Site-Specific Immobilization of β2-AR Using O6-Benzylguanine Derivative-Functionalized Supporter for High-Throughput Receptor-Targeting Lead Discovery.

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta2-adrenergic receptor (β2-AR), the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of β2-AR on the resin. The immobilized β2-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to β2-AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting β2-AR in Fructus Perillae. We concluded that O6-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.