Eicosanoid receptors as therapeutic targets for asthma.

Eicosanoids comprise a group of oxidation products of arachidonic and 5,8,11,14,17-eicosapentaenoic acids formed by oxygenases and downstream enzymes. The two major pathways for eicosanoid formation are initiated by the actions of 5-lipoxygenase (5-LO), leading to leukotrienes (LTs) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), and cyclooxygenase (COX), leading to prostaglandins (PGs) and thromboxane (TX). A third group (specialized pro-resolving mediators; SPMs), including lipoxin A4 (LXA4) and resolvins (Rvs), are formed by the combined actions of different oxygenases. The actions of the above eicosanoids are mediated by approximately 20 G protein-coupled receptors, resulting in a variety of both detrimental and beneficial effects on airway smooth muscle and inflammatory cells that are strongly implicated in asthma pathophysiology. Drugs targeting proinflammatory eicosanoid receptors, including CysLT1, the receptor for LTD4 (montelukast) and TP, the receptor for TXA2 (seratrodast) are currently in use, whereas antagonists of a number of other receptors, including DP2 (PGD2), BLT1 (LTB4), and OXE (5-oxo-ETE) are under investigation. Agonists targeting anti-inflammatory/pro-resolving eicosanoid receptors such as EP2/4 (PGE2), IP (PGI2), ALX/FPR2 (LXA4), and Chemerin1 (RvE1/2) are also being examined. This review summarizes the contributions of eicosanoid receptors to the pathophysiology of asthma and the potential therapeutic benefits of drugs that target these receptors. Because of the multifactorial nature of asthma and the diverse pathways affected by eicosanoid receptors, it will be important to identify subgroups of asthmatics that are likely to respond to any given therapy.

The affinity, intrinsic activity and selectivity of a structurally novel EP2 receptor agonist at human prostanoid receptors.

BACKGROUND AND PURPOSE: Prostanoid EP2 receptor agonists exhibit several activities including ocular hypotension, tocolysis and anti-inflammatory activity. This report describes the affinity and selectivity of a structurally novel, non-prostanoid EP2 receptor agonist, PGN-9856, and its therapeutic potential. EXPERIMENTAL APPROACH: The pharmacology of a series of non-prostanoid EP2 receptor agonists was determined according to functional and radioligand binding studies, mostly using human recombinant prostanoid receptor transfectants. The selectivity of PGN-9856, as the preferred compound, was subsequently determined by using a diverse variety of non-prostanoid target proteins. The therapeutic potential of PGN-9856 was addressed by determining its activity in relevant primate cell, tissue and disease models. KEY RESULTS: PGN-9856 was a selective and high affinity (pKi ≥ 8.3) ligand at human recombinant EP2 receptors. In addition to high affinity binding, it was a potent and full EP2 receptor agonist with a high level of selectivity at EP1 , EP3 , EP4 , DP, FP, IP and TP receptors. In cells overexpressing human recombinant EP2 receptors, PGN-9856 displayed a potency (pEC50 ≥ 8.5) and a maximal response (increase in cAMP) comparable to that of the endogenous agonist PGE2 . PGN-9856 exhibited no appreciable affinity (up 10 µM) for a range of 53 other receptors, ion channels and enzymes. Finally, PGN-9856 exhibited tocolytic, anti-inflammatory and long-acting ocular hypotensive properties consistent with its potent EP2 receptor agonist properties. CONCLUSIONS AND IMPLICATIONS: PGN-9856 is a potent, selective and efficacious prostanoid EP2 receptor agonist with diverse potential therapeutic applications: tocolytic, anti-inflammatory and notably anti-glaucoma.

Synthesis, molecular modelling studies and biological evaluation of new oxoeicosanoid receptor 1 agonists.

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC50) and efficacy (Emax) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles.

In vivo α-hydroxylation of a 2-alkylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid in monkeys.

We have developed a selective indole antagonist (230) targeting the OXE receptor for the potent eosinophil chemoattractant 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid), that may be useful for the treatment of eosinophilic diseases such as asthma. In previous studies we identified ω2-oxidation of the hexyl side chain of racemic 230 as a major metabolic route in monkeys, but also obtained evidence for another pathway that appeared to involve hydroxylation of the hexyl side chain close to the indole. The present study was designed to investigate the metabolism of the active S-enantiomer of 230 (S230) and to identify the novel hydroxy metabolite and its chirality. Following oral administration, S230 rapidly appeared in the blood along with metabolites formed by a novel and highly stereospecific α-hydroxylation pathway, resulting in the formation of αS-hydroxy-S230. The chirality of α-hydroxy-S230 was determined by the total synthesis of the relevant diastereomers. Of the four possible diastereomers of α-hydroxy-230 only αS-hydroxy-S230 has significant OXE receptor antagonist activity and only this diastereomer was found in significant amounts in blood following oral administration of S230. Other novel metabolites of S230 identified in plasma by LC-MS/MS were αS,ω2-dihydroxy-S230 and glucuronides of S230 and ω2-hydroxy-S230. Thus the alkyl side chain of S230, which is essential for its antagonist activity, is also the major target of the metabolic enzymes that terminate its antagonist activity. Modification of this side chain might result in the development of related antagonists with improved metabolic stability and efficacy.

Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.

The endogenous ligands for the LT, lipoxin (LX) and oxoeicosanoid receptors are bioactive products produced by the action of the lipoxygenase family of enzymes. The LT receptors BLT1 and BLT2 , are activated by LTB4 and the CysLT1 and CysLT2 receptors are activated by the cysteinyl-LTs, whereas oxoeicosanoids exert their action through the OXE receptor. In contrast to these pro-inflammatory mediators, LXA4 transduces responses associated with the resolution of inflammation through the receptor FPR2/ALX (ALX/FPR2). The aim of the present review is to give a state of the field on these receptors, with focus on recent important findings. For example, BLT1 receptor signalling in cancer and the dual role of the BLT2 receptor in pro- and anti-inflammatory actions have added more complexity to lipid mediator signalling. Furthermore, a cross-talk between the CysLT and P2Y receptor systems has been described, and also the presence of novel receptors for cysteinyl-LTs, such as GPR17 and GPR99. Finally, lipoxygenase metabolites derived from ω-3 essential polyunsaturated acids, the resolvins, activate the receptors GPR32 and ChemR23. In conclusion, the receptors for the lipoxygenase products make up a sophisticated and tightly controlled system of endogenous pro- and anti-inflammatory signalling in physiology and pathology.

Expression and function of OXE receptor, an eicosanoid receptor, in steroidogenic cells.

Hormonal regulation of steroidogenesis involves arachidonic acid (AA) metabolism through the 5-lipoxygenase pathway. One of the products, 5-hydroperoxy-eicosatetraenoic acid (5-HpETE), acts as a modulator of the activity of the steroidogenic acute regulatory (StAR) protein promoter. Besides, an oxoeicosanoid receptor of the leukotriene receptor family named OXE-R is a membrane protein with high affinity and response to 5-HpETE, among other AA derivatives. The aim of our work was to elucidate whether this receptor may be involved in steroidogenesis. RT-PCR and western blot analysis demonstrated the presence of the mRNA and protein of the receptor in human H295R adrenocortical cells. The treatment of H295R or MA-10 cells (murine Leydig cell line) with 8Br-cAMP together with docosahexaenoic acid (DHA, an antagonist of the receptor) partially reduced StAR induction and steroidogenesis. On the contrary, 5-oxo-ETE - the prototypical agonist, with higher affinity and potency on the receptor - increased cAMP-dependent steroid production, StAR mRNA and protein levels. These results lead us to conclude that AA might modulate StAR induction and steroidogenesis, at least in part, through 5-HpETE production and activation of a membrane receptor, such as the OXE-R.

17(R),18(S)-epoxyeicosatetraenoic acid, a potent eicosapentaenoic acid (EPA) derived regulator of cardiomyocyte contraction: structure-activity relationships and stable analogues.

17(R),18(S)-epoxyeicosatetraenoic acid [17(R),18(S)-EETeTr], a cytochrome P450 epoxygenase metabolite of eicosapentaenoic acid (EPA), exerts negative chronotropic effects and protects neonatal rat cardiomyocytes against Ca(2+)-overload with EC(50) ≈ 1-2 nM. Structure-activity studies revealed that a cis-Δ(11,12)- or Δ(14,15)-olefin and a 17(R),18(S)-epoxide are minimal structural elements for antiarrhythmic activity whereas antagonist activity was often associated with the combination of a Δ(14,15)-olefin and a 17(S),18(R)-epoxide. Compared with natural material, the agonist and antagonist analogues are chemically and metabolically more robust and several show promise as templates for future development of clinical candidates.

Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations.

BACKGROUND AND PURPOSE: The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP3 receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated. EXPERIMENTAL APPROACH: Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted. KEY RESULTS: L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP3 antagonists. For ligands of low to moderate lipophilicity, potency increments for EP3 and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC50 of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H1 antagonism above a pA2 limit of 8.0. In contrast, L-798106 (EP3), L-826266 (EP3, TP) and the lipophilic H1 antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP3) had a faster onset. Agonism and antagonism on the vas deferens EP3 system were overall much faster, although trends were similar. CONCLUSIONS AND IMPLICATIONS: High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP3 antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP3 antagonists is discussed.

Identification of novel peptide agonists from a random peptide library for a 5-oxo-ETE receptor, a receptor for bioactive lipids.

A combinatorial peptide library contains an enormous combination of amino acid sequences and drug candidates, but an effective screening strategy to identify a variety of bioactive peptides has yet to be established. In this article, a random hexapeptide library was screened to identify novel peptide ligands for a 5-oxo-ETE receptor (OXER), which is a G-protein-coupled receptor for bioactive lipids, by using an OXER-Gi1alpha fusion protein. We successfully identified 2 hexapeptides-Ac-HMQLYF-NH2 and Ac-HMWLYF-NH(2)-that exhibited agonistic activity. Although the corresponding affinities were relatively low (EC50 values of 146 and 6.7 microM, respectively), the activities were confirmed by other independent cell-based assay methods, namely, intracellular calcium mobilization and cell chemotaxis. This study demonstrates that a combinatorial peptide library may be screened using a [35S]GTPgammaS binding assay with G-protein-coupled receptor (GPCR)-Galpha fusion proteins, in general, and that of peptide ligands can be obtained even for nonpeptide receptors.

Studies of synergistic and antagonistic combinations of conventional cytotoxic agents with the multiple eicosanoid pathway modulator LY 293111.

The arachidonic acid metabolic pathway is currently under active investigation as a promoter of malignancy and several molecules have been synthesized to block either the cyclooxygenase or lipoxygenase branches. LY 293111 is an oral agent known to be a leukotriene B4 antagonist, a 5-lipoxygenase inhibitor and a peroxisome proliferator-activated receptor (PPAR)-gamma agonist with cytotoxic properties in cell lines. We have studied this agent with classical chemotherapeutic agents in a 72-h culture with cell lines using median-effect analysis as a measure of antagonism or synergy. LY 293111 displays global synergy with the active metabolite of irinotecan, SN-38, in the majority of cell lines, synergistic to additive effects with gemcitabine in bladder cancer cell lines, and synergism with 5'-DFUR (the active metabolite of capecitabine) in two breast cancer and one sarcoma cell line. These effects occur at clinically attainable concentrations. The addition of a proteosome inhibitor to the LY 293111 and SN-38 combination markedly enhanced the cytotoxic effects in the sarcoma cell line. As the toxicity of LY 293111 in man is not hematological, this agent may have a role in combination therapy of selected malignancies.