Design and Development of Microsomal Prostaglandin E2 Synthase-1

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Design and Development of Microsomal Prostaglandin E2 Synthase-1 Inhibitors: Challenges and Future Directions Andreas Koeberle, Stefan A. Laufer, and Oliver Werz J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01750 • Publication Date (Web): 20 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Journal of Medicinal Chemistry

Design and Development of Microsomal Prostaglandin E2 Synthase-1 Inhibitors: Challenges and Future Directions Andreas Koeberle,*,†, Stefan A. Laufer,§ and Oliver Werz*,†

†

Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, 07743 Jena, Germany

§

Department of Pharmaceutical Chemistry, Pharmaceutical Institute, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

ACS Paragon Plus Environment

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ABSTRACT

Microsomal prostaglandin E2 synthase (mPGES)-1 is responsible for the massive PGE2 formation during inflammation. Increasing evidence reveals mPGES-1 inhibitors as a safe alternative to non-steroid anti-inflammatory drugs. The first selective mPGES-1 inhibitors recently entered clinical trials. Major challenges for drug development have been the high plasma protein binding of lead structures, interspecies discrepancies, nuisance inhibition, the sophisticated enzyme assays and limited structural information about the mPGES-1 inhibitor binding site. Since most of these drawbacks could be solved during the last few years, we are standing at the threshold of a new era of mPGES-1-targeting anti-inflammatory drugs. This perspective introduces mPGES-1 as key player within the network of eicosanoid biosynthesis and summarizes our current understanding of its structure and mechanism. Moreover, we present high-throughput and in silico screening techniques and discuss the structure-activity relationship and pharmacological potential of major mPGES-1 inhibitor classes in light of recent insights from pharmacophore models and co-crystallization studies.


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INTRODUCTION Microsomal prostaglandin E2 synthase (mPGES)-1 catalyzes the terminal step in the biosynthesis of prostaglandin (PG)E2 from arachidonic acid (Fig.1).1 The lipid mediator PGE2 is secreted from cells, binds to G protein-coupled receptors (EP1-4) and promotes inflammatory processes, fever and pain but is also important for the resolution of inflammation, the protection of the gastrointestinal mucosa, natriuresis, blood pressure regulation and ovulation.1-3 Two other PGE2 synthases have been identified besides mPGES-1. The constitutively expressed cytosolic PGE2 synthase (cPGES) is predominantly responsible for homeostatic PGE2 production,4 whereas the inducible mPGES-1 governs PGE2 synthesis during acute and chronic inflammation, for example, in arthritis, atherosclerosis and cancer.1,5 The biological function of mPGES-2 is poorly understood.3,4 Current anti-inflammatory, analgesic and anti-febrile therapies resort to non-steroidal antiinflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX) and thus suppress the formation of PGE2 and other COX-derived PGs with important physiological functions.5,6 Longterm treatment with NSAIDs is associated with severe side effects such as gastrointestinal bleeding, ulceration and perforation, which could be ascribed to the inhibition of the isoenzyme COX-1.5 Isoform-selective COX-2 inhibitors (coxibs) were consequently developed. COX-2 is induced by pro-inflammatory stimuli, functionally coupled to mPGES-1 and together with mPGES-1 responsible for the massive production of PGE2 and other PGs during inflammation.1,7 Unlike originally expected, coxibs were not free of gastrointestinal side effects, and their longterm intake has later on been associated in large-scale clinical trials with an increased risk for myocardial infarction, stroke, systemic and pulmonary hypertension, congestive heart failure, and sudden cardiac death.6 These cardiovascular complications are considered to arise from a


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lack of COX-2-derived prostacyclin (PGI2) with anti-thrombotic, vasoprotective and vasodilatory properties. After several coxibs have been withdrawn from the market, such as rofecoxib (VIOXX®) in 2004, mPGES-1 moved into the focus of interest as target for anti-inflammatory drug development.8 Inhibitors of mPGES-1 have been expected to be less afflicted with side effects than classical NSAIDs and coxibs because they exclusively interfere with pro-inflammatory PGE2 formation without affecting the production of other PGs.1 The intensive research on mPGES-1 in the following years revealed comparable efficiency of interference with mPGES-1 and COX-2 in diverse animal models of inflammation,2,3,5,9,10 though also differences became evident. For example, inhibition of mPGES-1 has been claimed to be less efficient in relieving pain compared to COX because PGI2 and other PGs also contribute to pain hypersensitivity.11 Large efforts have been spent to recognize side effects resulting from selective inhibition of mPGES-1. The controversial discussion about the pharmacological potential of mPGES-1 as safe drug target reached its peak after inhibition of mPGES-1 has been shown to redirect the COX product and mPGES-1 substrate PGH2 towards the biosynthesis of other PGs.3,11 The blockage of mPGES-1 either elevated levels of thromboxane (Tx)B2, PGI2 and/or PGD2 or was without effect depending on the cell type- and tissue-specific expression pattern of PG synthases. The redirection of PGH2 is not necessarily detrimental. The increase of PGI2 production, for example, might even be advantageous for the cardiovascular safety of mPGES-1 inhibitors.12 Meanwhile, multiple cellular and animal studies have drawn a more complete picture about the physiological interrelations of mPGES-1 as described in several excellent reviews.1,2,12-14 In contrast to classical NSAIDs and coxibs, inhibition of mPGES-1 does not evoke gastrointestinal complications and apparently also lacks renal and cardiovascular adverse effects, at least in


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healthy, non-challenged individuals. Syndromes of cardiovascular inflammation are even impaired by inhibition of mPGES-1.12,15-17 Physiological functions of mPGES-1 have been reported in the renal, cardiovascular and immune system, especially under distinct stress and pathophysiological conditions, such as an acute water loading, high salt diet, atherosclerosis or cancer.12,13,18-22 Taken together, current evidence suggests that mPGES-1 inhibitors are efficient in the treatment of inflammatory diseases and are less afflicted with side effects compared to NSAIDs and other anti-inflammatory drugs (such as glucocorticoids), proposing mPGES-1 as promising target for the treatment of chronic inflammatory diseases.

BIOSYNTHESIS OF PGE2

The biosynthesis of PGE2 is initiated by phospholipases (PL)A2, which cleave the sn-2 ester bond of phospholipids and release arachidonic acid from membranes upon activation (Fig. 1).7,23 More than 30 different PLA2 enzymes have been identified so far, which differ in their phospholipid headgroup and fatty acid specificity.24 The isoenzyme cytosolic phospholipase (cPL)A2α possesses specificity for arachidonic acid-containing phosphatidylcholines and is activated by Ca2+-influx and phosphorylation through diverse kinases including mitogenactivated protein kinases, protein kinase C and Ca2+/calmodulin-dependent protein kinase.25 cPLA2α essentially contributes to the massive eicosanoid production during inflammation but is also involved in regulating the basal availability of arachidonic acid during homeostasis. The cellular concentration of free arachidonic acid is tightly limited by an interplay of PLA2s (which release sn-2 fatty acids from phospholipids), lysophospholipid acyltransferases (which incorporate sn-2 fatty acids into phospholipids) and alternative arachidonic acid-generating


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systems such as the successive degradation of phosphatidylinositols by phospholipase C, diacylglycerol lipase and monoacylglycerol lipase.24,26,27

Figure 1. Biosynthesis of PGE2 and other bioactive oxylipins. Polyunsaturated fatty acids, such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are released by PLA2s from membrane phospholipids, in particular phosphatidylcholines. Enzymatic oxygenation of AA by either COX, lipoxygenases (LOs) or cytochrome P450 monooxygenases (P450)

and

further

metabolization

yields

pro-inflammatory/non-resolving

and

anti-

inflammatory/pro-resolving lipid mediators. For PGE2 biosynthesis, AA is converted by COX to PGH2 and subsequently isomerized to PGE2 by PGE2 synthases. EET, epoxyeicosatrienoic acid;


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HDoHEs, hydroxydocosahexaenoic acids; HEPE, hydroxyeicosapentaenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LT, leukotriene; LX, lipoxine; SPM, specialized pro-resolving mediator.

Enzymatic oxygenation of arachidonic acid yields a variety of bioactive lipid mediators with both pro-inflammatory/non-resolving and anti-inflammatory/pro-resolving properties.7 Three major pathways are subdivided according to the respective key oxygenase: COX, lipoxygenase (LO) and cytochrome P450 monooxygenase (Fig. 1). The membrane-bound protein COX catalyzes both the dioxygenation of arachidonic acid to PGG2 and the subsequent reduction of PGG2 to the instable endoperoxide PGH2. COX is located at the luminal side of the endoplasmic reticulum and on the inner and outer membrane of the nucleus.28 The isoenzyme COX-1 is constitutively expressed in most tissues and contributes to homeostatic PGE2 production.7 Only few tissues (such as kidney and brain) express COX-2 at a basal level, though this isoenzyme is induced by pro-inflammatory stimuli and mitogens in a variety of cell types, which renders COX-2 a key enzyme for PGE2 production in inflammation.7 The COX product PGH2 is isomerized by prostaglandin synthases to PGE2, PGD2, PGF2α, PGI2 and TxA2 (Fig. 1).1,23 Prostaglandin synthases are cell type- and tissue-specifically expressed and shape the multifaceted prostanoid profiles.4,29 Three PGE2 synthase isoenzymes have been identified so far: cPGES, mPGES-1 and mPGES-2. cPGES is ubiquitously and constitutively expressed with few exceptions, functionally coupled to COX-1 and mainly considered responsible for homeostatic PGE2 synthesis.4,30 Deletion of cPGES is perinatal lethal and associated with severe morphological defects.4 It is difficult to estimate the extent to which


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biological functions ascribed to cPGES are related to its PGE2 synthase activity because cPGES (alternative name: p23) is also a co-chaperone of heat shock protein 90.31 mPGES-2 is an integral protein of the Golgi, which can be released as active enzyme into the cytosol by proteolytic cleavage, and is constitutively expressed in diverse organs, especially in brain, heart, skeletal muscle, kidney and liver.4 The biological relevance of mPGES-2 has been a mystery as knockout studies neither found an obvious phenotype nor altered PGE2 levels in diverse tissues.32 It has been speculated that mPGES-2 binds heme in vivo, which might shift the enzyme activity from isomerization to degradation of PGH2 as shown in vitro.33 Recent findings suggest a role of mPGES-2 in substituting for cPGES and mPGES-1 under pathophysiological conditions, e.g., in Alzheimer’s Disease.34 mPGES-1 has been recognized as key enzyme for inflammation, fever, pain and diseases with inflammatory component such as arthritis, atherosclerosis, stroke, neurodegenerative diseases and cancer.1 In concert with COX-2, mPGES-1 synthesizes large amounts of PGE2 during inflammation (Fig. 1). Although constitutively expressed at a low level in some organs (e.g., the reproductive system, lung, gastric mucosa and kidney), mPGES-1 is in first line an inducible enzyme which is strongly upregulated in inflamed tissues and overexpressed in tumors.1 mPGES-1 is bound to the endoplasmic reticulum and preferentially accepts PGH2 from COX-2.1 Colocalization studies suggest that COX-1 takes over the role of COX-2 in supplying PGH2 to mPGES-1 under certain conditions, for example, in distinct regions of the kidney.35 Moreover, PGH2 has been shown to be provided by neighboring cells, which is astonishing in light of its short half-life.36 COX-2 and mPGES-1 are often co-induced because their promotors share binding sites for the transcription factors early growth response protein (EGR)-1 and nuclear factor (NF)-κB.1,5 Despite similar mRNA expression kinetics, protein expression of mPGES-1


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tends to be delayed compared to COX-237 suggesting differences in either mRNA processing or translation. Deletion or inhibition of mPGES-1 induces complex changes in the prostanoid profile due to redirection of PGH2 to other PG synthases.3,11 The expression pattern of PG synthases differs between tissues and experimental conditions and thereby determines whether levels of distinct prostanoids (PGD2, PGF2α, PGI2, TxA2) either increase or remain unaltered upon blockage of mPGES-1. Indirect effects on the eicosanoid profile, which are not necessarily limited to COXderived prostanoids, might result from impaired EP receptor signaling when PGE2 levels decline. EP receptors initiate feedback mechanisms,38 regulate the activation and expression of key enzymes in eicosanoid biosynthesis7,38 and represent key players for eicosanoid class switching from pro-inflammatory to pro-resolving lipid mediators during the resolution of inflammation.39 Furthermore, systemic changes in the lipid mediator profile might be the consequence of an altered recruitment and/or activation of immune and other eicosanoid-producing cells. Along these lines, the small number of lipidomic studies conducted so far revealed extensive changes of the (non-prostanoid) eicosanoid profile by deletion of mPGES-1.3,19,40 One of the major challenges in future mPGES-1 research will be to combine comprehensive eicosanoid profiling with network science approaches to rapidly address safety concerns of mPGES-1 inhibitors.

STRUCTURE AND MECHANISM OF MPGES-1

mPGES-1 (16 kDa, 152 amino acids) is a member of the MAPEG (membrane-associated proteins involved in eicosanoid and glutathione metabolism) superfamily to which also 5-


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lipoxygenase- activating protein (FLAP), leukotriene C4 synthase and the microsomal glutathione transferases (MGST)1/2/3 belong.41 The development of early 3D homology models of mPGES-1 was based on MGST1 which shares the highest sequence identity to mPGES-1 (39%).42-44 The first structure of mPGES-1 was determined at low-resolution by electron crystallography in 2008.45 mPGES-1 forms homotrimers with each subunit consisting of four transmembrane helices like other members of the MAPEG family (Fig. 2A). The center of the mPGES-1 trimer consists of a funnel-shaped cavity, which opens towards the cytoplasm and expands well into the transmembrane region (Fig. 2B). A

B

C

Figure 2. Crystal structure of human mPGES-1 in complex with glutathione, modified according to ref46 (A) Organization of the mPGES-1 trimer. (B) Surface structure of mPGES-1 (left) and interior of mPGES-1 showing the funnel-shaped cavity (right). (C) Structure of the active site at the interface between two monomers. The crystal structure of human mPGES-1 in complex with its essential cofactor glutathione (GSH) was recently resolved at high-resolution (1.2 Å).46 The mPGES-1 trimer contains three active site cavities which are formed by the transmembrane helices 1, 2 and 4 and the cytoplasmic (C)-domain of the adjacent monomer (Fig. 2C). The positively charged C-domain (20 amino acids) connects transmembrane helices 1 and 2, consists of two loops and a helix and


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is not present in other MAPEG members like FLAP and leukotriene C4 synthase.47,48 The active sites are located at the interface between the monomers and have their entrance near to the cytoplasmic side of the transmembrane-spanning region.46 Since COX generates PGH2 at the luminal side of the endoplasmic reticulum, PGH2 has to diffuse through the membrane for transfer to mPGES-1.45 PGH2 is believed to enter the active site pocket with its peroxofuran head group.46 The two flexible aliphatic chains of PGH2 protrude from the pocket and might be inserted into the membrane, or they interact with the membrane/cytosol interface. The cofactor GSH is bound within the active site in a bent conformation and coordinated by hydrogen bonds and π-stacking interactions (Fig. 2C).46 The size of the active site entrance differs between humans and rodents (mouse/rat but not guinea pig) depending on the so-called gate keepers - a number of certain amino acids situated in transmembrane helix 4 and the C-domain. Mouse and rat mPGES-1 contain more bulky, sterically hindered and/or aromatic amino acids in these positions than the human enzyme (human: Thr131, Leu135, Ala138 and potentially Arg52 and His53; rat: Val131, Phe135, Phe138, Lys52 and Arg53).46,49 As consequence, several potent inhibitors of human mPGES-1, such as phenanthrene imidazoles, failed to suppress rat and mouse enzymes2,10 as discussed in detail below. Jegerschöld et al. proposed that mPGES-1 might switch between two conformations45 as previously described for leukotriene C4 synthase: a closed conformation without access to the active site (which was actually determined by electron crystallography) and an open confirmation for substrate loading.50 The high-resolution crystal structure of mPGES-1 shows the enzyme in the open confirmation and neither supports nor disproves the hypothesis of a conformational change.46 Other differences between the highresolution and low-resolution structure of mPGES-1 are related to the C-domain and the GSH


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binding mode, which is why caution should be used when interpreting findings from computational models based on the former mPGES-1 structure. The top of the cone-shaped cavity in the center of the mPGES-1 trimer is in close proximity to the active sites.46 The central cavity and the active site pockets are separated by the side chain of Arg73 but would merge to a large super pocket for an alternative conformation of the amino acid. Still elusive is whether conformational changes of Arg73 are of physiological relevance. Several potential functions have been discussed for the super pocket including i) a mechanism for the polar GSH to access the active site within the phospholipid bilayer and ii) an element of cooperativity,46 through which the 1:3-site reactivity of mPGES-1 might be explained.51 Site-directed mutagenesis and crystallographic studies provided detailed insights into the catalytic mechanism of mPGES-1.46 Deprotonation of GSH is critical for all catalytically active MAPEG family members (Scheme 1),45,46,52 though the subsequent stabilization of the thiolate seems to be differentially realized between them.48 There is strong evidence for mPGES-1 that Ser127 (rather than Arg126) stabilizes the GSH thiolate by forming a hydrogen bridge. Two mechanisms are currently discussed for the cleavage of the PGH2 peroxide bond.46 The thiolate might act as base and abstract the proton in 9-position, which results in the opening of the peroxide bond under formation of an unstable reaction intermediate with an alcoholate group in 11-position. Subsequent protonation of the alcoholate yields PGE2. In an alternative scenario, the GSH thiolate acts as nucleophile and directly attacks the peroxide bond of PGH2 under formation of a mixed sulfide. Deprotonation in 9-position and cleavage of the S-O bond lead to the same alcoholate intermediate as proposed for the first mechanism. Both alternatives require a proton acceptor nearby which either takes over the proton from carbon-9 or the sulfhydryl proton from


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GSH during the regeneration of thiolate. Sjörgen et al. suggested that the catalytic base is deprotonated Asp49 and that its basicity is increased through the proximate Arg126.46

Scheme 1. Hypothetic catalytic mechanism of mPGES-1. Two alternative mechanisms are currently discussed for the isomerization of PGH2 by mPGES-1. The thiolate of GSH might either function as base and abstract the proton at C9 of PGH2 or may nucleophilically attack its endoperoxide bond under formation of an S-O bond.


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EXPERIMENTAL HIGH THROUGHPUT SCREENING OF MPGES-1 INHIBITORS

Suppression of massive PGE2 biosynthesis by inhibition of mPGES-1 is a reasonable pharmacological strategy for intervention with many diseases where PGE2 is strongly upregulated. Accordingly, drugs that target mPGES-1 are considered as valuable alternatives to NSAIDs/coxibs with improved selectivity. They may offer the potential of a better safety profile and thus, lower risk of side effects that are usually associated with NSAIDs and coxibs,1,3,5,10,53 but this awaits extensive evaluation in humans. Until today, there is no selective mPGES-1 inhibitor available for clinical use, but just recently the first clinical trial with an mPGES-1 inhibitor has been reported (discussed below).54 Experimental biological approaches to discover mPGES-1 inhibitors are essentially pursued using cell-free mPGES-1 activity assays. Most of these assays are based on microsomes derived from mPGES-1-(over)expressing cell lines such as human A549, transfected HEK293, and HeLa cells or murine RAW264.7 macrophages, or recombinant human mPGES-1 is used as enzyme source. The reaction is initiated by exogenous addition of the substrate PGH2 (or [3H]-PGH2).2,53 The direct conversion of PGH2 to PGE2 by mPGES-1 can be monitored by RP-HPLC (at 195 nm or radiometric detection),41,55 ELISA, or by NADH produced by 15-hydroxy-PG dehydrogenase in a PGE2-dependent manner.56,57 These cell-free assays allow to analyze the direct interference of a given test compound with mPGES-1 activity and are suitable for inhibitor screening approaches. Inclusion of the detergent triton-X100 (0.1%) in the assays helps to exclude nuisance inhibition, which is relevant for highly lipophilic compounds that may form colloid-like aggregates which in turn inhibit mPGES-1 without specific interaction.58


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Various types of high throughput screening (HTS) systems for identification of mPGES-1 inhibitors were reported that essentially differ with respect to the analysis of PGE2. For instance, in 2005, Massé et al. presented an automated 13-step HTS method for mPGES-1 inhibitors using EIA for PGE2 quantification.59 Later, Goedken et al. described a 96-well plate-based competition assay applying homogenous time-resolved fluorescence for PGE2 monitoring

60

while Spahiu et

al. presented an assay with thiobarbituric acid-based detection of PGH2 and GSH.61 Because PGH2 is quite unstable and decomposes to a mixture of PGE2, PGD2 and PGF2α nonenzymatically within minutes at room temperature in aqueous buffers, most of the methods applied short enzymatic reaction times of 0.5 to 1 min at low temperatures, i.e., 4 °C. Unreacted PGH2 substrate is afterwards chemically decomposed to PGF2α. Anderson et al. recently reported a 384-well automated assay for room temperature analysis of mPGES-1 inhibitors.62 Finally, a fluorescent mPGES-1 activity assay, miniaturized to 1536-well plates, was introduced, where PGH2 is generated in situ by COX-2 from arachidonic acid, and PGE2 is then detected by coupling through 15-PGDH and diaphorase.63 In contrast to HTS-compatible cell-free assays, cell-based test systems (using isolated cells or whole blood assays) are less suitable for mPGES-1 inhibitors screening attempts, because the direct mPGES-1-mediated transformation of PGH2 to PGE2 within the cellular environment cannot be unequivocally monitored. Thus, exogenous supplementation of instable PGH2 as mPGES-1 substrate to intact cells is not feasible. Most cells express also cPGES and/or mPGES2 that contribute to cellular PGE2 synthesis as well.64 Also, suppression of PGE2 formation in intact cells by a given compound may be the consequence of inhibition of other targets including PLA2, COX enzymes and upstream signaling molecules, or might be related to effects on the


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expression of PGE2-synthesizing enzymes or unspecific detrimental modulation on cellular functionality.3

VIRTUAL SCREENING OF MPGES-1 INHIBITORS

De novo designs of mPGES1 inhibitors follow either ligand- or structure based design. H N

Cl O

H N Cl

H N

O

N

O

O S

Cl

N

F O S HO O

1 Cl N

O

O

Cl

O

2

5

HO

N+ OO

O O2N

N

OH

F

F

N

N N

O OH

3

6

HOOC

N S Cl

O

O

N

CF3

N O

4

O

O

HO N O

NH S

O

N N O

O

N H

O

7

O

8

Scheme 2. Exemplary structures of mPGES-1 inhibitors identified by virtual screening

Ligand-based approaches


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Rörsch et. al reported a multistep virtual screening protocol based on a 2D pharmacophore model and an ASINEX library of 360,169 structures.65 Special focus was placed on non-acidic hits. As a result, compound 1 with an IC50 of 0.5 µM was identified. Another pharmacophore-based virtual screening approach focused on acidic compounds and made use of NCI and Specs databases of 450,000 structures.66 Waltenberger et al. were able to identify submicromolar inhibitors (IC50 = 0.4 and 0.5 µM) in cell free assays, e.g., compounds 2 and 3. Whereas poor drug-like properties have to be expected for 3, compound 2 might be a good starting point for chemical modifications. The urea structure allows straight forward synthetic strategies. Hamza et al. constructed a pharmacophore model derived from the bonding pattern of two known mPGES1 inhibitors.67 In total, 2.1 million structures (including isomers, conformers, tautomers, etc.) were filtered. This approach led to 21 virtual ligands which were experimentally investigated for inhibition of mPGES-1. As a result, two single-digit micromolar hits (compound 4 with IC50 = 3.5 µM and compound 5 with 4.6 µM) were characterized. Both structures are, however, quite similar to already known inhibitors.

Structure-based approaches The lack of x-ray structures for mPGES-1 in its proposed open conformation led De Simone et al. to use the closely related structure of MGST-1 for the structure-based design of mPGES-1 inhibitors, which finally succeeded in compound 6 with an IC50 of 3.2 µM.68 Copper-catalyzed click chemistry allowed rapid access to a small library of triazine-structures. He et al. relied on a MGST-based model as well.69 Thus, by screening a 3D structure library of 197,211 compounds (SPECS library), the group was finally able to identify compounds (including compound 7) with


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IC50 values as low as 4 nM. Several other rather diverse structures with nanomolar activity impressively underline the potential of this approach. In 2013, Sjörgen et al. published the first high (1.2 Ǻ) resolution x-ray structure of mPGES-1 (4AL0).46 After having such high quality structural data, de novo design and virtual screening may open a new avenue to potent and selective mPGES-1 inhibitors. In a first attempt, Lauro et al. used docking studies to generate a small set of dihydropyrimidine-2-ones.70 The most potent derivate was compound 8 with an IC50 of 4.16 µM. In conclusion, both ligand- and structure-based approaches promise novel drug-like hits, with very good inhibitory activity against mPGES-1. Ligand-based virtual screening has already proven success yielding further optimizable leads.65 The recently published high quality x-ray structure may open an avenue for high quality hits from structure-based approaches as well. By combining 3D library searches and powerful docking methods, we may expect a rapid increase in structurally diverse nanomolar mPGES-1 inhibitor candidates in the near future.“

STRUCTURE-ACTIVITY-RELATIONSHIP OF MPGES-1 INHIBITORS

Initial approaches in identification of small molecules that interfere with mPGES-1 revealed endogenous fatty acids including arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and corresponding lipid mediators such as leukotriene C4, PGJ2 and 15-deoxy-∆12,14-PGJ2 as direct mPGES-1 inhibitors with moderate potency.71. Also, well-recognized NSAIDs (e.g., N-[2cyclohexyloxy-4-nitrophenyl] methanesulfonamide (NS-398),72 sulindac, dimethylcelecoxib) were found to inhibit mPGES-1, some of them were active in the low micromolar range,3 and


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recent attempts in modifying the structure of the NSAIDs lonazolac and indomethacin led to mPGES-1 inhibitors with IC50 = 0.16 and 0.9 µM, respectively, devoid of COX-1 inhibition.73 To date, numerous structurally distinct chemotypes and series have been identified as inhibitors of mPGES-1. The major challenges in the design and development of synthetic mPGES-1 inhibitors were and are still related in finding candidates with (I) high potency against mPGES-1, (II) marked selectivity over COX enzymes and other PG synthases, (III) low plasma protein binding and efficiency in whole blood, (VI) and lack of interspecies differences, i.e., efficacy against rodent mPGES-1 in order to allow for in vivo studies in mice or rats.


19


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Cl S

NC N F

N

COOH N

N H NC

COOH

9

10

N

11

Cl Cl Cl Cl

12

N H

F

13

Cl

O

Cl

N

N

N Br

N H

N H

Cl N 14

F

Cl Cl

NH S O O Cl

N O

N H

15

16 O

N

N H O

S

O

Cl

HN

17

O

F3C

18

O

N H F3C

Cl

O

O N H

O

NH NH

20

H N

19

N

N NH

Cl

N

N N

NH

F

F F O

OH

N

HN

O

N O

HN

Cl

N F3C

O

N

OH O

CF3

N H

O 21

F F

Scheme 3. Structures of mPGES-1 inhibitors.

The FLAP inhibitor 9 (MK-886),74 an indole-carboxylic acid derivative carrying lipophilic pchlorobenzyl, isopropyl, and tert-butylthio residues, was the first synthetic mPGES-1 inhibitor described,75 with, however, low potency in cell-free assays (IC50 rat mPGES-1 = 2.4 µM76 and human mPGES-1 = 1.6 µM77) as compared to FLAP antagonism (IC50 = 2.5 nM74). Moreover,


20

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the efficiency of 9 is strongly reduced in the presence of plasma protein or in cell-based assays (i.e., whole blood assay),77,78 and the compound also blocks COX-1 (IC50 = 8 µM).79 Attempts to obtain more potent and selective inhibitors based on the core structure of compound 9 led to even more lipophilic derivatives where the isopropyl residue in 5-position of the indole was replaced by halogenated biphenyl residues, exemplified by compound 10 that inhibited mPGES-1 (cell-free) with IC50 = 3 nM, and greater that 100-fold selectivity over FLAP, mPGES-2, and thromboxane synthase.77 PGE2 synthesis in cell-based assays was repressed by these compounds in the low micromolar range but due to the high lipophilicity of the biphenyl residue, strong plasma protein binding caused failure of mPGES-1 inhibition in whole blood, and thus, anti-inflammatory efficacy in vivo was not further explored.77 HTS at Merck Frosst led to the discovery of three different chemotypes that were explored as mPGES-1 inhibitors in vitro and in vivo: (I) phenanthrene imidazoles, (II) 2,4-biarylimidazoles, and (III) trisubstituted urea derivatives. Among the phenanthrene imidazoles, the lead 11 (MF63)80 that carries a 2,6-dicyano-substituted phenyl ring in 2-position of the imidazole was most promising.80 Besides high potency against human and guinea pig mPGES-1 in cell-free assays (IC50 = 1.3 and 0.9 nM, respectively) and >1000-fold selectivity over other prostanoid synthases, compound 11 is still markedly active in human whole blood assays (IC50 = 1.3 µM). Although compound 11 exhibited analgesic activity in guinea pigs after oral application of 30 - 100 mg/kg, it failed to inhibit mPGES-1 from rat and mouse.80 Nevertheless, knock-in of human mPGES-1 into mice revealed selective suppression of PGE2 in vivo along with reduced LPS-induced pyresis, hyperalgesia, and iodoacetate-induced osteoarthritic pain while being devoid of NSAIDlike gastrointestinal toxicity.81 Further improvements resulted in derivatives with up to 10-fold higher efficiency in human whole blood (IC50 = 0.14 µM) and better efficacy in a guinea pig


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Page 22 of 64

hyperalgesia model (i.e., at 14 mg/kg, p.o.).82 The current development status of these promising inhibitors is unknown. For the 2,4-biarylimidazoles, structural optimization of the hit compound 12 (IC50 = 660 nM) led to potent and selective human mPGES-1 inhibitors exemplified by compound 13 with IC50 = 1 nM (cell-free).83 Inhibition of rodent mPGES-1 was not reported. However, the required modifications, in particular the insertion of hydrophobic phenyl alkyne residues, increased the overall lipophilicity and thus caused a serum shift and loss of efficiency in whole blood assays (IC50 = 1600 nM).83 Follow-up studies or further preclinical analysis on these mPGES-1 inhibitors have not been reported yet. The third class of mPGES-1 inhibitors that originated from the HTS at Merck Frosst are trisubstituted urea derivatives where incorporation of large hydrophobic residues, i.e., aryl alkynes, yielded compounds, exemplified by compound 14, that potently inhibited human mPGES-1 in cell-free assays (IC50 = 1 - 3 nM) with, however, strong loss of potency in the whole blood assay (IC50 = 2.1 µM).84 Inhibition of rat and murine mPGES-1 and antiinflammatory or analgesic efficacy in vivo were not addressed. HTS performed at Pfizer identified two structural classes of mPGES-1 inhibitors: (I) dioxobenzo-thiazinones and (II) benzoxazoles. Wang et al. presented a hit compound with a benzo-thiopyran S-dioxide core that moderately inhibited human mPGES-1 (IC50 = 1.68 µM) with selectivity over COX-2 (IC50 > 90 µM).85 Replacement of the benzo-thiopyran by dioxobenzo-thiazinone, a scaffold found in oxicame-type COX inhibitors, led to potent mPGES1 inhibition, exemplified by compound 15 with IC50 = 16 nM in cell-free assays. Compound 15, decorated with a 3,4-dichloro-biphenyl moiety also repressed PGE2 synthesis in intact cells (0.42


22

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µM) with >200-fold selectivity over COX-2, but high lipophilicity and tendency for protein binding again impaired PGE2 suppression in human whole blood (IC50 about 5 µM).85 The second HTS hit with a benzoxazole core yielded to a series of orally active, selective benzoxazole piperidinecarboxamides with IC50 = 2-3 nM for cell-free mPGES-1.86 Structural optimization aiming at reducing lipophilicity led to good potency in whole blood (IC50 = 53 - 240 nM). The respective lead compounds (10 mg/kg; p.o.) reduced PGE2 synthesis in vivo in a carrageenan-induced air pouch model in guinea pig without lowering 6-keto-PGF1α. Compound 16 (PF-4693627)86 exhibited most favorable in vitro potency and selectivity, in vivo efficacy and promising pharmacokinetic and preclinical safety profile. This compound was advanced to clinical studies for the treatment of inflammation in osteoarthritis and rheumatoid arthritis.86 The sulfone-containing back up compound 17 displayed the best overall profile with improved solubility and metabolic stability and may serve as valuable alternative to compound 16.87 Detailed SAR studies on imidazoquinolines focusing on 2- and 7-substituents led to potent mPGES-1 inhibitors (IC50 = 9.1 nM) with high selectivity (>1000-fold) over COX-1/2.88,89 Compound 18 (IC50 = 4.1 nM), equipped with phenyl residues in 2- and 7-position turned out as optimal candidate for further development that effectively inhibited cell-based mPGES-1 activity (IC50 = 33 nM) with good mPGES-1 selectivity (> 700-fold), excellent in vitro absorption, distribution, metabolism and excretion (ADME) profile, and good oral absorption in a rat pharmacokinetic study.90 Another set of potent mPGES-1 inhibitors based on 2-aryl substituted quinazolin-4(3H)-one, pyrido[4,3-d]pyrimidin-4(3H)-one

and

pyrido[2,3-d]pyrimidin-4(3H)-one

scaffolds

were

recently presented.91 The lead compounds blocked mPGES-1 activity in cell-free and A549 cellular assays at low nanomolar concentrations and showed good potency also in human whole


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blood assay (< 400 nM). Lead compound 19 (GRC27864) was selective over other prostanoid synthases and effectively repressed PGE2 synthesis in clinically relevant inflammatory settings.91 Finally, several recent patents have presented promising scaffolds that potently inhibit mPGES-1 including 5-phenylimidazole-2-amides exemplified by compound 2092

and benzimidazole

amines like compound 2193 with IC50 values for mPGES-1 (cell-free assays) in the single-digit nanomolar or even subnanomolar range. Of interest, compound 20 inhibited PGE2 synthesis also in intact A549 cells and in human whole blood with a remarkably low IC50 value of only 12 nM. Among a series of benzene-fused 5-membered heterocyclic derivatives, compound 21 blocked isolated human mPGES-1 with IC50 = 0.1 nM and mPGES-1 in intact A549 cells with IC50 = 1.2 nM.94 Quite recently, the first clinical trial with the mPGES-1 inhibitor LY3023703 (structure not disclosed) by Lilly has been published.95 In a multiple ascending dose study, 48 subjects received either LY3023703, celecoxib (400 mg), or placebo once per day over 28 days. LY3023703 inhibited LPS-stimulated PGE2 synthesis ex vivo > 90% after 30 mg dosing, being even superior over celecoxib and in contrast to celecoxib without reducing PGI2 synthesis.54 Taken together, starting from well-recognized core structures (e.g., compound 9) and hits identified by HTS, a variety of structurally different mPGES-1 inhibitors were developed and refined. Unfortunately, for most of these leads, significant drawbacks in the preclinical development of mPGES-1 inhibitors were obvious, such as high lipophilicity (required for potent mPGES-1 affinity) that caused loss of potency in biological systems (i.e., indole carboxylic acids, 2,4-biarylimidazoles, trisubstituted ureas, and benzo-thiopyran S-dioxides) and interspecies-differences (i.e., phenanthrene imidazoles) that impeded in vivo studies for evaluation of efficacy and proof-of-concept. However, recent developments such as the imidazoquinolines and benzoxazole piperidinecarboxamides (e.g., compound 16) and


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benzimidazole amides/amines have circumvented these drawbacks. These compounds exhibit an excellent preclinical profile and represent promising candidates for clinical studies.

PHARMACOPHORE MODELS AND NATURAL PRODUCTS AS SOURCES FOR NOVEL MPGES-1 INHIBITORS

Besides HTS and targeted design of novel chemotypes, two additional approaches have been pursued in order to identify mPGES-1 inhibitors, that is, (I) in silico screening of virtual compound libraries based on pharmacophore models (see above), and (II) exploitation of antiinflammatory natural products as scaffolds for mPGES-1 inhibitors. Numerous natural products from plants with anti-inflammatory properties have long been recognized as efficient repressors of PGE2 biosynthesis in intact cells, seemingly due to COX-1/2 inhibition. Detailed analysis of their molecular targets and modes of action revealed mPGES-1 as primary point of attack providing unique chemotypes for development of mPGES-1 inhibitors.3,5 Most of these mPGES-1-active natural products include lipophilic acidic molecules including (I) acylphloroglucinols such as myrtucommulone A (22),96 hyperforin (23),97 arzanol (24),98 (II) (poly)phenols such as epigallocatechin-3-gallate (EGCG (25),99 curcumin (26),100 the depside perlatolic acid (27) and the depsidone physodic acid (28),101 carnosol (29) and carnosic acid (30),102 (III) quinones such as embelin (31),103 and (IV) tetra- or pentatcyclic triterpene acids such as boswellic, tirucallic and lupeolic acids (compounds 32, 33, 34).104,105


25


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O O O

OHHO

OHHO

O

O O

O

OH

HO

O

23

OH 22

OH OH

O HO

O

OH O

HO

OH

O

O

OH

OH

HO OH

O

OH

26 O

O

25

24

OH

OH

OH

HO

OH O

O O

OH O

O O O

29

O

O OH

OH

OH

27

O

O

HO

O

28

OH

HO

HO HOOC 30

HO O 31

HOOC H

H OH H

HO HOOC

O

O

H

O

H 32

33

O HOOC

H H 34

Scheme 4. Structures of natural product mPGES-1 inhibitors

The IC50 values of most of these natural products for mPGES-1 are in the range of 0.2 to 10 µM, and some of them have been demonstrated to suppress PGE2 levels in vivo, connected to antiinflammatory activity. For more detailed review see reference.3 Inhibition of mPGES-1 by these natural products may rationalize the anti-inflammatory properties of remedies containing them,


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and they may also serve as novel templates for drug development. In fact, for some of these natural products (e.g., compound 22), chemical derivatization and SAR studies yielded promising leads with higher potencies and more favorable drug-like chemical properties (see below, dual mPGES-1/5-LO inhibitors).

DUAL INHIBITORS OF MPGES-1 AND 5-LIPOXYGENASE

Although high selectivity is generally one of the primary aims in the development of mPGES-1 inhibitors, the targeted discovery of agents that besides mPGES-1 also interfere with 5-LO has been pursued in parallel, and various chemical scaffolds have been identified that dually suppress mPGES-1 and 5-LO. Such simultaneous suppression of PGE2 and leukotrienes might be a valuable pharmacological strategy to intervene with inflammatory disorders and is expected to have beneficial effects over single interference, not only in terms of better efficacy but also in view of a reduced incidence of side effects.5,13 The well-recognized shunting of arachidonic acidderived lipid mediator biosynthesis towards leukotrienes due to suppression of PG formation by COX inhibitors (which cause NSAID-induced asthma) can be circumvented by dual COX/5-LO inhibitors that had been developed already 20 years ago.106,107 The disadvantage of dual COX/5LO inhibitors, however, concerns the suppression of beneficial prostanoids such as antithrombotic and vasodilatory PGI2, but also of gastrointestinal-protective PGE2. Accordingly, agents that mainly suppress the formation of pro-inflammatory ones (i.e., “inducible” PGE2, leukotriene B4 and cysteinyl-leukotrienes) among all eicosanoids may ideally have an exceptional benefit for a safe therapy of inflammation; dual mPGES-1/5-LO inhibitors may have this potential.


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Licofelone (35), first proposed as dual COX/5-LO inhibitor based on its suppressive effects on eicosanoid synthesis in cellular studies,106 directly inhibits mPGES-1 without affecting COX-2 and only moderately interferes with COX-1.78 Detailed molecular studies showed that compound 35 acts primarily on FLAP, but hardly on 5-LO, to suppresses cellular leukotriene formation.108 Although the overall potency of compound 35 to inhibit PGE2 and leukotriene synthesis in intact cells is moderate (low micromolar to submicomolar range) as compared to advanced and specific mPGES-1 or 5-LO inhibitors, respectively, it exhibited strong anti-inflammatory efficacy in a panel of animal models without typical side effects of NSAIDs (reviewed in reference

109

).

Replacement of the carboxylic acid moiety of compound 35 by sulfonimide led to diarylpyrrolizine sulfonimides (compound 36) with improved potencies against mPGES-1 and comparable efficiency in the suppression of cellular 5-LO product synthesis.110


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H N Cl

N

N COOH 35

O 36

S

O

O

Cl

S 37

N H S

N

COOH

N

COOH O

N

O

Cl 38

39 H N

N

S

N

S

COOH

OH

40

COOEt HO

O2N

O O S N

N N H

Cl 41

HN Cl 43

42 O

O OH HO

O

COOH

O

O N

S N

O

H N

N

Cl

OH HO

OH HO

OH

O

O

44

OH HO

OH

O

45

Scheme 5. Structures of dual mPGES-1/5-LO inhibitors

The first dual 5-LO/mPGES-1 inhibitors were reported in 2008 and meanwhile several different chemical series as well as natural products have been introduced that block 5-LO product and PGE2 formation without lowering the level of other prostanoids. Numerous α-alkyl- and α-arylsubstituted derivatives of pirinixic acid, a synthetic lipid-lowering agonist of PPARα, were presented as dual 5-LO/mPGES-1 inhibitors starting with the lead compound 37 (IC50 = 1.3 and 1 µM for mPGES-1 and 5-LO, respectively).111 Target-oriented design focusing on the α-


29


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substituent and the residue in 6-position of the pyrimidyl core yielded promising leads including the α-n-hexyl substituted pirinixic acid 38,112 compound 39 with a bulky α-naphthyl residue,113 the α-n-hexyl derivative 40 carrying two phenethoxy moieties in 4- and 6-position of pyrimidin113 and compound 41, a 2-aminothiazole-featured α-n-hexyl pirinixic acid.114 For many of these compounds, marked anti-inflammatory efficiency was demonstrated in vivo (carrageenan-induce pleurisy in rats and zymosan-induced peritonitis in mice) which was associated with reduced exudate levels of leukotrienes and PGE2. Only marginal loss of potency in intact cells versus cell-free assays was obvious and COX enzymes were hardly affected by these compounds. Compound 41 is the most advanced pirinixic acid derivative that inhibits 5-LO and mPGES-1 with IC50 of 0.3 and 0.4 µM, respectively.114 For detailed review on pirinxic acid derivatives related to mPGES-1 and 5-LO see reference 115. A series of non-acidic benzo[g]indole-3-carboxylates were identified as dual mPGES-1/5-LO inhibitors exemplified by compound 42 that suppressed 5-LO and mPGES-1 in cell-free assays with IC50 = 0.086 and 0.6 µM, respectively. Compound 42 also efficiently suppressed PGE2 generation in intact A549 cells and potently inhibited 5-LO product synthesis in neutrophils or human whole blood with only 3-5-fold loss of potency versus crude mPGES-1 and 5-LO in cellfree assays, without significant inhibition of other enzymes within the arachidonic acid cascade. Moreover, in carrageenan-induced mouse paw edema and rat pleurisy, compound 42 exhibited anti-inflammatory properties and reduced leukotriene B4 and PGE2 levels in the respective exudates.116,117 Recently, 6-nitro-3-(m-tolylamino)benzo[d]isothiazole-1,1-dioxide analogues were shown to dually inhibit 5-LO and mPGES-1, exemplified by compound 43 with IC50 values of 0.6 µM,


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2.1 µM, respectively.118 Whether or not these compounds are active in cell-based assays or in animal models of inflammation remains to be investigated. Interestingly, several natural mPGES-1 inhibitors were found to interfere also with 5-LO, often along with minor effects on COX enzymes (see reference

3

and references therein for more

details). For some of them (i.e., compound 22, 31 and 26), synthetic derivatives were prepared and SAR analysis revealed promising leads with higher potencies against mPGES-1 and/or 5LO119-121 suitable for further preclinical analysis. For instance, following a straightforward modular strategy, 28 analogues of compound 22 have been synthesized. The replacement of the syncarpic acid moieties by indandione and substitution of the isopropyl carbonyl residue by nhexanoyl yielded compound 44 with 12.5-fold higher potency (IC50 = 0.08 µM) over the parental compound 22 (IC50 = 1 µM). For 5-LO, a 33-fold improvement in potency was obtained for compound 45 for which the isopropyl carbonyl residue of compound 22 (IC50 = 15 µM) was replaced by n-hexanoyl and the isopropyl at the methylene bridges by phenyl (IC50 = 0.46 µM).119

CO-CRYSTAL STRUCTURS OF MPGES-1 IN COMPLEX WITH INHIBITORS

Backbone amide hydrogen/deuterium exchange kinetics were applied to map the mPGES-1 inhibitor binding sites of compound 9, the phenanthrene imidazole 11, the 3-benzoylamido-Nnapthyl-benzamide 46 and the anti-inflammatory lipid mediator 15-deoxy-∆12,14-PGJ2.122,123 Crystal structures of human mPGES-1 are available in complex with the 2,4-biarylimidazole 47, compound 11, the compound 9 analogue 10, the indole-2-carboxylic acid 48,124 and the 2-(2,6-


31


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Page 32 of 64

(dichlorophenyl)amino)benzoimidazole 49.125 All mPGES-1 inhibitors studied so far seemingly bind to the active site of the enzyme. Allosteric inhibitors of mPGES-1 have not yet been reported. They might be disfavored by the exceptionally rigid structure of mPGES-1 which is stabilized by interhelix hydrogen bonds.46 According to co-crystal structural data, mPGES-1 inhibitors i) interact with transmembrane helix 4 from one monomer and transmembrane helix 1 from the other monomer, ii) place their head groups or cores in a groove above the GSH cofactor and iii) let the hydrophobic tails protrude from the active site cavity.124,125 The headgroups/cores of the inhibitors form salt-bridges to Arg52 and/or hydrogen bonds to His53, other amino acids and/or structural water. The hydrophobic tails are bound by hydrophobic and van der Waals interactions.

Cl

H N

Br O

HN

N

Cl

N H

F

N

O 46

47

F3C HO

COOH

O

N

O

N

NH

N Cl

NH

O

F 48

F

F

Cl

NH

49 O

Scheme 6. mPGES-1 inhibitors used in structural studies


32

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Differences in the inhibitor binding modes are based i) on the orientation of the inhibitor in the active site, ii) the binding regions occupied by the inhibitor within the pocket and iii) the concrete amino acid interaction partners.124 The 2,4-biarylimidazole 47, for instance, extends with its 2-chloro-6-fluorophenyl group into a hydrophobic grove above bound GSH and forms hydrogen bonds with His53, Ser127, Thr131 and a structured water molecule via imidazole and pyridine nitrogens (Fig. 3A).124 The importance of the hydrogen bond network for inhibitory activity was confirmed by chemical modification of the central imidazole.83 A

B

C Figure 3. Crystal structure of human mPGES-1 in complex with GSH and compound 47 (A), 10 (B) and 48 (C) as previously published.124 The interaction of the inhibitor with the active site is shown.


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The phenanthrene imidazole 11 has a similar binding mode to compound 47: the distant binding pocket above GSH is occupied by the dicyanophenyl group, and imidazole forms a hydrogen bond to His53 and a structured water molecule.124 In agreement with the crystal structure of mPGES-1, replacement of imidazole by heteroaromatic rings lacking hydrogen bond donors was detrimental.80 The planar chlorophenanthrene core of compound 11 is attached to the flat hydrophobic surface of transmembrane helix 4 and exposed to the solvent on its other side.124 Luz et al. speculated that the poor inhibitory activity of compound 11 in mice and rats compared to humans also depends on protein/inhibitor interactions124 and not only on the access to the active site, which is limited by gate keeper amino acids as discussed above.46,49 His53 and Pro124 of human mPGES-1 are exchanged in mice and rat enzyme by Arg. The longer Arg53 side chain is expected to clash with the inhibitor and potentially disturb the hydrogen bonding geometry, whereas the exchange of Pro124 is believed to disrupt the flat surface to which the planar phenanthrene binds. The compound 9-derived analogue 10 is more centrally located than compounds 11 and 47 and orientated parallel to the transmembrane helices (Fig. 3B).124 The carboxylate forms a pair of salt bridges to Arg52 and a hydrogen bond to a structural water molecule interacting with His53. Disruption of the salt bridges by esterification or amidation abolished mPGES-1 inhibition as expected.77 The chlorophenyl moiety is directed towards GSH with the chlorine located at the top of the GSH thiol.124 The biphenylen system extends along the transmembrane helices and binds to a hydrophobic cavity. Diverse substitutions of the methyl group in 3-position of the indole are tolerated,77 which can be explained by a nearby hydrophobic groove; the same which is also traversed by the alkyne of compound 47.124


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The indole-2-carboxylic acid 48 places its isopropoxyphenyl-headgroup into the cavity proximate to GSH and the catalytic amino acid Ser127 (Fig. 3C).124 The carboxylate forms a salt bridge to Arg52 and a hydrogen bond to a bridging water molecule similar to compound 10. The tert-butylphenyl-residue binds to a shallow hydrophobic groove and is directed towards the interior of the membrane. Taken together, the crystal structure of mPGES-1 and the subsequent co-crystal structures in complex with diverse inhibitors represent milestones in mPGES-1 research.124,125 They give important insights into the active and inhibitor binding sites and provide a molecular basis for previously reported SARs. Together with future docking and QSAR studies based on the novel structural information, we believe that they will essentially promote the directed design of mPGES-1 inhibitors and likely initiate a next generation lead discovery of mPGES-1 inhibitors by high-throughput in silico screening. Nonetheless, mPGES-1 inhibitor design will remain a challenging task. Small structural variations apparently have complex consequences on the orientation of the inhibitor in the binding pocket as well as on the organization of structured water und thus the hydrogen bond networks. Hence, not all SARs can readily be explained from the mPGES-1 crystal structure. For example, the gain in inhibitory potency of 47 by replacement of imidazole with triazole is not obvious.83 The authors speculate about indirect effects with impact on neighboring hydrogen bonds or on the exposition of the inhibitor to the solvent.124

FUTURE DIRECTIONS

Drugs targeting mPGES-1 did not enter the market so far despite enormous efforts upon the discovery of mPGES-1 in 1999.41 After the failure of coxibs,6 academia increasingly gained


35


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interest in mPGES-1 as key enzyme for a growing number of diseases with inflammatory component.2,3,5,9,10 Many pharmaceutical companies initiated extensive mPGES-1 drug development programs. The reasons for the still lacking breakthrough are multifaceted. On the one hand, mPGES-1-derived PGE2 displays a central role within the network of eicosanoids, which not only impacts disease but also physiological processes, such as platelet aggregation and the salt and water balance.12,20-22 Safety concerns arose from shunting phenomena after blockage of mPGES-13,11 and were substantiated by controversial findings from mPGES-1 knockout studies implying a protective function of mPGES-1 against cancer,13 hypertension, cardiac malignancies and renal hyperosmotic stress.12 Critical voices questioning the safety of mPGES-1 inhibitors slowly fall silent after an increasing number of animal studies were published during the last few years with an overall favorable outcome.2 Despite these encouraging results, it remains indispensable to carefully investigate the complex effects of mPGES-1 inhibitors on the eicosanoid network and interpret them in terms of efficacy and safety. Combination of eicosanoid profiling (not only related to prostanoids) and network scientific approaches promise organ, tissue and disease-dependent network models for correlating eicosanoid patterns with biological responses. Liquid chromatographic and mass spectrometric instrumentations/methodologies required for such a comprehensive eicosanoid analysis became available during the last decade and now allows analysis of dozens to hundreds of different lipid mediators in a single run.126,127 Also disease models first had to be established for studying the potency and safety of mPGES-1 inhibitors, since preclinical mouse and rat models were only of limited value in light of the interspecies differences of mPGES-1 inhibitors.2,49 Alternative models in guinea pigs or humanized mice are now available and have already been used for characterizing promising pre-clinical mPGES-1 inhibitor candidates.80,81,86


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Despite all these enthusiasm, mPGES-1 inhibitors, even highly selective ones, will not be free of side effects as predetermined by the pleiotropic functions of mPGES-1.1,2,128 However, mPGES1 inhibitors are meanwhile widely accepted to possess a favorable safety profile compared to other anti-inflammatory and analgesic therapeutics, such as NSAIDs and coxibs.2,3,13 mPGES-1 inhibitors interfere with PGE2 synthesis without lowering the biosynthesis of physiologically relevant PGs.5 Moreover, they do not completely abolish PGE2 signaling but preferentially suppress the excessive PGE2 formation related to inflammation while maintaining basal PGE2 levels required for homeostatic processes.5 In this regard, inhibition of mPGES-1 might even be superior to targeting EP receptors, another emerging strategy for selective interference with PGE2 signaling. In particular, EP4 and EP2 are considered as promising drug targets.129,130 However, EP receptors cannot be simply distinguished into those relevant for disease or homeostasis as reflected by the current efforts in developing both agonists and antagonists of EP4 depending on the respective disease. Comparative studies of mPGES-1 inhibitors and EPreceptor agonists/antagonists regarding efficacy and safety have unfortunately not been performed so far but would be of great interest. Network scientists even go one step further and aim to readjust the dysregulated eicosanoid network upon disease by combining high affinity targeting of mPGES-1 with moderate inhibition of compensatory targets such as COX-1 and/or 5-LO/FLAP.3 Such an approach is realized in compound 35 (currently in phase III clinical trials for osteoarthritis), which represents a combined inhibitor of mPGES-1, COX-1 and FLAP.78,108,131,132 Compound 35 possesses an advantageous safety profile without apparent gastrointestinal and cardiovascular toxicity and represents the most advanced mPGES-1-targeting drug to date.132


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Apart from discussions about the potential of mPGES-1 as drug target, the development of mPGES-1 inhibitors was compromised by the lack of structural information about the mPGES-1 inhibitor binding site. Diverse mPGES-1 inhibitors bind to the active site as shown by hydrogen/deuterium exchange kinetics.122 Initial structural insights were obtained by computational homology models and a low-resolution electron crystallography structure.2,45 High-resolution crystal structures of mPGES-1 in complex with inhibitors were only published in 2015, which is not surprising since crystallization and structure determination of membrane proteins is a challenging task, and techniques have to be adapted for each protein.124,125 The recent breakthrough in crystallizing mPGES-1 allows for reinterpretation of the SARs assessed so far, which will induce a new era in rational mPGES-1 inhibitor design driven by docking as well as further crystallographic studies. One of the major drawbacks for mPGES-1 inhibitor design during the last years have been the often unfavorable physicochemical properties of mPGES-1 inhibitors and interspecies differences. The latter hampered preclinical evaluation of efficacy in routine animal models of inflammation such as mouse peritonitis or rat air pouch and adjuvant-induced arthritis, but use of guinea pigs instead of mice or rats or knock-in of human mPGES-1 into mice helped to overcome these hurdles. The general structure of mPGES-1 inhibitors consists of a lipophilic tail and a (polar) headgroup/core with either negatively charge and/or hydrogen bond donors/acceptors. Such amphiphilic molecules resemble in their structure PGH2, the substrate of mPGES-1, and reflect the nature of the inhibitor binding site which often overlaps with the active center.46 Lipophilic mPGES-1 inhibitors are prone to aggregation and non-specific protein binding, which i) bears the risk of false positive results by nuisance inhibition, ii) predestinates for a failure in clinically relevant test systems (such as whole blood), and iii) is deleterious for


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the efficacy in vivo due to poor oral availability. Enormous efforts have been spent for overcoming these limitations, though with mixed success. In particular, non-acidic mPGES-1 inhibitors succeeded in the phenanthrene imidazole 11,80,133 the 2-pyridyl-benzo[1,3]oxazole 16,86 and the 2-aryl-substituted pyrido[4,3-d]pyrimidin-4(3H)-one derivative 19 (91 or not published).

These compounds combine potent and selective inhibition of mPGES-1 with

efficient PGE2-lowering and anti-inflammatory activity in animal models of inflammation; compound 19 was advanced to clinical trials; the outcome has not been published so far. The first clinical trial with the mPGES-1 inhibitor LY302370395 revealed potent inhibition of PGE2 synthesis ex vivo with superiority over celecoxib and no reduction of PGI2 formation.54 In conclusion, a set of potential clinical candidates is meanwhile available, which have been well characterized in preclinical studies or even clinical trials and show a favorable pharmacological profile. The time has come to dare the next step and transfer promising preclinical mPGES-1 inhibitor candidates to clinical trials. The outcome of these studies will essentially shape the fate of mPGES-1 as drug target and conclusively answer one of the most exciting questions of pharmacological mPGES-1 research since its beginnings, namely those of the superior safety of mPGES-1 as drug target.


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ANCILLARY INFORMATION Author information Corresponding Author *Phone: +49-3641-949815. Fax: +49-3641-949802. E-mail: [email protected] (AK). *Phone: +49-3641-949801. Fax: +49-3641-949802. E-mail: [email protected] (OW).

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgment Peter Keck PhD for figure preparation and Sebastian Haase for scheme preparation.

ABBREVIATIONS COX, cyclooxygenase; cPGES, cytosolic prostaglandin E2 synthase; C-domain, cytoplasmic domain; FLAP, 5-LO-activating protein; GSH, glutathione; HTS, high throughput screening; LO, lipoxygenase; MAPEG, membrane-associated proteins involved in eicosanoid and glutathione metabolism; MGST, microsomal glutathione transferase; (m)PGES, (microsomal) prostaglandin E2 synthase; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PL, phospholipase; Tx, thromboxane.


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Biographies Andreas Koeberle received his PhD in Medicinal Chemistry/Analytics at the University of Tuebingen, Germany, in 2009. After postdoctoral studies at the University of Tokyo, Japan, he joined the Chair of Medicinal/Pharmaceutical Chemistry at the University of Jena, Germany, where he established the institutional lipidomics facility in 2012. At the present, he is junior group leader with research focus on bioactive lipid profiling and medicinal chemistry. He coauthored more than 40 original articles and reviews focusing on the discovery, design, synthesis and biological characterization of mPGES-1 inhibitors. He is recipient of the KlausGrohe-Award for Medicinal Chemistry, the Bionorica Global Research Initiative and the young scientist award of the European Society for Lipid Mediators. Stefan A. Laufer studied Pharmacy and completed his PhD from Regensburg University (Supervisor: Prof. G. Dannhardt). After postdoctoral research in Frankfurt, he took a position in the pharmaceutical industry, but maintained lectureships at Frankfurt and later Mainz University, where he finished his habilitation in 1997. Since 1999, he has been full professor (chair) for pharmaceutical and medicinal chemistry at Tübingen University. He is cofounder/spokesman of CAIR Biosciences and ICEPHA (Interfaculty center for pharmacogenomics and pharma research), since 2012 vice president of the DPhG and president elect for 2016-9. He received the Phoenix Pharmacy Science Award in 2006 and became 2014 a permanent member (membro correspondente) of the Brazilian Academy of Sciences (ABC). His research interests are protein kinase inhibitors and eicosanoid modulators.


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Oliver Werz studied pharmacy and performed his PhD studies in Pharmaceutical Chemistry at University Tuebingen. He received his PhD in 1996 and was subsequently postdoc at University Frankfurt (Germany) and Karolinska Institute, Stockholm (Sweden). Werz became professor for Pharmaceutical Analytics at University Tuebingen in 2005. Since 2010 he is professor for Pharmaceutical/Medicinal Chemistry at the University of Jena (Germany) and director of the Institute of Pharmacy since 2013. His research is focused on the cell biology of lipid mediators in inflammation and on the development of anti-inflammatory synthetic compounds and natural products. Werz is supported by the Deutsche Forschungsgemeinschaft and the Jena School for Microbial Communication (JSMC). He received the Phoenix Pharmacy Science Award 1999 and 2009 and the Dr. Willmar Schwabe Award 2008.


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Design and Development of Microsomal Prostaglandin E2 Synthase-1

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