Prostanoid receptor EP2 as a therapeutic target.

Although very little bioactivity or biology is included in the patent literature, a brief summary on patented EP2 antagonists is presented due to the relatively limited number of chemical scaffolds available so far. Two pharmaceutical companies; Pfizer and Bayer Schering Pharma have identified small molecule antagonists for the EP2 receptor. Representative structures 16-22 are illustrated in Figure 6 . Pfizer has created 3-azetidine carboxylic acids and 3-azetidine carboxamides ( 16-17 ) with potent EP2 inhibition activity. 111 Compound 15 ( Figure 4 ; Table 2 ) emerged as a lead compound from the general structure 16 . Bayer Schering Pharma has also developed four distinct classes of antagonists represented by 18-21 , 112 - 115 but in vivo data is not available for any lead structure from these scaffolds 18-22 . Emory University also created a class of compounds represented by 22 , which display potent EP2 antagonist activity, 116 from which 13 ( Figure 4 ) has emerged as a lead compound. Several compounds from each class ( 16-22 ) show EP2 potency in the range of low nanomolar to low micromolar level ( Figure 6 ), and are available as backups for development into therapeutically useful compounds. Due to the large number of EP2 agonists published in the patent literature and scholarly articles, they are not highlighted here. 122 - 125

Inflammation plays a vital role in the pathogenesis of many chronic neurodegenerative 1 , 2 and peripheral diseases. 3 - 6 An acute injury to the brain or to the body induces inflammatory pathways as an immune response. Common features of inflammation include upregulation of cytokines and chemokines, activation of microglia (in the central nervous system) or macrophages (in the periphery), induction of cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) and oxidative-stress. 1 , 2 All of these features are manifested in patients as well as animal models of chronic neurodegenerative diseases such as Alzheimer’s disease (AD), 7 , 8 Parkinson’s disease (PD), 9 , 10 epilepsy, 11 - 14 amyotrophic lateral sclerosis (ALS), 15 and traumatic brain injury (TBI). 16 , 17 The inflammatory features are also evident in patients with other diseases such as rheumatoid arthritis (RA), 4 chronic obstructive pulmonary disease (COPD) 5 and inflammatory bowel diseases. 3 Prolonged activation of these inflammatory pathways results in a secondary damage to the neighboring neurons in the brain, and to the cells and tissues in the body exacerbating the disease pathology. Thus, inhibition of inflammatory pathways with small molecule inhibitors should provide benefit to patients by reducing the severity of the diseases. It is important to note that the inflammatory responses are critical for survival and homeostasis. However, more than one inflammatory pathway or mediator is activated simultaneously during and after an injury to maintain homeostasis and aid in repair. There is also cross-talk among some of these inflammatory mediators (cytokines, iNOS and COX-2). An important issue that is often difficult to address is whether these inflammatory mediators act independently or in concert to fight against potential pathogens or clearing the debris from the tissue. As a result, it will be ambitious to comprehend an anti-inflammatory therapeutic agent that blocks a single inflammatory target or pathway resulting in alleviation of an entire inflammatory cascade without interfering with compensatory mechanisms. However, such a therapeutic agent might mitigate the severity of the inflammation and disease progression to some extent. Nonetheless, small molecular agents that block a single biological target such as COX-2, iNOS, cytokines and glia are currently being pursued by investigators for anti-inflammatory therapy. The cyclooxygenase enzymes (COX-1 and COX-2) have been widely explored for anti-inflammatory therapy. 18 COX-1 is constitutively expressed throughout the body and is involved in a range of physiological functions including cellular homeostasis and protection of gastrointestinal integrity. 19 , 20 COX-2 is rapidly induced or upregulated after a variety of stimuli or injuries and remains upregulated in chronic inflammatory diseases. Both COX-1 and COX-2 isoforms activate prostaglandin signaling ( Figure 1 ). Several COX-1 selective inhibitors (a.k.a. non steroidal anti-inflammatory drugs (NSAIDs)) have been shown to produce on-target mediated side-effects, mainly gastrointestinal tract and renal toxicity. 21 It has been hypothesized that selective inhibition of COX-2 by small molecules could potentially eliminate COX-1 mediated toxicities, because COX-2 is induced only upon an insult or injury in inflamed joints and tissues. 22 , 23 Thus, several selective COX-2 inhibitors were discovered. 24 , 25 For example, celecoxib (Celebrex), rofecoxib (Vioxx) and valdecoxib (Bextra) have been clinically proven as anti-inflammatory and pain relievers for chronic indications such as osteoarthritis and rheumatoid arthritis. 26 , 27 Surprisingly, although elevated levels of COX-2 were observed in AD patients and COX-2 levels were correlated with progression of AD, 28 COX-2 inhibitors and NSAIDs have not shown any cognitive benefits to patients with AD. The data suggest that COX-2 inhibitors could have an adverse effect on AD pathogenesis. 29 , 30 Moreover, chronic use of COX-2 drugs like rofecoxib and valdecoxib for arthritis and other diseases also resulted in adverse cardiovascular side effects including myocardial infarction and stroke in a significant number of patients. 31 Consequently, rofecoxib and valdecoxib were withdrawn from the USA market. These side effects could impose severe restrictions on the future use of COX-2 drugs, particularly for elderly patients with AD, who are already at increased risk for heart disease. It was demonstrated that inhibition of the prostanoid receptor IP, which is activated by COX-2 derived prostaglandin PGI 2 (see Figure 1 ), is responsible for myocardial infarction, stroke and atherosclerosis. 32 , 33 Thus, future generations of anti-inflammatory therapy should be directed to target specific prostaglandin synthases or prostanoid receptors downstream of COX-2 rather than the generic block of the entire COX-2 signaling cascade ( Figure 1 ).

Creation and use of EP2 genetic knockout models provided a wealth of information regarding the function of EP2 receptor in physiological and pathophysiological conditions. However, it is important to note that EP2 knockout mice exhibited significant cognitive and social memory deficits, impaired spatial learning and prepulse inhibition, and heightened anxiety, suggesting an association between the EP2 receptor and long term hippocampal depression and learning and memory. 87 , 88 The loss of EP2 also has functional ramifications for reproduction, as EP2-lacking mice have decreased ovulation number and fertilization rate compared to wild type mice, and EP2 null females produce lower than normal litter sizes. 89 Other functional roles of EP2 identified by genetic manipulation studies in pregnant EP2 mice include arterial dilatation, salt-sensitive hypertension during pregnancy, and reduced female fertility. 90 Reports such as these may have contributed to the paucity in antagonistic drug discovery approaches for this receptor. However, it is unlikely that these developmental compromises will be observed with small molecule inhibitors. Thus, use of a highly specific EP2 receptor antagonist for longer periods of time may provide a definitive answer to whether EP2 antagonism has a therapeutic advantage in comparison to genetically manipulated mice.

In comparison to other prostanoid receptors, relatively fewer efforts have appeared towards discovery and development of small molecule modulators of EP2. 34 , 38 There are only two classes of agonists currently available. Representative members of these classes that are widely used and chemically optimized are shown in Figure 4 . The first class of agonists is composed of molecules that are structurally related to the endogenous ligand 1 , a non selective EP2 agonist with EP2 K i = 38 nM determined by a competitive binding assay. 75 The synthetic agonist 2 (butaprost) is about 63-fold less potent than 1 with an EP2 K i = 2400 nM. The agonist 2 is selective for EP2 over other prostanoid receptors in receptor binding assays, but was equally effective in activating IP receptors in a functional assay (see Table 1 ). 75 In a separate study, 2 was shown to be only 3 to 4-fold selective for EP2 over EP3 and EP4 receptors, but very selective against the IP receptor in binding assays. 104 The ester group of 2 is unstable and is expected to be cleaved metabolically to produce acid 3 ( Figure 4 ). The acid 3 is more potent than 2 with an EP2 K i of 73 nM. By comparison, 3 is only 2-fold less potent than 1 , however, the free acid 3 has lost selectivity to EP2 binding over IP receptor binding (IP K i = 870 nM). In a potency assay, 3 showed nearly the same activity for EP2 and IP receptors with an EC 50 of 32 nM and 25 nM, respectively ( Table 1 ). 75 The other agonist in the same class represented as compound 4 57 ( Figure 4 ) appears very selective for the EP2 receptor over other prostanoid receptors. The affinity of 4 to EP2 is nearly 10-fold higher than that of 1 and 20-fold higher than that of 3 ( Table 1 ). However, in functional assays, it has nearly the same potency as 1 , and roughly a 10-fold higher potency than 3 . 57 Compound 4 is found to be unstable as the free acid, but its stability is markedly improved when it is converted to the lysine salt. 57 Compound 5 shows a higher binding affinity for EP2 ( K i = 1.7 nM), a 22-fold higher affinity than 1 and 42-fold higher affinity than 3 . Compound 5 also shows ~700 to 1500-fold selectivity for EP2 over other prostanoid receptors such as EP1, EP3, EP4 and IP. 57 In a functional assay, it has about an 18-fold higher potency than 2 and 3 ( Table 1 ). The non-selective EP2 agonist AH-13205 105 , 106 ( 6) ( Figure 4 ) is also a member of this class, but because of its low potency it has not received much attention. The second class of EP2-agonists are pyridyl-sulfonamides. Compound 7 is the initial lead compound in the class, and has high binding affinity to EP2 (K i = 50 nM) with high selectivity for EP2 over other prostanoid receptors (16-fold selectivity against DP1 and 50 to 60-fold selectivity against EP1, EP3, EP4 and IP receptors). 60 A related compound in this class, 8 also displays high binding affinity to EP2 and at least a 270-fold selectivity against EP1, EP3 and EP4 receptors. The potency of 8 is equal to 1 in a cAMP determination assay ( Table 1 ). Importantly, a prodrug of 8 , taprenepag isopropyl ( 9 ) has been developed as a clinical candidate ( vide supra ). 83 The only class of EP2-allosteric potentiators reported, represented by 10 ( Figure 4 ) was developed recently at Emory University. Compound 10 potentiates EP2 receptors with EC 50 = 7.8 μM in a cAMP assay and also displays selective potentiation of EP2 receptors versus EP4 and β2-AR receptors. 46 Allosteric potentiators are designed to function in the presence of high PGE 2 concentrations that are expected to be present during tissue injury. Two drawbacks of these potentiators are that they are weakly active and poorly water soluble for dosing into animal models. However, 10 may be useful for in vitro studies. 46 The only EP2 antagonist widely used for probing the roles of EP2 is AH-6809 107 ( 11) ( Figure 4 ). 11 is commercially available, but it shows weak antagonistic activity (EP2 K i = 1150 nM) and very poor selectivity against other prostanoid receptors such as EP1, EP3 and DP1 ( Table 2 ). 104 Recently, Pfizer revealed the azetidine-carboxylic acid derivative PF-04418948 108 ( 15) ( Figure 4 ) as the first of a new class of selective EP2 antagonists with an IC 50 of 16 nM and Schild K B of 1.8 nM (a concentration required to cause a twofold increase of the EC 50 of PGE 2 ). 108 This antagonist exhibits >10,000-fold selectivity for the EP2 receptor over other prostanoid receptors (i.e. EP1, EP3, EP4, DP1 and IP) ( Table 2 ) and does not show significant effects on a diverse panel of other GPCRs. 108 However, brain penetration of this antagonist has not been disclosed and thus it is unclear whether this compound could be useful for in vivo evaluation in mouse models of CNS disease. Nonetheless, 15 has been shown to attenuate butaprost-induced vasodilatory blood flow in rats, demonstrating that it has the requisite plasma pharmacokinetics and tissue penetration to be useful as an in vivo probe for a variety of peripheral diseases. 108 Independently from Pfizer, Emory University identified a novel cinnamic acid amide class of EP2 antagonists, and has investigated them for proof of concept in mouse models of status epilepticus. 85 , 86 These compounds are unique in structure and do not belong to any other class of agonists or antagonists previously reported. Importantly, the Emory EP2 antagonists do not possess a carboxylic acid, which is intrinsic to the majority of the EP2-agonists and -antagonists discovered thus far ( Figure 4 ). Two representatives in this class of EP2 antagonists are 12 and 13 whose structures are shown in Figure 4 . 85 The prototype compound 12 has an EP2 Schild K B of 2.4 nM and displays 550-4750-fold selectivity for EP2 over EP1, EP3, EP4 and IP, but only 14-fold selectivity against the DP1 receptor ( Table 2 ). 85 Another lead compound in this class of EP2 receptor antagonists is 13 , which has an EP2 Schild K B of 17.8 nM, and displays greater than 300-fold selectivity against EP3, EP4 and IP receptors. Compound 13 shows a 100-fold selectivity against EP1, FP and TP receptors, but only 10-fold selectivity against the DP1 receptor. 86 Moreover, 13 shows a plasma half-life ( t 1/2 ) of 1.6 h and brain to plasma ratio of 1.6 in mice. These two Emory compounds ( 12 and 13 ) did not show significant off-target activity against a diverse panel of 47 enzymes and receptors including COX, NOS, PDE, adrenergic receptors, serotonin receptors and cytochrome P450 enzymes. So far 12 and 13 have only shown significant off-target activity at the serotonin 5-HT2B receptor with an IC 50 2.6 μM and 7.5 μM respectively. 86 , 109 These IC 50 values far exceed their respective EP2 Schild K B values (1080-fold higher for 12 , 420-fold higher for 13) . The data obtained thus far on these novel EP2 receptor antagonists suggest that 13 may serve as a valuable probe for proof of concept studies in CNS and other neurodegenerative animal models where EP2 receptors may mediate deleterious effects. Very recently, Emory university group discovered a second class of antagonists that are carbamothioylacrylamides. 110 A lead compound, TG6-129 110 ( 14 ) ( Figure 4 ) displayed EP2 potency at low nanomolar concentrations (EP2 Schild K B = 8.8 nM). Unlike 12 and 13 , compound 14 has shown high selectivity against DP1 (1660-fold) and EP4 (440-fold) receptors, but only 22-fold selectivity against the IP receptor. This compound did not cross the blood brain barrier and thus it might be useful as a probe for a variety of peripheral disease models shown in Figure 3 .

PGE 2 is a major product of increased COX-2 activity in the brain of rodents and human patients following various insults. It activates four G protein-coupled receptors (EP1, EP2, EP3 and EP4). EP2 receptor is expressed in various parts of the body and brain and plays context-dependent, beneficial, and deleterious roles. Thus, concerns are inevitable about whether EP2 receptor is druggable with small molecules, and if so, whether activation or inhibition is a better therapeutic strategy. Recent studies with mouse models of ischemic stroke and inflammatory neurodegenerative disease model of status epilepticus ( vide supra ) suggest both agonism and antagonism strategies may be explored for therapeutic development. Studies also suggest that the cell type from which EP2 receptors originate may affect the role of the receptor. For example, in the brain, EP2 receptors are expressed in neurons and glia. Thus, it is also possible that EP2 of neuronal origin may act, mainly through PKA activation, in an autocrine fashion to protect neurons from acute brain injury (as in stroke, or just after status epilepticus). The signaling from dying neurons, in conjunction with several other proinflammatory mediators produced during acute brain injury may activate EP2 receptors in microglia and astrocytes, which, predominantly via the Epac activation pathway, may exacerbate paracrine toxicity leading to neurodegeneration. Thus, as illustrated in Figure 7 , an appropriate therapeutic approach may be to use an EP2 agonist or an allosteric potentiator immediately after brain injury to maximize the beneficial effects. An alternative therapeutic strategy would be to use an EP2 antagonist, starting several hours to days after brain insult to break the glia-induced vicious inflammatory cycle, as a first line or adjunct therapeutic agent in patients with neurodegenerative diseases such as epilepsy, AD, PD, TBI and ALS. As recently described by Weiss et al., 117 it is not always true that the phenotype of small molecular inhibitors of a target mimics the phenotype of genetic knockout animals lacking the same target. There are several receptors and enzymes including a few kinases (e.g. Aurora B) known to show divergence. EP2 knockout mice were created many years ago and have been used extensively to explore the role of EP2 in a variety of disease models. However, EP2 receptor antagonists have been reported recently and have been explored in limited neurodegenerative disease models. Thus they should be examined for on-target mediated effects on healthy mice during chronic dosing (2-3 months) in order to characterize memory deficits, fertility complications, formation and loss of synapses in the hippocampus ( vide supra ). This experiment would yield valuable information to rule in or out whether an antagonism of EP2 is therapeutically beneficial. There are several highly characterized EP2 specific agonists now available ( Table 1 ), but a limited number of selective antagonists ( Table 2 ) that are useful for CNS and other disease models. Other than steroids, NSAIDs, and COX-2 inhibitors, currently, there is no other FDA approved anti-inflammatory therapeutic agent available which works through a specific inflammatory target. However, selective inhibitors of microsomal prostaglandin-E-synthase (mPGES) 118 and antagonists of prostanoid receptor EP4 119 are currently under preclinical evaluation. These agents (mPGSE-1 and EP4 inhibitors) may also alleviate some of the adverse side effects caused by COX-2 inhibitors. However, experiments designed to reduce inflammation or neurodegeneration (or both) in chronic inflammatory or neurodegenerative disease models (examples; arthritis, AD, epilepsy) using COX-2, and EP2 or EP4 or mPEGS-1 selective inhibitors head-to-head, is worth pursuing. Although the available EP2 agonists and antagonists are selective for EP2 in binding and in vitro functional assays ( Table 1 , 2 ), in vivo target-engagement studies 120 , 121 are lacking to ascertain whether the observed efficacy is mediated through the binding of EP2 modulators to the receptor in vivo . Demonstration of target engagement is a formidable task for EP2 receptor, and is complicated by the fact that there is no specific biomarker produced solely by the EP2 receptor, and lack of available radio-labeled agonists or antagonists that specifically bind to EP2 in vivo . Nonetheless, this study is very important, and if target engagement can be demonstrated, it will strengthen the published results and advance the EP2 receptor as a target for drug discovery.

Both COX-1 and COX-2 catalyze the conversion of arachidonic acid into endoperoxide intermediate prostaglandin-H 2 (PGH 2 ), which is then transformed by a variety of cell specific prostaglandin synthases to produce five prostaglandins (PGD 2 , PGE 2 , PGF 2 , PGI 2 and TXA 2 ). These natural ligands activate eleven different G protein-coupled prostanoid receptors (DP1, DP2, EP1, EP2, EP3, EP4, FPα, β, IP and TPα, β). 34 These prostanoid receptors are differentially expressed in various cells and tissues and show a physiological and pathological role in a variety of conditions. 35 Based on structural and functional properties, these prostanoid receptors are classified into three major groups. The first group called relaxant receptors, consists of DP1, EP2, EP4 and IP receptors, because they mediate smooth muscle relaxation through cyclic AMP (cAMP) production. The second group known as contractile receptors, consists of EP1, FP and TP receptors, because they induce smooth muscle contraction by intracellular Ca 2+ mobilization. The third group known as inhibitory receptors, has only two members (EP3 and DP2), that inhibit the production of cAMP. Activation of the DP2 receptor has been shown to increase intracellular Ca 2+ levels as well ( Figure 1 ). Additional articles regarding the detailed description of prostanoid receptor properties and function have been published recently. 34 , 36 - 38 Thus, in this perspective article, we focus specifically on the EP2 receptor and the binding of the natural ligand prostaglandin-E 2 (PGE 2 ) 39 ( 1 ) ( Figure 4 ) and synthetic analogs. Both a beneficial and deleterious role of EP2 has been suggested in central nervous system (CNS) and peripheral diseases by making use of EP2 knockout mouse models and selective small molecular agonists and antagonists developed for EP2 (see below).

A significant body of evidence suggests that EP2 activates downstream signaling either by a G protein-coupled or by G protein-independent mechanism. 91 - 95 As shown briefly in Figure 5 , activation of EP2 by PGE 2 stimulates adenylate cyclase ( via Gαs) resulting in cAMP, which mediates events either through protein kinase A (PKA), 95 or exchange protein activated (Epac). 96 , 97 PKA activates transcription factors such as cAMP-responsive element binding protein (CREB), which is involved in neuronal survival and neurogenesis. 98 , 99 Epac initiates downstream effectors Rap1/2 to regulate neuronal differentiation, neuronal excitability, learning, memory and social interactions. 100 - 102 It has been shown that there is cross-talk between PKA and Epac and that PKA and Epac work synergistically to activate CREB. However, more often they work antagonistically. Generally, it is believed that PKA activation is associated with neuronal survival, whereas Epac activation leads to neuronal injury. EP2 also regulates other downstream effectors independent of G protein-coupling via β-arrestin to promote or exacerbate disease conditions including cancer proliferation and metastasis ( Figure 3 and Figure 5 ). Thus, EP2 is a complex target displaying pleotrophic roles depending on the circumstances. For simplicity, only a few downstream pathways are described here. However, it is important to remember that some of the downstream effectors shown in Figure 5 are also influenced by other G protein-coupled receptors, like EP4. 91 - 95