Use of luminescent proximity-based techniques to investigate IL-23 receptor complex formation, cytokine binding and activation in living cells
Publication Date
July 18, 2023
Creator
Abstract
The cytokine IL-23 is a pro-inflammatory messenger molecule, involved in upregulating the immune response after infection by fungal and bacterial pathogens. The pathway is also crucial to the induction and maintenance of several auto-inflammatory diseases, including Psoriatic Arthritis, Plaque Psoriasis, Crohn’s Disease and Ulcerative Colitis. Over the last decade anti-IL23 antibody drugs have been developed and shown to be effective treatments for these conditions. However, despite the proven efficacy of drugs targeting the IL23 pathway, little is known about the ligand binding and activation mechanism of the receptor, which is made up of the receptor chains IL23R and IL12Rβ1. Further pharmacological characterisation of the receptor could enable the targeting of the receptor with alternative modalities such as small molecule or peptide antagonists.
In this project, proximity-based techniques are utilised to quantify the interaction of IL-23 with its receptor and to probe how engagement of ligand leads to activation of signalling. Initially a NanoBRET ligand binding assay was created and used to quantify the interaction between a fluorescently tagged version of IL-23 and the full-length N-terminally NanoLuc labelled receptor components IL23R and IL12Rβ1, expressed individually and together in the membranes of living cells. An intra-receptor NanoBRET assay was also used to measure the formation of receptor complexes and changes in the relative positions of the Nterminal domains of the receptor. The results from these experiments demonstrated that the IL-23 receptor complex forms in the absence of ligand, with subsequent binding of cytokine to the high affinity binding site initiating a potent change in the conformation of the N-terminal domains of the receptor.
Further study of receptor heteromer formation using split luciferase complementation and measurement of receptor activation with a phosphoSTAT3 AlphaLISA assay, confirmed that receptor components formed inactive complexes in the absence of ligand, that could be activated through addition of IL-23. This technique also allowed the induction of ligand independent signalling complexes through the high affinity crosslinking of the receptor Nterminal domains in certain conformations.
The assays developed were then utilised to characterise receptor antagonists including a cyclic peptide from an emerging class of IL23R inhibitors, which was subsequently developed into a probe specific to the IL23R:IL-23 interface. Finally, the effect of several disease relevant mutations of IL23R on ligand binding, conformational change and activation were tested, demonstrating that the mechanism of action of the C115Y mutation is disruption of the interaction between IL23R and IL-23.
Item Type
ethesis
Thesis Type
PhD
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
72518
UoN Repository URI
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