Abstract

The binding of a ligand to a specific target is an essential requirement for endogenous messengers as well as for exogenous molecules to exert physiologically or pharmacologically relevant effects. In the drug development process, binding assays play a critical role in the assessment of the affinity of test compounds to specific targets. 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 gammaaminobutyric acid (GABA)_B1b receptor using an unlabeled ligand.

GABA_B receptors are the G-protein-coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. The GABA_B receptor is one of the most important therapeutic targets in the treatment for spasticity.

The membrane fraction of a Chinese hamster ovary (CHO) cell stably expressing the GABA_B1b subunit was isolated. The potent, selective antagonist CGP 54626 was selected as the specific ligand. The detection of CGP 54626 was optimized by liquid chromatography coupled to mass spectrometry (LCMS), 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 test compound), followed by afiltration and wash steps, the plates were subsequently dried and remaining CGP 54626 bound to the GABA_B1b receptor was eluted and quantified by LC-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 K_D of CGP 52432 was determined. The K_off 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 exists, as well as to determine the K_D’s.