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

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 (Gαs) 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-Gαs signaling cascade by chromatographic assays using GTP as an indicator. The immobilized miniature exhibited specificity to β2AR and Gαs 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 Gαs-cpHalo-β2AR column. Agonists strengthened the binding affinity and kinetics of GTP with Gαs, 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.