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Successful Identification of Glycine Transporter Inhibitors Using an Adaptation of a Functional Cell-Based Assay

^* To whom correspondence should be addressed. E-mail: kkopec{at}cephalon.com.

	Abstract

Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), 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 the 384well format, standard laboratory automation, and cryopreserved CHOK1 cells stably overexpressing human GlyT1a transporter (CHOK1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K_m, V_max, Z' factor analysis, and IC₅₀ value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 mM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 mM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC₅₀ values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this costeffective method. (Journal of Biomolecular Screening XXXX:xx-xx)

First published on October 19, 2009
Journal of Biomolecular Screening 2009, doi:10.1177/1087057109347779


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