An Allosteric Assay for Identifying Ligands Binding to β₂ Adrenergic Receptor by Surface-Enhanced Raman Scattering...

The development of conventional orthosteric drugs targeting G protein-coupled receptors (GPCRs) is often hindered by off-target effects and low subtype selectivity. Allosteric ligands enable precise control of receptor signaling through selective conformational changes, highlighting their critical importance as a promising therapeutic strategy and as a key frontier in modern drug discovery. To enhance GPCR ligand screening efficacy, surface-enhanced Raman spectroscopy (SERS), with sensitivity and narrow spectral peaks, was employed to outline a methodology based on allosteric aptamer binding and stabilizing conformations of the β₂ adrenergic receptor (β₂AR) for identifying GPCR ligands by fabricating a sandwich-type SERS sensor. The method involved immobilization of β₂AR on magnetic plasmonic nanoparticles by label-free click chemistry, probing and stabilization of the allosteric conformational changes by RNA aptamer, and amplification and detection of the signal by SERS-active nanoparticles. The success of the allosteric β₂AR assay was confirmed through multiple analytical techniques, including SERS, SEM, UV-vis spectroscopy, and particle size analysis. This methodology demonstrates broad applicability, characterized by rapid response, high sensitivity, and excellent selectivity. BI167107 could be detected within a range of 10^-9-10^-6 M, with a detection limit of 49 pM. Such a sandwich biosensor successfully identified the agonist, antagonist, and allosteric inhibitor of β₂AR from the ligand complex in both aqueous solution and serum. This method is expected to become an alternative for high-throughput and multiplexed drug screening when it comes to a compound library.