A high-throughput screening system for G-protein-coupled receptors using β-lactamase enzyme complementation...

Area of Science:

• Pharmacology
• Biochemistry
• Molecular Biology

Background:

• G-protein-coupled receptors (GPCRs) are crucial drug targets.
• Monitoring GPCR activation is essential for drug discovery.
• Existing methods may lack efficiency or specificity.

Purpose of the Study:

• To develop a novel cell-based assay for monitoring GPCR activation.
• To utilize β-lactamase enzyme fragment complementation (EFC) technology for this purpose.
• To screen a natural compound library for β2-adrenoceptor (β2AR) agonists.

Main Methods:

• Fused inactive β-lactamase fragments to β-arrestin and GPCR.
• Generated a stable cell line (HEK/293-β2a2) expressing these fusion proteins.
• Performed high-throughput screening (HTS) of HPLC-fractionated natural compounds on the cell line.
• Validated hits by measuring cyclic adenosine monophosphate (cAMP) production.

Main Results:

• The HEK/293-β2a2 cell line showed dose-dependent responses to β2AR agonists/antagonists.
• Isoproterenol and propranolol EC50/IC50 values were determined.
• HTS yielded a Z' factor of 0.68.
• Three potent β2AR agonists were identified from the natural compound library.

Conclusions:

• Established a cell-based HTS assay for monitoring GPCR activation.
• The assay relies on β-arrestin and GPCR interaction via β-lactamase EFC.
• This system is effective for identifying drug leads from natural compound libraries.