A cell-based affinity mass spectrometry strategy for the rapid discovery of active components from Chinese herbal...

As compilations of natural products, Traditional Chinese medicines (TCMs) formulae constitute a rich resource for novel drug discovery. Nevertheless, their complex composition brought about significant challenges in identifying bioactive constituents. Chai-Gui Decoction (CGD) is a combination of two classic prescriptions commonly employed in gynecological therapeutics, and its active ingredients remain uncharacterized.

This study aimed to establish a cell-based affinity mass spectrometry strategy for the rapid discovery of the active components from complex TCM formulae. This study chose CGD as an example and the G protein-coupled estrogen receptor (GPER) as a target.

A stable HEK293-T cell line overexpressing GPER-1 were established and incubated with CGD to execute ligand screening. Cell membrane fragments were harvested, and the ligand-bound components were isolated and identified by using UPLC-QTOF/MS. The theoretical binding affinities of the fished ligands for GPER were calculated via molecular docking. The compounds exhibiting the highest theoretical affinity were further managed to binding validation using a cellular thermal shift assay (CETSA), and their activity was confirmed through cell-based experiments.

A novel fishing assay was developed, which integrated ligand-receptor binding in live cells, cell membrane extraction and lysis, and liquid chromatography-mass spectrometry identification. Seven compounds from the CGD extract demonstrated significantly higher detection levels in GPER-overexpressing cells compared to vehicle control cells. Molecular docking analyses further indicated high binding affinities between these compounds and the GPER protein. Among them, the two compounds with the highest predicted affinity, 16α-Hydroxytrametenolic acid (16α-HTA) and Alisol A, were confirmed to bind GPER by altering its thermal stability in CETSA. Subsequent cellular investigations revealed that 16α-HTA and Alisol A reduced GPER expression at a low dose and suppressed AKT phosphorylation independently of EGFR.

The present study proposes a feasible strategy integrating cell extraction, UPLC-QTOF/MS analysis, molecular docking simulation, CETSA and cell-based experiments, which enables the effective exploration and validation of ligands for protein targets within complex mixtures.