Visualizing Ligand Binding to a GPCR In Vivo Using NanoBRET.

Area of Science:

• Pharmacology and Molecular Biology
• Oncology
• Biotechnology

Background:

• Drug efficacy relies on in vivo molecular target engagement, which is not adequately assessed by current methods quantifying organ drug levels.
• Assessing drug binding in solid tumors is challenging due to disrupted vasculature and hypoxia, hindering therapeutic potential evaluation.
• G protein-coupled receptors (GPCRs) are critical drug targets, but their in vivo binding is difficult to measure directly.

Purpose of the Study:

• To develop and validate a novel in vivo method for directly quantifying drug-target binding.
• To assess the therapeutic potential of drugs targeting malignant tumors by measuring direct ligand binding.
• To overcome limitations in evaluating drug efficacy in challenging tumor microenvironments.

Main Methods:

• Utilized triple-negative human breast cancer cells engineered to express β2-adrenoceptors tagged with the NanoLuc bioluminescence protein.
• Developed a bioluminescence resonance energy transfer (BRET) assay for direct in vivo measurement of ligand binding.
• Applied the BRET approach in a mouse model of human breast cancer.

Main Results:

• Successfully demonstrated the ability to directly quantify ligand binding to a GPCR in vivo using the BRET approach.
• The method provides a direct measure of drug engagement with its molecular target within a living organism.
• Validated the technique in a relevant preclinical model of malignant breast cancer.

Conclusions:

• The developed BRET-based method enables direct in vivo quantification of drug-target binding, particularly for GPCRs.
• This approach offers a significant advancement for drug discovery and development, especially for challenging targets like those in solid tumors.
• The technique holds promise for more accurate assessment of therapeutic potential and personalized medicine strategies in oncology.