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Journal of Biomolecular Screening
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Validation of a Fluorescent Imaging Plate Reader Membrane Potential Assay for High-Throughput Screening of Glycine Transporter Modulators

Elfrida R. Benjamin

Purdue Pharma Discovery Research, Cranbury, New Jersey

Joanne Skelton

Veridex, Warren, New Jersey

Denise Hanway

Shakira Olanrewaju

Farhana Pruthi

Victor I. Ilyin

Purdue Pharma Discovery Research, Cranbury, New Jersey

Daniel Lavery

Chromocell Corporation, North Brunswick, New Jersey

Sam F. Victory

TransTech Pharma, High Point, North Carolina

Kenneth J. Valenzano

Purdue Pharma Discovery Research, Cranbury, New Jersey

A fluorescent imaging plate reader (FLIPR) membrane potential (Vm) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT2) in a stable rGlyT2-HEK cell line. Data show that glycine activation of rGlyT2 consistently results in a concentration-dependent Vm response on the FLIPR that is blocked by the potent and selective GlyT2 antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [3H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT2 physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT2 inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based Vm assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT2.

Key Words: glycine • transporter • membrane potential • fluorescent imaging plate reader • electrogenic

Journal of Biomolecular Screening, Vol. 10, No. 4, 365-373 (2005)
DOI: 10.1177/1087057104274090
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[Abstract] [PDF]