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 the inhibition of human EAAT2 was(WAY-211686) (IC 50 ϭ 190 Ϯ 10 nM). WAY-213613 was the most selective of the compounds examined, with IC 50 values for inhibition of EAAT1 and EAAT3 of 5 and 3.8 M, respectively, corresponding to a 59-and 45-fold selectivity toward EAAT2. An identical rank order of potency [WAY-213613 (35 Ϯ 7 nM) Ͼ WAY-213394 (92 Ϯ 13 nM) ϭ WAY-212922 (95 Ϯ 8 nM) ϭ WAY-211686 (101 Ϯ 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 nonsubstrates by evaluating both the stimulation of currents in EAAT2-injected oocytes and the heteroexchange of D-[ 3 H]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.Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Glutamate transmission is mediated via interaction with the ligand-gated ion channel receptors, termed the ionotropic receptors, and the seven-transmembrane domain G-protein-coupled receptors, termed metabotropic glutamate receptors (Barnard, 1997;Schoepp et al., 1999). Activation of these receptors is responsible for the physiological actions of glutamate, whereas paradoxically, overstimulation of the ionotropic receptors contributes to the excitotoxic actions attributed to glutamate. Therefore, synaptic glutamate levels must be tightly regulated to maintain the integrity of synaptic transmission and to limit or prevent the pathophysiological activity of this excitatory neurotransmitter.A family of high-affinity Na ϩ -dependent glutamate transporters expressed in the plasma membranes of both neurons and astroglia is responsible for the clearance of extracellular glutamate by mediating the cellular uptake of glutamate in a Article, publication date, and citation information can be found at

Characterization of the Tritium-Labeled Analog of L-threo-β-Benzyloxyaspartate Binding to Glutamate Transporters

“…One of the folded-type glutamate analogs, L-CCG-III (Nakamura et al, 1993) 3 H]ETB-TBOA binding to EAAT2 only at a concentration of 1 mM, although it was less potent in our preparations than in other reports (Shimamoto et al, 1998 (Aprico et al, 2001). Recently EAAT2-preferring blockers have been reported (Dunlop et al, 2003(Dunlop et al, , 2005Bunch et al, 2006), but their effects on EAAT4 and EAAT5 are unknown. Characterization of these compounds in the binding assay developed here may further elucidate their specificity.…”

Neuroprotective Effects of the Novel Glutamate Transporter Inhibitor (–)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic Acid, Which Preferentially Inhibits Reverse Transport (Glutamate Release) Compared with Glutamate Reuptake

“…The effects of (Ϫ) These data question the hypothesis that synaptosomal EAATs correspond only to EAAT2/GLT1 transporters (Dunlop et al, 1999(Dunlop et al, , 2005Tan et al, 1999) and identify our novel compounds as interesting pharmacological tools for further investigating the nature of presynaptic transporters (Danbolt, 2001). …”

Fragmental Modeling of Human Glutamate Transporter EAAT1 and Analysis of its Binding Modes by Docking and Pharmacophore Mapping

“…As substrate inhibitors the set includes: 4-methylglutamate isomers (4)(5), [42] l-threo-b-hydroxyaspartate (THA, 6), [6] l-threo-4-hydroxyglutamate (7), [42] l-sulphate-O-serine (l-SOS, 8), [9] three cyclopropyl derivatives (namely, l-CCG-III, 9, d-CCG-I, 10, and l-CCG-IV, 11), [44] four pyrrolidine dicarboxylates (namely, t-2,4 PDC, 12, 2,4MPDC, 13, l-3,4MPDC, 14, 4-Methyl-2,4 PDC, 15), [42] and cis-aminocyclobutyl dicarboxylate (cis-ABCD, 16). [9] Figure 2 includes the nontransported blockers which can be divided in three main groups: b-hydroxyaspartate derivatives (17)(18)(19)(20)(21)(22)(23)(24)(25)(26), [45,46] aspartic acid amides (27)(28)(29)(30), [47] and diaminopropionic acid analogues (DAPAs, 31-32). [48] The biological activities were measured as IC 50 (mm) by the inhibition of l-glutamate uptake in cells transiently expressing EAAT1 (as compiled in Table 2).…”