Radiofluorination of non-activated aromatic prosthetic groups for synthesis and evaluation of fluorine-18 labelled ghrelin(1-8) analogues.

The growth hormone secretagogue receptor 1a (GHSR) is differentially expressed in various disease states compared to healthy tissues and thus is a target for molecular imaging. The endogenous ligand for the GHSR is ghrelin, a 28 amino acid peptide with a unique octanoyl group on the serine-3 residue. A recently reported ghrelin analogue revealed the successful use of fluorine-containing, polycyclic aromatic groups in place of the octanoyl side chain, thereby providing potential access to new 18F-PET imaging probes. The peptide [Inp1,Dpr3(6-FN),1Nal4,Thr8]ghrelin(1-8) amide (1) showed sub-nanomolar receptor affinity (IC50 = 0.11 nM) toward the GHSR making it the strongest affinity ghrelin analogue reported to date. However, attempts to label such non-activated aromatic groups with fluoride-18 through conventional substitution methods resulted in low radiochemical yields, impractical for use in vivo. Since larger, non-activated aromatic groups appear to be of value for incorporating fluorine into ghrelin(1-8) analogues, an additional peptide bearing a 4'-fluorobiphenyl-4-carboxyl (4'-FBC) group in place of the octanoyl side chain was also of interest. Herein, we describe the radiosynthesis of [Inp1,Dpr3([18F]6-FN),1Nal4,Thr8]ghrelin(1-8) amide ([18F]1) and [Inp1,Dpr3([18F]4'-FBC),1Nal4,Thr8]ghrelin(1-8) amide ([18F]2) using a prosthetic group approach from iodonium ylide precursors as well as initial in vitro and in vivo evaluation of [18F]1 as a potential PET tracer for targeted imaging of the GHSR.

A Decade's Progress in the Development of Molecular Imaging Agents Targeting the Growth Hormone Secretagogue Receptor.

The growth hormone secretagogue receptor 1a (GHSR), also called the ghrelin receptor, is a G protein-coupled receptor known to play an important metabolic role in the regulation of various physiological processes, including energy expenditure, growth hormone secretion, and cell proliferation. This receptor has been implicated in numerous health issues including obesity, gastrointestinal disorders, type II diabetes, and regulation of body weight in patients with Prader-Willi syndrome, and there has been growing interest in studying its mechanism of behavior to unlock further applications of GHSR-targeted therapeutics. In addition, the GHSR is expressed in various types of cancer including prostate, breast, and testicular cancers, while aberrant expression has been reported in cardiac disease. Targeted molecular imaging of the GHSR could provide insights into its role in biological processes related to these disease states. Over the past decade, imaging probes targeting this receptor have been discovered for the imaging modalities PET, SPECT, and optical imaging. High-affinity analogues of ghrelin, the endogenous ligand for the GHSR, as well as small molecule inhibitors have been developed and evaluated both in vitro and in pre-clinical models. This review provides a comprehensive overview of the molecular imaging agents targeting the GHSR reported to the end of 2019.

Discovery of Ghrelin(1-8) Analogues with Improved Stability and Functional Activity for PET Imaging.

The highest affinity ghrelin-based analogue for fluorine-18 positron emission tomography, [Inp1,Dpr3(6-FN),1Nal4,Thr8]ghrelin(1-8) amide (1), has remarkable subnanomolar receptor affinity (IC50 = 0.11 nM) toward the growth hormone secretagogue receptor 1a (GHSR). However, initial in vivo PET imaging and biodistribution of [18F]1 in mice demonstrated an unfavorable pharmacokinetic profile with rapid clearance and accumulation in liver and intestinal tissue, prompting concerns about the metabolic stability of this probe. The aims of the present study were to examine the proteolytic stability of ghrelin analogue 1 in the presence of blood and liver enzymes, structurally modify the peptide to improve stability without impeding the strong binding affinity, and measure the presently unknown functional activity of ghrelin(1-8) analogues. The in vitro stability and metabolite formation of 1 in human serum and liver S9 fraction revealed a metabolic soft spot between amino acids Leu5 and Ser6 in the peptide sequence. A focused library of ghrelin(1-8) analogues was synthesized and evaluated in a structure-activity-stability relationship study to further understand the structural importance of the residues at these positions in the context of stability and receptor affinity. The critical nature of l-stereochemistry at position 5 was identified and substitution of Ser6 with l-2,3-diaminopropionic acid led to a novel ligand with substantially improved in vitro stability while maintaining subnanomolar GHSR affinity. Despite the highly modified nature of these analogues compared to human ghrelin, ghrelin(1-8) analogues were found to recruit all G protein subtypes (Gαq/11/13/i1/oB) known to associate with GHSR as well as ß-arrestins with low micromolar to nanomolar potencies. The study of these analogues demonstrates the ability to balance desirable ligand properties, including affinity, stability, and potency to produce well-rounded candidate molecules for further in vivo evaluation.

Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging.

The proteolytically-activated G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2), is implicated in various cancers and inflammatory diseases. Synthetic ligands and in vitro imaging probes targeting this receptor have been developed with low nanomolar affinity, however, no in vivo imaging probes exist for PAR2. Here, we report the strategic design, synthesis, and biological evaluation of a series of novel 4-fluorobenzoylated PAR2-targeting peptides derived from 2f-LIGRLO-NH2 (2f-LI-) and Isox-Cha-Chg-Xaa-NH2 (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the 19F-standard of an 18F-labeled probe for potential use in in vivo imaging. We found that several of the 4-fluorobenzoylated peptides from the 2f-LI-family exhibited PAR2 selectivity with moderate potency (EC50 = 151-252 nM), whereas several from the Isox-family exhibited PAR2 selectivity with high potency (EC50 = 13-42 nM). Our lead candidate, Isox-Cha-Chg-Ala-Arg-Dpr(4FB)-NH2 (EC50 = 13 nM), was successfully synthesized with fluorine-18 with a radiochemical yield of 37%, radiochemical purity of >98%, molar activity of 20 GBq/µmol, and an end of synthesis time of 125 min. Biodistribution studies and preliminary PET imaging of the tracer in mice showed predominantly renal clearance. This 18F-labeled tracer is the first reported PAR2 imaging agent with potential for use in vivo. Future work will explore the use of this tracer in cancer xenografts and inflammation models involving upregulation of PAR2 expression.

Cellular Uptake of a Fluorescent Ligand Reveals Ghrelin O-Acyltransferase Interacts with Extracellular Peptides and Exhibits Unexpected Localization for a Secretory Pathway Enzyme.

Ghrelin O-acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHSR, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies support the interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site. Our work provides a new understanding of GOAT's catalytic mechanism, establishes that GOAT can interact with ghrelin and other peptides located outside the cell, and raises the possibility that other peptide hormones may exhibit similar complexity in their intercellular and organismal-level signaling pathways.