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

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 1.01×106 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.

Screening for New Inhibitors of Glycine Transporter 1 and 2 by Means of MS Binding Assays

“…The determined IC 50 value was then transferred into K i values (inhibition constant of the test compound) using the Cheng-Prusoff equation K i = IC 50 /(1 + [L]/K d ), with [L] = reporter ligand concentration in competition experiment and K d = dissociation constant of the reporter ligand for the target protein. For GlyT1 competition experiments an Org24598 concentration of 15 nm and a K d = 16.8 � 2.2 nm [32] and for GlyT2 competition experiments an Org25543 concentration of 10 nm and a K d = 7.45 � 0.55 nm [33] were used. Finally, for the determined K i values of the hit compounds the corresponding pK i values were calculated.…”

“…[37] and Org25543 (N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5dimethoxy-4-(phenylmethoxy)benzamide) as hydrochloride (purity � 99 %, HPLC), were purchased from Tocris (Bristol, UK). [ 2 H 5 ] Org24598 [32] and [ 2 H 7 ]Org25543 [33] were synthesized in-house. All other test compounds were either synthesized in-house or were acquired by purchase from common supplier.…”

“…The binding affinities (pK i ) of the six hit compounds SC70-o, SC70-p, SC71-a, SC71-f, SC71-m and SC107-f were determined in competitive GlyT2 MS Binding Assays, which were performed as described recently. [33] Of each hit compound one representative inhibition curve out of three independent competition experiments is depicted in Figure 5 and the obtained pK i values are listed in Table 6. Compounds SC70-o, SC70-p, SC71-a, SC71-f and SC71-m had been primarily developed as fluorescent ligands of the GABA transporters mGAT1-mGAT4.…”

“…Membrane preparations of CHO-K1 cells stably expressing hGlyT1c and HEK 293 cells stably expressing hGlyT2 were prepared and applied as recently described. [32,33] General procedure of compound and library screening A total of 2439 compounds were available for screening which were either tested as single compounds (α-amino acid) or as sublibraries each with 16 compounds (all other compounds) in competitive binding assays. For the following steps 10-fold concentrated solutions in incubation buffer of the single compounds or sublibraries of the applied concentrations were prepared to use for the experiments.…”

“…For this screening, the recently published GlyT1 and GlyT2 MS Binding Assays based on the selective GlyT1 and GlyT2 inhibitors Org24598 and Org25543, respectively, as reporter ligands should be used (see Figure 1 ). [ 32 , 33 ] Of the aforementioned compound library those compounds, representing α‐amino acids, should be studied individually, all other screened in sublibraries consisting of 16 compounds. In case of the individually studied compounds, hits would, of course, become directly apparent.…”