title: Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters.
abstract: Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-beta-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their respective benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC(50) values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC(50) values for EAAT1-3 were 33, 6.2, and 15 microM, respectively). Electrophysiological analyses revealed that TBOA analogs block the transport-associated currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that observed in the uptake assay. However, the kinetics of TFBTBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters.
authors.affiliation: Suntory Institute for Bioorganic Research, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan. shimamot@sunbor.or.jp
authors.forename: Keiko
authors.initials: K
authors.lastname: Shimamoto
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Sakai
authors.forename: Kiyo
authors.initials: K
authors.lastname: Takaoka
authors.forename: Noboru
authors.initials: N
authors.lastname: Yumoto
authors.forename: Terumi
authors.initials: T
authors.lastname: Nakajima
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shigeri
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000037:antagonists & inhibitors*; D026905:Amino Acid Transport Systems; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000494:pharmacology*; D019556:COS Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D027341:Excitatory Amino Acid Transporter 1 / Q000037:antagonists & inhibitors; D027342:Excitatory Amino Acid Transporter 2 / Q000037:antagonists & inhibitors; D050580:Excitatory Amino Acid Transporter 3; D050582:Excitatory Amino Acid Transporter 5; D050491:Glutamate Plasma Membrane Transport Proteins; D008297:Male; D051379:Mice; D010786:Photoreceptor Cells; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D027981:Symporters / Q000037:antagonists & inhibitors
chemical_list: D027322:Amino Acid Transport System X-AG; D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050582:Excitatory Amino Acid Transporter 5; D050491:Glutamate Plasma Membrane Transport Proteins; C493399:Slc1a1 protein, mouse; C493398:Slc1a1 protein, rat; C493396:Slc1a2 protein, mouse; C493395:Slc1a2 protein, rat; C493393:Slc1a3 protein, mouse; C493392:Slc1a3 protein, rat; C493387:Slc1a7 protein, mouse; C484554:Slc1a7 protein, rat; D027981:Symporters; C120673:benzyloxyaspartate; D001224:Aspartic Acid