A survey of stapling methods to increase affinity, activity, and stability of ghrelin analogues

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor which regulates various important physiological and pathophysiological processes in the body such as energy homeostasis, growth hormone secretion and regulation of appetite. As a result, it has been postulated as a potential therapeutic target for the treatment of cancer cachexia and other metabolic disorders, as well as a potential imaging agent target for cancers and cardiovascular diseases. Ghrelin is the primary high affinity endogenous ligand for GHSR and has limited secondary structure in solution, which makes it proteolytically unstable. This inherent instability in ghrelin can be overcome by incorporating helix-inducing staples that stabilize its structure and improve affinity and activity. We present an analysis of different stapling methods at positions 12 and 16 of ghrelin(1-20) analogues with the goal of increasing proteolytic stability and to retain or improve affinity and activity towards the GHSR. Ghrelin(1-20) analogues were modified with a wide range of chemical staples, including a lactam staple, triazole staple, hydrocarbon staple, Glaser staple, and xylene-thioether staple. Once synthesized, the receptor affinity and α-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 improved affinity towards GHSR. Ghrelin(1-20) analogues with a lactam, triazole, and hydrocarbon staple resulted in helical analogues with stronger affinity towards GHSR 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 and for their metabolic stability through serum stability analysis.