Thermodynamic Profiling and Fragment Screening of GPCRs Using Grating-Coupled Interferometry

Abstract G protein-coupled receptors (GPCRs) represent one of the most important target classes in drug discovery, yet many remain pharmacologically underexplored due to limited biological knowledge and technical challenges associated with their characterisation. Here, we evaluate grating-coupled interferometry (GCI) as a versatile optical biosensor platform for GPCR research. Using the human adenosine A2A receptor (A2AR) as a model system, we demonstrate that GCI provides high-quality kinetic data that correlates with data obtained using well-established Biacore technology. When properly set up, the waveRAPID injection method enables fast and reliable determination of binding kinetics and affinities from single-concentration injections. We further extend this approach to determine thermodynamic parameters for diverse A2AR ligands using minimal protein consumption, by combining waveRAPID injections with the fast temperature changes available on the WAVEdelta system. Finally, we performed a kinetic fragment screening using a 704-member fragment library to identify specific A2AR binders. Nano differential scanning fluorimetry (nanoDSF) was subsequently applied to confirm binding for the identified hits. Collectively, this study establishes GCI-based analysis as a powerful and information-rich platform for kinetic, thermodynamic, and fragment-based discovery on GPCR targets, which is particularly valuable for early-stage drug discovery.