Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Multipathway In Vitro Pharmacological Characterization of Specialized Proresolving G Protein-Coupled Receptors.

Specialized proresolving mediators (SPMs) and their cognate G protein-coupled receptors are implicated in autoimmune disorders, including chronic inflammation, rheumatoid arthritis, systemic scleroderma, and lupus erythematosus. To date, six G protein-coupled receptors (GPCRs) have been paired with numerous endogenous and synthetic ligands. However, the function and downstream signaling of these receptors remains unclear. To address this knowledge gap, we systematically expressed each receptor in a human embryonic kindney 293 (HEK293)-Flp-In-CD8a-FLAG cell system. Each receptor was pharmacologically characterized with both synthetic and putative endogenous ligands across different signaling assays, covering both G protein-dependent (Gs, Gi, and Gq) and independent mechanisms (ß-arrestin2 recruitment). Three orphan GPCRs previously identified as SPM receptors (GPR 18, GPR32 and GPR37) failed to express in HEK 293 cells. Although we were unsuccessful in identifying an endogenous ligand for formyl peptide receptor 2 (FPR2)/lipoxin A4 receptor (ALX), with only a modest response to N-formylmethionine-leucyl-phenylalanine (fMLP), we did reveal clear signaling bias away from extracelluar signal-related kinase (ERK) 1/2 phosphorylation for the clinically tested agonist N-(2-{[4-(1,1-difluoroethyl)-1,3-oxazol-2-yl]methyl}-2H-1,2,3-triazol-4-yl)-2-methyl-5-(3-methylphenyl)-1,3-oxazole-4-carboxamide (ACT-389949), adding further evidence for its poor efficacy in two phase I studies. We also identified neuroprotectin D1 as a new leukotriene B4 receptor 1 (BLT1) agonist, implying an alternative target for the neuroprotective effects of the ligand. We confirmed activity for resolvin E1 (RvE1) at BLT1 but failed to observe any response at the chemerin1 receptor. This study provides some much-needed clarity around published receptor-ligand pairings but indicates that the expression and function of these SPM GPCRs remains very much context-dependent. In addition, the identification of signaling bias at FPR2/ALX may assist in guiding design of new FPR2/ALX agonists for the treatment of autoimmune disorders. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to comprehensibly show how several natural mediators and synthetic ligands signal through three specialized proresolving mediator GPCRs using multiple ligands from different classes across four-six endpoint signaling assays. This study discovers new ligand pairings, refutes others, reveals poly-pharmacology, and identifies biased agonism in formyl peptide receptor 2/lipoxin A4 receptor pharmacology. This study highlights the potential of these receptors in treating specific autoimmune diseases, including rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus.

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms.

INTRODUCTION: A potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are complicated by non-selective deletion of Gpr21. RESEARCH DESIGN AND METHODS: We hypothesized that a Gpr21-selective knockout mouse model, coupled with type 2 diabetes patient samples, would clarify these issues and enable clear assessment of GPR21 as a potential therapeutic target. RESULTS: High-fat feeding studies in Gpr21-/- mice revealed improved glucose tolerance and modest changes in inflammatory gene expression. Gpr21-/- monocytes and intraperitoneal macrophages had selectively impaired chemotactic responses to monocyte chemoattractant protein (MCP)-1, despite unaltered expression of Ccr2. Further genotypic analysis revealed that chemotactic impairment was due to dysregulated monocyte polarization. Patient samples revealed elevated GPR21 expression in peripheral blood mononuclear cells in type 2 diabetes, which was correlated with both %HbA1c and fasting plasma glucose levels. CONCLUSIONS: Collectively, human and mouse data suggest that GPR21 influences both glucose homeostasis and MCP-1/CCL2-CCR2-driven monocyte migration. However, a Gpr21-/- bone marrow transplantation and high-fat feeding study in mice revealed no effect on glucose homeostasis, suggesting that there is no (or limited) overlap in the mechanism involved for monocyte-driven inflammation and glucose homeostasis.

New Advances in Targeting the Resolution of Inflammation: Implications for Specialized Pro-Resolving Mediator GPCR Drug Discovery.

Chronic inflammation is a component of numerous diseases including autoimmune, metabolic, neurodegenerative, and cancer. The discovery and characterization of specialized pro-resolving mediators (SPMs) critical to the resolution of inflammation, and their cognate G protein-coupled receptors (GPCRs) has led to a significant increase in the understanding of this physiological process. Approximately 20 ligands, including lipoxins, resolvins, maresins, and protectins, and 6 receptors (FPR2/ALX, GPR32, GPR18, chemerin1, BLT1, and GPR37) have been identified highlighting the complex and multilayered nature of resolution. Therapeutic efforts in targeting these receptors have proved challenging, with very few ligands apparently progressing through to preclinical or clinical development. To date, some knowledge gaps remain in the understanding of how the activation of these receptors, and their downstream signaling, results in efficient resolution via apoptosis, phagocytosis, and efferocytosis of polymorphonuclear leukocytes (mainly neutrophils) and macrophages. SPMs bind and activate multiple receptors (ligand poly-pharmacology), while most receptors are activated by multiple ligands (receptor pleiotropy). In addition, allosteric binding sites have been identified signifying the capacity of more than one ligand to bind simultaneously. These fundamental characteristics of SPM receptors enable alternative targeting strategies to be considered, including biased signaling and allosteric modulation. This review describes those ligands and receptors involved in the resolution of inflammation, and highlights the most recent clinical trial results. Furthermore, we describe alternative mechanisms by which these SPM receptors could be targeted, paving the way for the identification of new therapeutics, perhaps with greater efficacy and fidelity.

Acetylcholine Muscarinic M4 Receptors as a Therapeutic Target for Alcohol Use Disorder: Converging Evidence From Humans and Rodents.

BACKGROUND: Alcohol use disorder (AUD) is a major socioeconomic burden on society, and current pharmacotherapeutic treatment options are inadequate. Aberrant alcohol use and seeking alters frontostriatal function. METHODS: We performed genome-wide RNA sequencing and subsequent quantitative polymerase chain reaction and receptor binding validation in the caudate-putamen of human AUD samples to identify potential therapeutic targets. We then back-translated our top candidate targets into a rodent model of long-term alcohol consumption to assess concordance of molecular adaptations in the rat striatum. Finally, we adopted rat behavioral models of alcohol intake and seeking to validate a potential therapeutic target. RESULTS: We found that G protein-coupled receptors were the top canonical pathway differentially regulated in individuals with AUD. The M4 muscarinic acetylcholine receptor (mAChR) was downregulated at the gene and protein levels in the putamen, but not in the caudate, of AUD samples. We found concordant downregulation of the M4 mAChR, specifically on dopamine D1 receptor-expressing medium spiny neurons in the rat dorsolateral striatum. Systemic administration of the selective M4 mAChR positive allosteric modulator, VU0467154, reduced home cage and operant alcohol self-administration, motivation to obtain alcohol, and cue-induced reinstatement of alcohol seeking in rats. Local microinjections of VU0467154 in the rat dorsolateral striatum reduced alcohol self-administration and cue-induced reinstatement of alcohol seeking. CONCLUSIONS: Collectively, these results identify the M4 mAChR as a potential therapeutic target for the treatment of AUD and the D1 receptor-positive medium spiny neurons in the dorsolateral striatum as a key site mediating the actions of M4 mAChR in relation to alcohol consumption and seeking.

Drug-receptor kinetics and sigma-1 receptor affinity differentiate clinically evaluated histamine H3 receptor antagonists.

The histamine H3 receptor is a G protein-coupled receptor (GPCR) drug target that is highly expressed in the CNS, where it acts as both an auto- and hetero-receptor to regulate neurotransmission. As such, it has been considered as a relevant target in disorders as varied as Alzheimer's disease, schizophrenia, neuropathic pain and attention deficit hyperactivity disorder. A range of competitive antagonists/inverse agonists have progressed into clinical development, with pitolisant approved for the treatment of narcolepsy. Given the breadth of compounds developed and potential therapeutic indications, we assessed the comparative pharmacology of six investigational histamine H3 agents, including pitolisant, using native tissue and recombinant cells. Whilst all of the compounds tested displayed robust histamine H3 receptor inverse agonism and did not differentiate between the main H3 receptor splice variants, they displayed a wide range of affinities and kinetic properties, and included rapidly dissociating (pitolisant, S 38093-2, ABT-239) and slowly dissociating (GSK189254, JNJ-5207852, PF-3654746) agents. S 38093-2 had the lowest histamine H3 receptor affinity (pKB values 5.7-6.2), seemingly at odds with previously reported, potent in vivo activity in models of cognition. We show here that at pro-cognitive and anti-hyperalgesic/anti-allodynic doses, S 38093-2 preferentially occupies the mouse sigma-1 receptor in vivo, only engaging the histamine H3 receptor at doses associated with wakefulness promotion and neurotransmitter (histamine, ACh) release. Furthermore, pitolisant, ABT-239 and PF-3654746 also displayed appreciable sigma-1 receptor affinity, suggesting that this property differentiates clinically evaluated histamine H3 receptor antagonists and may play a role in their efficacy.

Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research.

Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.

G Protein-Coupled Receptors Targeting Insulin Resistance, Obesity, and Type 2 Diabetes Mellitus.

G protein-coupled receptors (GPCRs) continue to be important discovery targets for the treatment of type 2 diabetes mellitus (T2DM). Many GPCRs are directly involved in the development of insulin resistance and ß-cell dysfunction, and in the etiology of inflammation that can lead to obesity-induced T2DM. This review summarizes the current literature describing a number of well-validated GPCR targets, but also outlines several new and promising targets for drug discovery. We highlight the importance of understanding the role of these receptors in the disease pathology, and their basic pharmacology, which will pave the way to the development of novel pharmacological probes that will enable these targets to fulfill their promise for the treatment of these metabolic disorders.

Isoform-Specific Biased Agonism of Histamine H3 Receptor Agonists.

The human histamine H3 receptor (hH3R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H3R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH3R splice variants (hH3R445 and hH3R365) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3ß (GSK3ß) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hH3R365 is more permissive in its signaling than hH3R445: 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH3R365 was completely unable to stimulate GSK3ß phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G protein-coupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants.

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy.

Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [(3)H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2(PGD2). The binding kinetics of QAW039 determined directly using [(3)H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 10(7)M(-1)min(-1)and 0.048 minute(-1), respectively. Importantly, thekoffof QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [(35)S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2concentrations, which may be clinically relevant.

Label-Free Kinetics: Exploiting Functional Hemi-Equilibrium to Derive Rate Constants for Muscarinic Receptor Antagonists.

Drug receptor kinetics is as a key component in drug discovery, development, and efficacy; however, determining kinetic parameters has historically required direct radiolabeling or competition with a labeled tracer. Here we present a simple approach to determining the kinetics of competitive antagonists of G protein-coupled receptors by exploiting the phenomenon of hemi-equilibrium, the state of partial re-equilibration of agonist, antagonist, and receptor in some functional assays. Using functional [Ca(2+)]i-flux and extracellular kinases 1 and 2 phosphorylation assays that have short incubation times and therefore are prone to hemi-equilibrium "behaviors," we investigated a wide range of structurally and physicochemically distinct muscarinic acetylcholine receptor antagonists. Using a combined operational and hemi-equilibrium model of antagonism to both simulate and analyze data, we derived estimates of association and dissociation rates for the test set of antagonists, identifying both rapidly dissociating (4-DAMP, himbacine) and slowly dissociating (tiotropium, glycopyrrolate) ligands. The results demonstrate the importance of assay incubation time and the degree of receptor reserve in applying the analytical model. There was an excellent correlation between estimates of antagonist pK(B), k(on), and k(off) from functional assays and those determined by competition kinetics using whole-cell [(3)H]N-methylscopolamine binding, validating this approach as a rapid and simple method to functionally profile receptor kinetics of competitive antagonists in the absence of a labeled tracer.

Investigating the molecular mechanisms through which FTY720-P causes persistent S1P1 receptor internalization.

BACKGROUND AND PURPOSE: The molecular mechanism underlying the clinical efficacy of FTY720-P is thought to involve persistent internalization and enhanced degradation of the S1P1 receptor subtype (S1P1R). We have investigated whether receptor binding kinetics and ß-arrestin recruitment could play a role in the persistent internalization of the S1P1R by FTY720-P. EXPERIMENTAL APPROACH: [(3) H]-FTY720-P and [(33) P]-S1P were used to label CHO-S1P1/3Rs for binding studies. Ligand efficacy was assessed through [(35) S]-GTPγS binding and ß-arrestin recruitment. Metabolic stability was evaluated using a bioassay measuring intracellular Ca(2+) release. CHO-S1P1/3R numbers were determined, following FTY720-P treatment using flow cytometry. KEY RESULTS: The kinetic off-rate of [(3) H]-FTY720-P from the S1P1R was sixfold slower than from the S1P3R, and comparable to [(33) P]-S1P dissociation from S1P1/3Rs. S1P and FTY720-P stimulated [(35) S]-GTPγS incorporation to similar degrees, but FTY720-P was over 30-fold less potent at S1P3Rs. FTY720-P stimulated a higher level of ß-arrestin recruitment at S1P1Rs, 132% of the total recruited by S1P. In contrast, FTY720-P was a weak partial agonist at S1P3R, stimulating just 29% of the total ß-arrestin recruited by S1P. Internalization experiments confirmed that cell surface expression of the S1P1R but not the S1P3R was reduced following a pulse exposure to FTY720-P, which is metabolically stable unlike S1P. CONCLUSIONS AND IMPLICATIONS: FTY720-P and S1P activation of the S1P1R results in receptor internalization as a consequence of an efficient recruitment of ß-arrestin. The combination of slow off-rate, efficacious ß-arrestin recruitment and metabolic stability all contribute to FTY720-P's ability to promote prolonged S1P1R internalization and may be critical factors in its efficacy in the clinic.