DL-threo-b-Benzyloxyaspartate, A Potent Blocker of Excitatory Amino Acid Transporters

DL-threo-b-Benzyloxyaspartate (DL-TBOA), a novel derivative of DL-threo-b-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki 5 42 mM) with almost the same potency as DL-threo-b-hydroxyaspartate (Ki 5 58 mM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki 5 5.7 mM) was much more potent than that of dihydrokainate (Ki 5 79 mM), which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DLTBOA without a significant change in the maximum current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 mM and 116 nM, respectively. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiological roles of transporters. Glutamate acts as an excitatory neurotransmitter in the mammalian central nervous system as well as a potent neurotoxin. The termination of neurotransmission is mediated by sodium-dependent high affinity glutamate/aspartate transporters. Glutamate transporters also play an important role in maintaining the extracellular glutamate concentration below neurotoxic levels and therefore contribute to the prevention of neuronal damage from excessive activation of glutamate receptors (Nicholls and Attwell, 1990; Rothstein et al., 1996; Gegelashvili and Schousboe, 1997; Kanai, 1997; Tanaka et al., 1997). Some serious neuronal diseases, such as epilepsy, amyotrophic lateral sclerosis, Alzheimer’s diseases, and cellular damage from a stroke, may be linked to the failure of transporters. Five subtypes of EAATs (EAAT1–5) have been cloned from mammalian tissues (Kanai and Hediger, 1992; Pines et al., 1992; Storck et al., 1992; Tanaka, 1993; Shashidharan et al., 1993, 1994; Arriza et al., 1994, 1997; Kawakami et al., 1994; Manfras et al., 1994; Fairman et al., 1995; Inoue et al., 1995). These transporters couple the electrochemical gradient of three cotransported sodium ions and one countertransported potassium ion to that of glutamate (Zerangue and Kavanaugh, 1996). A proton also is cotransported. In addition, a substrate-dependent chloride conductance provides a potential mechanism for dampening cell excitability (Fairman et al., 1995). The transporter subtypes notably differ in the magnitude of this chloride flux relative to the flux of glutamate (Fairman et al., 1995; Wadiche et al., 1995; Arriza et al., 1997). ABBREVIATIONS: EAAT, excitatory amino acid transporter; EAAC, excitatory amino acid carrier; THA, threo-b-hydroxyaspartate; TBzOAsp, threo-b-benzoyloxyaspartate; TBOA, threo-b-benzyloxyaspartate; t-2,4-PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; L-CCG-III, (2S,19S,29R)2-(2-carboxycyclopropyl)glycine; L-CCG-IV, (2S,19R,29S)-2-(2-carboxycyclopropyl)glycine; 2S4R4MG, (2S,4R)-4-methylglutamate; PCR, polymerase chain reaction; DHKA, dihydrokainate; KA, kainate; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; mGluR; metabotropic glutamate receptor. 0026-895X/98/020195-07$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:195–201 (1998). 195 at A PE T Jornals on M ay 5, 2017 m oharm .aspeurnals.org D ow nladed from Inhibitors of glutamate transporters are essential for elucidation of the intrinsic properties and physiological roles of transporters. A number of pharmacological agents have been shown to inhibit glutamate transport (Ferkany and Coyle, 1986; Bridges et al., 1991, 1993, 1994; Nakamura et al., 1993; Robinson et al., 1993; Arriza et al., 1994; Yamashita et al., 1995; Vandenberg et al., 1997). Most of them indeed act as competitive substrates, inducing a transport current and a substrate-dependent chloride flux. Blocker-type inhibitors, which are not transportable, inhibit the transport of glutamate while letting transporters to be electrically silent. Moreover, they also suppress voltage-dependent pre-steady state charge movements, allowing kinetic information on the transporters to be obtained (Wadiche et al., 1995). KA and DHKA block EAAT2 in the low-micromolar range, whereas '10 mM is required to block transport by EAAT1, EAAT3, and EAAT4 (Arriza et al., 1994; Fairman et al., 1995). Recently, 2S4R4MG was shown to be a very potent blocker for EAAT2 and a substrate for EAAT1 (Vandenberg et al., 1997). However, these compounds also activate ionotropic glutamate receptors (Gu et al., 1995). As such, they can be valuable pharmacological tools for the study of EAAT2 in heterologous expression systems but cannot be used to determine the physiological role of this transporter in complex preparations. THA and t-2,4-PDC were found to be blockers for EAAT5 (Arriza et al., 1997) and substrates for EAAT1–4 (Arriza et al., 1994; Fairman et al., 1995). THA also was demonstrated to be a ligand for N-methyl-D-aspartate receptors (Jane et al., 1994); therefore, pharmacological agents that are able to block EAATs without being transported and without affecting glutamate receptors are needed. Our approach to the development of blockers of EAATs was the synthesis of derivatives of THA. We recently demonstrated that DL-TBzOAsp is a competitive blocker for bovine EAAT1 (glutamate/aspartate transporter type) (Lebrun et al., 1997). However, because DL-TBzOAsp has an ester bond, it is unstable in aqueous solution (ester cleavage or acyl migration); therefore, we synthesized a more stable ethertype derivative, DL-TBOA. DL-TBOA showed potent inhibitory activity on the [C]glutamate uptake in COS-1 cells expressing human EAAT1 or EAAT2. Moreover, it proved to be highly selective for EAATs versus the glutamate receptors. Electrophysiological analysis demonstrated that DLTBOA is a competitive blocker for human EAAT1 and EAAT2; it is indeed the most potent blocker for both EAAT1 and EAAT2 described to date. Experimental Procedures Materials. L-Glutamate was obtained from Nacalai Tesque (Kyoto, Japan). THA was from Sigma Chemical (St. Louis, MO). t-2,4-PDC, DHKA, and 2S4R4MG were from Tocris Cookson (Bristol, UK). L-[C]Glutamate, [H]CGS 19755 (cis-4-phosphono-methyl-2piperidine carboxylic acid), [H]KA, and [H]AMPA were from DuPont-New England Nuclear (Botson, MA). L-CCG-III and L-CCG-IV were synthesized as described previously (Shimamoto et al., 1991). DL-TBOA was synthesized in the same manner as DL-TBzOAsp, except for the use of benzyl bromide instead of acyl chloride (Lebrun et al., 1997). The structure and purity (.95%) of the compound were confirmed with 400-MHz NMR. Stock solutions (100 mM) of the inhibitors, except for DL-TBzOAsp, were made in 0.1 M NaOH and stored at 220°. Stock solutions for DL-TBzOAsp were made in 50% dimethylsulfoxide without NaOH. DL-TBOA was stable for $1 week at room temperature; no noticeable decomposition was observed by