identi®ed as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2 In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic aciddependent production of prostaglandin E 2 (PGE 2 ) with at least a 1,000 fold selectivity for COX-2 (IC 50 =41+14 nM) over COX-1 (IC 50 450 mM). Indomethacin was a potent inhibitor of both COX-1 (IC 50 =18+3 nM) and COX-2 (IC 50 =26+6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B 2 (TXB 2 ) by Ca 2+ ionophore-challenged human platelets (IC 50 450 mM and 4.1+1.7 nM, respectively). 3 DFU caused a time-dependent inhibition of puri®ed recombinant human COX-2 with a K i value of 140+68 mM for the initial reversible binding to enzyme and a k 2 value of 0.11+0.06 s 71 for the ®rst order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62+26 mM and 0.06+0.01 s 71 , respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t 1/2 =1 ± 3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4 Inhibition of puri®ed recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC 50 =63+5 mM at 0.1 mM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5 DFU inhibited lipopolysaccharide (LPS)-induced PGE 2 production (COX-2) in a human whole blood assay with a potency (IC 50 =0.28+0.04 mM) similar to indomethacin (IC 50 =0.68+0.17 mM). In contrast, DFU was at least 500 times less potent (IC 50 497 mM) than indomethacin at inhibiting coagulationinduced TXB 2 production (COX-1) (IC 50 =0.19+0.02 mM). 6 In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 mM), DFU inhibited COX-1 with an IC 50 value of 13+2 mM as compared to 20+1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac4indomethacin*naproxen4nimesulide* meloxicam*piroxicam4NS-398*SC-576664SC-581254CGP 28238*etodolac4L-745,3374DFU. 7 DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED 50 of 1.1 mg kg 71 vs 2.0 mg kg 71 for indomethacin) and hyperalgesia (ED 50 of 0.95 mg kg 71 vs 1.5 mg kg 71 for indomethacin). The compound was also e ective at reversing LPS-induced pyrexia in rats (ED 50 =0.76 mg kg 71 vs 1.1 mg kg 71 for indomethacin). 8 In a sensitive model in which 51 Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no signi®cant e ect was detected after oral...

5-Lipoxygenase-activating protein (FLAP) is an 1 S-kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates S-LO has not been determined. We have expressed high levels of human FLAP in Spodopteru frugiperda (39) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds ['2sI]L-739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5-LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.5-Lipoxygenase-activating protein; 5-Lipoxygenase; Arachidonic acid; Photoaffinity labeling; Baculovirus

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-EJB 93 0154/6 5-Lipoxygenase (5-LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5-LO catalyzing both the synthesis of (SS)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) from arachidonic acid and the subsequent synthesis of LTA, from 5-HPETE. We have previously expressed both human 5-LO and human FLAP to high levels in Spodopteru frugiperdu (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5-L0, we compared cellular 5-LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5-LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5-LO as a substrate, and increases the efficiency with which 5-LO converts 5-HPETE to LTA,. LT synthesis in cells coexpressing FLAP and 5-LO is inhibited by 3-[l-(p-chlorophenyl)-5-isopropyl-3-~e~-buty1t~o-1~-indo1-2-y1]-2,2-dimethy1-prop~oic acid (MK-886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5-LO by acting as an arachidonic acid transfer protein. jects, and these levels are increased during an asthmatic attack (Tagari et al., 1990). Consequently, compounds which inhibit the synthesis of LTs or act as LT receptor antagonists are currently being developed as potential therapeutic agents. The first two steps in the synthesis of LTs from arachidonic acid, the oxygenation of arachidonic acid to (5S)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) followed by the conversion of 5-HPETE to the unstable epoxide LT&, are catalyzed by the enzyme 5-lipoxygenase (Rouzer et al., 1986). In neutrophils, LTA, is subsequently metabolized to the proinflammatory compound LTB, by the enzyme LTA, hydrolase, while in other cell types, including macrophages, eosinophils, and epithelial cells, LTA, is converted to the peptidoleukotrienes LTC4, LTD4, and LTE4 in a series of enzymatic reactions (Maycock et al., 1989). In response to a variety of agents which stimulate cellular LT synthesis, including calcium ionophore A231 87 (Rouzer and Kargman, 1988), thapsigargin (Wong et al., 1991), IgE (Wong et al., 1992), and fMetLeuPhe (Kargman et al., 1991), 5-LO translocates from the soluble to a membrane compartment. This specific membrane association of 5-LO appears to be an early step in the cellular synthesis of LTs, and may be required for the efficient utilization of arachidonic acid, which is released from membrane phospholipids by phospholipases.The direct inhibition of 5-LO or blocking of the specific membrane association of the enzyme represent alternative approaches to the inhibition of LT synthesis. Inhibitors based on indole and quinoline moieties and hybrid compounds containing both indole and quinoline groups have been shown to inhibit and reverse the membrane association of 5-LO Evans et al., 1991;Brideau et al.,

Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) onto PGHS-1 utilizing the known crystal structure of PGHS-1 shows that the only residues impinging directly on the active site that were not conserved in the two enzymes are His 513 and Ile 523 of PGHS-1 (Arg 499 and Val 509 of PGHS-2). These residues of human PGHS-1 were each mutated to the corresponding PGHS-2 residues (His 513 3 Arg and Ile 523 3 Val) and a double mutant (His 513 3 Arg,Ile 523 3 Val) containing both residues was also constructed. The mutant enzyme forms were expressed in COS-7 cells, and their properties were compared with those of the normal isoforms using microsomal membranes. The mutated enzyme forms all had apparent K m values within 1.4-fold that of the wild type enzyme, and the specific activity of the mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are selective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile 523 3 Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His 513 3 Arg,Ile 523 3 Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibition was observed. In contrast, the single HR mutation did not increase the sensitivity to inhibition by the selective PGHS-2 inhibitors. The potency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5-and 13-fold in the HR and HR-IV mutants, respectively. All the results indicate that mutations of His 513 and Ile 523 residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and restore time-dependent inhibition. They also suggest that the corresponding Arg 499 and Val 509 residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.Prostaglandins are derived from arachidonic acid and act as mediators of pain, fever, and other inflammatory responses (1). Prostaglandin G/H synthase (PGHS)1 converts arachidonic acid into prostaglandin G 2 by the addition of molecular oxygen (a cyclooxygenase step) and then catalyzes the conversion of prostaglandin G 2 to prostaglandin H 2 by a peroxidase reaction (2, 3). Prostaglandin H 2 is the precursor to the formation of all prostaglandins, thromboxane, and prostacyclin. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and indomethacin, abrogate prostaglandin synthesis through inhibition of the cyclooxygenase reaction of PGHS (4).A second isoform of PGHS has been discovered (PGHS-2) that is induced in inflammatory situations in response to cytokines or growth factors (5-10). This has lead to the development of selective PGHS-2 inhibitors, which have demonstrated that inhibition of PGHS-2 alone is suffici...