Title:
Rapid Screening of G Protein-Coupled Receptors in Yeast
Rapid Screening of G Protein-Coupled Receptors in Yeast
Author(s)
Yasi, Emily A.
Advisor(s)
Peralta-Yahya, Pamela
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Abstract
G protein-coupled receptors (GPCRs) comprise the largest family of proteins in humans and are the target of 1/3 of FDA approved drugs. All 800 human GPCRs can be considered druggable, however, ~400 remain “orphans”, or lack ligands. Over 300 orphan receptors are olfactory receptors (ORs), that are not only expressed in your nose, but ectopically expressed throughout your body and are implicated in diseases. High-throughput screening of GPCRs continues to advance receptor deorphanization and drug discovery. Here, the yeast Saccharomyces cerevisiae was used as a heterologous host to develop high-throughput GPCR-based assays. First, a fluorescence-based GPCR assay was used to deorphanize olfactory ORs ectopically expressed in the colon. ORs in the colon could be interacting with microbial metabolites and influence human physiology. This work identified ligands for OR10S1 and OR2T4, for the first time, and found an alternative ligand for OR2A7. Now these receptors can be studied to understand their downstream effects in the colon. Additionally, the OR-based assay can elucidate endogenous ligands for the colon ORs. Second, a luminescence-based GPCR assay was used to identify new ligands for serotonin receptor 4. The assay had a dynamic range of 32-fold and a screening speed of 1 compound per second, 30 times faster and 3 times better dynamic range than the fluorescent base screen. Optimization of the assay reporter accelerated the yeast screening assay for drug discovery purposes. This work uses a drug target for irritable bowel syndrome, and allows for a screening rate of one compound per second. Screening of a commercial natural products led to the discovery that the antimicrobials can activate the serotonin receptor 4 endogenously expressed in colon epithelial cells. Altogether, this body of work advances yeast-based GPCR assay technology with applications in deorphanization and drug discovery.
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Date Issued
2020-05-18
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Text
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Dissertation