Authors

P Leznicki¹; W Keene¹; R Satchell¹; S Best¹; S Pollack¹; 
¹ Sygnature Discovery, UK

Discussion

Affinity and kinetics of compound binding to a protein of interest (POI) are fundamental parameters that guide any drug discovery effort. Their characterisation is, however, often hindered by technical challenges such as lack of active, purified POI that could be used in biophysical assays. This is particularly true for membrane proteins, including plasma membrane-localised receptors, which are notoriously difficult to purify in an active, correctly folded state. Binding of radioactively labelled ligands to POIs present in membrane fractions derived from cells that express them, overcomes this problem and has been widely used in receptor-ligand interaction studies. However, environmental concerns, high cost, time-line considerations and the need of generating a radioactive derivative of a ligand limit the utility of this approach. For these reasons, non-radioactive, label-free approaches have been investigated as a substitute for radioactivity-based ligand detection. Advances in mass spectrometry allow it to be used for detection of picomolar concentrations of compounds and as such mass spectrometry has recently been successfully used in receptor-ligand interaction studies. The application of mass spectrometry to these assays additionally offers increased specificity over radioactivity approaches with mass spectrometry having the potential of detecting the unlabelled ligand as opposed to the radioactive tracer. Here, we have quantified affinities and kinetic parameters of several ligands’ interaction with adenosine A2A receptor. Our results are in good agreement with published studies that relied on either mass spectrometry or radioligand binding. Hence, we provide additional support for the validity of using mass spectrometry for the detection of unlabelled ligands in receptor-ligand binding studies and highlight the capabilities that Sygnature Discovery can offer in this area.