Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators.
A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) 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 GlyT(2) 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 V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).
Benjamin
Elfrida R
ER
Purdue Pharma Discovery Research, Cranbury, New Jersey 08512, USA. elfrida.benjamin@pharma.com
Skelton
Joanne
J
Hanway
Denise
D
Olanrewaju
Shakira
S
Pruthi
Farhana
F
Ilyin
Victor I
VI
Lavery
Daniel
D
Victory
Sam F
SF
Valenzano
Kenneth J
KJ
D026921:Amino Acid Transport Systems, Neutral / Q000187:drug effects* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000032:analysis* / Q000494:pharmacology; D002460:Cell Line; D017931:DNA Primers; D050487:Glycine Plasma Membrane Transport Proteins; D006801:Humans; D008564:Membrane Potentials; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
C433813:4-benzyloxy-3,5-dimethoxy-N-((1-dimethylaminocyclopentyl)methyl)benzamide; D026921:Amino Acid Transport Systems, Neutral; D001549:Benzamides; D017931:DNA Primers; D050487:Glycine Plasma Membrane Transport Proteins