Identification, chemical synthesis, and receptor binding of a reptilian gecko ghrelin.

Ghrelin is known to be a gastrointestinal peptide hormone in vertebrates. It has a unique posttransrational modification, octanoylation, at the Ser side chain of the third position. In this study, we identified the genes encoding ghrelin and its receptor from the Schlegel's Japanese gecko Gekko japonicus. The C-terminal residue of gecko ghrelin was His, although the chemical synthesis method for the O-octanoyl peptide with a C-terminal His residue has not yet been well-established. Acyl-ghrelin has been synthesized using a Ser derivative without side chain protecting group in the solid-phase peptide synthesis, although this synthetic strategy has not yet been well-established. Here we show the efficient synthetic method with minimal side reactions, and G. japonicus ghrelin could be obtained in good yield. This would be useful and applicable to the synthesis of ghrelin from other animal species. The gecko ghrelin receptor was expressed in HEK 293 cells, which was fully responsive to the synthetic gecko ghrelin. These results indicate that the ghrelin system similar to mammals also exists in a reptilian gecko, G. japonicus.

Unusual orthologs shed new light on the binding mechanism of ghrelin to its receptor GHSR1a.

The gastric peptide ghrelin has important functions in energy metabolism and cellular homeostasis by activating growth hormone secretagogue receptor type 1a (GHSR1a). The N-terminal residues of ghrelin orthologs from all vertebrates are quite conserved; however, in orthologs from Cavia porcellus and Phyllostomus discolor, Ser2 and Leu5 are replaced by a smaller Ala and a positively charged Arg, respectively. In the present study, we first demonstrated that the hydrophobic Leu5 is essential for the function of human ghrelin, because Ala replacement caused an approximately 100-fold decrease in activity. However, replacement of Leu5 by an Arg residue caused much less disruption; further replacement of Ser2 by Ala almost restored full activity, although the [S2A] mutation itself showed slight detriments, implying that the positively charged Arg5 in the [S2A,L5R] mutant might form alternative interactions with certain receptor residues to compensate for the loss of the essential Leu5. To identify the responsible receptor residues, we screened GHSR1a mutants in which all conserved negatively charged residues in the extracellular regions and all aromatic residues in the ligand-binding pocket were mutated separately. According to the decrease in selectivity of the mutant receptors towards [S2A,L5R]ghrelin, we deduced that the positively charged Arg5 of the ghrelin mutant primarily interacts with the essential aromatic Phe286 at the extracellular end of the sixth transmembrane domain of GHSR1a by forming cation-π and π-π interactions. The present study provided new insights into the binding mechanism of ghrelin with its receptor, and thus would facilitate the design of novel ligands for GHSR1a.

Biotinylated non-ionic amphipols for GPCR ligands screening.

We present herein the synthesis of biotin-functionalized polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG)8-alkyne that resulted in formation of the biotinylated polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized polymers was done using both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based on their ability to bind the immobilized receptor. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin-GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved Asp99 of GHSR1a probably interacts with the essential Arg6 of LEAP2. After five conserved Phe residues in the predicted ligand-binding pocket of human GHSR1a were mutated, three of them were identified as important for GHSR1a binding to LEAP2. According to a structural model of GHSR1a, we deduced that the adjacent Phe279 and Phe312 might interact with the essential Phe4 of LEAP2, while Phe119 might interact with the aromatic Trp5 of LEAP2. The present study provided new insights into the interaction of LEAP2 with its receptor, and would facilitate the design of novel ligands for GHSR1a in future studies.

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

Early Detection of Growth Hormone Secretagogue Receptor Antagonists Exploiting Their Atypical Behavior in Competitive Assays.

We report the proof-of-concept of a bioaffinity format designed for the early detection of growth hormone secretagogue receptor (GHS-R1a) antagonists in urine samples. We exploit here their atypical behavior in competitive experiments with labeled ghrelin (GHR), namely, the strong promoting effect on the GHR/GHS-R1a interaction at low molar ratios GHR/antagonist. The antagonists potentiate the GHR/GHS-R1a interaction, and they display the same effect on the interaction of GHS-R1a with other agonists listed as doping agents. The developed assay allows the estimation of affinity constants of ligand/receptor and antagonist/receptor binding and is amenable to optical, electrochemical, and mass-sensitive detection. The estimated affinity constants for GHR/GHS-R1a and antagonist/GHS-R1a in the absence of G proteins are in good agreement with recently reported data.

Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex.

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 protein activation. 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.

Clarifying the Ghrelin System's Ability to Regulate Feeding Behaviours Despite Enigmatic Spatial Separation of the GHSR and Its Endogenous Ligand.

Ghrelin is a hormone predominantly produced in and secreted from the stomach. Ghrelin is involved in many physiological processes including feeding, the stress response, and in modulating learning, memory and motivational processes. Ghrelin does this by binding to its receptor, the growth hormone secretagogue receptor (GHSR), a receptor found in relatively high concentrations in hypothalamic and mesolimbic brain regions. While the feeding and metabolic effects of ghrelin can be explained by the effects of this hormone on regions of the brain that have a more permeable blood brain barrier (BBB), ghrelin produced within the periphery demonstrates a limited ability to reach extrahypothalamic regions where GHSRs are expressed. Therefore, one of the most pressing unanswered questions plaguing ghrelin research is how GHSRs, distributed in brain regions protected by the BBB, are activated despite ghrelin's predominant peripheral production and poor ability to transverse the BBB. This manuscript will describe how peripheral ghrelin activates central GHSRs to encourage feeding, and how central ghrelin synthesis and ghrelin independent activation of GHSRs may also contribute to the modulation of feeding behaviours.

Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

C-terminus of a hexapeptidic ghrelin receptor inverse agonist can switch peptide behavior from inverse agonism to agonism.

Subtle changes in the sequence at the N-terminus and in the aromatic core of hexapeptidic ghrelin receptor inverse agonists can switch behavior from inverse agonism to agonism, but the C-terminal role of the sequence is unclear. Thus, analogs of the ghrelin receptor inverse agonist KbFwLL-NH2 (b = ß-(3-benzothienyl)-d-alanine) were synthesized by solid phase peptide synthesis in order to identify the influence of aromaticity, charge, and hydrophobicity. Potency and efficacy of the hexapeptides were evaluated in inositol triphosphate turnover assays. Notably, modifications directly at the C-terminal Leu(6) could influence peptide efficacy leading to decreased constitutive activity. High hydrophobicity at the C-terminal position was of importance for elevated inverse agonist activity, the introduction of charged amino acids led to decreased potency. In contrast, structure-activity relationship studies of Leu(5) located closer to the aromatic core revealed an agonism-inducing position. These findings imply that amino acids with possible cation-π or π-π interactions and a suitable orientation at the C-terminus of the aromatic core induce agonism. Receptor binding studies showed that most peptides bind to the receptor at a concentration of 1 µM and modification directly at the C-terminus is generally more accepted than Leu(5) substitution. Interestingly, this observation is not dependent on the type of modification. These studies reveal another switch region of the short ghrelin receptor ligand pointing out the sensitivity of the ghrelin receptor binding pocket.

Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor.

The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, µ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.

Sequence, genomic organization and expression of ghrelin receptor in grass carp, Ctenopharyngodon idellus.

The growth hormone secretagogue-receptor (GHS-R) is an endogenous receptor for the gut hormone ghrelin. Here we report the identification and characterization of GHS-R1a in grass carp, Ctenopharyngodon idellus. The full-length GHS-R1a cDNA contained a 1803-bp coding domain sequence which encoded a peptide of 360 amino acid residues. Comparison analysis revealed that the amino acid sequences of GHS-R1a were highly conserved in vertebrates and shared 97% amino acid identity with zebrafish (Danio rerio), 96% with jian carp (Cyprinus carpio var. Jian) and 93% with goldfish (Carassius auratus). The GHS-R1a showed the highest level of mRNA expression in the pituitary, followed by the brain and liver, and the lowest expression was observed in the hindgut. Intraperitoneally injected with grass carp ghrelin (50, 100 and 150ng/g body weight (BW)), grass carp showed greater mRNA expression of GHS-R1a in the pituitary compared with saline injected at 0.5h postinjection. It was observed that food deprivation could promote the expression of ghrelin and GHS-R1a in the pituitary, demonstrating that nutritional status can influence the expression of both ghrelin and GHS-R1a in the pituitary. After a 2- or 4-week fast, plasma growth hormone (GH) increased, was positively correlated with ghrelin and GHS-R1a mRNA expression levels in the pituitary. These results suggested that the involvement of ghrelin/GHS-R1a systems in mediating the effects of nutritional status and ghrelin on growth processes in grass carp.

Heterodimerization with Its splice variant blocks the ghrelin receptor 1a in a non-signaling conformation: a study with a purified heterodimer assembled into lipid discs.

Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.

Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop.

Based on a rare, natural Glu for Ala-204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2b (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for the Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided phenotypes similar to those of the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of extracellular loop 2b to form an extended α-helix was increased from 15% in the wild type to 89 and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal ion site, stabilizing an α-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding that constrains this segment and thereby conceivably the movements of transmembrane domain V relative to transmembrane domain III inhibits the high constitutive signaling.

High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs.

Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates G(q) in the absence of agonist, as assessed through guanosine 5'-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with µ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.

The 1,2,4-triazole as a scaffold for the design of ghrelin receptor ligands: development of JMV 2959, a potent antagonist.

Ghrelin is a 28-residue peptide acylated with an n-octanoyl group on the Ser 3 residue, predominantly produced by the stomach. Ghrelin displays strong growth hormone (GH) releasing activity, which is mediated by the activation of the so-called GH secretagogue receptor type 1a (GHS-R1a). Given the wide spectrum of biological activities of Ghrelin in neuroendocrine and metabolic pathways, many research groups, including our group, developed synthetic peptide, and nonpeptide GHS-R1a ligands, acting as agonists, partial agonists, antagonists, or inverse agonists. In this highlight article, we will focus on the discovery of a GHS-R1a antagonist compound, JMV 2959, which has been extensively studied in different in vitro and in vivo models. We will first describe the peptidomimetic approach that led us to discover this compound. Then we will review the results obtained with this compound in different studies in the fields of food intake and obesity, addictive behaviors, hyperactivity and retinopathy.

Antiproliferative activity on human prostate carcinoma cell lines of new peptidomimetics containing the spiroazepinoindolinone scaffold.

Peptidomimetics containing the spiroazepinoindolinone scaffold were designed and synthesized in order to ascertain their antiproliferative activity on the DU-145 human prostatic carcinoma cell line. Ethyl 2'-oxa-1,2,3,5,6,7-hexahydrospiro[4H-azepine-4,3'-3H-indole]-1'-carboxylate scaffold was functionalized at nitrogen azepino ring with Aib-(l/d)Trp-OH dipeptides. Combining the different stereochemistries of the scaffold and the tryptophan, diastereoisomeric peptidomimetics were prepared and tested. Their biological activity was evaluated by proliferation studies proving that the isomer containing S spiroazepino-indolinone scaffold and l tryptophan is the most active compound. Docking studies confirmed that the active peptidomimetic could bind the GHSR-1a receptor with docking scores comparable with those of well-known agonists even though with a somewhat different binding mode.

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

Acylated and unacylated ghrelin binding to membranes and to ghrelin receptor: towards a better understanding of the underlying mechanisms.

The O-octanoylation of human ghrelin is a natural post-translational modification that enhances its binding to model membranes and could potentially play a central role in ghrelin biological activities. Here, we aimed to clarify the mechanisms that drive ghrelin to the membrane and hence to its receptor that mediates most of its endocrinological effects. As the acylation enhances ghrelin lipophilicity and that ghrelin contains many basic residues, we examined the electrostatic attraction and/or hydrophobic interactions with membranes. Using various liposomes and buffer conditions in binding, zeta potential and isothermal titration calorimetry studies, we found that whereas acylated and unacylated ghrelin were both electrostatically attracted towards the membrane, only acylated ghrelin penetrated into the headgroup and the lipid backbone regions of negatively charged membranes. The O-acylation induced a 120-fold increase in ghrelin local concentration in the membrane. However, acylated ghrelin did not deeply penetrate the membrane nor did it perturb its organisation. Conformational studies by circular dichroism and attenuated total reflection Fourier transformed infrared as well as in silico modelling revealed that both forms of ghrelin mainly adopted the same structure in aqueous, micellar and bilayer environments even though acylated ghrelin structure is slightly more α-helical in a lipid bilayer environment. Altogether our results suggest that membrane acts as a "catalyst" in acylated ghrelin binding to the ghrelin receptor and hence could explain why acylated and unacylated ghrelin are both full agonists of this receptor but in the nanomolar and micromolar range, respectively.

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.