High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

Kenshi Yaginuma,Haruko Takeyama,Wataru Aoki,Shunsuke Aburaya,Yuta Ohtani,Masato Kogawa,Mitsuyoshi Ueda,Yohei Nishikawa,Masahito Hosokawa,Natsuko Miura

doi:10.1038/s41598-019-47388-x

Kenshi Yaginuma, Haruko Takeyama + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-47388-x

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Abstract

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

Highlights

G-protein coupled receptors (GPCRs) are seven-transmembrane receptors representing the largest membrane protein family in humans[1]
We have previously developed a functional assay system using combined mammalian cells, which produce NanoLuc in response to the activation of human glucagon-like peptide 1 receptor (hGLP1R) and yeast cells that secrete randomized peptides[13]; its throughput was low because we carried out functional assays using 96-well plates
We compared hGLP1R/LacZ-293 and hGLP1R/ NLuc-293 cell lines constructed in a previous study13. hGLP1R/NLuc-293 is a cell line that produced NanoLuc in response to the activation of hGLP1R

Summary

Introduction

G-protein coupled receptors (GPCRs) are seven-transmembrane receptors representing the largest membrane protein family in humans[1]. In a previous study, we constructed a hGLP1R/NLuc-293 reporter cell line[13], which secretes NanoLuc[14] in response to the activation of the human glucagon-like peptide 1 receptor (hGLP1R)[15]. Such a system facilitates easy and sensitive detection of GPCRs activities, in turn facilitating the identification of agonists or antagonists of target GPCRs. In the search for novel peptide ligands, designing a ligand library is an important process. Such an approach permits the easy construction of a soluble peptide ligand library at relatively low cost

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Results

Conclusion