The lobby is separated from the active site proper by a constriction site comprising the conserved residues, Arg-120, Tyr-355, and Glu-524 (Figure ?Figure11). The active site is located in a hydrophobic channel that runs from the constriction site to the catalytic tyrosine (Tyr-385), then bends sharply and terminates in an alcove near Gly-533 at the top of the active site.24 Site-directed mutagenesis has been very useful in defining critical Bioymifi interactions between inhibitors and residues in the active site and, in some cases, has predicted novel binding modes in advance of the solution of protein-inhibitor structures.9 Open in a separate window Figure 1 Stereo view of the structure of COX-2 based on the crystal structure with indomethacin (INDO) shown in the active site. The constriction site residues (E524, Y355, and R120) are shown in gray. Lobby and secondary shell residues that were the subject of mutagenesis to their COX-1 counterparts are shown in magenta. L472 is highlighted in yellow. The molecular basis for the selectivity of inhibitors for the individual COX enzymes has been of special interest from a biochemical and pharmacological point of view. Several years ago, our laboratory reported that neutral derivatives of certain arylcarboxylic acid-containing NSAIDs, such as indomethacin, are highly selective COX-2 inhibitors.25 Inhibition of COX by the various ester and amide derivatives contrasts sharply with that of their parent carboxylic acids, which are frequently more potent inhibitors of COX-1 than COX-2. Site-directed mutagenesis indicates that the constriction site residues, Tyr-355 and Glu-524, are important for neutral NSAID derivative binding, while interactions with Tyr-355 and Arg-120 are required for the carboxylic acid-containing indomethacin.25 Although hydrogen-bonding and ion-pairing interactions at the constriction site are different between indomethacin and its ester/amide derivatives, it is unlikely that these residues solely account for the COX-2-selectivity of the neutral derivatives since the constriction site residues are conserved in both proteins. The generality of COX-2-selective inhibition by indomethacin amides or esters implies the existence of novel molecular interactions outside of the primary residues of the cyclooxygenase active site. Thus, we undertook a study of the importance of lobby or second-shell residues in the binding and inhibition of COX-2 by this class of molecules. The results revealed a subtle substitution of a second-shell residue (Leu-472 in COX-2 Met-472 Bioymifi in COX-1) that makes a significant contribution to inhibition of COX-2 by indomethacin amides/esters. Experimental Procedures Materials Arachidonic acid (AA) was from NuChek Prep (Elysian, MN). 1-[14C]-AA was from PerkinElmer (Boston, MA). All inhibitors were either purchased from Sigma-Aldrich (St. Louis, MO) or synthesized as described in Mouse monoclonal to Rab25 the Supporting Information. Site-directed mutagenesis was performed on.