A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G 2 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 time-dependent inhibition by nimesulide and piroxicam.The cyclooxygenase (COX) 1 activity of prostaglandin endoperoxide synthase catalyzes the incorporation of two molecules of O 2 into arachidonic acid to yield the hydroperoxy endoperoxide, prostaglandin G 2 (PGG 2 ) (1, 2). PGG 2 diffuses from the cyclooxygenase active site and binds at the peroxidase active site where it is reduced to the hydroxy endoperoxide, PGH 2 , the precursor to prostaglandins, thromboxane, and prostacyclin (3). Two COX isoforms exist that differ in expression pattern, mode of regulation, and biological function (4). COX-1 is generally considered the homeostatic form of the enzyme as it is constitutively expressed in a number of tissues, whereas COX-2 is sensitive to induction in many tissues by a broad range of physiological and pathological stimuli (5). Inhibition of COX enzymes by non-steroidal anti-inflammatory drugs (NSAIDs) accounts for their anti-inflammatory and analgesic activities, as well as their gastrointestinal toxicity (6). Development of selective COX-2 inhibitors has reduced the gastrointestinal liability (7).Structural and functional analysis is providing an increasingly detailed picture of the molecular determinants of COXsubstrate and COX-inhibitor interactions (3). COX-1 and COX-2 have very similar structures characterized by a membrane-binding domain comprised of amphipathic helices forming the entrance to a long hydrophobic channel (8 -10). This channel leads deep inside the protein, and at its upper end comprises the cyclooxygenase active site. The cyclooxygenase active site is separated from the opening near the membran...

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

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A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.