L-Glutamate acts as the principal excitatory neurotransmitter in the mammalian central nervous systems. The extracellular glutamate concentrations are tightly controlled by a transport system that limits the activation of receptors during signaling and that maintains below excitotoxic level. Non-transportable blockers have been required as indispensable tools for the investigation of transporters. We have been developing novel blockers by two approaches. One is the screening from conformationally constrained glutamate analogs and the other is the synthesis of THA (threo-β-hydroxyaspartates) derivatives. We found CCG-III and CMP-III (MPDC) are substrates while CPG-II is a blocker for EAAT1. Superimposition studies using CMP-III (MPDC) as a template provided a putative pharmacophre, in which the distal carbaxylate is close to α-amino group. Although the functional groups of CPG-II were able to fit with the proposed model, the bulky five-membered ring moiety seemed to overhang the pharmacophore. We guess such a bulky substituent plays an important role in blocker activity. On the other hand, both the L- and D-THA are excellent substrates of EAAT1-4 and blockers for EAAT5. We considered that introduction of a bulky substituent on the β-hydroxy group would be useful to develop blockers. Among the synthesized THA derivatives, L-TBOA (L-threo-β-benzyloxyaspartate) is, so far, the most potent blocker for all EAAT subtypes (EAAT1-5). The functional groups of the folded form of glutamate are well superimposed with those of the extended form of aspartate. If L-TBOA takes the extended conformation, its benzyl group was well overlapped with the bulky substituents of dihydrokainate and CPG-II. Next, we synthesized L-TBOA analogs with functional groups suitable for linking to an affinity column such as amino and biotinyl groups. Although para-substituted analogs lost the activity, meta-substituted analogs inhibited [^<14>C]Glu uptake with almost comparable potency as TBOA.
