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.

Structural basis of human ghrelin receptor signaling by ghrelin and the synthetic agonist ibutamoren.

The hunger hormone ghrelin activates the ghrelin receptor GHSR to stimulate food intake and growth hormone secretion and regulate reward signaling. Acylation of ghrelin at Ser3 is required for its agonistic action on GHSR. Synthetic agonists of GHSR are under clinical evaluation for disorders related to appetite and growth hormone dysregulation. Here, we report high-resolution cryo-EM structures of the GHSR-Gi signaling complex with ghrelin and the non-peptide agonist ibutamoren as an investigational new drug. Our structures together with mutagenesis data reveal the molecular basis for the binding of ghrelin and ibutamoren. Structural comparison suggests a salt bridge and an aromatic cluster near the agonist-binding pocket as important structural motifs in receptor activation. Notable structural variations of the Gi and GHSR coupling are observed in our cryo-EM analysis. Our results provide a framework for understanding GHSR signaling and developing new GHSR agonist drugs.

Molecular recognition of an acyl-peptide hormone and activation of ghrelin receptor.

Ghrelin, also called "the hunger hormone", is a gastric peptide hormone that regulates food intake, body weight, as well as taste sensation, reward, cognition, learning and memory. One unique feature of ghrelin is its acylation, primarily with an octanoic acid, which is essential for its binding and activation of the ghrelin receptor, a G protein-coupled receptor. The multifaceted roles of ghrelin make ghrelin receptor a highly attractive drug target for growth retardation, obesity, and metabolic disorders. Here we present two cryo-electron microscopy structures of Gq-coupled ghrelin receptor bound to ghrelin and a synthetic agonist, GHRP-6. Analysis of these two structures reveals a unique binding pocket for the octanoyl group, which guides the correct positioning of the peptide to initiate the receptor activation. Together with mutational and functional data, our structures define the rules for recognition of the acylated peptide hormone and activation of ghrelin receptor, and provide structural templates to facilitate drug design targeting ghrelin receptor.

Identification of a Putative ß-Arrestin Superagonist of the Growth Hormone Secretagogue Receptor (GHSR).

Ghrelin is a pleiotropic feeding hormone which also has a pivotal role in the central nervous system. Upon the activation of its receptor, growth hormone secretagogue receptor (GHSR), the Gαq/11 -mediated and the ß-arrestin-mediated signaling pathways are activated. As the ß-arrestin pathway is a potential drug target, there is a strong need for ß-arrestin-biased GHSR modulators. Activation of the ß-arrestin pathway should inhibit the Gαq/11 -mediated calcium flux through internalization of the receptor. Hence, we used the antagonistic activity in the calcium assay as the first screening for the ß-arrestin activation. By conducting the second screening assay for the ß-arrestin activation based on extracellular signal regulated kinase (ERK) 1/2 phosphorylation, we discovered a putative ß-arrestin-biased superagonist. The activity of the compound was not completely blocked with the competitive antagonist, which implies that the effect is mediated, at least partly, by allosteric binding of the compound.

Allosteric modulation of ghrelin receptor signaling by lipids.

The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.

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.

In silico strategy for detailing the binding modes of a novel family of peptides proven as ghrelin receptor agonists.

Ghrelin is a peptide hormone involved in multiple functions, including growth hormone release stimulation, food intake regulation, and metabolic and cytoprotective effect. A novel family of peptides with internal cycles was designed as ghrelin analogs and the biological activity of two of them (A228 and A233) was experimentally studied in-depth. In this work, an in silico strategy was developed for describing and assessing the binding modes of A228 and A233 to GHS-R1a (ghrelin receptor) comparing it with ghrelin and GHRP-6 peptides. Several reported structures of different G protein coupled receptors were used as templates, to obtain a good quality model of GHS-R1a. The best model was selected by preliminary molecular docking with ghrelin and GHRP-6. Docking was used to estimate peptide orientations in the binding site of the best model, observing a superposition of its N-terminal and its first aromatic residue. To test the complex stability in time, the C-terminal fragments of each peptide were added and the complexes were inserted a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, performing a molecular dynamic simulation for 100 ns using the CHARMM36 force field. Despite of the structural differences, the studied peptides share a common binding mode; the N-terminal interacts with E124 and the aromatic residue close to it, with the aromatic cluster (F279, F309, and F312). A preliminary pharmacophore model, consisting in a positive charged amine and an aromatic ring at an approximate distance of 0.79 nm, can be proposed. The results here described could represent a step forward in the efficient search of new ghrelin analogs.

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.

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.

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.

Identification of two teaghrelins in Shy-jih-chuen oolong tea.

Teaghrelins are unique acylated flavonoid tetraglycosides originally identified in Chin-shin oolong tea, and proposed to be potential oral analogs of ghrelin. Two acylated flavonoid tetraglycosides were isolated from Shy-jih-chuen oolong tea, and their chemical structures were determined to be quercetin and kaempferol 3-O-[α-L-arabinopyranosyl(1 â†’ 3)][2"-O-(E)-p-coumaroyl] [ß-D-glucopyranosyl(1 â†’ 3)-α-L-rhamnopyranosyl(1 â†’ 6)]-ß-D-glucoside. These two compounds were extremely similar to the two teaghrelins (teaghrelin-1 and teaghrelin-2) in Chin-shin oolong tea by simply replacing a glucopyranosyl group with an arabinopyranosyl group. Molecular modeling showed that the two putative teaghrelins identified in Shy-jih-chuen docked to and interacted with the ghrelin receptor as well as teaghrelin-1 and teaghrelin-2. Mixture of these two putative teaghrelins was shown to enhance the release of growth hormone from primary anterior pituitary cells of rats. The results suggest that two teaghrelins, named teaghrelin-3 and teaghrelin-4, are present in Shy-jih-chuen oolong tea and possess biological activities analogous to teaghrelins in Chin-shin oolong tea. PRACTICAL APPLICATIONS: According to this study, teaghrelin-3 and teaghrelin-4 may be regarded as active ingredients for the quality control of Shy-jih-chuen oolong tea. The content of teaghrelins may serve as a key factor for the farmers to select new tea plants in their next propagation of Shy-jih-chuen cultivar. Crude water extract of Shy-jih-chuen oolong tea containing teaghrelins is considered to be an adequate food supplement or additive in functional food products.

Rational Design, Synthesis, and Pharmacological Characterization of Novel Ghrelin Receptor Inverse Agonists as Potential Treatment against Obesity-Related Metabolic Diseases.

A new chemotype of ghrelin inverse agonists was discovered through chimeric design based on molecular scaffolds known as growth-hormone secretagogue receptor (GHSR) modulators but with divergent pharmacodynamic and pharmacokinetic properties. The structure-activities/properties exploration led to compound 47, which displayed potent human GHSR antagonism and inverse agonism in cellular assays (IC50 = 68 nM, EC50 = 29 nM), moderate oral bioavailability, and notable brain penetration in rat ( F = 27%, B/ P ratio = 1.9). First in vivo studies demonstrated effective reduction of food intake after oral or parenteral administration to mouse (78% at 1 h and 38% at 8 h, respectively). Further preclinical studies are needed to evaluate the most suited mode of administration with the aim of promoting a first central-acting ghrelin inverse agonist molecule to development, which would represent a significant step toward therapeutic agents to treat metabolic disorders related to obesity, such as type 2 diabetes mellitus.

Identification and Pharmacological Profile of an Indane Based Series of Ghrelin Receptor Full Agonists.

Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease, and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF-1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and functional capacity. Ghrelin further acts on inflammation, appetite, and adipogenesis and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity.

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.

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40-50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

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.

Bridging computational modeling with amino acid replacements to investigate GHS-R1a-peptidomimetic recognition.

The ghrelin receptor, also referred to as the growth hormone secretagogue receptor 1a (GHS-R1a), is a G protein-coupled receptor (GPCR) primarily expressed in the brain and pituitary. The wide spectrum of biological functions of GHS-R1a has rendered it a target for therapeutic drugs and for molecular imaging agents, for a variety of diseases. An improved understanding of the binding mechanism of a ligand to GHS-R1a would provide guidance on ligand design. This study investigates the binding of G-7039, a peptidomimetic agonist, to the GHS-R1a. A series of computational studies including homology modeling, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were carried out in conjunction with amino acid replacements on G-7039. The results suggest that the first three residues on the N-terminal segment of the peptidomimetic are bound to three hydrophobic sub-pockets in the receptor binding site, with the driving force for binding mainly from hydrophobic interactions. It has been reported that a charge-charge interaction between the positively charged terminal amine of the agonist and Glu124 on the receptor serves as an anchor point for binding. However, our studies suggest that this interaction is not strong enough to anchor a ligand to the ghrelin receptor in the absence of hydrophobic interactions. The resulting computational model provides insight into structure activity relationship analysis for the ghrelin receptor and will assist in future ligand design.

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.

Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics.

Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems.