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Excitatory amino acid transporters (EAATs) play a pivotal role in maintaining glutamate homeostasis in the mammalian central nervous system, with the EAAT-2 subtype thought to be responsible for the bulk of the glutamate uptake in forebrain regions. A complete elucidation of the functional role of EAAT-2 has been hampered by the lack of potent and selective pharmacological tools. In this study, we describe the synthesis and biological activities of novel aryl-ether, biaryl-, and fluorene-aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT-2. Compound (16) represents one of the most potent (IC50=85+/-5 nM) and selective inhibitors of EAAT-2 identified to date.

The pharmacological profile of a novel glutamate transport inhibitor, WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY-855 inhibited glutamate uptake mediated by all three subtypes in a concentration-dependent manner, with preferential inhibition of the CNS-predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 μM, respectively, while EAAT1 activity was inhibited by 50% at 100 μM (IC50 values determined in oocytes were 1.3 μM (EAAT2), 52.5 μM (EAAT3) and 125.9 μM (EAAT1)). Application of WAY-855 to EAAT-expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]D-aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY-855 inhibited D-aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY-855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY-855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes. British Journal of Pharmacology (2003) 140, 839–846. doi:10.1038/sj.bjp.0705509

A dihydroquinolinone moiety was found to be a potent serotonin reuptake inhibitor pharmacophore when combined with certain amines. This fragment was coupled with selected D2 ligands to prepare a series of dual acting compounds with attractive in vitro profiles as dopamine D2 partial agonists and serotonin reuptake inhibitors. Structure–activity studies revealed that the linker plays a key role in contributing to D2 affinity, function, and SRI activity.

A 5-fluoro-tetrahydrocarbazole serotonin reuptake inhibitor (SRI) building block was combined with a variety of linkers and dopamine D2 receptor ligands in an attempt to identify potent D2 partial agonist/SRI molecules for treatment of schizophrenia. This approach has the potential to treat a broader range of symptoms compared to existing therapies. Selected compounds in this series demonstrate high affinity for both targets and D2 partial agonism in cell-based and in vivo assays.

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of 3 H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT 6 ) receptors expressed in Chinese hamster ovary (CHO)–Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT 6 agonists and antagonists using intact CHO–Dukx/5-HT 6 cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT 6 membranes. K i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 ( K i = 1.9 nM) > methiothepin ( K i = 6.2 nM) > mianserin ( K i = 74.3 nM) > 5-methoxytryptamine (5-MeOT, K i = 111 nM) > 5-HT ( K i = 150 nM) > ritanserin ( K i = 207 nM) > 5-carboxamidotryptamine (5-CT, K i = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z ′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.

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 N(4)-[4-(2-bromo-4,5-difluorophenoxy)phenyl]-L-asparagine (WAY-213613) (IC(50) = 85 +/- 5 nM)N(4)-(2'-methyl-1,1'-biphenyl-4-yl)-L-asparagine (WAY-213394) (IC(50) = 145 +/- 22 nM) = N(4)-[7-(trifluoromethyl)-9H-fluoren-2-yl]-L-asparagine (WAY-212922) (IC(50) = 157 +/- 11 nM) = 3-{[(4'-chloro-2-methyl-1,1'-biphenyl-4-yl)carbonyl]amino}-L-alanine (WAY-211686) (IC(50) = 190 +/- 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 microM, respectively, corresponding to a 59- and 45-fold selectivity toward EAAT2. An identical rank order of potency [WAY-213613 (35 +/- 7 nM)WAY-213394 (92 +/- 13 nM) = WAY-212922 (95 +/- 8 nM) = WAY-211686 (101 +/- 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.