Synthesis and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[11C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one, a potential PET tracer for growth hormone secretagogue receptor (GHSR)

The peptide hormone ghrelin mediates through action on its receptor, the growth hormone secretagogue receptor (GHSR), and is known to play an important role in a variety of metabolic functions including appetite stimulation, weight gain, and suppression of insulin secretion. In light of the fact that obesity is one of the major health problems plaguing the modern society, the ghrelin signaling system continues to remain an important and attractive pharmacological target for the treatment of obesity. In vivo imaging of the GHSR could shed light on the mechanism by which ghrelin affects feeding behavior and thus offers a new therapeutic perspective for the development of effective treatments. Recently, a series of piperidine-substituted quinazolinone derivatives was reported to be selective and potent GHSR antagonists with high binding affinities. Described herein is the synthesis, in vitro, and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[(11)C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one ([(11)C]1), a potential PET radioligand for imaging GHSR.     

N-(arylacetyl)-biphenylalanines as potent VLA-4 antagonists

A series of potent N-(aralkyl-, arylcycloalkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4 antagonists was prepared by rapid analogue methods using solid-phase chemistry. Further optimization led to several highly potent compounds (IC(50) <1 nM). Evaluation of rat pharmacokinetic revealed generally high clearance.     

Associations of growth hormone secretagogue receptor (GHSR) genes polymorphisms and protein structure changes with carcass traits in sheep

Growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor that binds ghrelin, plays an important role in the central regulation of pituitary growth hormone secretion, food intake, and energy homeostasis. Ghrelin receptor (GHSR) modulates many physiological effects and therefore is a candidate gene for sheep production performance. Polymorphism of the GHSR gene was detected by PCR-SSCP and DNA sequencing methods in 463 individuals. Two different structures in protein and nine single nucleotide polymorphisms (SNPs) were identified. The evaluation of the associations between these SSCP patterns with carcass traits suggests a positive effect of genotype TT and B structure on carcass weight, and body length (P<0.05). In addition, the animal with TC had greater abdominal fat than those with TT and CC (P<0.05) while CC genotype contributed to low blood cholesterol (P=0.04). The results confirm the hints suggesting that GHSR is a preferential target for further investigation on mutations that influence carcass trait variations.     

Ghrelin Is Produced in Taste Cells and Ghrelin Receptor Null Mice Show Reduced Taste Responsivity to Salty (NaCl) and Sour (Citric Acid) Tastants

Background

The gustatory system plays a critical role in determining food preferences, food intake and energy balance. The exact mechanisms that fine tune taste sensitivity are currently poorly defined, but it is clear that numerous factors such as efferent input and specific signal transduction cascades are involved.

Methodology/Principal Findings

Using immunohistochemical analyses, we show that ghrelin, a hormone classically considered to be an appetite-regulating hormone, is present within the taste buds of the tongue. Prepro-ghrelin, prohormone convertase 1/3 (PC 1/3), ghrelin, its cognate receptor (GHSR), and ghrelin-O-acyltransferase (GOAT , the enzyme that activates ghrelin) are expressed in Type I, II, III and IV taste cells of mouse taste buds. In addition, ghrelin and GHSR co-localize in the same taste cells, suggesting that ghrelin works in an autocrine manner in taste cells. To determine a role for ghrelin in modifying taste perception, we performed taste behavioral tests using GHSR null mice. GHSR null mice exhibited significantly reduced taste responsivity to sour (citric acid) and salty (sodium chloride) tastants.

Conclusions/Significance

These findings suggest that ghrelin plays a local modulatory role in determining taste bud signaling and function and could be a novel mechanism for the modulation of salty and sour taste responsivity.     

Endogenous ghrelin‐O‐acyltransferase (GOAT) acylates local ghrelin in the hippocampus

Ghrelin is an appetite‐stimulating peptide. Serine 3 on ghrelin must be acylated by octanoate via the enzyme ghrelin‐O‐acyltransferase (GOAT) for the peptide to bind and activate the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). Interest in GHSR1a increased dramatically when GHSR1a mRNA was demonstrated to be widespread in the brain, including the cortex and hippocampus, indicating that it has multifaceted functions beyond the regulation of metabolism. However, the source of octanoylated ghrelin for GHSR1a in the brain, outside of the hypothalamus, is not well understood. Here, we report the presence of GOAT and its ability to acylate non‐octanoylated ghrelin in the hippocampus. GOAT immunoreactivity is aggregated at the base of the dentate granule cell layer in the rat and wild‐type mouse. This immunoreactivity was not affected by the pharmacological inhibition of GHSR1a or the metabolic state‐dependent fluctuation of systemic ghrelin levels. However, it was absent in the GHSR1a knockout mouse hippocampus, pointing the possibility that the expression of GHSR1a may be a prerequisite for the production of GOAT. Application of fluorescein isothiocyanate (FITC)‐conjugated non‐octanoylated ghrelin in live hippocampal slice culture (but not in fixed culture or in the presence of GOAT inhibitors) mimicked the binding profile of FITC‐conjugated octanoylated ghrelin, suggesting that extracellularly applied non‐octanoylated ghrelin was acylated by endogenous GOAT in the live hippocampus while GOAT being mobilized out of neurons. Our results will advance the understanding for the role of endogenous GOAT in the hippocampus and facilitate the search for the source of ghrelin that is intrinsic to the brain.

     

Apo-ghrelin receptor forms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of DRD2 agonism

We identified subsets of neurons in the brain that coexpress the dopamine receptor subtype-2 (DRD2) and the ghrelin receptor (GHSR1a). Combination of FRET confocal microscopy and Tr-FRET established the presence of GHSR1a:DRD2 heteromers in hypothalamic neurons. To interrogate function, mice were treated with the selective DRD2 agonist cabergoline, which produced anorexia in wild-type and ghrelin?/? mice; intriguingly, ghsr?/? mice were refractory illustrating dependence on GHSR1a, but not ghrelin. Elucidation of mechanism showed that formation of GHSR1a:DRD2 heteromers allosterically modifies canonical DRD2 dopamine signaling resulting in Gβγ subunit-dependent mobilization of [Ca2?](i) independent of GHSR1a basal activity. By targeting the interaction between GHSR1a and DRD2 in wild-type mice with a highly selective GHSR1a antagonist (JMV2959) cabergoline-induced anorexia was blocked. Inhibiting dopamine signaling in subsets of neurons with a GHSR1a antagonist has profound therapeutic implications by providing enhanced selectivity because neurons expressing DRD2 alone would be unaffected.     

Parallel solid-phase synthesis of vitronectin receptor (alphavbeta3) inhibitors

A combinatorial approach for rapid optimization of a vitronectin receptor (alphavbeta3) inhibitor lead was accomplished by solid-phase synthesis. Orthogonally bis protected 2,3-diaminopropionic acid was used to immobilize the C-terminus of the molecule. Selective deprotection and functionalization of the alpha-amino group followed by acyl resorcinol scaffold attachment and N-terminus diversification was used to explore structure activity relationship (SAR).     

A relationship between motilin and growth hormone secretagogue receptors

The motilin receptor (MR) belongs to a family of Class I G protein-coupled receptors that also includes growth hormone secretagogue receptor (GHSR). Their potentially unique structure and the molecular basis of their binding and activation are not yet clear. We previously reported that the perimembranous residues in the predicted extracellular loops and amino-terminal tail of the MR were important for responses to the natural peptide ligand, motilin, and the transmembrane domains of the MR were important for a non-peptidyl ligand, erythromycin. We also reported that the perimembranous residues in the second extracellular loop of the GHSR were critical for natural ligand ghrelin binding and activity. The MR is 52% identical to GHSR, with 86% sequence identity in the transmembrane domains. In the current work, to gain insight into a relationship between MR and GHSR, we studied functional responses to motilin, erythromycin and ghrelin of expression cells of chimeric constructs of MR and GHSR and co-expression cells of both MR and GHSR. We also generated human MR transgenic mice, and clarified a relationship between motilin and ghrelin. MR(1-62)/GHSR(68-366) construct responded only to ghrelin, MR(1-102)/GHSR(108-366) responded to ghrelin and erythromycin, and MR(1-129)/GHSR(135-366) and MR(1-178)/GHSR(184-366) responded to erythromycin, while GHSR(1-183)/MR(179-412) responded to neither motilin, erythromycin nor ghrelin. MR and GHSR co-expression cells have no additional responses to these ligands. Motilin or erythromycin administration to human MR transgenic mice resulted in a decrease of serum acyl-ghrelin levels, while MR and GHSR mRNA expression in the gastrointestinal tracts were not changed. These data suggested that in species expressing both motilin-MR and ghrelin-GHSR, there is a compensatory relationship in vivo.     

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G_q/11, G_i/o, and G_12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca²⁺ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.     

中华鳖GHSR基因SNPs的筛选及生长性状的关联分析

为探究GHSR基因多态性对中华鳖(Pelodiscus sinensis)生长相关性状的影响,采用直接测序法在GHSR基因5'侧翼和3'侧翼上筛选SNPs位点,共检测到5个单核苷酸多态性位点:A335T、G397T、A527G、A13482C和T13526A。随机选取同批繁殖的1冬龄200只中华鳖用直接测序法进行SNPs位点的分型,并分析与生长性状的相关性。检测结果显示,所有SNP位点均符合Hardy-Weinberg平衡状态(p>0.05)。方差分析显示,A336T位点的AT、TT基因型的体重、背甲长、背甲宽和裙边宽4项生长数据均显著高于AA基因型。A13482C位点的AC基因型的体重、背甲长、背甲宽和裙边宽4项数据均显著高于AA基因型(p<0.05)。研究表明,本实验在GHSR基因上获得的这些SNP位点可能影响着中华鳖的生长性状或与之紧密连锁,可为中华鳖分子辅助育种提供助力与参考。     

Mutually opposite signal modulation by hypothalamic heterodimerization of ghrelin and melanocortin-3 receptors

Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number of examples implicate a profound functional and physiological relevance of homo- or hetero-oligomeric GPCRs. The ghrelin (growth hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have orexigenic effects on the hypothalamic control of body weight. Because in vitro studies indicate heterodimerization of GHSR and MC3R, we investigated their functional interplay. Combined in situ hybridization and immunohistochemistry indicated that the vast majority of GHSR-expressing neurons in the arcuate nucleus also express MC3R. In vitro coexpression of MC3R and GHSR promoted enhanced melanocortin-induced intracellular cAMP accumulation compared with activation of MC3R in the absence of GHSR. In contrast, agonist-independent basal signaling activity and ghrelin-induced signaling of GHSR were impaired, most likely due to interaction with MC3R. By taking advantage of naturally occurring GHSR mutations and an inverse agonist for GHSR, we demonstrate that the observed enhanced MC3R signaling capability depends directly on the basal activity of GHSR. In conclusion, we demonstrate a paradigm-shifting example of GPCR heterodimerization allowing for mutually opposite functional influence of two hypothalamic receptors controlling body weight. We found that the agonist-independent active conformation of one GPCR can determine the signaling modalities of another receptor in a heterodimer. Our discovery also implies that mutations within one of two interacting receptors might affect both receptors and different pathways simultaneously. These findings uncover mechanisms of important relevance for pharmacological targeting of GPCR in general and hypothalamic body weight regulation in particular.     

The discovery of sulfonylated dipeptides as potent VLA-4 antagonists

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.     

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

Discovery of a new class of ghrelin receptor antagonists

A series of benzodiazepine antagonists of the human ghrelin receptor GHSR1a were synthesized and their antagonism and metabolic stability were evaluated. The potency of these analogs was determined using a functional aequorin (Euroscreen) luminescent assay measuring the intracellular Ca(2+) concentration, and their metabolic stability was measured using an in vitro rat and human S9 hepatocyte assay. These efforts led to the discovery of a potent ghrelin antagonist with good rat pharmacokinetic properties.     

MRAP2 inhibits β-arrestin recruitment to the ghrelin receptor by preventing GHSR1a phosphorylation

The melanocortin receptor accessory protein 2 (MRAP2) is essential for several physiological functions of the ghrelin receptor growth hormone secretagogue receptor 1a (GHSR1a), including increasing appetite and suppressing insulin secretion. In the absence of MRAP2, GHSR1a displays high constitutive activity and a weak G-protein–mediated response to ghrelin and readily recruits β-arrestin. In the presence of MRAP2, however, G-protein–mediated signaling via GHSR1a is strongly dependent on ghrelin stimulation and the recruitment of β-arrestin is significantly diminished. To better understand how MRAP2 modifies GHSR1a signaling, here we investigated the role of several phosphorylation sites within the C-terminal tail and third intracellular loop of GHSR1a, as well as the mechanism behind MRAP2-mediated inhibition of β-arrestin recruitment. We show that Ser²⁵² and Thr²⁶¹ in the third intracellular loop of GHSR1a contribute to β-arrestin recruitment, whereas the C-terminal region is not essential for β-arrestin interaction. Additionally, we found that MRAP2 inhibits GHSR1a phosphorylation by blocking the interaction of GRK2 and PKC with the receptor. Taken together, these data suggest that MRAP2 alters GHSR1a signaling by directly impacting the phosphorylation state of the receptor and that the C-terminal tail of GHSR1a prevents rather than contribute to β-arrestin recruitment.

The “hunger hormone” ghrelin is secreted by X/A cells of the oxyntic mucosa of the stomach in response to a low energetic state, which leads to an increase in appetite (1, 2) and prevents hypoglycemia (3, 4). Ghrelin is the agonist of the growth hormone secretagogue receptor 1a (GHSR1a), a G-protein–coupled receptor (GPCR) expressed in the brain and in multiple peripheral organs including the heart and the endocrine pancreas. Activation of GHSR1a by ghrelin in hypothalamic agouti-related protein (AgRP) neurons potently stimulates feeding (5, 6, 7). In pituitary somatotrophs, GHSR1a stimulation promotes growth hormone release (8, 9, 10). Finally, in cardiomyocytes, ghrelin increases cell survival and contractility (11, 12) while in the endocrine pancreas the hormone inhibits insulin secretion (13, 14).GHSR1a primarily couples to Gα_q/11, thus stimulating the production of intracellular inositol triphosphate (IP) 3. Like other GPCRs, agonist stimulation results in phosphorylation of GHSR1a by kinases, including GPCR kinase 2 (GRK2) and PKC (15), and β-arrestin recruitment. Notably, GHSR1a contains several phosphorylation sites within the C-terminal tail, some of which have been shown to be important for β-arrestin recruitment (16). However, although other putative phosphorylation sites are present in the third intracellular loop (ICL3) of GHSR1a, their role in β-arrestin recruitment has not yet been described.When expressed in heterologous cells, GHSR1a displays a high constitutive activity and a limited ghrelin-stimulated responses (17).Both constitutive- and agonist-stimulated GHSR1a signaling are regulated by the single transmembrane melanocortin receptor accessory protein 2 (MRAP2), which functions to drastically reduce GHSR1a constitutive activity and increase ghrelin-stimulated responses (17). Additionally, MRAP2 significantly inhibits ghrelin-induced β-arrestin recruitment to GHSR1a (17). As such, MRAP2 is essential for several physiological functions of ghrelin including its orexigenic activity (18) and its insulinostatic actions (14). Global or AGRP neuron–targeted deletion of MRAP2 abrogates the effect of ghrelin on food intake (18) and global or pancreatic δ-cell-targeted deletion of MRAP2 prevents ghrelin-mediated inhibition of insulin secretion (14).Although expressed in AGRP neurons and pancreatic δ-cells (thus promoting G-protein coupling and inhibiting β-arrestin-dependent signaling), MRAP2 is not present in every GHSR1a-expressing tissue. Consequently, it is possible that β-arrestin signaling plays an important role in the physiological function of ghrelin in tissues where MRAP2 is absent. Whereas, the inhibition of β-arrestin recruitment to GHSR1a by MRAP2 is well established and the domains of MRAP2 required for this function have been identified (17), the molecular mechanism by which MRAP2 alters GHSR1a signaling is not yet understood. In this study, we investigated the importance of GHSR1a phosphorylation for β-arrestin recruitment and the mechanism involved in MRAP2-mediated inhibition of β-arrestin recruitment.     

Discovery of galectin ligands in fully randomized combinatorial one-bead-one-compound (glyco)peptide libraries

The involvement of human lectins (galectins) in disease progression accounts for the interest to design potent inhibitors. Three fully randomized hexa(glyco)peptide libraries were prepared using the portion mixing method combined with ladder synthesis. On-bead screening with fluorescently labelled galectin-1 and -3 yielded a series of lead structures, whose inhibitory activity on carbohydrate-dependent galectin binding was tested in solution by solid-phase and cell assays. The various data obtained define the library approach as a facile route for the discovery of selective (glyco)peptide-based galectin inhibitors.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Discovery of indazole aldosterone synthase (CYP11B2) inhibitors as potential treatments for hypertension

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.     

Synthesis and pharmacological evaluation of indolinone derivatives as novel ghrelin receptor antagonists

The ghrelin receptor is a G-protein-coupled receptor (GPCR) widely expressed in the brain, stomach and the intestine. It was firstly identified during studies aimed to find synthetic modulators of growth hormone (GH) secretion. GHSR and its endogenous ligand ghrelin were found to be involved in hunger response. Through food intake regulation, they could affect body weight and adiposity. Thus GHSR antagonists rapidly became an attractive target to treat obesity and feeding disorders. In this study we describe the biological properties of new indolinone derivatives identified as a new, chiral class of ghrelin antagonists. Their synthesis as well as the structure-activity relationship will be discussed herein. The in vitro identified compound 14f was a potent GHSR1a antagonist (IC(50)=7nM). When tested in vivo, on gastric emptying model, 14f showed an inhibitory intrinsic effect when given alone and it dose dependently inhibited ghrelin stimulation. Compound 14f also reduced food intake stimulated both by fasting condition (high level of endogenous ghrelin) and by icv ghrelin. Moreover this compound improved glucose tolerance in ipGTT test.