The Design and Synthesis of Ghrelin Analogues as Non‐Invasive GHS‐R1a Imaging Probes

The field of molecular imaging is constantly growing and evolving in order to provide the best possible healthcare for patients in various stages of disease and therapy. Molecular imaging aims to locate specific markers of disease by selectively targeting the markers of interest with high selectivity and visualizing the accumulation using external detection. The growth hormone secretagogue receptor-1a (GHS-R1a) has been shown to be involved in various important biological functions such as energy homeostasis and cardiac contractility. GHS-R1a has shown involvement in proliferation, migration and cell invasion of specific cancer subtypes. Therefore, targeting GHS-R1a is an important marker of different disease states and would be advantageous to selectively target for diagnostic and therapeutic purposes. This thesis will document the development of peptide-based molecular imaging agents capable of targeting GHS-R1a with high affinity designed off the structure of ghrelin, the endogenous ligand for GHS-R1a. Chapter 2 discusses the synthesis and evaluation of gallium-69/71 and gallium-68 labelled ghrelin(1-19) analogues. The first generation of ghrelin analogues was designed to detect GHS-R1a by positron emission tomography (PET). Chelation of gallium had a positive effect on binding affinity to GHS-R1a resulting in an IC50 comparable to natural ghrelin(1-28). Preclinical evaluation of HT1080/GHSR-1a xenografts showed higher SUVR values than the HT1080 xenograft with no GHS-R1a. Chapter 3 discusses the second generation of ghrelin analogues that were further truncated to eight amino acids. A structural activity study investigated residues 1, 3, 4, and 8 to determine whether amino acid substitutions produce the best binding affinity GHS-R1a. The optimized ghrelin analogue has 12-fold higher binding affinity to GHS-R1a than natural ghrelin. New radiochemical syntheses were reported for a 6-[ 18F]-fluoro-2-pentafluorophenylnaphthoate prosthetic group. The lead peptide analogue was radiolabelled in a 3% radiochemical yield and resulted in the first fluorine-18 labelled ghrelin(1-8) analogue with greater affinity to GHS-R1a. ii The final chapter describes the effects of targeting GHS-R1a with a dimerized ghrelin(1-8) peptide. Dimerizing other peptide targeting entities has increased binding affinity to the target however, this is not the case found with ghrelin. The in vitro kinetics were evaluated using fluorescence microscopy in GHS-R1a expressing cells. All three chapters discuss the systematic modification of an endogenous peptide ligand into a high affinity, PET imaging agent through classical methods of peptide modification and radiochemistry

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.

Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues