Received for publication February 14, 2005.
Revised July 5, 2005.
Accepted for publication July 13, 2005.

Characterization of Novel Aryl-Ether, Biaryl and Fluorene Aspartic Acid and Diaminopropionic Acid Analogs as Potent Inhibitors of the High-Affinity Glutamate Transporter EAAT2

Abstract

In this study, we describe the pharmacological characterization of novel aryl-ether, biaryl and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT2, the predominant glutamate transporter in forebrain regions. The rank order of potency determined for inhibition of human EAAT2 was WAY-213613 (N⁴-[4-(2-bromo-4,5-difluorophenoxy)phenyl]-L-asparagine) (IC₅₀, 85 ± 5 nM) > WAY-213394 (N⁴-(2'-methyl-1,1'-biphenyl-4-yl)-L-asparagine) (145 ± 22 nM) = WAY-212922 (N⁴-[7-(trifluoromethyl)-9H-fluoren-2-yl]-L-asparagine) (157 ± 11 nM) = WAY-211686 (3-{[(4'-chloro-2-methyl-1,1'-biphenyl-4-yl)carbonyl]amino}-L-alanine) (190 ± 10 nM). WAY-213613 was the most selective of the compounds examined with IC₅₀s for inhibition of EAAT1 and EAAT3 of 5 and 3.8 µM, respectively, corresponding to a 59- and 45-fold selectivity toward EAAT2. An identical rank order of potency (WAY-213613 (35 ± 7 nM) > WAY-213394 (92 ± 13 nM) = WAY-212922 (95 ± 8 nM) = WAY-211686 (101 ± 20 nM)) was observed for the inhibition of glutamate uptake in rat cortical synaptosomes, consistent with the predominant contribution of EAAT2 to this activity. Kinetic studies with each of the compounds in synaptosomes revealed a competitive mechanism of inhibition. All compounds were determined to be non-substrates by evaluating both the stimulation of currents in EAAT2-injected oocytes and the heteroexchange of D-[³H]aspartate from cortical synaptosomes. WAY-213613 represents the most potent and selective inhibitor of EAAT2 identified to date. Taken in combination with its selectivity over ionotropic and metabotropic glutamate receptors this compound represents a potential tool for the further elucidation of EAAT2 function.

Key words: Glutamate, Amino Acid

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