[DOT 2019] Validation of a Label-Free Mass Spectrometry-Based Binding Assay for the Gamma-Aminobutyric Acid B1b/2 Receptor

[DOT 2019] Validation of a Label-Free Mass Spectrometry-Based Binding Assay for the Gamma-Aminobutyric Acid B1b/2 Receptor
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DOT 2019


The binding of a ligand to a specific target is an essential requirement for endogenic messengers as well as for exogenic molecules in order to exert physiologically or  oharmacologically relevant effects. In the drug development process, binding assays play a critical role in the assessment of the affinity of test compounds to specific targets1. Traditionally, binding assays have been performed using either radioactive, fluorescent, or luminescence labeled ligands. Our aim was to determine the feasibility of using a mass spectrometry-based readout in order to study the binding kinetics of the gamma-aminobutyric acid (GABA) B1b/2 receptor using an unlabeled ligand.
GABAB receptors are the G-protein-coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system2. The GABAB receptor is one of the most important therapeutic targets in the treatment for spasticity.
The membrane fraction of a Chinese hamster ovary cell stably expressing the GABAB1b/2 subunits were isolated. The well-known antagonist CGP 54626 was selected as the specific ligand. The detection of CGP 54626 was optimized by liquid chromatography coupled to mass spectrometry (UHPLC-MS/MS), an ultra-high performance liquid chromatography (Infinity 1290 and CTC autosampler, Agilent) using a C18 column which was coupled to a triple quadrupole
linear ion traps (QTRAP 5500, Sciex). As per “gold standard” radioligand binding assays  identical steps were followed which included, incubation of ligand and receptor (in the presence or absence of a test compound), followed by a filtration and wash steps, the plates were  subsequently dried and remaining CGP 54626 bound to the GABAB1b/2 receptor was eluted and quantified by UHPLC-MS/MS. IC50’s of Baclofen, CGP 52432 and GABA were determined
and conformed with data generated by in-house radioligand binding assays. Finally, the equilibrium dissociation constant KD of CGP 52432 was determined. The Koff of CGP 52432, was determined by both a displacer technique and dilution method.
Our work clearly demonstrates that mass spectrometry-based binding assays are a viable alternative to traditionally labeled binding assays and will allow for the development of novel binding assays for targets for which no labeled
ligands exist as well as to determine the KD’s.