Anne Chateauneuf

IDENTIFICATION OF A GABAB RECEPTOR SUBUNIT, GB2, REQUIRED FOR FUNCTIONAL GABAB RECEPTOR ACTIVITY

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant γ-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situhybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2

The recombinant human prostaglandin D2 (PGD2) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD2 were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (KD) of 2.5 and 109 nM. This revealed that the affinity of PGD2 for CRTH2 is eight times less than its affinity for the DP receptor. Equilibrium competition binding assays revealed that of the compounds tested, only PGD2 and several related metabolites bound with high affinity to CRTH2 (Ki values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD2>13,14‐dihydro‐15‐keto PGD2>15‐deoxy‐Δ12,14‐PGJ2>PGJ2>Δ12‐PGJ2>15(S)‐15 methyl‐PGD2. This is in sharp contrast with the rank order of potency obtained at DP : PGD2>PGJ2>Δ12‐PGJ2>15‐deoxy‐Δ12,14‐PGJ2 >>>13,14‐dihydro‐15‐keto‐PGD2. Functional studies demonstrated that PGD2 activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin‐sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G‐protein Gαi/o. PGD2 and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid‐differentiated HL‐60 and AML 14.3D10, also endogenously express CRTH2.

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

Abstract: Galanin is a 29‐ or 30‐amino acid peptide with wide‐ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I‐galanin show that recombinant human GALR2 binds with high affinity to human galanin (KD = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK‐293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammons horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2–13.1, respectively.

Discovery of a potent and selective agonist of the prostaglandin EP4 receptor

Analogues of PGE(2) wherein the hydroxycyclopentanone ring has been replaced by a lactam have been prepared and evaluated as ligands for the EP(4) receptor. An optimized compound (19a) shows high potency and agonist efficacy at the EP(4) receptor and is highly selective over the other seven known prostaglandin receptors.

Structure–Activity Relationship of Biaryl Acylsulfonamide Analogues on the Human EP3 Prostanoid Receptor

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.

Structure-activity relationship of triaryl propionic acid analogues on the human EP3 prostanoid receptor.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Triaryl compounds bearing an ortho-substituted propionic acid moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinities of key compound on all eight human prostanoid receptors is reported.

Potent and selective 5-LO inhibitor bearing benzothiophene pharmacophore: Discovery of MK-5286 ☆

The strategy and SAR studies that led to the discovery of a novel potent and orally available 5-lipoxygenase (5-LO) inhibitor 3-(4-fluorophenyl)-6-({4-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1H-1,2,3-triazol-1-yl}methyl)-1-benzothiophene-2-carboxamide ((S)-2l or MK-5286) were described.