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

Abstract

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.