Label-free LC-MS based assay to characterize small molecule compound binding to cells

Area of Science:

• Pharmacology and Analytical Chemistry
• Biochemistry and Molecular Biology

Background:

• Characterizing small molecule binding to live cells is crucial for pharmacological ligand assessment.
• Existing protein-based binding assays can be challenging for certain target classes, such as G protein-coupled receptors (GPCRs) and ion channels.

Purpose of the Study:

• To develop and validate a novel, label-free cell binding assay using liquid chromatography-mass spectrometry (LC-MS).
• To enable quantitative characterization of ligand binding to cell surface targets in a physiologically relevant context.

Main Methods:

• A label-free LC-MS based assay was developed, utilizing centrifugation to separate bound and unbound ligands.
• The assay was applied to study a G protein-coupled receptor (GPCR) using an antagonist as a probe and competitor ligands.
• The method was also validated using an ion channel target and its agonists.

Main Results:

• The GPCR binding assay demonstrated specificity, saturability, and fast equilibrium.
• Competition binding analysis showed good correlation between ligand inhibition potency and binding affinity.
• Binding affinity determined for the ion channel target was consistent with results from functional assays.

Conclusions:

• The developed LC-MS cell binding assay is a versatile and quantitative method for characterizing small molecule ligand interactions with cell surface targets.
• This approach is particularly valuable for targets where traditional protein-based assays are difficult to implement.
• The assay provides a physiologically relevant environment for assessing ligand binding, aiding in drug discovery and development.