A general method for quantifying ligand binding to unmodified receptors using Gaussia luciferase

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Accurate measurement of ligand binding to cell surface receptors is crucial for biological and pharmacological research.
• Existing resonance energy transfer (RET) assays often require receptor tagging, which can interfere with ligand binding.
• A tag-free method is needed to reliably study ligand-receptor interactions without altering native binding properties.

Purpose of the Study:

• To develop and validate a novel tag-free system for measuring ligand-receptor interactions in live cells.
• To utilize Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer (BRET) donor for enhanced assay sensitivity and performance.
• To demonstrate the broad applicability of the assay for various receptor types and drug discovery applications.

Main Methods:

• Development of a GLuc-based extracellular surface biosensor for BRET measurements.
• Detection of bystander BRET between the biosensor and fluorescent ligands bound to unmodified receptors.
• Application of the assay for equilibrium and kinetic binding measurements, including competitive binding assays with unlabeled ligands.
• Utilizing the assay for high-throughput screening (HTS) to identify novel receptor ligands.

Main Results:

• The GLuc-based BRET assay successfully measured ligand-receptor interactions in live cells without receptor tagging.
• The assay demonstrated high sensitivity and broad applicability across different plasma membrane receptors.
• Successful identification of novel α1 adrenergic receptor ligands through high-throughput screening.
• The system proved effective for both equilibrium and kinetic binding studies.

Conclusions:

• The developed GLuc-based BRET biosensor offers a simple, sensitive, and cost-efficient platform for studying ligand-receptor interactions.
• This tag-free approach overcomes limitations of existing methods, enabling more accurate drug characterization and development.
• The assay is suitable for diverse applications, including high-throughput screening and the discovery of new therapeutic agents.