The growth hormone secretagogue receptor, GHSR, is a G protein-coupled receptor which regulates various important physiological and pathophysiological processes in the body. The 28 amino acid peptide ghrelin is the primary high affinity endogenous ligand for GHSR and has been used as a structural template for the development of therapeutic and imaging agents. Linear peptides, like ghrelin, have limited secondary structure in solution and are often proteolytically unstable. This inherent instability in ghrelin-like peptides can be overcome by incorporating helix-inducing staples that stabilize their structure and improve affinity.

We present an analysis of different stapling methods at positions 12 and 16 of ghrelin(1-20) analogues, a previously identified optimal staple location, with the goal of increasing proteolytic stability and to retain or improve affinity towards GHSR. Ghrelin(1-20) analogues were modified with a wide range of chemical staples, including a lactam staple, triazole staple, Glaser staple, bis-thioether staple, and hydrocarbon staple. Once synthesized, the analogue affinity and alpha-helicity were measured using competitive binding assays and circular dichroism spectroscopy, respectively.

Generally, an increase in alpha-helicity using a flexible staple linker led to an improved affinity towards GHSR. Ghrelin(1-20) analogues with a lactam and a triazole staple resulted in helical analogues, 26% and 35% helical character, with stronger affinity towards GHSR, IC50 = 0.94 nM and 1.65 nM, than unstapled ghrelin(1-20), a compound that lacks helical character.

Compounds were also investigated for their agonist activity through β-arrestin 1 & 2 recruitment BRET assays. Incorporating these staples into the structure of ghrelin(1-20) can provide the required stability and affinity to make a robust therapeutic or imaging agent targeting GHSR.