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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M-channels (M-current), encoded by KCNQ2/3 K(+) channel genes, have emerged as novel drug targets for a number of neurological disorders. The lack of direct high throughput assays combined with the low throughput of conventional electrophysiology (EP) has impeded rapid screening and evaluation of K(+)-channel modulators. Development of a sensitive and efficient assay for the direct measurement of M-current activity is critical for identifying novel M-channel modulators and subsequent investigation of their therapeutic potential. Using a stable CHO cell line expressing rat KCNQ2/3 K(+) channels confirmed by EP, we have developed and validated a nonradioactive rubidium (Rb(+)) efflux assay in a 96-well plate format. The Rb(+) efflux assay directly measures the activity of functional channels by atomic absorption spectroscopy using the automated Ion Channel Reader (ICR) 8000. The stimulated Rb(+) efflux from KCNQ2/3-expressing cells was blocked by the channel blockers XE991 and linopirdine with IC(50) values of 0.15 microM and 1.3 microM, respectively. Twelve compounds identified as KCNQ2/3 openers were further assessed in this assay, and their EC(50) values were compared with those obtained with EP. A higher positive correlation coefficient between these two assays (r = 0.60) was observed than that between FlexStation membrane potential and EP assays (r = 0.23). To simplify the assay and increase the throughput, we demonstrate that EC(50) values obtained by measuring Rb(+) levels in the supernatant are as robust and consistent as those obtained from the ratio of Rb(+) in supernatant/lysate. By measuring the supernatant only, the throughput of ICR8000 in an eight-point titration is estimated to be 40 compounds per day, which is suitable for a secondary confirmation assay.

PTHs) as potent lead structure. Here we report the comparison of PTHs to selected curcumin derivatives and approved tetracyclines for their tau antiaggregatory properties and their affinity to fibrillar beta-amyloid in several assays and cellular models. The most potent fluorescent compounds were evaluated for beta-amyloid/tau PHF selectivity by fluorescence microscopy of human AD probes. Methods: Lead Optimization of PTHs. Several diverse PTH analogues were obtained by variation of the substituents R1-R4 to explore a preliminary SAR. Fluorescent substituents were incorporated without significantly compromising in vitro activity. Results: Tau-biased ligands with enhanced affinity to tau paired helical filaments over beta-amyloid were identified by a panel of protein aggregation assays and fluorescence microscopy of human AD preparations. Conclusions: Phenylthiazolylhydrazides are potent inhibitors of tau-aggregation. The fluorescence microscopy of human AD preparation suggests a binding to growth-relevant sites of tau aggregates.Background: Tau is the major microtubule-associated protein in neurones, which functions in the formation and maintenance of axons by influencing microtubule organization. Increased tau phosphorylation is also a salient feature of Alzheimer's disease (AD) and tauopathies such as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). In these neurodegenerative disorders, normally soluble tau is present as paired-helical filaments (PHFs), which in turn aggregate to form neurofibrillary tangles (NFTs). p53 and p73 are tumor suppressor proteins, which induces cell cycle arrest or apoptosis. Methods: Here we explore the effects of p53 family members (p53 and p73␣) on tau phosphorylation in a mammalian cell culture model by western blotting. We also investigate the expression levels of p53 and p73 in human post-mortem samples from AD and control cases. Results: Normally, p53 and p73 are maintained at low levels inside cells. However, we and others have reported an increase in p53 immunoreactivity, but not p73 immunoreactivity in AD. Furthermore, p53 -/mice display a reduction in tau phosphorylation. Consistent with this, we have demonstrated that p53 induces tau (human 2N4R) phosphorylation at the AT8/tau-1 epitope in HEK293a cells and that p73␣, a related p53 homologue, also induces tau (human 2N4R) phosphorylation at the AT-8/tau-1 epitope as well as the PHF-1 epitope in HEK293a cells. In contrast, we find that the N-terminally truncated transcriptionally inactive or ⌬N isoform of p73␣ fails to induce tau (human 2N4R) phosphorylation. Conclusions: These results suggest that p53 and indeed p73 induce tau phosphorylation in a cell culture model of neurodegeneration and that the effects of p73 and perhaps p53 on tau are dependent on the transcriptional activity of these proteins. Interestingly, p53, but not p73 was upregulated in AD. However, p73 has been reported to be aberrantly expressed in the nucleus of hippocampal neurons in AD -p73 expression co-localizing with NFTs....

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