Discovery of 9-Cyclopropylethynyl-2-((S)-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (GLPG1205), a Unique GPR84 Negative Allosteric Modulator Undergoing Evaluation in a Phase II Clinical Trial
- Frédéric LabéguèreFrédéric LabéguèreGalapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, FranceMore by Frédéric Labéguère
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- Luke Alvey
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- Annick HagersAnnick HagersGalapagos NV, Generaal De Wittelaan L11 A3, 2800 Mechelen, BelgiumMore by Annick Hagers
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- Emanuelle WakselmanEmanuelle WakselmanGalapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, FranceMore by Emanuelle Wakselman
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- Philippe Clement-LacroixPhilippe Clement-LacroixGalapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, FranceMore by Philippe Clement-Lacroix
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- Laura JenkinsLaura JenkinsCentre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United KingdomMore by Laura Jenkins
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- Graeme MilliganGraeme MilliganCentre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United KingdomMore by Graeme Milligan
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- Stephen FletcherStephen FletcherGalapagos NV, Generaal De Wittelaan L11 A3, 2800 Mechelen, BelgiumMore by Stephen Fletcher
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- Romain Gosmini*Romain Gosmini*Email: [email protected]. Telephone: +33149424778.Galapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, FranceMore by Romain Gosmini
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Abstract
GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit 1, identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 (36). Compared with the initial hit, 36 showed improved potency in a guanosine 5′-O-[γ-thio]triphosphate assay, exhibited metabolic stability, and lacked activity against phosphodiesterase-4. This novel pharmacological tool allowed investigation of the therapeutic potential of GPR84 inhibition. At once-daily doses of 3 and 10 mg/kg, GLPG1205 reduced disease activity index score and neutrophil infiltration in a mouse dextran sodium sulfate-induced chronic inflammatory bowel disease model, with efficacy similar to positive-control compound sulfasalazine. The drug discovery steps leading to GLPG1205 identification, currently under phase II clinical investigation, are described herein.
Introduction
ARTICLE SECTIONS
GPR84 is a G-protein-coupled receptor (GPCR) that couples almost exclusively to pertussis-toxin sensitive Gi/o pathways. (1) Whereas GPR84 has no unanimously accepted cognate ligand, medium-chain free fatty acids (MCFAs, carbon chain lengths of 9–14) have been known as agonists for over a decade (1,2) with decanoic acid (C10), undecanoic acid (C11), and lauric acid (C12) being the most effective. (3) An increasing range of synthetic GPR84 agonist ligands have been described, including embelin, 3,3′-diindolylmethane (DIM), 6-n-octylaminouracil (6-OAU), (3) and 2-(hexylthiol)pyrimidine-4,6 diol (2-HTP) (4,5) (Figure 1). These synthetic GPR84 ligands are more potent than the MCFAs, with particular derivatives and structural relatives of 6-OAU being the most potent agonists of GPR84 to date. (4−6)
Figure 1
Figure 1. Chemical structures of known GPR84 agonists MCFA C10, embelin, 2-HTP, and DIM derivatives and the GPR84 antagonists PBI-4050 and 6-OAU. Abbreviations: 2-HTP, 2-(hexylthiol)pyrimidine-4,6 diol; 6-OAU, 6-n-octylaminouracil; DIM, 3,3′-diindolylmethane; MCFA, medium-chain free fatty acids.
GPR84 is primarily expressed in innate immune cells (neutrophils, (1,7) monocytes, (1) macrophages, (1,8) dendritic cells, (9) and microglia (8,10)) and splenic T and B cells. (11) Expression of GPR84 is highly upregulated by inflammatory stimuli in these innate immune cells and adipocytes, (12) suggesting an immunoregulatory role for the receptor. (1,7,10−13) In polymorphonuclear leukocytes and macrophages, activation of GPR84 results in chemotactic responses and enhances the release of cytokines (interleukin [IL]-8, IL-12, and tumor necrosis factor α [TNF-α]). (1,3) In addition, activation of this receptor in neutrophils and macrophages triggers the release of reactive oxygen species. (14)
GPR84 is suspected to be involved in inflammatory bowel disease (IBD), as increased levels of GPR84 messenger RNA (mRNA) have been observed in intestinal mucosal biopsies of patients with IBD (either ulcerative colitis or Crohn’s disease). (15) Additionally, GPR84 may be involved in fibrotic processes, as one recent study observed that GPR84 knock-out mice display significantly reduced kidney fibrosis when compared with wild-type mice in an adenine-induced chronic kidney disease model. (16) Moreover, in a mouse bleomycin-induced pulmonary fibrosis model, treatment with the nonspecific GPR84 antagonist PBI-4050 reduced histological lesions in the lung by 47% compared with vehicle. (16) Other animal disease models have indicated a role for GPR84 in chronic pain, (3) reflux esophagitis, (17) acute myeloid leukemia, (18) obesity, (19) and Alzheimer’s disease. (9) As such, GPR84 has emerged as a potential drug target, particularly for inflammatory and fibrotic diseases.
To date, only one low potency, nonspecific antagonist of GPR84 has been reported (PBI-4050), which inhibits GPR84 Gα/i activation initiated by either sodium decanoate or embelin. (16) In this article, we describe the discovery of GLPG1205 (36), a potent and selective GPR84 modulator, which demonstrates efficacy in in vivo IBD models and has been progressed in phase IIa clinical trials for the treatment of ulcerative colitis.
Results and Discussion
ARTICLE SECTIONS
During the course of our investigations, there was limited structural information available to provide support with molecular modeling tools for identifying GPR84 antagonists. The closest homologs of GPR84 with resolved structures had sequence identities below 30%, thereby preventing homology models being developed with sufficient accuracy to enable structure-based design. Furthermore, ligand-based approaches were limited due to the absence of reference antagonists. These issues led us to rely on a high-throughput screen (HTS) approach to identify GPR84 antagonists.
A functional assay based on measurement of cyclic adenosine 3′,5′-monophosphate (cAMP) was selected to detect antagonists from a collection of approximately 200 000 diverse compounds. The comparison of assay robustness between the cAMP assay and the orthogonal assay ([35S]guanosine 5′-O-[γ-thio]triphosphate (GTPγS) binding) led us to use the GTPγS for hit confirmation and concentration–response determination. The GTPγS assay measures the level of G protein activation following agonist occupancy of a GPCR by determining the binding of the nonhydrolyzable analogue [35S]GTPγS to Gα subunits using membranes derived from a HEK293 cell line stably overexpressing human GPR84.
Through HTS, the commercially available compound 2-(benzylamino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1) (Table 1) was identified as an appropriate starting point with submicromolar potency (IC50 = 735 nM) combined with favorable druglikeness properties, (20) such as moderate lipophilicity (XlogP3 = 2.4), polar surface area (PSA = 63.2), and molecular weight (MW = 363.4).
Table 1. Properties of Hit Compound 1a
| hGPR84 GTPγS IC50 (nM) | 735 |
| PDE4A IC50 (nM) | 553 |
| aqueous solubility 2% DMSO (μg/mL) | |
| pH 3/pH 7.4 | >73/24 |
| liver microsomal stability | |
| CLint MIC scaled (L h–1 kg–1) | mouse: 10.4 (n = 2) |
| human: 1.9 (n = 2) | |
| hepatocyte stability | |
| CLint HEP scaled (L h–1 kg–1) | mouse: 10.6 (n = 2) |
| human: 1 (n = 2) | |
| intestinal permeability Caco-2 cells | |
| Papp A2B (10–6 cm/s)/ER | 15/2.0 (n = 2) |
| hCYP inhibition | |
| % inhibition at 10 μM | <33 with CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4 (midazolam and testosterone) |
| rat PK 5 mg/kg po | |
| Cmax (ng/mL) | 1269 |
| AUC0–inf (ng·h/mL) | 2588 |
| F (%) | 50 |
| rat PK 1 mg/kg iv | |
| T1/2 (h) | 0.89 |
| CL (L h–1 kg–1); CL (% LBF) | 1.06; 21.6 |
| Vss (L/kg) | 1.27 |
a
Abbreviations: AUC0–inf, area under curve (from time 0 to infinity); CLint, intrinsic clearance; Cmax, maximum plasma concentration; DMSO, dimethyl sulfoxide; ER, efflux ratio; hCYP, human cytochrome P450; F, oral bioavailability; GTPγS, guanosine 5′-O-[γ-thio]triphosphate; HEP, hepatocyte; hGPR84, human G-protein-coupled receptor 84; IC50, half maximal inhibitory concentration; iv, intravenous; LBF, liver blood flow; MIC, microsomal; MW, molecular weight; Papp A2B, apparent permeability from apical to basal chambers; PDE4A, phosphodiesterase 4; PK, pharmacokinetics; po, per os; PSA, polar surface area; T1/2, half-life; Vss, apparent volume of distribution at steady state.
Compound 1 showed moderate in vitro metabolic clearance in both mouse and human microsomes and hepatocytes (Table 1). In vivo intravenous (iv) dosing at 1 mg/kg in Sprague Dawley rats confirmed a moderate plasma clearance and revealed a moderate volume of distribution and short terminal half-life (Table 1).
The favorable physicochemical parameters of 1 translated into high apparent permeability in Caco-2 cells with a low efflux ratio (Table 1). When administered orally to Sprague Dawley rats at 5 mg/kg in polyethylene glycol (PEG) 200/H2O (60/40; v/v), high plasma exposure (Cmax = 1269 ng/mL and AUC0–inf = 2588 ng·h/mL) was obtained for 1 with a 50% oral bioavailability (Table 1). The hit series did not show marked inhibition of either cytochrome P450 (CYP) or human ether-à-go-go-related gene (hERG).
At the start of our investigations, the presence of two methoxy groups in the 9 and 10 positions of the isoquinoline moiety raised some concerns, as such groups might play the role of the catechol ether structural motif common to most of the phosphodiesterase 4 (PDE4A) inhibitors (21,22) and introduce a PDE4A inhibitory component to the hit series already reported for 9 and 10 substituted dialkoxy analogues of pyrimido[6,1-a]isoquinolin-4-ones. (23)
PDE4 inhibitors possess a broad range of anti-inflammatory effects but have pronounced nausea and emesis side effects. As the aim of this project was to generate selective antagonists allowing us to unequivocally assess the therapeutic potential of GPR84, we wanted to show less than 50% inhibition over PDE4A at 10 μM. PDE4A inhibitory activity of 1 was confirmed (IC50 = 553 nM; Table 1). This led us to implement a PDE4A biochemical assay to assess the inhibitory activity of the compounds and gain SAR (structure–activity relationship) understanding to remove this residual PDE4A activity.
Initial SAR investigations focused on modifications of the benzyl amine moiety. Potency was abolished by either replacement of the nitrogen atom by a carbon atom (2) or introduction of a carbon–carbon double bond in place of the amino-methyl linker (3) (Table 2). A more favorable replacement was identified with the introduction of an oxygen atom, leading to an encouraging 5-fold increase in potency (4), compared with 1 (Table 2). We decided to consolidate the investigations with the oxygen atom in place.
Table 2. hGPR84 GTPγS, PDE4A, Human Neutrophil Migration, and Solubility Profiles of 1 Derivativesa
a
Abbreviations: ASOL, aqueous solubility; Cap, capric acid; Compd, compound; hGTPγS, human guanosine 5′-O-[γ-thio]triphosphate; Hum neutro, human neutrophil migration assay; IC50, half maximal inhibitory concentration; IP1, inositol monophosphate; PDE4A, phosphodiesterase 4.
Improved potency (IC50 = 21 nM, Table 2) was observed with the introduction of an aliphatic chain that is common to the MCFA endogenous ligands (5), although solubility was drastically reduced by the lipophilic side chain (aqueous solubility [ASOL] pH 7.4 = 3 μg/mL).
Various lipophilic spacers were introduced (6 and 7) and showed decreased potency compared with 4 (Table 2). With this ether linkage in place, introduction of a fluorine in the ortho position (8) had limited impact on potency, compared with 4. Further assessment of PDE4A inhibitory activity showed no selectivity improvement. Moreover, 8 showed decreased metabolic stability in mouse microsomes (Table 3), compared with 1.
Table 3. hGTPγS, PDE4A, and Mouse Liver Microsomal Stability of Substructural Derivativesa
a
Abbreviations: Compd, compound; CLint, intrinsic clearance; hGTPγS, human guanosine 5′-O-[γ-thio]triphosphate; IC50, half maximal inhibitory concentration; LMS, liver microsomal stability; Me, methyl group; PDE4A, phosphodiesterase 4.
We probed the scope of polarity introduction which, if tolerated, could be beneficial for further improvement of metabolic stability. The addition of an ethylene glycol spacer (9) restored good potency (IC50 = 63 nM) and gave a 150-fold increase compared with the match paired analogue (7) (Table 2). This disparity indicated a strong contribution from the oxygen atom introduced, possibly resulting from an H-bond interaction. Compound 9 showed a reduced inhibitory activity on PDE4A (Table 2), leading to a 30-fold ratio selectivity. Internal investigations identified that GPR84 agonists such as embelin, DIM, and sodium decanoate were able to induce human neutrophil migration. GPR84 antagonists were able to specifically block neutrophil chemotaxis induced by GPR84 agonists (Supplemental Figure 4). The embelin-induced neutrophil chemotaxis assay was used as an inflammatory disease relevant cell assay to determine the inhibitory activity of our GPR84 antagonists. Assessment of 9 showed a good translation of the antagonist effect on a target based GTPγS assay toward the human neutrophil migration assay (IC50 = 128 nM; Table 2). Comparing the matched pairs 10 and 8 showed that the introduction of one nitrogen atom had only a moderate impact on potency; however, a positive impact was observed on metabolic stability (Table 3) and its selectivity toward PDE4A (IC50 = 1088; Table 2).
Replacement of the phenyl ring with a more polar tetrahydrofuran was tolerated for the 2-substituted heterocycle (11) and found inactive for the 3-substituted analogue (12) (Table 2). This result is associated with the favorable impact observed on potency with the oxygen introduction (9) and may be indicative of a specific interaction of GPR84 with an oxygen atom in this position. In addition to increased potency, the position of oxygen in 11 improved selectivity against PDE4A (IC50 = 2165 nM; Table 2) and solubility (9, ASOL pH 7.4 = 26 μg/mL and 11, ASOL pH 7.4 = 72 μg/mL). The potency of 9, in the human neutrophil assay (IC50 = 128 nM; Table 2), prompted investigation into the tolerance of further polarity. Replacement of the phenyl ring of 9 with a 2-pyridyl ring increased both the potency toward the target and the selectivity ratio over PDE4A (13, GTPγS IC50 = 8 nM and selectivity ratio = 73).
Sequence differences between human and mouse GPR84 (mouse GPR84 shares 85% with its human ortholog at the amino acid level) prompted us to assess activity of the hit series on mouse GPR84 receptor assays. At this stage of investigation, no cell line stably overexpressing the mouse receptor for the GTPγS assay was available. An assay measuring inositol monophosphate (IP1) using transiently transfected HEK293 cells with either the human or mouse recombinant GPR84 and a variant of a chimeric Gq protein was developed to allow direct comparison on mouse and human receptor under similar assay conditions. We confirmed the high inhibitory activity of 13 in the IP1-based hGPR84 assay (hGPR84 IP1 IC50 = 11 nM) and observed an approximate 60-fold reduction in the IP1-based mouse GPR84 assay (mGPR84 IP1 IC50 = 636 nM).
Initial investigations demonstrated that a high potency in the nanomolar range could be reached in the human GTPγS assay, which translated into potent inhibition of human neutrophil migration. Further optimization comprised the consolidation of the attractive profile of the series, with the aim to improve the selectivity ratio by producing a compound that was inactive in the PDE4A assay or had at least a 100-fold selectivity.
The presence of the catechol ether moiety in known potent inhibitors of PDE4A suggested a key role for this functional group in the enzyme pharmacophore, prompting further investigation of the impact of modifications in this region. The presence of the two methoxy substituents was found to be crucial to PDE4A activity but not mandatory for maintaining GPR84 inhibitory activity, as illustrated with the unsubstituted analogue 14 (Table 3). Removal of either methoxy group markedly decreased PDE4A activity for the corresponding analogues (15 and 16; Table 3). Of particular interest were the 9-methoxy substituted analogues, which showed a less than 10-fold loss of GPR84 activity in three matched pairs (17 and 10; 18 and 11; 15 and 8; Table 3). The favorable selectivity trend was found to be well conserved when applied to the 2-fluoropyridine analogue (17 and 10; Table 3). Despite decreased metabolic stability in mouse liver microsomes observed for the 9-substituted analogue 17 compared with the dimethoxy substituted analogue 10 (Table 3), the gain in selectivity and the attractiveness of the undocumented 9-methoxy derivatives led us to continue in this direction.
Introduction of the phenoxyethyl group (19) led to a 20-fold increase in potency compared with the moderately potent starting point 18 (Table 4), a marked improvement from the previous 6-fold increase in potency for the dimethoxy substituted matched pair analogues 11 and 9. Additionally, 19 achieved good selectivity with an inhibitory activity above 10 μM in the PDE4A assay (Table 4). Within the chemical series, the preliminary results observed from liver microsomal stability data and the lipophilicity of the compounds seemed to point toward a favorable XlogP3 threshold below 3. Subsequent SAR optimization aimed to keep lipophilicity with XlogP3 below 3, retain the key β-oxygen, and determine the most suitable moieties for demonstrating a good metabolic profile while maintaining acceptable potency and selectivity.
Table 4. hGTPγS, PDE4A, Liver Microsomal Stability, Hepatocyte Stability, PPB, and XlogP3 Profiles for Substructural Derivativesa
a
Abbreviations: CLint, intrinsic clearance; H, human; HEP, hepatocyte; hGTPγS, human guanosine 5′-O-[γ-thio]triphosphate; IC50, half maximal inhibitory concentration; M, mouse; MIC, microsomal; PPB, plasma protein binding; PDE4A, phosphodiesterase 4; R, rat.
Replacement of the 2-tetrahydrofuryl with the homologous tetrahydro-2H-pyranyl moiety improved potency (20; Table 4) with a moderate increase in lipophilicity. However, a significant reduction in lipophilicity was achieved with the introduction of the 1,4-dioxane (21) with similar potency compared with 20 and improved metabolic stability in mouse and human liver microsomes and hepatocytes. The attractive absorption, distribution, metabolism, and excretion (ADME) profile obtained by introduction of the dioxane moiety prompted attempts to mitigate the associated reduced potency by adding moderate lipophilicity with fused heterocycles on the dioxane. This was achieved through fusion of a phenyl ring to produce 22 with increased potency (IC50 = 50 nM; Table 4) but with severely reduced stability in mouse, rat, and human liver microsomes.
To reduce the lipophilicity, a 2-pyridyl ring was fused to the dioxane to give 23. However, the introduction of the nitrogen in the ortho position of the key β-oxygen resulted in reduced potency compared with 22. Introducing the nitrogen to the ortho position of the other oxygen in the dioxane ring had a positive impact on potency (24) but was accompanied by a high intrinsic liver microsomal clearance in mice (Table 4). Assessment of the metabolic stability of compounds 18–24 in hepatocytes showed a good correlation of intrinsic clearance between liver microsomes and both mouse and human hepatocytes, indicating a major contribution in phase I metabolism (Table 4).
With the high potency of 24, further optimization investigations focused on modifications of the 9 position with opportunistic introduction in one step of diverse and exotic commercially available moieties (Table 5). Assessment of the tolerability of this region to substantial modulation was investigated through the introduction of a negatively charged chain and a very bulky moiety. Interestingly, substitution of the methyl group with an acetic acid group from the potent compound 22 led only to a moderate 5-fold decrease in potency (25; Table 5). Replacement of the methoxy group with 2-cyanophenyl (26) was neutral (IC50 = 212 nM; Table 5) when compared with 21. The compound was unstable in dog, monkey, and human liver microsomes, but moderate clearance was observed in rodents. Furthermore, 26 demonstrated good permeability in Caco-2 cells with low efflux and high systemic exposure in rat at 5 mg/kg (AUC0–Z of 2645 ng·h/mL [6.37 μM·h] following oral dosing in PEG200/water [60/40; v/v]) providing an interesting opportunity to evaluate the capability of the lead series to modulate inflammatory bowel disease (IBD).
Table 5. hGTPγS, XlogP3, Metabolic Stability, and Permeability Profiles of 25, 26, and 27a
a
Abbreviations: CLint, intrinsic clearance; Compd, compound; ER, efflux ratio; hGTPγS, human guanosine 5′-O-[γ-thio]triphosphate; IC50, half maximal inhibitory concentration; MIC, minimum inhibitory concentration; Papp A2B, apparent permeability from apical to basal chambers.
To assess in vivo efficacy, 26 was studied in a mouse chronic dextran sodium sulfate (DSS)-induced colitis model, a well-validated model for IBD. (24,25) The DSS treatment significantly increased the disease activity index (DAI) score (sum of scores for weight loss, stool consistency, and fecal blood) reflecting active colitis. As expected, once-daily treatment with the positive control cyclosporine (administered at 25 mg/kg, once daily [qd]) diminished the DAI score in diseased mice. Once-daily treatment with 26 at 10 mg/kg qd reduced the DAI score at 8–11 days (p < 0.05 at day 9 versus diseased mice) (Figure 2). Encouraged by these in vivo data, subsequent optimization of the lead series was aimed at improving in vivo potency and metabolic stability in dog and human.
Figure 2
Figure 2. Disease activity index score in a mouse DSS-induced chronic colitis model. Diseased mice were administered with 4% DSS in drinking water for 4 days, followed by 3 days of regular drinking water and a new cycle of DSS. In addition they were orally administered 26 at 10 mg/kg once daily (qd), cyclosporine at 25 mg/kg qd (positive control), or vehicle for 11 consecutive days. Control mice were administered water alone as a negative control. Data are the mean ± SEM. No scores were measured on days 5 and 6.
A 33-fold gain in potency was achieved by the addition of a bulky phenylpro-2-yn-1-ol moiety to the highly potent compound 24 to produce 27 with a subnanomolar potency on GPR84 (IC50 = 0.6 nM; Table 5). This result confirmed the option of replacing the oxygen linker at the 9 position with a carbon atom, as with 26. These additions, of both the negatively charged chain and bulky moiety, indicated a broad SAR tolerance in this region of the molecule. Further modification of this region was pursued with the aim of maintaining overall lipophilicity below the defined threshold for the series while benefiting from a gain in potency.
The high potency of 27 was associated with increased lipophilicity (Table 5), so further modifications aimed to achieve a good balance of these properties. The moderately potent 21 (Table 4) was selected as a starting compound as it afforded a 2 log margin for lipophilicity and required only an approximate 10-fold gain in potency to produce an attractive target molecule. Removal of the 9-methoxy substituent (28; Table 6) had a minor impact on the lipophilicity and the inhibitory activity (Table 6). Introduction of groups with growing size to maintain lipophilicity in the targeted range improved the potency (29, 30, and 31; Table 6). Introduction of a 2 ethylpyridyl moiety maintained a moderate lipophilicity and gave a remarkable potency (32) in human GTPγS and IP1 assays and reduced orthology shift in the mouse IP1 assay. However, while the compound showed low clearance in human microsomes (intrinsic clearance (CLint) scaled of 1.5 L h–1 kg–1), it was highly cleared in mouse liver microsomes (CLint scaled of 31 L h–1 kg–1) precluding further in vivo evaluation of the compound.
Table 6. hGTPγS, XlogP3, PDE4A, IP1, Liver Microsomal Stability, and Human Neutrophil Migration Profiles of Substructural Derivativesa
a
Abbreviations: CLint, intrinsic clearance; Compd, compound; D, dog; H, human; hGTPγS, human guanosine 5′-O-[γ-thio]triphosphate; Hum neutro, human neutrophil migration; IC50, half maximal inhibitory concentration; IP1, inositol monophosphate; LMS, liver microsomal stability; M, mouse; MIC, microsomal; PDE4A, phosphodiesterase; R, rat.
We decided to further exploit the initial path identified with compound 27 (Table 5). Starting from a decreased level of lipophilicity, due to the removal of the fused pyridyl moiety, the phenyl ring linked to the secondary alcohol was replaced with an isobutyl group limiting the lipophilicity at XlogP3 = 2.3. Compound 33 showed good potency (IC50 = 25 nM; Table 6); however, in spite of a moderate lipophilicity, the compound was found to be insufficiently stable in rat liver microsomes (CLint scaled of 15.3 L h–1 kg–1). Compound 34 was designed to remove the propargylic alcohol as a putative metabolite site and introduce a basic benzyl amine moiety. These modifications retained potency (IC50 = 22 nM; Table 6), but 34 showed elevated liver microsomal clearances in both mouse and human (in mouse, CLint scaled of 26 L h–1 kg–1 and in human, CLint scaled of 5.3 L h–1 kg–1).
The addition of a simple cyclopropyl ring into the alkyne moiety maintained both acceptable lipophilicity and potency (IC50 = 55 nM; Table 6), and the resulting compound 35 was devoid of PDE4A activity. The high potency of the compound was confirmed in a human IP1 assay (IC50 = 2 nM) and was found to be 10-fold less potent in the same assay in mice (IC50 = 21 nM). In accordance with previous data, the high potency of 35 in GPR84 functional assays corresponded to high potency in human neutrophil migration assays (IC50 = 17; Table 6). A rat neutrophil migration assay was successfully developed and confirmed the high inhibitory activity of the compound (IC50 = 119 nM) and the shift in activity from human to rodent. Additionally, 35 showed acceptable metabolic stability in human, rat, and mouse liver microsomes (CLint scaled of 2.2, 1.4 and 9.7, respectively).
The attractive profile of 35, with good potency in rat and human in vitro assays and improved metabolic stability in human liver microsomes, led to further in vivo studies. High plasma exposure was observed following oral administration at 5 mg/kg in rats with an AUC0–inf of 10246 ng·h/mL and a Cmax of 2423 ng/mL at 1.7 h. In a mouse DSS-induced chronic IBD model, once-daily treatment with 35 at 1, 3, or 10 mg/kg dose-dependently reduced the DAI score; 35 at 10 mg/kg was found to have similar efficacy as cyclosporine at 25 mg/kg, the positive control (Figure 3).
Figure 3
Figure 3. Disease activity index score in a mouse DSS-induced chronic colitis model. Diseased mice were administered with 4% DSS in drinking water for 4 days, followed by 3 days of regular drinking water and a new cycle of DSS. In addition they were orally administered 35 at 1*, 3† or 10‡ mg/kg qd, cyclosporine at 25 mg/kg qd (positive control), or vehicle, for 11 consecutive days. Control mice were administered water alone as a negative control. Data are the mean ± SEM. No scores were measured on days 5 and 6.
The attractive profile of the compound instigated efforts to isolate and characterize the two enantiomers of 35 (Table 7). When profiled in terms of ADME and biological assays, the two enantiomers (S) 36 and (R) 37 showed moderate differences favoring the (S) enantiomer 36 (Table 7). Compound 36 was approximately 5-fold more potent in human neutrophil migration assays (Table 7) but only slightly more potent in human GTPγS and rat and dog neutrophil migration assays (Table 7). Both enantiomers appeared stable in hepatocytes when incubated at 1 μM, with a low predicted hepatic clearance for rat, mouse, dog, and human (Table 7). The two enantiomers did not show marked differences in binding to plasma protein when assessed in four different species in an in vitro equilibrium dialysis PPB assay (tested at 5 μM) (Table 7). The assessment of the CYP inhibitory potential of the two enantiomers was markedly more favorable for 36. The IC50 was determined using human liver microsomes with probe substrates for CYP1A2, 2C9, 2C19, 2D6, and 3A4 (Table 7). The IC50 value for 36 for each of the CYP isoenzymes was >33 μM (Table 7), whereas the (R) enantiomer showed micromolar inhibition of the CYP2C9 isoform (IC50 = 1.85 μM; Table 7) and moderate inhibition of CYP2C19 (IC50 = 18 μM; Table 7). In addition, there was no evidence for time-dependent inhibition of CYP3A4 by 36 (human liver microsomes, midazolam, and testosterone used as probe substrates to assess remaining enzyme activity) and the IC50 was >50 μM, both with and without preincubation.
Table 7. Profiles of the Two Enantiomers of 35a
| 36 (S) | 37 (R) | ||
|---|---|---|---|
| hGPR84 (GTPγS IC50, nM) | 54 | 102 | |
| human neutrophil assay (IC50, nM) | 11 | 51 | |
| rat neutrophil assay (IC50, nM) | 111 | 153 | |
| dog neutrophil assay (IC50, nM) | 75 | 111 | |
| aqueous solubility 2% DMSO (μg/mL) | |||
| pH 3/pH 7.4 | 113/113 | 113/113 | |
| thermodynamic solubility (μg/mL) | |||
| pH 3/pH 7.4 | 12.6/7.75 | 17.6/9.49 | |
| liver microsomal stability 1 μM | |||
| CLint MIC scaled (L h–1 kg–1) | mouse | 8.0 | 5.9 |
| rat | 4.0 | 4.97 | |
| dog | 5.34 | 3.16 | |
| human | <1.16 | 0.962 | |
| hepatocyte stability 1 μM | |||
| T1/2 (min)/CLint HEP scaled (L h–1 kg–1) | mouse | >200/<4.37 | >200/<4.37 |
| rat | >200/<2.0 | >188/<2.12 | |
| dog | >200/<1.45 | >151/<1.92 | |
| human | >200/<1.28 | >200/<1.28 | |
| intestinal permeability Caco-2 cells | |||
| Papp A2B (×10–6 cm/s)/ER | 41.9/1.05 | 31.2/1.07 | |
| CYP inhibition HLM IC50 (μM) | 1A2 | >100 | >100 |
| 2C19 | >100 | 18.2 | |
| 2D6 | >100 | 31.6 | |
| 2C9 | 34.6 | 1.85 | |
| 3A4 mid | >50 | >50 | |
| 3A4 test | >50 | >50 | |
| PPB % bound | mouse | 97.8 | 98.3 |
| rat | 97.7 | 96.3 | |
| human | 97.2 | 94.1 | |
| dog | 93.5 | 96.7 | |
a
Abbreviations: CLint, intrinsic clearance; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; ER, efflux ratio; GTPγS, guanosine 5′-O-[γ-thio]triphosphate; HEP, hepatocyte; HLM, human liver microsome; IC50, half maximal inhibitory concentration; IP1, inositol monophosphate; MIC, microsomal; Papp A2B, apparent permeability from apical to basal chambers; PPB, plasma protein binding.
The pharmacokinetic profile (PK) of 36 was assessed in three species (mouse, rat, and dog) after an iv administration at 1 mg/kg (in mouse) or 0.5 mg/kg (in rat and dog) and an oral administration in fasted animals at 5 mg/kg (in mouse) or 2.5 mg/kg (in rat and dog). PEG200/NaCl 0.9% (60/40; v/v) was used as formulation for the iv route and PEG200/water (60/40; v/v) for the po route. A high oral exposure was obtained in all three species (Table 8), giving a bioavailability of 100%. A high total clearance was observed in dogs but did not preclude its progression owing to acceptable clearances in other species. The use of the recently introduced unbound clearance parameter shows that this is due to the lower PPB in dog (93.5% of binding) compared with mouse, rat, and human, leading to a lower unbound clearance (CLunb = 18.5). The volume of distribution was low in mice and high in dogs.
Table 8. Pharmacokinetic Parameters of 36 in Different Speciesa
| mouse | rat | dog | |||||
|---|---|---|---|---|---|---|---|
| 1 mg/kg iv (n = 3) | 5 mg/kg po (n = 3) | 0.5 mg/kg iv (n = 3) | 2.5 mg/kg po (n = 3) | 0.5 mg/kg iv (n = 3) | 2.5 mg/kg po (n = 3) | ||
| C0 or Cmax | (ng/mL) | 1227 | 4593 | 395 | 1164 | NC | 558 |
| Tmax | (h) | 0.5 | 1.5 | 0.5 | |||
| AUC(0–24h) | (ng·h/mL) | 1054 | 15349 | 710 | 4469 | 410 | 2232 |
| CL | (L h–1 kg–1)/(% LBF) | 0.9/11 | 0.7/14 | 1.2/48 | |||
| CLunb | (L h–1 kg–1) | 40.9 | 30.4 | 18.5 | |||
| Vss | (L/kg) | 0.6 | 1.2 | 2.0 | |||
| t1/2 | (h) | 0.6 | 1.9 | 1.0 | 1.3 | 1.4 | 2.0 |
| F | (%) | >100 | >100 | >100 | |||
a
Abbreviations: AUC, area under curve; CL, clearance; CLunb, clearance unbound calculated using PPB data (Table 7); Cmax, maximum plasma concentration; C0, concentration extrapolated at T0 after a bolus iv dose; F, bioavailability; LBF, liver blood flow; t1/2, half-life; Tmax, time at Cmax; Vss, volume of distribution.
In competition studies, 36 was found to block agonist effects of both modulators considered as orthosteric ligands, such as C10 and embelin, and allosteric ligands, such as PSB-16671 (Figures 1 and 4A). In parallel to the progression of 36, additional potent compounds with modified substitutions in position 9 and different side chains in the northern region of the compound were identified. Compound 38, with a phenethyl chain in position 9 and a 2-ethoxypyrazine moiety in the northern region (Figure 4B), reached a high potency level in the hGTPγS assay (IC50 = 5 nM). In addition to being one of the most potent analogues in the GTPγS assay, the straightforward synthetic introduction of a tritium atom into the molecule from the corresponding 3-chlorophenetyl derivative guided the selection of 38 as a target for generating a radiolabeled ligand ([3H]38 was prepared by Quotient Bioresearch). Compound 36 competed well with its analogue [3H]38 with a pKi of 7.52. Increasing concentrations of C10 were unable to displace the [3H]38 ligand, suggesting an allosteric binding site for the GPR84 antagonists (Figure 4C). In the same competition experiment, PSB-16671, an allosteric agonist from the DIM series, showed only moderate displacement at a very high dose (Figure 4C). In a Schild experiment, further characterization of the interaction between the two ligands showed 36 to act dose-dependently as a noncompetitive and reversible blocker of PSB-16671, with a decreased maximum effect rather than markedly increasing the half maximal effective concentration (EC50) (Figure 4D). These results, combined with previous studies showing that [3H]38 binding was not affected by mutation of key residue Arg172, (26) indicate that 36 acts as a negative allosteric modulator of the known GPR84 agonists.
Figure 4
Figure 4. (A) Effect of increasing concentrations of 36 on effects of orthosteric and allosteric GPR84 agonists (GTPyS assay with agonists used at EC80). (B) Structure of tritiated ligand 38. (C) Ability of various concentrations of C10, PSB-16671, and 36 to compete for binding with [3H]38 to GPR84 (competition binding + 0.2 nM [3H]38). (D) Schild analysis showing inhibition of PSB-16671 activity with 36 at concentrations of 10 nM, 30 nM, 100 nM, and 300 nM.
In a mouse DSS-induced chronic IBD model, once-daily treatment with 36 at 3 or 10 mg/kg reduced the DAI score (Figure 5A). In the colon, compound 36 also reduced neutrophil infiltration induced by DSS, a pharmacodynamics marker in relation to in vitro neutrophil chemotaxis activity (Figure 5B). The pharmacodynamics marker was reversed by 36 at 3 mg/k and 10 mg/kg, with a similar dose effect to DAI. This provides a mechanistic insight, clearly indicating the capacity of 36 to reduce neutrophil migration toward the inflamed colonic tissue. This activity was associated with a Cmax of 1837 ng/mL and an exposure level (AUC0–24h) of 5556 ng·h/mL (Table 9).
Figure 5
Figure 5. Diseased mice (DSS-induced chronic colitis model) were administered with 4% DSS in drinking water for 4 days, followed by 3 days of regular drinking water and a new cycle of DSS. In addition they were orally administered 36 at 3* and 10† mg/kg qd, sulfasalazine 20 mg/kg qd (positive control) or vehicle, for 11 consecutive days. Control mice were administered water alone as a negative control. (A) Disease activity index score. Data are the mean ± SEM. No scores were measured on days 5 and 6. (B) Neutrophil density (number of cells per square millimeter of colon area, as determined by immunohistochemistry assay with anti-Ly6G/Ly6C antibodies).
Table 9. Pharmacokinetic Parameters of 36 at Steady State in Female BALB/c Mice after Multiple Oral Doses of 3 or 10 mg kg–1 day–1 a
| 3 mg kg–1 day–1 (n = 2 or 3) | 10 mg kg–1 day–1 (n = 2 or 3) | ||
|---|---|---|---|
| Cmax | (ng/mL) | 1837 | 8140 |
| Cmax/dose | 612 | 814 | |
| Tmax | (h) | 1 | 1 |
| AUC(0–24h) | (ng·h/mL) | 5556 | 50387 |
| AUC(0–24h)/dose | 1852 | 5039 | |
| t1/2 | (h) | 2.9 | 2.3 |
a
Abbreviations: AUC, area under curve; Cmax, maximum plasma concentration; Tmax, time to Cmax; t1/2, half-life.
Compound 36 was tested in three independent mouse DSS-induced chronic IBD studies at 1, 3, or 10 mg/kg to confirm the efficacy and to define the minimal efficacious dose (MED). The dose of 1 mg/kg dose was inactive; however the dose of 3 mg/kg was constantly found active in all three studies and thus was considered as the MED.
Compound 36 was further profiled in a diverse set of selectivity assays. Compound 36 was fully selective over close homologs GPR43 (FFA2R) and GPR41 (FFA3R) (no inhibition up to 10 μM in [Ca2+]i flux assay) and >100-fold selective over 123 other GPCRs on a Millipore GPCR Profiler panel. Compound 36 was remarkably selective over a kinase panel, displaying only weak inhibitory activity against maternal embryonic leucine zipper kinase (MELK; 57% inhibition at a concentration of 10 μM) out of a selection of 152 kinases (Reaction Biology, Malvern, PA, USA); this inhibitory activity was not considered to be clinically relevant. Similar high selectivity was also observed against a diverse panel of 67 enzymes focusing mostly on ion channels and transporters. Compound 36 was considered to present a low cardiovascular risk due to the minimal impact observed on the amplitude of the IKr current in the in vitro hERG manual patch clamp assay and the lack of effect on hemodynamic and electrocardiographic parameters in telemetered monkeys. Compound 36 was assessed in the full Ames test, up to concentrations of 5000 μg/plate, in the presence and absence of S9 fraction, leading to the conclusion that 36 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Compound 36 did not have any genotoxic effect in vitro in the mouse lymphoma assay or in vivo in the bone marrow micronucleus test in rats. Compound 36 did not show any teratogenic or embryotoxic effects in rats, up to the highest tolerated maternal dose of 50 mg kg–1 day–1. In addition, no phototoxic effects with 36 have been observed in vivo in rats and mice.
Chemistry
ARTICLE SECTIONS
GLPG1205 Synthesis
GLPG1205 (36) was synthesized in nine linear steps that all proceeded with yields higher than 70% and involved readily available starting material (Scheme 1).
Scheme 1
Scheme 1. Synthesis of (S)-GLPG1205 (36)a
aReagents and conditions: (a) urea, AcOH, conc HCl, water, reflux; (b) conc HBr, reflux; (c) AllylBr, K2CO3, DMF; (d) Na, EtOH, diethyl malonate, reflux; (e) POCl3, 50 °C; (f) [(2R)-1,4-dioxan-2-yl]methanol, tBuOK, DCM; (g) Pd(PPh3)4, K2CO3, MeOH/THF; (h) PhN(SO3CF3)2, Et3N, DCM; (i) ethynylcyclopropane, Pd(PPh3)4, CuI, Et3N, DMF.
Starting from 3-methoxyphenethylamine, refluxing urea in acetic acid overnight led to the urea analogue. Demethylation was carried out upon overnight reflux with hydrogen bromide to lead to the corresponding phenol derivative which was further protected by introduction of an allyl group. Condensation with diethyl malonate afforded the hexahydropyrimidine-2,4,6-trione which underwent regioselective cyclization reaction with treatment with POCl3. Subsequent O-alkylation with commercially available or easily prepared (R)-(1,4-dioxan-2-yl)methanol (27) followed by deprotection of the allyl group led to the phenol intermediate. The phenol intermediate was converted into the corresponding triflate for further use in Sonogashira coupling reaction with ethynylcyclopropane, providing GLPG1205. The purity of all compounds was determined by liquid chromatography–mass spectrometry (LCMS) and 1H NMR. Minor adaptations of this synthetic route afforded 9,10-dimethoxy derivatives starting from 3,4-dimethoxyphenethylamine.
Conclusion
ARTICLE SECTIONS
The progression of the commercially available hit compound 1 to the clinical candidate 36 was achieved by structure optimization to improve potency (GTPγS assay), metabolic stability, lipophilicity, and selectivity against PDE4A. In a mouse DSS-induced chronic IBD model, treatment with 36 reduced both the DAI score and neutrophil infiltration at oral doses of 3 and 10 mg/kg qd, with an efficacy similar to the positive control compound sulfasalazine. A phase I first-in-human study (28) and a phase IIa study in IBD (ulcerative colitis; NCT02337608) (29) confirmed favorable PK, safety, and tolerability profiles for 36, but the compound failed to meet the primary efficacy end point in the latter, and as GLPG1205 is the first in class GPR84 antagonist tested in clinical study, additional translational studies would contribute to the further understanding of results. (29) The biology of GPR84 has been further investigated and a potential role in fibrosis described in different organs including the kidney (16) and liver. (30) Moreover, GLPG1205 (36) has demonstrated reduced lung fibrosis in two mouse models in a therapeutic setting. (31) GLPG1205 (36) is currently being evaluated in a phase II study in patients with idiopathic pulmonary fibrosis. Additional safety, pharmacological, PK, and clinical data will be reported in due course.
Experimental Section
ARTICLE SECTIONS
Chemistry. Methods
All reagents were of commercial grade and were used as received without further purification unless otherwise stated. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases unless otherwise specified. Column chromatography was performed on silica gel 60 (35–70 μm). Thin layer chromatography was carried out using precoated silica gel F-254 plates (thickness 0.25 mm). Microwave heating was performed with a Biotage Initiator apparatus (Biotage, Uppsala, Sweden). Celpure P65 (Merck KgaA, Darmstadt, Germany) is a filtration aid and commercial product (CAS number 61790-53-2). 1H NMR spectra were recorded on a Bruker DPX 400 NMR spectrometer (400 MHz) (Bruker, Billerica, MA, USA) or a Bruker Avance 300 NMR spectrometer (300 MHz) (Bruker, Billerica, MA, USA). Chemical shifts (δ) for 1H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) or the appropriate residual solvent peak, i.e., CHCl3 (δ 7.27), as internal reference. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), quintuplet (quin), multiplet (m), and broad (br). Waters Acquity UPLC (Waters, Elstree, U.K.) with Waters Acquity PDA detector (Waters, Elstree, U.K.) and SQD mass spectrometer (Waters, Elstree, U.K.) were used to generate UV and MS chromatograms as well as MS spectra. Columns used were the following: UPLC BEH C18 1.7 μm, 2.1 mm × 5 mm, VanGuard Precolumn (Waters, Elstree, U.K.) with Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 30 mm column or Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm column. LC–MS analyses were conducted using the following two methods. Method 1: solvent A, H2O–0.05% NH4OH; solvent B, MeCN–0.05% NH4OH; flow rate, 0.8 mL/min; start 5% B, final 95% B in 1.55 min, linear gradient. Method 2: solvent A, H2O–0.1% HCO2H; solvent B, MeCN–0.1% HCO2H; flow rate, 0.8 mL/min; start 5% B, final 95% B in 1.55 min, linear gradient. All final compounds reported were analyzed using one of these analytical methods, and purity is greater than 95% or otherwise indicated in the Supporting Information. Autopurification system from Waters was used for LC–MS purification. LC–MS columns used: Waters XBridge Prep OBD C18 5 μm, 30 mm × 100 mm (preparative column) and Waters XBridge BEH C18 5 μm, 4.6 mm × 100 mm (analytical column) (Waters, Elstree, U.K.). All the methods used MeCN/H2O gradients. MeCN and H2O contained either 0.1% formic acid or 0.1% diethylamine.
2-(3-Methoxyphenyl)ethylurea
A solution of 3-methoxyphenethylamine (100 g, 661.3 mmol), urea (157.3 g, 2619.0 mmol), glacial AcOH (36 mL), and concentrated HCl (12 mL) in H2O (800 mL) was heated under reflux for 5 days. The reaction mixture was cooled to room temperature (RT), and the precipitate obtained was filtered off, washed with water, and dried under vacuum to give 2-(3-methoxyphenyl)ethylurea as a white solid (92 g, 72% yield). 1H NMR (400 MHz, methanol-d4) δ 7.18 (t, J = 8.1 Hz, 1H), 6.83–6.71 (m, 3H), 3.77 (s, 3H), 3.37–3.31 (m, 2H), 2.74 (t, J = 7.2 Hz, 2H).
2-(3-Hydroxyphenyl)ethylurea
A solution of 2-(3-methoxyphenyl)ethylurea (92 g, 473.6 mmol) in concentrated HBr (600 mL) was heated under reflux overnight. The reaction was cooled to RT and basified by addition of NaHCO3 and extracted with EtOAc. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude 2-(3-hydroxyphenyl)ethylurea as a light brown solid (67 g) which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 7.07 (td, J = 7.2, 1.8 Hz, 1H), 6.65–6.55 (m, 3H), 5.89 (t, J = 5.8 Hz, 1H), 3.16 (dt, J = 7.7, 6.3 Hz, 2H), 2.57 (t, J = 7.3 Hz, 2H). LCMS: m/z 181 [M + H]+.
2-(3-Allyloxyphenyl)ethylurea
To a solution of crude 2-(3-hydroxyphenyl)ethylurea (67 g, 371.0 mmol) and K2CO3 (154 g, 1115.0 mmol) in DMF (400 mL) was added allyl bromide (75 mL, 743.6 mmol), and the reaction was stirred for 2.5 days. DMF was concentrated under reduced pressure and then the residue was dissolved in EtOAc, washed with a saturated aqueous solution of Na2CO3 followed by brine, then dried over MgSO4. Concentration under reduced pressure gave crude 2-(3-allyloxyphenyl)ethylurea as a colorless solid (79 g) which was used in the next step without further purification. 1H NMR (400 MHz, methanol-d4) δ 7.97 (s, 1H), 7.22–7.13 (m, 1H), 6.84–6.71 (m, 3H), 6.13–5.97 (m, 1H), 5.39 (dq, J = 17.3, 1.7 Hz, 1H), 5.23 (dq, J = 10.6, 1.6 Hz, 1H), 4.52 (ddt, J = 5.1, 3.5, 1.6 Hz, 2H), 3.34 (d, J = 7.0 Hz, 2H), 2.98 (d, J = 0.6 Hz, 2H), 2.74 (t, J = 7.3 Hz, 2H). LCMS: m/z 221 [M + H]+.
1-[2-(3-Allyloxyphenyl)ethyl]hexahydropyrimidine-2,4,6-trione
To a stirred solution of sodium (16 g, 718.0 mmol) in EtOH (1.0 L), diethyl malonate (109.1 mL, 718.0 mmol) was added, and the reaction mixture was heated under reflux for 1 h. Crude 2-(3-allyloxyphenyl)ethylurea (72 g, 359.0 mmol) in EtOH (200 mL) was added, and the reaction mixture was heated under reflux for 12 h. The reaction was cooled to RT, 1 M aq HCl was added, and the precipitate obtained was filtered off, washed with water, and dried under vacuum to give 1-[2-(3-allyloxyphenyl)ethyl]hexahydropyrimidine-2,4,6-trione as a yellow solid (82 g, 79% yield). 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.25–7.16 (m, 1H), 6.87–6.72 (m, 3H), 6.05 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H), 5.41 (dq, J = 17.3, 1.6 Hz, 1H), 5.28 (dq, J = 10.5, 1.4 Hz, 1H), 4.53 (dt, J = 5.3, 1.5 Hz, 2H), 4.15–4.04 (m, 2H), 3.61 (s, 2H), 2.92–2.82 (m, 2H). LCMS: m/z 289 [M + H]+.
9-Allyloxy-2-chloro-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one
A solution of 1-[2-(3-allyloxyphenyl)ethyl]hexahydropyrimidine-2,4,6-trione (50 g, 173.4 mmol) in POCl3 (100 mL) was stirred at 50 °C for 3 days. POCl3 was concentrated under pressure, and the residue was dissolved in dichloromethane (DCM) and quenched with a saturated aqueous solution of NaHCO3. The organic layer was washed with water, dried over MgSO4, filtered, and concentrated under reduced pressure to give crude 9-allyloxy-2-chloro-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one as a yellow solid (46 g, 92% yield). 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 8.8 Hz, 1H), 6.94 (dd, J = 8.8, 2.6 Hz, 1H), 6.85–6.80 (m, 1H), 6.67 (s, 1H), 6.04 (ddt, J = 17.2, 10.5, 5.2 Hz, 1H), 5.43 (dq, J = 17.3, 1.6 Hz, 1H), 5.34 (dq, J = 10.5, 1.4 Hz, 1H), 4.62 (dt, J = 5.3, 1.5 Hz, 2H), 4.26–4.19 (m, 2H), 3.01 (t, J = 6.7 Hz, 2H). LCMS: m/z 289 [M + H]+.
9-Allyloxy-2-[[(2S)-1,4-dioxan-2-yl]methoxy]-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one
To a solution of (R)-2-hydroxymethyl[1,4]dioxane (1.82 g, 15.4 mmol) in anhydrous DCM (100 mL) under N2 at 0 °C, NaH (618 mg, 15.4 mmol, 60% in mineral oil) was added and stirred for 15 min. 9-Allyloxy-2-chloro-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (3.43 g, 11.9 mmol) was added, and the reaction mixture was stirred and warmed up to RT overnight. Saturated NH4Cl was added, and the organic layer was washed with water, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel eluting with 5% MeOH in DCM to afford 9-allyloxy-2-[[(2S)-1,4-dioxan-2-yl]methoxy]-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one as a yellow solid (2.90 g, 66% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.7 Hz, 1H), 7.02–6.93 (m, 2H), 6.54 (s, 1H), 6.06 (ddt, J = 17.3, 10.5, 5.2 Hz, 1H), 5.42 (dq, J = 17.3, 1.7 Hz, 1H), 5.29 (dq, J = 10.6, 1.5 Hz, 1H), 4.67 (dt, J = 5.3, 1.5 Hz, 2H), 4.30–4.20 (m, 2H), 4.06–3.98 (m, 2H), 3.85 (dddd, J = 10.0, 5.9, 4.4, 2.6 Hz, 1H), 3.78 (td, J = 10.7, 10.1, 2.8 Hz, 2H), 3.70–3.56 (m, 2H), 3.49 (td, J = 10.8, 2.7 Hz, 1H), 3.38 (dd, J = 11.4, 9.9 Hz, 1H), 2.97 (t, J = 6.5 Hz, 2H). LCMS: m/z 371 [M + H]+.
2-[[(2S)-1,4-Dioxan-2-yl]methoxy]-9-hydroxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one
To a suspension of 9-allyloxy-2-[[(2S)-1,4-dioxan-2-yl]methoxy]-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (31.2 g, 84.2 mmol) in a mixture of DCM/MeOH (300 mL/300 mL), K2CO3 (23.27 g, 168.4 mmol) and Pd(PPh3)4 (4.86 g, 4.21 mmol) were added, and the reaction mixture was degassed before stirring at RT overnight. After completion, the reaction was quenched with H2O, the aqueous layer was separated, and the pH was adjusted to pH 1 with 2 M aq HCl, and the precipitate formed was filtered off, washed with water, and dried to afford 2-[[(2S)-1,4-dioxan-2-yl]methoxy]-9-hydroxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one as a yellow solid (18.62 g, 67% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.83 (d, J = 8.6 Hz, 1H), 6.77 (dd, J = 8.6, 2.5 Hz, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.44 (s, 1H), 4.28–4.18 (m, 2H), 4.03–3.94 (m, 2H), 3.89–3.72 (m, 3H), 3.69–3.56 (m, 2H), 3.49 (td, J = 10.8, 2.7 Hz, 1H), 3.37 (dd, J = 11.4, 9.9 Hz, 1H), 2.91 (t, J = 6.5 Hz, 2H). LCMS: m/z 331 [M + H]+.
[2-[[(2S)-1,4-Dioxan-2-yl]methoxy]-4-oxo-6,7-dihydropyrimido[6,1-a]isoquinolin-9-yl] Trifluoromethanesulfonate
A solution of 2-[[(2S)-1,4-dioxan-2-yl]methoxy]-9-hydroxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (23.25 g, 70.5 mmol), N-phenyl-bis(trifluoromethanesulfonimide) (27.69 g, 77.5 mmol), and Et3N (17.5 mL, 126.8 mmol) in DCM (800 mL) under N2 was stirred at RT for 5 h. The reaction mixture was concentrated, and the crude product was purified by recrystallization from iPrOH to afford [2-[[(2S)-1,4-dioxan-2-yl]methoxy]-4-oxo-6,7-dihydropyrimido[6,1-a]isoquinolin-9-yl] trifluoromethanesulfonate as a white solid (24.5 g, 75% yield). 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.7 Hz, 1H), 7.31 (dd, J = 8.7, 2.6 Hz, 1H), 7.27–7.23 (m, 1H), 6.37 (s, 1H), 4.50–4.35 (m, 2H), 4.24 (dd, J = 7.1, 5.8 Hz, 2H), 4.02–3.93 (m, 1H), 3.90–3.69 (m, 4H), 3.65 (ddd, J = 11.7, 10.6, 3.0 Hz, 1H), 3.48 (dd, J = 11.5, 10.1 Hz, 1H), 3.07 (t, J = 6.5 Hz, 2H). LCMS: m/z 463 [M + H]+.
9-(2-Cyclopropylethynyl)-2-[[(2S)-1,4-dioxan-2-yl]methoxy]-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (36)
To a solution of [2-[[(2S)-1,4-dioxan-2-yl]methoxy]-4-oxo-6,7-dihydropyrimido[6,1-a]isoquinolin-9-yl] trifluoromethanesulfonate (42 g, 90.8 mmol) in anhydrous DMF (840 mL), cyclopropylacetylene (15.38 mL, 181.6 mmol) was added followed by Et3N (31.5 mL, 227.0 mmol), and the mixture was degassed. Pd(PPh3)2Cl2 (3.19g, 4.5 mmol) was added with CuI (3.46 g, 18.2 mmol), and the reaction mixture was heated at 50 °C overnight. The reaction mixture was concentrated and the crude was purified by flash chromatography on silica gel eluting with 1–3% MeOH in DCM to afford 9-(2-cyclopropylethynyl)-2-[[(2S)-1,4-dioxan-2-yl]methoxy]-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one as a light brown solid (30.2 g, 88% yield). 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 8.2 Hz, 1H), 7.32 (dd, J = 8.2, 1.6 Hz, 1H), 6.31 (s, 1H), 4.45–4.32 (m, 2H), 4.16 (dd, J = 7.0, 5.9 Hz, 2H), 4.00–3.90 (m, 1H), 3.87–3.57 (m, 5H), 3.45 (dd, J = 11.5, 10.1 Hz, 1H), 2.93 (t, J = 6.5 Hz, 2H), 1.45 (tt, J = 8.2, 5.0 Hz, 1H), 0.94–0.85 (m, 2H), 0.85–0.75 (m, 2H). LCMS: 0.70 min, m/z 379.5 [M + H]+. Chromatographic conditions: Waters Alliance HPLC, column Daicel Chiralpak (Chiral Technologies, West Chester, PA, USA) IG 4.6 mm i.d. × 250 mm, 5 μm particle size, wavelength 330 nm, flow rate 0.8 mL/min, column temperature 25 °C, injection volume 10 μL, run time 65 min, isocratic elution (85/15) tert-butyl methyl ether/ethanol, retention time 32.87 min.
Biology. HTS
The cAMP assay measures the level of Gi protein activation following agonist occupancy of GPR84, in the presence of forskolin to stimulate production of cAMP, and adenylate cyclase and 3-isobutyl-1-methylxanthine (IBMX) to block phosphodiesterase activity. HEK293T samples stably transfected with recombinant human GPR84 (Multiscreen; Multispan, Hayward, CA, USA) were seeded at a density of 2500 cells/well in a 384-well plate in suspension on the day of the assay in assay buffer (1× Hanks’ balanced salt solution [HBSS]), 20 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.5 mM IBMX). GPR84 was stimulated with capric acid as the natural ligand at EC85 (30.5 μM), and tested compounds were added simultaneously for 30 min before the addition of 1.5 μM forskolin for 30 min. Cells were lysed and analyzed for cAMP level using a competitive immunoassay in homogeneous time-resolved fluorescence (HTRF) format (Cisbio, Bedford, MA, USA). Fluorescence signals were read on a PerkinElmer ViewLux1430 microplate imager (PerkinElmer, Boston, MA, USA).
Recombinant Human GPR84 GTPγS Binding Assay
The assay was performed in a 96-well plate where the following reagents were added. First, 50 μL of hit compound from HTS was added into the assay plate, followed by addition of 20 μL of DIM at EC80 concentration (concentration which gives 80% of the activity of GPR84) and was incubated with 30 μL of mixture consisting of membranes derived from stable cell line overexpressing recombinant human GPR84, [35S]GTPγS, and scintillation proximity assay (SPA) beads (PerkinElmer, Boston, MA, USA; RPNQ0001). All components were diluted in assay buffer containing 20 mM HEPES, pH 7.4; 5 mM MgCl2; 250 mM NaCl; 0.05% bovine serum albumin (BSA); 75 μg/mL saponin. Reactions were incubated at room temperature for 90 min followed by centrifugation. Plates were read on a Topcount reader (Molecular Devices, Sunnyvale, CA, USA) immediately after centrifugation.
IP1 Assay
The IP1 assay using transiently transfected cells allowed the development of similar assays with overexpression of either mouse or human GPR84. IP1 activity was measured in HEK293T cells transiently transfected with a variant of Gq protein (GNAO1 transcription variant 1) and GPR84 using the Cisbio IP-One assay, which detected the accumulation of IP1, a stable downstream metabolite of IP3 induced by activation of a phospholipase C (PLC) cascade.
HEK293T cells were transiently transfected in suspension with a variant of Gq protein and recombinant mouse or human GPR84 using Jet-PEI (Polyplus-transfection, Sebastien Brant, France) and seeded at a density of 25 000 cells/well in a 384-well plate. After overnight incubation, the transfection mix was removed and replaced by cell culture medium for 24 h. The cells were then treated with test compounds for 30 min before addition of capric acid at EC80 for 1 h for the human assay. As transfection of mouse GPR84 resulted in IP1 activity in the absence of capric acid stimulation, compounds were tested in inverse agonist mode without addition of capric acid. IP1 was measured using competitive immunoassay in HTRF format (Cisbio, Bedford, CA, USA) according to manufacturer’s instructions. Fluorescence signal was read on PerkinElmer EnVision (PerkinElmer, Boston, MA, USA).
PDE4A Inhibition
The IC50 value for human PDE4A was determined in a HTRF based cAMP assay. PDE4A present in a HEK293 cell lysate containing overexpressed PDE4A hydrolyzes cAMP into 5′AMP. The amount of cAMP present is measured in a competitive immunoassay, which is based on competition between native cAMP and d2-labeled cAMP for binding to an anti-cAMP monoclonal antibody labeled with cryptate. The specific signal (i.e., energy transfer) is inversely proportional to the concentration of cAMP.
Human Neutrophil Migration
Freshly isolated human neutrophils were diluted in a suspension of 8.9 × 106 cells per mL. 20 μL of compound solution in chemotaxis buffer was added to the 180 μL cell suspension. The mixture was incubated at 37 °C for 30 min with intermediate resuspension of the cells after 15 min. Following this, 70 μL of cell suspension was transferred to the upper compartment of a Corning HTS Transwell 96 permeable support system with 5.0 μm pore size polycarbonate membrane (Corning, Corning, NY, USA; catalog no. 3387). The receiver well of the Transwell system was then filled with 200 μL of chemotaxis buffer containing compound and chemotactic agent (embelin). After incubation at 37 °C in 5% CO2 for 1 h, the upper plate of the Transwell system was removed and the cell suspension in the receiver plate is transferred to a 96-well V-bottom plate. 50 μL of DPBS was added to the receiver plate to prevent remaining cells from drying out. The V-bottom plate was centrifuged for 6 min at 1500 rpm. The supernatant was removed, and the cells were resuspended in 50 μL DPBS. The cells were then transferred back to the receiver plate of the Transwell system. After this, 100 μL of ATPlite solution (PerkinElmer, Boston, MA, USA; catalog no. 436110) was added to the cells. The plate was incubated for 10 min in the dark while shaking. 170 μL of cell lysate was then transferred to a white 96-well plate, and luminescence was measured. The detected luminescent signal was considered as linearly related to the number of cells having migrated from the upper well to the receiver well.
Rat Neutrophil Migration
Freshly isolated rat neutrophils were diluted in a suspension of 8.9 × 106 cells per mL. 10 μL of compound solution in chemotaxis buffer was added to the 90 μL cell suspension. The mixture was incubated at 37 °C for 30 min with intermediate resuspension of the cells after 15 min. Following this, 75 μL of cell suspension was transferred to the upper compartment of a Corning HTS Transwell 96 permeable support system with 5.0 μm pore size polycarbonate membrane (Corning, Corning, NY, USA; catalog no. 3387). The receiver well of the Transwell system was then filled with 200 μL of chemotaxis buffer containing compound and chemotactic agent (embelin). After incubation at 37 °C in 5% CO2 for 1 h, the upper plate of the Transwell system was removed and 70 μL of CellTiter-Glo substrate (Promega, Madison, WI, USA; catalog no. G755B) was added to a 96-well V-bottom plate. The plate was incubated for 10 min in the dark while shaking. 180 μL of cell lysate was then transferred to a white 96-well plate, and luminescence was measured. The detected luminescent signal was considered as linearely related to the number of cells having migrated from the upper well to the receiver well.
Liver Microsomal Stability (LMS) Assay
The microsomal stability assay was performed by incubation of test compound at 1 μM, 0.2% DMSO in phosphate buffer with microsomes (0.5 mg/mL) from mouse, rat, dog, or human (Xeno-Tech, Kansas City, KS, USA), and cofactors with final concentrations of 0.6 U/mL glucose-6-phosphate dehydrogenase, 3.3 mM MgCl2, 3.3 mM glucose-6-phosphate, and 1.3 mM nicotinamide adenine dinucleotide phosphate (NADP)+. Before addition of the microsomes (time zero) and after 30 min of incubation at 37 °C with shaking, the reaction was stopped and proteins were precipitated with an excess of acetonitrile containing an internal standard. The samples were mixed, centrifuged, and filtered, and the supernatant was analyzed by liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS). The instrument responses (peak areas/IS peak area) were referenced to the zero-time point samples (considered as 100%) in order to determine the percentage of compound remaining. Verapamil (1 μM) and warfarin (1 μM) were used as reference compounds, as unstable and stable compounds, respectively.
In vitro scaled intrinsic clearance (CLint scaled) was calculated from the half-life using the following equations:
| (1) | Intrinsic clearance equation: | ||||
| (2) | Scaled intrinsic clearance equation: | ||||
Hepatocyte Stability Assay
The hepatocyte stability assay was performed by incubation of test compound at 1 μM, 0.03% DMSO in modified Krebs–Henseleit buffer with suspension of pooled cryopreserved hepatocytes (BioIVT, Hicksville, NY, USA) from mouse, rat, dog, or human (BioIVT, Hicksville, NY, USA) at 0.5 million viable hepatocytes/mL.
Before adding the hepatocytes (time zero) and after 10, 20, 45, 90, 120, and 180 min of incubation (n = 2) at 37 °C while gently shaking, the reaction was stopped and proteins were precipitated with an excess of acetonitrile containing an internal standard. The samples were mixed, centrifuged, and filtered, and the supernatant was analyzed by liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS). The instrument responses (peak areas/IS peak area) were referenced to the zero-time point samples (considered as 100%) in order to determine the percentage of compound remaining. Testosterone (1 μM) and 7-hydroxycoumarin (1 μM) were used as phase I and phase II reaction controls, while caffeine was used as a negative control.
In vitro intrinsic clearance (CLint) was calculated from half-life using the following equations:
Plasma Protein Binding Assay
Plasma protein binding was determined by equilibrium dialysis using the Pierce Red device plate with inserts (Thermo Fisher Scientific, Waltham, MA, USA). Test compound at 5 μM (0.5% DMSO) spiked in freshly thawed human, rat, mouse, or dog plasma (Bioreclamation INC, Westbury, NY, USA) was dialyzed against phosphate-buffered saline (PBS, pH 7.4) at 37 °C under shaking for 4 h. Proteins were precipitated with an excess of methanol, and samples were mixed and centrifuged, and the supernatant was analyzed by liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS). Addition of compound peak areas in the buffer chamber and the plasma chamber was considered to be 100% compound. The percentage bound to plasma proteins was derived from these results with the formula
Acebutolol and nicardipine are included in the assay design as low and high binding controls.
Intestinal Permeability on Caco-2 Cells
Caco-2 cells were obtained from European Collection of Cell Cultures (ECACC) and used after a 21-day cell culture in 24-well Transwell plates.
GLPG1205 and the references (vinblastine and propranolol) were prepared in protein-free Hanks’ balanced salt solution containing 25 mM HEPES (pH 7.4) at a concentration of 10 μM and added to either the apical or basolateral chambers of a Transwell plate assembly.
Before the experiment, the integrity of the monolayer was checked by measuring the transepithelial resistance. Lucifer yellow (LY) was added to the donor buffer in all wells to assess integrity of the cell layers by monitoring LY permeation. As LY cannot freely permeate lipophilic barriers, a high degree of LY transport indicates poor integrity of the cell layer.
After a 1 h incubation at 37 °C, aliquots were taken from both apical (A) and basolateral (B) chambers and added to acetonitrile containing analytical internal standard (carbamazepine) in a 96-well plate. Concentrations of compound in the samples were measured by LC–MS/MS.
Apparent permeability (Papp) values were calculated from the relationship
where Vdonor is the volume of the apical chamber at 125 μL, Vacceptor is the volume of the basolateral chamber at 600 μL, Tinc is the incubation time of 3600 s, and the surface area for the 24-well Transwell is 0.33 cm2. The efflux ratios, as an indication of active efflux from the apical cell surface, were calculated using the ratio of Papp(B → A)/Papp(A → B).
CYP Direct Inhibition Assay
A 5 mM stock solution of compound was prepared in methanol. This stock was serially diluted 1:3 in methanol and then added in duplicates to a mixture containing 50 mM potassium phosphate buffer, pH 7.4, and human liver microsomes (BD Gentest, BD, Wokingham, U.K.). A total of seven different concentrations (0.14–100 μM in final reaction mixture; 2% methanol) of compound were prepared in duplicates.
After prewarming for 5 min at 37 °C, the 20 min preincubation was started by adding a probe substrate to the first concentration range (first replica) and a cofactor mix (7. 65 mg/mL glucose-6-phosphate, 1.7 mg/mL NADP, 6 U/mL of glucose-6-phosphate dehydrogenase) to the second replica. Final concentrations of cofactor mix components were as follows: 1.56 mg/mL glucose-6-phosphate, 0.34 mg/mL NADP, 1.2 U/mL of glucose-6-phosphate dehydrogenase.
After preincubation, the reaction was finally started by adding the cofactor mix to the first replica and adding the probe substrate to second replica (opposite from preincubation).
After incubation at 37 °C, the reaction (aliquot of 50 μL) was terminated with 150 μL of acetonitrile/methanol (2:1) solution with internal standard (diclofenac). Samples were centrifuged and the supernatant fractions analyzed by LC–MS/MS.
The instrument responses (ratio of compound and internal standard peak areas) were referenced to those for solvent controls (assumed as 100%) in order to determine the percentage reduction in probe metabolism.
Percent of control activity versus concentration plots were generated and fitted using GraphPad Prism software to determine IC50 for both substrate- and cofactor preincubations, as well as the IC50 fold shift.
hERG Manual Patch Clamp Assay
Whole-cell patch-clamp recordings were performed using an EPC10 amplifier controlled by Pulse version 8.77 software (HEKA, Ludwigshafen, Germany). The external bathing solution contained 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 5 mM glucose, 10 mM HEPES, pH 7.4. The internal patch pipet solution contained 100 mM K gluconate, 20 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM Na2ATP, 2 mM glutathione, 11 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 mM HEPES, pH 7.2. Compound was perfused using an automate scientific pressurized superfusion system coupled to a Biologic micromanifold.
All recordings were performed on HEK293 cells stably expressing hERG channels. Cells were cultured on 12 mm round coverslips (German glass; Bellco, Vineland, NJ, USA) anchored in the recording chamber using two platinum rods (Goodfellow, Coraopolis, PA, USA). hERG currents were evoked using an activating pulse to +40 mV for 1000 ms followed by a tail current pulse to −50 mV for 2000 ms, and holding potential was −80 mV. Pulses were applied every 15 s, and all experiments were performed at room temperature. For the concentration response, peak tail current amplitude was measured during the voltage step to −50 mV.
PK Mouse
This study was performed with 42 naïve male CD1 mice, weighing 18–20 g upon arrival (4–5 weeks old). The first group of mice were dosed iv via a bolus in the tail vein with a dose level of 1 mg/kg, and the second group of mice was dosed orally as a single esophageal gavage with a dose level of 5 mg/kg.
Formulations were prepared the day before administration and kept at room temperature, protected from light. For the iv route, compound was formulated in PEG 200 and saline (60/40; v/v) as a clear solution. For the oral route, compound was formulated in PEG 200/purified water (60/40; v/v) as a homogeneous suspension. Before the oral dosing, the animals were deprived of food for at least 12 h before compound administration and 4 h after administration. All animals had free access to tap water.
From all animals, approximately 500 μL blood samples were collected by intracardiac puncture and placed into tubes containing Li-heparin as anticoagulant. Blood samples were collected at the following time points: iv, 0.05, 0.25, 0.5, 1, 3, 5, and 8 h after dosing (n = 3 mice per sampling time); po, 0.25, 0.5, 1, 3, 5, 8, and 24 h after dosing (n = 3 mice per sampling time). Each mouse was sampled once and then euthanized.
Blood was kept on ice. Within 1 h after sampling, blood was centrifuged at 5000 rpm for 10 min at 4 °C. Immediately after centrifugation, the resulting plasma samples were collected into polypropylene tubes and were kept frozen at −20 °C pending bioanalysis.
Plasma samples were assayed by LC–MS/MS with a non-good-laboratory-practice (non-GLP)-validated method. Compound 36 plasma concentrations were calculated against a calibration curve consisting of eight levels with a 3 log amplitude. Back-calculated values for quality control (three levels prepared in duplicate) were used for accepting or rejecting the entire batch. The lower limit of quantification was 4 ng/mL with a plasma volume of 25 μL.
Plasma proteins were precipitated with an excess of methanol containing the internal standard, and the corresponding supernatant was injected onto a C18 column. Analytes were eluted out of the HPLC system by increasing the percentage of the organic mobile phase. An API5500 QTrap mass spectrometer (AB Sciex, Sciex, Warrington, U.K.) was used for the simultaneous detection and quantification of 36.
PK parameters were calculated from the mean of individual plasma concentrations by noncompartmental analysis using WinNonlin software (Pharsight, version 5.2; Certara, Princeton, NJ, USA).
PK Rat
This study was performed with six naïve male Sprague Dawley rats, weighing 180–200 g upon arrival (6–8 weeks old). Catheterized rats (in the jugular vein) were used to avoid interindividual variability. Three rats per group was a sufficient number to carry out statistical calculations.
For the iv route, compound was formulated in PEG 200 and NaCl for injection (60/40; v/v) as a clear solution. For the oral route, compound was formulated in PEG 200 and purified water (60/40; v/v) as a clear solution.
Rats (n = 3) were dosed iv via a bolus in the tail vein. Three additional rats were dosed orally as a single gavage. The iv bolus and the oral administration were given with a dose volume of 5 mL/kg. Actual dose volumes were based on the individual body weights.
From all animals, approximately 200 μL blood samples were collected via a catheter into the jugular vein and placed into tubes containing Li-heparin as anticoagulant. Blood samples were collected at the following time points: iv, 0.05, 0.25, 0.5, 1, 3, 5, 8, and 24 h after dosing; po, 0.25, 0.5, 1, 3, 5, 8, and 24 h after dosing. Each rat was sampled seven or eight times (at each sampling time), depending on the route of administration. Just after each sampling, the same volume of physiological serum with sodium heparin (50 UI/mL) was injected into the catheter to avoid blocking.
Blood was kept on ice. Within 1 h after sampling, blood was centrifuged at 5000 rpm for 10 min at 4 °C. Immediately after centrifugation, the resulting plasma samples were collected into polypropylene tubes and were kept frozen at −20 °C pending bioanalysis.
Plasma samples were assayed by LC–MS/MS with a non-GLP-validated method. Plasma concentrations were calculated against a calibration curve consisting of eight levels with a 3 log amplitude. Back-calculated values for quality control (three levels prepared in duplicate) were used for accepting or rejecting the whole batch.
Plasma proteins were precipitated with an excess of methanol containing the internal standard, and the corresponding supernatant was injected on a C18 column. Analytes were eluted out the HPLC system by increasing the percentage of the organic mobile phase. An API5500 QTrap mass spectrometer (ABSciex, Sciex, Warrington, U.K.) was used.
PK parameters were calculated for each individual rat by noncompartmental analysis using WinNonlin software (Pharsight, version 5.2; Certara, Princeton, NJ, USA).
PK Dog
This study was performed with three non-naïve male Beagle dogs. Formulations were prepared the day of administration.
Dogs (n = 3) were dosed iv via a 10 min infusion via a catheter with a dose level of 0.5 mg/kg (12 mL kg–1 h–1). After a washout of 3 days, they were dosed orally as a single gavage with a dose level of 2.5 mg/kg in PEG200/water. The oral dose of 2.5 mg/kg was given with a dose volume of 5 mL/kg. Actual dose volumes were based on the individual body weights. Approximately 250 g of Harlan Teklad 2027 diet was distributed daily to each animal. The food was given 4 h after the iv administration. Before administration by the po route, animals were fasted for a period of at least 12 h before treatment, and food was given just after the 4 h blood sampling. All animals had free access to tap water.
From all animals approximately 0.5 mL blood samples were taken without anesthetic from a jugular or cephalic vein into tubes containing lithium heparin as anticoagulant. Blood samples were collected at the following time points: iv, at predose, 0.083, 0.167 (end of infusion), 0.5, 1, 2, 4, 6, 8, 10, and 24 h after start of infusion; po, at predose and then 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, and 24 h after dose administration.
Plasma samples were assayed by LC–MS/MS with a non-GLP-validated method. Compound 36 plasma concentrations were calculated against a calibration curve consisting of eight levels with a 3 log amplitude. Back-calculated values of the quality control samples (three levels prepared in duplicate) were used for accepting or rejecting the whole batch. The lower limit of quantification was 4 ng/mL with a plasma volume of 25 μL.
Plasma proteins were precipitated with an excess of methanol containing the internal standard, and the corresponding supernatant was injected on a C18 column. Analytes were eluted out the high-performance liquid chromatography (HPLC) system by increasing the percentage of the organic mobile phase. An API5500 QTrap mass spectrometer (ABSciex, Sciex, Warrington, U.K.) was used for the detection and quantification of 36.
PK parameters were calculated for each individual dog by noncompartmental analysis using WinNonlin software (Pharsight, version 5.2; Certara, Princeton, NJ, USA).
Negative Allosteric Modulation of Known GPR84 Agonists by GLPG1205. Membrane Preparation
Membranes were generated from Flp-In T-REx 293 cells treated with 100 ng/mL doxycycline to induce expression of FLAG-GPR84-Gαi2. (32) Cells were washed with ice-cold phosphate buffered saline, removed from dishes by scraping, and centrifuged at 3000 rpm for 5 min at 4 °C. Pellets were resuspended in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA; pH 7.5) containing a protease inhibitor mixture (Roche Applied Science, West Sussex, U.K.) and homogenized with a 5 mL hand-held homogenizer. This material was centrifuged at 1500 rpm for 5 min at 4 °C, and the supernatant was further centrifuged at 50 000 rpm for 30 min at 4 °C. The resulting pellet was resuspended in TE buffer, and protein content was assessed using a BCA protein assay kit (Pierce, Fisher Scientific, Loughborough, U.K.).
Competition Binding Assay
Assays were performed using 0.2 nM [3H]38, binding buffer (phosphate-buffered saline with 0.5% fatty acid free bovine serum albumin; pH 7.4) and the indicated concentrations of test compounds, in a total assay volume of 500 μL. Binding was initiated by the addition of membranes (5 μg of protein per tube), and incubations were carried out at 25 °C for 1 h. Reactions were terminated by the addition of ice-cold phosphate buffered saline and vacuum filtration through GF/C glass filters using a 24-well Brandel cell harvester (Alpha Biotech, Glasgow, U.K.). Each reaction tube was washed three times with ice-cold PBS. The filters were allowed to dry for 2–3 h and then placed in 3 mL of Ultima Gold XR. Radioactivity was quantified by liquid scintillation spectrometry.
Reaction Biology Kinase Selectivity Profiling
Inhibition of 152 kinases by 10 μM compound was determined in a Reaction Biology proprietary nanoliter radioactive HotSpot assay in duplicate. The principle of the assay consists of the measurement of incorporated 33P into a specific substrate for each kinase when the substrate is phosphorylated by the enzyme using [33P]-γ-ATP and ATP.
Millipore GPCR Profiler Selectivity Profiling
Free intracellular [Ca2+] flux assays were conducted to test compound agonist and antagonist activities against 123 GPCRs selected to cover as many GPCR families as possible. Percentage activation was determined upon initial addition of compound followed by 10 min incubation at 25 °C (in absence of agonist). Following this initial incubation, reference agonists were added at EC80 concentration allowing determination of percentage inhibition of the agonist induced signal caused by compound.
Dextran Sulfate Sodium (DSS)-Induced Colitis Model
Balb/cJ female mice (Janvier Labs, Le Genest-Saint-Isle, France) were housed in a dedicated in house animal facility under specific pathogen-free conditions according to the Federation for Laboratory Animal Science Associations guidelines. The study was performed according to ethical guidelines approved by the Animal Institutional Care and Use Committee of Galapagos controlled by the French Authorities (Agreement No. C93-063-06, DDPP, Seine Saint Denis). Animals were housed in filter top cages, provided with filtered tap water and standard chow ad libitum, and maintained at 22 ± 2 °C in 55 ± 10% humidity on a 12 h light/dark cycle. The randomization of mice in the different groups was based on body weight (BW) at study commencement. A model of chronic colitis was induced in 8-week-old mice (BW 25 g) with three cycles, each consisting of 4% DSS administered in drinking water for 4 days, followed by 3 days of regular drinking water. The mice (n = 10 per group) were orally administered vehicle (PEG200/H20 [60/40; v/v]) or 1, 3, or 10 mg/kg of compounds 26, 35, 36 qd from day 1 of DSS administration and throughout the dosing period. Compound 36 was tested in three independent experiments. Two positive controls were used in these different studies: cyclosporine (CsA) at 25 mg/kg qd po in PEG200/water (60/40; v/v) or sulfazalazine at 20 mg/kg qd po in 0.5% methylcellulose. Clinical parameters were measured every other day. DAI (a combined score of body weight loss, stool consistency, and rectal bleeding scores) was recorded daily. Each criterion was graded from 0 to 4. Animals were decapitated after brief isoflurane anesthesia. At necropsy at day 17 of the experiment, the complete colon was removed and rinsed with sterile PBS. A segment of 5 mm of distal colon was collected for histology analysis. Histology was performed on formalin-fixed (4% buffered formaldehyde for 3 h). After fixation, samples were transferred into a sucrose solution prior to being frozen in isopentane. Three series of cryosections within a distance of 1 mm were performed in duplicate. One series of sections were stained with a Goldner trichrome for colon lesion evaluation, and scoring was performed. Neutrophil cell infiltration was assayed by immunohistochemistry using anti-Ly6G/Ly6C (clone NIMP-R14, Santa Cruz, Dallas, TX, USA). Neutrophil identification was followed by manual quantification of cell number per surface. The neutrophil density was expressed as a number of cells per mm2 of colon area. Blood was sampled at day 4 at four time points: 0, 1, 3, and 6 h (and assuming 24 h is equal to the predose sample). Plasma was prepared and 36 was quantified using LC–MS to assess steady state PK. Statistical analysis was performed with Prism 5.03 software (GraphPad, San Diego, CA, USA) with a one-way ANOVA performed on the arcsinh(log)-transformed neutrophil density data and a two-way ANOVA performed on the arcsinh(log)-transformed DAI data. Dunnett’s post-hoc multiplicity correction procedure was applied to the neutrophil density data, and Bonferonni post-hoc multiplicity correction procedure was applied to the DAI data.
Supporting Information
ARTICLE SECTIONS
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00272.
General methods and synthetic routes used in the investigation of compounds; synthetic procedures and analytical data for all compounds (PDF)
Molecular formula strings and some data (CSV)
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Author Information
ARTICLE SECTIONS
- Graeme Milligan - Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom;
http://orcid.org/0000-0002-6946-3519
This study and the medical writing support were funded by Galapagos.
Acknowledgments
ARTICLE SECTIONS
Medical writing assistance in the preparation of this manuscript was provided by Emily Fisher, Alexander Bowen and Samuel McCracken of CircleScience (an Ashfield Company, part of UDG Healthcare plc) and funded by Galapagos. The authors thank Sebastien Richard for his support in the development of the analytical chemistry section.
| ADME | absorption, distribution, metabolism, excretion |
| ASOL | aqueous solubility |
| AUC0–inf | area under the curve (from time 0 to infinity) |
| BEH | ethylene bridged hybrid |
| BSA | bovine serum albumin |
| BW | body weight |
| cAMP | cyclic adenosine 3′,5′-monophosphate |
| Cap | capric acid |
| CAS | Chemical Abstracts Service |
| CL | clearance |
| CLint | intrinsic clearance |
| CLunb | unbound clearance |
| Cmax | maximum plasma concentration |
| compd | compound |
| cpm | counts per minute |
| C0 | concentration extrapolated at time 0 after a bolus intravenous dose |
| D | dog |
| DAI | disease activity index |
| DIM | 3,3′-diindolylmethane |
| DMSO | dimethyl sulfoxide |
| DSS | dextran sodium sulfate |
| ECACC | European Collection of Cell Cultures |
| EC50 | half maximal effective concentration |
| EC80 | concentration of agonist leading to 80% maximal response |
| EGTA | ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid |
| ER | efflux ratio |
| F | oral bioavailability |
| GPCR | G-protein-coupled receptor |
| GTPγS | guanosine 5′-O-[γ-thio]triphosphate |
| h | hour |
| HBSS | Hanks’ balanced salt solution |
| hCYP | human cytochrome P450 |
| HEP | hepatocyte |
| HEPES | (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid |
| hERG | human ether-à-go-go-related gene |
| hGPR84 | human G-protein-coupled receptor 84 |
| hGTPγS | human guanosine 5′-O-[γ-thio]triphosphate |
| HLM | human liver microsome |
| HTRF | homogeneous time-resolved fluorescence |
| HTS | high-throughput screen |
| Hum neutro | human neutrophil migration assay |
| H | human |
| IBD | inflammatory bowel disease |
| IBMX | 3-isobutyl-1-methylxanthine |
| IC50 | half maximal inhibitory concentration |
| IL | interleukin |
| IP1 | inositol monophosphate |
| iv | intravenous |
| LCMS/MS | liquid chromatography–mass spectrometry/mass spectrometry |
| LMS | liver microsomal stability |
| M | mouse |
| MCFA | medium-chain free fatty acid |
| Me | methyl group |
| MED | minimal efficacious dose |
| MELK | maternal embryonic leucine zipper kinase |
| MIC | microsomal |
| min | minute |
| mRNA | messenger ribonucleic acid |
| MW | molecular weight |
| NMR | nuclear magnetic resonance |
| OBD | optical bed density |
| Papp A2B | apparent permeability from apical to basal chambers |
| PDA | photodiode array |
| PDE4A | phosphodiesterase 4 |
| PEG | polyethylene glycol |
| PK | pharmacokinetics |
| PPB | plasma protein binding |
| po | per os |
| POCl3 | phosphoryl chloride |
| PSA | polar surface area |
| qd | once daily |
| R | rat |
| rpm | revolutions per minute |
| RT | room temperature |
| SAR | structure–activity relationship |
| SPA | scintillation proximity assay |
| SQD | single quadrupole detection |
| T1/2 | half-life |
| Tmax | time at maximum plasma concentration |
| TNF-α | tumor necrosis factor α |
| UPLC | ultraperformance liquid chromatography |
| UV | ultraviolet |
| Vss | apparent volume of distribution at steady state |
References
ARTICLE SECTIONS
This article references 32 other publications.
- 1Wang, J.; Wu, X.; Simonavicius, N.; Tian, H.; Ling, L. Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84. J. Biol. Chem. 2006, 281 (45), 34457– 34464, DOI: 10.1074/jbc.M608019200[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFKgur%252FI&md5=2bbe13a202723f62e59eab0b08a25878Medium-chain Fatty Acids as Ligands for Orphan G Protein-coupled Receptor GPR84Wang, Jinghong; Wu, Xiaosu; Simonavicius, Nicole; Tian, Hui; Ling, LeiJournal of Biological Chemistry (2006), 281 (45), 34457-34464CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Free fatty acids (FFAs) play important physiol. roles in many tissues as an energy source and as signaling mols. in various cellular processes. Elevated levels of circulating FFAs are assocd. with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cAMP prodn., and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive Gi/o pathway. In addn., we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated prodn. of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metab. to immunol. regulation.
- 2Davenport, A. P.; Alexander, S. P.; Sharman, J. L.; Pawson, A. J.; Benson, H. E.; Monaghan, A. E.; Liew, W. C.; Mpamhanga, C. P.; Bonner, T. I.; Neubig, R. R.; Pin, J. P.; Spedding, M.; Harmar, A. J. International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations for new pairings with cognate ligands. Pharmacol. Rev. 2013, 65 (3), 967– 986, DOI: 10.1124/pr.112.007179[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVyrtLvN&md5=561149f6eb486cf3aea0bb255a9cbf39International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations for new pairings with cognate ligandsDavenport, Anthony P.; Alexander, Stephen P. H.; Sharman, Joanna L.; Pawson, Adam J.; Benson, Helen E.; Monaghan, Amy E.; Liew, Wen Chiy; Mpamhanga, Chidochangu P.; Bonner, Tom I.; Neubig, Richard R.; Pin, Jean Philippe; Spedding, Michael; Harmar, Anthony J.Pharmacological Reviews (2013), 65 (3), 967-986CODEN: PAREAQ; ISSN:1521-0081. (American Society for Pharmacology and Experimental Therapeutics)A review. In 2005, the International Union of Basic and Clin. Pharmacol. Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) published a catalog of all of the human gene sequences known or predicted to encode G protein-coupled receptors (GPCRs), excluding sensory receptors. This review updates the list of orphan GPCRs and describes the criteria used by NC-IUPHAR to recommend the pairing of an orphan receptor with its cognate ligand(s). The following recommendations are made for new receptor names based on 11 pairings for class A GPCRs: hydroxycarboxylic acid receptors [HCA1 (GPR81) with lactate, HCA2 (GPR109A) with 3-hydroxybutyric acid, HCA3 (GPR109B) with 3-hydroxyoctanoic acid]; lysophosphatidic acid receptors [LPA4 (GPR23), LPA5 (GPR92), LPA6 (P2Y5)]; free fatty acid receptors [FFA4 (GPR120) with omega-3 fatty acids]; chemerin receptor (CMKLR1; ChemR23) with chemerin; CXCR7 (CMKOR1) with chemokines CXCL12 (SDF-1) and CXCL11 (ITAC); succinate receptor (SUCNR1) with succinate; and oxoglutarate receptor [OXGR1 with 2-oxoglutarate]. Pairings are highlighted for an addnl. 30 receptors in class A where further input is needed from the scientific community to validate these findings. Fifty-seven human class A receptors (excluding pseudogenes) are still considered orphans; information has been provided where there is a significant phenotype in genetically modified animals. In class B, six pairings have been reported by a single publication, with 28 (excluding pseudogenes) still classified as orphans. Seven orphan receptors remain in class C, with one pairing described by a single paper. The objective is to stimulate research into confirming pairings of orphan receptors where there is currently limited information and to identify cognate ligands for the remaining GPCRs. Further information can be found on the IUPHAR Database website.
- 3Suzuki, M.; Takaishi, S.; Nagasaki, M.; Onozawa, Y.; Iino, I.; Maeda, H.; Komai, T.; Oda, T. Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor. J. Biol. Chem. 2013, 288 (15), 10684– 10691, DOI: 10.1074/jbc.M112.420042[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlvFSisr8%253D&md5=63a395e01eb2083bfb7d4a1195c88b1aMedium-chain Fatty Acid-sensing Receptor, GPR84, Is a Proinflammatory ReceptorSuzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, TomiichiroJournal of Biological Chemistry (2013), 288 (15), 10684-10691CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiol. roles have not yet been clarified. Here, the authors show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. The authors also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [35S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated prodn. of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the i.v. injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. The results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation assocd.-disease.
- 4Liu, Y.; Zhang, Q.; Chen, L. H.; Yang, H.; Lu, W.; Xie, X.; Nan, F. J. Design and synthesis of 2-alkylpyrimidine-4,6-diol and 6-alkylpyridine-2,4-diol as potent GPR84 agonists. ACS Med. Chem. Lett. 2016, 7 (6), 579– 583, DOI: 10.1021/acsmedchemlett.6b00025[ACS Full Text
], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XltFGht7c%253D&md5=52f50ffae4ab4bbfd79f9eeda0bb063bDesign and Synthesis of 2-Alkylpyrimidine-4,6-diol and 6-Alkylpyridine-2,4-diol as Potent GPR84 AgonistsLiu, Yang; Zhang, Qing; Chen, Lin-Hai; Yang, Hui; Lu, Wei; Xie, Xin; Nan, Fa-JunACS Medicinal Chemistry Letters (2016), 7 (6), 579-583CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)A series of alkylpyrimidine-4,6-diol derivs. were designed and synthesized as novel GPR84 agonists based on a high-throughput screening (HTS) hit I. 6-Nonylpyridine-2,4-diol was identified as the most potent agonist of GPR84 reported so far, with an EC50 of 0.189 nM. These novel GPR84 agonists will provide valuable tools for the study of the physiol. functions of GPR84. - 5Zhang, Q.; Yang, H.; Li, J.; Xie, X. Discovery and characterization of a novel small-molecule agonist for medium-chain free fatty acid receptor G protein-coupled receptor 84. J. Pharmacol. Exp. Ther. 2016, 357 (2), 337– 344, DOI: 10.1124/jpet.116.232033[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlWhu7fE&md5=8a2f4113fddcb35cdd39f99b9470e0c7Discovery and characterization of a novel small-molecule agonist for medium-chain free fatty acid receptor G protein-coupled receptor 84Zhang, Qing; Yang, Hui; Li, Jing; Xie, XinJournal of Pharmacology and Experimental Therapeutics (2016), 357 (2), 337-344CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clin. applications. Here, we report the screen of 160,000 small- mol. compds. with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Ga16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compd. may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization.
- 6Pillaiyar, T.; Kose, M.; Namasivayam, V.; Sylvester, K.; Borges, G.; Thimm, D.; von Kugelgen, I.; Muller, C. E. 6-(Ar)alkylamino-substituted uracil derivatives: lipid mimetics with potent activity at the orphan G protein-coupled receptor 84 (GPR84). ACS omega 2018, 3 (3), 3365– 3383, DOI: 10.1021/acsomega.7b02092[ACS Full Text], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVyqu7g%253D&md5=184964bac823f7cd34d4de98da0cef536-(Ar)Alkylamino-Substituted Uracil Derivatives: Lipid Mimetics with Potent Activity at the Orphan G Protein-Coupled Receptor 84 (GPR84)Pillaiyar, Thanigaimalai; Koese, Meryem; Namasivayam, Vigneshwaran; Sylvester, Katharina; Borges, Gleice; Thimm, Dominik; von Kuegelgen, Ivar; Mueller, Christa E.ACS Omega (2018), 3 (3), 3365-3383CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)GPR84, a Gi protein-coupled receptor that is activated by medium-chain (hydroxy)fatty acids, appears to play an important role in inflammation, immunity, and cancer. Recently, 6-octylaminouracil has been reported to act as an agonist at GPR84. Here, we describe the synthesis of 69 derivs. and analogs which represent new compds. They were evaluated in (a) cyclic adenosine monophosphate accumulation and (b) β-arrestin assays in human GPR84-expressing cells. Potent nonbiased as well as G protein-biased agonists were developed, e.g., 6-hexylamino-2,4(1H,3H)-pyrimidinedione (PSB-1584, EC50 5.0 nM (a), 3.2 nM (b), bias factor: 0) and 6-((p-chloro- and p-bromo-phenylethyl)amino)-2,4(1H,3H)-pyrimidinedione (PSB-16434, EC50 7.1 nM (a), 520 nM (b), bias factor: 1.9 = 79-fold Gi pathway-selective; I, PSB-17365, EC50 2.5 nM (a), 100 nM (b), bias factor 1.3 = 20-fold selective), which were selective vs. other free fatty acid-activated receptors. Compds. II and I were found to be metabolically stable upon incubation with human liver microsomes. A pharmacophore model was created on the basis of structurally diverse lipidlike GPR84 agonists.
- 7Yousefi, S.; Cooper, P. R.; Potter, S. L.; Mueck, B.; Jarai, G. Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes. Journal Leukocyte Biol. 2001, 69 (6), 1045– 1052, DOI: 10.1189/jlb.69.6.1045[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXkslWrurs%253D&md5=03d1eb21f619cdd4790ace3c2b571d23Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytesYousefi, Shida; Cooper, Paul R.; Potter, Suzanne L.; Mueck, Beatrice; Jarai, GaborJournal of Leukocyte Biology (2001), 69 (6), 1045-1052CODEN: JLBIE7; ISSN:0741-5400. (Federation of American Societies for Experimental Biology)The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. The authors used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was detd. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homolog. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot expts. revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR anal., the authors showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.
- 8Lattin, J. E.; Schroder, K.; Su, A. I.; Walker, J. R.; Zhang, J.; Wiltshire, T.; Saijo, K.; Glass, C. K.; Hume, D. A.; Kellie, S.; Sweet, M. J. Expression analysis of G protein-coupled receptors in mouse macrophages. Immunome Res. 2008, 4, 5, DOI: 10.1186/1745-7580-4-5[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3czktlyjtw%253D%253D&md5=d0f33a92e85b59309930de7b0bfb36ecExpression analysis of G Protein-Coupled Receptors in mouse macrophagesLattin Jane E; Schroder Kate; Su Andrew I; Walker John R; Zhang Jie; Wiltshire Tim; Saijo Kaoru; Glass Christopher K; Hume David A; Kellie Stuart; Sweet Matthew JImmunome research (2008), 4 (), 5 ISSN:.BACKGROUND: Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). RESULTS: Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. CONCLUSION: The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.
- 9Audoy-Remus, J.; Bozoyan, L.; Dumas, A.; Filali, M.; Lecours, C.; Lacroix, S.; Rivest, S.; Tremblay, M. E.; Vallieres, L. GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer’s disease. Brain, Behav., Immun. 2015, 46, 112– 120, DOI: 10.1016/j.bbi.2015.01.010[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitFCntbo%253D&md5=4b643287374786cdde611ffbb3002101GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer's diseaseAudoy-Remus, Julie; Bozoyan, Lusine; Dumas, Aline; Filali, Mohammed; Lecours, Cynthia; Lacroix, Steve; Rivest, Serge; Tremblay, Marie-Eve; Vallieres, LucBrain, Behavior, and Immunity (2015), 46 (), 112-120CODEN: BBIMEW; ISSN:0889-1591. (Elsevier)Microglia surrounds the amyloid plaques that form in the brains of patients with Alzheimer's disease (AD), but their role is controversial. Under inflammatory conditions, these cells can express GPR84, an orphan receptor whose pathophysiol. role is unknown. Here, we report that GPR84 is upregulated in microglia of APP/PS1 transgenic mice, a model of AD. Without GPR84, these mice display both accelerated cognitive decline and a reduced no. of microglia, esp. in areas surrounding plaques. The lack of GPR84 affects neither plaque formation nor hippocampal neurogenesis, but promotes dendritic degeneration. Furthermore, GPR84 does not influence the clin. progression of other diseases in which its expression has been reported, i.e., exptl. autoimmune encephalomyelitis (EAE) and endotoxic shock. We conclude that GPR84 plays a beneficial role in amyloid pathol. by acting as a sensor for a yet unknown ligand that promotes microglia recruitment, a response affecting dendritic degeneration and required to prevent further cognitive decline.
- 10Bouchard, C.; Page, J.; Bedard, A.; Tremblay, P.; Vallieres, L. G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions. Glia 2007, 55 (8), 790– 800, DOI: 10.1002/glia.20506[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2s3jtVemsw%253D%253D&md5=5bc25664243312ac66f94244ab598c9dG protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditionsBouchard Caroline; Page Julie; Bedard Andreanne; Tremblay Pierrot; Vallieres LucGlia (2007), 55 (8), 790-800 ISSN:0894-1491.G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.
- 11Venkataraman, C.; Kuo, F. The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking. Immunol. Lett. 2005, 101 (2), 144– 153, DOI: 10.1016/j.imlet.2005.05.010[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVKjs77L&md5=abef6f196008a5055ab99e9213c0d55dThe G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinkingVenkataraman, Chandrasekar; Kuo, FrederickImmunology Letters (2005), 101 (2), 144-153CODEN: IMLED6; ISSN:0165-2478. (Elsevier B.V.)The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biol. function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-γ prodn. in GPR84-/- mice compared to wild-type mice. Augmented IL-4 prodn. in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-γ steady-state mRNA in GPR84-/- T cells. In addn., Th2 effector cells generated in vitro from GPR84-/- mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 prodn. between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.
- 12Nagasaki, H.; Kondo, T.; Fuchigami, M.; Hashimoto, H.; Sugimura, Y.; Ozaki, N.; Arima, H.; Ota, A.; Oiso, Y.; Hamada, Y. Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes. FEBS Lett. 2012, 586 (4), 368– 372, DOI: 10.1016/j.febslet.2012.01.001[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xit1yqu7w%253D&md5=8fecdea5b404e472d6f66a9ed22cd88bInflammatory changes in adipose tissue enhance expression of GPR G-protein coupled receptor84, a medium-chain fatty acid receptorNagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, YojiFEBS Letters (2012), 586 (4), 368-372CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)In this study we aimed to identify the physiol. roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, coculture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity.
- 13Nicol, L. S.; Dawes, J. M.; La Russa, F.; Didangelos, A.; Clark, A. K.; Gentry, C.; Grist, J.; Davies, J. B.; Malcangio, M.; McMahon, S. B. The role of G-protein receptor 84 in experimental neuropathic pain. J. Neurosci. 2015, 35 (23), 8959– 8969, DOI: 10.1523/JNEUROSCI.3558-14.2015[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVOqsLrJ&md5=4d7a16fa2471aaaa6ed9395ad1215544The role of G-protein receptor 84 in experimental neuropathic painNicol, Louise S. C.; Dawes, John M.; La Russa, Federica; Didangelos, Athanasios; Clark, Anna K.; Gentry, Clive; Grist, John; Davies, John B.; Malcangio, Marzia; McMahon, Stephen B.Journal of Neuroscience (2015), 35 (23), 8959-8969CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiol. function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mech. or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter mol. 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array anal. revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addn., the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger assocd. with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired.
- 14Sundqvist, M.; Christenson, K.; Holdfeldt, A.; Gabl, M.; Mårtensson, J.; Björkman, L.; Dieckmann, R.; Dahlgren, C.; Forsman, H. Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R. Biochim. Biophys. Acta, Mol. Cell Res. 2018, 1865 (5), 695– 708, DOI: 10.1016/j.bbamcr.2018.02.008[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjsFaju7w%253D&md5=5f80256de9cc62715bf953312937170eSimilarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2RSundqvist, Martina; Christenson, Karin; Holdfeldt, Andre; Gabl, Michael; Maartensson, Jonas; Bjoerkman, Lena; Dieckmann, Regis; Dahlgren, Claes; Forsman, HuameiBiochimica et Biophysica Acta, Molecular Cell Research (2018), 1865 (5), 695-708CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small mols. that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), resp. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naive cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.
- 15Arijs, I.; De Hertogh, G.; Machiels, K.; Van Steen, K.; Lemaire, K.; Schraenen, A.; Van Lommel, L.; Quintens, R.; Van Assche, G.; Vermeire, S.; Schuit, F.; Rutgeerts, P. Mucosal gene expression of cell adhesion molecules, chemokines, and chemokine receptors in patients with inflammatory bowel disease before and after infliximab treatment. Am. J. Gastroenterol. 2011, 106 (4), 748– 761, DOI: 10.1038/ajg.2011.27[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkt1ehsr8%253D&md5=f4483700084ba9b4a7dddae654ecd0e2Mucosal Gene Expression of Cell Adhesion Molecules, Chemokines, and Chemokine Receptors in Patients With Inflammatory Bowel Disease Before and After Infliximab TreatmentArijs, Ingrid; De Hertogh, Gert; Machiels, Kathleen; Van Steen, Kristel; Lemaire, Katleen; Schraenen, Anica; Van Lommel, Leentje; Quintens, Roel; Van Assche, Gert; Vermeire, Severine; Schuit, Frans; Rutgeerts, PaulAmerican Journal of Gastroenterology (2011), 106 (4), 748-761CODEN: AJGAAR; ISSN:0002-9270. (Nature Publishing Group)OBJECTIVES: Inflammatory bowel disease (IBD) is characterized by a continuous influx of leukocytes into the gut wall. This migration is regulated by cell adhesion mols. (CAMs), and selective antimigration therapies have been developed. This study investigated the effect of infliximab therapy on the mucosal gene expression of CAMs in IBD. METHODS: Mucosal gene expression of 69 leukocyte/endothelial CAMs and E-cadherin was investigated in 61 IBD patients before and after first infliximab infusion and in 12 normal controls, using Affymetrix gene expression microarrays. Quant. reverse transcriptase-PCR (qRT-PCR), immunohistochem., and western blotting were used to confirm the microarray data. RESULTS: When compared with control colons, the colonic mucosal gene expression of most leukocyte/endothelial adhesion mols. was upregulated and E-cadherin gene expression was downregulated in active colonic IBD (IBDc) before therapy, with no significant colonic gene expression differences between ulcerative colitis and colonic Crohn's disease. Infliximab therapy restored the upregulations of leukocyte CAMs in IBDc responders to infliximab that paralleled the disappearance of the inflammatory cells from the colonic lamina propria. Also, the colonic gene expression of endothelial CAMs and of most chemokines/chemokine receptors returned to normal after therapy in IBDc responders, and only CCL20 and CXCL1-2 expression remained increased after therapy in IBDc responders vs. control colons. When compared with control ileums, the ileal gene expression of MADCAM1, THY1, PECAM1, CCL28, CXCL1, -2, -5, -6, and -11, and IL8 was increased and CD58 expression was decreased in active ileal Crohn's disease (CDi) before therapy, and none of the genes remained dysregulated after therapy in CDi responders vs. control ileums. This microarray study identified a no. of interesting targets for antiadhesion therapy including PECAM1, IL8, and CCL20, besides the currently studied α4β7 integrin-MADCAM1 axis. CONCLUSIONS: Our data demonstrate that many leukocyte/endothelial CAMs and chemokines/chemokine receptors are upregulated in inflamed IBD mucosa. Controlling the inflammation with infliximab restores most of these dysregulations in IBD. These results show that at least part of the mechanism of anti-tumor necrosis factor-α therapy goes through downregulation of certain adhesion mols. Am J Gastroenterol 2011; 106:748-761; doi:10.1038/ajg.2011.27; published online 15 Feb. 2011.
- 16Gagnon, L.; Leduc, M.; Thibodeau, J. F.; Zhang, M. Z.; Grouix, B.; Sarra-Bournet, F.; Gagnon, W.; Hince, K.; Tremblay, M.; Geerts, L.; Kennedy, C. R. J.; Hebert, R. L.; Gutsol, A.; Holterman, C. E.; Kamto, E.; Gervais, L.; Ouboudinar, J.; Richard, J.; Felton, A.; Laverdure, A.; Simard, J. C.; Letourneau, S.; Cloutier, M. P.; Leblond, F. A.; Abbott, S. D.; Penney, C.; Duceppe, J. S.; Zacharie, B.; Dupuis, J.; Calderone, A.; Nguyen, Q. T.; Harris, R. C.; Laurin, P. A newly discovered antifibrotic pathway regulated by two fatty acid receptors: GPR40 and GPR84. Am. J. Pathol. 2018, 188 (5), 1132– 1148, DOI: 10.1016/j.ajpath.2018.01.009[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVGqs7w%253D&md5=47fff3190f70beafa69897d9d89f206dA Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid ReceptorsGagnon, Lyne; Leduc, Martin; Thibodeau, Jean-Francois; Zhang, Ming-Zhi; Grouix, Brigitte; Sarra-Bournet, Francois; Gagnon, William; Hince, Kathy; Tremblay, Mikael; Geerts, Lilianne; Kennedy, Christopher R. J.; Hebert, Richard L.; Gutsol, Alex; Holterman, Chet E.; Kamto, Eldjonai; Gervais, Liette; Ouboudinar, Jugurtha; Richard, Jonathan; Felton, Alexandra; Laverdure, Alexandre; Simard, Jean-Christophe; Letourneau, Sylvie; Cloutier, Marie-Pier; Leblond, Francois A.; Abbott, Shaun D.; Penney, Christopher; Duceppe, Jean-Simon; Zacharie, Boulos; Dupuis, Jocelyn; Calderone, Angelino; Nguyen, Quang T.; Harris, Raymond C.; Laurin, PierreAmerican Journal of Pathology (2018), 188 (5), 1132-1148CODEN: AJPAA4; ISSN:0002-9440. (Elsevier B.V.)Numerous clin. conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiol. pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are assocd. with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiol. processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, resp., we uncovered an antifibrotic pathway involving these receptors. In expts. using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity obsd. in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising mol. targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compd. that may be effective for managing inflammatory and fibrosis-related diseases.
- 17Abdel-Aziz, H.; Schneider, M.; Neuhuber, W.; Meguid Kassem, A.; Khailah, S.; Muller, J.; Gamal Eldeen, H.; Khairy, A.; Khayyal, M. T.; Shcherbakova, A.; Efferth, T.; Ulrich-Merzenich, G. GPR84 and TREM-1 signaling contribute to the pathogenesis of reflux esophagitis. Mol. Med. 2015, 21 (1), 1011– 1024, DOI: 10.2119/molmed.2015.00098[Crossref], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKlsLbI&md5=d626c0592ea9ac67a3700ec6b2138383GPR84 and TREM-1 signaling contribute to the pathogenesis of reflux esophagitisAbdel-Aziz, Heba; Schneider, Mathias; Neuhuber, Winfried; Kassem, Abdel Meguid; Khailah, Saleem; Mueller, Juergen; Eldeen, Hadeel Gamal; Khairy, Ahmed; Khayyal, Mohamed T.; Shcherbakova, Anastasiia; Efferth, Thomas; Ulrich-Merzenich, GudrunMolecular Medicine (Manhasset, NY, United States) (2015), 21 (), 1011-1024CODEN: MOMEF3; ISSN:1528-3658. (Feinstein Institute for Medical Research)Gastro-esophageal reflux disease (GERD) is one of the most common disorders in gastroenterol. Patients present with or without increased acid exposure indicating a nonuniform etiol. Thus, the common treatment with proton pump inhibitors (PPIs) fails to control symptoms in up to 40% of patients. To further elucidate the pathophysiol. of the condition and explore new treatment targets, transcriptomics, proteomics and histol. methods were applied to a surgically induced subchronic reflux esophagitis model in Wistar rats after treatment with either omeprazole (PPI) or STW5, a herbal prepn. shown to ameliorate esophagitis without affecting refluxate pH. The normal human esophageal squamous cell line HET-1A and human endoscopic biopsies were used to confirm our findings to the G-protein-coupled receptor (GPR) 84 in human tissue. Both treatments reduced reflux-induced macroscopic and microscopic lesions of the esophagi as well as known proinflammatory cytokines. Proteomic and transcriptomic analyses identified CINC1-3, MIP-1/3α, MIG, RANTES and interleukin (IL)-1β as prominent mediators in GERD. Most regulated cyto-/chemokines are linked to the TREM-1 signaling pathway. The fatty acid receptor GPR84 was upregulated in esophagitis but significantly decreased in treated groups, a finding supported by Western blot and immunohistochem. in both rat tissue and HET-1A cells. GPR84 was also found to be significantly upregulated in patients with grade B reflux esophagitis. The expression of GPR84 in esophageal tissue and its potential involvement in GERD are reported for the first time. IL-8 (CINC1-3) and the TREM-1 signaling pathway are proposed, besides GPR84, to play an important role in the pathogenesis of GERD.
- 18Dietrich, P. A.; Yang, C.; Leung, H. H.; Lynch, J. R.; Gonzales, E.; Liu, B.; Haber, M.; Norris, M. D.; Wang, J.; Wang, J. Y. GPR84 sustains aberrant beta-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis. Blood 2014, 124 (22), 3284– 3294, DOI: 10.1182/blood-2013-10-532523[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVSgs73L&md5=3da07da89bd6da728d09aa26b273d659GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesisDietrich, Philipp A.; Yang, Chen; Leung, Halina H. L.; Lynch, Jennifer R.; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D.; Wang, Jianlong; Wang, Jenny YingziBlood (2014), 124 (22), 3284-3294CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)β-Catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacol. manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray anal. demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML.
- 19Du Toit, E.; Browne, L.; Irving-Rodgers, H.; Massa, H. M.; Fozzard, N.; Jennings, M. P.; Peak, I. R. Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich diets. Eur. J. Nutr. 2018, 57 (5), 1737– 1746, DOI: 10.1007/s00394-017-1456-5[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmsFGjsbs%253D&md5=5c76ae9693f9d1e78aed516c0f5e3567Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich dietsDu Toit, Eugene; Browne, Liam; Irving-Rodgers, Helen; Massa, Helen M.; Fozzard, Nicolette; Jennings, Michael P.; Peak, Ian R.European Journal of Nutrition (2018), 57 (5), 1737-1746CODEN: EJNUFZ; ISSN:1436-6207. (Springer)Purpose: Although there is good evidence showing that diets rich in medium chain fatty acids (MCFAs) have less marked obesogenic and diabetogenic effects than diets rich in long chain fatty acids (LCFAs), the role of the pro-inflammatory, medium chain fatty acid receptor (GPR84) in the etiol. of obesity and glucose intolerance is not well characterised. We set out to det. whether GPR84 expression influences obesity and glucose intolerance susceptibility in MCFA and LCFA rich diet fed mice. Methods: Wild type (WT) and GPR84 knockout (KO) mice were fed a control, MCFA or LCFA diet, and body mass, heart, liver and epididymal fat mass was assessed, as well as glucose tolerance and adipocyte size. Results: LCFA diets increased body mass and decreased glucose tolerance in both WT and GPR84 KO animals while MCFA diets had no effect on these parameters. There were no differences in body wt. when comparing WT and GPR84 KO mice on the resp. diets. Glucose tolerance was also similar in WT and GPR84 KO mice irresp. of diet. Liver mass was increased following LCFA feeding in WT but not GPR84 KO mice. Hepatic triglyceride content was increased in GPR84 KO animals fed MCFA, and myocardial triglyceride content was increased in GPR84 KO animals fed LCFA. Conclusions: GPR84 deletion had no effects on body wt. or glucose tolerance in mice fed either a high MCFA or LCFA diet. GPR84 may influence lipid metab., as GPR84 KO mice had smaller livers and increased myocardial triglyceride accumulation when fed LCFA diets, and increased liver triglyceride accumulation in responses to increased dietary MCFAs.
- 20Mignani, S.; Rodrigues, J.; Tomas, H.; Jalal, R.; Singh, P. P.; Majoral, J. P.; Vishwakarma, R. A. Present drug-likeness filters in medicinal chemistry during the hit and lead optimization process: how far can they be simplified?. Drug Discovery Today 2018, 23 (3), 605– 615, DOI: 10.1016/j.drudis.2018.01.010[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvgtlCksw%253D%253D&md5=a61e00552a35b455199f0a8d351d0522Present drug-likeness filters in medicinal chemistry during the hit and lead optimization process: how far can they be simplified?Mignani Serge; Rodrigues Joao; Tomas Helena; Jalal Rachid; Singh Parvinder Pal; Vishwakarma Ram A; Majoral Jean-PierreDrug discovery today (2018), 23 (3), 605-615 ISSN:.During the past decade, decreasing the attrition rate of drug development candidates reaching the market has become one of the major challenges in pharmaceutical research and drug development (R&D). To facilitate the decision-making process, and to increase the probability of rapidly finding and developing high-quality compounds, a variety of multiparametric guidelines, also known as rules and ligand efficiency (LE) metrics, have been developed. However, what are the 'best' descriptors and how far can we simplify these drug-likeness prediction tools in terms of the numerous, complex properties that they relate to?
- 21Kodimuthali, A.; Jabaris, S. S. L.; Pal, M. Recent advances on phosphodiesterase 4 inhibitors for the treatment of asthma and chronic obstructive pulmonary disease. J. Med. Chem. 2008, 51 (18), 5471– 5489, DOI: 10.1021/jm800582j[ACS Full Text], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1alsbg%253D&md5=d4502dc5ce4b8b17d5b1d4d853403768Recent Advances on Phosphodiesterase 4 Inhibitors for the Treatment of Asthma and Chronic Obstructive Pulmonary DiseaseKodimuthali, Arumugam; Jabaris, S. Sugin Lal; Pal, ManojitJournal of Medicinal Chemistry (2008), 51 (18), 5471-5489CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review.
- 22Niu, M.; Dong, F.; Tang, S.; Fida, G.; Qin, J.; Qiu, J.; Liu, K.; Gao, W.; Gu, Y. Pharmacophore modeling and virtual screening for the discovery of new type 4 cAMP phosphodiesterase (PDE4) inhibitors. PLoS One 2013, 8 (12), e82360, DOI: 10.1371/journal.pone.0082360[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlsVCju74%253D&md5=d1b6be4406df69aea27e763c04522d42Pharmacophore modeling and virtual screening for the discovery of new type 4 cAMP phosphodiesterase (PDE4) inhibitorsNiu, Miaomiao; Dong, Fenggong; Tang, Shi; Fida, Guissi; Qin, Jingyi; Qiu, Jiadan; Liu, Kangbo; Gao, Weidong; Gu, YueqingPLoS One (2013), 8 (12), e82360/1-e82360/15, 15 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Type 4 cAMP phosphodiesterase (PDE4) inhibitors show a broad spectrum of anti-inflammatory effects in almost all kinds of inflamed cells, by an increase in cAMP levels which is a pivotal second messenger responsible for various biol. processes. These inhibitors are now considered as the potential drugs for treatment of chronic inflammatory diseases. However, some recently marketed inhibitors e.g., roflumilast, have shown adverse effects such as nausea and emesis, thus restricting its use. In order to identify novel PDE4 inhibitors with improved therapeutic indexes, a highly correlating (r = 0.963930) pharmacophore model (Hypo1) was established on the basis of known PDE4 inhibitors. Validated Hypo1 was used in database screening to identify chem. with required pharmacophoric features. These compds. are further screened by using the rule of five, ADMET and mol. docking. Finally, twelve hits which showed good results with respect to following properties such as estd. activity, calcd. drug-like properties and scores were proposed as potential leads to inhibit the PDE4 activity. Therefore, this study will not only assist in the development of new potent hits for PDE4 inhibitors, but also give a better understanding of their interaction with PDE4. On a wider scope, this will be helpful for the rational design of novel potent enzyme inhibitors.
- 23Oxford, A. W.; Jack, D. Derivatives of Pyrimido[6,1-A]Isoquinolin-4-One. WO/2000/058309, Mar 29, 2000; https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2000058309.Google ScholarThere is no corresponding record for this reference.
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- 25Wirtz, S.; Neufert, C.; Weigmann, B.; Neurath, M. F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2007, 2 (3), 541– 546, DOI: 10.1038/nprot.2007.41[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFGntbnL&md5=7a3c29eaca8be756e3d866e23e151fdfChemically induced mouse models of intestinal inflammationWirtz, Stefan; Neufert, Clemens; Weigmann, Benno; Neurath, Markus F.Nature Protocols (2007), 2 (3), 541-546CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chem. induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In the DSS model, mice are subjected several days to drinking water supplemented with DSS, which seems to be directly toxic to colonic epithelial cells of the basal crypts. The procedures for the hapten models of colitis and acute DSS colitis can be accomplished in about 2 wk but the protocol for chronic DSS colitis takes about 2 mo.
- 26Mahmud, Z. A.; Jenkins, L.; Ulven, T.; Labéguère, F.; Gosmini, R.; De Vos, S.; Hudson, B. D.; Tikhonova, I. G.; Milligan, G. Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84. Sci. Rep. 2017, 7 (1), 17953, DOI: 10.1038/s41598-017-18159-3[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzjslSqug%253D%253D&md5=fe3d58f70952bef231505deb9bbb9de0Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84Mahmud Zobaer Al; Jenkins Laura; Hudson Brian D; Milligan Graeme; Ulven Trond; Labeguere Frederic; Gosmini Romain; Labeguere Frederic; De Vos Steve; Tikhonova Irina GScientific reports (2017), 7 (1), 17953 ISSN:.Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine(172), located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [(3)H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine(172). These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.
- 27Kim, H. Y.; Kuhn, R. J.; Patkar, C.; Warrier, R.; Cushman, M. Synthesis of dioxane-based antiviral agents and evaluation of their biological activities as inhibitors of Sindbis virus replication. Bioorg. Med. Chem. 2007, 15 (7), 2667– 2679, DOI: 10.1016/j.bmc.2007.01.040[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXivVagtro%253D&md5=67f56b313ec33524f54720fc702d6bd1Synthesis of dioxane-based antiviral agents and evaluation of their biological activities as inhibitors of Sindbis virus replicationKim, Ha Young; Kuhn, Richard J.; Patkar, Chinmay; Warrier, Ranjit; Cushman, MarkBioorganic & Medicinal Chemistry (2007), 15 (7), 2667-2679CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)The crystal structure of the Sindbis virus capsid protein contains one or two solvent-derived dioxane mols. in the hydrophobic binding pocket. A bis-dioxane antiviral agent was designed by linking the two dioxane mols. with a three-carbon chain having R,R connecting stereochem., and a stereospecific synthesis was performed. This resulted in an effective antiviral agent that inhibited Sindbis virus replication with an EC50 of 14 μM. The synthesis proceeded through an intermediate (R)-2-hydroxymethyl-[1,4]-dioxane, which unexpectedly proved to be a more effecting antiviral agent than the target compd., as evidenced by its EC50 of 3.4 μM as an inhibitor of Sindbis virus replication. Both compds. were not cytotoxic in uninfected BHK cells at concns. of 1 mM.
- 28Vanhoutte, F.; Dupont, S.; Van Kaem, T.; Gouy, M.-H.; Gheyle, L.; Blanque, R.; Brys, R.; Vandeghinste, N.; Haazen, W.; van’t Klooster, G.; Beetens, J. Human safety, pharmacokinetics and pharmacodynamics of the GPR84 antagonist GLPG1205, a potential new approach to treat IBD. United Eur. Gastroenterol. J. 2014, 2 (1S), A224, (Abstract P0341)Google ScholarThere is no corresponding record for this reference.
- 29Galapagos Reports Results with GLPG1205 in Ulcerative Colitis. Galapagos Press Release January 26, 2016. https://cws.huginonline.com/G/133350/PR/201601/1981193_5.html (accessed Apr 3, 2019).Google ScholarThere is no corresponding record for this reference.
- 30Puengel, T.; De Vos, S.; Hundertmark, J.; Kohlhepp, M.; Guldiken, N.; Pujuguet, P.; Auberval, M.; Marsais, F.; Shoji, K. F.; Saniere, L.; Trautwein, C.; Luedde, T.; Strnad, P.; Brys, R.; Clement-Lacroix, P.; Tacke, F. The medium-chain fatty acid receptor GPR84 mediates myeloid cell infiltration promoting steatohepatitis and fibrosis. J. Clin. Med. 2020, 9 (4), E1140, DOI: 10.3390/jcm9041140[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovF2hur0%253D&md5=e9977d9275af8aad56c1715aa362eaf0The medium-chain fatty acid receptor GPR84 mediates myeloid cell infiltration promoting steatohepatitis and fibrosisPuengel, Tobias; De Vos, Steve; Hundertmark, Jana; Kohlhepp, Marlene; Guldiken, Nurdan; Pujuguet, Philippe; Auberval, Marielle; Marsais, Florence; Shoji, Kenji F.; Saniere, Laurent; Trautwein, Christian; Luedde, Tom; Strnad, Pavel; Brys, Reginald; Clement-Lacroix, Philippe; Tacke, FrankJournal of Clinical Medicine (2020), 9 (4), 1140CODEN: JCMOHK; ISSN:2077-0383. (MDPI AG)Medium-chain fatty acids (MCFAs) have been assocd. with anti-steatotic effects in hepatocytes. Expression of the MCFA receptor GPR84 (G protein-coupled receptor 84) is induced in immune cells under inflammatory conditions and can promote fibrogenesis. We aimed at deciphering the role of GPR84 in the pathogenesis of non-alc. steatohepatitis (NASH), exploring its potential as a therapeutic target. GPR84 expression is upregulated in liver from patients with non-alc. fatty liver disease (NAFLD), correlating with the histol. degree of inflammation and fibrosis. In mouse and human, activated monocytes and neutrophils upregulate GPR84 expression. Chemotaxis of these myeloid cells by GPR84 stimulation is inhibited by two novel, small mol. GPR84 antagonists. Upon acute liver injury in mice, treatment with GPR84 antagonists significantly reduced the hepatic recruitment of neutrophils, monocytes, and monocyte-derived macrophages (MoMF). We, therefore, evaluated the therapeutic inhibition of GPR84 by these two novel antagonists in comparison to selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, in three NASH mouse models. Pharmacol. inhibition of GPR84 significantly reduced macrophage accumulation and ameliorated inflammation and fibrosis, to an extent similar to selonsertib. In conclusion, our findings support that GPR84 mediates myeloid cell infiltration in liver injury and is a promising therapeutic target in steatohepatitis and fibrosis.
- 31Saniere, L.; Marsais, F.; Jagerschmidt, C.; Meurisse, S.; Cuzic, S.; Shoji, K.; Clement-Lacroix, P.; Van Osselaer, N.; De Vos, S. Characterization of GLPG1205 in mouse fibrosis models: a potent and selective antagonist of GPR84 for treatment of idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 2019, 199, A1046, DOI: 10.1164/ajrccm-conference.2019.199.1_MeetingAbstracts.A1046
- 32Mancini, S.J.; Al Mahmud, Z.; Jenkins, L.; Bolognini, D.; Newman, R.; Barnes, M.; Edye, M. E.; McMahon, S. B.; Tobin, A.; Milligan, G. On-target and off-target effects of novel orthosteric and allosteric activators of GPR84. Sci. Rep. 2019, 9 (1), 1861, DOI: 10.1038/s41598-019-38539-1[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cfis1amsA%253D%253D&md5=1bd20c2443890cb3691d558cb8511c5fOn-target and off-target effects of novel orthosteric and allosteric activators of GPR84Mancini Sarah J; Mahmud Zobaer Al; Jenkins Laura; Bolognini Daniele; Tobin Andrew B; Milligan Graeme; Newman Robert; Barnes Matt; Edye Michelle E; McMahon Stephen BScientific reports (2019), 9 (1), 1861 ISSN:.Many members of the G protein-coupled receptor family, including examples with clear therapeutic potential, remain poorly characterised. This often reflects limited availability of suitable tool ligands with which to interrogate receptor function. In the case of GPR84, currently a target for the treatment of idiopathic pulmonary fibrosis, recent times have seen the description of novel orthosteric and allosteric agonists. Using 2-(hexylthiol)pyrimidine-4,6 diol (2-HTP) and di(5,7-difluoro-1H-indole-3-yl)methane (PSB-16671) as exemplars of each class, in cell lines transfected to express either human or mouse GPR84, both ligands acted as effective on-target activators and with high co-operativity in their interactions. This was also the case in lipopolysaccharide-activated model human and mouse immune cell lines. However in mouse bone-marrow-derived neutrophils, where expression of GPR84 is particularly high, the capacity of PSB-16671 but not of 2-HTP to promote G protein activation was predominantly off-target because it was not blocked by an antagonist of GPR84 and was preserved in neutrophils isolated from GPR84 deficient mice. These results illustrate the challenges of attempting to study and define functions of poorly characterised receptors using ligands that have been developed via medicinal chemistry programmes, but where assessed activity has been limited largely to the initially identified target.
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Abstract
Figure 1
Figure 1. Chemical structures of known GPR84 agonists MCFA C10, embelin, 2-HTP, and DIM derivatives and the GPR84 antagonists PBI-4050 and 6-OAU. Abbreviations: 2-HTP, 2-(hexylthiol)pyrimidine-4,6 diol; 6-OAU, 6-n-octylaminouracil; DIM, 3,3′-diindolylmethane; MCFA, medium-chain free fatty acids.
Figure 2
Figure 2. Disease activity index score in a mouse DSS-induced chronic colitis model. Diseased mice were administered with 4% DSS in drinking water for 4 days, followed by 3 days of regular drinking water and a new cycle of DSS. In addition they were orally administered 26 at 10 mg/kg once daily (qd), cyclosporine at 25 mg/kg qd (positive control), or vehicle for 11 consecutive days. Control mice were administered water alone as a negative control. Data are the mean ± SEM. No scores were measured on days 5 and 6.
Figure 3
Figure 3. Disease activity index score in a mouse DSS-induced chronic colitis model. Diseased mice were administered with 4% DSS in drinking water for 4 days, followed by 3 days of regular drinking water and a new cycle of DSS. In addition they were orally administered 35 at 1*, 3† or 10‡ mg/kg qd, cyclosporine at 25 mg/kg qd (positive control), or vehicle, for 11 consecutive days. Control mice were administered water alone as a negative control. Data are the mean ± SEM. No scores were measured on days 5 and 6.
Figure 4
Figure 4. (A) Effect of increasing concentrations of 36 on effects of orthosteric and allosteric GPR84 agonists (GTPyS assay with agonists used at EC80). (B) Structure of tritiated ligand 38. (C) Ability of various concentrations of C10, PSB-16671, and 36 to compete for binding with [3H]38 to GPR84 (competition binding + 0.2 nM [3H]38). (D) Schild analysis showing inhibition of PSB-16671 activity with 36 at concentrations of 10 nM, 30 nM, 100 nM, and 300 nM.
Figure 5
Figure 5. Diseased mice (DSS-induced chronic colitis model) were administered with 4% DSS in drinking water for 4 days, followed by 3 days of regular drinking water and a new cycle of DSS. In addition they were orally administered 36 at 3* and 10† mg/kg qd, sulfasalazine 20 mg/kg qd (positive control) or vehicle, for 11 consecutive days. Control mice were administered water alone as a negative control. (A) Disease activity index score. Data are the mean ± SEM. No scores were measured on days 5 and 6. (B) Neutrophil density (number of cells per square millimeter of colon area, as determined by immunohistochemistry assay with anti-Ly6G/Ly6C antibodies).
Scheme 1
Scheme 1. Synthesis of (S)-GLPG1205 (36)aaReagents and conditions: (a) urea, AcOH, conc HCl, water, reflux; (b) conc HBr, reflux; (c) AllylBr, K2CO3, DMF; (d) Na, EtOH, diethyl malonate, reflux; (e) POCl3, 50 °C; (f) [(2R)-1,4-dioxan-2-yl]methanol, tBuOK, DCM; (g) Pd(PPh3)4, K2CO3, MeOH/THF; (h) PhN(SO3CF3)2, Et3N, DCM; (i) ethynylcyclopropane, Pd(PPh3)4, CuI, Et3N, DMF.
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- 1Wang, J.; Wu, X.; Simonavicius, N.; Tian, H.; Ling, L. Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84. J. Biol. Chem. 2006, 281 (45), 34457– 34464, DOI: 10.1074/jbc.M608019200[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFKgur%252FI&md5=2bbe13a202723f62e59eab0b08a25878Medium-chain Fatty Acids as Ligands for Orphan G Protein-coupled Receptor GPR84Wang, Jinghong; Wu, Xiaosu; Simonavicius, Nicole; Tian, Hui; Ling, LeiJournal of Biological Chemistry (2006), 281 (45), 34457-34464CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Free fatty acids (FFAs) play important physiol. roles in many tissues as an energy source and as signaling mols. in various cellular processes. Elevated levels of circulating FFAs are assocd. with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cAMP prodn., and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive Gi/o pathway. In addn., we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated prodn. of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metab. to immunol. regulation.
- 2Davenport, A. P.; Alexander, S. P.; Sharman, J. L.; Pawson, A. J.; Benson, H. E.; Monaghan, A. E.; Liew, W. C.; Mpamhanga, C. P.; Bonner, T. I.; Neubig, R. R.; Pin, J. P.; Spedding, M.; Harmar, A. J. International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations for new pairings with cognate ligands. Pharmacol. Rev. 2013, 65 (3), 967– 986, DOI: 10.1124/pr.112.007179[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVyrtLvN&md5=561149f6eb486cf3aea0bb255a9cbf39International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations for new pairings with cognate ligandsDavenport, Anthony P.; Alexander, Stephen P. H.; Sharman, Joanna L.; Pawson, Adam J.; Benson, Helen E.; Monaghan, Amy E.; Liew, Wen Chiy; Mpamhanga, Chidochangu P.; Bonner, Tom I.; Neubig, Richard R.; Pin, Jean Philippe; Spedding, Michael; Harmar, Anthony J.Pharmacological Reviews (2013), 65 (3), 967-986CODEN: PAREAQ; ISSN:1521-0081. (American Society for Pharmacology and Experimental Therapeutics)A review. In 2005, the International Union of Basic and Clin. Pharmacol. Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) published a catalog of all of the human gene sequences known or predicted to encode G protein-coupled receptors (GPCRs), excluding sensory receptors. This review updates the list of orphan GPCRs and describes the criteria used by NC-IUPHAR to recommend the pairing of an orphan receptor with its cognate ligand(s). The following recommendations are made for new receptor names based on 11 pairings for class A GPCRs: hydroxycarboxylic acid receptors [HCA1 (GPR81) with lactate, HCA2 (GPR109A) with 3-hydroxybutyric acid, HCA3 (GPR109B) with 3-hydroxyoctanoic acid]; lysophosphatidic acid receptors [LPA4 (GPR23), LPA5 (GPR92), LPA6 (P2Y5)]; free fatty acid receptors [FFA4 (GPR120) with omega-3 fatty acids]; chemerin receptor (CMKLR1; ChemR23) with chemerin; CXCR7 (CMKOR1) with chemokines CXCL12 (SDF-1) and CXCL11 (ITAC); succinate receptor (SUCNR1) with succinate; and oxoglutarate receptor [OXGR1 with 2-oxoglutarate]. Pairings are highlighted for an addnl. 30 receptors in class A where further input is needed from the scientific community to validate these findings. Fifty-seven human class A receptors (excluding pseudogenes) are still considered orphans; information has been provided where there is a significant phenotype in genetically modified animals. In class B, six pairings have been reported by a single publication, with 28 (excluding pseudogenes) still classified as orphans. Seven orphan receptors remain in class C, with one pairing described by a single paper. The objective is to stimulate research into confirming pairings of orphan receptors where there is currently limited information and to identify cognate ligands for the remaining GPCRs. Further information can be found on the IUPHAR Database website.
- 3Suzuki, M.; Takaishi, S.; Nagasaki, M.; Onozawa, Y.; Iino, I.; Maeda, H.; Komai, T.; Oda, T. Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor. J. Biol. Chem. 2013, 288 (15), 10684– 10691, DOI: 10.1074/jbc.M112.420042[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlvFSisr8%253D&md5=63a395e01eb2083bfb7d4a1195c88b1aMedium-chain Fatty Acid-sensing Receptor, GPR84, Is a Proinflammatory ReceptorSuzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, TomiichiroJournal of Biological Chemistry (2013), 288 (15), 10684-10691CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiol. roles have not yet been clarified. Here, the authors show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. The authors also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [35S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated prodn. of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the i.v. injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. The results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation assocd.-disease.
- 4Liu, Y.; Zhang, Q.; Chen, L. H.; Yang, H.; Lu, W.; Xie, X.; Nan, F. J. Design and synthesis of 2-alkylpyrimidine-4,6-diol and 6-alkylpyridine-2,4-diol as potent GPR84 agonists. ACS Med. Chem. Lett. 2016, 7 (6), 579– 583, DOI: 10.1021/acsmedchemlett.6b00025[ACS Full Text], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XltFGht7c%253D&md5=52f50ffae4ab4bbfd79f9eeda0bb063bDesign and Synthesis of 2-Alkylpyrimidine-4,6-diol and 6-Alkylpyridine-2,4-diol as Potent GPR84 AgonistsLiu, Yang; Zhang, Qing; Chen, Lin-Hai; Yang, Hui; Lu, Wei; Xie, Xin; Nan, Fa-JunACS Medicinal Chemistry Letters (2016), 7 (6), 579-583CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)A series of alkylpyrimidine-4,6-diol derivs. were designed and synthesized as novel GPR84 agonists based on a high-throughput screening (HTS) hit I. 6-Nonylpyridine-2,4-diol was identified as the most potent agonist of GPR84 reported so far, with an EC50 of 0.189 nM. These novel GPR84 agonists will provide valuable tools for the study of the physiol. functions of GPR84.
- 5Zhang, Q.; Yang, H.; Li, J.; Xie, X. Discovery and characterization of a novel small-molecule agonist for medium-chain free fatty acid receptor G protein-coupled receptor 84. J. Pharmacol. Exp. Ther. 2016, 357 (2), 337– 344, DOI: 10.1124/jpet.116.232033[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlWhu7fE&md5=8a2f4113fddcb35cdd39f99b9470e0c7Discovery and characterization of a novel small-molecule agonist for medium-chain free fatty acid receptor G protein-coupled receptor 84Zhang, Qing; Yang, Hui; Li, Jing; Xie, XinJournal of Pharmacology and Experimental Therapeutics (2016), 357 (2), 337-344CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clin. applications. Here, we report the screen of 160,000 small- mol. compds. with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Ga16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compd. may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization.
- 6Pillaiyar, T.; Kose, M.; Namasivayam, V.; Sylvester, K.; Borges, G.; Thimm, D.; von Kugelgen, I.; Muller, C. E. 6-(Ar)alkylamino-substituted uracil derivatives: lipid mimetics with potent activity at the orphan G protein-coupled receptor 84 (GPR84). ACS omega 2018, 3 (3), 3365– 3383, DOI: 10.1021/acsomega.7b02092[ACS Full Text], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVyqu7g%253D&md5=184964bac823f7cd34d4de98da0cef536-(Ar)Alkylamino-Substituted Uracil Derivatives: Lipid Mimetics with Potent Activity at the Orphan G Protein-Coupled Receptor 84 (GPR84)Pillaiyar, Thanigaimalai; Koese, Meryem; Namasivayam, Vigneshwaran; Sylvester, Katharina; Borges, Gleice; Thimm, Dominik; von Kuegelgen, Ivar; Mueller, Christa E.ACS Omega (2018), 3 (3), 3365-3383CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)GPR84, a Gi protein-coupled receptor that is activated by medium-chain (hydroxy)fatty acids, appears to play an important role in inflammation, immunity, and cancer. Recently, 6-octylaminouracil has been reported to act as an agonist at GPR84. Here, we describe the synthesis of 69 derivs. and analogs which represent new compds. They were evaluated in (a) cyclic adenosine monophosphate accumulation and (b) β-arrestin assays in human GPR84-expressing cells. Potent nonbiased as well as G protein-biased agonists were developed, e.g., 6-hexylamino-2,4(1H,3H)-pyrimidinedione (PSB-1584, EC50 5.0 nM (a), 3.2 nM (b), bias factor: 0) and 6-((p-chloro- and p-bromo-phenylethyl)amino)-2,4(1H,3H)-pyrimidinedione (PSB-16434, EC50 7.1 nM (a), 520 nM (b), bias factor: 1.9 = 79-fold Gi pathway-selective; I, PSB-17365, EC50 2.5 nM (a), 100 nM (b), bias factor 1.3 = 20-fold selective), which were selective vs. other free fatty acid-activated receptors. Compds. II and I were found to be metabolically stable upon incubation with human liver microsomes. A pharmacophore model was created on the basis of structurally diverse lipidlike GPR84 agonists.
- 7Yousefi, S.; Cooper, P. R.; Potter, S. L.; Mueck, B.; Jarai, G. Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes. Journal Leukocyte Biol. 2001, 69 (6), 1045– 1052, DOI: 10.1189/jlb.69.6.1045[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXkslWrurs%253D&md5=03d1eb21f619cdd4790ace3c2b571d23Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytesYousefi, Shida; Cooper, Paul R.; Potter, Suzanne L.; Mueck, Beatrice; Jarai, GaborJournal of Leukocyte Biology (2001), 69 (6), 1045-1052CODEN: JLBIE7; ISSN:0741-5400. (Federation of American Societies for Experimental Biology)The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. The authors used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was detd. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homolog. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot expts. revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR anal., the authors showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.
- 8Lattin, J. E.; Schroder, K.; Su, A. I.; Walker, J. R.; Zhang, J.; Wiltshire, T.; Saijo, K.; Glass, C. K.; Hume, D. A.; Kellie, S.; Sweet, M. J. Expression analysis of G protein-coupled receptors in mouse macrophages. Immunome Res. 2008, 4, 5, DOI: 10.1186/1745-7580-4-5[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3czktlyjtw%253D%253D&md5=d0f33a92e85b59309930de7b0bfb36ecExpression analysis of G Protein-Coupled Receptors in mouse macrophagesLattin Jane E; Schroder Kate; Su Andrew I; Walker John R; Zhang Jie; Wiltshire Tim; Saijo Kaoru; Glass Christopher K; Hume David A; Kellie Stuart; Sweet Matthew JImmunome research (2008), 4 (), 5 ISSN:.BACKGROUND: Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). RESULTS: Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. CONCLUSION: The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.
- 9Audoy-Remus, J.; Bozoyan, L.; Dumas, A.; Filali, M.; Lecours, C.; Lacroix, S.; Rivest, S.; Tremblay, M. E.; Vallieres, L. GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer’s disease. Brain, Behav., Immun. 2015, 46, 112– 120, DOI: 10.1016/j.bbi.2015.01.010[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitFCntbo%253D&md5=4b643287374786cdde611ffbb3002101GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer's diseaseAudoy-Remus, Julie; Bozoyan, Lusine; Dumas, Aline; Filali, Mohammed; Lecours, Cynthia; Lacroix, Steve; Rivest, Serge; Tremblay, Marie-Eve; Vallieres, LucBrain, Behavior, and Immunity (2015), 46 (), 112-120CODEN: BBIMEW; ISSN:0889-1591. (Elsevier)Microglia surrounds the amyloid plaques that form in the brains of patients with Alzheimer's disease (AD), but their role is controversial. Under inflammatory conditions, these cells can express GPR84, an orphan receptor whose pathophysiol. role is unknown. Here, we report that GPR84 is upregulated in microglia of APP/PS1 transgenic mice, a model of AD. Without GPR84, these mice display both accelerated cognitive decline and a reduced no. of microglia, esp. in areas surrounding plaques. The lack of GPR84 affects neither plaque formation nor hippocampal neurogenesis, but promotes dendritic degeneration. Furthermore, GPR84 does not influence the clin. progression of other diseases in which its expression has been reported, i.e., exptl. autoimmune encephalomyelitis (EAE) and endotoxic shock. We conclude that GPR84 plays a beneficial role in amyloid pathol. by acting as a sensor for a yet unknown ligand that promotes microglia recruitment, a response affecting dendritic degeneration and required to prevent further cognitive decline.
- 10Bouchard, C.; Page, J.; Bedard, A.; Tremblay, P.; Vallieres, L. G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions. Glia 2007, 55 (8), 790– 800, DOI: 10.1002/glia.20506[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2s3jtVemsw%253D%253D&md5=5bc25664243312ac66f94244ab598c9dG protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditionsBouchard Caroline; Page Julie; Bedard Andreanne; Tremblay Pierrot; Vallieres LucGlia (2007), 55 (8), 790-800 ISSN:0894-1491.G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.
- 11Venkataraman, C.; Kuo, F. The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking. Immunol. Lett. 2005, 101 (2), 144– 153, DOI: 10.1016/j.imlet.2005.05.010[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVKjs77L&md5=abef6f196008a5055ab99e9213c0d55dThe G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinkingVenkataraman, Chandrasekar; Kuo, FrederickImmunology Letters (2005), 101 (2), 144-153CODEN: IMLED6; ISSN:0165-2478. (Elsevier B.V.)The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biol. function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-γ prodn. in GPR84-/- mice compared to wild-type mice. Augmented IL-4 prodn. in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-γ steady-state mRNA in GPR84-/- T cells. In addn., Th2 effector cells generated in vitro from GPR84-/- mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 prodn. between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.
- 12Nagasaki, H.; Kondo, T.; Fuchigami, M.; Hashimoto, H.; Sugimura, Y.; Ozaki, N.; Arima, H.; Ota, A.; Oiso, Y.; Hamada, Y. Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes. FEBS Lett. 2012, 586 (4), 368– 372, DOI: 10.1016/j.febslet.2012.01.001[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xit1yqu7w%253D&md5=8fecdea5b404e472d6f66a9ed22cd88bInflammatory changes in adipose tissue enhance expression of GPR G-protein coupled receptor84, a medium-chain fatty acid receptorNagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, YojiFEBS Letters (2012), 586 (4), 368-372CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)In this study we aimed to identify the physiol. roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, coculture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity.
- 13Nicol, L. S.; Dawes, J. M.; La Russa, F.; Didangelos, A.; Clark, A. K.; Gentry, C.; Grist, J.; Davies, J. B.; Malcangio, M.; McMahon, S. B. The role of G-protein receptor 84 in experimental neuropathic pain. J. Neurosci. 2015, 35 (23), 8959– 8969, DOI: 10.1523/JNEUROSCI.3558-14.2015[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVOqsLrJ&md5=4d7a16fa2471aaaa6ed9395ad1215544The role of G-protein receptor 84 in experimental neuropathic painNicol, Louise S. C.; Dawes, John M.; La Russa, Federica; Didangelos, Athanasios; Clark, Anna K.; Gentry, Clive; Grist, John; Davies, John B.; Malcangio, Marzia; McMahon, Stephen B.Journal of Neuroscience (2015), 35 (23), 8959-8969CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiol. function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mech. or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter mol. 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array anal. revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addn., the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger assocd. with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired.
- 14Sundqvist, M.; Christenson, K.; Holdfeldt, A.; Gabl, M.; Mårtensson, J.; Björkman, L.; Dieckmann, R.; Dahlgren, C.; Forsman, H. Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R. Biochim. Biophys. Acta, Mol. Cell Res. 2018, 1865 (5), 695– 708, DOI: 10.1016/j.bbamcr.2018.02.008[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjsFaju7w%253D&md5=5f80256de9cc62715bf953312937170eSimilarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2RSundqvist, Martina; Christenson, Karin; Holdfeldt, Andre; Gabl, Michael; Maartensson, Jonas; Bjoerkman, Lena; Dieckmann, Regis; Dahlgren, Claes; Forsman, HuameiBiochimica et Biophysica Acta, Molecular Cell Research (2018), 1865 (5), 695-708CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small mols. that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), resp. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naive cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.
- 15Arijs, I.; De Hertogh, G.; Machiels, K.; Van Steen, K.; Lemaire, K.; Schraenen, A.; Van Lommel, L.; Quintens, R.; Van Assche, G.; Vermeire, S.; Schuit, F.; Rutgeerts, P. Mucosal gene expression of cell adhesion molecules, chemokines, and chemokine receptors in patients with inflammatory bowel disease before and after infliximab treatment. Am. J. Gastroenterol. 2011, 106 (4), 748– 761, DOI: 10.1038/ajg.2011.27[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkt1ehsr8%253D&md5=f4483700084ba9b4a7dddae654ecd0e2Mucosal Gene Expression of Cell Adhesion Molecules, Chemokines, and Chemokine Receptors in Patients With Inflammatory Bowel Disease Before and After Infliximab TreatmentArijs, Ingrid; De Hertogh, Gert; Machiels, Kathleen; Van Steen, Kristel; Lemaire, Katleen; Schraenen, Anica; Van Lommel, Leentje; Quintens, Roel; Van Assche, Gert; Vermeire, Severine; Schuit, Frans; Rutgeerts, PaulAmerican Journal of Gastroenterology (2011), 106 (4), 748-761CODEN: AJGAAR; ISSN:0002-9270. (Nature Publishing Group)OBJECTIVES: Inflammatory bowel disease (IBD) is characterized by a continuous influx of leukocytes into the gut wall. This migration is regulated by cell adhesion mols. (CAMs), and selective antimigration therapies have been developed. This study investigated the effect of infliximab therapy on the mucosal gene expression of CAMs in IBD. METHODS: Mucosal gene expression of 69 leukocyte/endothelial CAMs and E-cadherin was investigated in 61 IBD patients before and after first infliximab infusion and in 12 normal controls, using Affymetrix gene expression microarrays. Quant. reverse transcriptase-PCR (qRT-PCR), immunohistochem., and western blotting were used to confirm the microarray data. RESULTS: When compared with control colons, the colonic mucosal gene expression of most leukocyte/endothelial adhesion mols. was upregulated and E-cadherin gene expression was downregulated in active colonic IBD (IBDc) before therapy, with no significant colonic gene expression differences between ulcerative colitis and colonic Crohn's disease. Infliximab therapy restored the upregulations of leukocyte CAMs in IBDc responders to infliximab that paralleled the disappearance of the inflammatory cells from the colonic lamina propria. Also, the colonic gene expression of endothelial CAMs and of most chemokines/chemokine receptors returned to normal after therapy in IBDc responders, and only CCL20 and CXCL1-2 expression remained increased after therapy in IBDc responders vs. control colons. When compared with control ileums, the ileal gene expression of MADCAM1, THY1, PECAM1, CCL28, CXCL1, -2, -5, -6, and -11, and IL8 was increased and CD58 expression was decreased in active ileal Crohn's disease (CDi) before therapy, and none of the genes remained dysregulated after therapy in CDi responders vs. control ileums. This microarray study identified a no. of interesting targets for antiadhesion therapy including PECAM1, IL8, and CCL20, besides the currently studied α4β7 integrin-MADCAM1 axis. CONCLUSIONS: Our data demonstrate that many leukocyte/endothelial CAMs and chemokines/chemokine receptors are upregulated in inflamed IBD mucosa. Controlling the inflammation with infliximab restores most of these dysregulations in IBD. These results show that at least part of the mechanism of anti-tumor necrosis factor-α therapy goes through downregulation of certain adhesion mols. Am J Gastroenterol 2011; 106:748-761; doi:10.1038/ajg.2011.27; published online 15 Feb. 2011.
- 16Gagnon, L.; Leduc, M.; Thibodeau, J. F.; Zhang, M. Z.; Grouix, B.; Sarra-Bournet, F.; Gagnon, W.; Hince, K.; Tremblay, M.; Geerts, L.; Kennedy, C. R. J.; Hebert, R. L.; Gutsol, A.; Holterman, C. E.; Kamto, E.; Gervais, L.; Ouboudinar, J.; Richard, J.; Felton, A.; Laverdure, A.; Simard, J. C.; Letourneau, S.; Cloutier, M. P.; Leblond, F. A.; Abbott, S. D.; Penney, C.; Duceppe, J. S.; Zacharie, B.; Dupuis, J.; Calderone, A.; Nguyen, Q. T.; Harris, R. C.; Laurin, P. A newly discovered antifibrotic pathway regulated by two fatty acid receptors: GPR40 and GPR84. Am. J. Pathol. 2018, 188 (5), 1132– 1148, DOI: 10.1016/j.ajpath.2018.01.009[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVGqs7w%253D&md5=47fff3190f70beafa69897d9d89f206dA Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid ReceptorsGagnon, Lyne; Leduc, Martin; Thibodeau, Jean-Francois; Zhang, Ming-Zhi; Grouix, Brigitte; Sarra-Bournet, Francois; Gagnon, William; Hince, Kathy; Tremblay, Mikael; Geerts, Lilianne; Kennedy, Christopher R. J.; Hebert, Richard L.; Gutsol, Alex; Holterman, Chet E.; Kamto, Eldjonai; Gervais, Liette; Ouboudinar, Jugurtha; Richard, Jonathan; Felton, Alexandra; Laverdure, Alexandre; Simard, Jean-Christophe; Letourneau, Sylvie; Cloutier, Marie-Pier; Leblond, Francois A.; Abbott, Shaun D.; Penney, Christopher; Duceppe, Jean-Simon; Zacharie, Boulos; Dupuis, Jocelyn; Calderone, Angelino; Nguyen, Quang T.; Harris, Raymond C.; Laurin, PierreAmerican Journal of Pathology (2018), 188 (5), 1132-1148CODEN: AJPAA4; ISSN:0002-9440. (Elsevier B.V.)Numerous clin. conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiol. pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are assocd. with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiol. processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, resp., we uncovered an antifibrotic pathway involving these receptors. In expts. using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity obsd. in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising mol. targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compd. that may be effective for managing inflammatory and fibrosis-related diseases.
- 17Abdel-Aziz, H.; Schneider, M.; Neuhuber, W.; Meguid Kassem, A.; Khailah, S.; Muller, J.; Gamal Eldeen, H.; Khairy, A.; Khayyal, M. T.; Shcherbakova, A.; Efferth, T.; Ulrich-Merzenich, G. GPR84 and TREM-1 signaling contribute to the pathogenesis of reflux esophagitis. Mol. Med. 2015, 21 (1), 1011– 1024, DOI: 10.2119/molmed.2015.00098[Crossref], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKlsLbI&md5=d626c0592ea9ac67a3700ec6b2138383GPR84 and TREM-1 signaling contribute to the pathogenesis of reflux esophagitisAbdel-Aziz, Heba; Schneider, Mathias; Neuhuber, Winfried; Kassem, Abdel Meguid; Khailah, Saleem; Mueller, Juergen; Eldeen, Hadeel Gamal; Khairy, Ahmed; Khayyal, Mohamed T.; Shcherbakova, Anastasiia; Efferth, Thomas; Ulrich-Merzenich, GudrunMolecular Medicine (Manhasset, NY, United States) (2015), 21 (), 1011-1024CODEN: MOMEF3; ISSN:1528-3658. (Feinstein Institute for Medical Research)Gastro-esophageal reflux disease (GERD) is one of the most common disorders in gastroenterol. Patients present with or without increased acid exposure indicating a nonuniform etiol. Thus, the common treatment with proton pump inhibitors (PPIs) fails to control symptoms in up to 40% of patients. To further elucidate the pathophysiol. of the condition and explore new treatment targets, transcriptomics, proteomics and histol. methods were applied to a surgically induced subchronic reflux esophagitis model in Wistar rats after treatment with either omeprazole (PPI) or STW5, a herbal prepn. shown to ameliorate esophagitis without affecting refluxate pH. The normal human esophageal squamous cell line HET-1A and human endoscopic biopsies were used to confirm our findings to the G-protein-coupled receptor (GPR) 84 in human tissue. Both treatments reduced reflux-induced macroscopic and microscopic lesions of the esophagi as well as known proinflammatory cytokines. Proteomic and transcriptomic analyses identified CINC1-3, MIP-1/3α, MIG, RANTES and interleukin (IL)-1β as prominent mediators in GERD. Most regulated cyto-/chemokines are linked to the TREM-1 signaling pathway. The fatty acid receptor GPR84 was upregulated in esophagitis but significantly decreased in treated groups, a finding supported by Western blot and immunohistochem. in both rat tissue and HET-1A cells. GPR84 was also found to be significantly upregulated in patients with grade B reflux esophagitis. The expression of GPR84 in esophageal tissue and its potential involvement in GERD are reported for the first time. IL-8 (CINC1-3) and the TREM-1 signaling pathway are proposed, besides GPR84, to play an important role in the pathogenesis of GERD.
- 18Dietrich, P. A.; Yang, C.; Leung, H. H.; Lynch, J. R.; Gonzales, E.; Liu, B.; Haber, M.; Norris, M. D.; Wang, J.; Wang, J. Y. GPR84 sustains aberrant beta-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis. Blood 2014, 124 (22), 3284– 3294, DOI: 10.1182/blood-2013-10-532523[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVSgs73L&md5=3da07da89bd6da728d09aa26b273d659GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesisDietrich, Philipp A.; Yang, Chen; Leung, Halina H. L.; Lynch, Jennifer R.; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D.; Wang, Jianlong; Wang, Jenny YingziBlood (2014), 124 (22), 3284-3294CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)β-Catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacol. manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray anal. demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML.
- 19Du Toit, E.; Browne, L.; Irving-Rodgers, H.; Massa, H. M.; Fozzard, N.; Jennings, M. P.; Peak, I. R. Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich diets. Eur. J. Nutr. 2018, 57 (5), 1737– 1746, DOI: 10.1007/s00394-017-1456-5[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmsFGjsbs%253D&md5=5c76ae9693f9d1e78aed516c0f5e3567Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich dietsDu Toit, Eugene; Browne, Liam; Irving-Rodgers, Helen; Massa, Helen M.; Fozzard, Nicolette; Jennings, Michael P.; Peak, Ian R.European Journal of Nutrition (2018), 57 (5), 1737-1746CODEN: EJNUFZ; ISSN:1436-6207. (Springer)Purpose: Although there is good evidence showing that diets rich in medium chain fatty acids (MCFAs) have less marked obesogenic and diabetogenic effects than diets rich in long chain fatty acids (LCFAs), the role of the pro-inflammatory, medium chain fatty acid receptor (GPR84) in the etiol. of obesity and glucose intolerance is not well characterised. We set out to det. whether GPR84 expression influences obesity and glucose intolerance susceptibility in MCFA and LCFA rich diet fed mice. Methods: Wild type (WT) and GPR84 knockout (KO) mice were fed a control, MCFA or LCFA diet, and body mass, heart, liver and epididymal fat mass was assessed, as well as glucose tolerance and adipocyte size. Results: LCFA diets increased body mass and decreased glucose tolerance in both WT and GPR84 KO animals while MCFA diets had no effect on these parameters. There were no differences in body wt. when comparing WT and GPR84 KO mice on the resp. diets. Glucose tolerance was also similar in WT and GPR84 KO mice irresp. of diet. Liver mass was increased following LCFA feeding in WT but not GPR84 KO mice. Hepatic triglyceride content was increased in GPR84 KO animals fed MCFA, and myocardial triglyceride content was increased in GPR84 KO animals fed LCFA. Conclusions: GPR84 deletion had no effects on body wt. or glucose tolerance in mice fed either a high MCFA or LCFA diet. GPR84 may influence lipid metab., as GPR84 KO mice had smaller livers and increased myocardial triglyceride accumulation when fed LCFA diets, and increased liver triglyceride accumulation in responses to increased dietary MCFAs.
- 20Mignani, S.; Rodrigues, J.; Tomas, H.; Jalal, R.; Singh, P. P.; Majoral, J. P.; Vishwakarma, R. A. Present drug-likeness filters in medicinal chemistry during the hit and lead optimization process: how far can they be simplified?. Drug Discovery Today 2018, 23 (3), 605– 615, DOI: 10.1016/j.drudis.2018.01.010[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvgtlCksw%253D%253D&md5=a61e00552a35b455199f0a8d351d0522Present drug-likeness filters in medicinal chemistry during the hit and lead optimization process: how far can they be simplified?Mignani Serge; Rodrigues Joao; Tomas Helena; Jalal Rachid; Singh Parvinder Pal; Vishwakarma Ram A; Majoral Jean-PierreDrug discovery today (2018), 23 (3), 605-615 ISSN:.During the past decade, decreasing the attrition rate of drug development candidates reaching the market has become one of the major challenges in pharmaceutical research and drug development (R&D). To facilitate the decision-making process, and to increase the probability of rapidly finding and developing high-quality compounds, a variety of multiparametric guidelines, also known as rules and ligand efficiency (LE) metrics, have been developed. However, what are the 'best' descriptors and how far can we simplify these drug-likeness prediction tools in terms of the numerous, complex properties that they relate to?
- 21Kodimuthali, A.; Jabaris, S. S. L.; Pal, M. Recent advances on phosphodiesterase 4 inhibitors for the treatment of asthma and chronic obstructive pulmonary disease. J. Med. Chem. 2008, 51 (18), 5471– 5489, DOI: 10.1021/jm800582j[ACS Full Text], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1alsbg%253D&md5=d4502dc5ce4b8b17d5b1d4d853403768Recent Advances on Phosphodiesterase 4 Inhibitors for the Treatment of Asthma and Chronic Obstructive Pulmonary DiseaseKodimuthali, Arumugam; Jabaris, S. Sugin Lal; Pal, ManojitJournal of Medicinal Chemistry (2008), 51 (18), 5471-5489CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review.
- 22Niu, M.; Dong, F.; Tang, S.; Fida, G.; Qin, J.; Qiu, J.; Liu, K.; Gao, W.; Gu, Y. Pharmacophore modeling and virtual screening for the discovery of new type 4 cAMP phosphodiesterase (PDE4) inhibitors. PLoS One 2013, 8 (12), e82360, DOI: 10.1371/journal.pone.0082360[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlsVCju74%253D&md5=d1b6be4406df69aea27e763c04522d42Pharmacophore modeling and virtual screening for the discovery of new type 4 cAMP phosphodiesterase (PDE4) inhibitorsNiu, Miaomiao; Dong, Fenggong; Tang, Shi; Fida, Guissi; Qin, Jingyi; Qiu, Jiadan; Liu, Kangbo; Gao, Weidong; Gu, YueqingPLoS One (2013), 8 (12), e82360/1-e82360/15, 15 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Type 4 cAMP phosphodiesterase (PDE4) inhibitors show a broad spectrum of anti-inflammatory effects in almost all kinds of inflamed cells, by an increase in cAMP levels which is a pivotal second messenger responsible for various biol. processes. These inhibitors are now considered as the potential drugs for treatment of chronic inflammatory diseases. However, some recently marketed inhibitors e.g., roflumilast, have shown adverse effects such as nausea and emesis, thus restricting its use. In order to identify novel PDE4 inhibitors with improved therapeutic indexes, a highly correlating (r = 0.963930) pharmacophore model (Hypo1) was established on the basis of known PDE4 inhibitors. Validated Hypo1 was used in database screening to identify chem. with required pharmacophoric features. These compds. are further screened by using the rule of five, ADMET and mol. docking. Finally, twelve hits which showed good results with respect to following properties such as estd. activity, calcd. drug-like properties and scores were proposed as potential leads to inhibit the PDE4 activity. Therefore, this study will not only assist in the development of new potent hits for PDE4 inhibitors, but also give a better understanding of their interaction with PDE4. On a wider scope, this will be helpful for the rational design of novel potent enzyme inhibitors.
- 23Oxford, A. W.; Jack, D. Derivatives of Pyrimido[6,1-A]Isoquinolin-4-One. WO/2000/058309, Mar 29, 2000; https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2000058309.Google ScholarThere is no corresponding record for this reference.
- 24Sina, C.; Gavrilova, O.; Förster, M.; Till, A.; Derer, S.; Hildebrand, F.; Raabe, B.; Chalaris, A.; Scheller, J.; Rehmann, A.; Franke, A.; Ott, S.; Häsler, R.; Nikolaus, S.; Fölsch, U. R.; Rose-John, S.; Jiang, H. P.; Li, J.; Schreiber, S.; Rosenstiel, P. G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation. J. Immunol. 2009, 183 (11), 7514– 7522, DOI: 10.4049/jimmunol.0900063[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVehtrrN&md5=98fd06777b79a924befb00cdc5ee1b6bG Protein-Coupled Receptor 43 Is Essential for Neutrophil Recruitment during Intestinal InflammationSina, Christian; Gavrilova, Olga; Foerster, Matti; Till, Andreas; Derer, Stefanie; Hildebrand, Friederike; Raabe, Bjoern; Chalaris, Athena; Scheller, Juergen; Rehmann, Ateequr; Franke, Andre; Ott, Stephan; Haesler, Robert; Nikolaus, Susanna; Foelsch, Ulrich R.; Rose-John, Stefan; Jiang, Hui-Ping; Li, Jun; Schreiber, Stefan; Rosenstiel, PhilipJournal of Immunology (2009), 183 (11), 7514-7522CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)Mol. danger signals attract neutrophilic granulocytes (polymorphonuclear leukocytes (PMNs)) to sites of infection. The G protein-coupled receptor (GPR) 43 recognizes propionate and butyrate and is abundantly expressed on PMNs. The functional role of GPR43 activation for in vivo orchestration of immune response is unclear. The authors examd. dextrane sodium sulfate (DSS)-induced acute and chronic intestinal inflammatory response in wild-type and Gpr43-deficient mice. The severity of colonic inflammation was assessed by clin. signs, histol. scoring, and cytokine prodn. Chemotaxis of wild-type and Gpr43-deficient PMNs was assessed through transwell cell chemotactic assay. A reduced invasion of PMNs and increased mortality due to septic complications were obsd. in acute DSS colitis. In chronic DSS colitis, Gpr43-/- animals showed diminished PMN intestinal migration, but protection against inflammatory tissue destruction. No significant difference in PMN migration and cytokine secretion was detected in a sterile inflammatory model. Ex vivo expts. show that GPR43-induced migration is dependent on activation of the protein kinase p38α, and that this signal acts in cooperation with the chemotactic cytokine keratinocyte chemoattractant. Interestingly, shedding of L-selectin in response to propionate and butyrate was compromised in Gpr43-/- mice. These results indicate a crit. role for GPR43-mediated recruitment of PMNs in contg. intestinal bacterial translocation, yet also emphasize the bipotential role of PMNs in mediating tissue destruction in chronic intestinal inflammation.
- 25Wirtz, S.; Neufert, C.; Weigmann, B.; Neurath, M. F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2007, 2 (3), 541– 546, DOI: 10.1038/nprot.2007.41[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFGntbnL&md5=7a3c29eaca8be756e3d866e23e151fdfChemically induced mouse models of intestinal inflammationWirtz, Stefan; Neufert, Clemens; Weigmann, Benno; Neurath, Markus F.Nature Protocols (2007), 2 (3), 541-546CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chem. induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In the DSS model, mice are subjected several days to drinking water supplemented with DSS, which seems to be directly toxic to colonic epithelial cells of the basal crypts. The procedures for the hapten models of colitis and acute DSS colitis can be accomplished in about 2 wk but the protocol for chronic DSS colitis takes about 2 mo.
- 26Mahmud, Z. A.; Jenkins, L.; Ulven, T.; Labéguère, F.; Gosmini, R.; De Vos, S.; Hudson, B. D.; Tikhonova, I. G.; Milligan, G. Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84. Sci. Rep. 2017, 7 (1), 17953, DOI: 10.1038/s41598-017-18159-3[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzjslSqug%253D%253D&md5=fe3d58f70952bef231505deb9bbb9de0Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84Mahmud Zobaer Al; Jenkins Laura; Hudson Brian D; Milligan Graeme; Ulven Trond; Labeguere Frederic; Gosmini Romain; Labeguere Frederic; De Vos Steve; Tikhonova Irina GScientific reports (2017), 7 (1), 17953 ISSN:.Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine(172), located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [(3)H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine(172). These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.
- 27Kim, H. Y.; Kuhn, R. J.; Patkar, C.; Warrier, R.; Cushman, M. Synthesis of dioxane-based antiviral agents and evaluation of their biological activities as inhibitors of Sindbis virus replication. Bioorg. Med. Chem. 2007, 15 (7), 2667– 2679, DOI: 10.1016/j.bmc.2007.01.040[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXivVagtro%253D&md5=67f56b313ec33524f54720fc702d6bd1Synthesis of dioxane-based antiviral agents and evaluation of their biological activities as inhibitors of Sindbis virus replicationKim, Ha Young; Kuhn, Richard J.; Patkar, Chinmay; Warrier, Ranjit; Cushman, MarkBioorganic & Medicinal Chemistry (2007), 15 (7), 2667-2679CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)The crystal structure of the Sindbis virus capsid protein contains one or two solvent-derived dioxane mols. in the hydrophobic binding pocket. A bis-dioxane antiviral agent was designed by linking the two dioxane mols. with a three-carbon chain having R,R connecting stereochem., and a stereospecific synthesis was performed. This resulted in an effective antiviral agent that inhibited Sindbis virus replication with an EC50 of 14 μM. The synthesis proceeded through an intermediate (R)-2-hydroxymethyl-[1,4]-dioxane, which unexpectedly proved to be a more effecting antiviral agent than the target compd., as evidenced by its EC50 of 3.4 μM as an inhibitor of Sindbis virus replication. Both compds. were not cytotoxic in uninfected BHK cells at concns. of 1 mM.
- 28Vanhoutte, F.; Dupont, S.; Van Kaem, T.; Gouy, M.-H.; Gheyle, L.; Blanque, R.; Brys, R.; Vandeghinste, N.; Haazen, W.; van’t Klooster, G.; Beetens, J. Human safety, pharmacokinetics and pharmacodynamics of the GPR84 antagonist GLPG1205, a potential new approach to treat IBD. United Eur. Gastroenterol. J. 2014, 2 (1S), A224, (Abstract P0341)Google ScholarThere is no corresponding record for this reference.
- 29Galapagos Reports Results with GLPG1205 in Ulcerative Colitis. Galapagos Press Release January 26, 2016. https://cws.huginonline.com/G/133350/PR/201601/1981193_5.html (accessed Apr 3, 2019).Google ScholarThere is no corresponding record for this reference.
- 30Puengel, T.; De Vos, S.; Hundertmark, J.; Kohlhepp, M.; Guldiken, N.; Pujuguet, P.; Auberval, M.; Marsais, F.; Shoji, K. F.; Saniere, L.; Trautwein, C.; Luedde, T.; Strnad, P.; Brys, R.; Clement-Lacroix, P.; Tacke, F. The medium-chain fatty acid receptor GPR84 mediates myeloid cell infiltration promoting steatohepatitis and fibrosis. J. Clin. Med. 2020, 9 (4), E1140, DOI: 10.3390/jcm9041140[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovF2hur0%253D&md5=e9977d9275af8aad56c1715aa362eaf0The medium-chain fatty acid receptor GPR84 mediates myeloid cell infiltration promoting steatohepatitis and fibrosisPuengel, Tobias; De Vos, Steve; Hundertmark, Jana; Kohlhepp, Marlene; Guldiken, Nurdan; Pujuguet, Philippe; Auberval, Marielle; Marsais, Florence; Shoji, Kenji F.; Saniere, Laurent; Trautwein, Christian; Luedde, Tom; Strnad, Pavel; Brys, Reginald; Clement-Lacroix, Philippe; Tacke, FrankJournal of Clinical Medicine (2020), 9 (4), 1140CODEN: JCMOHK; ISSN:2077-0383. (MDPI AG)Medium-chain fatty acids (MCFAs) have been assocd. with anti-steatotic effects in hepatocytes. Expression of the MCFA receptor GPR84 (G protein-coupled receptor 84) is induced in immune cells under inflammatory conditions and can promote fibrogenesis. We aimed at deciphering the role of GPR84 in the pathogenesis of non-alc. steatohepatitis (NASH), exploring its potential as a therapeutic target. GPR84 expression is upregulated in liver from patients with non-alc. fatty liver disease (NAFLD), correlating with the histol. degree of inflammation and fibrosis. In mouse and human, activated monocytes and neutrophils upregulate GPR84 expression. Chemotaxis of these myeloid cells by GPR84 stimulation is inhibited by two novel, small mol. GPR84 antagonists. Upon acute liver injury in mice, treatment with GPR84 antagonists significantly reduced the hepatic recruitment of neutrophils, monocytes, and monocyte-derived macrophages (MoMF). We, therefore, evaluated the therapeutic inhibition of GPR84 by these two novel antagonists in comparison to selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, in three NASH mouse models. Pharmacol. inhibition of GPR84 significantly reduced macrophage accumulation and ameliorated inflammation and fibrosis, to an extent similar to selonsertib. In conclusion, our findings support that GPR84 mediates myeloid cell infiltration in liver injury and is a promising therapeutic target in steatohepatitis and fibrosis.
- 31Saniere, L.; Marsais, F.; Jagerschmidt, C.; Meurisse, S.; Cuzic, S.; Shoji, K.; Clement-Lacroix, P.; Van Osselaer, N.; De Vos, S. Characterization of GLPG1205 in mouse fibrosis models: a potent and selective antagonist of GPR84 for treatment of idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 2019, 199, A1046, DOI: 10.1164/ajrccm-conference.2019.199.1_MeetingAbstracts.A1046
- 32Mancini, S.J.; Al Mahmud, Z.; Jenkins, L.; Bolognini, D.; Newman, R.; Barnes, M.; Edye, M. E.; McMahon, S. B.; Tobin, A.; Milligan, G. On-target and off-target effects of novel orthosteric and allosteric activators of GPR84. Sci. Rep. 2019, 9 (1), 1861, DOI: 10.1038/s41598-019-38539-1[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cfis1amsA%253D%253D&md5=1bd20c2443890cb3691d558cb8511c5fOn-target and off-target effects of novel orthosteric and allosteric activators of GPR84Mancini Sarah J; Mahmud Zobaer Al; Jenkins Laura; Bolognini Daniele; Tobin Andrew B; Milligan Graeme; Newman Robert; Barnes Matt; Edye Michelle E; McMahon Stephen BScientific reports (2019), 9 (1), 1861 ISSN:.Many members of the G protein-coupled receptor family, including examples with clear therapeutic potential, remain poorly characterised. This often reflects limited availability of suitable tool ligands with which to interrogate receptor function. In the case of GPR84, currently a target for the treatment of idiopathic pulmonary fibrosis, recent times have seen the description of novel orthosteric and allosteric agonists. Using 2-(hexylthiol)pyrimidine-4,6 diol (2-HTP) and di(5,7-difluoro-1H-indole-3-yl)methane (PSB-16671) as exemplars of each class, in cell lines transfected to express either human or mouse GPR84, both ligands acted as effective on-target activators and with high co-operativity in their interactions. This was also the case in lipopolysaccharide-activated model human and mouse immune cell lines. However in mouse bone-marrow-derived neutrophils, where expression of GPR84 is particularly high, the capacity of PSB-16671 but not of 2-HTP to promote G protein activation was predominantly off-target because it was not blocked by an antagonist of GPR84 and was preserved in neutrophils isolated from GPR84 deficient mice. These results illustrate the challenges of attempting to study and define functions of poorly characterised receptors using ligands that have been developed via medicinal chemistry programmes, but where assessed activity has been limited largely to the initially identified target.
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