The Ghrelin Receptor and its Appetite for Highly Potent Ghrelin Analogues
Abstract
The growth hormone secretagogue receptor-1a (GHS-R1a) is expressed in many human tissues, most notably the hypothalamus, and causes an increase in appetite upon activation by its endogenous ligand, ghrelin. GHS-R1a is differentially expressed in malignant compared to benign tumours. Therefore, there is an interest in developing GHS-R1a-targeted peptides as novel drugs to modulate signaling for diseases such as cancer cachexia and obesity and to image the receptor for disease diagnosis and following progression.
Chapter 2 discusses a fluorescently labelled ghrelin analogue for imaging GHS-R1a in ex vivo biopsy analysis and in vivo distribution studies. The analogue was created through side-chain cyclization resulting in an improved affinity and stability compared to natural ghrelin. This stapled peptide was used as a cancer cell-specific fluorescent stain.
G7039, a peptidomimetic ghrelin agonist (IC50 5.2 nM/EC50 0.18 nM), underwent structure-activity relationship studies to generate improved ligands and positron emission tomography (PET) agents. The first generation peptidomimetic (Chapter 3) [1-Nal4,Lys5 (4-fluorobenzoyl-4-FB)]G7039 (IC50 69 nM/EC50 1.1 nM) was radiolabelled with 18F in a radiochemical yield of 48%, radio purity of ≥ 99%, and molar activity of ≥ 34 GBq/μmol. Despite success in radiolabelling, its solubility (cLogP = 8.76) and binding affinity needed improvement.
The second generation peptidomimetic (Chapter 4) [Tyr4,Lys5(2-fluoropropionyl (2-FP)]G7039 (IC50 0.28 nM/EC50 0.12 nM) had improved binding and lipophilicity (cLogP = 4.36). Labeling of this ligand was low yielding, however, a unique H-bond interaction was identified with molecular docking.
The third generation (Chapter 5) required a modified prosthetic group (2-FP to ammonium methyltrifluoroborate-AMBF3) to radiolabel in higher yields, resulting in [Tyr4,Lys5(AMBF3)]G7039 (IC50 0.85 nM). This compound was radiolabelled in a single step and with improved radiochemical data.
Finally, in Chapter 6 a homobivalent G7039 ligand (IC50high 0.43 nM:IC50low 0.42 pM /EC50 1.8-2.1 nM) was found to bind to GHS-R1a homo-oligomer and was designed to study GHS-R1a homo-oligomerization. Differential signalling with the GHS-R1a homo-oligomer was observed by measuring cellular signals such as b-arrestin, ERK, and gene reporters. A successful series of GHS-R1a targeting probes have been synthesized and characterized with applications driven towards imaging and therapy.