A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine...

Area of Science:

• Pharmacology and Drug Discovery
• Biochemistry and Molecular Biology

Background:

• Proximal readout assays are crucial for evaluating drug candidates targeting G protein-coupled receptors (GPCRs).
• Traditional methods using radiolabeled nucleotides (e.g., [35S]GTPγS) for studying GPCR-G protein interactions often suffer from quality issues.
• There is a need for a robust, novel proximal functional assay for routine characterization of histamine receptor ligands.

Purpose of the Study:

• To develop and validate a novel proximal functional assay for histamine receptor (H1R, H2R, H3R, H4R) ligand characterization.
• To compare the performance of the new assay with established radiolabeled nucleotide binding assays.
• To investigate the influence of minimal G protein expression levels on assay performance.

Main Methods:

• Application of the split-NanoLuc technology combined with engineered minimal G (mini-G) proteins to four histamine receptor subtypes.
• Real-time monitoring of functional responses upon receptor activation.
• Evaluation of mini-G sensors using standard agonists and antagonists, with comparison to [35S]GTPγS binding assays.

Main Results:

• The split-NanoLuc assay accurately determined ligand potencies and efficacies for all histamine receptor subtypes, consistent with literature data.
• Improved dynamic ranges (Z' factor) and signal spans were observed for all receptor subtypes compared to the [35S]GTPγS assay.
• A positive correlation between signal amplitude and mini-G protein expression was found for H2R, but not H1R or H3R; histamine pEC50 values remained consistent across expression levels.

Conclusions:

• The split-NanoLuc/mini-G protein assay is a highly effective and reliable tool for the routine functional characterization of histamine receptor ligands.
• This novel assay surpasses traditional methods in terms of dynamic range and signal span, offering significant advantages for drug discovery.
• The assay's robustness and consistency facilitate accurate assessment of ligand activity across different experimental conditions.