Author: Delna Joseph
Joseph, Delna, 2025 Discovering Novel Endogenous Ligands For G-protein- Coupled Receptor (GPCRS) Using High Throughput Screening Assays, Flinders University, College of Medicine and Public Health
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The G-protein coupled receptor family is one of the most important classes of cellular receptors and are targets for around one third of approved drugs. The G-protein coupled bile
acid receptor GPCR (GPBAR1, also known as TGR5) is a promising drug target because of its control of incretin biology and metabolism. The purpose of this thesis is to establish a
simple and flexible, high-throughput procedure for discovering and evaluating ligands for GPCRs, and to apply this to the study of naturally modified (glycosylated) bile acid ligands
for the GPBAR1/TGR5. Two different reporter assays were established in HEK293T to measure GPCR ligand activity: the PRESTO-Tango β-Arrestin-TA/TRE-luciferase assay, and the cAMP/CRE luciferase assay. For the PRESTO-Tango assay, we first modified the Tango β-Arrestin helper plasmid to carry a hygromycin resistance (HygR) gene so that double-stable HEK293T cell lines could be generated that carry both the β-Arrestin-TA plasmid and the pTRE-Luciferase plasmid. To test the system, these two components were transfected in combination with various GPCR expression plasmids and then relevant ligands were applied (e.g. the serotonin receptor, HTR1A and its ligand 5HT were used in early validation experiments). The generation of double stable cells lines was a 2-step process: first single-stable lines carrying pTRE-luc were screened for low background expression and high inducibility. Once an optimal (low background/high inducibility) line was identified, this was stably transfected with the β-Arrestin-TA-hygromycinR plasmid. The resulting dual stable lines were tested for vector integration by genomic PCR. While dual stable lines were identified, their functional validation could not be completed within the project period. Thus, further studies of TGR5 ligands were performed using transient transfections only. The activity of TGR5 with its optimal bile acid ligand LCA was compared in the PRESTO-Tango
system (with the pTRE-luciferase reporter gene) and the cAMP/pCRE-luciferase system. Dose-response graphs showed that the latter assay system was more sensitive for measuring
receptor associated responses. Finally, novel pipelines were developed and tested to analyse the effects of endogenous bile acid glycosylation on TGR5 activity. Due to time constraints, only pilot studies were performed using these pipelines and results remain to be confirmed. Overall, the project established tools and protocols for future screening of novel endogenous ligands of TGR5 and other GPCRs. Future steps include validating the dual stable cell lines and developing triple-stable cell lines, comparing the luciferase-based assays to kinetic cAMP assays, and developing cell-based systems to measure GLP-1 secretion to link TGR5 activity with physiology.
Keywords: GPCRs, Highthroughput screening assay, PRESTO- Tango, bile acid, diabetices
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Professor Robyn Meech