Purification-free immobilization of dopamine D2 receptor and glucocorticoid receptor by alkyne–azide cycloaddition for affinity chromatography-based drug screening

Immobilized protein-based affinity chromatography offers a promising alternative for high-throughput drug screening, yet its effectiveness is often hampered by conventional immobilization techniques as they result in functional loss of the protein due to random orientation and multi-step purification. Herein, we developed a universal strategy for fabricating highly efficient chromatographic methods by integrating genetic code expansion with alkyne-azide click cycloaddition in the immobilization of dopamine D2 receptor (DRD2) and glucocorticoid receptor (GR) strain-promoted alkyne-azide cycloaddition (SPAAC). The site-specific incorporation of p -azidophenylalanine ( p AzF) into the two receptors using an engineered E. coli C321.ΔA strain enabled direct, oriented, and covalent immobilization of them onto dibenzocyclooctyne (DBCO)-functionalized silica by a copper-free click reaction without the need for prior purification. The resulting immobilized DRD2 p AzF and GR p AzF stationary phases were thoroughly characterized by XPS, SEM/EDS, and immunofluorescence, confirming successful immobilization and preserved receptor activity. These exhibited exceptional specificity, stability (>30 days), and reproducibility (RSD<1%). Applications immobilized DRD2 and GR in analyzing Wendan Decoction identified liquiritigenin, neohesperidin, and naringin as active components of the prescription. Among them, liquiritigenin, neohesperidin were characterized as dual-target ligands. Determination of their binding affinities to the receptors by frontal analysis enables calculation of drug-like efficiency indices (SEI, BEI, LLE). Cellular assays in HT22 cells further confirmed their bioactivities and target specificity to the receptors. This work is possible to advance the discovery of potential leads from natural products as it has the properties of streamlining the construction of reliable receptor chromatography methods, integrating the screening with binding affinity measurement, and preliminary drug-likeness evaluation.