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

Marina D Childs; Arundhasa Chandrabalan; Derian Hodgson; Rithwik Ramachandran; Leonard G Luyt

doi:10.1021/acsptsci.3c00088

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

ACS Pharmacol Transl Sci. 2023 Jun 12;6(7):1075-1086. doi: 10.1021/acsptsci.3c00088. eCollection 2023 Jul 14.

Authors

Marina D Childs¹, Arundhasa Chandrabalan², Derian Hodgson¹, Rithwik Ramachandran², Leonard G Luyt^{1

3

4}

Affiliations

¹ Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 3K7, Canada.
² Department of Physiology and Pharmacology, University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5C1, Canada.
³ Departments of Medical Imaging and Oncology, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 3K7, Canada.
⁴ London Regional Cancer Program, Lawson Health Research Institute, 800 Commissioners Road East, London, Ontario, N6A 4L6, Canada.

PMID: 37470019
PMCID: PMC10353549 (available on 2024-06-12)
DOI: 10.1021/acsptsci.3c00088

Abstract

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 (Gα_{q/11/13/i1/oB}) known to associate with GHSR as well as β-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.