A CotB1p-mediated one-step immobilization strategy for constructing GPCR affinity chromatography to screen multi-target natural product ligands.

G protein-coupled receptors (GPCRs) are critical targets for drug discovery, yet the screening of GPCR-binding ligands from complex natural products remains challenging due to the lack of mild and universal immobilization strategies for these fragile membrane proteins. Here, we reported CotB1p-mediated one-step immobilization strategy for the construction of GPCR affinity chromatography columns to screen natural product ligands. Three asthma-related GPCRs β2-adrenergic receptor (β2AR), muscarinic M3 receptor (M3R), and cysteinyl leukotriene receptor 1 (CysLT1R) were genetically fused with the silica-binding peptide CotB1p. These fusion proteins were then directly immobilized onto macroporous silica via simple affinity adsorption, a process that avoids harsh chemical crosslinking and preserves receptor binding activity. The prepared affinity columns showed good specificity, stability, and repeatability, with retention time RSDs of 1.56%, 0.98%, and 1.68% for the β2AR, M3R, and CysLT1R columns, respectively. The three affinity columns were applied to screen ligands from a representative natural product prescription, followed by LC-MS/MS identification. Amygdalin, ephedrine/pseudoephedrine, scopolamine, atropine, forsythoside A, and phillyrin were identified as receptor-binding constituents selectively retained by different GPCR columns. Nonlinear chromatography enabled the evaluation of ligand-receptor interactions, with association constants ranging from 4.33 × 104-5.08 × 105 M⁻¹ .Molecular docking and molecular dynamics simulations further supported the possible binding modes and relative conformational stability of the ligand-receptor complexes. In addition, cellular assays in human airway smooth muscle cells indicated that the screened compounds could reduce TNF-α-induced inflammatory cytokine production. Overall, this CotB1p-mediated one-step immobilization strategy provides a mild and efficient approach for preparing functional GPCR affinity stationary phases, offering a useful analytical platform for target-resolved screening of natural product ligands against multiple GPCRs.