Stepwise Frontal Analysis Coupled with Affinity Chromatography: A Fast and Reliable Method for Potential Ligand...

Area of Science:

• Pharmacology
• Biochemistry
• Analytical Chemistry

Background:

• Mahuang-Fuzi-Xixin Decoction (MFXD) is a traditional remedy for asthma, but its active components targeting the beta2-adrenoceptor (β2-AR) are not fully understood.
• Identifying these components is crucial for understanding MFXD's therapeutic mechanisms and for drug discovery.

Purpose of the Study:

• To identify the specific components within MFXD that interact with the β2-AR.
• To develop and validate a method for screening bioactive compounds targeting the β2-AR using an immobilized receptor.
• To elucidate the binding affinities and interactions of identified compounds with the β2-AR.

Main Methods:

• Immobilization of haloalkane dehalogenase (Halo)-tagged β2-AR onto modified microspheres to create an affinity stationary phase.
• Stepwise frontal analysis using the affinity stationary phase to determine association constants of known ligands and MFXD components.
• Radioligand binding assay and molecular docking analysis to validate findings and understand binding interactions.

Main Results:

• The immobilized β2-AR system successfully screened MFXD, identifying ephedrine, aconifine, karakoline, and chasmanine as bioactive compounds targeting the receptor.
• Ephedrine and chasmanine showed significant association constants (2.94±0.02×10⁴ M⁻¹ and 4.60±0.15×10⁴ M⁻¹, respectively) with the β2-AR.
• Molecular docking confirmed that these compounds bind to the same pocket on the β2-AR as known agonists, involving key amino acid residues.

Conclusions:

• Ephedrine and chasmanine are likely the primary MFXD components responsible for its therapeutic effects in asthma via the β2-AR pathway.
• Immobilized β2-AR coupled with stepwise frontal analysis offers a robust and efficient method for drug discovery and screening of receptor-binding compounds.