Therapeutic validation of an orphan G protein‐coupled receptor: The case of GPR84

Despite the importance of members of the GPCR superfamily as targets of a broad range of effective medicines many GPCRs remain poorly characterised. GPR84 is an example. Expression of GPR84 is strongly up regulated in immune cells in a range of pro‐inflammatory settings and clinical trials to treat idiopathic pulmonary fibrosis are currently ongoing using ligands with differing levels of selectivity and affinity as GPR84 antagonists. Although blockade of GPR84 may potentially prove effective also in diseases associated with inflammation of the lower gut there is emerging interest in defining if agonists of GPR84 might find utility in conditions in which regulation of metabolism or energy sensing is compromised. Here, we consider the physiological and pathological expression profile of GPR84 and, in the absence of direct structural information, recent developments and use of GPR84 pharmacological tool compounds to study its broader role and biology. LINKED ARTICLES This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc

Despite the importance of members of the GPCR superfamily as targets of a broad range of effective medicines many GPCRs remain poorly characterised.
GPR84 is an example. Expression of GPR84 is strongly up regulated in immune cells in a range of pro-inflammatory settings and clinical trials to treat idiopathic pulmonary fibrosis are currently ongoing using ligands with differing levels of selectivity and affinity as GPR84 antagonists. Although blockade of GPR84 may potentially prove effective also in diseases associated with inflammation of the lower gut there is emerging interest in defining if agonists of GPR84 might find utility in conditions in which regulation of metabolism or energy sensing is compromised. Here, we consider the physiological and pathological expression profile of GPR84 and, in the absence of direct structural information, recent developments and use of GPR84 pharmacological tool compounds to study its broader role and biology. GPR84 is a poorly characterised G i -coupled, class A GPCR mainly expressed by immune cells, including monocytes, macrophages and neutrophils in the periphery, and microglia in the brain (Wojciechowicz & Ma'ayan, 2020). Because GPR84 transcript is upregulated in many pro-inflammatory conditions, potential therapeutic opportunity in targeting this receptor in inflammatory conditions, including ulcerative colitis and fibrotic diseases, has been suggested Suzuki et al., 2013;Vermeire et al., 2017; Abbreviations: 2-HTP, 2-(hexylthio)pyrimidine-4,6-diol; 6-OAU, 6-n-octylaminouracil; APP-PS1, a murine model of Alzheimer's disease; DIM, 3,3′-diindolylmethane; GLPG1205, 9-cyclopropylethynyl-2-((S)-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one; IBD, inflammatory bowel disease; ICL3, intracellular loop 3; MCFAs, medium-chain fatty acids; NAFLD, non-alcoholic fatty liver disease; PAM, positive allosteric modulator; PMA, phorbol myristate acetate; PMN, polymorphonuclear leukocyte; PNL, partial sciatic nerve ligation. Sara Marsango and Natasja Barki contributed equally to this work. Wojciechowicz & Ma'ayan, 2020). Identified almost two decades ago (Wittenberger, Schaller, & Hellebrand, 2001;Yousefi, Cooper, Potter, Mueck, & Jarai, 2001), human GPR84 encodes a protein of 396 amino acids that shares 85% identity with the protein encoded by murine Gpr84. The extensive intracellular loop 3 (ICL3) provides most of the differences between these orthologues, while the predicted transmembrane domains have only 14 amino acid differences. Although it is widely accepted that medium-chain fatty acids (MCFAs), particularly those with 10-12 carbon acids (decanoic acid [C10], undecanoic acid [C11], and lauric acid [C12]), can bind to and activate GPR84 (Nikaido, Koyama, Yoshikawa, Furuya, & Takeda, 2015;Southern et al., 2013;Suzuki et al., 2013;Wang, Wu, Simonavicius, Tian, & Ling, 2006), officially, GPR84 remains an orphan receptor (Sharman et al., 2011) Bradley et al., 2018). Because of this, we will avoid ascribing absolute agonist affinity values unless these have been generated by a reliable method, such as mathematical analysis of operational models (Ehlert, 2005) of the extent of allosteric modulation between ligands (Al Mahmud et al., 2017) that bind to distinct sites of GPR84 (Table 1) and limit comments to relative potency of compounds where these have been assessed in parallel using the same experimental system, for example, direct measures of EC 50 employing functional assays of second messenger regulation or G-protein activation in cell lines transfected to express an orthologue of GPR84. As such, levels of MCFAs circulating in the plasma (approx. 0.5 mM) might be too low to substantially activate GPR84 in vivo (Al Mahmud et al., 2017;Mancini et al., 2019).
Here, as well as discussing the expression profile of GPR84 in physiological and pathophysiological conditions, we will consider the portfolio of available ligands that can be used as tool compounds to study the function and biology of GPR84 and how limitations of these are currently restricting a full understanding of the therapeutic potential of this receptor. As noted above, although MCFAs certainly can activate GPR84, the modest potency of these in native systems has promoted efforts to identify and characterise other, more potent agonists. Moreover, as GPR84 is linked to various inflammatory diseases, efforts have also been made to identify antagonists to assess if these might have therapeutic utility in such settings.
It is assumed here that MCFAs act as orthosteric agonists, although while GPR84 remains classified as an orphan GPCR, this must remain a presumptive definition ( Figure 1 and Table 1). Of key importance is that, although MCFAs do activate GPR84, equivalent fatty acid amides do not (Nikaido et al., 2015), and the requirement for the acid function was also observed when decanoic acid (C10) was replaced with its ester, methyl decanoate (Al Mahmud et al., 2017).
Although this defines the importance of the acid function for binding and/or activation, in the absence of suitable atomic-level structures, the potential orientation of the MCFA in the binding site remains a matter of conjecture. In class A GPCRs, the orthosteric binding site is typically a deep pocket leading from the extracellular side of the receptor with which endogenously produced ligands interact (Congreve, de Graaf, Swain, & Tate, 2020). By contrast, binding sites that are topographically distinct from the orthosteric site are generically described as allosteric (Congreve et al., 2020). Given that other class A GPCRs that respond to either short-chain (free fatty acid receptors 2 and 3) or long-chain (free fatty acid receptors 1 and 4) fatty acids have specific arginine residues as part of the orthosteric binding pocket that act to co-ordinate the carboxylate of the appropriate fatty acid (Milligan, Shimpukade, Ulven, & Hudson, 2017), Al Mahmud et al. (2017) assessed whether this might also be true for GPR84, even though this receptor is not closely related to any of the currently IUPHAR-accepted free fatty acid receptors (Alexander, Christopoulos et al., 2019). A chimeric homology model that noted and took into account the high similarity of the second extracellular loop of GPR84 with that of rhodopsin, for which atomic-level structures were known, suggested that Arg 172 from this region would potentially face into the binding pocket and anchor the fatty acid carboxylate (Tikhonova, 2017). Mutation of this residue to alanine, and indeed even to lysine, abolished function of MCFAs. By contrast, the potency of a known allosteric agonist (see later) of GPR84, 3,3′-diindolylmethane (DIM), was unaffected by these mutations (Al Mahmud et al., 2017). Co-ordination of the fatty acid carboxylate by this arginine implied that the alkyl chain of the fatty acid would penetrate down into the orthosteric cavity of the receptor with the carboxylate at the extracellular interface. In contrast, earlier mutagenesis and homology modelling studies by Nikaido et al. (2015) concluded that the opposite orientation of the fatty acid was more likely, with the carboxylate reaching downwards into the receptor. Mutagenesis and homology modelling studies by Köse et al. (2020) have supported this orientation, showing the fatty acid carboxylate in the binding cleft and forming hydrogen-bond interactions with Tyr 69 , Asn 104 , and Asn 357 . In this model, Arg 172 contributes indirectly to agonist-receptor interaction, possibly by playing a role in initial ligand recognition (Köse et al., 2020). It also appears that MCFAs with a hydroxyl group at the 2-or 3-position can activate GPR84 more potently than non-hydroxylated MCFAs (Suzuki et al., 2013). Hydroxylation at other positions on the fatty acid alkyl chain is also consistent with activation of the receptor (Kaspersen, Jenkins, Dunlop, Milligan, & Ulven, 2017).  Wang et al., 2006Nagasaki et al., 2012Suzuki et al., 2013Southern et al., 2013Nikaido et al., 2015Pillaiyar et al., 2017Wei, Tokizane, Konishi, Yu, & Kiyama, 2017Al Mahmud et al., 2017Recio et al., 2018Puengel et al., 2020Lucy et al., 2019 Embelin Human: 89-200 nM b 0.63 μM a 0.4 μM c Mouse: 220 nM b -Recombinant system -Human monocyte-derived macrophages -Mouse peritoneal macrophages -Human blood-derived neutrophils -Mouse blood-derived neutrophils -Mouse primary cultured microglia -Human monocyte -Rat neutrophils Hakak, Unett, Gatlin, & Liaw, 2007Southern et al., 2013Al Mahmud et al., 2017Wei et al., 2017Pillaiyar et al., 2017Gaidarov et al., 2018Puengel et al., 2020 6-OAU Human: 14-438 nM b 1.74-11 μM c 512 nM a -Recombinant system -Mouse primary cultured microglia -Bone marrow-derived macrophages -Human peripheral polymorphonuclear leukocyte -U937 cells differentiated into macrophage-like cells -Human monocytes Suzuki et al., 2013Liu et al., 2016Zhang, Yang, Li, & Xie, 2016Wei et al., 2017Recio et al., 2018Lucy et al., 2019PSB-1584 Human: 5 nM b 3.2 nM c F I G U R E 1 Chemical structures of low MW GPR84 ligands. The chemical structure of some widely used ligands with actions at GPR84 is shown. The alphabetical labelling system (A-O) is used to define which ligands were employed in studies highlighted in Figure 2 and Tables 1 and 2 2 | ORTHOSTERIC AGONISTS 2.1 | Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) is a natural product derived from the plant Embelia ribes, used in traditional Chinese medicine for treatment of diverse conditions, such as gastrointestinal and inflammatory diseases (Gaidarov et al., 2018). It was first described in the patent literature as a potent and efficacious GPR84 agonist using inhibition of cAMP assays (Hakak et al., 2007). Although appreciated for a period of time to have such activity by those working in the field, it was a number of years before this was confirmed in the peer-reviewed literature (Al Mahmud et al., 2017;Southern et al., 2013;Wei et al., 2017).
Embelin consists of a polar dihydroxybenzoquinone head group and an 11-carbon alkyl chain tail ( Figure 1). Truncation of the alkyl chain length to 7 or 8 carbons results in increased potency, whilst agonist activity is lost in derivatives containing very short (C3) or very long (C15) alkyl chains (Gaidarov et al., 2018). Embelin has a range of biological activities including inhibition of the X-linked inhibitor of apoptosis protein and activation of caspase 9, as well as antioxidant properties (Nikolovska-Coleska et al., 2004). Even at other GPCRs, embelin has been noted to have as potent actions, as at GPR84, but rather as a blocker; for example, at the chemokine receptor CXCR2 (K i = 93 nM) and the adenosine A 3 receptor (K i = 1.4 μM). However, at least at these receptors, the C7 derivative reportedly lacks activity (Gaidarov et al., 2018). As such, embelin and derivatives have been used to characterise roles of GPR84 in a variety of settings (Table 1)  2.2 | 6-OAU (6-(octylamino) pyrimidine-2,4(1H,3H)dione) 6-OAU (6-(octylamino) pyrimidine-2,4(1H,3H)-dione) is a further example of a GPR84 agonist characterised by a polar head group and alkyl tail ( Figure 1 and Table 1) (Recio et al., 2018;Suzuki et al., 2013;Wei et al., 2017). 6-OAU is able to induce chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and to promote production of the pro-inflammatory cytokine IL-8 from PMNs, and TNF-α, IL-6, IL-12B, as well as the chemokines CCL2, CCL5 and CXCL1 from bone marrow-derived macrophages previously treated with LPS (Recio et al., 2018;Suzuki et al., 2013). These were clearly GPR84-mediated inflammatory responses as they were absent in cells isolated from homozygous GPR84 knockout mice and when such macrophages were treated with a selective GPR84 antagonist (see later) (Recio et al., 2018). In such LPS-stimulated bone marrow-derived macrophages, treatment with 6-OAU enhanced phosphorylation of PKB (Akt) and the ERK1/2 and promoted p65 nuclear translocation (Recio et al., 2018). A series of analogues of 6-OAU have also been generated by modifying the uracil head to incorporate alternative groups (Pillaiyar et al., 2018). Among 66 reported new derivatives in this study, a few have been considered in detail, based on improved potency, selectivity and metabolic stability. 6-Hexylamino-2,4(1H,3H)pyrimidinedione (PSB-1584) ( Figure 1) is reportedly some 100 times more potent than 6-OAU in activating GPR84 (Pillaiyar et al., 2018).
Homology modelling and docking studies have attempted to provide insights into the structural basis for the higher potency of uracil derivatives, particularly when compared with embelin and decanoic acid (Köse et al., 2020). These studies predicted a stronger interaction of uracil derivatives with residues Asn 357 and Asn 104 , which may form part of the binding pocket of GPR84, as suggested initially by Köse et al. (2020) and Nikaido et al. (2015). However, while of interest, the dopamine D 3 receptor bound by the antagonist eticlopride, which was used to develop the GPR84 homology model, is far removed from GPR84 in terms of overall sequence similarity, and thus, these predic-  2.4 | DL-175 (3-(2-((4-chloronaphthalen-1-yl)oxy) ethyl)pyridine 1-oxide) DL-175 (3-(2-((4-chloronaphthalen-1-yl)oxy)ethyl)pyridine 1-oxide) was identified using a virtual screen based on comparisons with 6-OAU (Figure 1). This would suggest it also should be an orthosteric agonist (Lucy et al., 2019). Using a recombinant cell system, DL-175 showed comparable potency and efficacy to 6-OAU in inhibiting cAMP accumulation; however, it was ineffective in an arrestin recruitment assay, indicating a marked bias for G-protein signalling (Table 1) (Lucy et al., 2019). Interestingly, although not inducing signals in macrophages derived from GPR84 knockout mice (Lucy et al., 2019), DL-175 generated a distinct signalling profile from 6-OAU in both primary murine bone marrow-derived macrophages and phorbol myristate acetate (PMA)-differentiated human U937 macrophage-like cells (Lucy et al., 2019). This may reflect the distinct bias of DL-175 away from arrestin interactions as this would be anticipated to limit potential desensitisation. Interestingly, when cells were pretreated with CMPD101, a combined GPCR kinase GRK2 and GRK3 inhibitor (Ikeda, Kaneko, & Fujiwara, 2007), the response to 6-OAU now resembled that of DL-175 (Lucy et al., 2019). Finally, in contrast with 6-OAU, DL-175 failed to promote chemotaxis of M 1 -polarised U937 macrophages (Lucy et al., 2019). Despite these interesting characteristics, DL-175 is rapidly metabolised when exposed to mouse hepatocytes, suggesting that it will be of limited use for in vivo studies (Lucy et al., 2019).

| EXPRESSION PROFILE OF GPR84
Transcriptome studies have revealed that GPR84 is expressed primarily in a subset of peripheral immune cells (Figure 2). Following initial identification on peripheral blood leukocytes (Yousefi et al., 2001), F I G U R E 2 GPR84 modulation in inflammatory conditions. Upper panel: Activation of GPR84 by orthosteric and allosteric ligands generates pro-inflammatory responses in different cell types. Lower panel: GPR84 inhibition reduces responses associated with inflammation, fibrosis, and metabolic diseases in multiple cell types and tissues (letters indicate use of ligands illustrated in Figure 1) Sundqvist et al., 2018;Suzuki et al., 2013;Wang et al., 2006). Relatively lower levels of GPR84 mRNA transcript have been reported in other immune cells, including dendritic cells, T cells, and B cells (Suzuki et al., 2013;Wang et al., 2006), and in microglial cells in the CNS (Gautier et al., 2012;Hickman et al., 2013;Recio et al., 2018).
Although limited, expression has been detected outside the immune system. For example, some studies have found GPR84 mRNA in adipocytes, epithelial cells, fibroblast, and podocytes (Abdel-Aziz et al., 2016;Gagnon et al., 2018;Nagasaki et al., 2012). Receptor expression in many of these tissues could possibly be due to immune cells resident in these tissues, but this has yet not been confirmed. Additionally, up-regulation was also observed in models of chronic inflammation, including diabetes and atherosclerosis (Recio et al., 2018). Consistent with the notion that GPR84 is a pro-inflammatory receptor, increase in GPR84 expression has also been reported in colonic tissues and blood samples of patients with IBD (Arijs et al., 2011;Planell et al., 2017). As previously noted, it remains unclear to what extent GPR84 up-regulation in these tissues can be attributed to immune cell infiltration. However, the studies of Arijs et al. (2011) have been quoted as a driver leading to the assessment of whether blockade of GPR84 with the antagonist GLPG1205 might improve clinical observations in patients with ulcerative colitis (Labéguère et al., 2020).
Although up-regulation of GPR84 has been primarily studied in immune cells, a limited number of studies have also observed up-regulation in fat cells (Figure 2). GPR84 expression in human adipocytes is significantly up-regulated following acute exposure to IL-33, TNF-α, or IL-1β. This increase, however, was reported to be transitory, since the increase in receptor mRNA expression was not present following 24 h of exposure (Muredda, Kepczynska, Zaibi, Alomar, & Trayhurn, 2018;Zaibi, Kepczynska, Harikumar, Alomar, & Trayhurn, 2018). Similar observations have been made with treatment of mouse 3T3-L1 adipocytes with TNF-α and LPS and in human adipose-derived stem cells (Nagasaki et al., 2012). Up-regulation of GPR84 mRNA was also observed in vivo, specifically in fat pads of mice fed with a high-fat diet. Interestingly, a correlation was reported between receptor mRNA up-regulation and increase in specific staining for macrophages, suggesting infiltration of immune cells as the likely cause of receptor up-regulation (Nagasaki et al., 2012). An increase in GPR84 mRNA transcripts has also been reported in liver biopsies of patients with non-alcoholic fatty liver disease (NAFLD).
This increase, however, is associated with inflammation rather than fat accumulation in the liver (Puengel et al., 2020). In line with other reports, higher levels of GPR84 mRNA have been observed in fibroblasts, podocytes, proximal tubule epithelial cells, and macrophages under fibrotic conditions Grouix et al., 2018;Li et al., 2018). Interestingly, this up-regulation is also accompanied by up-regulation of various pro-fibrotic and inflammatory biomarkers, and treatment with PBI-4050 significantly reduced the expression of these markers Grouix et al., 2018;Li et al., 2018). Such observations underpin the clinical trials of both PBI-4050 (Khalil et al., 2019) and more recently GLPG1205 (Labéguère et al., 2020) in idiopathic pulmonary fibrosis.
Unsurprisingly, promising initial results have promoted ideas that blockade of GPR84 might be of more general use in a wider range of fibrotic conditions (Nguyen et al., 2020;Wojciechowicz & Ma'ayan, 2020). The exact mechanism responsible for disease-induced GPR84 up-regulation in many in vivo studies remains to be identified.
Nevertheless, these studies further highlight the potential role of the receptor in disease conditions primarily associated with inflammation.
Furthermore, observations of GPR84 up-regulation are not limited to peripheral diseases but have also been demonstrated in neuroinflammatory conditions in the CNS. Although low-level expression of GPR84 was reported in the brain of healthy adult mice in early studies (Bouchard et al., 2007), inflammatory stimuli induce significant up- Not surprisingly, given the strong up-regulation of GPR84 mRNA in such conditions, it has been suggested that GPR84 could also be a useful marker for activation of glial cells following CNS damage. In addition to up-regulation of GPR84 mRNA following TLR stimulation of microglia and astrocytes in vitro, GPR84 up-regulation has also been noted in vivo, for example, following La Crosse virus infection, which results in considerable neuronal cell death and inflammation in the CNS (Madeddu et al., 2015). Similarly, Gamo et al. (2008)  mRNA and anti-inflammatory macrophage markers (Arg-1 and cytokine Il-10) were also reported in spinal cord and sciatic nerve after partial sciatic nerve ligation (PNL) (Nicol et al., 2015). This study provides a rationale for further investigating the potential role of Interestingly, this study also emphasised how GPR84 might be involved in mitochondrial metabolism. Loss of GPR84 led to impaired mitochondria and increased oxidative stress. Despite the observed differences in measures of glucose tolerance, these findings support the notion that activation of GPR84 may be beneficial as a therapeutic strategy for metabolic dysfunction associated with obesity. This is consistent with another study reporting anti-atherosclerotic effects of GPR84 agonism. Activation of GPR84 by embelin leads to release of PGE 2 from human macrophages. GPR84 agonism also up-regulates expression of the cholesterol transporters ABCA1 and ABCG1 and induces a significant increase in apolipoprotein A-I-mediated efflux of cholesterol (Gaidarov et al., 2018). As opposed to inflammatory conditions, where inhibiting GPR84 function may serve a potential therapeutic benefit, agonism of GPR84 may be beneficial for its anti-atherosclerotic properties.