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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Conventional orthosteric drugs targeting G protein-coupled receptors (GPCRs) face challenges with off-target effects and limited subtype selectivity.
• Allosteric ligands offer precise control over receptor signaling by inducing specific conformational changes, representing a key strategy in modern drug discovery.
• Developing efficient screening methods for GPCR ligands, particularly allosteric modulators, is crucial for advancing therapeutic strategies.

Purpose of the Study:

• To develop a sensitive and selective biosensor for identifying GPCR ligands, focusing on allosteric modulators.
• To utilize surface-enhanced Raman spectroscopy (SERS) for high-throughput screening of ligands targeting the β2 adrenergic receptor (β2AR).
• To establish a methodology for detecting allosteric binding and stabilizing receptor conformations.

Main Methods:

• Fabrication of a sandwich-type SERS sensor involving immobilization of β2AR on magnetic plasmonic nanoparticles via click chemistry.
• Probing and stabilization of β2AR allosteric conformational changes using RNA aptamers.
• Signal amplification and detection using SERS-active nanoparticles for quantitative analysis.

Main Results:

• The developed SERS biosensor demonstrated high sensitivity and selectivity for detecting β2AR ligands.
• The assay successfully identified agonists, antagonists, and allosteric inhibitors of β2AR in both aqueous solutions and serum.
• A detection limit of 49 pM was achieved for the compound BI167107 within a concentration range of 10^-9 to 10^-6 M.

Conclusions:

• The novel SERS-based sandwich biosensor provides a rapid, sensitive, and selective method for GPCR ligand screening.
• This methodology holds promise as an alternative for high-throughput and multiplexed screening of compound libraries.
• The approach effectively identifies various types of GPCR ligands, including allosteric modulators, paving the way for advanced drug discovery.