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Cyclic adenosine 3’,5’-monophosphate is an important second messenger molecule that regulates many downstream signaling pathways in cells. Detection of cAMP levels relies on screenings of cell lysates or the use of genetically encoded biosensors for detection in living cells. Genetically encoded biosensors are, however, primarily used for bioimaging and rarely in high-throughput screenings of potential drug candidates. Here, we describe a quantitative fluorescence-based imaging method based on measurements of single living cells. We used a genetically encoded Epac149 biosensor to investigate cAMP production in living cells following ligand stimulation. The study revealed a dependence of the measured cAMP levels on the expression level of the biosensor in transiently transfected cells. While the biosensor maintained linearity of the signal at high expression levels, the linearity of the biosensor was lost at lower expression levels due to a deficit of the biosensor compared to the maximum possible production of cAMP in the cells. This problem was circumvented by establishment of a stable cell line with constitutive expression of the biosensor. We established dose response curves by stimulation with the β1-adrenergic receptor partial agonist denopamine and observed up to 1.48-fold difference in the cellular response as well as up to 4.27-fold difference in LogEC50 values between cells with insufficient and sufficient biosensor expression. Careful characterization and control of the biosensor expression is therefore important in order to conduct quantitative analysis of the cAMP production and it allows the use of genetically encoded biosensor to be applied in high-throughput screenings.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling.

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 β2-adrenergic receptor (β2AR) 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 β2/β1AR-selectivity, and/or substantially slower β2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the β2AR, 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.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6–TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6–TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

INTRODUCTIONTransmembrane signaling of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) is mediated by ligand-dependent conformational changes that are transmitted from the extracellular ligand binding site to the intracellular side of the receptor to allow coupling with transducers. One hallmark of GPCR activation is the outward movement of the cytoplasmic end of transmembrane domain 6 (TM6) that opens up an intracellular cavity to accommodate the Gα subunit, leading to nucleotide exchange and activation of the G protein. Comparison of family A and family B receptor-Gs protein complex structures has revealed substantial differences in the conformational changes of TM6 upon activation. In family B GPCRs, TM6 shows a disruption of the helical fold and the formation of a sharp kink. This differs from the gradual bending in TM6 observed in family A GPCRs.RATIONALEDespite the recent surge of determined GPCR–G protein complex structures, the activation mechanism of family B receptors remains poorly understood. The missing conserved structural motifs found in family A GPCRs together with the marked differences in the conformation of TM6 in the active state suggest distinct activation mechanisms between family B and family A GPCRs. In particular, the disruption of the helical fold and the unraveling of the extracellular end of TM6 suggest that the energy required to produce the fully active-state of family B GPCRs is higher than for family A GPCRs. We investigated the functional impact of these structural differences by comparing the structure and function of a prototypical family B receptor, the glucagon receptor (GCGR), with the β2 adrenergic receptor (β2AR), a family A GPCR.RESULTSWe present the cryo–electron microscopy structure of the GCGR-Gs complex bound to an engineered soluble glucagon derivative. The structure shows that full activation of GCGR leads to a disruption in the α-helix of TM6 typical for family B GPCRs. Analysis of the functional consequence of this helix break on receptor-mediated G protein dissociation and guanosine triphosphate (GTP) turnover reveals that GCGR exhibits a substantially lower guanine nucleotide exchange factor (GEF) activity in comparison with the family A receptor β2AR. Characterization of G protein association, guanosine diphosphate (GDP) release, and GTP binding kinetics shows that the receptor-mediated GDP dissociation and GTP binding of Gs are slower for GCGR than for β2AR. Measurements of ligand-dependent conformational alterations of GCGR by means of fluorescence and double electron-electron resonance spectroscopy show that agonist binding alone is insufficient to promote TM6 opening, in contrast to previously studied family A GPCRs, including β2AR. The outward movement of TM6 of GCGR is only observed upon interaction with Gs, suggesting that TM6 activation is only triggered by the engagement of the α5 helix of Gαs. Furthermore, TM6 of GCGR remains in the active state for a prolonged time after disengagement of Gs, which might contribute to the persistent and sustained cyclic adenosine monophosphate (cAMP) signaling previously observed for this receptor. A comprehensive comparison of the G protein activation kinetics for a number of other family A and family B GPCRs shows that family B receptors are in general less efficient than family A GPCRs in triggering G protein signaling.CONCLUSIONOur findings provide evidence for distinct activation mechanisms between family A and family B GPCRs. We propose that formation of the helix break and the sharp kink in TM6 of GCGR requires overcoming a higher energy barrier than the bending and outward movement of TM6 in family A receptors. Because of this kinetic barrier, ligand binding alone is not sufficient to stabilize the outward movement of TM6 but promotes the initial formation of the receptor–G protein complex and subsequent full engagement of the G protein at a slower time scale. Once activated by the insertion of the α5 helix of Gαs into the receptor core, as seen in the nucleotide-free complex structure, TM6 stays in the active conformation long after full disengagement of the G protein. This may be responsible for the previously described sustained and prolonged signaling of GCGR. Our comprehensive comparison of the G protein activation kinetics of family A and family B receptors suggests that the activation mechanism described for GCGR is generalizable to other family B GPCRs.

Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABAB caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABAB dimers, and have important implications for rational drug design that targets these receptors. Cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors, combined with cellular signalling assays, shed light on the mechanisms that underpin signal transduction mediated by these receptors.

Most G protein-coupled receptors (GPCRs) recruit β-arrestins and internalize upon agonist stimulation. For the μ-opioid receptor (μ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for μ-OR recruitment of β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in β-arrestin2 recruitment and μ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced μ-OR internalization and β-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of β-arrestin2 recruitment to the plasma membrane upon μ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein–coupled receptors. The G(q/11) subfamily consists of G(q), G(11), G(14), and G(16) proteins, of which all but G(16) are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, G(s), G(i/o), and G(12/13), but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits G(q/11/14), the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359–binding site in G(q) and G(s). We found that the activity of a G(s) mutant with a G(q)-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in G(s). In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive G(s), G(i/o), and G(12/13) inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gα(s) inhibitor, FR900359-sensitive Gα(s) mutants may prove useful in studies where delicate control over Gα(s) signaling would be of the essence.

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 β

The glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.

A series of Gs protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β2-Adrenergic receptor (β2AR) 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 β2AR. 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 β2AR. 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 β2AR. 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.

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 β2-adrenergic receptor (β(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 β(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.

International audience; G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized β2 -adrenergic receptor (β2 AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of β2 AR and stabilize the receptor in an active conformation. Within Nb80, the complementarity-determining region 3 (CDR3) is responsible for most of the binding interactions. Hence, we hypothesized that peptidomimetics of the CDR3 loop might be sufficient for binding to the receptor, inhibiting the interaction of β2 AR with intracellular GPCR interacting proteins (e.g., G proteins). Based on previous crystallographic data, a set of peptidomimetics were synthesized that, similar to the Nb80 CDR3 loop, adopt a β-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial inhibition of cAMP production.

G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized β

Since its discovery in 1992, mGluR5 has attracted significant attention and been linked to several neurological and psychiatric diseases. Ligand development was initially focused on the orthosteric binding pocket, but lack of subtype selective ligands changed the focus to the transmembrane allosteric binding pocket. This strategy has resulted in several drug candidates in clinical testing. In the present article we explore the orthosteric and allosteric binding pockets in terms of structure and ligand recognition across the mGluR subtypes and groups, and discuss the clinical potential of ligands targeting these pockets. We have performed binding mode analyses of non- and group-selective orthosteric ligands based on molecular docking in mGluR crystal structures and models. For the analysis of the allosteric binding pocket we have combined data from all mGluR5-mutagenesis studies, collectively reporting five negative allosteric modulators and 47 unique mutations, and compared it to the closest related homolog, mGluR1.

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.

The crystal structure of the β2-adrenergic receptor (β2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (β2AR-Gsempty). Unfortunately, the β2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the β2AR and GDP-bound Gs protein (β2AR-GsGDP) that may represent an intermediate on the way to the formation of β2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the β2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the β2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.

The surname of author Toon Laeremans was misspelled ‘Laermans’. This error has been corrected online.

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 A crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

A series of compounds based on the mGluR5-selective ligand 2-methyl-6-(phenylethynyl)pyridine (MPEP) were designed and synthesized. The compounds were found to be either structural analogues of MPEP, substituted monomers, or dimeric analogues. All compounds retained mGluR5 selectivity with only weak or no activity at other mGluRs or iGluRs. The substituted analogue, 1,3-bis(pyridin-2-ylethynyl)benzene (19), is a potent negative modulator at mGluR5, whereas all other compounds lost potency relative to MPEP and showed that activity is highly dependent on the position of the nitrogen atom in the pyridine moieties. A homology modeling and ligand docking study was used to understand the binding mode and the observed selectivity of compound 19.

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further investigation.

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 SAR features have been further explored for (2-benzhydryl-4-phenyl-thiazol-5-yl)acetic acids as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. The introduction of a nitrogen or a methyl substituent in the benzhydrylic position offer two alternative drugable scaffolds attractive for unsymmetrically substituted derivatives. An imidazole analogue lacks activity due to formation of a favored coplanar intramolecular hydrogen bond. The pyrimidine derivative 18 represents a potent and selective compound that will be subject to continued investigations.

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.

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.

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.

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-[35S]thiotriphosphate ([35S]GTPγS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB-1893 and MPEP had no effects alone in GTPγS 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 [3H]L-AP4 binding, a two-fold decrease in KD but not in Bmax was observed with 100 μM SIB-1893, whereas MPEP affected neither parameter. Finally, SIB-1893 and MPEP failed to displace [3H]L-AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.

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 β(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.

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 β(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.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl)pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB [1,3-bis(pyridin-2-ylethynyl)-benzene, nitrogen atoms in ortho positions], with an IC(50) value in the nanomolar range, is significantly more potent than the 3- and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand-receptor interactions that may explain the potency differences of 2-, 3-, and 4-BisPEB. Residues Ile651, Pro655, Tyr659, Asn747, Trp785, Phe788, Tyr792, Ser809, and Ala810 were found to have critical roles for the activity of one or more of the three BisPEBs and the reference compound MPEP. The mutagenesis data suggest that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3- and 4-BisPEB (IC(50), 1-2.5 μM). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between MPEP and mGluR5. Potential binding modes of 2-, 3-, and 4-BisPEB obtained by molecular docking to the mGluR5 homology model provide a structural context for the reported major mutational effects.

Initial investigations towards the synthesis and glutamate receptor activity of triazolyl amino acids are undertaken.

Udgivelsesdato: 2010-Feb-1 Structure-activity relationships have been established by exploring the eastern and western side of 5-thiazolyleacetic acids as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Benzhydryl motifs in the 2-position of the thiazole was found to be most advantageous. The 4-thiazole position should either carry 3- or 4-fluorophenyl rings or a 4-pyridyl suitably substituted in the flanking 2-position. Several compounds with single digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained. The compound series display a good PK profile and selectivity over a large number of other targets.

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.

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.

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.

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.