Unlocking GPCR–ligand interactions: Measuring binding affinities with thermal shift assay

Abstract G protein‐coupled receptors (GPCRs) constitute the largest transmembrane protein superfamily, with over 800 representatives in the human genome. Recognized as pivotal targets in pharmacological research and drug discovery, these receptors play a crucial role in advancing therapeutics. Understanding the molecular mechanisms of receptor–ligand interactions is imperative for drug discovery applications. However, experimental procedures for measuring ligand binding are complicated by various factors, including the transmembrane nature of the receptors and the high cost associated with specialized instruments and consumables. Here we introduce an application of the thermal shift assay (TSA) to measuring ligand binding affinities for GPCRs. TSA is a cost‐effective and user‐friendly method that detects changes in protein stability induced by alterations in environmental conditions. Employing the human A 2A adenosine receptor as a representative GPCR, we determined binding constants for four orthosteric ligands and allosteric sodium using three mathematical models for TSA data approximation and analysis. Models were additionally validated by two antagonists of cysteinyl leukotriene GPCR (CysLT 1 R), used as antiasthmatic drugs. Our results suggest that the TSA approach demonstrates a high degree of reproducibility and agreement with existing literature data, thereby affirming its suitability for investigating GPCR interactions with various types of ligands.