Simultaneous Multiple MS Binding Assays Addressing D₁ and D₂ Dopamine Receptors

Abstract

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D and D dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D and D receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D and D receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays.

MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand

“…As the determination of binding affinities is in general (e. g., no living cells are needed) less elaborate than the performance of functional assays and binding affinities by being direct proportional to the Gibbs free energy (▵ G 0 ) are required as basis for molecular modelling studies, we aimed at the development of a binding assay addressing GlyT2. To this end we intended to use the concept of MS Binding Assays, which was established in our group during the last years and was employed already for GlyT1 and several other target proteins [28–39] . MS Binding Assays (as described in Section 2 3…”

A Library Screening Strategy Combining the Concepts of MS Binding Assays and Affinity Selection Mass Spectrometry

“…This strategy has been successfully applied to several membrane integrated drug targets such as neurotransmitter transporters (Zepperitz et al, 2006; Grimm et al, 2015; Ackermann et al, 2019), G protein-coupled receptors (Massink et al, 2015; Neiens et al, 2015; Chen et al, 2017), or ligand gated ion channels (Sichler et al, 2018). Furthermore, as a consequence of the superior selectivity of mass spectrometric detection (in comparison with LSC), MS Binding Assays enable binding experiments for multiple targets with corresponding selective reporter ligands simultaneously in the same binding sample (Schuller et al, 2017; Neiens et al, 2018). As the concept of MS Binding Assays has been established as alternative to radioligand binding assays, it is—in the same way as the latter—primarily suited for affinity determination of single compounds, but it is basically not restricted to this application.…”

Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters

“…This means MS Binding Assays offer excellent conditions to perform multiple binding experiments in which binding affinities at different targets are characterized simultaneously in one and the same binding sample when different reporter ligands with sufficient selectivity for the individual targets are used. This strategy, termed “Simultaneous Multiple MS Binding Assays”, was recently introduced in a study by Schuller et al., where it was successfully applied to perform saturation as well as competition experiments simultaneously at D 1 and D 2 dopamine receptors . Simultaneous Multiple MS Binding Assays are of particular value when it is necessary to characterize binding affinities of test compounds at various targets, as is the case when several related targets contribute to the intended therapeutic effect, or when subtype selectivity has to be determined.…”

“…This strategy,termed" Simultaneous Multiple MS Binding Assays", was recently introduced in as tudy by Schuller et al,where it was successfully applied to perform saturation as well as competition experiments simultaneously at D 1 and D 2 dopamine receptors. [21] Simultaneous Multiple MS BindingA ssays are of particular value when it is necessary to characterize bindinga ffinitiesoftest compounds at varioustargets, as is the case when several relatedt argets contributet o the intendedt herapeutic effect, or when subtype selectivity has to be determined.…”