ALXR Agonists and the Resolution of Inflammation - Journal of

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FPR2/ALXR Agonists and the Resolution of Inflammation Olivier Corminboeuf* and Xavier Leroy Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland ABSTRACT: The resolution of inflammation (RoI), once believed to be a passive process, has lately been shown to be an active and delicately orchestrated process. During the resolution phase of acute inflammation, novel mediators, including lipoxins and resolvins, which are members of the specialized pro-resolving mediators of inflammation, are produced. FPR2/ALXR, a receptor modulated by some of these lipids as well as by peptides (e.g., annexin A1), has been shown to be one of the receptors involved in the RoI. The aim of this perspective is to present the concept of the RoI from a medicinal chemistry point of view and to highlight the effort of the research community to discover and develop antiinflammatory/pro-resolution small molecules to orchestrate inflammation by activation of FPR2/ALXR.

1. INTRODUCTION The critical role of inflammatory processes in health and disease has long been recognized, culminating in the design and development of successful anti-inflammatory therapies. Until recently, little was known about the mechanism that leads to the return to homeostasis, complete resolution of inflammation, and restoration of normal tissue function. The resolution of inflammation (RoI) has long been considered as a passive process, and it is therefore not surprising that antiinflammatory therapies have focused on strategies to decrease or neutralize the level of pro-inflammatory mediators and/or inhibit the recruitment of leukocytes and their activation. Recently, several investigations have provided strong evidence that the RoI is not a passive but an active mechanism orchestrated by specialized pro-resolving mediators (SPMs), such as lipoxins (LX), resolvins (Rv), protectins (PD), and maresins (Mar), signaling via specific receptors (Figure 1).1−6 In fact, regulatory pathways built into the inflammatory response allow for a careful balance between inducing a sufficiently strong response to the injury and limiting damage to endogenous tissues, thus permitting resolution of inflammation. SPMs possess dual function, combining both anti-inflammatory and pro-resolving activities: they inhibit release of proinflammatory cytokines, limit infiltration of neutrophils, enhance macrophage uptake, and stimulate nonphlogistic (i.e., without eliciting an inflammatory response) clearance of apoptotic neutrophils and microbial particles by efferocytosis (from the Latin term efferre, meaning “to carry to the grave”). Over the past few years, it has been shown in multiple animal models that pro-resolution-based approaches have potential for the treatment of multiple inflammatory conditions.7−11 Still today, many of the detailed molecular mechanisms and biological events that regulate the resolution of inflammation remain to be elucidated. This perspective discusses the concept of the RoI as a new opportunity for drug development. The role © XXXX American Chemical Society

Figure 1. Schematic representation of the ideal sequence in an inflammatory process leading to complete resolution of inflammation. The inflammatory response to an injury or infection is rapidly initiated by the release of pro-inflammatory molecules, initiating the acute phase of the inflammatory response. SPMs are synthesized early on in the process to serve, via activation of their receptors, as braking signals to control leukocyte trafficking. Through activation of these receptors, apoptosis and efferocytosis are induced in a tightly orchestrated way, allowing for the resolution of inflammation and restoration of tissue function. A defect in SPMs signaling might not allow for the inflammation to resolve and can lead to chronic inflammation and/or tissue scarring. Adapted with permission from ref 12. Copyright 2007 Federation of American Societies for Experimental Biology.

of FPR2/ALXR will be reviewed through its host- or pathogenderived ligands. Specifically, the endeavors of the research community to discover small molecules designed to activate the RoI process via activation of FPR2/ALXR will be highlighted, with an emphasis on the patent literature. To conclude, the therapeutic impact of activating the resolution process through FPR2/ALXR will be discussed.

2. INFLAMMATORY RESPONSE Localized acute inflammation is a component of the host’s physiologic protective reaction to tissue injury and infection by Received: July 11, 2014

A

dx.doi.org/10.1021/jm501051x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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invading microbial pathogens.13 Although this inflammatory response to a range of injurious stimuli is protective to the host with the return to homeostasis as the final goal, if kept uncontrolled it can cause a wide range of acute, chronic, and systemic inflammatory disorders. 2.1. Initiation. At the beginning of the inflammatory response, phospholipase enzymes [e.g., phospholipase A2 (cPLA2)] mediate the release of free polyunsaturated fatty acids (PUFA), including arachidonic acid (AA), from phospholipids (Figure 2, in blue). AA has long been recognized to be involved in the initiation phase of inflammation, governing the early events in host protection.15 2.2. Propagation. A series of biosynthetic pathways involving 5-LO15−17 and cyclooxygenases, mainly the inflammation-induced isoform COX-2, are initiated, rapidly releasing AA-derived hydroxylated metabolites, such as leukotriene B4 (LTB4) or leukotriene C4 (LTC4) and prostaglandin E2 (PGE2) or prostaglandin D2 (PGD2), which act as pro-inflammatory lipid mediators (Figure 2, in red). Each of these potent lipid mediator exerts its specialized inflammation-related actions by acting on specific G-protein coupled receptors (GPCRs) that are present in the membranes of the pertinent cell types, launching a series of signaling cascades.5,6,15 Activation of these GPCRs by pro-inflammatory lipids directly affects the expression levels of multiple enzymes such as 5-LO16,17 or COX-218−21 that are key players in inflammation, not only at its initiation but also in its acceleration. Thus, the biosynthesis and release of potent chemotactic agents such as LTB4 promote neutrophil recruitment (Figure 3) to the inflamed tissue, whereas the formation of PGE2 and PGD222 further accelerates the inflammatory response, ultimately resulting in a condition of acute inflammation (Figure 2, in red). Polymorphonuclear cells (PMNs) represent a first-line immune defense by migrating to sites of injury or infection (Figure 3). However, their multiple defense mechanisms, which are required for elimination of the offending micro-organisms, need to be tightly regulated. Excessive or improper activity of PMNs could lead to tissue damage and contribute to the pathogenesis of various inflammatory diseases.23 Therefore, a finely tuned balance of immune defense to avoid host damage is of the utmost importance. Despite its critical host-protective function, acute inflammation is not sustainable over an extended period of time, giving rise to disruptive conditions of chronic inflammation that are possibly accountable for the pathogenesis of a wide range of diseases. 2.3. Lipid Mediator Class Switching. Successful resolution requires activation of endogenous programs that switch from production of pro-inflammatory molecules toward specialized pro-resolving molecules. These SPMs are generated early on upon inflammation to orchestrate its fate. Remarkably, on top of its fundamental role in the initiation and progression of inflammation, AA is also implicated in the biosynthesis of anti-inflammatory and pro-resolving lipid mediators (Figure 2).24,25 In addition, PGE2 and PGD2 stimulate the expression of 15-LO, switching, at the site of inflammation, the lipid mediator synthetic process from proinflammatory prostaglandins to pro-resolving lipoxins, such as LXA4. Thus, although COX-2-derived pro-inflammatory eicosanoids PGE2 and PGD2 are commonly viewed as harmful, they are also critical for positive feed-forward regulation of antiinflammatory SPMs circuits.26

Figure 2. Inflammatory response: onset (blue), propagation (red), and lipoxin-based resolution (green) mechanisms.14,15 In the propagation phase, arachidonic acid (AA) is converted either by 5-lipoxygenase (5LO) or cyclooxygenase-2 (COX-2), leading to leukotriene A4 (LTA4) and prostaglandin H2 (PGH2), respectively, which are further processed to leukotriene C4 (LTC4)/leukotriene B4 (LTB4) and prostaglandin D2 (PGD2)/prostaglandin E2 (PGE2), respectively, leading to acute inflammation by activating, among others, Bleukotriene 1 and 2 (BLT1/2) or cysteinyl leukotriene 1 and 2 (CysLT1/2). In the resolution phase, lipoxin and aspirin-triggered lipoxin are biosynthesized from AA, which is metabolized by 15lipoxygenase to form 15S-H(p)HETE, which is further metabolized to lipoxin A4 (LXA4) by sequential action of 5-LO and lipoxin hydrolase (LxH). Alternatively, AA is converted to LTA4 by 5-LO, and subsequent transformation by 12-lipoxygenase (12-LO) generates LXA4. The acetylation of the COX-2 active site by aspirin does not inhibit the enzyme activity but triggers the conversion of AA in 15RHETE that is further transformed by 5-LO in 15-epi-LXA4.

Actually, a number of endogenous lipid mediators acting in this manner have been identified,26 suggesting a lipid mediator class switch2 from the initial actions of pro-inflammatory lipid mediators, i.e., leukotriene and prostaglandins, to the antiinflammatory and pro-resolving actions of SPMs. Each family of these SPMs exerts specific events, including jamming recruitment of neutrophils, promoting the recruitment and activation of monocytes, and mediating the nonphlogistic phagocytosis and lymphatic clearance of apoptotic neutrophils by activated macrophages (Figure 3). Eventually, through the collective actions of these mediators, the resolution of inflammation is accomplished, and homeostasis is achieved.27 An important category of such SPMs is the class of lipoxins (LXA4 and LXB4),28,29 formed in humans to a large extent via trans-cellular biosynthesis through the sequential actions of 5LO and 12-LO or 15-LO-initiated interaction with 5-LObearing cells (Figure 2). The epimeric aspirin-triggered lipoxins (ATLN),30−32 AT-LXA4 and AT-LXB4, have actions comparable to those of the lipoxins but are formed by COX-2 in the presence of aspirin (Figure 2). The biosynthetic pathways and biological functions of the lipoxins and aspirin-triggered lipoxins have been described in numerous reviews.26,28,29,33 2.4. Resolution. The resolution phase of inflammation is initiated by the previously described class switch in lipid mediators’ biosynthesis. This switch generates SPMs that are able to influence apoptosis34 and efferocytosis,35 two landmarks of the RoI. Through specific receptor engagement by these SPMs and induction of downstream signaling, macrophages promote resolution of inflammation by (i) efficiently engulfing dying cells, thus avoiding cellular disruption and release of inflammatory contents, and (ii) producing anti-inflammatory mediators that dampen pro-inflammatory responses. B



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resolution,53,54 as do lipoxygenase (LO) inhibitors27,42 and many other nonsteroidal anti-inflammatory drugs.41,53 Importantly, introducing resolution indices also pinpointed the actions of SPMs in shortening resolution times for inflammation and showed that induction of RoI is possible. This represents new opportunities to develop innovative therapies to treat inflammatory diseases that can be attributed to a failure of resolution.12 Since SPMs were early considered to signal via specific receptors,1−3 modulating them by stable analogues or by small molecules mimicking their actions rapidly appeared as an attractive therapeutic approach that already led, for example, to a phase II clinical trial completed for RX-10045,55 an analogue of RvE1. Indeed, it is also now well-appreciated that inflammation plays a key role in many chronic diseases such as arthritis and asthma. It is increasingly apparent that diseases such as cancer, coronary heart disease, or Alzheimer’s disease have an inflammatory component.56−60 Moreover, following the original report that SPMs stimulate bacterial killing and clearance,61 it has been proposed that SPMs-based new strategies for the management of infectious diseases would profit from (1) their ability to lower antibiotic use and (2) the fact that they are not immunosuppressive agents, representing a substantial advantage over many antiinflammatory drugs.58 FPR2/ALXR has been revealed to be one of the receptors activated by SPMs and to be involved in the RoI. It is the topic of the next section, where the efforts to develop drugs by activating this receptor are described.

If apoptotic cells are not ingested rapidly, necrosis often develops, and products generating pro-inflammatory signals are released. Inflammation can thus be prolonged by a failure of neutrophils to undergo timely apoptosis or by a failure of macrophages to clear apoptotic cells.36 In summary, controlled neutrophil apoptosis and subsequent efferocytosis is a critical point in resolving inflammation, and macrophages play a central role.37 In addition, resolvin E1 (RvE1) has been also shown to stimulate endogenous LXA4 production,2 illustrating that the pro-resolving cascade is a feed-forward mechanism. This previously unknown pro-resolving cascade may be useful in the development of novel therapeutics that stimulate resolution of inflammation. Moreover, it has been shown that pro-resolving mediators such as resolvins and specifically LXA4 stimulate additional anti-inflammatory molecules in vivo such as interleukin-10 (IL-10).38

4. FPR2/ALXR AS A TARGET FOR THE ROI Our understanding of the mechanisms controlling inflammation and its resolution has made major progress over the past decade.62−64 Following this, new and innovative approaches to treat inflammation are in clinical development. A recent example is the reported efficacy of a lipoxin stable analogue,65 previously described to be active on human cells and in animal models,66 in topical treatment of eczema in humans.67 These developments have prompted research to identify small molecules able to orchestrate inflammation through specific receptors, such as FPR2/ALXR, a member of the formyl peptide receptor (FPR) family. The FPR family was extensively characterized throughout the 1980s as a pertussis toxin-sensitive GPCR.68 The human family was successfully cloned in the 1990s,69 with three genes encoding the FPR1, FPR2/ALXR, and FPR3 proteins clustered on chromosome 19q13.3. This nomenclature will be used here whenever referring to the human system. The FPR family has significant evolutionary divergence across mammalian species, with differential gene expansion particularly notable in the mouse.70 The murine fpr gene family is located on chromosome 17 and consists of seven members, with the orthologue of human FPR1 designated here as mFPR1, and the orthologues of human FPR2/ALXR designated here as mFPR2-RS1/RS2. FPR1 and FPR2/ALXR have been shown to have very similar cellular distribution and to be expressed both on PMN and mononuclear cells. This profile is mirrored by the murine orthologues, suggesting that they may share physiological roles across species.71 FPR1, the first receptor of the family to be discovered, was identified for its ability to transduce the chemotactic effect of a synthetic Escherichia coli-derived N-formyl-methionyl-leucylphenylalanine peptide (fMLF).72−74 FPR1 has been shown to

Figure 3. Schematic representation of the time course of the inflammatory response and resolution. The beginning of an acute inflammatory response is characterized by edema formation and migration of PMNs to the site of inflammation. Later in the response, monocytes and macrophages accumulate at the inflammatory site and promote the resolution of inflammation. Adapted with permission from ref 13. Copyright 1999 Elsevier.

3. ROI: AN OPPORTUNITY FOR THERAPEUTIC TREATMENT Classically, the treatment of nonresolving inflammation involves the inhibition of pro-inflammatory mediators (e.g., by modulating 5-LO,16,17 COX-2,18−21 BLT1/2, or CysLT1/239). In many cases, such approaches are not as effective as anticipated because they are lacking, or even inhibiting, the pro-resolution component of the inflammatory mechanism. The introduction of precise definitions for resolution mechanisms and resolution indices12,27,40,41 permitted the first demonstration that certain agents can be resolution-toxic42 or resolution-friendly, enhancing the resolution pathways and mechanisms.27,42−52 For example, aspirin,27,43,44 statins,45−47 pioglitazone,48 and some LTA4-H inhibitors49,50 are resolutionfriendly by inducing synthesis of SPMs, which might explain some of their effects. Glucocorticoids51,52 and isoflurane42 have also been reported to be resolution-friendly. In contrast, although they reduce the magnitude of inflammation, COX-2 inhibitors are also resolution-toxic. They block biosynthesis of SPMs and thereby delay C



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Further work from the Serhan group90 indicated that LXA4 action in both HL-60 cells and PMN required the expression of a 7-transmembrane domain receptor and that LXA4 is its preferred ligand. Since this receptor is required for LXA4mediated actions in myeloid cells, the term LXA4R, rather than FPR2/ALXR or FPRL1, has been proposed.91−94 The nomenclature has been recently redefined and clarified by the international union of basic and clinical pharmacology (IUPHAR) as FPR2/ALXR,79,95,96 and recent studies challenging LXA4 as a ligand for FPR2/ALXR97−100 were performed, pointing at the difficulty in assessing these studies due to the lack of a positive control for LXA4-induced responses and asking for caution in the interpretation of the results, which were also based on the use of commercial LXA4 without validation of the physical integrity prior to testing.79 Lipoxins have emerged as prominent chemical mediators whose synthesis is switched on during an inflammatory response. They can function as braking signals in inflammation by promoting nonphlogistic phagocytosis of apoptotic neutrophils by macrophages and thereby driving the resolution phase of inflammation.101 Both LXA4 and AT-LXA4 act on FPR2/ALXR (for a review see, ref 102) and GPR32 and exhibit potent anti-inflammatory and pro-resolving actions.103 Besides LXA4, other endogenous pro-resolution ligands are known. Their properties (pro-resolutive/anti-inflammatory/pro-inflammatory) are versatile, often contradictory, and concentrationdependent.77 This might be due to their lack of selectivity since their signaling is often not strictly FPR2/ALXR mediated. It is worth noting that AT-LXA4 (Figure 4) has been reported to rapidly isomerize to its corresponding inactive trans isomer85 and that the rapid conversion of lipoxins in vivo hinders their use as effective pharmaceutical agents.29,104−107 Accumulating evidence indicating that lipoxins are potential anti-inflammatory agents that serve as an endogenous braking signal in the inflammatory process has driven the search for stable analogues,30,89,108−113 culminating in the discovery of ZK-142 at Berlex-Schering,106 which was later licensed to Bayer. Topical efficacy for native LXA4 has been demonstrated, among others, in models of skin inflammation,66 keratitis,114 and uveitis.115 The clinical potential of LXA4 as anti-inflammatory and anti-hyperplasia drug targeting the skin has been demonstrated,116,117 and the first human studies indicating efficacy of LXA4 or stable analogues in the inhalation treatment of asthma have been published,112 followed by studies showing its efficacy in the topical treatment of infantile eczema.67 4.1.2. Annexin A1: Toward Selective Peptides. Annexin A1 (AnxA1), originally termed lipocortin 1, is a glucocorticoidregulated 346 amino acid protein. In human embryonic kidney293 (HEK-293) cells, AnxA1 interacts with FPR2/ALXR specifically.118,119 AnxA1 is present in the nervous system and is involved in nociceptive sensation.120,121 It has also analgesic effects in inflammatory pain through an interaction with FPR2/ ALXR.122 In a recent extended review,123 AnxA1 has been suggested to be a potential mediator for anti-nociception. Upon calcium binding, AnxA1 undergoes a conformational change resulting in the exposure of its unique N-terminal domain, which can then interact with FPR2/ALXR. This portion of the protein is susceptible to cleavage by proteolytic enzymes, leading to AnxA1 inactivation, thus driving the design of stable annexin-based peptides such as AC2−26. AC2−26 can activate and desensitize all three FPR family members at similar concentration in HEK-293 cells.124 The limited potency of AC2−26 on FPR2/ALXR combined with a

play a critical role in immune response, as demonstrated by the fact that fpr1−/− mice were more susceptible to Listeria monocytogenes infection than were wild-type mice.75 More recently, a prominent role for the mouse FPR system has been suggested in the regulation of hepatic inflammatory responses after lipopolysaccharide (LPS)-induced liver injury. Deletion of mFPR1 or mFPR-RS2/RS1 was associated with deregulation of the inflammatory response, with increased inflammation and more severe liver injury.76 FPR2/ALXR is a promiscuous receptor that is also activated by lipids as peptides,77,78 thus making FPR2/ALXR a rather unusual GPCR.79 FPR2/ALXR activation translates in effects ranging from pro-inflammatory to anti-inflammatory and proresolution, resulting in potent opposite effects depending on the ligand. Moreover, constitutive dimerization of the FPR system was shown to be ligand-dependent, indicating that agonist binding and its dimerization state contribute to the signaling signature of this system.80 These unique findings bring a better understanding on the complexity of the FPR2/ALXR signaling and provide a better basis for the development of novel therapeutic approaches,81 and this versatile and central role renders FPR2/ALXR an attractive and challenging therapeutic target. 4.1. Natural Pro-resolution Agonists and Analogues. Since an extended review of the FPR2/ALXR ligands is available,77 we focus here on the natural pro-resolution ligands that resulted in the discovery of FPR2/ALXR and/or served as a starting point to develop new pharmacological agents. 4.1.1. Lipoxins: From Ligand Discovery and Receptor Identification to Clinical Settings. In 1984, Serhan, Hamberg, and Samuelsson reported the first isolation of the natural eicosanoid LXA4 (Figure 4) from human leukocytes as a product originating from AA via the lipoxygenase pathway (Figure 2).82 The term lipoxin was proposed since these trihydroxytetraenes arise from the interactions of multiple distinct lipoxygenase pathways83 shown to occur via trans-cellular and cell−cell interactions (reviewed in ref 24).15,84

Figure 4. Lipoxin A4 and AT-LXA4, an epimer of LXA4 resulting from the interaction of AA with the covalently aspirin-bound COX-2.85

The original suggestion for a role of G-proteins in lipoxin’s effect on PMN cells was made in 1990 using pertussis toxin.86 The first indication showing a specific recognition site for LXA4 on PMNs using tritium-labeled LXA4 was made in 1992.87 Later, it was shown that the receptor is inducible in human promyelocytic leukemia-60 (HL-60) cells.88 In a collaboration with Berlex pharmaceuticals, the Serhan group identified a human 7-transmembrane receptor cDNA (pINF114) that encoded a functional high-affinity lipoxin A4 receptor.89 Chinese hamster ovary (CHO) cells transfected with this cDNA displayed specific 3H-LXA4 high-affinity binding and functional responses to LXA4 (including the stimulation of signal-transduction events), suggesting that the encoded protein is a candidate for a LXA4 receptor in myeloid cells. D



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lack of selectivity toward FPR1 favored the further development of AnxA1-derived peptides such as AnxA12−50 with better selectivity for FPR2/ALXR and suitable anti-inflammatory and pro-resolving effects in various animal models.125 4.1.3. Resolvin D1: Toward Stable Analogues. Resolvin D1 (RvD1) is a lipid biosynthesized from docosahexaenoic acid (DHA) that induces the resolution of inflammation. Its aspirintriggered epimer (Figure 5) displays potent and stereoselective anti-inflammatory properties, and its actions on PMNs were shown to be pertussis toxin-sensitive.3,126−128

Figure 6. WKYMVm-NH2, often referred to as WKYMVm or simply as the W-peptide.

FPR1-transfected cells, confirming it as a highly selective FPR2/ ALXR agonist and differentiating it from fMLF and WKYMVmNH2. MMK-1 was shown to directly compete with 3H-LXA4 on PMNs and to be efficacious in reducing PMN infiltration in an air pouch model.137 4.2.3. TIPMFVPESTSKLQKFTSWFM-Amide (CGEN-855A). The 21 amino acid peptide derivative TIPMFVPESTSKLQKFTSWFM-amide (CGEN-855A)138 was reported by researchers at Compugen Ltd. using a computational platform designed to predict novel peptide GPCR agonists generated by cleavage of secreted proteins. In vivo, the compound displayed anti-inflammatory activity in an air pouch model and provided protection against ischemia−reperfusion-mediated injury to the myocardium. These activities were accompanied by inhibition of PMN recruitment to the injured organ.138 A collaboration between Compugen Ltd. and Merck Serono for the development of the Compugen Ltd. peptide was initiated in 2008 and transferred to BiolineRX in late 2011,139 where it was reported to be in preclinical development.140 4.2.4. Summary. Several peptidic or lipidic ligands for FPR2/ ALXR have been described,77 and only a few of them, often used as references in the studies described in this perspective, have been reported here. Table 1 summarizes their FPR2/ALXR vs FPR1 selectivity profile as represented by their potency for calcium release induction. 4.3. Small Molecule FPR2/ALXR Agonists. The first reports on small molecule agonists of FPR2/ALXR date back to 2004, both from the pharmaceutical industry and academic groups. A review summarizing the research efforts on both FPR1 and FPR2/ALXR as published in peer-reviewed journals, without addressing the patent literature, was recently released.141 The reader is referred to this excellent review for details on modeling, whereas we aim here at reviewing the effort toward the development of FPR2/ALXR small molecule agonists with a special emphasis on the patent literature. 4.3.1. Pharmaceutical Industry. 4.3.1.1. Arena Pharmaceuticals. To the knowledge of the authors, the first report of nonpeptidic FPR2/ALXR agonists was made in 2004 from research performed at Arena Pharmaceuticals. Upon preincubation of eosinophils with these FPR2/ALXR specific agonists, LL-37-induced degranulation was suppressed. For neutrophils, preincubation suppressed TNFα-induced IL-8 release. A specific compound, named AR234245142 (structure not disclosed), has been reported to suppress ovalbumin-induced airway hyperresponsiveness in vivo. It also decreased mortality in a mouse model of bleomycin-induced pulmonary fibrosis, suggesting a use of this novel series of FPR2/ALXR agonists for the treatment of human asthma and chronic obstructive pulmonary disease (COPD).142

Figure 5. Biosynthesis of RvD1 and AT-RvD1. RvD1 and AT-RvD1 are both generated from DHA. 15-LO converts DHA into 17-S-H(p)DHA, whereas aspirin-acetylated COX-2 generates predominately R-containing 17-R-H(p)DHA. The 17-hydro(peroxy) products of DHA are then converted rapidly by 5-LO into the corresponding epoxide, whose enzymatic hydrolysis leads to the formation of RvD1 and AT-RvD1, respectively.3

Screening systems to deorphanize RvD1 identified FPR2/ ALXR as one of the receptors for RvD1, with the other being GPR32. This was confirmed using a β-arrestin-based ligand receptor system.103 In macrophages, RvD1 was shown to stimulate cAMP/PKA signaling in an FPR2/ALXR-dependent manner,129 and a stable RvD1 analogue was shown to promote corneal allograft survival.130 Recently, in an acute lung injury (ALI) model, RvD1 was shown to stimulate alveolar fluid clearance in the FPR2/ALXR pathway.131 4.2. Synthetic Peptides. Since reviews on synthetic FPR2/ ALXR peptide ligands were recently published,77,102 only the peptide agonists used as reference for the work on small molecules or those that reached clinical development are discussed here. 4.2.1. WKYMVM-NH2 and WKYMVm-NH2. WKYMVM-NH2 (Figure 6) was initially identified by screening of a synthetic peptide library for stimulation of the formation of inositol phosphate.132 Modification of the methionine at the C-terminus with the D-type amino acid yielded WKYMVm-NH2 which exhibited increased effectiveness in biological assays such as, for example, inositol phosphate hydrolysis.133 It was found to activate both FPR1 and FPR2/ALXR with high potency.134 4.2.2. LESIFRSLLFRVM (MMK1). The 13 amino acid peptide LESIFRSLLFRVM (MMK1)135 was identified using a yeastbased complementation screen. It was confirmed to be a selective FPR2/ALXR agonist in HEK-293 cells carrying Gα16 and stably expressing FPR2/ALXR, where its activity, as determined by measuring transient Ca2+ flux, was below 10 nM, whereas it was above 10 μM in the FPR1-transfected cells.135 In a calcium mobilization assay using RBL-2H3 transfected cells, it was reported to have a FPR2/ALXR EC50 Ca2+ of 56.7 nM136 and to induce minimal Ca2+ mobilization in E



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Table 1. Schematic Description of the Potency for Calcium Release Induction of a Selection of FPRs Agonists as Interpreted from Sometimes Contradictory Dataa FPR2/ALXR FPR1 a

fMLF

LXA4

RvD1

WKYMVm-NH2

WKYMVM-NH2

MMK1

AC2−26

+ ++++

++++ −

++++ −

++++ ++++

++++ +++

++++ +

+ +

A lack of agonistic effect is represented by −, whereas + represents an agonistic effect whose activity is represented by the number of +.

4.3.1.2. Acadia Pharmaceuticals. Acadia patented a series of compounds and claimed their use as selective FPR2/ALXR agonists for the treatment of pain and inflammation.143 Three classes of compounds (Figure 7) were presented, including, for example, compound 1, which was described with a FPR2/ALXR EC50 Ca2+ of 1260 nM, compound 2, with a FPR2/ALXR EC50 Ca2+ of 1585 nM, and compound 3, with a FPR2/ALXR EC50 Ca2+ of 50 nM. It was further shown that, when given intraperitoneally, compound 3 dose-dependently prevented thermal hyperalgia induced by carrageenan in male Sprague−Dawley (M-SD) rats. Compound 3 also dose-dependently prevented edema formation induced by carrageenan in M-SD rats with an efficacy comparable to that of ibuprofen. More recently, a publication by Biovitrum (see Section 4.3.1.5) described compound 3 with an FPR2/ALXR EC50 Ca2+ of 35 nM, whereas its enantiomer is described with a similar activity (30 nM).144

Switching the position of the chlorine from para to ortho delivered analogue 7, which was inactive, both in calcium release and PMN migration assays. The original hit 4 and the optimized pyrazolone 6 showed similar FPR2/ALXR-mediated Ca2+ flux activity (30 and 44 nM, respectively). These compounds were reported as having no significant activity in a FPR1 counterscreen. Later, this reported selectivity was challenged in a report from Daiichi Sankyo146 (see Section 4.3.1.4) and Biovitrum144 (see Section 4.3.1.5).

Figure 8. Selected examples from the Amgen FPR2/ALXR agonists.145 Upon topical administration, the poorly soluble 4 was shown to be efficacious in reducing ear swelling in mice, whereas the inactive 5 did not show efficacy. The optimized and soluble 6 was suitable for oral administration and showed efficacy in the same model. The key role of the aniline moiety substitution is exemplified by comparing 6 to 7. Compounds 4 and 6 were originally reported as having no activity on FPR1, but this has since been challenged,144,146 possibly explaining some of the later findings with 6.147

In a functional cell-based assay, 6 showed a dose-dependent inhibition of PMN migration, irrespective of the nature of the stimulant (IL-8 or fMLF). On the basis of a better solubility, 6 was selected for further profiling in the rat. Following iv administration at 0.5 mg/kg, the compound showed a relatively low clearance (0.126 L/h/kg) and a half-life of 2.78 h. Significant levels of 6 were detected in the systemic circulation such that the bioavailability following po administration (2 mg/ kg) was calculated to be 91%. Compound 6 was tested in a mouse ear inflammation model showing, at the 50 mg/kg dose po, a reduction in edema by 58%, which is similar to that of the positive control dexamethasone at 1 mg/kg iv. The original hit 4, not suitable for po application, was tested topically, where it demonstrated a 52% reduction in ear swelling (25 μg/ear). Under the same conditions, 16-phenoxy-lipoxin showed a 69% reduction. The authors suggested that the differential effects of the pyrazolone and the 16-phenoxy-lipoxin on PMN migration may be based on the different signal transduction events triggered by these different classes of agonists. This is not unprecedented for other GPCRs, as shown by differences observed between synthetic and endogenous agonists.148 Amgen developed a second series of agonists based on the benzimidazolone hit 8 (Figure 9) identified from a new screening campaign as a weak FPR2/ALXR agonist (FPR2/ ALXR EC50 Ca2+ = 6390 nM).147 Replacement of the benzimidazolone by a benzimidazole and subsequent contrac-

Figure 7. Examples of the three classes of compounds covered by the Acadia patent.143 Compound 3, for which no activity on the rat FPR system is reported, was shown to be efficacious in different rat models of inflammation.

4.3.1.3. Amgen. In 2006, Amgen reported the results of an HTS screen of their compounds library, identifying pyrazolone 4 as an agonist of FPR2/ALXR (Figure 8).145 A detailed SAR on the phenyl-urea moiety demonstrated that the steric and electronic nature of the para-substituent R2 on the phenyl group had a major impact on FPR2/ALXR activity. Alternating the position of the substituent resulted in a dramatic loss of potency, whereas disubstituted ring systems showed that subtle modification in the substitution pattern strongly altered the activity. Further studies suggested that replacing the phenyl group by heterocyclic moieties were not well-tolerated. Further SAR studies identified chloropyrazolone derivative 6, often referred as compound 43 in the literature (Figure 8). F



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tion of the piperidine ring to a pyrrolidine allowed benzimidazole 9 (FPR2/ALXR EC50 Ca2+ = 34 nM) to be identified, whereas the (S)-enantiomer of 9 was 100-fold less active (FPR2/ALXR EC50 Ca2+ = 3500 nM). According to the report, both enantiomers were inactive on FPR1 (EC50 > 10 000 nM). As for 4 and 6, (R)-9 showed a dose-dependent inhibition of PMN migration, irrespective of the nature of the stimulant, whereas its less active S-enantiomer had no effect on migration. Until this point, all of the effects observed by Amgen were antiinflammatory, but, when tested for secretion of IL-6 from human whole blood, both FPR2/ALXR agonists 9 and 6 stimulated IL-6 secretion above levels induced by IL-1β alone, suggesting a pro-inflammatory effect. 8 and the S-enantiomer of 9 did not cause such an effect. The authors concluded that this pro-inflammatory effect was FPR2/ALXR-mediated, questioning how FPR2/ALXR might serve as a therapeutic target. Put in the context of the discovery that 6 is a FPR1−FPR2/ALXR dual agonist (see above and below), we suggest that this questioning should be reviewed and that the selectivity profile of 9 might also be challenged. It should also be pointed out that IL-6 is important in host defense and that in this context IL-6 may not always be viewed as a pro-inflammatory molecule but may play an integral part in the resolution and clearance of microbial infection.149

other experimental differences, i.e., actual receptor expression levels may be a likely explanation. However, the critical remarks of Evi Kostenis on the use of Gα16 should be taken into account as well.153 Nevertheless, 6 remains the first potent, orally active compound that showed agonistic activity for both FPR2/ ALXR and mFPR-RS1/RS2, making it a suitable tool to study the function of FPRs in neutrophil migration in vivo. Studies were undertaken by scientists at Daiichi Sankyo150 to compare 6 to fMLF, which has the unique feature of inducing crossdesensitization in neutrophils in vitro. Cross-desensitization is the heterologous desensitization of chemoattractant receptors, that is, stimulation of neutrophils with one chemoattractant renders the cells unresponsive to subsequent stimulation with unrelated chemoattractants. fMLF, which stimulates FPR1, had been shown to desensitize not only FPR1 but also C5aR and CXCR2, and so it inhibited neutrophil responses, such as calcium mobilization and chemotaxis, which are induced by C5a or IL-8.154−158 There is a hierarchy in the ability to induce crossdesensitization among chemoattractants, with a rank order of fMLF > C5a > IL-8.155,157 Therefore, stimulation of FPR1 appears to have a dominant effect on the induction of crossdesensitization in neutrophils. Prior to the discovery of the property of 6, the phenomenon of cross-desensitization could be studied only in human cells in vitro due to the lack of suitable compounds since fMLF activity on mFPR1 is weaker than that on FPR1 and its peptidic nature made it difficult for in vivo studies to be performed. In the Daiichi Sankyo study,150 it was shown that neutrophils stimulated with 6 lost their chemotactic response to chemoattractants in vitro and that oral administration of 6 to mice inhibited LPS-induced neutrophil migration into the airway, possibly through the induction of cross-desensitization. These results are consistent with the idea that the agonist for mFPR1 and mFPR-RS1/RS2 induced cross-desensitization in neutrophils and attenuated neutrophil migration into the airway. These results also revealed that a FPR1 and FPR2/ALXR agonist may act as a functional antagonist for multiple chemoattractant receptors. Because 6 has agonistic activity for both FPR1 and FPR2/ ALXR, it remains unclear how each receptor is involved in the induction of cross-desensitization in neutrophils in vitro and in the inhibition of neutrophil migration in vivo. Previous studies using cells transfected with a combination of FPR1 and other chemoattractant receptors indicated that cross-desensitization induced by fMLF in vitro was mediated by FPR1.154,159,160 The authors speculated that FPR2/ALXR might also be capable of inducing cross-desensitization in neutrophils because it has been reported that some FPR2/ALXR-selective agonists induced cross-desensitization of CCR5 and CXCR4 in human monocytes and cells transfected with FPR2/ALXR in combination with CCR5 or CXCR4.161−163 In addition, the FPR2/ALXR-dependent inhibitory effect of 6 on neutrophil migration in the IL-1β induced air pouch assay described in a study using FPR2/ALXR-deficient mice164 implies that FPR2/ALXR-mediated cross-desensitization is operative in vivo as a mechanism of inhibition of neutrophil migration by 6. In a subsequent article, the same authors confirmed the specificity of compound 6 on a panel of 341 receptors, channels, transporters, or enzymes, where only cytochrome P450 2D6 and dopamine transporter were partially inhibited.151 The inhibitory mechanism of 6 was further investigated using the chemotactic

Figure 9. Compound 8, the Amgen hit originating from a screening campaign that led to the discovery of 9, described as a probe to better understand the role of the FPR2/ALXR receptors.147

4.3.1.4. Daiichi Sankyo. In a detailed study of the agonistic activity of 6 for FPR1 and FPR2/ALXR and their mouse homologous receptors mFPR1, mFPR-RS1/RS2, respectively, researchers at Daiichi Sankyo146 reported 6 as a FPR1 and FPR2/ALXR dual agonist, both for human (FPR1 EC50 Ca2+ = 65 nM; FPR2/ALXR EC50 Ca2+ = 22 nM) and mouse receptors (mFPR1 EC50 Ca2+ = 350 nM; mFPR-RS1/RS2 EC50 Ca2+ = 890 nM).146,150,151 The dual agonistic activity of 6 on FPR1 and FPR2/ALXR was further confirmed by receptor internalization studies, where 6 showed a pattern similar to that of WKYMVM and fMLF. This confirms that 6 is a potent and dual FPR1 and FPR2/ALXR agonist and led to a proposal to reconsider the Amgen data and their biological interpretation. The reported discrepancy could be due to the use of different types of G-proteins in the respective aequorin assay: Amgen scientists used Gα15, whereas Daiichi Sankyo scientists used Gα16, which might influence the sensitivity of the calcium signal in the aequorin assay. Nevertheless, although receptors are known to selectively interact with Gα16,152 there is no direct experimental evidence with regard to FPR2/ALXR and FPR1. Because significant differences in Gα16/Gα15 coupling (efficiencies) were not demonstrated by Offermanns et al.,152 G



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Figure 10. Five structurally new hits (10−14) identified in a library screen by their ability to induce a transient rise in intracellular Ca2+ in FPR2transfected cells144 and 15, a FPR2 agonist discovered earlier165 and used as comparator. In further studies, 11, 13, and Amgen compound 6 were shown to activate neutrophils preferentially through FPR1.

over 50 000 compounds. From the more than 500 hits that were confirmed as agonists, five structurally diverse FPR2/ALXR agonist hits (Figure 10, 10−14) were selected and further investigated together with compounds identified earlier: Amgen compound 6, Acadia compounds 1, both compounds (R)- and (S)-3, as well as compound 15, discovered earlier (see Section 4.3.2.1, where 15 is discussed in detail) and often referred to as Quin-C1 in the literature.165 All of these agonists were found to activate neutrophil superoxide production. Compounds 11, 13, and Amgen compound 6 were further studied and were shown to be able to activate both FPR2/ALXR and FPR1. On the basis of desensitization studies in neutrophils, the authors suggest that all three compounds activate neutrophils preferentially through FPR1. 4.3.1.6. Allergan. Allergan appears to be one of the most active players in developing FPR2/ALXR small molecule modulators. It started in 2011 with the publication of a patent covering FPR2/ALXR agonists or antagonists of formulas I and II (Figure 11).166 All 90 compounds with a given EC50 were aniline derivatives covered by formula II and, according to the chemistry, were either cis-endo or cis-exo.

responses of human peripheral blood neutrophils toward various chemoattractants, such as IL-8, C5a, and LTB4, by evaluation of calcium flux, chemotaxis, and receptor expression. Pretreatment of human neutrophils with 6 dose-dependently inhibited calcium mobilization in human neutrophils stimulated with IL8, C5a, or LTB4, suggesting that pretreatment with 6 heterologously desensitized chemoattractant-stimulated calcium mobilization in human neutrophils. Also, pretreatment with 6 attenuated the chemotactic responses of human neutrophils against IL-8, C5a, or LTB4 in a concentration-dependent manner. In a flow cytometric analysis, it was shown that 6 also reduced the expression of the chemoattractant receptors CXCR1, CXCR2, C5aR, and BLT1 on neutrophils, whereas CD11b, one of the surface activation markers on neutrophils, was increased by incubation with 6. These findings strongly suggest that 6 induced heterodesensitization (cross-desensitization) of chemoattractant receptors in human neutrophils. The significant effect of 6 on the expression of receptors might be attributed to either FPR1 or FPR2/ALXR since both FPR1154,155,157,159 and FPR2/ALXR161−163 have been found to induce cross-desensitization. The authors propose the induction of chemoattractant receptor cross-desensitization in neutrophils as a novel approach for the treatment of neutrophil-related inflammation. A crossdesensitization inducer is speculated to be an effective drug because it can inhibit responses to multiple chemoattractants simultaneously. Compounds such as 6 may work as functional antagonist for multiple chemoattractant receptors and may have therapeutic benefit on neutrophil-related inflammation in humans. To elucidate the roles of FPR1 and FPR2/ALXR in the induction of cross-desensitization, more active and, particularly, more selective ligands for each receptor are needed. 4.3.1.5. Biovitrum. Biovitrum144 reported small molecule FPR2/ALXR agonists originating from screening a library of

Figure 11. Formulas I and II of the Allergan patent covering derivatives of cycloalkyl- and cycloalkenyl-1,2-dicarboxyclic acid having FPR2/ ALXR agonist or antagonist activity.166 H



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The chirality of the agonist plays a major role on the activity, as shown by acid (−)-16 and (+)-16 obtained by chiral chromatography separation of the racemate (Figure 12). The (+)-isomer 16 was shown to be over 100 times more active than (−)-isomer 16, whose absolute stereochemistry was determined by X-ray crystallography. Tentative modification of the aniline moiety revealed the importance of the NH moiety, as shown by the strong loss in activity upon N-methylation (17). From these examples, it appears that 4-substitution is a requirement, with halogen as the favored substitution and bromide being the most exemplified one. The examples with a single ortho or meta substitution indicate low-activity compounds (Figure 12).

Figure 13. Enantiomers (−)-20 and (+)-20 show the preponderant role of the stereochemistry in the amide subseries. Substitution is tolerated, as shown by 21 and 22, whereas compounds 23−26 show that neither the ester, alcohol, nitrile, nor the hydroxylamine are tolerated.166

Figure 12. Pure enantiomers (−)-16 and (+)-17 obtained by chiral chromatography separation show the key role of chirality. Compounds 17−19 exemplify the tight SAR observed on the aniline moiety.166

The acid can be replaced by an amide group, where a chiral preference is also observed. For instance, (−)-isomer 20, obtained by chiral separation, is solely responsible for the activity (Figure 13). The presence of a double bond on the bicyclic moiety had no major impact, and the cyclopropyl could be replaced by a dimethyl moiety with minor loss of activity. Monosubstitution of the amide moiety was tolerated, as shown by 21, and the NH of this amide was not essential, as shown by bis-alkylated amine 22. Other described modification of the acid moiety resulted in a drastic loss of activity: an ester induced a 100-fold loss of activity, as shown by comparing 23 to 21. Alcohol ((±)-24), nitrile (25), or hydroxylamine (26) did not seem to be tolerated either. In 2013, a follow-up patent of Allergan covering polycylic pyrrolidine-2,5-dione of formula III (Figure 14) as FPR2/ALXR modulators was published, with 12 cyclized analogues of this class of compound exemplified.167 These new compounds can be seen as cyclized derivatives of the previously claimed acids.166 In 2012, a patent of Allergan covering four 3,4-dihydroisoquinoline-phenylurea derivatives (Figure 15) having FPR2/ ALXR activity was published.168 As for most of the previous Allergan exemplified compounds, they all contain an aniline moiety. The same year, a patent of Allergan169 covering dihydroisoquinoline derivatives as FPR2/ALXR modulators was published. Among the phenylurea derivatives exemplified, mostly as racemate (such as 34), 32 had an EC50 given, allowing for the presumption that the (R)-enantiomer is preferred, as displayed for 35 (Figure 16). Researchers at Allergan also reported dihydronaphthalene and naphthalene derivatives (Figure 17) as FPR2/ALXR modulators in a patent published in 2012 and covering

Figure 14. Formula III of the Allergan patent covering polycyclic pyrrolidine-2,5-dione derivatives as FPR2/ALXR modulators167 with 27−29, selected among the most active examples and focusing on the tolerability of the R1 position.

Figure 15. Four examples covered by the Allergan patent covering 3,4dihydroisoquinoline-2(1H)-yl-3-phenylurea derivatives having FPR2/ ALXR agonist or antagonist activity.168

compounds of formula IV.170 From the 12 compounds for which an FPR2/ALXR EC50 is given, 36 is the most active example and is also described as one of the preferred compounds. Substituted amino acid derivatives (Figure 18) have been covered by Allergan as FPR2/ALXR modulators in a patent with 98 examples published in 2013.171 An article commenting on this discovery and showing selected representatives with their reported activities was published recently.172 Regarding the stereochemistry, both R and S compounds are covered, but a I



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Figure 19. Selected aryl urea derivatives covered in an Allergan patent with 29 examples. 41 shows the importance of the chiral information, whereas 42 represents the most active compound exemplified.173

Figure 16. Representative examples of the Allergan patent covering novel 1-(1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)urea derivatives as FPR2/ALXR modulators.169

Figure 17. Formula IV of the Allergan patent covering dihydronaphthalene and naphthalene derivatives as FPR2/ALXR modulator, with 36 as the most active example.170

Figure 20. Formula V of a patent covering 2,5-dioxoimidazolin-1-yl-3phenylurea derivatives as FPR2/ALXR modulators,174 with 43 as the most active compound among the 96 examples. Formula VI of a followup patent,175 with example 44 shown, which is 10-fold less active than the direct analogue 45 covered by the previous patent.

strong preference for the (S) enantiomers is shown, for example, by 37, reported at 0.88 nM, whereas the (R) enantiomer is reported at 1229 nM. There seems to be a parallel between this series and the bicyclic compounds reported earlier: parasubstituted anilines are preferred, and modifications of the acid are possible, with carboxamide 38 as the preferred modification, whereas ester induced loss of activity, as shown by 39. It is of interest to note that a methyl ketone is a suitable alternative, as shown by 40.

Recently, (2-ureidoacetamido)alkyl derivatives (Figure 21) were described as FPR2/ALXR modulators.176 The reported examples show that phosphonic diesters such as 46 and 47, phosphonic monoesters such as 48, sulfonic acids such as 49, hydroxyl isoxazoles such as 50, and tetrazoles such as 51 or 52 are tolerated. Different substituents at the chiral center were also exemplified. 4.3.1.7. Actelion. The most common feature of the FPR2/ ALXR agonists described to date is the presence of an aniline moiety, preferably substituted in the para position. Such anilines are known to possibly lead to genotoxicity and thus FPR2/

Figure 18. Selected examples from the Allergan patent covering amide derivatives on N-urea-substituted amino acids as FPR2/ALXR modulators. As previously observed for other classes of compounds, acids and carboxamides were well-tolerated, as shown by 37 and 38. Ester such as 39 induced some loss of activity, whereas a ketone was tolerated (40).171

Shorter urea derivatives (Figure 19) were described later in a patent with 29 examples.173 Not surprisingly, here again the stereochemistry played a role, as shown by 41, where the (S) compound is described as the most active enantiomer (51 vs 1713 nM). The most potent compound, 42, is described with an FPR2/ALXR EC50 of 8 nM. 2,5-Dioxoimidazolidine phenylurea derivatives (Figure 20) were published simultaneously, covering the compounds described by the following formula V with 96 examples and example 43 as the most active compound with an FPR2/ALXR EC50 of 0.5 nM.174 In a follow-up patent,175 imidazolidine-2,4diones of formula VI were reported. Direct comparison is possible between compound 44 reported with an FPR2/ALXR EC50 of 56 nM and compound 45 from the previous patent reported with an FPR2/ALXR EC50 of 6.3 nM.

Figure 21. Selected examples displaying acid bioisosters of (2ureidoacetamido)alkyl derivatives covered as FPR2/ALXR modulators by an Allergan patent.176 J



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In a patent covering oxazolyl-methyl ether derivatives (Figure 25),184 high agonistic activities were obtained only for oxazolyl groups having the nitrogen atom between the two substituted carbon atoms, as, for example, 59 with an FPR2/ALXR EC50 Ca2+ of 1.7 nM, something that was not the case in the previously reported derivatives. It was also demonstrated that the ether derivative 59 is remarkably more stable in plasma (t1/2 > 24 h) than the corresponding keto derivative 60 (t1/2 < 5 h) covered by a previous patent.180 No FPR2/ALXR EC50 Ca2+ was reported for 60. The same is true for a patent covering hydroxylated aminotriazole derivatives (Figure 25),185 where the plasma stability of example 61, the direct analogue of 60, is also reported to be above 24 h.

ALXR agonists without an aniline moiety would be preferrable.177,178 In 2009, two patents of Actelion were published simultaneously, both covering novel nonaniline-containing FPR2/ ALXR agonists (Figure 22). They cover aminopyrazoles of formula VII and aminotriazole derivatives of formula VIII, with FPR2/ALXR EC50’s Ca2+ given for 61179 and 23 compounds, respectively.180 Both patents were reviewed later, and examples 53 and 54 were described in the literature with a comment indicating that these patents are a clear indication that FPR2/ ALXR is an emerging potential therapeutic target.181

Figure 22. Novel nonaniline-containing aminopyrazoles179 53 and aminotriazoles180 54 covered by formulas VII and VIII of two Actelion patents covering FPR2/ALXR agonists.

Figure 25. Compound 59, covered by an Actelion patent on oxazolylmethyl ether derivatives,184 as well as compound 61, covered by a separate patent on hydroxylated aminotriazole derivatives185 were reported to have an improved plasma stability over that of the ketoanalogue 60.

The replacement of the central pyrazole or triazole was the object a follow-up patent covering oxazole and thiazole derivatives of formula IX, with 55 (Figure 23) as the most active compound with a FPR2/ALXR EC50 Ca2+ of 0.3 nM.182

In addition to these classes of compounds, a patent of Actelion covering bridged bicyclic compounds (Figure 26) was published in 2010.186 The similarity of these compounds with the Allergan compounds (Figures 11−13) is striking, with the exception that the arrangement of the substituent is cis in the Allergan examples and trans in the Actelion ones. From the 422 examples, all with an FPR2/ALXR EC50 Ca2+, 62 with an EC50 of 0.07 nM is the most active compound. Many of the examples do not contain an aniline moiety, such as example 63 or 64, whose optimized derivatives have been the topic of further patents by Actelion (see below).

Figure 23. Derivative 55, the most active compound covered by an Actelion patent on oxazole and thiazole derivatives of formula IX as FPR2/ALXR agonists.182

The modifications of the extremities of the triazole-based FPR2/ALXR agonists were exemplified in a series of follow-up patents.183−185 In a patent covering fluorinated aminotriazole derivatives,183 56 (Figure 24), reported with an FPR2/ALXR EC50 Ca2+ of 2.4 nM, was shown to have a superior covalent binding profile when compared to those of compounds of the original triazole covering patent,180 indicating a lower risk for adverse effects. Figure 26. Derivative 62, the most active compound of an Actelion patent covering bridged spiro [2.4] heptane derivatives as FPR2/ALXR agonists. 63 and 64 are two representatives of nonaniline-containing agonists.186

A patent with a single example (65, Figure 27), covering a 1(p-tolyl)cyclopropyl-substituted bridged spiro[2,4]heptane derivative, is the result of optimization around compound 64 (Figure 26).187 Another similar patent with six examples covering fluorinated bridged spiro[2,4]heptane derivatives188 describes enantiopure example 66 (Figure 27) as one of the most active compounds with an FPR2/ALXR EC50 Ca2+ of 2.0

Figure 24. Compound 56, covered by an Actelion patent on fluorinated aminotriazole derivatives, is reported to have improved covalent binding.183 K



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In 2005, a patent covering compounds of formula X was published, with 15 (Figure 29) exemplified among nine other derivatives.165

nM and an improved bioavailability in rat (12%) compared to its nonfluorinated analogue 67, which was covered in the previous patent and showed no bioavailability.186

Figure 29. Formula X of the patent covering, among others, 15, often referred to as Quin-C1 in the literature.136

Figure 27. Derivative 65, the unique example of a patent covering 1-(ptolyl)cyclopropyl-substituted bridged spiro[2.4]heptane derivatives as FPR2/ALXR agonist.187 66 is an example from a patent covering fluorinated derivatives shown to have an improved bioavailability, as shown by comparison with the nonfluorinated analogue 67.

In a calcium mobilization assay using RBL-2H3 transfected cells, a dose−response curve allowed the FPR2/ALXR EC50 Ca2+ for 15 (1.41 μM), MMK1 (56.7 nM), and WKYMVm (4.45 nM) to be determined. However, 15 was more efficacious than MMK1 in stimulating calcium mobilization, which was markedly reduced upon pertussis toxin pretreatment, indicating Gαi protein-mediated signaling. In addition to stimulating calcium mobilization, 15 also induced phosphorylation of the MAP kinases ERK1 and ERK2. Importantly, similar to MMK1, 15 was shown to induce minimal Ca2+ mobilization in FPR1-transfected cells, making it a selective FPR2/ALXR agonist and differentiating it from fMLF and WKYMVm. On the other hand, 15 might be seen as similar to WKYMVm since it also effectively stimulated FPR2/ALXR internalization. Nevertheless, it showed only partial competition with the WKYMVm peptide for binding to FPR2/ALXR, suggesting that 15 interacts with FPR2/ALXR on a site that is different from, but probably adjacent to, the WKYMVm binding site such that partial competition can be achieved. Using human neutrophils, 15 was shown to be able to induce their chemotaxis and to induce their degranulation (as measured by β-glucuronidase release) in a dose-dependent manner. In contrast to the MMK1 peptide, 15 was unable to stimulate potent superoxide production at a concentration up to 100 μM. The authors suggest that those ligands may behave differently in functional assays because of their intrinsic ability to induce different receptor conformational changes. This is supported by data showing that lipoxin A4 and several peptide agonists for FPR2/ALXR activate selected functions of neutrophils, with no clear correlation to their binding affinity and potency in other functional assays,192 or are able to induce superoxide production without Ca2+ mobilization.193,194 The authors further suggest that ligand-specific receptor conformation change is a property that may be explored for designing novel ligands capable of stimulating selected functions of FPR2/ALXR. In an effort to study the SAR around quinazolinone derivative 15, a series of substituted derivatives as modulating agents for FPR2/ALXR was designed.195 Most modification at Ar1 and Ar2 delivered inactive compounds. Studies of the substitution of the aryl at the 2 position of the quinazolinone backbone showed the critical importance of the aromatic ring substitution: removal of the 4-methoxy of 15 (reported with an FPR2/ALXR EC50 Ca2+ of 470 nM) or addition of a 2-methoxy induced full loss of activity. Only 4-methyl (1480 nM) and 4-nitro (1110 nM) were partially tolerated. Of particular interest in this perspective is the complete reversal of bioactivity when the methoxy group of 15

A subsequent patent of Actelion covered ester derivatives of the same bridged spiro[2,4]heptane.189 EC50’s are reported for all 78 examples, with compound 68 (Figure 28) being the most active with an EC50 of 0.4 nM, and 37 nonaromatic aminecontaining compounds, with 69 being the most active with an EC50 of 3.7 nM.

Figure 28. Compound 68 is the most active compound covered by a patent on ester derivatives of bridged spiro[2.4]heptane. Compound 69 is the most active nonaniline-containing derivative.189

4.3.2. Academic Research. 4.3.2.1. Shanghai Institute of Materia Medica and the University of Illinois. In the same year as the report from Arena, a library screen of 15 760 synthetic compounds and 400 natural compounds was performed by a joint effort of the University of Illinois and the Shanghai Institute of Materia Medica.136 Using the previous finding that transfected cells expressing FPR1 or FPR2/ALXR could respond to some of their respective agonists with NF-κB activation,190,191 an NF-κB-driven luciferase reporter-based assay was designed for the HTS with the purpose of discovering agonists that could selectively stimulate neutrophil functions. By selecting from among the 56 hits those stimulating the luciferase reporter only in the presence of FPR2/ALXR, three quinazolinone-based hits of formula X, where A represents an oxygen, were discovered (Figure 29). The most promising one, 15 (often referred to as Quin-C1 in the literature and already described in Section 4.3.1.5), was further characterized in calcium mobilization assays as well as neutrophil functional assays. L



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was replaced by a free hydroxyl, resulting in 70 (Figure 30), an antagonist for FPR2/ALXR with no activity in a calcium release assay but with dose-dependent inhibition (IC50 in the double digit micromolar range) of 2 nM WKYMVm-stimulated calcium mobilization. 70 was also shown to dose-dependently suppress chemotaxis (IC50 in the low double digit micromolar range) in 100 nM 15 and 10 nM WKYMVm-stimulated RBL-FPR2/ ALXR cells.

Figure 31. Compound 71, the most active FPR2/ALXR agonist covered by formula XI.199

Following these findings and aiming at the direct discovery of new selective FPR2/ALXR agonists, 6000 synthetic compounds were screened for their ability to activate Ca2+ mobilization in RBL cell lines transfected with human FPR2/ALXR.200 Several compounds (Figure 32) were identified as selective FPR2/ ALXR agonists and were shown to activate intracellular Ca2+ mobilization and chemotaxis in human neutrophils. The specificity of these compounds was verified by analyzing their ability to activate Ca2+ mobilization in HL-60 cells transfected with human FPR2/ALXR or FPR1. It has to be emphasized that, except for 80 which is based on the same quinazolinone backbone as 15136 (Figure 29), the chemotypes present in all discovered FPR2/ALXR agonists had not been previously reported. From the 20 analogues selected based on scaffold 72, five were shown to be active and selective for FPR2/ALXR, all having the para-methoxy group, which seemed to be essential for activity in this class of compounds, where 73 was the most active (FPR2/ALXR EC50 Ca2+ = 100 nM). From the 39 analogues selected based on scaffolds 74 and 75, eight were shown to be active and selective for FPR2/ALXR and showed the same trend regarding the aniline substitution, with 76 being the best derivative at around 2000 nM. From the 12 analogues selected based on scaffold 77, five were shown to be selective for FPR2/ALXR but displayed low efficacy. The authors concluded by proposing that the development of FPR2/ALXR agonists represents an important avenue to pursue for therapeutic purposes in oncology, as a vaccine adjuvant, or, more generally, as immune modulators to enhance phagocyte host defense against pathogens.

Figure 30. Compound 70, the des-methylated analogue of 15, acting as an antagonist for FPR2/ALXR.195

In binding studies, 15 has been shown to have an IC50 of 92 nM. In competition studies with [125I]WKYMVm in membranes prepared form RBL-FPR2/ALXR cells, 70 could not completely displace the binding of the peptide to FPR2/ALXR at 100 μM, and an IC50 value of 6653 nM was derived from maximal, but not complete, displacement of the radioligand. Despite this, it could be shown that it specifically interacts with FPR2/ALXR but not FPR1. These results are of interest since, based on the broad spectrum of ligand specificity, it is predicted that multiple binding sites exist in FPR2/ALXR to allow for its interaction with both peptides and lipids. 15 was initially identified as an agonist selective for FPR2/ ALXR, and its role in mice had to be established. In RBLtransfected cells, both mFPR1 and mFPR-RS1/RS2 were able to mediate 15-induced calcium mobilization (mFPR1 EC50 Ca2+ = 35 nM; mFPR-RS1/RS2 EC50 Ca2+ = 166 nM). The identification of this species difference shows the importance of characterizing FPR2/ALXR agonists in the relevant species to interpret pharmacological data. In a mouse model of bleomycin-induced lung injury, intraperitoneal application of 15 significantly reduced the neutrophil and lymphocyte counts in bronchoalveolar lavage fluid. Since the compound did not improve lung fibrosis when the treatment was started 5 days after bleomycin challenge, it has been suggested that the protection may be attributed to the antiinflammatory effects of 15.196 Those experimental data suggest the feasibility of using FPR2/ALXR small molecule agonists to treat certain inflammatory disorders. 4.3.2.2. University Consortium. This section reports the work from different universities working in collaboration, including groups from the Montana State University, the University of Illinois, the Altai State Technical University, the Commissariat à l′Energie Atomique in Grenoble, the Università degli Studi di Firenze, the Università degli Studi di Roma, the Università degli Studi di Bari, and the University of Gothenburg. A work originally designed to identify molecules that activate human neutrophil reactive oxygen species (ROS) production or TNF-α inducing compounds197 led to the discovery of selective FPR2/ALXR agonists.198 A patent covering compounds of formula XI (Figure 31) with 85 examples has been published, with 71 as the most active compound with an FPR2/ALXR EC50 Ca2+ of 300 nM in Ca2+ release on human neutrophils.199

Figure 32. Screening hits and derivatives representing, except for 80, new chemotypes for FPR2/ALXR agonists.200 M



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chosen based on structural features present in most of the lowmolecular-weight synthetic known FPR2/ALXR agonists. Bombesin-related neuromedin B receptor tryptophan antagonist 87 (Figure 35) was identified as the best compound for its ability to induce intracellular Ca2+ mobilization in FPR2/ALXRtransfected HL-60 cells. Testing of additional analogues allowed for the identification of amino acid-based agonists that were potent on FPR2/ALXR but not as selective vs FPR1, with some falling under the 2005 Acadia patent.143 Analogue testing also allowed for the discovery of new, nonpeptoid agonists such as 91, the most active FPR2/ALXR agonist from the series. In a following work,208 a series of 22 tryptophan derivatives was prepared as pure enantiomers; nine of them were found to be FPR2/ALXR-specific agonists. Four of them were present as enantiomeric pairs and showed enantiomeric preference for their FPR2/ALXR activity, but, interestingly, not always for the same enantiomer, as best exemplified by 88 and 89 (Figure 35), suggesting an important role of the polar pyridine in the binding mode, as supported by modeling studies.208 It is of high interest to note that removal of the methyl group of 87 is sufficient to obtain the FPR2/ALXR-selective agonist 90 that maintains its FPR2/ALXR β-arrestin recruitment capability.

In a follow-up work, 10 further mixed FPR1−FPR2/ALXR agonists were reported in which, of specific interest, slight modifications of the dual FPR1−FPR2/ALXR agonist 81 led to selective FPR1 agonist 82 (Figure 33).201

Figure 33. Derivative 81, a dual FPR1−FPR2/ALXR agonist, and 82, a selective FPR1 agonist.201

Looking for new FPR2/ALXR agonist chemotypes, Cilibrizzi202 initiated studies selecting functionalized pyridazinones, which can be considered as enlarged analogues of Amgen compound 6 (Figure 8). From the series of compounds made, 83 (Figure 34) was found to be a selective FPR2/ALXR agonist (EC50 = 2400 nM, with no activity observed on FPR1), whereas all other active compounds based on this scaffold were dual FPR1 and FPR2/ALXR agonists, pointing to the difficulties in finding selective FPR2/ALXR agonists. Slight modification, such as in 84, brings dual compounds. Substitution of the oxygen atom in the amide bridge with a sulfur atom led to a thioamide, which was inactive, presumably due to the steric bulkiness of the sulfur.203 Further studies on these 2-arylacetamide pyridazin-3(2H)ones confirmed that the arylacetamide moiety is essential for agonist activity and showed that modification of the methyl group on the pyridazinone led to substantial loss of activity. On the other side, a functionalized spacer could be introduced, such as in 85, retaining good activity but with no improved selectivity.204 During optimization of these pyridazinones, mixed FPR1/ FPR2/ALXR agonists showing enantiomeric preferences were identified.205 The most active FPR2/ALXR agonist, (R)-86, had an FPR2/ALXR EC50 Ca2+ of 89 nM with a 5-fold selectivity toward FPR1, whereas its enantiomer, (S)-86, was almost 100fold less active at 7000 nM. A strong correlation between EC50 values obtained for calcium mobilization and β-arrestin recruitment was reported. In a follow-up work, a new series of chiral C5 pyridazine derivatives was reported together with their corresponding aromatic nonchiral analogues, confirming that the stereogenic center at C5 of the pyridazine ring plays only a marginal role.206 In 2011, 32 ligands of 24 unrelated GPCRs were screened to possibly identify novel FPR agonists.207 These 32 ligands were

Figure 35. Tryptophan derivative 87, identified as a mixed FPR1− FPR2/ALXR agonist by screening selected GPCR ligands. Derivatives such as 88 or 89 show that enantiomeric preference is substitutiondependent. Slight modification allowed for improvement of selectivity, such as for 90, obtained by removal of the methyl group of 87.207,208

4.3.2.3. University of Osaka. Since the response of neutrophils or monocytes to calpain inhibitors mimics the response to chemoattractants such as fMLF, the authors tested the hypothesis that members of the FPR systems may be involved in cell activation by calpain inhibitors. In a HEK-293 cell system stably expressing FPR2/ALXR, they showed that 92 and 93 (Figure 36) display a stimulus-specific increase in cytoplasmic free Ca2+, whereas no stimulus was observed in the FPR1-transfected system.209,210 In a FPR2/ALXR-transfected HL-60 cell-based assay, the reported activity could not be reproduced, possibly due to the different types of G-proteins used in the assay.141 4.3.2.4. Torrey Pines Institute for Molecular Studies and the University of New Mexico. While screening for FPR2/ALXR antagonists using a duplex flow cytometry and mixture-based positional scanning libraries, 94 (Figure 37, EC50 = 144 nM) was identified as an FPR2/ALXR agonist. It is of interest to note that

Figure 34. 83, a selective FPR2/ALXR agonist, and its analogues 84, a dual FPR1-FPR2/ALXR agonist, and 85, a mixed agonist. The importance of the chiral information is evidenced by 86.202−205 N



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receptor using synthetic agonists to resolve persistent neutrophilic inflammation without compromising essential host defense mechanisms.214 5.2. Cystic Fibrosis (CF). The main cause of morbidity and mortality in CF is progressive lung destruction as a result of persistent bacterial infection and inflammation, coupled with reduced capacity for epithelial repair. Since the levels of LXA4 have been reported to be reduced in bronchoalveolar lavages of patients with CF, the ability of LXA4 to trigger epithelial repair was investigated. It was shown that LXA4 triggered an increase in migration, proliferation, and wound repair of both non-CF and CF airway epithelia.215 The fact that these responses were completely abolished by FPR2/ALXR antagonists and FPR2/ ALXR siRNA suggests that providing FPR2/ALXR agonists to CF patients might be a potential therapeutic approach. 5.3. Rheumatoid Arthritis (RA). RA is one of the most common chronic inflammatory diseases. It is associated with severe inflammation of the joints, and, still today, 50% of RA patients remain dependent on glucocorticoid treatment, even with all of its associated side effects.216,217 Since AnxA1 is a glucocorticoid-induced molecule, it made sense that efficacy was observed in an RA setting.218 More recently, the discovery that AnxA1 is a glucocorticoid-induced molecule signaling through FPR2/ALXR has increased interest for FPR2/ALXR as a therapeutic target for RA. The current knowledge of the biological activities of AnxA1, the role of its receptor, and the relevance to RA pathogenesis was reviewed recently.219 On the basis of the AnxA1−FPR2/ALXR−RA connection, Amgen compound 6 was investigated in models of RA, where treatment with 6 before or after the onset of arthritis significantly reduced clinical disease severity. In addition to these data, it was demonstrated that compound 6 also exhibited anti-inflammatory effects in relevant humans cells, suggesting that FPR ligands may represent novel therapeutic agents with which to ameliorate inflammation and bone damage in RA.220 5.4. Inflammatory Bowel Disease (IBD). IBD represents a set of chronic diseases that are caused by inflammation of the intestine, such as, for example, Crohn’s disease or ulcerative colitis. The cause of IBD is not yet clear, but the host immune system is closely associated with the pathogenesis and progress of IBD.221 Since the WKYMVm-NH2 peptide was shown to have therapeutic effect in a sepsis model via inhibition of the production of inflammatory cytokines, it was investigated in a mouse model of ulcerative colitis.222 WKYMVm-NH2, injected subcutaneously every 12 h over 60 h, had a therapeutic effect on ulcerative colitis by inhibiting epithelial permeability and modulating the cytokine profile. This effect was strongly inhibited by a FPR2/ALXR antagonist, indicating the crucial role of FPR2/ALXR in this effect and supporting the potential therapeutic utility of FPR2/ALXR agonists in the management of chronic intestinal inflammatory diseases. 5.5. CNS Diseases. The binding of SAA, Aβ42, and PrP106−126 to FPR2/ALXR suggests that this receptor may play a crucial role in pro-inflammatory aspects of systemic amyloidosis, Alzheimer’s disease (AD), and prion diseases. On the other hand, LXA4 shows an inhibitory effect on the expression of pro-inflammatory chemokines via FPR2/ALXR and may represent a strategy in the treatment of acute and chronic brain inflammation. Moreover, a resolution pathway has been shown to exist in the brain and to be deregulated in post-mortem hippocampal tissue from AD patients, and it has been suggested that

Figure 36. 92 and 93, two calpain inhibitors, as agonists of FPR2/ ALXR.209,210

replacement of the propyl chain by an isopropyl generated the FPR2/ALXR antagonist 95 (IC50 = 81 nM).211

Figure 37. 94, an FPR2/ALXR agonist identified by flow cytometry. Replacement of the propyl chain of 94 by and isopropyl chain delivered the FPR2/ALXR antagonist 95.211

5. THERAPEUTIC IMPACT The mechanisms by which resolution of inflammation occurs and the key biochemical pathways associated with the return to homeostasis clearly open many new avenues for potential therapeutic interventions in a wide range of diseases associated with unresolved inflammation. Timely resolution of inflammation is essential to maintain tissue integrity, and FPR2/ALXR appears to be capable of mediating both pro- and anti-inflammatory signals. The wide range of agonists and responses of FPR2/ALXR suggests that this receptor may represent a unique target for therapeutic drug design. Balancing the inflammation process, not overshooting it, will be the key for successful new and innovative therapeutics to emerge from this field of research.212 FPR2/ALXR has been found to interact with a series of structurally diverse pro- and anti-inflammatory ligands associated with different diseases, including amyloidosis, Alzheimer’s disease (AD), prion disease, and HIV. Understanding how this receptor recognizes such diverse ligands, which ligands are the most important in vivo, and how they contribute to disease pathogenesis and host defense are basic questions currently under investigation that could lead to new therapeutic applications,213 and only a selection will be discussed here. 5.1. Chronic Obstructive Pulmonary Disease (COPD). Neutrophilic inflammation persists in COPD despite the best current therapies and is particularly resistant to inhaled glucocorticosteroids. New strategies to counteract this inflammation in COPD by focusing on the anti-inflammatory role of FPR2/ALXR receptors have been proposed, highlighting this receptor as an emerging target in the pathogenesis of COPD because suspected FPR2/ALXR endogenous agonists such as serum amyloid A (SAA) are enriched in COPD.214 SAA initiates lung inflammation via FPR2/ALXR, and it is proposed that there is an imbalance in endogenous FPR2/ALXR receptor agonist in the inflamed COPD lung environment that opposes protective anti-inflammatory and pro-resolution pathways. These insights open the possibility of targeting FPR2/ALXR O



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stimulating the resolution pathway might be a promising therapy for AD.59 Using mFPR1 and mFPR-RS2/RS1 deficient mice, the effects on inflammation, bacterial growth, and mortality in a mouse model of pneumococcal meningitis were investigated. Taken together, the results of this study suggest that the FPR system plays an important role in the innate immune responses against Streptococcus pneumoniae within the CNS.223 5.6. Oncology. Following a study in which the growth of the melanoma cell line B16-F10 in wild-type mice could be significantly inhibited by treatment with the WKYMVm-NH2 peptide, it has been proposed that agonists of the FPR system might represent a novel antitumor therapy.224 5.7. Pain. FPR2/ALXR was shown to be expressed on spinal astrocytes, and spinal delivery of LXA4 attenuates inflammationinduced pain. 225 The authors proposed that targeting mechanisms that counter-regulate the spinal consequences of persistent peripheral inflammation provides a novel endogenous mechanism by which chronic pain may be controlled. Pain is further supported as an indication based on the Acadia report with compound 3, which dose-dependently prevented thermal hyperalgia induced by carrageenan in male Sprague− Dawley (M-SD) rats.143

The authors declare the following competing financial interest(s): Olivier Corminboeuf and Xavier Leroy are employees of Actelion Pharmaceuticals Ltd. and inventors on patents assigned to Actelion Pharmaceuticals Ltd. Biographies Olivier Corminboeuf majored in Chemistry at the University of Fribourg and performed his Ph.D. thesis in organic chemistry in the group of Prof. Philippe Renaud in Switzerland, developing new concepts for metal-based enantioselective transformation. He held a postdoctoral position in the group of Prof. Larry Overman at UC Irvine, working on a unified strategy for enantioselective total synthesis of diterpene-based marine products. Dr. Corminboeuf is now a Director in Drug Discovery Chemistry at Actelion Pharmaceuticals Ltd. Xavier Leroy majored in Biochemistry at the UTC, France, and performed his Ph.D. thesis at the Laboratory of Molecular & Cellular Biology, ISAMOR, France, led by Pr. Michel Branchard. He held a postdoctoral position at Novartis, Switzerland, before joining Axovan Pharmaceuticals Ltd. as Head of Molecular Biology & Applied Research. Dr. Xavier Leroy is now an Associate Director in Drug Discovery Biology at Actelion Pharmaceuticals Ltd.

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ABBREVIATIONS USED 15S-H(p)ETE, 15(S)-hydroperoxyeicosatetraenoic acid; 15RHETE, 15(R)-hydroxyeicosatetraenoic; 17R-H(p)DHA, 17(R)hydro(pero)xy-docosahexaenoic acid; 17S-H(p)DHA, 17(S)hydro(pero)xy-docosahexaenoic acid; AA, arachidonic acid; AD, Alzheimer’s disease; ALI, acute lung injury; AnxA1, annexin A1; ATLN, aspirin-triggered lipoxin; AT-LXA4, aspirin-triggered lipoxin A4 ((5S,6R,7E,9E,11Z,13E,15R)-5,6,15-trihydroxyicosa7,9,11,13-tetraenoic acid); AT-LXB4, aspirin-triggered lipoxin B4 ((5S,6E,8Z,10E,12E,14R,15R)-5,14,15-trihydroxyicosa6,8,10,12-tetraenoic acid); AT-RvD1, aspirin-triggered resolvin D1 ((4Z,7S,8R,9E,11E,13Z,15E,17R,19Z)-7,8,17-trihydroxydocosa-4,9,11,13,15,19-hexaenoic acid); BLT, B-leukotriene; Ca2+, calcium2+; CD11b, cluster of differentiation molecule 11b; CF, cystic fibrosis; CHO, chinese hamster ovary; CNS, central nervous system; COPD, chronic obstructive pulmonary disease; COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; CysLT, cysteinyl leukotriene; DHA, docosahexaenoic acid; EC50, half effective concentration; fMLF, N-formyl-methionylleucyl-phenylalanine; FPR, formyl peptide receptor; GPCR, Gprotein coupled receptor; HEK cells, human embryonic kidney cells; HIV, human immunodeficiency virus; HL cells, human promyelocytic leukemia cells; HTS, high-throughput screening; IBD, inflammatory bowel disease; IL-1β, interleukin-1β; IL-8, interleukin-8; IUPHAR, international union of basic and clinical pharmacology; LL-37, also known as hCAP18, is the 37 amino acid C-terminal part of human cationic antimicrobial protein (hCAP); LO, lipoxygenase; LPS, lipopolysaccharide; LTA4, leukotriene A4; LTA4-H, leukotriene A4 hydrolase; LTB4, leukotrienes B4; LTC4, leukotrienes C4; LX, lipoxins; LXA4, lipoxin A4 ((5S,6R,7E,9E,11Z,13E,15S)-5,6,15-trihydroxyicosa7,9,11,13-tetraenoic acid); LXB4, lipoxin B4 ((5S,6E,8Z,10E,12E,14R,15S)-5,14,15-trihydroxyicosa-6,8,10,12-tetraenoic acid); LxH, lipoxin hydrolase; Mar, maresins; M-SD rat, male Sprague−Dawley rat; NF-κB, nuclear factor kappa-light-chainenhancer of activated B cells; PD, protectins; PGD 2 , prostaglandin D2; PGE2, prostaglandin E2; PGH2, prostaglandin H2; PMN, polymorphonuclear; PUFA, polyunsaturated fatty acids; RA, rheumatoid arthritis; RBL cells, rat basophil leukemia cells; RoI, resolution of inflammation; ROS, reactive oxygen

6. FUTURE DIRECTIONS The recognition of the proactive nature of the resolution of inflammation has revealed alternative therapeutic paradigms based on resolving acute inflammation and preventing the onset of chronic inflammation.12,226 This is further supported by the anti-inflammatory/protective role of FPR2/ALXR demonstrated in vitro and in vivo by knocking down this receptor in human cells and in mice.103,164,223 Most of the small molecule FPR2/ALXR agonists reported here were discovered using calcium release as a rapid readout. The ability to select those compounds able to modulate landmarks of the RoI, such as stimulating nonphlogistic activation of monocytes or macrophages, efferocytosis, or apoptosis, will be key for the development of therapeutic molecules that carry the functional capabilities of the endogenous molecules. Moreover, the role of IL-10 and receptor cross-desensitization deserve further studies. Besides FPR2/ALXR presented here, other targets involved in the resolution process present opportunities for drug development (reviewed in ref 62). Even if the current knowledge regarding SPMs is only partial, it can be predicted that, based on the fact that the RoI is a complex, actively regulated process, a change in the paradigm for how acute and chronic inflammatory conditions are treated is under way. Moreover, the recent report on selective microRNA regulation through activation of pro-resolving receptors should be further investigated.227,228 A move away from anti-inflammatory substances toward substances that induce resolution mechanisms has been predicted.12,229 Nevertheless, future studies are still warranted to clarify whether the pro-resolving strategy will contribute to healing human inflammatory diseases and their complications.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 61 565 66 70. E-mail: olivier.corminboeuf@ actelion.com. P



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(20) Rajakariar, R.; Yaqoob, M. M.; Gilroy, D. W. COX-2 in inflammation and resolution. Mol. Interventions 2006, 6, 199−207. (21) FitzGerald, G. A. COX-2 and beyond: approaches to prostaglandin inhibition in human disease. Nat. Rev. Drug Discovery 2003, 2, 879−890. (22) Lawrence, T.; Willoughby, D. A.; Gilroy, D. W. Antiinflammatory lipid mediators and insights into the resolution of inflammation. Nat. Rev. Immunol. 2002, 2, 787−795. (23) Borregaard, N.; Theilgaard-Monch, K.; Cowland, J. B.; Stahle, M.; Sorensen, O. E. Neutrophils and keratinocytes in innate immunitycooperative actions to provide antimicrobial defense at the right time and place. J. Leukocyte Biol. 2005, 77, 439−443. (24) Serhan, C. N. Lipoxin biosynthesis and its impact in inflammatory and vascular events. Biochim. Biophys. Acta 1994, 1212, 1−25. (25) Ryan, A.; Godson, C. Lipoxins: regulators of resolution. Curr. Opin. Pharmacol. 2010, 10, 166−172. (26) Spite, M.; Serhan, C. N. Novel lipid mediators promote resolution of acute inflammation: impact of aspirin and statins. Circ. Res. 2010, 107, 1170−1184. (27) Schwab, J. M.; Chiang, N.; Arita, M.; Serhan, C. N. Resolvin E1 and protectin D1 activate inflammation−resolution programmes. Nature 2007, 447, 869−874. (28) Serhan, C. N. Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution. Prostaglandins, Leukotrienes Essent. Fatty Acids 2005, 73, 141−162. (29) Petasis, N. A.; Akritopoulou-Zanze, I.; Fokin, V. V.; Bernasconi, G.; Keledjian, R.; Yang, R.; Uddin, J.; Nagulapalli, K. C.; Serhan, C. N. Design, synthesis and bioactions of novel stable mimetics of lipoxins and aspirin-triggered lipoxins. Prostaglandins, Leukotrienes Essent. Fatty Acids 2005, 73, 301−321. (30) Takano, T.; Fiore, S.; Maddox, J. F.; Brady, H. R.; Petasis, N. A.; Serhan, C. N. Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors. J. Exp. Med. 1997, 185, 1693−1704. (31) Clish, C. B.; O’Brien, J. A.; Gronert, K.; Stahl, G. L.; Petasis, N. A.; Serhan, C. N. Local and systemic delivery of a stable aspirintriggered lipoxin prevents neutrophil recruitment in vivo. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8247−8252. (32) Hachicha, M.; Pouliot, M.; Petasis, N. A.; Serhan, C. N. Lipoxin (LX)A4 and aspirin-triggered 15-epi-LXA4 inhibit tumor necrosis factor 1α-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis. J. Exp. Med. 1999, 189, 1923−1930. (33) Schwab, J. M.; Serhan, C. N. Lipoxins and new lipid mediators in the resolution of inflammation. Curr. Opin. Pharmacol. 2006, 6, 414− 420. (34) El Kebir, D.; Jozsef, L.; Pan, W.; Wang, L.; Petasis, N. A.; Serhan, C. N.; Filep, J. G. 15-Epi-lipoxin A4 inhibits myeloperoxidase signaling and enhances resolution of acute lung injury. Am. J. Respir. Crit. Care Med. 2009, 180, 311−319. (35) Prieto, P.; Cuenca, J.; Traves, P. G.; Fernandez-Velasco, M.; Martin-Sanz, P.; Bosca, L. Lipoxin A4 impairment of apoptotic signaling in macrophages: implication of the PI3K/Akt and the ERK/Nrf-2 defense pathways. Cell Death Differ. 2010, 17, 1179−1188. (36) Nathan, C.; Ding, A. Non-resolving inflammation. Cell 2010, 140, 871−882. (37) El Kebir, D.; Filep, J. Targeting neutrophil apoptosis for enhancing the resolution of inflammation. Cells 2013, 2, 330−348. (38) Souza, D. G.; Fagundes, C. T.; Amaral, F. A.; Cisalpino, D.; Sousa, L. P.; Vieira, A. T.; Pinho, V.; Nicoli, J. R.; Vieira, L. Q.; Fierro, I. M.; Teixeira, M. M. The required role of endogenously produced lipoxin A4 and annexin-1 for the production of IL-10 and inflammatory hyporesponsiveness in mice. J. Immunol. 2007, 179, 8533−8543. (39) Norel, X.; Brink, C. The quest for new cysteinyl-leukotriene and lipoxin receptors: recent clues. Pharmacol. Ther. 2004, 103, 81−94. (40) Bannenberg, G. L.; Chiang, N.; Ariel, A.; Arita, M.; Tjonahen, E.; Gotlinger, K. H.; Hong, S.; Serhan, C. N. Molecular circuits of

species; Rv, resolvins; RvD1, resolvin D1 ((4Z,7S,8R,9E,11E,13Z,15E,17S,19Z)-7,8,17-trihydroxydocosa-4,9,11,13,15,19-hexaenoic acid); RvE1, resolvin E1 ((5S,6Z,8E,10E,12R,14Z,16E,18R)-5,12,18-trihydroxyicosa-6,8,10,14,16-pentaenoic acid); SAA, serum amyloid A; SAR, structure−activity relationship; SPMs, specialized pro-resolving mediators; TNFα, tumor necrosis factor alpha

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