The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

ADVERTISEMENT RETURN TO ISSUEPREVLetterNEXTBinding of ATP-Sensitive Potassium Channel (KATP) Openers to Cardiac Membranes: Correlation of Binding Affinities with Cardioprotective and Smooth Muscle Relaxing PotenciesKarnail S. Atwal, Gary J. Grover, Nicholas J. Lodge, Diane E. Normandin, Sarah C. Traeger, Paul G. Sleph, Robert B. Cohen, Catherine C. Bryson, and Keneth E. J. DickinsonView Author Information Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000 Cite this: J. Med. Chem. 1998, 41, 3, 271–275Publication Date (Web):January 13, 1998Publication History Received10 November 1997Published online13 January 1998Published inissue 1 January 1998https://pubs.acs.org/doi/10.1021/jm970762dhttps://doi.org/10.1021/jm970762drapid-communicationACS PublicationsCopyright © 1998 American Chemical SocietyRequest reuse permissionsArticle Views198Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Anatomy,Membranes,Potassium,Rodent models,Screening assays Get e-Alerts

This study examined the pharmacological characteristics of binding sites for the potent K+ channel opener [3H]P1075, as well as the functional effects of P1075 on ionic currents and membrane potential, in ovine choroid plexus (OCP) cells. [3H]P1075 bound to OCP cells with a Kd of 26 +/- 4 nM and a Bmax of 10400 +/- 480 sites/cell. Labelled sites were stereoselective and inhibited by potassium channel openers with a rank order of potency: P1075 > BMS-182264, ((4-[[9cyanoimino)[(1,2,2-trimethylpropyl)amino]-methyl]amino]benz onitrile) > pinacidil >> nicorandil > diazoxide. The K(ATP) channel antagonist glyburide inhibited [3H]P1075 binding with a Ki of 2 microM. The presence of K(ATP) channels on OCP cells was examined by patch clamp and fluorescent (membrane-potential sensitive dye) techniques. In some cells, P1075 activated an outward potassium current which was blocked by glyburide. P1075 produced a glyburide-sensitive, concentration-dependent, hyperpolarization of OCP cells. Levcromakalim hyperpolarized more strongly than its 3R,4S enantiomer, BRL 38226 ((3R-trans)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)- 2H-1-benzopyran-6-carbonitrile) indicating a stereoselective interaction. These data indicate that epithelial OCP cells contain glyburide-sensitive K(ATP) channels.

[3H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC50 value for MgATP-supported binding (0.4 mM) was decreased approximately 10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [3H]P1075 bound to skeletal muscle with a Kd value of 37 +/- 3 nM and a Bmax value of 280 +/- 14 fmol/mg of protein. [3H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (Ki = 200-300 nM). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [3H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [3H]P1075 dissociation in a manner consistent with noncompetitive interaction.

Four carboxyl-containing, selective beta(3) adrenergic agonists and their ester or amide derivatives were evaluated for their ability to bind to human beta(1), beta(2), and beta(3) adrenergic receptors. Stimulatory effects on the beta(3) adrenergic receptor were also measured. We conclude that carboxyl-derived beta(3) functional selectivity Likely results, at least in part, from the effect of the carboxyl on binding selectivity.

Novel heterocyclic β3 adrenergic receptor agonists2 were prepared and evaluated for their ability to bind to human β1, β2, and β3 adrenergic receptors. Stimulatory effects on the β3 adrenergic receptor were also measured. BMS-187257 (4b) was found to be a potent and selective β3 agonist.

The syntheses and pharmacological activity of a series of 1,4-substituted indoles which function as nonpeptidic antagonists of the angiotensin II (AII) receptor are described. Compounds in this series are orally active and demonstrate long lasting antihypertensive activity.

Prodrug derivatization of a potent quinoline-4-carboxylic acid angiotensin II receptor antagonist was Undertaken as an approach to achieve improved oral activity. A dioxolenone carboxylic ester and an alkylated tetrazole prodrug both showed greater oral antihypertensive acivity in the salt-deplete spontaneously hypertensive rat and increased oral bioavailability relative to the parent compound.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Discovery of Sulfonamide Endothelin Antagonists and the Development of the Orally Active ETA Antagonist 5-(Dimethylamino)-N-(3,4-dimethyl-5- isoxazolyl)-1-naphthalenesulfonamidePhilip D. Stein, John T. Hunt, David M. Floyd, Suzanne Moreland, Kenneth E. J. Dickinson, Caroline Mitchell, Eddie C.-K. Liu, Maria L. Webb, Natesan Murugesan, and Cite this: J. Med. Chem. 1994, 37, 3, 329–331Publication Date (Print):February 1, 1994Publication History Published online1 May 2002Published inissue 1 February 1994https://doi.org/10.1021/jm00029a001RIGHTS & PERMISSIONSArticle Views403Altmetric-Citations125LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (406 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

We have synthesized a series of quinoline-4-carboxylic acids that are potent antagonists of the angiotensin II receptor. They show high receptor binding affinities and functional antagonism in rabbit aortic strips. They are also effective in blocking the hypertensive effects of AII in vivo.

1. This study compares the activity of BMS-180560 (2-butyl-1-chloro-1-[[1-[2-(2H-tetrazol-5-yl)phenyl]-1H-indol-4- yl]methyl]-1H-imidazole-5-carboxylic acid), an insurmountable angiotensin II (AII) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII-receptor activation. 2. BMS-180560 selectively inhibited [125I]-Sar1Ile8AII ([125I]SI-AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT1 receptors (Ki = 7.6 +/- 1.2 and 18.4 +/- 3.9 nM respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 +/- 1.3 microM). The Ki values of BMS-180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin. 3. BMS-180560 (3-300 nM) increased the KD of SI-AII for RASM cell AT1 receptors. Only at high concentrations of BMS-180560 (300 nM) were Bmax values decreased. 4. BMS-180560 inhibited AII-stimulated contraction of rabbit aorta with a calculated KB = 0.068 +/- 0.048 nM and decreased maximal AII-stimulated contraction at 1 nM BMS-180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 +/- 0.92 nM) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 +/- 0.13 nM. Contraction stimulated by endothelin-1, noradrenaline, KCl, or the TXA2 receptor agonist U-46619 were unaffected by BMS-180560 (1 nM). 5. AII stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nM. Losartan (30 nM) shifted the AII concentration-effect curves in a competitive manner whereas BMS-180560 (0.01 and 0.1 nM) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS-180560 could be reversed following washout although recovery took longer for BMS-180560. 6. In [3H]-myoinositol-labelled RASM cells, losartan (30 and 200 nM), shifted the EC50 for AII-stimulated [3H]-inositol monophosphaste formation to higher values, with no change in the maximal response. By contrast, EXP3174 (0.1 to 1 nM) decreased the maximal response in a concentration-dependent manner (17-55%). BMS-180560 (3 and 10 nM) increased the EC50 for AII and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS-180560 could be reversed by inclusion of losartan (200 nM) indicating that the inhibition was not irreversible. 7. In conclusion, BMS-180560 is a potent, specific, predominantly competitive, reversible All receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS-180560 which have no effect on receptor number, BMS-180560 produced insurmountable antagonism of AII-stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.

4-Alkyl-1,2,4-oxadiazinones were prepared by regiospecific alkylation of the corresponding 4H-oxadiazinones, which were synthesized by a trimethylaluminum mediated cyclization reaction. Alkylation was regiospecific and generally facile; in one example, however, an unusual fragmentation reaction occurred. A homochiral oxadiazineone was also prepared and alkylated under the described conditions. 4-Biphenylmethyl-1,2,4-oxadi-azinones were potent angiotensin-II receptor antagonists.

The vascular angiotensin (A) II receptor cDNA (AT1a) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-AT1a. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT1 receptor. Specific binding of sarcosine1-[125I]tyrosine4-isoleucine8-AII ([125I]SI-AII) to CHO-AT1a membranes reached equilibrium after 1 h at 25°C and was consistently greater than 95% of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII>AII>losartan>AI> >PD 123,177. AII (1 μM) treatment of CHO-AT1a cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 μM) did not effect adenylate cyclase activity in CHO-AT1a cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10 μM losartan. These results indicate that CHO-AT1a cells express functional AT1a receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-AT1a cells should provide a useful model for studies of AT1a receptor domains which are critical to signaling pathways.

A series of novel quinoxaline heterocycle containing angiotensin II receptor antagonist analogs were prepared. This heterocycle was coupled to the biphenyl moiety via an oxygen atom linker instead of a carbon atom. Many of these analogs exhibit very potent activity and long duration of effect. Interestingly, the N-oxide quinoxaline analog was more potent than the nonoxidized quinoxaline as in the comparison of compounds 5 vs 30. In order to improve oral activity, the carboxylic acid function of these compounds was converted to the double ester. This change did result in an improvement in oral activity as represented by compound 44.

Quinoxaline heterocycle containing angiotensin II receptor antagonist analogs were prepared. All five analogs reported here display potent antagonistic activities and most interestingly, quinozaline bis-N-oxide 10 exhibits very potent activities both in binding and functional assays.