Master of Science
Luyt, Leonard G.
The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor which regulates various important physiological and pathophysiological processes in the body. 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 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 analogue 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 an 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 assays.
Summary for Lay Audience
The ghrelin receptor is a receptor in our bodies that is responsible for our ability to feel hunger. This receptor is activated by a hormone called ghrelin, which is a peptide molecule made up of 28 amino acids. When the ghrelin receptor is activated by ghrelin, it produces appetite, releases growth hormone, and controls our metabolism. Due to its effects on stimulating appetite, ghrelin has been studied as a potential therapeutic for the treatment of diseases like cancer cachexia, which is the debilitating complication of cancer that causes wasting of the body. Furthermore, the ghrelin receptor has been identified as a potential biomarker for ovarian and prostate cancers, as this receptor is expressed differently in those cancers than in normal tissue. As a result, ghrelin’s peptide structure has been used as a template for the design of therapeutics and molecular imaging agents. However, ghrelin is very unstable to the enzymes in our bodies that are effective at breaking down peptides (proteases). Proteases are efficient at breaking down peptides when they have no secondary structure, however studies have demonstrated that they are not able to break down peptides if they are in an alpha-helical confirmation. Thus, peptide chemists have developed methods of inducing alpha-helicity into a peptide structure to improve peptides’ biological properties. One of these methods is using chemical staples to lock the peptide into the desired confirmation. The work in this thesis demonstrates introducing 5 different chemical staples into the structure of truncated ghrelin(1-20) compounds and seeing the effects the different staples have on producing helical compounds and their affinity towards the ghrelin receptor. The ability of these compounds to activate the receptor will also be discussed, as well as their stability in human serum.
Esteban, Juan, "A Survey of Stapling Methods to Increase the Affinity, Activity and Stability of Ghrelin Analogues" (2022). Electronic Thesis and Dissertation Repository. 8843.
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