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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.

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.

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.

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.

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.

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.

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.

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.