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

Neiens, Patrick; Simone, Angela De; Höfner, Georg; Wanner, Klaus T.

doi:10.1002/cmdc.201700737

Cited by 8 publications

(4 citation statements)

References 41 publications

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“…In the last step of the binding experiment, the target‐bound‐marker remaining on the filters has to be liberated and eluted. In former MS Binding Assays the solvents acetonitrile and methanol have already been shown to be suitable for this task [30–39] . We decided to select acetonitrile as elution solvent due to the fact that it is advantageous for the chromatography, especially for peak shapes, when the composition of the sample solvent of the final sample is identical with that of the mobile phase of the employed LC‐ESI‐MS/MS method which in the present case consists of a mixture of acetonitrile and 5 mM ammonium bicarbonate buffer in a ratio of 80 : 20 ( v / v ).…”

Section: Resultsmentioning

confidence: 99%

“…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…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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This study describes the first binding assay for glycine transporter 2 (GlyT2) following the concept of MS Binding Assays. The selective GlyT2 inhibitor Org25543 was employed as a reporter ligand and it was quantified with a highly sensitive and rapid LC‐ESI‐MS/MS method. Binding of Org25543 at GlyT2 was characterized in kinetic and saturation experiments with an off‐rate of 7.07×10−3 s−1, an on‐rate of 1.01×106 M−1 s−1, and an equilibrium dissociation constant of 7.45 nM. Furthermore, the inhibitory constants of 19 GlyT ligands were determined in competition experiments. The validity of the GlyT2 affinities determined with the binding assay was examined by a comparison with published inhibitory potencies from various functional assays. With the capability for affinity determination towards GlyT2 the developed MS Binding Assays provide the first tool for affinity profiling of potential ligands and it represents a valuable new alternative to functional assays addressing GlyT2.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

2019

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The primary objective of early drug development is to identify hits and leads for a target of interest. To achieve this aim, rapid, and reliable screening techniques for a huge number of compounds are needed. Mass spectrometry based binding assays (MS Binding Assays) represent a well-established technique for library screening based on competitive binding experiments revealing active sublibraries due to reduced binding of a reporter ligand and following hit identification for active libraries by deconvolution in further competitive binding experiments. In the present study, we combined the concepts of MS Binding Assays and affinity selection mass spectrometry (ASMS) to improve the efficiency of the hit identification step. In that case, only a single competitive binding experiment is performed that is in the first step analyzed for reduced binding of the reporter ligand and—only if a sublibrary is active—additionally for specific binding of individual library components. Subsequently, affinities of identified hits as well as activities of reduced sublibraries (i.e., all sublibrary components without hit) are assessed in additional competitive binding experiments. We exemplified this screening concept for the identification of ligands addressing the most widespread GABA transporter subtype in the brain (GAT1) studying in the beginning a library composed of 128 and further on a library of 1,280 well-characterized GAT1 inhibitors, drug substances, and pharmacological tool compounds. Determination of sublibraries' activities was done by quantification of bound NO711 as reporter ligand and hit identification for the active ones achieved in a further LC-ESI-MS/MS run in the multiple reaction monitoring mode enabling detection of all sublibrary components followed by hit verification and investigation of reduced sublibraries in further competitive binding experiments. In this way, we could demonstrate that all GAT1 inhibitors reducing reporter ligand binding below 50% at a concentration of 1 μM are detected reliably without generation of false positive or false negative hits. As the described strategy is apart from its reliability also highly efficient, it can be assumed to become a valuable tool in early drug research, especially for membrane integrated drug targets that are often posing problems in established screening techniques.

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“…Indirect AS–MS methods involve a four-step process (Figure ): (1) incubation of a target biomolecule, such as a protein, with a pool of small molecules, often hundreds or thousands; (2) separation of nonbinding compounds from the protein–ligand complexes; (3) dissociation of the protein–ligand complexes, and (4) identification of the ligands by MS. The different AS–MS methods mainly differ in how the protein–ligand complexes and the small molecules in solution are separated, some approaches include ultrafiltration, − immobilized protein AS, , magnetic beads, , and size-exclusion chromatography. − …”

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Solving the Competitive Binding Equilibria between Many Ligands: Application to High-Throughput Screening and Affinity Optimization

2020

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Modern small-molecule drug discovery relies on the selective targeting of biological macromolecules by low-molecular weight compounds. Therefore, the binding affinities of candidate drugs to their targets are key for pharmacological activity and clinical use. For drug discovery methods where multiple drug candidates can simultaneously bind to the same target, a competition is established, and the resulting equilibrium depends on the dissociation constants and concentration of all the species present. Such coupling between all equilibrium-governing parameters complicates analysis and development of improved mixture-based, high-throughput drug discovery techniques. In this work, we present an iterative computational algorithm to solve coupled equilibria between an arbitrary number of ligands and a biomolecular target that is efficient and robust. The algorithm does not require the estimation of initial values to rapidly converge to the solution of interest. We explored binding equilibria under ligand/receptor conditions used in mixture-based library screening by affinity selection–mass spectrometry (AS–MS). Our studies support a facile method for affinity-ranking hits. The ranking method involves varying the receptor-to-ligand concentration ratio in a pool of candidate ligands in two sequential AS–MS analyses. The ranking is based on the relative change in bound ligand concentration. The method proposed does not require a known reference ligand and produces a ranking that is insensitive to variations in the concentration of individual compounds, thereby enabling the use of unpurified compounds generated by mixture-based combinatorial synthesis techniques.

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

Cited by 8 publications

References 41 publications

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

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

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

Solving the Competitive Binding Equilibria between Many Ligands: Application to High-Throughput Screening and Affinity Optimization

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