A fluorescent ligand-binding alternative using Tag-lite® technology.

Area of Science:

• Pharmacology and Drug Discovery
• Molecular Biology
• Biochemistry

Background:

• G-protein-coupled receptors (GPCRs) are critical cell surface proteins involved in signal transduction.
• Approximately 40-50% of marketed drugs target GPCRs, highlighting their significance in drug discovery.
• Radiolabeled ligand binding assays are the current gold standard for GPCR drug screening but pose safety concerns.

Purpose of the Study:

• To introduce and validate Tag-lite, a nonradioactive, fluorescence-based technology for GPCR binding assays.
• To demonstrate the utility of Tag-lite for high-throughput screening (HTS) applications.
• To provide a safer and more flexible alternative to traditional radioligand assays.

Main Methods:

• Development and implementation of the Tag-lite fluorescence-based assay system.
• Testing the assay with various fluorescent ligands targeting diverse GPCRs, including chemokine, opioid, and cholecystokinin receptors.
• Utilizing recombinant cell lines (transient or stable) expressing SNAP-tagged GPCRs for binding studies.

Main Results:

• Selective binding of fluorescent ligands to multiple GPCR classes was successfully demonstrated.
• Measured affinity constants for known GPCR drugs aligned with published literature values.
• The Tag-lite assay proved reproducible with both adherent and suspension cells in various plate formats (96- and 384-well).

Conclusions:

• Tag-lite provides a robust, nonradioactive alternative for GPCR binding assays.
• The technology is highly suitable for high-throughput screening due to its flexibility, speed, and user-friendliness.
• Tag-lite facilitates miniaturization and broad application in GPCR drug discovery efforts.