Premium
MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand
Author(s) -
Ackermann Thomas M.,
Allmendinger Lars,
Höfner Georg,
Wanner Klaus T.
Publication year - 2021
Publication title -
chemmedchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.817
H-Index - 100
eISSN - 1860-7187
pISSN - 1860-7179
DOI - 10.1002/cmdc.202000342
Subject(s) - dissociation constant , affinities , chemistry , ligand (biochemistry) , ligand binding assay , transporter , stereochemistry , reaction rate constant , biochemistry , receptor , kinetics , physics , quantum mechanics , gene
Abstract 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 1.01×10 6 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.