Structure-activity relationship of cinnamic acylsulfonamide analogues on the human EP3 prostanoid receptor

Potent and selective antagonists of the human EP3 receptor have been identified. The structure-activity relationship of the chemical series was conducted and we found several analogues displaying sub-nanomolar K(i) values at the EP3 receptor and micromolar activities at the EP1, EP2 and EP4 receptors. The effect of added human serum albumin (HSA) on the binding affinity at the EP3 receptor was also investigated.     

Discovery of a series of acrylic acids and their derivatives as chemical leads for selective EP3 receptor antagonists

A series of acrylic acids and their structurally related compounds were evaluated for their binding affinity to four EP receptor subtypes (EP1-4). Starting from the initial hit 3, which was discovered in our in-house library, compounds 4 and 5 were identified as new chemical leads as candidates for further optimization towards a selective EP3 receptor antagonist. The identification process of these compounds and their pharmacokinetic profiles are presented.     

Synthesis, pharmacophore modeling and in vitro activity of 10,11-dihydrodibenzo[b,f]oxepine-4-carboxamide derivatives as novel and potent antagonists of the prostaglandin EP4 receptor

The construction of a EP(4) antagonists pharmacophore model and the discovery of a highly potent oxepinic series of EP(4) antagonists is discussed. Compound 1a exhibits an excellent selectivity profile toward EP(2) receptor subtype and low cytochrome P450 inhibition potential.     

Discovery of novel prostaglandin analogs of PGE2 as potent and selective EP2 and EP4 receptor agonists

Analogs of PGE(2) with introduction of diene groups at the omega-side chain have been synthesized and evaluated for their binding affinity for EP(2) and EP(4) receptors. An optimized analog (compound 9b) showed high potency and selectivity for the EP(4) receptor over other known receptors.     

Discovery of a potent and selective agonist of the prostaglandin EP4 receptor

Analogues of PGE(2) wherein the hydroxycyclopentanone ring has been replaced by a lactam have been prepared and evaluated as ligands for the EP(4) receptor. An optimized compound (19a) shows high potency and agonist efficacy at the EP(4) receptor and is highly selective over the other seven known prostaglandin receptors.     

Structure-activity relationship studies on ortho-substituted cinnamic acids, a new class of selective EP(3) antagonists

A series of novel ortho-substituted cinnamic acids have been synthesized, and their binding activity and selectivity on the four prostaglandin E(2) receptors evaluated. Many of them are very potent and selective EP(3) antagonists (K(i) 3-10 nM), while compound 9 is a very good and selective EP(2) agonist (K(i) 8 nM). The biological profile of the EP(2) agonist 9 in vivo and the metabolic profile of selected EP(3) antagonists are also reported.     

PGE1 and PGE2 modify platelet function through different prostanoid receptors

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.     

Simultaneous voltammetric measurement of ascorbic acid, epinephrine and uric acid at a glassy carbon electrode modified with caffeic acid

A stable electroactive thin film of poly(caffeic acid) has been deposited on the surface of a glassy carbon electrode by potentiostatic technique in an aqueous solution containing caffeic acid. Poly(caffeic acid) was used as a modified electrode for the detection of ascorbic acid (AA), epinephrine (EP), uric acid (UA) and their mixture by cyclic voltammetry. This modified electrode exhibits potent and persistent electron-mediating behavior followed by well-separated oxidation peaks towards AA, EP and UA with activation overpotential. For the ternary mixture containing AA, EP and UA, the three compounds can well separate from each other at the scan rate of 20 mVs(-1) with a potential difference of 156, 132 and 288 mV between AA and EP, EP and UA and AA and UA, respectively, which was large enough to determine AA, EP and UA individually and simultaneously. The catalytic peak current obtained, was linearly dependent on the AA, EP and UA concentrations in the range of 2.0 x 10(-5) to 1.0 x 10(-3) mol l(-1), 2.0 x 10(-6) to 8.0 x 10(-5) mol l(-1) and 5.0 x 10(-6) to 3.0 x 10(-4) mol l(-1), and the detection limits for AA, EP and UA were 7.0 x 10(-6), 2.0 x 10(-7) and 6.0 x 10(-7) mol l(-1), respectively. The modified electrode shows good sensitivity, selectivity and stability, and has been applied to the determination of EP in practical injection samples and that of EP, UA and AA simultaneously with satisfactory results.     

Characterization of PGE2 receptors in fetal and newborn lamb ductus arteriosus

Although the role of PGE2 in maintaining ductus arteriosus (DA) patency is well established, the specific PGE2 receptor subtype(s) (EP) involved have not been clearly identified. We used late gestation fetal and neonatal lambs to study developmental regulation of EP receptors. In the fetal DA, radioligand binding and RT-PCR assays virtually failed to detect EP1 but detected EP2, EP3D, and EP4 receptors in equivalent proportions. In the newborn lamb, DA total density was one-third of that found in the fetus and only EP2 was detected. Stimulation of EP2 and EP4 increased cAMP formation and was associated with DA relaxation. Though stimulation of EP3 inhibited cAMP formation, it surprisingly relaxed the fetal DA both in vitro and in vivo. This EP3-induced relaxation was specifically diminished by the ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide. In conclusion, PGE2 dilates the late gestation fetal DA through pathways that involve either cAMP (EP2 and EP4) or K(ATP) channels (EP3). The loss of EP3 and EP4 receptors in the newborn DA is consistent with its decreased responsiveness to PGE2.     

Design and synthesis of a highly selective EP4-receptor agonist. Part 1: 3,7-dithiaPG derivatives with high selectivity.

A series of 3,7-dithiaPGE(1) analogues 3, 4, 11, 16 and 19 were identified as highly selective EP4-receptor agonists starting from the chemical modification of 7-thiaPGE(1) analogue 1. EP4-receptor selectivity and agonist activity were maximized in 3 and 4.     

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.     

A novel series of potent and selective EP(4) receptor ligands: facile modulation of agonism and antagonism

A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.     

Sequence Analysis and Identification of New Isoform of EP4 Receptors in Different Atlantic Salmon Tissues (Salmo salar L.) and Its Role in PGE2 Induced Immunomodulation In Vitro

PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5’ untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ--prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.     

2,3-Diarylthiophenes as selective EP1 receptor antagonists

The synthesis and the EP(1) receptor binding affinity of 2,3-diarylthiophene derivatives are described. The evaluation of the structure-activity relationship (SAR) in this series led to the identification of compounds 4, 7, and 12a, which exhibit high affinity for the human EP(1) receptor and a selectivity greater than 100-fold against the EP(2), EP(3), EP(4), DP, FP, and IP receptors and greater than 25-fold versus the TP receptor. These three antagonists present good pharmacokinetics in rats and significant differences in the way they are distributed in the brain.     

PGE2 through the EP4 receptor controls smooth muscle gene expression patterns in the ductus arteriosus critical for remodeling at birth

The ductus arteriosus (DA) is a fetal shunt that directs right ventricular outflow away from pulmonary circulation and into the aorta. Critical roles for prostaglandin E(2) (PGE(2)) and the EP4 receptor (EP4) have been established in maintaining both the patency of the vessel in utero and in its closure at birth. Here we have generated mice in which loss of EP4 expression is limited to either the smooth muscle (SMC) or endothelial cells and demonstrated that SMC, but not endothelial cell expression of EP4 is required for DA closure. The genome wide expression analysis of full term wild type and EP4(-/-) DA indicates that PGE(2)/EP4 signaling modulates expression of a number of unique pathways, including those involved in SMC proliferation, cell migration, and vascular tone. Together this supports a mechanism by which maturation and increased contractility of the vessel is coupled to the potent smooth muscle dilatory actions of PGE(2).     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

Lactams as EP4 prostanoid receptor subtype selective agonists. Part 1: 2-Pyrrolidinones-stereochemical and lower side-chain optimization

A series of 7-[(5R)-substituted 2-oxo-1-pyrrolidinyl]-heptanoic acids were prepared, their isomeric purity determined, and pharmacologically evaluated. Lactams with affinity for the EP(4) receptor displayed agonist behavior. The lower side-chain of the lactam template could be substituted to afford ligands (e.g., 17, 24, 30, 31, and 33) of high potency and greater than 1000-fold affinity for EP(4) versus the other EP prostanoid receptors.     

Gastric mucosal protection against ethanol by EP2 and EP4 signaling through the inhibition of leukotriene C4 production

Prostaglandin (PG)E derivatives are widely used for treating gastric mucosal injury. PGE receptors are classified into four subtypes, EP(1), EP(2), EP(3), and EP(4). We have tested which EP receptor subtypes participate in gastric mucosal protection against ethanol-induced gastric mucosal injury and clarified the mechanisms of such protection. The gastric mucosa of anesthetized rats was perfused at 2 ml/min with physiological saline, agonists for EP(1), EP(2), EP(3), and EP(4), or 50% ethanol, using a constant-rate pump connected to a cannula placed in the esophagus. The gastric microcirculation of the mucosal base of anesthetized rats was observed by transillumination through a window made by removal of the adventitia and muscularis externa. PGE(2) and subtype-specific EP agonists were applied to the muscularis mucosae at the window. Application of 50% ethanol dilated the mucosal arterioles and constricted the collecting venules. Collecting venule constriction by ethanol was completely inhibited by PGE(2) and by EP(2) and EP(4) agonists (100 nM) but not by an EP(1) or an EP(3) agonist. Ethanol-induced mucosal injury was also inhibited by EP(2) and EP(4) agonists. When leukotriene (LT)C(4) levels in the perfusate of the gastric mucosa were determined by ELISA, intragastric ethanol administration elevated the LTC(4) levels sixfold from the basal levels. These elevated levels were significantly (60%) reduced by both EP(2) and EP(4) agonists but not by other EP agonists. Since LTC(4) application at the window constricted collecting venules strongly, and an LTC antagonist reduced ethanol-induced mucosal injury, reductions in LTC(4) generation in response to EP(2) and EP(4) receptor signaling may be relevant to the protective action of PGE(2). The present results indicate that EP(2) and EP(4) receptor signaling inhibits ethanol-induced gastric mucosal injury through cancellation of collecting venule constriction by reducing LTC(4) production.