Structure-Activity Study of Ghrelin(1-8) Resulting in High Affinity Fluorine-Bearing Ligands for the Ghrelin Receptor.

The ghrelin receptor, also known as the growth hormone secretagogue receptor 1a (GHS-R1a), is a G-protein-coupled receptor that is differentially expressed in healthy tissue and several cancers, including prostate, testicular, and ovarian. Selectively targeting the ghrelin receptor using fluorine-18 tagged entities would allow localization and visualization of ghrelin receptor expressing carcinomas using PET imaging. The endogenous ligand ghrelin, a 28 amino acid peptide with 3.1 nM affinity, has poor in vivo stability. Here we report on ghrelin(1-8) analogues bearing modifications at residues 1, 3, 4, and 8. The lead analogue, [Inp1,Dpr3(6-fluoro-2-naphthoate),1-Nal4,Thr8]ghrelin(1-8), possessed an IC50 value of 0.11 nM that is a 28-fold improvement compared to the natural ligand. A novel 6-fluoro-2-pentafluorophenyl naphthoate (PFPN) prosthetic group was synthesized to incorporate fluorine-18 for PET imaging. This is not only the highest affinity ghrelin analogue reported but also the shortest ghrelin analogue capable of binding GHS-R1a with better affinity than ghrelin(1-28).

Ghrelin receptor as a novel imaging target for prostatic neoplasms.

BACKGROUND: Ghrelin is a natural growth hormone secretagogue (GHS) that is co-expressed with its receptor GHSR in human prostate cancer (PCa) cells. Imaging probes that target receptors for ghrelin may delineate PCas from benign disease. The specificity of a novel ghrelin-imaging probe for PCa over normal tissue or benign disease was assessed. METHODS: A fluorescein-bearing ghrelin analogue was synthesized (fluorescein-ghrelin(1-18)), and its application for imaging was evaluated in a panel of PCa cell lines and human prostate tissue. Prostate core biopsy samples were collected from fresh surgery specimens of 13 patients undergoing radical prostatectomy. Ghrelin probe signal was detected and quantified in each sample using a hapten amplification technique and associated with pathological features. RESULTS: The ghrelin probe was taken up by GHSR-expressing LNCaP and PC-3 cells, and not in BPH cells that express low levels of GHSR. Binding was blocked by competition with excess unlabeled probe. The ghrelin probe signal was 4.7 times higher in PCa compared to benign hyperplasia tissue (P = 0.0027) and normal tissue (P = 0.0093). Furthermore, while the ghrelin probe signal was 1.9-fold higher in PIN compared to benign hyperplasia (P = 0.0022) and normal tissue (P = 0.0047), there was no significant difference in the signal of benign hyperplasia compared to normal tissue. CONCLUSION: The imaging probe fluorescein-ghrelin(1-18) is specific for PCa, and did not associate significantly with benign hyperplasia or normal prostate tissue. This data suggests that ghrelin analogues may be useful as molecular imaging probes for prostatic neoplasms in both localized and metastatic disease.

Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a.

Ghrelin is a 28-amino acid peptide hormone produced in the stomach. It binds to the growth hormone secretagogue receptor 1a (GHS-R1a), a class A G-protein-coupled receptor. In the present study, we describe the design, synthesis and characterization of a truncated, 18-amino acid analog of ghrelin conjugated to a fluorescent molecule, fluorocein isothiocyanate (FITC), through the addition of a lysine at its C terminus ([Dpr(octanoyl)(3), Lys(fluorescein)(19)]ghrelin(1-19)). Receptor binding affinity of this novel fluorescein-ghrelin(1-18) was similar to that of wild-type ghrelin and a synthetic GHS-R1a ligand, hexarelin. Live cell imaging in CHO/GHS-R1a cells demonstrated cell surface receptor labeling and internalization, and agonist activity of fluorescein-ghrelin(1-18) was confirmed by increased phosphorylation of ERK1/2. We also show that GHS-R1a protein is expressed primarily in the heart when compared to all other organs, suggesting high receptor density in the left ventricle. Finally, we demonstrate that fluorescein-ghrelin(1-18) binds specifically to heart tissue in situ, and its binding is displaced by both wt ghrelin and hexarelin. We have therefore developed a novel imaging probe, fluorescein-ghrelin(1-18), that can be used to image GHS-R1a in situ, for the purposes of investigating mechanisms of receptor trafficking or pharmacological agents that target GHS-R1a.