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5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that is a potent granulocyte chemoattractant, which induces the infiltration of eosinophils into human skin when injected intradermally. It could therefore be an important proinflammatory mediator in eosinophilic diseases such as asthma and allergic rhinitis, and the OXE receptor, which mediates its actions, is therefore an attractive drug target. Using a structure-based approach in which substituents mimicking the essential polar (C1-C5) and hydrophobic (C15-C20) regions of 5-oxo-ETE were incorporated on an indole scaffold, we identified two potent selective OXE antagonists with IC50 values of about 30 nM. Neither compound displayed agonist activity and both inhibited 5-oxo-ETE-induced chemotaxis and actin polymerization and were relatively resistant to metabolism by rat liver homogenates. The active enantiomers of these racemic antagonists were even more potent, with IC50 values of <10 nM. These selective OXE antagonists could potentially be useful therapeutic agents in allergic diseases such as asthma.

Prostaglandin D 2 (PGD 2 ) is a mast cell-derived mediator that seems to play a role in asthma and allergic diseases. It is the only primary prostanoid to activate human eosinophils, which it accomplishes through the DP 2 receptor/chemoattractant receptor-homologous molecule expressed on T helper cell type 2 (Th2) cells (CRTH2). In addition, PGD 2 has both pro-and antiinflammatory effects via the adenylyl cyclase-coupled DP 1 receptor. To attempt to identify potent and selective DP 2 receptor agonists we compared the abilities of a series of PGD 2 analogs to activate eosinophils via the DP 2 receptor with their abilities to stimulate adenylyl cyclase in platelets via the DP 1 receptor. All of the PGD 2 analogs tested stimulated CD11b expression and actin polymerization with a rank order of potency of 15R-methyl-PGD 2 Ͼ PGD 2 Ͼ 17-phenyl-18,19,20-trinor-PGD 2 Ͼ 15S-methyl-PGD 2 Ϸ 16,16-dimethyl-PGD 2 Ͼ 11-keto-fluprostenol. Surprisingly, 15R-methyl-PGD 2 , which has the unnatural R-configuration at carbon 15, was about 5 times more potent than PGD 2 and about 75 times more potent than 15S-methyl-PGD 2 . 15R-methyl-PGD 2 (EC 50 value of 1.7 nM) was also much more potent as an eosinophil chemoattractant than PGD 2 (EC 50 value of 10 nM) and 15S-methyl-PGD 2 (EC 50 value of 128 nM). Cross-desensitization experiments indicated that 15R-methyl-PGD 2 acts through the DP 2 receptor. None of the PGD 2 analogs tested elevated platelet cAMP by more than 20% of the maximal level in response to PGD 2 . However, in contrast to eosinophils, the most active was 15S-methyl-PGD 2 .In conclusion, 15R-methyl-PGD 2 is the most potent known DP 2 receptor agonist, and because of its selectivity and resistance to metabolism, should be a useful tool in probing the physiological role of this receptor in inflammatory diseases.

The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a potent chemoattractant for neutrophils and eosinophils, and its actions are mediated by the oxoeicosanoid (OXE) receptor, a member of the G proteincoupled receptor family. To define the requirements for activation of the OXE receptor, we have synthesized a series of 5-oxo-6E,8Z-dienoic acids with chain lengths between 12 and 20 carbons, as well as a series of 20-carbon 5-oxo fatty acids, either fully saturated or containing between one and five double bonds. The effects of these compounds on neutrophils (calcium mobilization, CD11b expression, and cell migration) and eosinophils (actin polymerization) were compared with those of 5-oxo-ETE. The C 12 and C 14 analogs were without appreciable activity, whereas the C 16 5-oxo-dienoic acid was a weak partial agonist. In contrast, the corresponding C 18 analog (5-oxo-18:2) was nearly as potent as 5-oxo-ETE. Among the C 20 analogs, the fully saturated compound had virtually no activity, whereas 5-oxo-6E-eicosenoic acid had only weak agonist activity. In contrast, 5-oxo-6E,8Z,11Z-eicosatrienoic acid (5-oxo-20:3) and its 8-trans isomer were approximately equipotent with 5-oxo-ETE in activating granulocytes. Because of the potent effects of 5-oxo-20:3, we investigated its formation from Mead acid (5Z,8Z,11Z-eicosatrienoic acid), which accumulates in dietary essential fatty acid deficiency, by neutrophils. The main Mead acid metabolite identified was 5-hydroxy-6,8,11-eicosatrienoic acid, followed by 5-oxo-20:3 and two 6-trans isomers of leukotriene B 3 . We conclude that optimal activation of the OXE receptor is achieved with 5-oxo-ETE, 5-oxo-18:2, and 5-oxo-20:3, and that the latter compound could potentially be formed under conditions of essential fatty acid deficiency.Metabolism of arachidonic acid by the 5-lipoxygenase (5-LO) pathway leads to the formation of leukotriene (LT) B 4 , LTC 4 , LTD 4 , and 5-HETE (Funk, 2001). LTB 4 , acting through the BLT 1 receptor, is a potent activator of neutrophils and lymphocytes. LTD 4 interacts with the cysteinyl-LT 1 and cysteinyl-LT 2 receptors to stimulate smooth muscle contraction, cytokine release from leukocytes, and various other responses. Although

SUMMARY5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant that is synthesized from the 5-lipoxygenase product 8,11, by the NADP + -dependent enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH), previously reported only in inflammatory cells. Because of their critical location at the interface of the lung with the external environment, we sought to determine whether epithelial cells could also synthesize this substance. We found that HEp-2, T84, A549, and BEAS-2B cells all synthesize 5-oxo-ETE in amounts comparable to leukocytes. The epithelial dehydrogenase is localized in the microsomal fraction, requires NADP + , and is selective for the S-isomer of 5-HETE, suggesting that it is identical to leukocyte 5-HEDH. Normal human bronchial epithelial cells have an even greater capacity to synthesize 5-oxo-ETE. H 2 O 2 dramatically stimulates its synthesis in association with increased levels of intracellular GSSG and NADP + . These responses were all blocked by removal of GSH/GSSG with N-ethylmaleimide, suggesting that H 2 O 2 stimulates 5-oxo-ETE synthesis by raising NADP + levels through activation of the GSH redox cycle. Airway smooth muscle cells can also synthesize 5-oxo-ETE, but to a lesser extent. These results suggest that epithelial cells may be a major source of 5-oxo-ETE under conditions of oxidative stress, which may contribute to eosinophil infiltration in allergic diseases.