Ghrelin Receptor: High Constitutive Activity and Methods for Developing Inverse Agonists

doi:10.1016/B978-0-12-381296-4.00006-3

Methods in Enzymology

Volume 485, 2010, Pages 103-121

https://doi.org/10.1016/B978-0-12-381296-4.00006-3 Get rights and content

Abstract

The ghrelin receptor is a G protein-coupled receptor (GPCR) mainly distributed in the brain, and also expressed in peripheral tissues. Remarkably, the ghrelin receptor possesses a naturally high constitutive activity representing 50% of its maximal activity. Its endogenous ligand ghrelin is the only known orexigenic gastrointestinal peptide and plays a central role in the regulation of appetite, food intake, and energy homeostasis.

Reducing the constitutive activity of the ghrelin receptor by inverse agonists is the strategy adopted by our group to develop anti-obesity drugs. Therefore, short peptides were synthesized and showed high inverse agonist potency toward the ghrelin receptor.

This review describes the methods used to synthesize the peptides and to evaluate their biological activity. Peptide synthesis was performed on solid phase using a Fmoc/tBu-strategy. Peptide potency was measured with a signal transduction assay, the inositol trisphosphate turnover assay, adapted to a receptor expressing constitutive activity.

Introduction

The ghrelin receptor (formerly the growth hormone secretagogue receptor, GHSR) was identified in 1996 as the receptor of GHS (growth hormone secretagogues) (Howard et al., 1996, Smith et al., 1999a, Smith et al., 1999b). The receptor is a typical G protein-coupled receptor (GPCR) with seven transmembrane helices, an extracellular N-terminus and an intracellular C-terminus. It is coupled to a G protein of the type G_αq/11 and belongs to the rhodopsin family of GPCR. The ghrelin receptor is well conserved in vertebrate species and presents typical conserved sites such as cysteines in the first two extracellular loops (Smith et al., 2001). It is mainly expressed in the pituitary gland, the hypothalamus, and the hippocampus but also in other tissues such as stomach, liver, and heart (Kojima & Kangawa, 2005, van der Lely et al., 2004).

The endogenous ligand ghrelin (from “ghre,” the word-root in Proto-Indo-European languages for “grow”) was isolated in 1999. It is a 28-amino acid peptide (Fig. 6.1), octanoylated on Ser³, and mainly produced in the oxyntic mucosa of the stomach. The N-terminal pentapeptide was identified as the minimal sequence necessary for activation of the ghrelin receptor (Bednarek et al., 2000). The n-octanoylation is carried out posttranslationally by ghrelin-O-acyltransferase (GOAT; Gutierrez et al., 2008, Yang et al., 2008) and is essential for binding the ghrelin receptor and for ghrelin circulation in blood (Bednarek et al., 2000, De Vriese & Delporte, 2007).

Physiologically, ghrelin is the only known orexigenic peptide produced in the gastrointestinal tract. It stimulates food intake, and upregulates energy homeostasis through the stimulation of NPY/AGRP-containing neurons in the arcuate nucleus of hypothalamus (Cummings, 2006, Nakazato et al., 2001).

Ghrelin receptor agonists were widely developed before the discovery of ghrelin and its receptor (Nargund et al., 1998). These molecules were called GHS as they were able to initiate the release of growth hormone, independently from the growth hormone releasing hormone (GHRH) pathway. The major role of ghrelin in the regulation of energy homeostasis orientates the research in the development of ghrelin receptor antagonists that may downregulate energy homeostasis and thus, decrease food intake and body weight (Chollet et al., 2009).

Section snippets

Constitutive Activity and Development of Ghrelin Receptor Inverse Agonists

GPCRs can be stimulated by different ligands like partial or full agonists, inverse agonists, and neutral antagonists. According to IUPHAR receptor nomenclature and drug classification (Neubig et al., 2003), an agonist binds its target receptor and generates a biological response. Conventionally, an agonist increases the receptor activity while an inverse agonist decreases it. A full agonist induces a maximal response by switching the receptor into its active conformation. In case of

Synthesis of Ghrelin Receptor Inverse Agonists: Solid-Phase Peptide Synthesis (SPPS)

In this section, we provide information about the synthesis of ghrelin receptor inverse agonists. All peptides are synthesized automatically or manually on solid phase.

Functional Assays for Ghrelin Receptor Inverse Agonists

The potency of a ligand is defined as its ability to activate or inhibit a receptor and can be evaluated by functional assays. Several functional assays are available for GPCRs. Most of them consist in measuring a component of the signal transduction cascade, generated by the GPCR in response to a ligand.

Briefly, a ligand induces a conformational change of the receptor, leading to the dissociation of the G protein subunits. Depending on the nature of the G subunit, different pathway can be

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Inverse agonist efficacy of selatogrel blunts constitutive P2Y12 receptor signaling by inducing the inactive receptor conformation
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In recent years, advancements in GPCR pharmacology and ligand efficacy have led to major breakthroughs in GPCR drug discovery. In particular, several studies highlighted the physiological relevance of the ligand-independent activation of many GPCRs (i.e., constitutive activity) and the therapeutic potential of inverse agonists in the treatment of diseases.[10–13] In line with this concept, we and others previously reported that the P2Y12 receptor triggered constitutive signaling in human platelets. [14,15]
Selatogrel is a potent inhibitor of adenosine diphosphate (ADP) binding to the P2Y12 receptor, preventing platelet activation. We have previously shown that the P2Y12 receptor constitutively activates Gi- and Go-protein-mediated signaling in human platelets.
Here, we report that selatogrel acts as an inverse agonist of the P2Y12 receptor. Specifically, using bioluminescence resonance energy transfer2 (BRET2) probes, selatogrel, ticagrelor, and elinogrel were shown to stabilize the inactive form of the G_αi/o-G_βγ complex in cells with recombinant expression of the P2Y12 receptor. In dose–response experiments, while selatogrel exhibited a maximal efficacy similar to ticagrelor, selatogrel was approximately 100-fold more potent than ticagrelor. Quantification of relative cyclic adenosine monophosphate (cAMP) levels in cells expressing the cAMP BRET1 sensor (CAMYEL probe) confirmed that selatogrel completely abolished the constitutive activity of the P2Y12 receptor. In agreement, selatogrel increased basal cAMP levels in human platelets, confirming inverse agonism on the endogenous human platelet P2Y12 receptor. In agreement with the biochemical phenotype of inverse agonism efficacy of selatogrel, the 2.8 Angstrom resolution cocrystal structure of selatogrel bound to the P2Y12 receptor confirmed that selatogrel stabilizes the inactive, basal state of the receptor. Selatogrel bound to pocket 1, spanning helix III to VII. Furthermore, the binding mode of selatogrel, suggesting steric overlap with the proposed binding site of ADP and the ADP analog 2-methylthioadenosine diphosphate (2MeSADP), agrees with the functional characterization of selatogrel preventing platelet activation by blocking ADP binding to the P2Y12 receptor.
Ghrelin: From a gut hormone to a potential therapeutic target for alcohol use disorder
2019, Physiology and Behavior
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According to the aforementioned preclinical and clinical data, GHS-R1a blockade appears to be a potential pharmacological approach to treat AUD. Given that GHS-R1a has high constitutive (ligand-independent) activity, inverse agonism of the receptor may exert more potent and desirable effects than pure competitive antagonism [129]. PF-5190457, an investigational drug acting as a GHS-R1a inverse agonist [130], is the first GHS-R1a blocker that has progressed to the human phases of medication development.
Alcohol use disorder (AUD) is a leading cause of morbidity and mortality worldwide. However, treatment options, including pharmacotherapies, are limited in number and efficacy. Accumulating evidence suggests that elements of the gut-brain axis, such as neuroendocrine pathways and gut microbiome, are involved in the pathophysiology of AUD and, therefore, may be investigated as potential therapeutic targets. One pathway that has begun to be examined in this regard is the ghrelin system. Here, we review preclinical and clinical data on the relationship between ghrelin and alcohol-related outcomes, with a special focus on the role of the ghrelin system as a treatment target for AUD. Observational studies indicate that endogenous ghrelin levels are positively associated with craving for alcohol, subjective responses to alcohol, and brain activity in response to alcohol cues. Knockout rodent models suggest that deletion of the ghrelin peptide or receptor gene leads to reduction of alcohol intake and other alcohol-related outcomes. Different research groups have found that ghrelin administration increases, while ghrelin receptor (GHS-R1a) blockade reduces alcohol intake and other alcohol-related outcomes in rodents. Ghrelin administration in heavy-drinking individuals increases alcohol craving and self-administration and modulates brain activity in response to alcohol reward anticipation. PF-5190457, a GHS-R1a blocker, has been shown to be safe and tolerable when co-administered with alcohol. Furthermore, preliminary results suggest that this compound may reduce cue-elicited craving for alcohol in heavy-drinking individuals – a finding in need of replication. Collectively, the existing literature supports further examination of the ghrelin system as a therapeutic target for AUD. More research is also needed to understand the biobehavioral and molecular mechanisms underlying ghrelin's functions and to examine different interventional approaches to target the ghrelin system for AUD treatment.
Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor
2019, Structure
The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif. This information combined with assignment of the secondary structure of ghrelin in its receptor-bound state was incorporated into Rosetta using an approach that accounts for flexible binding partners. The NMR data and models revealed an extended binding surface that was confirmed via mutagenesis. Our results agree with a growing evidence of peptides interacting via two sites at G protein-coupled receptors.
Pharmacological manipulation of the ghrelin system and alcohol hangover symptoms in heavy drinking individuals: Is there a link?
2018, Pharmacology Biochemistry and Behavior
Citation Excerpt :
Before secretion, a fraction of ghrelin undergoes post-translational modification, i.e., acylation via the ghrelin-O-acyltransferase (GOAT) enzyme (Kojima and Kangawa, 2005). This process enables acyl-ghrelin to bind to its receptor named the growth hormone secretagogue receptor 1a (GHS-R1a), a G protein-coupled receptor (GPCR) with high constitutive (ligand-independent) activity (Els et al., 2010; Holst et al., 2004; Howard et al., 1996). Ghrelin receptors are widely expressed in various brain regions (e.g., hypothalamus, pituitary gland, ventral tegmental area, amygdala, and hippocampus) as well as peripheral organs (e.g., gut, pancreas, thyroid, heart, and adipose tissue) (Cabral et al., 2017; Gnanapavan et al., 2002; Guan et al., 1997; Howick et al., 2017; Zigman et al., 2006).
Ghrelin, an orexigenic peptide synthesized in the stomach, is a key player in the gut-brain axis. In addition to its role in regulating food intake and energy homeostasis, ghrelin has been shown to modulate alcohol-related behaviors. Alcohol consumption frequently results in hangover, an underexplored phenomenon with considerable medical, psychological, and socioeconomic consequences. While the pathophysiology of hangover is not clear, contributions of mechanisms such as alcohol-induced metabolic/endocrine changes, inflammatory/immune response, oxidative stress, and gut dysbiosis have been reported. Interestingly, these mechanisms considerably overlap with ghrelin's physiological functions. Here, we investigated whether pharmacological manipulation of the ghrelin system may affect alcohol hangover symptoms. Data were obtained from two placebo-controlled laboratory studies. The first study tested the effects of intravenous (IV) ghrelin and consisted of two experiments: a progressive-ratio IV alcohol self-administration (IV-ASA) and a fixed-dose IV alcohol clamp. The second study tested the effects of an oral ghrelin receptor inverse agonist (PF-5190457) and included a fixed-dose oral alcohol administration experiment. Alcohol hangover data were collected the morning after each alcohol administration experiment using the Acute Hangover Scale (AHS). IV ghrelin, compared to placebo, significantly reduced alcohol hangover after IV-ASA (p = 0.04) and alcohol clamp (p = 0.04); PF-5190457 had no significant effect on AHS scores. Females reported significantly higher hangover symptoms than males following the IV-ASA experiment (p = 0.04), but no gender × drug condition (ghrelin vs. placebo) effect was found. AHS total scores were positively correlated with peak subjective responses, including ‘stimulation’ (p = 0.08), ‘sedation’ (p = 0.009), ‘feel high’ (p = 0.05), and ‘feel intoxicated’ (p = 0.03) during the IV-ASA. IV ghrelin blunted the positive association between alcohol sedation and hangover as shown by trend-level drug × sedation effect (p = 0.08). This is the first study showing that exogenous ghrelin administration, but not ghrelin receptor inverse agonism, affects hangover symptoms. Future research should investigate the potential mechanism(s) underlying this effect.
High metabolic in vivo stability and bioavailability of a palmitoylated ghrelin receptor ligand assessed by mass spectrometry
2015, Bioorganic and Medicinal Chemistry
The constitutive activity of the ghrelin receptor is of high physiological and pathophysiological relevance. In-depth structure–activity relationship studies revealed a palmitoylated ghrelin receptor ligand that displays an in vitro binding affinity in the low nanomolar range. Activity studies revealed inverse agonistic as well as antagonistic properties and in vitro metabolic analysis indicated a high stability in blood serum and liver homogenate. For metabolic testing in vivo, a combined approach of stable isotopic labeling and mass spectrometry-based analysis was established. Therefore, a heavy isotopic version of the peptide containing a ¹³C-labeled palmitic acid was synthesized and a 1:1 ratio of a ¹²C/¹³C-peptide mixture was injected into rats. Biological samples were analyzed by multiple reaction monitoring allowing simultaneous peptide detection and quantification. Measurements revealed a suitable bioavailability over 24 h in rat serum and subsequent high-resolution mass spectrometry investigations showed only negligible degradation and slow body clearance. Hence, this method combination allowed the identification and evaluation of a highly potent and metabolically stable ghrelin receptor ligand in vivo.
A link between ghrelin and major depressive disorder: a mini review
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Chapter Six - Ghrelin Receptor: High Constitutive Activity and Methods for Developing Inverse Agonists

Abstract

Introduction

Section snippets

Constitutive Activity and Development of Ghrelin Receptor Inverse Agonists

Synthesis of Ghrelin Receptor Inverse Agonists: Solid-Phase Peptide Synthesis (SPPS)

Functional Assays for Ghrelin Receptor Inverse Agonists

Anal. Biochem.

Physiol. Behav.

Biochem. Pharmacol.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem. Sci.

Trends Endocrinol. Metab.

Cell

Structure–function studies on the new growth hormone-releasing peptide, ghrelin: Minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a

J. Med. Chem.