A Novel Small Molecular Prostaglandin Receptor EP4 Antagonist, L001, Suppresses Pancreatic Cancer Metastasis.

Metastatic pancreatic cancer remains a major clinical challenge, emphasizing the urgent need for the exploitation of novel therapeutic approaches with superior response. In this study, we demonstrate that the aberrant activation of prostaglandin E2 (PGE2) receptor 4 (EP4) is a pro-metastatic signal in pancreatic cancer. To explore the therapeutic role of EP4 signaling, we developed a potent and selective EP4 antagonist L001 with single-nanomolar activity using a panel of cell functional assays. EP4 antagonism by L001 effectively repressed PGE2-elicited cell migration and the invasion of pancreatic cancer cells in a dose-dependent manner. Importantly, L001 alone or combined with the chemotherapy drug gemcitabine exhibited remarkably anti-metastasis activity in a pancreatic cancer hepatic metastasis model with excellent tolerability and safety. Mechanistically, EP4 blockade by L001 abrogated Yes-associated protein 1 (YAP)-driven pro-metastatic factor expression in pancreatic cancer cells. The suppression of YAP's activity was also observed upon L001 treatment in vivo. Together, these findings support the notions that EP4-YAP signaling axis is a vital pro-metastatic pathway in pancreatic cancer and that EP4 inhibition with L001 may deliver a therapeutic benefit for patients with metastatic pancreatic cancer.

[Activation Mechanism of Prostanoid Receptors -X-ray Crystallography of EP3 Receptor].

Prostanoids [prostaglandins (PGs) and thromboxanes (TXs)] are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G protein-coupled receptors (GPCRs). The nine subtypes of prostanoid receptors (DP1, DP2, EP1, EP2, EP3, EP4, FP, IP, TP) are involved in a wide range of functions, including inflammation, immune response, reproduction, and homeostasis of the intestinal mucosa and cardiovascular system. Among the prostanoid receptors, the structure of antagonist-bound DP2, which belongs to the chemoattractant receptor family, was previously determined. However, the mechanisms of prostanoid recognition and receptor activation remained elusive. To address this issue, we determined the crystal structures of antagonist-bound EP4 and PGE2-bound EP3. The EP3-PGE2 complex exhibits an active-like conformation, including outward movement of the cytoplasmic end of transmembrane (TM) 6 relative to the cytoplasmic end of TM6 of the EP4 complex. The carboxyl moiety of PGE2 is recognized through three hydrogen bonds formed by highly conserved residues: Y1142.65, T206Extracelluar loop 2 (ECL2), and R3337.40 (superscripts denote Ballesteros-Weinstein numbering). In addition, the ω-chain of PGE2 orients toward TM6, which appears to contribute to receptor activation. The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands. These findings should facilitate the development of subtype-selective and non-PG-like ligands.

Cryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to G Protein.

Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we report the cryoelectron microscopy (cryo-EM) structure of the EP4-heterotrimeric G protein (Gs) complex with the endogenous ligand at a global resolution of 3.3 Å. In this structure, compared with that in the inactive EP4 structure, the sixth transmembrane domain is shifted outward on the intracellular side, although the shift is smaller than that in other class A GPCRs bound to Gs. Instead, the C-terminal helix of Gs is inserted toward TM2 of EP4, and the conserved C-terminal hook structure formsthe extended state. These structural features are formed by the conserved residues in prostanoid receptors (Phe542.39 and Trp3277.51). These findings may be important for the thorough understanding of the G protein-binding mechanism of EP4 and other prostanoid receptors.

Prostaglandins and Bone.

Prostaglandins (PGs) are highly bioactive fatty acids. PGs, especially prostaglandin E2 (PGE2), are abundantly produced by cells of both the bone-forming (osteoblast) lineage and the bone-resorbing (osteoclast) lineage. The inducible cyclooxygenase, COX-2, is largely responsible for most PGE2 production in bone, and once released, PGE2 is rapidly degraded in vivo. COX-2 is induced by multiple agonists - hormones, growth factors, and proinflammatory factors - and the resulting PGE2 may mediate, amplify, or, as we have recently shown for parathyroid hormone (PTH), inhibit responses to these agonists. In vitro, PGE2 can directly stimulate osteoblast differentiation and, indirectly via stimulation of RANKL in osteoblastic cells, stimulate the differentiation of osteoclasts. The net balance of these two effects of PGE2 in vivo on bone formation and bone resorption has been hard to predict and, as expected for such a widespread local factor, hard to study. Some of the complexity of PGE2 actions on bone can be explained by the fact that there are four receptors for PGE2 (EP1-4). Some of the major actions of PGE2 in vitro occur via EP2 and EP4, both of which can stimulate cAMP signaling, but there are other distinct signaling pathways, important in other tissues, which have not yet been fully elucidated in bone cells. Giving PGE2 or agonists of EP2 and EP4 to accelerate bone repair has been examined with positive results. Further studies to clarify the pathways of PGE2 action in bone may allow us to identify new and more effective ways to deliver the therapeutic benefits of PGE2 in skeletal disorders.

Improved homology modeling of the human & rat EP4 prostanoid receptors.

BACKGROUND: The EP4 prostanoid receptor is one of four GPCRs that mediate the diverse actions of prostaglandin E2 (PGE2). Novel selective EP4 receptor agonists would assist to further elucidate receptor sub-type function and promote development of therapeutics for bone healing, heart failure, and other receptor associated conditions. The rat EP4 (rEP4) receptor has been used as a surrogate for the human EP4 (hEP4) receptor in multiple SAR studies. To better understand the validity of this traditional approach, homology models were generated by threading for both receptors using the RaptorX server. These models were fit to an implicit membrane using the PPM server and OPM database with refinement of intra and extracellular loops by Prime (Schrödinger). To understand the interaction between the receptors and known agonists, induced-fit docking experiments were performed using Glide and Prime (Schrödinger), with both endogenous agonists and receptor sub-type selective, small-molecule agonists. The docking scores and observed interactions were compared with radioligand displacement experiments and receptor (rat & human) activation assays monitoring cAMP. RESULTS: Rank-ordering of in silico compound docking scores aligned well with in vitro activity assay EC50 and radioligand binding Ki. We observed variations between rat and human EP4 binding pockets that have implications in future small-molecule receptor-modulator design and SAR, specifically a S103G mutation within the rEP4 receptor. Additionally, these models helped identify key interactions between the EP4 receptor and ligands including PGE2 and several known sub-type selective agonists while serving as a marked improvement over the previously reported models. CONCLUSIONS: This work has generated a set of novel homology models of the rEP4 and hEP4 receptors. The homology models provide an improvement upon the previously reported model, largely due to improved solvation. The hEP4 docking scores correlates best with the cAMP activation data, where both data sets rank order Rivenprost>CAY10684 > PGE1 ≈ PGE2 > 11-deoxy-PGE1 ≈ 11-dexoy-PGE2 > 8-aza-11-deoxy-PGE1. This rank-ordering matches closely with the rEP4 receptor as well. Species-specific differences were noted for the weak agonists Sulprostone and Misoprostol, which appear to dock more readily within human receptor versus rat receptor.

Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist.

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Synthesis and evaluation of 18F-labeled CJ-042794 for imaging prostanoid EP4 receptor expression in cancer with positron emission tomography.

The potent and selective prostanoid EP4 receptor antagonist CJ-042794 was radiolabeled with 18F, and evaluated for imaging EP4 receptor expression in cancer with positron emission tomography (PET). The fluorination precursor, arylboronic acid pinacol ester 4, was prepared in 4 steps with 42% overall yield. 18F-CJ-042794 was synthesized via a copper-mediated 18F-fluorination reaction followed by base hydrolysis, and was obtained in 1.5±1.1% (n=2) decay-corrected radiochemical yield. PET/CT imaging and biodistribution studies in mice showed that 18F-CJ-042794 was excreted through both renal and hepatobiliary pathways with significant retention in blood. The EP4-receptor-expressing LNCaP prostate cancer xenografts were clearly visualized in PET images with 1.12±0.08%ID/g (n=5) uptake value and moderate tumour-to-muscle contrast ratio (2.73±0.22) at 1h post-injection. However, the tumour uptake was nonspecific as it could not be blocked by co-injection of cold standard, precluding the application of 18F-CJ-042794 for PET imaging of EP4 receptor expression in cancer.

In vivo effects of two novel ALN-EP4a conjugate drugs on bone in the ovariectomized rat model for reversing postmenopausal bone loss.

UNLABELLED: Two alendronate-EP4 agonist (ALN-EP4a) conjugate drugs, C1 and C2, which differ in structure by a short linker molecule, were evaluated in ovariectomized (OVX) rats for their anabolic effects. We showed that C1 led to significant anabolic effects on cortical and trabecular bone while anabolic effects associated with C2 were minimal. INTRODUCTION: EP4as were covalently linked to ALN to create ALN-EP4a conjugate anabolic bone drugs, C1 and C2, which differ in structure by a short linker molecule in C1. When administered systemically, C1 and C2 are delivered to bone through targeted binding of ALN, where local hydrolytic enzymes liberate EP4a from ALN to exert anabolic effects. Here, we compare effects of C1 to C2 in a curative in vivo study. METHODS: Three-month-old female Sprague Dawley rats were OVX or sham operated and allowed to lose bone for 3 months. Animals were then treated via tail vein injections for 3 months and sacrificed. Treatment groups were as follows: C1L (5 mg/kg biweekly), C1H (5 mg/kg weekly), C2L (15 mg/kg monthly), C2H (15 mg/kg biweekly), OVX and sham control (phosphate-buffered saline (PBS) biweekly), and ALN/EP4a-unconjugated mixture (0.75 mg/kg each biweekly). RESULTS: MicroCT analysis showed that C1H treatment significantly increased vertebral bone mineral density (vBMD) and trabecular bone volume versus OVX controls while C2 treatments did not. Biomechanical testing showed that C1H treatment but not C2 treatments led to significant improvement in the load bearing abilities of the vertebrae compared to OVX controls. C1 stimulated endocortical bone formation and increased load bearing in femurs, while C2 did not. CONCLUSIONS: We showed that C1 led to significant anabolic effects on cortical and trabecular bone while anabolic effects associated with C2 were minimal. These results led us to hypothesize a mode of action by which presence of a linker is crucial in facilitating the anabolic effects of EP4a when dosed as a prodrug with ALN.

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-1ß genes expression in a time dependent manner and without cAMP upregulation.

The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis.

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Previously, we reported that in macrophages (Mϕs), infection with avirulent Mtb H37Ra resulted in inhibition of necrosis by an inhibitory effect on mitochondrial permeability transition via the PGE2 receptor EP2. However, human Mϕs also express EP4, a PGE2 receptor functionally closely related to EP2 that also couples to stimulatory guanine nucleotide binding protein, but the functional differences between EP2 and EP4 in Mtb-infected Mϕs have been unclear. EP4 antagonist addition to H37Ra-infected Mϕs inhibited the expression of cyclooxygenase 2 (COX2) and microsomal prostaglandin E synthase-1 (mPGES-1), which are involved in PGE2 production. Moreover, H37Ra infection induced PGE2 production through the Toll-like receptor (TLR) 2/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Induction of COX2 and mPGES-1 expression by TLR2 stimulation or Mtb infection was increased after additional stimulation with EP4 agonist. Hence, in Mtb-infected Mϕs, PGE2 production induced by pathogen recognition receptors/p38 MAPK signaling is up-regulated by EP4-triggered signaling to maintain an effective PGE2 concentration.

Discovery of a novel EP2/EP4 dual agonist with high subtype-selectivity.

A series of γ-lactam prostaglandin E(1) analogs bearing a 16-phenyl moiety in the ω-chain and aryl moiety in the α-chain were synthesized and biologically evaluated. Among the tested compounds, γ-lactam PGE analog 3 designed as a structural hybrid of 1 and 2 was discovered as the most optimized EP2/EP4 dual agonist with excellent subtype-selectivity (K(i) values: mEP2=9.3 nM, mEP4=0.41 nM). A structure-activity relationship study is presented.