A Rational Approach to the Design of Selective Substrates and Potent Nontransportable Inhibitors of the Excitatory Amino Acid Transporter EAAC1 (EAAT3). New Glutamate and Aspartate Analogues as Potential Neuroprotective Agents

Campiani, Giuseppe; Angelis, Meri De; Armaroli, Silvia; Fattorusso, Caterina; Catalanotti, Bruno; Ramunno, Anna; Nacci, Vito; Novellino, Ettore; Grewer, Christof; Ionescu, Dumitru; Rauen, Thomas; Griffiths, Roger; Sinclair, C. D.; Fumagalli, Elena; Mennini, Tiziana

doi:10.1021/jm015509z

Cited by 55 publications

(42 citation statements)

References 16 publications

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“…A major distinction between L-TBOA and WAY-213613 is the conformity of the former to all features with the exception of the hydrophobic center 4.11, whereas WAY-213613 fits all features with the exception of the hydrogen bond acceptor. We find that the newly described inhibitors presented in the current study lack a distal acidic group identified by others (Campiani et al, 2001(Campiani et al, , 2003 as an important pharmacophoric site corresponding to the hydrogen bond acceptor feature in our model. Moreover, the compounds reported in the current study share in common the distal hydrophobic center 4.11.…”

Section: Resultssupporting

confidence: 49%

“…It is important to note that in the current model, the folded form of the aspartate framework in TBOA is wellsuperimposed with the folded form of glutamate (Fig. 9), whereas others have proposed the extended form for the aspartate framework in their model (Koch et al, 1999;Campiani et al, 2001Campiani et al, , 2003. Each of these approaches has used different modeling scenarios, and although we have predicted the folded form of the aspartate framework, the other features of the molecule present a very similar configuration in all modeling studies.…”

Section: Resultsmentioning

confidence: 69%

“…Each of these approaches has used different modeling scenarios, and although we have predicted the folded form of the aspartate framework, the other features of the molecule present a very similar configuration in all modeling studies. Moreover, close inspection of the models predicted by Koch et al (1999) and Campiani et al (2001Campiani et al ( , 2003 suggests that these do not predict exactly the same configuration of the aspartate framework. Our model provides a plausible alternative hypothesis for EAAT binding and should be considered in the rational design of new compounds.…”

Section: Resultsmentioning

confidence: 96%

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

Dunlop

McIlvain²,

Carrick³

et al. 2005

Mol Pharmacol

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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

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Section: Resultssupporting

confidence: 49%

Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 96%

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

Dunlop

McIlvain²,

Carrick³

et al. 2005

Mol Pharmacol

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“…6A, left panel). This outward current was caused by inhibition of the sustained leak anion conductance of EAAC1 in the presence of intracellular SCN − (19,31). However, TBOA did not inhibit the inward anion current generated by 500 μM alanine (Fig.…”

Section: Properties Of Mixed Eaac1 Wt -Eaac1 R446qmentioning

confidence: 92%

Individual Subunits of the Glutamate Transporter EAAC1 Homotrimer Function Independently of Each Other

et al. 2005

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Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on transporter function. To do so, we coexpressed EAAC1 WT with the mutant transporter EAAC1 R446Q , which transports glutamine, but not glutamate. Application of 50 μM glutamate or 50 μM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 pA and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore, but could be explained by anion permeation through independently-functioning subunits. To further test the subunit independence, we coexpressed EAAC1 WT and EAAC1 H295K , a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1 WT , and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1 H295K and EAAC1 WT , respectively. Finally, we coexpressed EAAC1 WT with the pH-independent mutant transporter EAAC1 E373Q , showing two independent populations of transporters, one being pH dependent, the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits, but that the individual subunits in the trimer function independently of each other.Plasma membrane glutamate transporters actively remove glutamate from the synaptic cleft after excitatory neurotransmission is complete. Uptake into the cells surrounding the synapse against a glutamate concentration gradient is achieved by these transporters by coupling transmembrane glutamate movement to the cotransport of three sodium ions and one proton, and the countertransport of one potassium ion (1,2). In addition to the movement of ions across the membrane being directly coupled to glutamate transport, glutamate transporters also catalyze uncoupled transmembrane flux of anions (3). This anion conductance is thought to be an integral property of the transporters and is not mediated by indirect coupling of transport to a secondary anion channel (3-5).Address correspondence to: Christof Grewer, PhD, Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136; Phone: (305) 243-1021; Fax: (305) The mammalian glutamate transporters belong to a large family of membrane transport proteins that comprise also neutral amino acid transporters, such as the alanine serine cysteine transporters (ASCTs (6,7)), and dicarboxylate transporters (8,9). A large number of biochemical data from both mammalian (10,11) and bacterial glutamate transporters (12,13), as well as recent crystallographic evidence f...

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“…As pointed out earlier, there has been a wealth of information on the substrate specificity and structural requirements of GluT 19,26,34,[92][93][94][95][96][97][98][99][100][101]103,105,[107][108][109] even in relation to individual EAAT's 110,112,114,[179][180][181][182][183] yet this structure-activity data has not so far been satisfactorily matched with the protein architecture of the putative substrate-binding regions. Moreover, some of the EAAT's may form multimeric complexes 184) and most of them also act as chloride channels 185) ; for a review see ref.…”

Section: Unresolved Problems and Future Direc-tionsmentioning

confidence: 99%

Molecular Pharmacology of the Na+-Dependent Transport of Acidic Amino Acids in the Mammalian Central Nervous System.

Balcar

2002

Biological & Pharmaceutical Bulletin

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The crucial discovery of the excitatory properties of acidic amino acids [2][3][4][5] lead only gradually and with considerable delay, to the formulation of the hypothesis of the synaptic role of L-glutamate in the mammalian central nervous system (CNS) 6-9) (for reviews of early history of amino acid research, see [10][11][12][13] ). It may never be possible to identify any single experimental study after which L-glutamate became unequivocally accepted as the most important excitatory synaptic transmitter in the brain and spinal cord, but, it is certain that the discovery of the "high affinity uptake" accumulating L-glutamate into "synaptosomes" 14,15) (for reviews see 13,16,17) ) was an important step in the development of the concept of the glutamatergic neurotransmission in the CNS. As a putative mechanism for the necessary removal and inactivation of extracellular L-glutamate it provided a target for pharmacological studies and helped to establish a neurochemical link between L-glutamate and the mechanisms of synaptic transmission. 13,18,19) Most of the latter experiments were carried out in vitro using tissue "mini-slices" prepared from the rat cerebral cortex or cat spinal cord (for details of the methodology see 20,21) ). Rather than disrupting the nervous tissue by homogenization, the technique was using what might be called "neurochemical dissection". In more specific terms, it examined a large number of compounds, mostly glutamate and aspartate analogues but also a range of drugs, for possible effects on L-glutamate transport (GluT). Such approach had a potential to yield valuable information on the molecular properties of the substrate-recognition site on GluT,19) but, also, by testing neuroactive compounds with known sites of actions in brain, it helped to formulate hypotheses defining the most probable functions of the GluT in the mechanisms of the excitatory synaptic transmission.The pharmacology and biochemistry of GluT was later extensively investigated in cultured glial cells [22][23][24] (for a review see 25) ) but it was the data obtained in anaesthetised animals, using compounds (D-and L-threo-3-hydroxyaspartate, DL-t3OHA) identified earlier as powerful specific inhibitors of [ 3 H]L-glutamate uptake in brain slices, 26) that provided the first convincing evidence that GluT could influence the excitatory actions of L-glutamate in vivo, at least in the cat spinal cord.27) Furthermore, if the inhibition of GluT in vivo caused an increase in the extracellular concentrations of L-glutamate then, given the neurotoxic nature of excessive glutamatergic excitation, 28,29) similar inhibition of GluT applied to functioning synapses should, under suitable experimental conditions, result in the appearance of neurodegeneration. Such experiment was indeed performed and it was shown that an injection of DL-t3OHA into the rat striatum could cause the death of brain cells in vivo. 30) HETEROGENEITY OF GLUTAMATE TRANSPORTDuring the 1980's and early 1990's, it became apparent that, in order to account for th...

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A Rational Approach to the Design of Selective Substrates and Potent Nontransportable Inhibitors of the Excitatory Amino Acid Transporter EAAC1 (EAAT3). New Glutamate and Aspartate Analogues as Potential Neuroprotective Agents

Cited by 55 publications

References 16 publications

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

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

Individual Subunits of the Glutamate Transporter EAAC1 Homotrimer Function Independently of Each Other

Molecular Pharmacology of the Na+-Dependent Transport of Acidic Amino Acids in the Mammalian Central Nervous System.

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