A label-free strategy for immobilization of GPCRs using site-specific encoded non-natural amino acids to develop a selectively chromatographic approach for pursuing potential ligands binding to 5-hydroxytryptamine 1A receptor.

G protein-coupled receptors (GPCRs) are one of the most prominent targets for drug discovery. Immobilizing GPCRs has proven to be an effective strategy for expanding the utility of GPCRs into nonbiological contexts. However, traditional strategies of immobilizing GPCRs have been severely challenged due to the loss of receptor function. Here, we reported a novel and general approach to realize the label-free and site-selective immobilization of 5-hydroxytryptamine 1A receptor (5-HTR) and the application in developing a chromatographic method with improved specificity for pursuing 5-HTR ligands from natural products. This method involved the use of a clickable non-natural amino acid, O-allyl-L-tyrosine (O-ALTyr) to immobilize the receptor onto thiol-functionalized silica gels through a 'thiol-ene' click chemistry, which allowed us to avoid the purification step and directly immobilize 5-HTR on silica gels. The immobilized receptor was characterized using immunofluorescence assay, and receptor-ligand interaction analysis was conducted through frontal analysis. To test the feasibility of the immobilized 5-HTR in complex matrices, bioactive compounds in Ziziphi Spinosae Semen (ZSS) were screened and their interaction with the receptor was assessed using zonal elution. Our findings indicated that immobilizing the receptor through nnAAs effectively minimizes the chromatographic peak tailing and broadening of specific ligands, leading to a significant improvement in chromatographic performance. The association constants of the three ligands to 5-HTR were approximately one order of magnitude greater than those of Halo-tag attachment. These results demonstrated that the immobilized 5-HTR exhibits high specificity and the ability to recognize receptor ligands from complex matrices. This allowed us to identify magnoflorine (Mag) as a potential ligand of 5-HTR from ZSS extract. In vivo assay also proved that Mag presented a promising anxiolytic effect by promoting the expression of 5-HTR in mice brain. The above findings pointed to the fact that the immobilized 5-HTR affinity chromatographic strategy relying on the site-specific encoded non-natural amino acid is a powerful alternative for precisely determining the drug-protein interaction and discovering the specific ligand of GPCRs from complex matrixes.