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Abstract Leukotrienes (LT) are lipid mediators of the inflammatory response that play key roles in diseases such as asthma and atherosclerosis. The precursor leukotriene A 4 (LTA 4) is synthesized from arachidonic acid (AA) by 5‑lipoxygenase (5-LOX), a membrane-associated enzyme, with the help of 5‑lipoxygenase-activating protein (FLAP), a nuclear transmembrane protein. In lipoxygenases the main chain carboxylate of the C-terminus is a ligand for the non-heme iron and thus part of the catalytic center. We investigated the role of a lysine-rich sequence (KKK653–655) 20 amino acids upstream of the C-terminus unique to 5-LOX that might displace the main-chain carboxylate in the iron coordination sphere. A 5-LOX mutant in which KKK653–655 is replaced by ENL was transfected into HEK293 cells in the absence and presence of FLAP. This mutant gave ~20-fold higher 5-LOX product levels in stimulated HEK cells relative to the wild-type 5-LOX. Co-expression of the enzymes with FLAP led to an equalization of 5-LOX products detected, with wild-type 5-LOX product levels increased and those from the mutant enzyme decreased. These data suggest that the KKK motif limits 5-LOX activity and that this attenuated activity must be compensated by the presence of FLAP as a partner protein for effective LT biosynthesis. Graphical abstract Unlabelled Image Highlights • 5-Lipoxygenase (5-LOX) initiates the synthesis of Leukotrienes. • A tri-Lys sequence motif specific to 5-LOX is destabilizing in vitro. • Substitution of the motif results in highly elevated enzyme activity in a cellular context. • The helper protein 5-Lipoxgenase Activating Protein (FLAP) relieves auto-suppression of activity by the motif. • The tri-Lys motif confers a need for a 5-LOX accesory protein to achieve full enzymatic capacity. [ABSTRACT FROM AUTHOR]

Abstract: Many chemotherapeutic agents activate multiple signaling systems, including potentially emetogenic arachidonic acid metabolites. Of these messengers, the emetic role of the leukotriene family has been neglected. The aims of this study were to test the emetic potential of key leukotrienes (LTA4, LTB4, LTF4, and the cysteinyl leukotrienes LTC4, LTD4 and LTE4), and to investigate whether the leukotriene CysLT1 receptor antagonist pranlukast or mixed leukotriene CysLT1/2 receptor antagonist Bay u9773 can prevent the LTC4-induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30min. LTC4 and LTD4 were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05mg/kg, respectively. The other tested leukotrienes were either weakly emetic or ineffective at doses up to 4mg/kg. The relative emetogenic activities of the cysteinyl leukotrienes (LTC4 =LTD4 >LTE4) suggest that leukotriene CysLT2 receptors have a key role in emesis. However, pranlukast dose-dependently, and at 10mg/kg completely, blocked LTC4-induced vomiting, implicating a leukotriene CysLT1 receptor-mediated emetic effect. Bay u9773 dose-dependently reduced the percentage of animals vomiting, but did not significantly reduce vomiting frequency. Fos immunoreactivity, measured subsequent to LTC4-induced vomiting to define its putative anatomical substrates, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC4 (P <0.05) versus vehicle injections. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT1 and/or leukotriene CysLT2 receptors. [Copyright &y& Elsevier]

Abstract: The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA4-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties. [Copyright &y& Elsevier]

Abstract: The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA4-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency and oral bioavailability. [Copyright &y& Elsevier]

Abstract: Leukotriene B4 (LTB4) is a potent pro-inflammatory mediator that has been implicated in the pathogenesis of multiple diseases, including psoriasis, inflammatory bowel disease, multiple sclerosis and asthma. As a method to decrease the level of LTB4 and possibly identify novel treatments, inhibitors of the LTB4 biosynthetic enzyme, leukotriene A4 hydrolase (LTA4-h), have been explored. Here we describe the discovery of a potent inhibitor of LTA4-h, arylamide of glutamic acid 4f, starting from the corresponding glycinamide 2. Analogs of 4f are then described, focusing on compounds that are both active and stable in whole blood. This effort culminated in the identification of amino alcohol 12a and amino ester 6b which meet these criteria. [Copyright &y& Elsevier]

Context Myocardial infarction (MI) is the leading cause of death in the world. Variants in the 5-lipoxygenase–activating protein (FLAP) gene are associated with risk of MI. Objective To determine the effect of an inhibitor of FLAP on levels of biomarkers associated with MI risk. Design, Setting, and Patients A randomized, prospective, placebo-controlled, crossover trial of an inhibitor of FLAP (DG-031) in MI patients who carry at-risk variants in the FLAP gene or in the leukotriene A4 hydrolase gene. Of 268 patients screened, 191 were carriers of at-risk variants in FLAP (87%) or leukotriene A4 hydrolase (13%). Individuals were enrolled in April 2004 and were followed up by designated cardiologists from a university hospital in Iceland until September 2004. Interventions Patients were first randomized to receive 250 mg/d of DG-031, 500 mg/d of DG-031, 750 mg/d of DG-031, or placebo. After a 2-week washout period, patients received DG-031 if they had received placebo first or placebo if they had received DG-031 first. Treatment periods lasted for 4 weeks. Main Outcome Measures Changes in levels of biomarkers associated with risk of MI. Results In response to 750 mg/d of DG-031, production of leukotriene B4 was significantly reduced by 26% (95% confidence interval [CI], 10%-39%; P = .003) and myeloperoxidase was significantly reduced by 12% (95% CI, 2%-21%; P = .02). The higher 2 doses of DG-031 produced a nonsignificant reduction in C-reactive protein (16%; 95% CI, -2% to 31%; P = .07) at 2 weeks. However, there was a more pronounced reduction (25%; 95% CI, 5%-40%; P = .02) in C-reactive protein at the end of the washout period that persisted for another 4 weeks thereafter. The FLAP inhibitor DG-031 was well tolerated and was not associated with any serious adverse events. Conclusion In patients with specific at-risk variants of 2 genes in the leukotriene pathway, DG-031 led to significant and dose-dependent suppression of biomarkers that are associated with increased risk of MI events. [ABSTRACT FROM AUTHOR]

Human 5-lipoxygenase is a non-heme into protein which possesses 5-oxygenase, leukotriene A4 synthase and pseudoperoxidase activities and which undergoes a rapid irreversible inactivation during these reactions. The inactivation of the enzyme was dependent on the structural characteristics of the substrate for the reaction, on O2 concentration and on exposure to phospholipids and calcium. The apparent first-order rate constant for enzyme inactivation (km) was 0.6 min-1 during the oxygenation of arachidonic acid in air-saturated buffer containing phosphatidylcholine vesicles and Ca2+. The rate of enzyme inactivation was dependent on the substrate for the reaction and was about threefold slower during the oxygenation of 5,8-icosadienoic acid and 12(S)-hydroxyicosatetraenoic acid compared with arachidonic acid. Lowering the O2 concentration to 60 µM during the oxygenation of arachidonic acid also caused a 2.5-fold decrease in km without affecting the initial rate of the reaction resulting in an increase in both 5 hydroperoxyicosatetraenoic acid (5-HPETE) and leukotriene A3 accumulation. The concentration of O2 for half-maximal activity (initial rate and product accumulation) was approximately 10 µM. In contrast, the activity and the rate of inactivation during the leukotriene A4 synthase reaction with exogenous 5HPETE (km = 2.0 min-1) were independent of O2 concentration. A rapid inactivation of the enzyme was also observed during aerobic incubation with phosphatidylcholine vesicles and Ca2+ in the absence of substrate, with a sequential loss of the oxygenase α1/2 = 0.5 min) and pseudoperoxidase (t1/2 = 7 min) activities. Protection against this turnover-independent inactivation was observed in the presence of the selective reversible 5-lipoxygenase inhibitor L-739.010 ([1S, 5R] 3-cyano-1-(3-furyl)-6-(6-[3-(3α-hydroxy-6,8-dioxyabicyclo [3.2.1] octanyl))] pyridin-2-ylmethoxy) naphthalene) and by prior treatment of vesicles with sodium borohydride and, to a lesser extent, by glutathione peroxidase. The results show that the inactivation of 5-lipoxygenase in phospholipid vesicles is dependent on the structure of the unsaturated fatty acid substrate for the reaction, on the concentration of oxygen and on a turnover-independent oxidation at the active-site leading to the sequential loss of the oxygenase and pseudoperoxidase activities of the enzyme. [ABSTRACT FROM AUTHOR]