Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors

Area of Science:

• Pharmacology
• Biochemistry
• Molecular Biology

Background:

• Cannabinoid receptors (CBRs) are key in pain, inflammation, and energy balance.
• Existing methods for detecting Gαi protein signaling are challenging.
• Developing novel therapeutics targeting CBRs requires better understanding of receptor signaling.

Purpose of the Study:

• To develop a robust membrane-based assay for Gαi protein signaling.
• To enable reliable detection and characterization of cannabinoid receptor activity.
• To facilitate higher-throughput screening of Gαi-coupled receptors.

Main Methods:

• Developed a membrane-based Gαi signaling system using HEK293TR cells overexpressing CBRs and a Gαi-CASE biosensor.
• Utilized Bioluminescence Resonance Energy Transfer (BRET) to monitor Gαi protein activation.
• Applied the assay in a 384-well format to profile reference cannabinoid compounds.

Main Results:

• The assay successfully detected agonist and inverse agonist activity of cannabinoid ligands.
• Characterized potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists.
• Results aligned with whole-cell assays and literature values.

Conclusions:

• The novel membrane-based Gi-CASE NanoBRET system provides a reliable method for Gαi protein signaling assays.
• This assay is applicable to various Gαi-coupled GPCRs, including orphan receptors.
• Enables real-time, higher-throughput measurements of receptor activation for drug discovery.