Design, synthesis and characterization of a fluorescently labeled functional analog of full-length human ghrelin.

Human ghrelin receptor (GHSR) is a recognized prospective target in the diagnosis and therapy of multiple cancer types. To gain a better understanding of this receptor signaling system, we have synthesized a novel full-length ghrelin analog that is fluorescently labeled at the side-chain of a C-terminal cysteine extension. This analog exhibited nanomolar affinity and potency for the ghrelin receptor. It shows comparable efficacy with that of endogenous ghrelin. The fluorescently-labeled ghrelin analog is a valuable tool for in vitro imaging of cell lines that express ghrelin receptor.

Side Chain Orientation of Tryptophan Analogues Determines Agonism and Inverse Agonism in Short Ghrelin Peptides.

We describe two synthetic amino acids with inverted side chain stereochemistry, which induce opposite biological activity. Phe4 is an important part of the activation motif of ghrelin, and in short peptide inverse agonists such as KwFwLL-NH2 , the aromatic core is necessary for inactivation of the receptor. To restrict indole/phenyl mobility and simultaneously strengthen the interaction between peptide and receptor, we exchanged the natural monoaryl amino acids for diaryl amino acids derived from tryptophan. By standard solid-phase peptide synthesis, each of them was inserted into ghrelin or in the aromatic core of the inverse agonist. Both ghrelin analogues showed nanomolar activity, indicating sufficient space to accommodate the additional side chain. In contrast, diaryl amino acids in the inverse agonist had considerable influence on receptor signaling. Whereas the introduction of Wsf maintains inverse agonism of the peptide, Wrf shifts the receptor more to active states and can induce agonism depending on its introduction site.

A Compact and Synthetically Accessible Fluorine-18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper-Free Click Chemistry.

A new fluorine-containing azadibenzocyclooctyne (ADIBO-F) was designed using a synthetically accessible pathway. The fluorine-18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21-35 % radiochemical yield in 30 minutes of synthetic time. ADIBO-F has been incorporated into azide-functionalized, cancer-targeting peptides through a strain-promoted alkyne-azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide-based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptor 1a (GHSR-1a) and gastrin-releasing peptide receptor (GRPR), with IC50 values of 9.7 and 0.50 nm, respectively. This is a new and rapid labelling option for the incorporation of fluorine-18 into biomolecules for PET imaging.

Chitosan-coated liposome dry-powder formulations loaded with ghrelin for nose-to-brain delivery.

The nose-to-brain delivery of ghrelin loaded in liposomes is a promising approach for the management of cachexia. It could limit the plasmatic degradation of ghrelin and provide direct access to the brain, where ghrelin's specific receptors are located. Anionic liposomes coated with chitosan in either a liquid or a dry-powder formulation were compared. The powder formulation showed stronger adhesion to mucins (89 ±â€¯4% vs 61 ±â€¯4%), higher ghrelin entrapment efficiency (64 ±â€¯2% vs 55 ±â€¯4%), higher enzymatic protection against trypsin (26 ±â€¯2% vs 20 ±â€¯3%) and lower ghrelin storage degradation at 25 °C (2.67 ±â€¯1.1% vs 95.64 ±â€¯0.85% after 4 weeks). The powder formulation was also placed in unit-dose system devices that were able to generate an appropriate aerosol characterized by a Dv50 of 38 ±â€¯6 µm, a limited percentage of particles smaller than 10 µm of 4 ±â€¯1% and a reproducible mass delivery (CV: 1.49%). In addition, the device was able to deposit a large amount of powder (52.04% w/w) in the olfactory zone of a 3D-printed nasal cast. The evaluated combination of the powder formulation and the device could provide a promising treatment for cachexia.

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs.

Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.

Prandial ghrelin attenuation provides evidence that des-acyl ghrelin may be an artifact of sample handling in human plasma.

BACKGROUND: Plasma acyl and des-acyl ghrelin are thought of as components of total ghrelin, but this has never been validated using ex vivo spiking experiments, human sample collection comparisons and fit-for-purpose translatable assays. RESULTS: Acyl ghrelin plasma stability was analyzed by LC-MS/MS and it revealed that acyl ghrelin is enzymatically and chemically converted to des-acyl ghrelin in the presence of active serine proteases and HCl. ELISAs with less than 30% total error were used to assess acyl ghrelin behavior in matched authentic human samples. Acyl and total ghrelin were not statistically different in 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride samples and acyl ghrelin losses in K(2)EDTA plasma were accounted for in des-acyl ghrelin formation. CONCLUSION: Acyl ghrelin is total ghrelin and des-acyl ghrelin should not be detectible in healthy human plasma under optimal sample handling and assaying conditions.

Variety of acyl modifications in mammalian ghrelins.

Ghrelin, a 28-amino acid-long peptide with an n-octanoyl modification at Ser(3), has been isolated from rat and human stomachs as an endogenous ligand for the growth hormone secretagogue receptor. It is very important to study the ghrelin from mammals (especially, domestic animals) that serve as human companions, food resources, and model organisms. We purified feline and caprine ghrelin and observed that the administration of synthetic ghrelin increased plasma growth hormone (GH) levels in cats and goats. Therefore, we believe that ghrelin may play important roles in GH release in mammals.

Synthesis of ghrelin: chemical synthesis and semisynthesis for large-scale preparation of modified peptides.

Most biologically active peptide hormones, including ghrelin, undergo numerous posttranslational modifications and play many crucial roles in nature. Medium- or large-scale preparation methods are required to understand their biological functions and potential applications in life sciences and the biomedical fields. Since ghrelin has an O-acyl modification in its Ser3, recombinant expression for its production has not solely been employed thus far. In this chapter, we provide two distinct protocols for the preparation of human ghrelin: a chemical synthesis method for medium-scale (up to hundreds of milligrams) and a semisynthesis method for large-scale (more than grams) preparation. Established Fmoc chemistry for solid-phase synthesis enables the highly efficient procedure for synthesizing ghrelin in the medium scale. Semisynthesis method, the coupling of chemically synthesized O-acylated ghrelin(1-7) with recombinantly expressed ghrelin(8-28), can be applied for larger scale preparation.

Clinical application of ghrelin for chronic respiratory diseases.

The discovery of ghrelin has resulted in the development of potential therapeutics for cachexia caused by multiple underlying diseases. When chronic respiratory diseases progress to their advanced stages, cachexia often occurs, thereby worsening the patient's prognosis. A small clinical trial that enrolled cachectic patients with chronic respiratory disease revealed that administration of ghrelin improved their nutritional status and exercise tolerance. Short-term administration of ghrelin was found to be safe and tolerated with adverse events, including suffusion, sleepiness, peristalsis, hunger, and sweating. Further large-scale and long-term clinical trials are needed.

Characterization of new stable ghrelin analogs with prolonged orexigenic potency.

Ghrelin, the only known peripherally produced and centrally acting peptide that stimulates food intake, is synthesized primarily in the stomach and acts through the growth hormone secretagogue receptor (GHS-R1a). In addition to its orexigenic effect, ghrelin stimulates the release of growth hormone (GH). In this study, we investigated the biological properties of full-length and shortened ghrelin analogs in which octanoylated Ser(3) is replaced with an octanoic acid moiety coupled to diaminopropionic acid (Dpr). Ghrelin analogs stabilized with Dpr(N-octanoyl) in position 3 and noncoded amino acids in position 1 (sarcosine) and/or position 4 (naphthylalanine or cyclohexylalanine) were found to possess affinities similar to those of ghrelin for cell membranes with transfected GHS-R1a. In vivo, the prolonged orexigenic effects of analogs containing Dpr(N-octanoyl)(3) compared with that of ghrelin in adult mice and a similar impact on GH secretion in young mice were found. Full-length [Dpr(N-octanoyl)(3)]ghrelin and its analogs with a noncoded amino acid in position 1 and/or 4 showed significantly prolonged stability in blood plasma compared with that of ghrelin. Ghrelin analogs with a prolonged orexigenic effect are potential treatments for GH deficiency or cachexia that accompanies chronic diseases. Desoctanoylated ghrelin analogs and N-terminal penta- and octapeptides of ghrelin did not show any biological activity.

Fluorine and rhenium substituted ghrelin analogues as potential imaging probes for the growth hormone secretagogue receptor.

In our effort to create imaging probes targeting the growth hormone secretagogue receptor (GHSR), we now report on the design and synthesis of fluorine and rhenium containing ghrelin analogues through modification of the n-octanoyl Ser-3 side chain. Fluorine analogues were designed whereby the fluorine atom is situated at the terminus of an aliphatic chain using diaminopropionic acid (Dpr) as residue-3. Truncated ghrelin(1-5) and ghrelin(1-14) fluorine-bearing analogues were prepared, the best of which had a 28 nM IC(50) for GHSR. Ghrelin(1-14) analogues were also prepared containing rhenium, as a surrogate metal for technetium-99m, with a cyclopentadienylrhenium tricarbonyl being situated at the terminus of the residue-3 side chain, yielding compounds the best of which had a 35 nM IC(50). This represents a rare case of incorporating rhenium into a peptide structure where the metal complex is required for biological activity. These fluorine and rhenium derivatives demonstrate the ability to modify the Ser-3 side chain of ghrelin in order to create imaging probes for the GHSR.