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Birgitte Holst - One of the best experts on this subject based on the ideXlab platform.
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Ghrelin Receptor version 2019 4 in the iuphar bps guide to pharmacology database
IUPHAR BPS Guide to Pharmacology CITE, 2019Co-Authors: Anthony P Davenport, Birgitte Holst, Matthias J Kleinz, Janet J Maguire, Bjørn SivertsenAbstract:The Ghrelin Receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee for the Ghrelin Receptor [18]) is activated by a 28 amino-acid peptide originally isolated from rat stomach, where it is cleaved from a 117 amino-acid precursor (GHRL, Q9UBU3). The human gene encoding the precursor peptide has 83% sequence homology to rat prepro-Ghrelin, although the mature peptides from rat and human differ by only two amino acids [70]. Alternative splicing results in the formation of a second peptide, [des-Gln14]Ghrelin with equipotent biological activity [48]. A unique post-translational modification (octanoylation of Ser3, catalysed by Ghrelin Ο-acyltransferase (MBOAT4, Q96T53) [127] occurs in both peptides, essential for full activity in binding to Ghrelin Receptors in the hypothalamus and pituitary, and for the release of growth hormone from the pituitary [56]. Structure activity studies showed the first five N-terminal amino acids to be the minimum required for binding [4], and Receptor mutagenesis has indicated overlap of the Ghrelin binding site with those for small molecule agonists and allosteric modulators of Ghrelin function [43]. In cell systems, the Ghrelin Receptor is constitutively active [44], but this is abolished by a naturally occurring mutation (A204E) that results in decreased cell surface Receptor expression and is associated with familial short stature [88].
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functionally biased signalling properties of 7tm Receptors opportunities for drug development for the Ghrelin Receptor
British Journal of Pharmacology, 2013Co-Authors: Bjørn Sivertsen, Nicholas D. Holliday, Andreas N. Madsen, Birgitte HolstAbstract:The Ghrelin Receptor is a 7 transmembrane (7TM) Receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism and energy expenditure, the Ghrelin Receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the Ghrelin Receptor is able to signal through several different signalling pathways including Gαq, Gαi/o, Gα12/13 and arrestin recruitment. These multiple signalling pathways allow for functionally biased signalling, where one signalling pathway may be favoured over another either by selective ligands or through mutations in the Receptor. In the present review, we have described how ligands and mutations in the 7TM Receptor may bias the Receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G–protein-dependent pathways. For the Ghrelin Receptor, both agonist and inverse agonists have been demonstrated to signal more strongly through the Gαq-coupled pathway than the Gα12/13-coupled pathway. Similarly a ligand that promotes Gαq coupling over Gαi coupling has been described and it has been suggested that several different active conformations of the Receptor may exist dependent on the properties of the agonist. Importantly, ligands with such biased signalling properties may allow the development of drugs that selectively modulate only the therapeutically relevant physiological functions, thereby decreasing the risk of side effects. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7
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an aromatic region to induce a switch between agonism and inverse agonism at the Ghrelin Receptor
Journal of Medicinal Chemistry, 2012Co-Authors: Sylvia Els, Jacek Mokrosinski, Thomas M. Frimurer, Thue W. Schwartz, Pia Steen Petersen, Enrico Schild, Tommarten Kilian, Constance Chollet, Birgitte HolstAbstract:The Ghrelin Receptor displays a high constitutive activity suggested to be involved in the regulation of appetite and food intake. Here, we have created peptides with small changes in the core binding motif -wFw- of the hexapeptide KwFwLL-NH(2) that can swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. Introduction of β-(3-benzothienyl)-d-alanine (d-Bth), 3,3-diphenyl-d-alanine (d-Dip) and 1-naphthyl-d-alanine (d-1-Nal) at position 2 resulted in highly potent and efficient inverse agonists, whereas the substitution of d-tryptophane at position 4 with 1-naphthyl-d-alanine (d-1-Nal) and 2-naphthyl-d-alanine (d-2-Nal) induces agonism in functional assays. Competitive binding studies showed a high affinity of the inverse agonist K-(d-1-Nal)-FwLL-NH(2) at the Ghrelin Receptor. Moreover, mutagenesis studies of the Receptor revealed key positions for the switch between inverse agonist and agonist response. Hence, only minor changes in the peptide sequence can decide between agonism and inverse agonism and have a major impact on the biological activity.
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In Vivo Characterization of High Basal Signaling from the Ghrelin Receptor
Endocrinology, 2009Co-Authors: Pia Steen Petersen, Manja Lang, Annette G. Beck-sickinger, Andreas N. Madsen, David P.d. Woldbye, Kristoffer L. Egerod, Chunyu Jin, Maria Rasmussen, Birgitte HolstAbstract:The Receptor for the orexigenic peptide, Ghrelin, is one of the most constitutively active 7TM Receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active Receptor is associated with change in signaling independent of the endogenous ligand. In the following study, we found that the expression of the Ghrelin Receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the Ghrelin Receptor in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P. This peptide selectively displays inverse agonism at the Ghrelin Receptor as compared with an inactive control peptide with just a single amino acid substitution. Food intake and body weight were significantly decreased in the group of rats treated with the inverse agonist, as compare...
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overlapping binding site for the endogenous agonist small molecule agonists and ago allosteric modulators on the Ghrelin Receptor
Molecular Pharmacology, 2009Co-Authors: Birgitte Holst, Jacek Mokrosinski, Thomas M. Frimurer, Tine Halkjaer, Karina B Cullberg, Christina Rye Underwood, Thue W. SchwartzAbstract:A library of robust Ghrelin Receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand Ghrelin and the hexapeptide growth hormone secretagogue GHRP-6) plus four nonpeptide agonists—the original benzolactam L-692,429 [3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2′-(1H-tetrazol-5-yl) (1,1′-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamide], the spiroindoline sulfonamide MK-677 [N-[1(R)-1, 2-dihydro-1-ethanesulfonylspiro-3H-indole-3,4′-piperidin)-1′-yl]carbonyl-2-(phenylmethoxy)-ethyl-2-amino-2-methylpropanamide], and two novel oxindole derivatives, SM-130686 [(+)-6-carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole] and SM-157740 [(±)-6-carbamoyl-3-(2, 4-dichlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole)]. The strongest mutational effect with respect to decrease in potency for stimulation of inositol phosphate turnover was for all six agonists the GluIII:09-to-Gln substitution in the extracellular segment of TM-III. Likewise, all six agonists were affected by substitutions of PheVI:16, ArgVI:20, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing Ghrelin9s maximal efficacy overlapped with the common mutational map for agonism but it was not identical with the map for the agonist property of these small-molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the nonpeptide agonists could be docked to satisfy many of their mutational hits. It is concluded that although each of the ligands in addition exploits other parts of the Receptor, a large, common binding site for both small-molecule agonists—including ago-allosteric modulators—and the endogenous agonist is found on the opposing faces of TM-III and -VI of the Ghrelin Receptor.
Thue W. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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an aromatic region to induce a switch between agonism and inverse agonism at the Ghrelin Receptor
Journal of Medicinal Chemistry, 2012Co-Authors: Sylvia Els, Jacek Mokrosinski, Thomas M. Frimurer, Thue W. Schwartz, Pia Steen Petersen, Enrico Schild, Tommarten Kilian, Constance Chollet, Birgitte HolstAbstract:The Ghrelin Receptor displays a high constitutive activity suggested to be involved in the regulation of appetite and food intake. Here, we have created peptides with small changes in the core binding motif -wFw- of the hexapeptide KwFwLL-NH(2) that can swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. Introduction of β-(3-benzothienyl)-d-alanine (d-Bth), 3,3-diphenyl-d-alanine (d-Dip) and 1-naphthyl-d-alanine (d-1-Nal) at position 2 resulted in highly potent and efficient inverse agonists, whereas the substitution of d-tryptophane at position 4 with 1-naphthyl-d-alanine (d-1-Nal) and 2-naphthyl-d-alanine (d-2-Nal) induces agonism in functional assays. Competitive binding studies showed a high affinity of the inverse agonist K-(d-1-Nal)-FwLL-NH(2) at the Ghrelin Receptor. Moreover, mutagenesis studies of the Receptor revealed key positions for the switch between inverse agonist and agonist response. Hence, only minor changes in the peptide sequence can decide between agonism and inverse agonism and have a major impact on the biological activity.
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overlapping binding site for the endogenous agonist small molecule agonists and ago allosteric modulators on the Ghrelin Receptor
Molecular Pharmacology, 2009Co-Authors: Birgitte Holst, Jacek Mokrosinski, Thomas M. Frimurer, Tine Halkjaer, Karina B Cullberg, Christina Rye Underwood, Thue W. SchwartzAbstract:A library of robust Ghrelin Receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand Ghrelin and the hexapeptide growth hormone secretagogue GHRP-6) plus four nonpeptide agonists—the original benzolactam L-692,429 [3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2′-(1H-tetrazol-5-yl) (1,1′-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamide], the spiroindoline sulfonamide MK-677 [N-[1(R)-1, 2-dihydro-1-ethanesulfonylspiro-3H-indole-3,4′-piperidin)-1′-yl]carbonyl-2-(phenylmethoxy)-ethyl-2-amino-2-methylpropanamide], and two novel oxindole derivatives, SM-130686 [(+)-6-carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole] and SM-157740 [(±)-6-carbamoyl-3-(2, 4-dichlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole)]. The strongest mutational effect with respect to decrease in potency for stimulation of inositol phosphate turnover was for all six agonists the GluIII:09-to-Gln substitution in the extracellular segment of TM-III. Likewise, all six agonists were affected by substitutions of PheVI:16, ArgVI:20, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing Ghrelin9s maximal efficacy overlapped with the common mutational map for agonism but it was not identical with the map for the agonist property of these small-molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the nonpeptide agonists could be docked to satisfy many of their mutational hits. It is concluded that although each of the ligands in addition exploits other parts of the Receptor, a large, common binding site for both small-molecule agonists—including ago-allosteric modulators—and the endogenous agonist is found on the opposing faces of TM-III and -VI of the Ghrelin Receptor.
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Identification of an efficacy switch region in the Ghrelin Receptor responsible for interchange between agonism and inverse agonism
The Journal of biological chemistry, 2007Co-Authors: Birgitte Holst, Jacek Mokrosinski, Manja Lang, Erik Brandt, Rie Nygaard, Thomas M. Frimurer, Annette G. Beck-sickinger, Thue W. SchwartzAbstract:The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active Ghrelin Receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by Receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM Receptor activation.
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Ghrelin Receptor inverse agonists identification of an active peptide core and its interaction epitopes on the Receptor
Molecular Pharmacology, 2006Co-Authors: Birgitte Holst, Manja Lang, Erik Brandt, Thomas M. Frimurer, Anders Bach, Annette G Becksickinger, Andrew D Howard, Thue W. SchwartzAbstract:[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the Ghrelin Receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the Ghrelin Receptor demonstrated that Ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous Receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the Receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for the Ghrelin Receptor.
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nonpeptide and peptide growth hormone secretagogues act both as Ghrelin Receptor agonist and as positive or negative allosteric modulators of Ghrelin signaling
Molecular Endocrinology, 2005Co-Authors: Birgitte Holst, Erik Brandt, Anders Bach, Anders Heding, Thue W. SchwartzAbstract:Two nonpeptide (L692,429 and MK-677) and two peptide [GH-releasing peptide (GHRP)-6 and Ghrelin] agonists were compared in binding and in signal transduction assays: calcium mobilization, inositol phosphate turnover, cAMP-responsive element (CRE), and serum-responsive element (SRE) controlled transcription, as well as arrestin mobilization. MK-677 acted as a simple agonist having an affinity of 6.5 nm and activated all signal transduction systems with similar high potency (0.2-1.4 nm). L-692,429 also displayed a very similar potency in all signaling assays (25-60 nm) but competed with a 1000-fold lower apparent affinity for Ghrelin binding and surprisingly acted as a positive allosteric Receptor modulator by increasing Ghrelin's potency 4- to 10-fold. In contrast, the potency of GHRP-6 varied 600-fold (0.1-61 nm) depending on the signal transduction assay, and it acted as a negative allosteric modulator of Ghrelin signaling. Unexpectedly, the maximal signaling efficacy for Ghrelin was increased above what was observed with the hormone itself during coadministration with the nonendogenous agonists. It is concluded that agonists for the Ghrelin Receptor vary both in respect of their intrinsic agonist properties and in their ability to modulate Ghrelin signaling. A Receptor model is presented wherein Ghrelin normally only activates one Receptor subunit in a dimer and where the smaller nonendogenous agonists bind in the other subunit to act both as coagonists and as either neutral (MK-677), positive (L-692,429), or negative (GHRP-6) modulators of Ghrelin function. It is suggested that an optimal drug candidate could be an agonist that also is a positive modulator of Ghrelin signaling.
Lorenzo Leggio - One of the best experts on this subject based on the ideXlab platform.
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a population pharmacokinetic analysis of pf 5190457 a novel Ghrelin Receptor inverse agonist in healthy volunteers and in heavy alcohol drinkers
Clinical Pharmacokinectics, 2021Co-Authors: Enoch Cobbina, Lorenzo Leggio, Mary R Lee, Fatemeh AkhlaghiAbstract:The Ghrelin Receptor (GHS-R1a) is a potential target for alcohol use disorders. PF-5190457 is the first inverse agonist of GHS-R1a to progress to clinical development with potential to treat alcohol use disorder. We present a population pharmacokinetic model for PF-5190457 in non-heavy (alcohol consumption status = 0) and heavy alcohol drinkers (alcohol consumption status = 1), and identify relevant factors that can influence its pharmacokinetics. Plasma concentration–time data from non-heavy (n = 35) and heavy drinkers (n = 12) were pooled for the population pharmacokinetic model development. The influence of various covariates including alcohol consumption status was evaluated. The accuracy, precision, and robustness of the model were also evaluated using bootstrapping and visual predictive checks. A two-compartment model best described the pharmacokinetics of PF-5190457. The apparent volume of distribution of 44.5 L, apparent clearance of 72.0 L/h, apparent peripheral volume of distribution of 271 L, apparent distributional clearance of 28.7 L/h, and first-order absorption rate constant of 0.27/h were accurate and precise. The apparent volume of distribution was 3.8-fold higher (169 L) in heavy drinkers, and correlated with a lower maximum plasma concentration in heavy drinkers compared with non-heavy drinkers at the same dose; and a corresponding reduced incidence of somnolence in heavy drinkers at doses > 50 mg. This work provides an accurate, precise, and robust two-compartment model that describes the pharmacokinetics of PF-5190457 and suggests a possible link of PF-5190457 pharmacokinetics with somnolence. ClinicalTrials.gov identifier numbers NCT01247896 and NCT02039349
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endocrine effects of the novel Ghrelin Receptor inverse agonist pf 5190457 results from a placebo controlled human laboratory alcohol co administration study in heavy drinkers
Neuropharmacology, 2020Co-Authors: Mary R Lee, Enoch Cobbina, Lisa A Farinelli, Mehdi Farokhnia, Jillian T Battista, Fatemeh Akhlaghi, Anitha Saravanakumar, Lorenzo LeggioAbstract:Abstract Both animal and human work suggests that the Ghrelin system may be involved in the mechanisms that regulate the development and maintenance of alcohol use disorder. Previously, in a Phase 1b study, we tested pharmacological blockade of the growth hormone secretagogue Receptor 1a (GHS-R1a, also known as the Ghrelin Receptor), in heavy drinking individuals with PF-5190457, an orally bioavailable, potent and selective GHS-R1a inverse agonist. We report here the effects of PF-5190457 on endocrine blood concentrations of amylin, gastric inhibitory polypeptide, glucagon-like peptide 1, insulin, leptin, pancreatic polypeptide, peptide YY, thyroid stimulating hormone, free triiodothyronine (T3), thyroxine (T4), cortisol, prolactin, and glucose during PF-5190457 dosing, as compared to placebo, in absence of alcohol as well as during an alcohol challenge when PF-5190457 was on steady-state. Blood hormone levels were largely unaffected by PF-5190457, both during dosing and in the context of alcohol challenge. The safety-related relevance of these findings to further develop PF-5190547 in alcohol use disorder is discussed. Clinicaltrials.gov NCT02039349. This article is part of the special issue on ‘Neuropeptides’.
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development and validation of an assay for a novel Ghrelin Receptor inverse agonist pf 5190457 and its major hydroxy metabolite pf 6870961 by lc ms ms in human plasma
Journal of Chromatography B, 2019Co-Authors: Sravani Adusumalli, Lorenzo Leggio, Rohitash Jamwal, Fatemeh AkhlaghiAbstract:Abstract PF-5190457 is a selective and potent Ghrelin Receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL−1 tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min−1 and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1–1000 ng mL−1 and 2–250 ng mL−1 for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL−1, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ±15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.
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role of molybdenum containing enzymes in the biotransformation of the novel Ghrelin Receptor inverse agonist pf 5190457 a reverse translational bed to bench approach
Drug Metabolism and Disposition, 2019Co-Authors: Sravani Adusumalli, Lorenzo Leggio, Rohitash Jamwal, Scott R Obach, Tim F Ryder, Fatemeh AkhlaghiAbstract:(R)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) was identified as a potent and selective inverse agonist of the Ghrelin Receptor [growth hormone secretagogue Receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 or (R)-1-(2-(5-(2-hydroxy-6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethan-1-one was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenum-containing oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT PF-5190457 is a novel Ghrelin Receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies.
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Ghrelin Receptor deletion reduces binge like alcohol drinking in rats
Journal of Neuroendocrinology, 2019Co-Authors: Brendan J. Tunstall, George F. Koob, Leandro F. Vendruscolo, Lia J Zallar, Silvia Beurmann, Claire M Fraser, Lorenzo LeggioAbstract:Ghrelin is a gastric hormone that has been implicated in the neurobiology of alcohol drinking. We have recently developed a Ghrelin Receptor (growth hormone secretagogue Receptor; GHSR) knockout (KO) rat model, which exhibits reduced food consumption and body weight. In addition, recent preliminary work suggests that the gut-microbiome, which appears to interact with the Ghrelin system, may modulate alcohol drinking. In the present study, we investigated the effects of GHSR deletion on alcohol consumption utilising GHSR KO and wild-type (WT) rats in three separate alcohol consumption paradigms: (i) operant self-administration (30-minute sessions); (ii) drinking in the dark (DID) (4-hour sessions); and (iii) intermittent access (24-hour sessions). These paradigms model varying degrees of alcohol consumption. Furthermore, we aimed to investigate the gut-microbiome composition of GHSR KO and WT rats before and after alcohol exposure. We found that the GHSR KO rats self-administered significantly less alcohol compared to WT rats in the operant paradigm, and consumed less alcohol than WT in the initial stages of the DID paradigm. No genotype differences were found in the intermittent access test. In addition, we found a significant decrease in gut-microbial diversity after alcohol exposure in both genotypes. Thus, the present results indicate that the Ghrelin system may be involved in drinking patterns that result in presumably increased alcohol exposure levels. Furthermore, GHSR may constitute a potential pharmacological target for the reduction of binge-alcohol consumption. The potential functional role of the gut-microbiome in alcohol drinking, as well as interaction with the Ghrelin system, is an interesting topic for further investigation.
Antonio Torsello - One of the best experts on this subject based on the ideXlab platform.
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New trisubstituted 1,2,4-triazole derivatives as potent Ghrelin Receptor antagonists. 3. Synthesis and Pharmacological in Vitro and in Vivo Evaluations
Journal of Medicinal Chemistry, 2008Co-Authors: Aline Moulin, Didier Gagne, Luc Demange, Delphine Mousseaux, Joanne Ryan, Gilbert Berge, Vittorio Locatelli, Pierre Sanchez, Daniel Perissoud, Antonio TorselloAbstract:Ghrelin Receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the R-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.
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toward potent Ghrelin Receptor ligands based on trisubstituted 1 2 4 triazole structure 2 synthesis and pharmacological in vitro and in vivo evaluations
Journal of Medicinal Chemistry, 2007Co-Authors: Aline Moulin, Didier Gagne, Luc Demange, Delphine Mousseaux, Joanne Ryan, Gilbert Berge, Annie Heitz, Daniel Perrissoud, Vittorio Locatelli, Antonio TorselloAbstract:A series of Ghrelin Receptor ligands based on the trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the significance of the aminoisobutyryl (Aib) moiety, a common feature in numerous growth hormone secretagogues described in the literature. Potent agonist and antagonist ligands of the growth hormone secretagogue Receptor type 1a (GHS-R1a) were obtained, i.e., compounds 41 (JMV2894) and 17 (JMV3031). The best compounds were evaluated for their in vivo activity on food intake, after sc injection in rodents. Among the tested compounds, few of them were able to stimulate food intake and some others, i.e., compounds 4 (JMV2959), 17, and 52 (JMV3021), acted as potent in vivo antagonist of hexarelin-stimulated food intake. These compounds did not stimulate growth hormone secretion in rats and furthermore did not antagonize growth hormone secretion induced by hexarelin, revealing that it is possible to modulate foo...
Didier Gagne - One of the best experts on this subject based on the ideXlab platform.
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Ghrelin Receptor conformational dynamics regulate the transition from a preassembled to an active Receptor:Gq complex.
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Marjorie Damian, Séverine Denoyelle, Sophie Mary, Mathieu Maingot, Céline M'kadmi, Didier Gagne, Jean-philippe Leyris, Gérald Gaibelet, Laurent Gavara, Mauricio Garcia De Souza CostaAbstract:How G protein-coupled Receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified Ghrelin Receptor assembled into lipid discs. Combining Receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct Receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the Receptor. The first of these assemblies is a preassembled complex with the Receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the Receptor in its active conformation triggers G proteinactivation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the Ghrelin Receptor. These data provide direct evidence of a mechanism for Ghrelin Receptor-mediated Gq signaling in which transition of the Receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled Receptor: G protein complex, ultimately leading to G protein activation and signaling.
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activation of the Ghrelin Receptor is described by a privileged collective motion a model for constitutive and agonist induced activation of a sub class a g protein coupled Receptor gpcr
Journal of Molecular Biology, 2010Co-Authors: Nicolas Floquet, Jean-alain Fehrentz, Didier Gagne, Jacky Marie, Jeanlouis Baneres, Gilbert Berge, Celine Mkadmi, David Perahia, Jeanclaude Galleyrand, Jean MartinezAbstract:Abstract Three homology models of the human Ghrelin Receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic Receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C W LP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short Ghrelin derivatives Gly-Ser-Ser [octa]-Phe-NH 2 and Gly-Ser-Ser [octa]-Phe-Leu-NH 2 allowed the identification of a robust position of these peptides in the active site of the Receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 Ghrelin Receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide–Receptor complex suggested stabilization of the Trp276 residue and of the whole Receptor in the active state, preventing the motion observed along mode 16 computed for the unbound Receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.
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New trisubstituted 1,2,4-triazole derivatives as potent Ghrelin Receptor antagonists. 3. Synthesis and Pharmacological in Vitro and in Vivo Evaluations
Journal of Medicinal Chemistry, 2008Co-Authors: Aline Moulin, Didier Gagne, Luc Demange, Delphine Mousseaux, Joanne Ryan, Gilbert Berge, Vittorio Locatelli, Pierre Sanchez, Daniel Perissoud, Antonio TorselloAbstract:Ghrelin Receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the R-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.
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toward potent Ghrelin Receptor ligands based on trisubstituted 1 2 4 triazole structure 2 synthesis and pharmacological in vitro and in vivo evaluations
Journal of Medicinal Chemistry, 2007Co-Authors: Aline Moulin, Didier Gagne, Luc Demange, Delphine Mousseaux, Joanne Ryan, Gilbert Berge, Annie Heitz, Daniel Perrissoud, Vittorio Locatelli, Antonio TorselloAbstract:A series of Ghrelin Receptor ligands based on the trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the significance of the aminoisobutyryl (Aib) moiety, a common feature in numerous growth hormone secretagogues described in the literature. Potent agonist and antagonist ligands of the growth hormone secretagogue Receptor type 1a (GHS-R1a) were obtained, i.e., compounds 41 (JMV2894) and 17 (JMV3031). The best compounds were evaluated for their in vivo activity on food intake, after sc injection in rodents. Among the tested compounds, few of them were able to stimulate food intake and some others, i.e., compounds 4 (JMV2959), 17, and 52 (JMV3021), acted as potent in vivo antagonist of hexarelin-stimulated food intake. These compounds did not stimulate growth hormone secretion in rats and furthermore did not antagonize growth hormone secretion induced by hexarelin, revealing that it is possible to modulate foo...
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synthesis and pharmacological in vitro and in vivo evaluations of novel triazole derivatives as ligands of the Ghrelin Receptor 1
Journal of Medicinal Chemistry, 2007Co-Authors: Luc Demange, Didier Gagne, Damien Boeglin, Aline Moulin, Delphine Mousseaux, Joanne Ryan, Gilbert Berge, Annie Heitz, Daniel Perrissoud, Vittorio LocatelliAbstract:A new series of growth hormone secretagogue (GHS) analogues based on the 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and their ability to stimulate intracellular calcium release to the cloned hGHS-1a Ghrelin Receptor expressed in LLC PK-1 cells. We have synthesized potent ligands of this Receptor, some of them behaving as agonists, partial agonists, or antagonists. Some compounds among the most potent, i.e., agonist 29c (JMV2873), partial agonists including 21b (JMV2810), antagonists 19b (JMV2866) and 19c (JMV2844), were evaluated for their in vivo activity on food intake, after sc injection in rodents. Some compounds were found to stimulate food intake like hexarelin; some others were identified as potent hexarelin antagonists in this assay. Among the tested compounds, 21b was identified as an in vitro Ghrelin Receptor partial agonist, as well as a potent in vivo antagonist of hexarelin-stimulated food intake in rodents. Compound 21b was without effect on GH release from rat. However, in this series of compounds, it was not possible to find a clear correlation between in vitro and in vivo results.