Glycine: a long-sought novel ligand for GPR158.

G-protein-coupled receptors (GPCRs) are important drug targets with chemically diverse ligands and varying intracellular coupling partners. Recent work by Laboute et al. deorphanized GPR158 as a metabotropic glycine receptor (mGlyR), thereby providing evidence of a novel neuromodulatory system involving this non-canonical Class C receptor with an impact on cognition and affective states.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Glepaglutide, a novel glucagon-like peptide-2 agonist, has anti-inflammatory and mucosal regenerative effects in an experimental model of inflammatory bowel disease in rats.

BACKGROUND: Glucagon-like peptide-2 (GLP-2) enhances intestinal repair and attenuates inflammation in preclinical inflammatory bowel disease (IBD) models, making GLP-2 analogues attractive candidates for IBD therapy. Glepaglutide is a long-acting GLP-2 receptor agonist in clinical development for treatment of short bowel syndrome. Here, we investigated if glepaglutide is therapeutically beneficial in rats with small intestinal inflammation. METHODS: Small intestinal inflammation was induced with indomethacin in naive Wistar rats, followed by glepaglutide administration at different disease stages. Glepaglutide was administered in co-treatment and post-treatment regimens. Small intestinal length and concentrations of inflammatory markers α-1-acid glycoprotein and myeloperoxidase were used to assess anti-inflammatory effects. Small intestinal mass was evaluated to determine intestinotrophic effects. RESULTS: Glepaglutide co- and post-treatment significantly reduced severity of small intestinal inflammation, evidenced by reversed small intestinal shortening and decreased α-1-acid glycoprotein and/or myeloperoxidase concentration(s). Co- and post-treatment with glepaglutide also significantly increased small intestinal mass, indicating intestinal regenerative effects. Similar effects were observed in naive rats after glepaglutide treatment. CONCLUSION: Glepaglutide has anti-inflammatory and intestinotrophic effects without the need for pre-treatment in a rat model of small intestinal inflammation. Thus, glepaglutide is of potential clinical interest for patients with IBD.

Structural insights into differences in G protein activation by family A and family B GPCRs.

Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the ß2 adrenergic receptor (ß2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the ß2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

Probing the Existence of a Metastable Binding Site at the ß2-Adrenergic Receptor with Homobivalent Bitopic Ligands.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the ß2-adrenergic receptor (ß2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased ß2/ß1AR-selectivity, and/or substantially slower ß2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the ß2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

Gs protein peptidomimetics as allosteric modulators of the ß2-adrenergic receptor.

A series of Gs protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state ß2-adrenergic receptor (ß2AR) in complex with the Gs protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular Gs protein binding site of the ß2AR. Peptidomimetics were designed to mimic the 15 residue C-terminal α-helix of the Gs protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified ß2AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of ß2AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native Gs protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation.

A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors.

Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)-based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in living cells. We used the ß2-adrenergic receptor (ß(2)AR) as a representative Gs-coupled receptor and characterized two cell lines with different expression levels. Low receptor expression allowed detection of desensitization kinetics and delineation of partial agonism, whereas high receptor expression resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of ß(2)AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS.

Real-time trafficking and signaling of the glucagon-like peptide-1 receptor.

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.

cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors.

Cyclic adenosine monophosphate (cAMP) is a common second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of G(s)-coupled G protein-coupled receptors (GPCRs) and decreased by activation of G(i)-coupled GPCRs via the adenylyl cyclase. Many end-point assays for quantifying GPCR-mediated changes in intracellular cAMP levels exist. More recently, fluorescence resonance energy transfer (FRET)-based cAMP biosensors that can quantify intracellular cAMP levels in real time have been developed. These FRET-based cAMP biosensors have been used primarily in single cell FRET microscopy to monitor and visualize changes in cAMP upon GPCR activation. Here, a similar cAMP biosensor with a more efficient mCerulean/mCitrine FRET pair is described for use in the 384-well plate format. After cloning and expression in HEK293 cells, the biosensor is characterized in the 384-well plate format and used for measuring the signaling of the G(s)-coupled ß(2)-adrenergic receptor. The procedures described may be applied for other FRET-based biosensors in terms of characterization and conversion to the 384-well plate format.

A new metabotropic glutamate receptor agonist with in vivo anti-allodynic activity.

As part of the vital search towards improved therapeutic agents for the treatment of neuropathic pain, the central nervous system glutamate receptors have become a major focus of research. Outlined herein are the syntheses of two new biologically active 3'-cycloalkyl-substituted carboxycyclopropylglycines, utilizing novel synthetic chemistry. The reaction between substituted 1,2-dioxines and an aminophosphonate furnished the cyclopropane core in a single step with all required stereochemistry of pendant groups. In vitro binding assays at metabotropic glutamate receptors revealed selective activity. In vivo testing in a rodent model of neuropathic pain indicated one amino acid significantly and dose-dependently decreased mechanical allodynia.

The C-terminal tail of CRTH2 is a key molecular determinant that constrains Galphai and downstream signaling cascade activation.

Prostaglandin D(2) activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galpha(i) and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galpha(i) proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galpha(i) signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

Antagonism of the prostaglandin D2 receptor CRTH2 attenuates asthma pathology in mouse eosinophilic airway inflammation.

BACKGROUND: Mast cell-derived prostaglandin D2 (PGD2), may contribute to eosinophilic inflammation and mucus production in allergic asthma. Chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2), a high affinity receptor for prostaglandin D2, mediates trafficking of TH2-cells, mast cells, and eosinophils to inflammatory sites, and has recently attracted interest as target for treatment of allergic airway diseases. The present study involving mice explores the specificity of CRTH2 antagonism of TM30089, which is structurally closely related to the dual TP/CRTH2 antagonist ramatroban, and compares the ability of ramatroban and TM30089 to inhibit asthma-like pathology. METHODS: Affinity for and antagonistic potency of TM30089 on many mouse receptors including thromboxane A2 receptor mTP, CRTH2 receptor, and selected anaphylatoxin and chemokines receptors were determined in recombinant expression systems in vitro. In vivo effects of TM30089 and ramatroban on tissue eosinophilia and mucus cell histopathology were examined in a mouse asthma model. RESULTS: TM30089, displayed high selectivity for and antagonistic potency on mouse CRTH2 but lacked affinity to TP and many other receptors including the related anaphylatoxin C3a and C5a receptors, selected chemokine receptors and the cyclooxygenase isoforms 1 and 2 which are all recognized players in allergic diseases. Furthermore, TM30089 and ramatroban, the latter used as a reference herein, similarly inhibited asthma pathology in vivo by reducing peribronchial eosinophilia and mucus cell hyperplasia. CONCLUSION: This is the first report to demonstrate anti-allergic efficacy in vivo of a highly selective small molecule CRTH2 antagonist. Our data suggest that CRTH2 antagonism alone is effective in mouse allergic airway inflammation even to the extent that this mechanism can explain the efficacy of ramatroban.

Novel selective orally active CRTH2 antagonists for allergic inflammation developed from in silico derived hits.

Hits from an in silico derived focused library for CRTH2 were transformed into highly selective antagonists with favorable ADME properties. Oral administration of 4-bromo-2-(1-phenyl-1H-pyrazole-4-carbonyl)phenoxyacetic acid (19) inhibited peribronchial eosinophilia and mucus cell hyperplasia in a mouse model of allergic asthma, supporting the therapeutic potential of this novel compound class. In addition, this selective pharmacological tool compound provides further evidence for CRTH2 as a relevant therapeutic target for treatment of Th2- and eosinophil-related inflammation.

The metabotropic glutamate receptor 4 is internalized and desensitized upon protein kinase C activation.

1. The metabotropic glutamate receptor 4 (mGluR4) is a Galphai-coupled receptor that modulates glutamatergic neurotransmission. As mGluR4 expression and activation have been implicated in a number of pathological conditions and because the internalization and desensitization properties of this receptor are poorly understood, studies were designed to investigate these aspects of mGluR4 biology. 2. Neither agonist activation by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) nor L-glutamate caused mGluR4 internalization when cmyc-tagged mGluR4 was expressed in a human embryonic kidney 293 cell line as assessed by cell surface enzyme-linked immunosorbent and immunostaining assays. Instead, a modest increase in mGluR4 surface expression was observed and found to be receptor specific as the competitive antagonist alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked this effect. 3. In contrast, mGluR4 internalized when the protein kinase C (PKC) pathway was activated either by phorbol-12-myristate-13-acetate (PMA) or by the activation of the Galphaq-coupled, neurokinin 3 receptor (NK3R) when co-expressed. This process was PKC-dependent as the specific PKC inhibitor GF 109203X inhibited PMA and NK3R-mediated internalization. 4. PKC activation by PMA caused desensitization of mGluR4 as measured by forskolin-stimulated cAMP inhibition, whereas agonist activation had no effect on desensitization. 5. When mGluR4's coupling was redirected from adenylyl cyclase to phospholipase C by coexpression of a chimeric Galphaqo5 protein, mGluR4 both internalized and desensitized in response to its agonists. 6. These findings demonstrate that mGluR4 internalization and desensitization are agonist-independent unless pathways leading to the activation of PKC are induced.

On the mechanism of interaction of potent surmountable and insurmountable antagonists with the prostaglandin D2 receptor CRTH2.

Chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2) has attracted interest as a potential therapeutic target in inflammatory diseases. Ramatroban, a thromboxane A2 receptor antagonist with clinical efficacy in allergic rhinitis, was recently found to also display potent CRTH2 antagonistic activity. Here, we present the pharmacological profile of three ramatroban analogs that differ chemically from ramatroban by either a single additional methyl group (TM30642), or an acetic acid instead of a propionic acid side chain (TM30643), or both modifications (TM30089). All three compounds bound to human CRTH2 stably expressed in human embryonic kidney 293 cells with nanomolar affinity. [3H]Prostaglandin D2 (PGD2) saturation analysis reveals that ramatroban and TM30642 decrease PGD2 affinity, whereas TM30643 and TM30089 exclusively depress ligand binding capacity (Bmax). Each of the three compounds acted as potent CRTH2 antagonists, yet the nature of their antagonism differed markedly. In functional assays measuring inhibition of PGD2-mediated 1) guanosine 5'-O-(3-thio)triphosphate binding, 2) beta-arrestin translocation, and 3) shape change of human eosinophils endogenously expressing CRTH2, ramatroban, and TM30642 produced surmountable antagonism and parallel rightward shifts of the PGD2 concentration-response curves. For TM30643 and TM30089, this shift was accompanied by a progressive reduction of maximal response. Binding analyses indicated that the functional insurmountability of TM30643 and TM30089 was probably related to long-lasting CRTH2 inhibition mediated via the orthosteric site of the receptor. A mechanistic understanding of insurmountability of CRTH2 antagonists could be fundamental for development of this novel class of anti-inflammatory drugs.

Molecular pharmacology and therapeutic prospects of metabotropic glutamate receptor allosteric modulators.

The metabotropic glutamate receptors (mGluR) consist of a family of eight G-protein-coupled receptors that differ in their function, distribution and physiological roles within the central nervous system. In recent years substantial efforts have been made towards developing selective agonists and antagonists which have proven useful for elucidating their potential as novel targets for the treatment of psychiatric and neurological diseases. In the present review we will provide an update of the recent developments of functional allosteric modulators of the mGluR family and explore their therapeutic potential for anxiety/depression, schizophrenia, epilepsy/stroke, pain and Alzheimer's, Parkinson's and Huntington's diseases.

Identification of indole derivatives exclusively interfering with a G protein-independent signaling pathway of the prostaglandin D2 receptor CRTH2.

The anti-inflammatory drugs indomethacin and ramatroban, the latter showing clinical efficacy in treating allergic asthma, have been shown to act as a classic agonist and antagonist, respectively, of the G protein-coupled chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 receptor). Here, we report the identification of two indole derivatives 1-(4-ethoxyphenyl)-5-methoxy-2-methylindole-3-carboxylic acid and N(alpha)-tosyltryptophan (hereafter referred to as 1 and 2, respectively), which are structurally related to indomethacin and ramatroban but which selectively interfere with a specific G protein-independent signaling pathway of CRTH2. In whole-cell saturation-binding assays, 1 and 2 both increase the number of [(3)H]prostaglandin D2 (PGD2)-recognizing CRTH2 sites and the affinity of PGD2 for CRTH2. Enzyme-linked immunosorbent assays show that they do not alter the total number of CRTH2 receptors on the cell surface. Analysis of their binding mode indicates that unlike indomethacin or ramatroban, 1 and 2 can occupy CRTH2 simultaneously with PGD2. On a functional level, however, 1 and 2 do not interfere with PGD2-mediated activation of heterotrimeric G proteins by CRTH2. In contrast, both compounds inhibit PGD2-mediated arrestin translocation via a G protein-independent mechanism. In human eosinophils endogenously expressing CRTH2, 1 selectively decreases the efficacy but not the potency of PGD2-induced shape change, unlike ramatroban, which displays competitive antagonistic behavior. These data show for the first time that "antagonists" can cause markedly dissimilar degrees of inhibition for different effector pathways and suggest that it may be possible to develop novel classes of specific signal-inhibiting drugs distinct from conventional antagonists.

Positive allosteric modulation of the human metabotropic glutamate receptor 4 (hmGluR4) by SIB-1893 and MPEP.

We have identified 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) and 2-methyl-6-phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB-1893 and MPEP enhanced the potency and efficacy of L-2-amino-4-phophonobutyrate (L-AP4) in guanosine 5'-O-(3-[(35)S]thiotriphosphate ([(35)S]GTPgammaS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB-1893 and MPEP had no effects alone in GTPgammaS binding, effects were observed in the cell-based cAMP assay due to media-derived activation as indicated by CPPG inhibition. Positive modulation of the mGluR4 was a receptor-specific effect since SIB-1893 and MPEP had neither effects on mGluR2-expressing cells nor on the parent BHK cell line. In [(3)H]L-AP4 binding, a two-fold decrease in K(D) but not in B(max) was observed with 100 micro M SIB-1893, whereas MPEP affected neither parameter. Finally, SIB-1893 and MPEP failed to displace [(3)H]L-AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.