Andrew J. Brown

The putative cannabinoid receptor GPR55 plays a role in mechanical hyperalgesia associated with inflammatory and neuropathic pain

Abstract It has been postulated that the G protein‐coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB1 and CB2 receptors are effective analgesic and anti‐inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well‐validated GPR55 tool compounds, a GPR55 knockout (GPR55−/−) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55−/− mice revealed no obvious primary differences, compared with wild‐type (GPR55+/+) littermates. GPR55−/− mice were then tested in the models of adjuvant‐induced inflammation and partial nerve ligation. Following intraplantar administration of Freund’s complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55−/− mice up to 14 days post‐injection. Cytokine profiling experiments showed that at 14 days post‐FCA injection there were increased levels of IL‐4, IL‐10, IFNγ and GM‐CSF in paws from the FCA‐injected GPR55−/− mice when compared with the FCA‐injected GPR55+/+ mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55−/− mice. In the model of neuropathic hypersensitivity, GPR55−/− mice also failed to develop mechanical hyperalgesia up to 28 days post‐ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

Mating in mushrooms: increasing the chances but prolonging the affair

Finding a compatible mating partner is an essential step in the life cycle of most sexually reproducing organisms. Fungi have two or more mating types, and only cells of different mating type combine to produce diploid cells. In mushrooms, this is taken to extremes, with the occurrence of many thousands of mating types. But, having gone to such extraordinary lengths to ensure that almost any two mushroom mycelia in the wild can mate, cell fusion is not followed by nuclear fusion and true diploidy. Instead, the fused cells form a characteristic mycelium, known as the dikaryon, in which haploid nuclei are paired but actively prevented from fusing. The mating-type genes, which encode pheromones, pheromone receptors and homeodomain transcription factors, have crucial roles in regulating the complex developmental programme by which the dikaryon is formed.

Yeast assays for G-protein-coupled receptors

The functional coupling of heterologous G protein-coupled receptors (GPCRs) to the pheromone-response pathway of the budding yeast Saccharomyces cerevisiae is well established as an experimental system for ligand identification and for characterizing receptor pharmacology and signal transduction mechanisms. A number of groups have developed yeast strains using various modifications to this signaling pathway, especially manipulation of the G protein alpha subunit Gpa1p, to facilitate coupling of a wide range of mammalian GPCRs. The attraction of these systems is the simplicity and low cost of yeast cell culture enabling the assays to be set up rapidly in academic or industrial labs without the requirement for expensive technical equipment. Furthermore, haploid yeasts contain only a single GPCR capable of activating the pathway, which can be deleted and replaced with a mammalian GPCR providing a cell-based functional assay in a eukaryotic host free from endogenous responses. The yeast strains used for this purpose are highly engineered and may be covered by intellectual property for commercial applications in some countries. However, they can usually be obtained from the host labs for research purposes covered by a Material Transfer Agreement and/or licence where appropriate. The protocols herein assume that such strains have been acquired and begin with introduction of the heterologous GPCR into the engineered yeast cell. Assays are configured such that agonism of the GPCR leads to induction of a reporter gene and/or growth of the yeast. A number of parameters may be optimized to generate robust experimental formats, in high-density microtiter plates, that may be used for ligand identification and pharmacological characterization.

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB2 receptor (CB2R), but additional modulatory sites distinct from CB2R have recently been suggested to impact CB2R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB2R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB2R interfere with each others signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB2R, while it synergizes with CB2R in recruiting neutrophils to sites of inflammation.

Discovery of 2-[(2,4-Dichlorophenyl)amino]-N-[(tetrahydro- 2H-pyran-4-yl)methyl]-4-(trifluoromethyl)- 5-pyrimidinecarboxamide, a Selective CB2 Receptor Agonist for the Treatment of Inflammatory Pain

Selective CB2 receptor agonists are promising potential therapeutic agents for the treatment of inflammatory and neuropathic pain. A focused screen identified a pyrimidine ester as a partial agonist at the CB2 receptor with micromolar potency. Subsequent lead optimization identified 35, GW842166X, as the optimal compound in the series. 35 has an oral ED50 of 0.1 mg/kg in the rat FCA model of inflammatory pain and was selected as a clinical candidate for this indication.

Minireview: Recent Developments in the Physiology and Pathology of the Lysophosphatidylinositol-Sensitive Receptor GPR55

Emerging data suggest that off-target cannabinoid effects may be mediated via novel seven-transmembrane spanning/G protein-coupled receptors. Due to its cannabinoid sensitivity, the G protein-coupled receptor 55 (GPR55) was recently proposed as a candidate; however, GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, and the conflicting pharmacology, signaling, and functional data have prevented its classification as a novel cannabinoid receptor. Indeed, the most consistent and potent agonist to date is the noncannabinoid lysophospholipid, lysophosphatidylinositol. Here we present new human GPR55 mRNA expression data, providing supportive evidence of GPR55 expression in a vast array of tissues and cell types. Moreover, we summarize major recent developments in GPR55 research and aim to update the reader in the rapidly expanding fields of GPR55 pharmacology, physiology, and pathology.

Screening β-Arrestin Recruitment for the Identification of Natural Ligands for Orphan G-Protein–Coupled Receptors:

A variety of G-protein–coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated β-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward β-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased β-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter β-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of β-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. β-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through β-arrestin in response to their native ligand remain to be determined.

Uncovering the Pharmacology of the G Protein-Coupled Receptor GPR40: High Apparent Constitutive Activity in Guanosine 5′-O-(3-[35S]thio)triphosphate Binding Studies Reflects Binding of an Endogenous Agonist

In cells lacking expression of Ca2+-mobilizing G proteins, coexpression of human GPR40 and Gαq allowed medium- and long-chain fatty acids to elevate intracellular [Ca2+]. This was also observed when human embryonic kidney (HEK) 293 cells were transfected with a GPR40-Gαq fusion protein. The kinetic of elevation of intracellular [Ca2+] slowed with increasing fatty acid chain length, suggesting different ligand on-rates, whereas the addition of fatty acid-free bovine serum albumin reduced signals, presumably by binding the fatty acids. To allow effective ligand equilibration, GPR40-Gαq was used in guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays. After expression of GPR40-Gαq in HEK293 cells and membrane preparation basal binding of [35S]GTPγSinGαq immunoprecipitates was high and not elevated substantially by fatty acids. However, treatment of membranes with fatty acid-free bovine serum albumin reduced the basal [35S]GTPγS binding in a concentration-dependent manner and allowed the responsiveness and pharmacology at GPR40 of each of the fatty acids thiazolidinediones and a novel small-molecule agonist to be uncovered. Membranes of rat INS-1E cells that express GPR40 endogenously provided similar observations. The high apparent constitutive activity of GPR40-Gαq was also reversed by a small-molecule GPR40 antagonist, and basal [35S]GTPγS binding was prevented by the selective Gαq/Gα11 inhibitor YM-254890. The current studies provide novel insights into the pharmacology of GPR40 and indicate that G protein-coupled receptors which respond to fatty acids, and potentially to other lipid ligands, can be occupied by endogenous agonists before assay and that this may mask the pharmacology of the receptor and may be mistaken for high levels of constitutive activity.

A Selective Antagonist Reveals a Potential Role of G Protein–Coupled Receptor 55 in Platelet and Endothelial Cell Function

The G protein–coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca2+ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

Discovery of 1-[4-(3-Chlorophenylamino)-1-methyl-1H-pyrrolo[3,2-c]pyridin-7-yl]-1-morpholin-4-ylmethanone (GSK554418A), a Brain Penetrant 5-Azaindole CB2 Agonist for the Treatment of Chronic Pain

We report the synthesis and SAR of a series of novel azaindole CB(2) agonists. 6-Azaindole 18 showed activity in an acute pain model but was inactive in a chronic model. 18 is a Pgp substrate with low brain penetration. The template was redesigned, and the resulting 5-azaindole 36 was a potent CB(2) agonist with high CNS penetration. This compound was efficacious in the acute model and the chronic joint pain model.