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A survey of stapling methods to increase affinity, activity, and stability of ghrelin analogues
Juan J Esteban1, Julia R Mason1, Jakob Kaminski1
1Department of Chemistry, University of Western Ontario 1151 Richmond Street London Ontario N6A 3K7 Canada lluyt@uwo.ca.
Abstract:
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.
Insights
Stabilizing ghrelin (a growth hormone secretagogue receptor ligand) with chemical staples enhances its structure and affinity for therapeutic applications. Stapled ghrelin analogues show improved proteolytic stability and receptor binding.
Area of Science:
- Biochemistry
- Medicinal Chemistry
- Pharmacology
Background:
- The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor involved in energy homeostasis, appetite, and growth hormone secretion.
- GHSR is a therapeutic target for metabolic disorders and cancer cachexia, and an imaging target for cancers and cardiovascular diseases.
- Ghrelin, the endogenous GHSR ligand, is proteolytically unstable due to limited secondary structure.
Purpose of the Study:
- To enhance the proteolytic stability, affinity, and activity of ghrelin analogues for the GHSR.
- To investigate the impact of different chemical stapling methods on ghrelin structure and function.
- To develop stabilized ghrelin analogues as potential therapeutic agents or imaging agents.
Main Methods:
- Synthesis of ghrelin(1-20) analogues with various chemical staples (lactam, triazole, hydrocarbon, Glaser, xylene-thioether) at positions 12 and 16.
- Measurement of receptor affinity using competitive binding assays.
- Assessment of α-helicity via circular dichroism spectroscopy.
- Evaluation of agonist activity using β-arrestin recruitment BRET assays and metabolic stability through serum stability analysis.
Main Results:
- Incorporation of helix-inducing staples generally increased α-helicity and improved affinity for GHSR.
- Lactam, triazole, and hydrocarbon stapled ghrelin(1-20) analogues demonstrated significantly stronger GHSR affinity compared to unstapled ghrelin.
- Flexible staple linkers correlated with enhanced helical structure and receptor affinity.
Conclusions:
- Chemical stapling is an effective strategy to stabilize ghrelin and enhance its binding affinity to GHSR.
- Stabilized ghrelin analogues hold promise for therapeutic development in metabolic diseases and cancer cachexia.
- Further investigation into agonist activity and metabolic stability of stapled analogues is warranted.
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