MS Transport Assays for γ-Aminobutyric Acid Transporters—An Efficient Alternative for Radiometric Assays

Abstract

Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

Application of MS Transport Assays to the Four Human γ‐Aminobutyric Acid Transporters

“…[14] In the next step, the hBGT-1-, hGAT-2-, and hGAT-3-expressing COS-7 cells exhibiting the highest amount of ( 2 H 6 )GABA transport relative to that of non-transfected COS-7c ells were characterizedi ns aturation experiments according to the procedure established for hGAT-1, [14] illustrated in Scheme 1. To implement MS Transport Assays for the other human GABA transporter subtypes hGAT-2( SLC6A13), hGAT-3 (SLC6A11), andh BGT-1 (SLC6A12), we tried to benefita gain from COS-7 cells, as these cellswithstand all aspiration and dispensings teps during the transport assay managed by an automatic plate washerw ithout expensive and time-consuming pre-coating of cell culture plates (Scheme1).…”

“…[14] Withint he course of this study,w ep rotected the columns with ag uard column, which elongated the runtime from 3.0 to 3.6min. [14] Withint he course of this study,w ep rotected the columns with ag uard column, which elongated the runtime from 3.0 to 3.6min.…”

“…[11] The search for new potent and selective GATinhibitors serving as drug candidates or pharmacological tools requires screening techniques to identify inhibitors and to characterize their potency at each GATsubtype. [13] We recently published an LC-MS/MS-based alternative to characterize substrate transport into hGAT-1-expressing COS-7 cells, referred to as MS Transport Assays; [14] it uses deuterium-labeled ( 2 H 6 )GABA as as ubstitute for [ 3 H]GABAt ypically used in radiometric uptakea ssays. For the developmento fn ew drugs or pharmacological tools addressing GATs,s creening techniques to identifyn ew inhibitors and to characterize their potencya te ach GATs ubtypea re indispensable.…”

“…Binding assays that can be used to determine affinities of inhibitors are-in the case of the GABA transporters-only applicable for GAT1, [12] as only for this GATsubtypea re markers with sufficienta ffinity-a prerequisite fort his approach-available,w hereas this is not the case g-Aminobutyric acid (GABA)t ransporters (GATs)a re promising drug targets for various diseases associated with imbalances in GABAergic neurotransmission. [14] MS Transport Assays could be shown to be an excellent substitute for common radiometric uptake assays employing mammalianc ells in a9 6-well plate format. By now,t he technique by far dominating is based on radiolabeled GABA.…”

“…By now,t he technique by far dominating is based on radiolabeled GABA. [14] Third, the COS-7c ells employed in this approach allow,a saresult of their high surface adherence, the use of an automated 96-well plate washer system for all dispensing and washing steps, without any precoating of cell culture plates as is necessaryf or the corresponding [ 3 H]GABAassays described so far. In the present study,w e appliedt his approach to all four human GATsubtypes and de-termined the K M valuesf or GAT-mediated transport of ( 2 H 6 )GABA at each subtype.…”