Citation: Ting Li, Xinxin Zheng, Lejing Qu, Yuanyuan Ou, Sai Qiao, Xue Zhao, Yajun Zhang, Xinfeng Zhao, Qian Li. A chromatographic method for pursuing potential GPCR ligands with the capacity to characterize their intrinsic activities of regulating downstream signaling pathway[J]. Chinese Chemical Letters, ;2024, 35(10): 109792. doi: 10.1016/j.cclet.2024.109792 shu

A chromatographic method for pursuing potential GPCR ligands with the capacity to characterize their intrinsic activities of regulating downstream signaling pathway

Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China

zhaoxf@nwu.edu.cn

20185365@nwu.edu.cn

Received Date: 13 February 2024
Revised Date: 26 February 2024
Accepted Date: 19 March 2024
Available Online: 19 March 2024

Figures(5)

GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable. Affinity-based screening technique is a promising platform to uncover GPCR ligands. However, the intrinsic activities of them are seldom simultaneously determined during the screening. Taking beta2-adrenoceptor (β₂AR) as a probe, this work created a strategy for screening GPCR ligands with simultaneous characterization of their downstream G protein binding responses associated with GTP. The strategy included (ⅰ) the design and expression of a protein miniature formed by β₂AR and G protein α-subunit (Gα_s) using circularly permuted HaloTag (cpHalo) as a flexible linker; (ⅱ) immobilization of the miniature onto silica gel by a click dehalogenation reaction; (ⅲ) systematic characterization of the immobilized miniature by fluorescent and chromatographic studies, and (ⅳ) simulating of ligand-induced β₂AR-Gα_s signaling cascade by chromatographic assays using GTP as an indicator. The immobilized miniature exhibited specificity to β₂AR and Gα_s antibodies and ligands. The specificity is stable at least within fifteen days with the variation less than 1%. The intrinsic activities of β₂AR ligands were distinguished by the changes of GTP chromatographic behaviors on Gα_s-cpHalo-β₂AR column. Agonists strengthened the binding affinity and kinetics of GTP with Gα_s, while antagonist did not give any effect on them. With the intrinsic activity evaluation, we believe, it will improve the attributes of chromatographic methods for drug discovery efforts with minimizing false-positive results.
- Intrinsic efficacy,
- GPCR-G protein miniature,
- Circularly permuted HaloTag,
- High-performance receptor chromatography,
- GTP binding,
- Agonist/antagonist
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