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P2045
Validation of a Functional Screening Assay for Glycine Transporter 1 Inhibitors Using Cryopreserved Cells
Presenter Beth Ann McKenna, Cephalon, Inc., USA
Additional Authors: Jeffrey Thomas, Chrysanthe M. Spais, Sheryl L. Meyer, Bruce E. Jones, Jacquelyn Lyons, Beverly P. Holskin, Lisa Saville, Mark Ator, Emir Duzic, and Karla Kopec
Glycine transporter (GlyT1) function is typically measured by [3H] glycine uptake using lysis methods or SPA, both of which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of 384-well format, standard laboratory automation and stocks of cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated in advance of screening. Selection of the plate type was key in adapting the assay to 384-well format.  Plates from different manufacturers were compared for quenching by counting [3H] glycine directly spotted onto the plates.  Optical bottom 384-well Nunc plates gave superior signal with little quench as compared to other plates.  Development of the assay in these plates yielded a good signal to background ratio (25-fold) while using a low concentration (3 µCi/mL) of [3H] glycine. CHO-K1/hGlyT1a cells were evaluated for the time course of glycine uptake, Km, Vmax, DMSO sensitivity, Z’ analysis and IC50 value determination with reference GlyT1 inhibitors.  Both cultured and cryopreserved cells were successfully validated; however, the use of cryopreserved cells eliminated the potential of cell passage variability while serving as a source of readily available cells.  Pharmacology consistent with literature values was observed across a range of inhibitor potencies, with IC50 values that ranged from 50 ?M (sarcosine) to 1.6 nM (NFPS).  The Z’ value (0.6) indicated that the assay has acceptable variability and good dynamic range.  The Beckman Coulter SAMI workstation allowed for throughput of 60 x 384-well plates per day.  This assay was more economical than SPA protocols due to reduced radiolabel concentration and plate cost.  These results demonstrated that the assay can be used for the identification of small-molecule inhibitors of GlyT1.