Discovery of novel prostaglandin analogs as potent and selective EP2/EP4 dual agonists

To identify potent EP2/EP4 dual agonists with excellent subtype selectivity, a series of γ-lactam prostaglandin E analogs bearing a 16-phenyl ω-chain were synthesized and evaluated. Structural hybridization of 1 and 2, followed by more detailed chemical modification of the benzoic acid moiety, led us to the discovery of a 2-mercaptothiazole-4-carboxylic acid analog 3 as the optimal compound in the series. An isomer of this compound, the 2-mercaptothiazole-5-carboxylic acid analog 13, showed 34-fold and 13-fold less potent EP2 and EP4 receptor affinities, respectively. Structure activity relationship data from an in vitro mouse receptor binding assay are presented. Continued evaluation in an in vivo rat model of another 2-mercaptothiazole-4-carboxylic acid analog 17, optimized for sustained compound release from PLGA microspheres, demonstrated its effectiveness in a rat bone fracture-healing model following topical administration.     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 2: Optimization of the side chains to improve in vitro and in vivo potencies

A series of 3-[2-{[(3-methyl-1-phenylbutyl)amino]carbonyl}-4-(phenoxymethyl)phenyl]propanoic acid analogs were synthesized and evaluated for their in vitro potency. In most cases, introduction of one or two substituents into the two phenyl moieties resulted in the tendency of an increase or retention of in vitro activities. Several compounds, which showed excellent subtype selectivity, were evaluated for their inhibitory effect against PGE₂-induced uterine contraction in pregnant rats, which is thought to be mediated by the EP3 receptor subtype. The structure–activity relationships (SARs) are also discussed.     

Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

Prostaglandin E2 facilitates subcellular translocation of the EP4 receptor in neuroectodermal NE-4C stem cells

Prostaglandin E2 (PGE₂) is a lipid mediator released from the phospholipid membranes that mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). There is growing evidence for the important role of the PGE₂/EP4 signaling in the nervous system. Previous studies in our lab show that the expression of the EP4 receptor is significantly higher during the neurogenesis period in the mouse. We also showed that in mouse neuroblastoma cells, the PGE₂/EP4 receptor signaling pathway plays a role in regulation of intracellular calcium via a phosphoinositide 3-kinase (PI3K)-dependent mechanism. Recent research indicates that the functional importance of the EP4 receptor depends on its subcellular localization. PGE₂-induced EP4 externalization to the plasma membrane of primary sensory neurons has been shown to play a role in the pain pathway. In the present study, we detected a novel PGE₂–dependent subcellular trafficking of the EP4 receptor in neuroectodermal (NE-4C) stem cells and differentiated NE-4C neuronal cells. We show that PGE₂ induces EP4 externalization from the Golgi apparatus to the plasma membrane in NE-4C stem cells. We also show that the EP4 receptors translocate to growth cones of differentiating NE-4C neuronal cells and that a higher level of PGE₂ enhances its growth cone localization. These results demonstrate that the EP4 receptor relocation to the plasma membrane and growth cones in NE-4C cells is PGE₂ dependent. Thus, the functional role of the PGE₂/EP4 pathway in the developing nervous system may depend on the subcellular localization of the EP4 receptor.     

Prostaglandin E2 upregulates survivin expression via the EP1 receptor in hepatocellular carcinoma cells

AimsCyclooxygenase-2 (COX-2)-controlled production of prostaglandin E₂ (PGE₂) has been implicated in cell growth and metastasis in many cancers. Recent studies have found that COX-2 is co-expressed with survivin in many cancers. Survivin is a member of the inhibitor-of-apoptosis protein family. Some COX-2 inhibitors (e.g., celecoxib) can reduce the expression of survivin. However, little is known about the mechanism of PGE₂-mediated expression of survivin. This study was designed to uncover the effect of PGE₂ on survivin expression in hepatocellular carcinoma cells.Main methodsThe effects of PGE₂ and EP1 agonist on survivin expression were examined in HUH-7 and HepG2 cells. Plasmid transfection and EP1 siRNA were used to regulate the expression of COX-2 and the EP1 receptor protein.Key findingsPGE₂ treatment increased survivin expression 2.3-fold. COX-2 overexpression resulted in a similar level of survivin upregulation. However, this effect was suppressed by treatment with celecoxib. EP1 receptor transfection or treatment with a selective EP1 agonist mimicked the effect of PGE₂ treatment. Conversely, the PGE₂-induced upregulation of survivin was blocked by treatment with a selective EP1 antagonist or siRNA against the EP1 receptor. The phosphorylation of EGFR and Akt were elevated in EP1 agonist-treated cells, and both EGFR and PI3K inhibitors suppressed the upregulation of survivin induced by PGE₂ or EP1 agonist.SignificancePGE₂ regulates survivin expression in hepatocellular carcinoma cells through the EP1 receptor by activating the EGFR/PI3K pathway. Targeting the PGE₂/EP1/survivin signaling pathway may aid the development of new therapeutic strategies for both the prevention and treatment of this cancer.     

Prostaglandin E Receptor Subtypes in Cultured Rat Microglia and Their Role in Reducing Lipopolysaccharide-Induced Interleukin-1 β Production

Abstract : Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE₂, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE₂ influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE₂ and the EP1 agonist 17-phenyl trinor PGE₂ but not the EP3 agonist sulprostone elicited reversible intracellular [Ca²⁺] increases in microglia as measured by fura-2. PGE₂ and the EP2/EP4-specific agonists 11-deoxy-PGE₁ and 19-hydroxy-PGE₂ but not the EP4-selective agonist 1-hydroxy-PGE₁ induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1β production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE₂ and the EP2 agonists reduced IL-1β production. IL-1β production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1β production. Thus, the inhibitory effects of PGE₂ on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE₂ on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.     

Prostaglandin E2 EP2 Receptor Deletion Attenuates Intracerebral Hemorrhage-Induced Brain Injury and Improves Functional Recovery

Intracerebral hemorrhage (ICH) is a devastating type of stroke characterized by bleeding into the brain parenchyma and secondary brain injury resulting from strong neuroinflammatory responses to blood components. Production of prostaglandin E₂ (PGE₂) is significantly upregulated following ICH and contributes to this inflammatory response in part through its E prostanoid receptor subtype 2 (EP2). Signaling through the EP2 receptor has been shown to affect outcomes of many acute and chronic neurological disorders; although, not yet explored in the context of ICH. Wildtype (WT) and EP2 receptor knockout (EP2^−/−) mice were subjected to ICH, and various anatomical and functional outcomes were assessed by histology and neurobehavioral testing, respectively. When compared with age-matched WT controls, EP2^−/− mice had 41.9 ± 4.7% smaller ICH-induced brain lesions and displayed significantly less ipsilateral hemispheric enlargement and incidence of intraventricular hemorrhage. Anatomical outcomes correlated with improved functional recovery as identified by neurological deficit scoring. Histological staining was performed to begin investigating the mechanisms involved in EP2-mediated neurotoxicity after ICH. EP2^−/− mice exhibited 45.5 ± 5.8% and 41.4 ± 8.1% less blood and ferric iron accumulation, respectively. Furthermore, significantly less striatal and cortical microgliosis, striatal and cortical astrogliosis, blood–brain barrier breakdown, and peripheral neutrophil infiltration were seen in EP2^−/− mice. This study is the first to suggest a deleterious role for the PGE₂-EP2 signaling axis in modulating brain injury, inflammation, and functional recovery following ICH. Targeting the EP2 G protein-coupled receptor may represent a new therapeutic avenue for the treatment of hemorrhagic stroke.     

Muscarinic agonist, (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate: High affinity,but low subtype selectivity for human M1 – M5 muscarinic acetylcholine receptors

Novel quinuclidinyl N-phenylcarbamate analogs were synthesized, and binding affinities at M₁-M₅ muscarinic acetylcholine receptor (mAChR) subtypes were determined using Chinese hamster ovary (CHO) cell membranes stably expressing one specific subtype of human mAChR. Although not subtype selective, the lead analog (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate (3c) exhibited the highest affinity (K_i?=?2.0, 13, 2.6, 2.2, 1.8?nM) at each of the M₁-M₅ mAChRs, respectively. Based on results from the [³H]dopamine release assay using rat striatal slices, 3c acted as an agonist at mAChRs. The effect of 3c was inhibited by the nonselective mAChR antagonist, scopolamine, and 3c augmented release evoked by oxotremorine. A potent analog from the same scaffold, (±)-quinuclidin-3-yl-(4-methoxyphenethyl)(phenyl)-carbamate (3b) exhibited the greatest selectivity (17-fold) at M₃ over M₂ mAChRs. These analogs could serve as leads for further discovery of novel subtype-selective muscarinic ligands with the goal of providing therapeutics for substance use disorders and chronic obstructive pulmonary disease.     

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin promotes endothelial cell apoptosis through activation of EP3/p38MAPK/Bcl‐2 pathway

Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose‐dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)‐2 expression and its downstream prostaglandin (PG) production (mainly PGE₂ and 6‐keto‐PGF_1α) in HUVECs. Interestingly, inhibition of COX‐2, but not COX‐1, markedly attenuated TCDD‐triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE₂ receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD‐treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl‐2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl‐2 overexpression attenuated the apoptosis in TCDD‐treated HUVECs. TCDD increased EP3‐dependent Rho activity and subsequently promoted p38MAPK/Bcl‐2 pathway‐mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re‐endothelialization after femoral artery injury in wild‐type (WT) mice, but not in EP3^?/? mice. In summary, TCDD promotes endothelial apoptosis through the COX‐2/PGE₂/EP3/p38MAPK/Bcl‐2 pathway. Given the cardiovascular hazard of a COX‐2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD‐associated cardiovascular diseases.     

Heterocyclic 1,7-disubstituted indole sulfonamides are potent and selective human EP3 receptor antagonists

We have developed a pharmacophore model for the EP₃ receptor antagonists based on its endogenous ligand PGE₂. This ligand-based design yielded a series of novel peri-substituted [4.3.0] bicyclic aromatics featuring 1-alklyaryl 7-heterocyclic sulfonamide substituents. The synthesized molecules are potent antagonists of human EP₃ receptor in vitro and show inhibition of rat platelets aggregation. Optimized derivatives display high selectivity over IP, FP, and other EP receptor panels.     

Prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) suppresses natural killer cell function primarily through the PGE<Subscript>2</Subscript> receptor EP4

The COX-2 product prostaglandin E₂ (PGE₂) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE₂ production or PGE₂-mediated signaling through the PGE₂ receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE₂, but very little is known about the mechanism by which PGE₂ affects NK effector activity. We now report the direct effects of PGE₂ on the NK cell. Endogenous murine splenic NK cells express all four PGE₂ receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE₂, the EP4 agonist PGE₁-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE₂, the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE₁-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE₂ production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.     

Role of the Prostaglandin E2 EP1 Receptor in Traumatic Brain Injury

Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E₂ (PGE₂), leading to overactivation of a class of its cognate G-protein-coupled receptors, including EP1, which is considered a promising target for treatment of ischemic stroke. However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.     

Activation of prostaglandin E2-EP4 signaling reduces chemokine production in adipose tissue

Inflammation of adipose tissue induces metabolic derangements associated with obesity. Thus, determining ways to control or inhibit inflammation in adipose tissue is of clinical interest. The present study tested the hypothesis that in mouse adipose tissue, endogenous prostaglandin E₂ (PGE₂) negatively regulates inflammation via activation of prostaglandin E receptor 4 (EP4). PGE₂ (5–500nM) attenuated lipopolysaccharide-induced mRNA and protein expression of chemokines, including interferon-γ-inducible protein 10 and macrophage-inflammatory protein-1α in mouse adipose tissue. A selective EP4 antagonist (L161,982) reversed, and two structurally different selective EP4 agonists [CAY10580 and CAY10598] mimicked these actions of PGE₂. Adipose tissue derived from EP4-deficient mice did not display this response. These findings establish the involvement of EP4 receptors in this anti-inflammatory response. Experiments performed on adipose tissue from high-fat-fed mice demonstrated EP4-dependent attenuation of chemokine production during diet-induced obesity. The anti-inflammatory actions of EP4 became more important on a high-fat diet, in that EP4 activation suppressed a greater variety of chemokines. Furthermore, adipose tissue and systemic inflammation was enhanced in high-fat-fed EP4-deficient mice compared with wild-type littermates, and in high-fat-fed untreated C57BL/6 mice compared with mice treated with EP4 agonist. These findings provide in vivo evidence that PGE₂-EP4 signaling limits inflammation. In conclusion, PGE₂, via activation of EP4 receptors, functions as an endogenous anti-inflammatory mediator in mouse adipose tissue, and targeting EP4 may mitigate adipose tissue inflammation.     

Synthesis and evaluation of alkoxy-phenylamides and alkoxy-phenylimidazoles as potent sphingosine-1-phosphate receptor subtype-1 agonists

In the design of potent and selective sphingosine-1-phosphate receptor agonists, we were able to identify two series of molecules based on phenylamide and phenylimidazole analogs of FTY-720. Several designed molecules in these scaffolds have demonstrated selectivity for S1P receptor subtype 1 versus 3 and excellent in vivo activity in mouse. Two molecules PPI-4621 (4b) and PPI-4691 (10a), demonstrated dose responsive lymphopenia, when administered orally.     

Synthesis and pharmacological characterization of new neuronal nicotinic acetylcholine receptor ligands derived from Sazetidine-A

The enantiomers of two analogs of Sazetidine-A as well as several other novel biosteric analogues were synthesized. Their binding affinities at three major nAChRs subtypes and selectivity profiles were determined. Though many (S)-enantiomers of Sazetidine-A analogs have high binding affinities and good subtype selectivities, it is not a general rule that (S)-enantiomers are better than their (R) counterparts. Compound 11, of which the ethynyl group was replaced by its’ bioisostere—the triazole via click chemistry, showed a high binding affinity to α4β2 subtype (K_i = 1.3 nM) and better selectivity to the α4β2 subtype over α3β4 subtype with that of Sazetidine-A. The azide compound 15, a potential photoaffinity label, showed improved high selectivity and similar binding property profile with that of Sazetidine-A. The biaryl analog 17 exhibited a much lower affinity as compared to Sazetidine-A indicating the importance of a ‘long tail’ side chain for α4β2 nAChR binding.     

A potent neuromedin U receptor 2-selective alkylated peptide

Neuromedin U (NMU) mediates various physiological functions via NMUR1 and NMUR2 receptors. NMUR2 has been considered a promising treatment option for diabetes and obesity. Although NMU-8, a shorter peptide, has potent agonist activity for both receptors, it is metabolically unstable. Therefore, NMU-8 analogs modified with long-chain alkyl moieties via a linker were synthesized. An octadecanoyl analog (17) with amino acid substitutions [αMePhe¹⁹, Nle²¹, and Arg(Me)²⁴] and a linker [Tra-γGlu-PEG(2)] dramatically increased NMUR2 selectivity, with retention of high agonist activity. Subcutaneous administration of 17 induced anorectic activity in C57BL/6J mice. Owing to its high metabolic stability, 17 would be useful in clarifying the physiological role and therapeutic application of NMU.     

Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells

Both Wnt signaling and prostaglandin E₂ (PGE₂) play pivotal roles in bone development, remodeling, osteoporosis and prostate cancer (PCa) bone metastases. We investigated the effects of PGE₂ on Wnt signaling in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells. We demonstrate that low dose PGE₂ (0.1 μM) promotes Wnt signaling while higher doses of PGE₂ (1.0-10 μM) inhibit these same parameters in osteoblast-lineage cells. The differential effects of low vs high-dose PGE₂ on pre-osteoblasts may be attributed to dose-dependent modulation of prostaglandin receptor (EP) subtype expression; with lower doses increasing the expression the cAMP-stimulatory EP4 receptor subtype and higher doses increasing the expression of the cAMP-inhibitory EP3 receptor subtype. Moreover, we demonstrate that high expression levels of COX-2 and PGE₂ promote the secretion of Wnt inhibitors from prostate cancer cells. These data demonstrate that there are dose-dependent effects of PGE₂ on Wnt activation in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells which may have clinical implications in the management.