MS binding assays for GlyT1 based on Org24598 as nonlabelled reporter ligand

In this study an alternative to radioligand binding assays addressing the glycine transporter 1 (GlyT1) based on quantification of a nonlabelled reporter ligand by means of mass spectrometry (MS) is presented. The established MS Binding Assays employ the GlyT1 inhibitor Org24598 as reporter ligand for which a highly sensitive LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) method was developed. A validation of this LC-ESI-MS/MS method with respect to selectivity, linearity, accuracy and precision according to the FDA guidance demonstrated its reliability for quantification of Org24598 in binding experiments. For the implementation of GlyT1 binding experiments conditions in accordance to known GlyT1 radioligand binding assays and already known filtration based MS Binding Assays were chosen. In saturation experiments the affinity of Org24598 towards GlyT1 could be characterized with an equilibrium dissociation constant (K_d) of 16.8 ± 2.2 nM that is well in agreement with the affinity determined in radioligand binding assays. Finally, several known GlyT ligands were studied in competition experiments and the determined inhibition constants (K_i) compared with results from radioligand binding and uptake assays. The almost perfect correlation of the affinities obtained in the MS based binding experiments with results from literature clearly indicates that the established GlyT1 MS Binding Assays are a powerful substitute for the GlyT1 radioligand binding assays so far used for affinity profiling and screening. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.