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1.
Neurochem Res ; 46(3): 494-503, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33398639

ABSTRACT

The vesicular glutamate transporter (VGLUT) facilitates the uptake of glutamate (Glu) into neuronal vesicles. VGLUT has not yet been fully characterized pharmacologically but a body of work established that certain azo-dyes bearing two Glu isosteres via a linker were potent inhibitors. However, the distance between the isostere groups that convey potent inhibition has not been delineated. This report describes the synthesis and pharmacologic assessment of Congo Red analogs that contain one or two glutamate isostere or mimic groups; the latter varied in the interatomic distance and spacer properties to probe strategic binding interactions within VGLUT. The more potent inhibitors had two glutamate isosteres symmetrically linked to a central aromatic group and showed IC50 values ~ 0.3-2.0 µM at VGLUT. These compounds contained phenyl, diphenyl ether (PhOPh) or 1,2-diphenylethane as the linker connecting 4-aminonaphthalene sulfonic acid groups. A homology model for VGLUT2 using D-galactonate transporter (DgoT) to dock and identify R88, H199 and F219 as key protein interactions with Trypan Blue, Congo Red and selected potent analogs prepared and tested in this report.


Subject(s)
Congo Red/analogs & derivatives , Congo Red/metabolism , Vesicular Glutamate Transport Proteins/metabolism , Animals , Congo Red/pharmacology , Drug Design , Molecular Docking Simulation , Molecular Structure , Protein Binding , Rats , Structure-Activity Relationship , Vesicular Glutamate Transport Proteins/antagonists & inhibitors
2.
Exp Neurol ; 297: 62-72, 2017 11.
Article in English | MEDLINE | ID: mdl-28756201

ABSTRACT

Multiple mild traumatic brain injury (mmTBI), in certain cases, produces persistent symptoms. However, the molecular mechanisms underlying these symptoms remain unclear. Here, we demonstrate extended pathological changes in the rat brain following mmTBI. Using the lateral fluid percussion (LFP) technique we exposed adult male Wistar rats to a mild TBI (mTBI) once a week for four weeks and compared them to surgical shams. At 90days following the last TBI or sham procedure the animals were cognitively tested in the Morris Water Maze (MWM), euthanized, and the brains removed for immunohistochemistry. At 90days following the last mTBI, NRF-2 staining was significantly decreased in the hilus of the hippocampus and cortex on the injured side, but did not significantly differ from shams on the un-injured side. CD68 positive microglia were significantly increased in the ipsilateral corpus callosum, cortex, and internal capsule of injured animals. Reactive astrocytosis, determined by increased GFAP staining, was also evident in the corpus callosum, cortex, internal capsule and thalamus on the injured side. Interestingly, the corpus callosum thickness at the midline was decreased in injured animals and had evident demyelination when compared to sham animals. Despite these findings, there were no significant differences in neurological assessments at 90days following the last injury. In MWM testing there were not significant differences in the training phase, the time spent in the thigmotaxia zone, or the target quadrant during the probe trial. However, there were significant differences between shams and injured animals in platform zone crossings during the probe trial. These results demonstrate that repetitive head trauma may produce persistent, long-term pathological alterations in brain architecture that may be difficult to detect using standard cognitive and neurological assessments.


Subject(s)
Brain Concussion/pathology , Brain/pathology , Animals , Brain Concussion/psychology , Male , Maze Learning/physiology , Microglia/pathology , Rats , Rats, Wistar
3.
J Clin Neurosci ; 38: 37-42, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28117263

ABSTRACT

Prediction of post-concussive syndrome after apparent mild traumatic brain injury (TBI) and subsequent cognitive recovery remains challenging, with substantial limitations of current methods of cognitive testing. This pilot study aimed to determine if levels of micro ribonucleic acids (RNAs) circulating in plasma are altered following TBI, and if changes to levels of such biomarkers over time could assist in determination of prognosis after TBI. Patients were enrolled after TBI on presentation to the Emergency Department and allocated to three groups: A - TBI (physical trauma to the head), witnessed loss of consciousness, amnesia, GCS=15, a normal CT Brain and a recorded first pass after post-traumatic amnesia (PTA) scale; B TBI, witnessed LOC, amnesia, GCS=15, a normal CT brain and a PTA scale test fail and: C - TBI and initial GCS <13 on arrival to the ED. Venous blood was collected at three time points (arrival, day 5 and day 30). Isolation of cell-free total RNA was then assayed using a custom miRNA PCR array. Two micro-RNAs, mir142-3p and mir423-3p demonstrated potential clinical utility differentiating patients after mild head injury into those at greater risk of developing amnesia and therefore, post-concussive syndromes. In addition, these miRNA demonstrated a decrease in expression over time, possibly indicative of brain healing after the injury. Further evaluation of these identified miRNA markers with larger patient cohorts, correlation with clinical symptoms and analysis over longer time periods are essential next steps in developing objective markers of severity of TBI.


Subject(s)
Amnesia/blood , Brain Injuries, Traumatic/blood , MicroRNAs/blood , Post-Concussion Syndrome/blood , Adult , Aged , Amnesia/etiology , Amnesia/physiopathology , Biomarkers/blood , Brain Injuries, Traumatic/complications , Brain Injuries, Traumatic/physiopathology , Female , Humans , Male , Middle Aged , Pilot Projects , Post-Concussion Syndrome/etiology , Post-Concussion Syndrome/physiopathology , Prognosis , Young Adult
4.
Concussion ; 2(4): CNC44, 2017 Dec.
Article in English | MEDLINE | ID: mdl-30202588

ABSTRACT

AIM: A small but notable number of individuals who suffer a concussion report ongoing cognitive difficulties. This preliminary study investigated the efficacy of repetitive test application to discern cognitive impairment in those with ongoing symptoms. METHODS: Participants (n = 17) with continuing self-reported symptoms following a concussion (∼9 months postinjury) were compared with 17 age group matched controls for working memory and word-list learning. RESULTS: Both groups performed similarly after the first trial for both assessments. However, in subsequent trials, the postconcussion group performed significantly worse than controls. DISCUSSION: While further studies to understand the mechanisms are warranted, data from this preliminary study suggest that a repetitive test application may be useful to discern cognitive fatigue in individuals who report ongoing concerns following a concussion.

5.
Brain Inj ; 31(1): 49-56, 2017.
Article in English | MEDLINE | ID: mdl-27936960

ABSTRACT

PRIMARY OBJECTIVE: The purpose of this study was to investigate the effects of mild traumatic brain injury (mTBI) on multiple postural indices that characterize body sway behaviour. METHODS AND PROCEDURES: The body's centre of pressure (COP) displacement was recorded from 11 individuals with a history of mTBI (29.4 ± 6.7 years old) and 11 healthy controls (26.8 ± 3.7 years old) performing bipedal stance on a force platform for 120 seconds. Spatio-temporal (area, amplitude and mean velocity of the COP displacement) and frequency characteristics (frequency containing 80% of the power spectral density) of the body oscillation, as well as its dynamic characteristics (sample entropy estimate of the COP displacement) were extracted from COP signals. MAIN OUTCOMES AND RESULTS: All postural indices studied were significantly affected by mTBI (p < 0.010). Participants with a history of mTBI presented a larger, slower, and more random body oscillation compared to controls. CONCLUSION: The results suggest that (a) balance deficits can be recognized as an effect of mTBI; (b) balance deficits induced by mTBI are multi-dimensional, affecting all three domains included in this study; and


Subject(s)
Brain Concussion/physiopathology , Postural Balance/physiology , Adult , Female , Humans , Male , Young Adult
6.
Bioorg Med Chem Lett ; 24(3): 850-4, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-24424130

ABSTRACT

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.


Subject(s)
Dicarboxylic Acids/chemical synthesis , Dicarboxylic Acids/pharmacology , Naphthols/chemical synthesis , Neurotransmitter Agents/chemical synthesis , Neurotransmitter Agents/pharmacology , Quinolines/chemical synthesis , Vesicular Glutamate Transport Proteins/antagonists & inhibitors , Binding, Competitive/drug effects , Cyclization , Dicarboxylic Acids/chemistry , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Naphthols/chemistry , Naphthols/pharmacology , Neurotransmitter Agents/chemistry , Pregnanolone/chemistry , Pregnanolone/pharmacokinetics , Quinolines/chemistry , Quinolines/pharmacology , Reference Standards
7.
Bioorg Med Chem Lett ; 23(21): 5931-5, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-24042010

ABSTRACT

Microwave accelerated reaction system (MARS) technology provided a good method to obtain selective and open isoxazole ligands that bind to and inhibit the Sxc- antiporter. The MARS provided numerous advantages, including: shorter time, better yield and higher purity of the product. Of the newly synthesized series of isoxazoles the salicyl hydrazide 6 exhibited the highest level of inhibitory activity in the transport assay. A homology model has been developed to summarize the SAR results to date, and provide a working hypothesis for future studies.


Subject(s)
Amino Acid Transport System y+/antagonists & inhibitors , Isoxazoles/chemistry , Isoxazoles/pharmacology , Amino Acid Transport System y+/chemistry , Amino Acid Transport System y+/metabolism , Cell Line , Cystine/metabolism , Glutamic Acid/metabolism , Humans , Isoxazoles/chemical synthesis , Microwaves , Molecular Docking Simulation , Structural Homology, Protein
8.
Br J Pharmacol ; 165(1): 20-34, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21564084

ABSTRACT

System x(c)(-) is an amino acid antiporter that typically mediates the exchange of extracellular l-cystine and intracellular L-glutamate across the cellular plasma membrane. Studied in a variety of cell types, the import of L-cystine through this transporter is critical to glutathione production and oxidative protection. The exchange-mediated export of L-glutamate takes on added significance within the CNS, as it represents a non-vesicular route of release through which this excitatory neurotransmitter can participate in either neuronal signalling or excitotoxic pathology. When both the import of L-cystine and the export of L-glutamate are taken into consideration, system x(c)(-) has now been linked to a wide range of CNS functions, including oxidative protection, the operation of the blood-brain barrier, neurotransmitter release, synaptic organization, viral pathology, drug addiction, chemosensitivity and chemoresistance, and brain tumour growth. The ability to selectively manipulate system x(c)(-), delineate its function, probe its structure and evaluate it as a therapeutic target is closely linked to understanding its pharmacology and the subsequent development of selective inhibitors and substrates. Towards that goal, this review will examine the current status of our understanding of system x(c)(-) pharmacology and the structure-activity relationships that have guided the development of an initial pharmacophore model, including the presence of lipophilic domains adjacent to the substrate binding site. A special emphasis is placed on the roles of system x(c)(-) within the CNS, as it is these actions that are among the most exciting as potential long-range therapeutic targets.


Subject(s)
Amino Acid Transport System y+/metabolism , Cystine/metabolism , Glutamic Acid/metabolism , Amino Acid Transport System y+/genetics , Animals , Central Nervous System/metabolism , Gene Expression Regulation/physiology , Substrate Specificity
9.
Glia ; 59(11): 1684-94, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21766339

ABSTRACT

Prion protein (PrP) is expressed on a wide variety of cells and plays an important role in the pathogenesis of transmissible spongiform encephalopathies. However, its normal function remains unclear. Mice that do not express PrP exhibit deficits in spatial memory and abnormalities in excitatory neurotransmission suggestive that PrP may function in the glutamatergic synapse. Here, we show that transport of D-aspartate, a nonmetabolized L-glutamate analog, through excitatory amino acid transporters (EAATs) was faster in astrocytes from PrP knockout (PrPKO) mice than in astrocytes from C57BL/10SnJ wild-type (WT) mice. Experiments using EAAT subtype-specific inhibitors demonstrated that in both WT and PrPKO astrocytes, the majority of transport was mediated by EAAT1. Furthermore, PrPKO astrocytes were more effective than WT astrocytes at alleviating L-glutamate-mediated excitotoxic damage in both WT and PrPKO neuronal cultures. Thus, in this in vitro model, PrPKO astrocytes exerted a functional influence on neuronal survival and may therefore influence regulation of glutamatergic neurotransmission in vivo.


Subject(s)
Astrocytes/metabolism , Glutamate Plasma Membrane Transport Proteins/metabolism , Prions/physiology , Animals , Aspartic Acid/metabolism , Blotting, Western , Cell Survival , Cells, Cultured , Coculture Techniques , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2/metabolism , Flow Cytometry , Glutamic Acid/physiology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/physiology , Prions/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Sodium/physiology
10.
Bioorg Med Chem Lett ; 21(14): 4358-62, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21669531

ABSTRACT

Evidence was acquired prior to suggest that the vesicular glutamate transporter (VGLUT) but not other glutamate transporters were inhibited by structures containing a weakly basic α-amino group. To test this hypothesis, a series of analogs using a hydantoin (pK(a)∼9.1) isostere were synthesized and analyzed as inhibitors of VGLUT and the obligate cystine-glutamate transporter (system x(c)(-)). Of the hydantoin analogs tested, a thiophene-5-carboxaldehyde analog 2l and a bis-hydantoin 4b were relatively strong inhibitors of VGLUT reducing uptake to less than 6% of control at 5mM but few inhibited system x(c)(-) greater than 50% of control. The benzene-2,4-disulfonic acid analog 2b and p-diaminobenzene analog 2e were also good hydantoin-based inhibitors of VGLUT reducing uptake by 11% and 23% of control, respectively, but neither analog was effective as a system x(c)(-) inhibitor. In sum, a hydantoin isostere adds the requisite chemical properties needed to produce selective inhibitors of VGLUT.


Subject(s)
Amino Acid Transport System y+/antagonists & inhibitors , Hydantoins/chemistry , Vesicular Glutamate Transport Proteins/antagonists & inhibitors , Amino Acid Transport System y+/metabolism , Glutamic Acid/metabolism , Hydantoins/chemical synthesis , Hydantoins/pharmacology , Structure-Activity Relationship , Vesicular Glutamate Transport Proteins/metabolism
11.
Glia ; 59(10): 1387-401, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21590811

ABSTRACT

The system x(C)- (Sx(C)-) transporter functions to mediate the exchange of extracellular cystine (L-Cys(2)) and intracellular glutamate (L-Glu). Internalized L-Cys(2) serves as a rate-limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L-Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl-cAMP) and GSH levels on the expression of Sx(C)- were investigated in primary rat astrocyte cultures. Sx(C)- activity in dbcAMP-treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (∼threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx(C)- triggered by GSH depletion was only observed in the dbcAMP-treated phenotype and was distinct from the Nrf2-mediated response initiated by exposure to electrophiles. Changes in Sx(C)- activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx(C)- heterodimer, an increase in the V(max) for L-Glu uptake and was linked temporally to GSH levels. This induction of Sx(C)- was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co-administration of the cell-permeant thiols GSH-ethyl ester and N-acetylcysteine. These findings demonstrate that the expression of Sx(C)- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx(C)- activity in CNS injury and disease.


Subject(s)
Amino Acid Transport Systems/metabolism , Astrocytes/metabolism , Cystine/metabolism , Glutamic Acid/metabolism , Glutathione/metabolism , Amino Acid Transport Systems/genetics , Analysis of Variance , Animals , Animals, Newborn , Antimetabolites/pharmacology , Astrocytes/drug effects , Buthionine Sulfoximine/pharmacology , Cells, Cultured , Cerebral Cortex/cytology , Cyclic CMP/analogs & derivatives , Cyclic CMP/pharmacology , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , RNA, Messenger/metabolism , Rats , Reactive Oxygen Species/metabolism , Tritium/metabolism
12.
Bioorg Med Chem Lett ; 20(8): 2680-3, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20303751

ABSTRACT

A panel of amino acid analogs and conformationally-restricted amino acids bearing a sulfonic acid were synthesized and tested for their ability to preferentially inhibit the obligate cysteine-glutamate transporter system x(c)(-) versus the vesicular glutamate transporter (VGLUT). Several promising candidate molecules were identified: R/S-4-[4'-carboxyphenyl]-phenylglycine, a biphenyl substituted analog of 4-carboxyphenylglycine and 2-thiopheneglycine-5-sulfonic acid both of which reduced glutamate uptake at system x(c)(-) by 70-75% while having modest to no effect on glutamate uptake at VGLUT.


Subject(s)
Glycine/pharmacology , Sulfonic Acids/chemistry , Vesicular Glutamate Transport Proteins/drug effects , Glycine/chemistry , Molecular Conformation
13.
Bioorg Med Chem ; 18(1): 202-13, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19932968

ABSTRACT

Analogues of amino methylisoxazole propionic acid (AMPA), were prepared from a common intermediate 12, including lipophilic analogues using lateral metalation and electrophilic quenching, and were evaluated at System xc-. Both the 5-naphthylethyl-(16) and 5-naphthylmethoxymethyl-(17) analogues adopt an E-conformation in the solid state, yet while the former has robust binding at System xc-, the latter is virtually devoid of activity. The most potent analogues were amino acid naphthyl-ACPA 7g, and hydrazone carboxylic acid, 11e Y=Y'=3,5-(CF(3))(2), which both inhibited glutamate uptake by the System xc- transporter with comparable potency to the endogenous substrate cystine, whereas in contrast the closed isoxazolo[3,4-d] pyridazinones 13 have significantly lower activity. A preliminary pharmacophore model has been constructed to provide insight into the analogue structure-activity relationships.


Subject(s)
Amino Acid Transport System y+/metabolism , Cell Membrane Permeability/drug effects , Isoxazoles/chemistry , Isoxazoles/pharmacology , Amino Acid Transport System y+/antagonists & inhibitors , Amino Acid Transport System y+/chemistry , Amino Acids/chemistry , Amino Acids/pharmacology , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Glutamic Acid/metabolism , Humans , Hydrazones/chemistry , Hydrazones/pharmacology , Models, Molecular , Molecular Structure , Protein Binding , Structure-Activity Relationship
14.
Toxicol Pathol ; 37(5): 644-60, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19638440

ABSTRACT

Residency in cities with high air pollution is associated with neuroinflammation and neurodegeneration in healthy children, young adults, and dogs. Nonsteroidal anti-inflammatory drugs may offer neuroprotection. The authors measured the plasma concentrations of 3-nitrotyrosine and the cerebro-spinal-fluid concentrations of prostaglandin E2 metabolite and the oligomeric form of amyloid derived diffusible ligand; measured the mRNA expression of cyclooxygenase-2, interleukin 1beta, CD14, and Aquaporin-4 in target brain areas; and evaluated brain MRI, cognition, and neuropathology in 8 dogs treated with a preferential cyclooxygenase-2 inhibitor (Nimesulide) versus 7 untreated litter-matched Mexico City dogs. Nimesulide significantly decreased nitrotyrosine in plasma (p < .0001), frontal gray IL1beta (p = .03), and heart IL1beta (p = .02). No effect was seen in mRNA COX2, amyloid, and PGE2 in CSF or the MRI white matter lesions. All exposed dogs exhibited olfactory bulb and frontal accumulation of Abeta(42) in neurons and blood vessels and frontal vascular subcortical pathology. White matter hyperintense MRI frontal lesions were seen in 4/6 non-treated and 6/8 treated dogs. Nonsteroidal anti-inflammatory drugs may offer limited neuroprotection in the setting of severe air pollution exposures. The search for potentially beneficial drugs useful to ameliorate the brain effects of pollution represents an enormous clinical challenge.


Subject(s)
Air Pollution/adverse effects , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase 2/metabolism , Dogs/metabolism , Sulfonamides/pharmacology , Amyloid beta-Peptides/metabolism , Animals , Aquaporin 4/metabolism , Brain/anatomy & histology , Brain/pathology , Chi-Square Distribution , Cyclooxygenase 2/genetics , Cyclooxygenase 2 Inhibitors/pharmacokinetics , Frontal Lobe/metabolism , Immunohistochemistry , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Lipopolysaccharide Receptors/metabolism , Magnetic Resonance Imaging , Mexico , Nasal Mucosa/metabolism , Ozone/adverse effects , Pilot Projects , Prostaglandins E/metabolism , Statistics, Nonparametric , Sulfonamides/pharmacokinetics , Tyrosine/metabolism
15.
J Proteome Res ; 7(2): 570-8, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18179165

ABSTRACT

Attempts to characterize recombinant integral membrane proteins (IMPs) by mass spectrometry are frequently hindered by several factors including the detergents required for extraction and purification that interferes with analysis, poor solubility, incomplete digestion, and limited identification of the transmembrane domain-spanning peptides. The goal of this study was to examine and develop methods for purification of an IMP that are amenable to downstream digestion of the protein and peptide analysis by mass spectrometry. In this study, we have overexpressed a candidate IMP, the vesicular glutamate transporter 1 (VGLUT1) in Pichia pastoris and examined conditions for the efficient affinity purification, in-solution digestion, and analysis of the protein. Analysis of the intact purified protein without detergent was performed by MALDI-TOF mass spectrometry. The purified IMP was digested with trypsin, and the resulting peptides were identified. A method that utilizes differential solubility and ionization properties of hydrophobic and hydrophilic peptides was developed. Large hydrophobic peptides were only detected in solutions containing 50% formic acid. Ionization of hydrophilic peptides was suppressed in formic acid, but they produced a strong signal in 50% acetonitrile. Eighty-seven percent sequence coverage of the protein was obtained with only one large hydrophobic peptide that remained unidentified. The results demonstrate a simple method to purify and digest a recombinant IMP for analysis by mass spectrometry.


Subject(s)
Pichia/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Vesicular Glutamate Transport Protein 1/metabolism , Affinity Labels , Amino Acid Sequence , Animals , Histidine , Molecular Sequence Data , Oligopeptides , Phosphorylation , Proteomics , Rats , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Vesicular Glutamate Transport Protein 1/analysis , Vesicular Glutamate Transport Protein 1/biosynthesis , Vesicular Glutamate Transport Protein 1/genetics
16.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 1): o144-5, 2008 Dec 17.
Article in English | MEDLINE | ID: mdl-21581603

ABSTRACT

The title compound, C(26)H(23)N(5)O(8), was prepared and its structure investigated to further develop a working hypothesis for the essential binding pharmacophore for ligands of the System Xc- transporter [Patel et al. (2004 ▶). Neuropharmacology, 46, 273-284]. The hydrazone group displays an E geometry and the isoxazole double bond and C=N group of the hydrazone are in an s-cis relationship. The secondary amino NH group forms an intra-molecular N-H⋯O hydrogen bond to a ring nitro group. There is a dihedral angle of 44.27 (5)° between the isoxazole plane and the hydrazone group plane.

17.
Bioorg Med Chem Lett ; 17(18): 5125-8, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17662605

ABSTRACT

Quinoline-2,4-dicarboxylic acids (QDCs) bearing lipophilic substituents in the 6- or 7-position were shown to be inhibitors of the glutamate vesicular transporter (VGLUT). Using the arrangement of the QDC lipophilic substituents as a template, libraries of X(1)X(2)EF and X(1)X(2)EW tetrapeptides were synthesized and tested as VGLUT inhibitors. The peptides QIEW and WNEF were found to be the most potent. Further stereochemical deconvolution of these two peptides showed dQlIdElW to be the best inhibitor (K(i)=828+/-252 microM). Modeling and overlay of the tetrapeptide inhibitors with the existing pharmacophore showed that H-bonding and lipophilic residues are important for VGLUT binding.


Subject(s)
Oligopeptides/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/antagonists & inhibitors , Models, Molecular
18.
Nature ; 433(7021): 73-7, 2005 Jan 06.
Article in English | MEDLINE | ID: mdl-15635412

ABSTRACT

Glutamate is the principal excitatory neurotransmitter in the nervous system. Inactivation of synaptic glutamate is handled by the glutamate transporter GLT1 (also known as EAAT2; refs 1, 2), the physiologically dominant astroglial protein. In spite of its critical importance in normal and abnormal synaptic activity, no practical pharmaceutical can positively modulate this protein. Animal studies show that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, brain tumours and epilepsy. Using a blinded screen of 1,040 FDA-approved drugs and nutritionals, we discovered that many beta-lactam antibiotics are potent stimulators of GLT1 expression. Furthermore, this action appears to be mediated through increased transcription of the GLT1 gene. beta-Lactams and various semi-synthetic derivatives are potent antibiotics that act to inhibit bacterial synthetic pathways. When delivered to animals, the beta-lactam ceftriaxone increased both brain expression of GLT1 and its biochemical and functional activity. Glutamate transporters are important in preventing glutamate neurotoxicity. Ceftriaxone was neuroprotective in vitro when used in models of ischaemic injury and motor neuron degeneration, both based in part on glutamate toxicity. When used in an animal model of the fatal disease ALS, the drug delayed loss of neurons and muscle strength, and increased mouse survival. Thus these studies provide a class of potential neurotherapeutics that act to modulate the expression of glutamate neurotransmitter transporters via gene activation.


Subject(s)
Anti-Bacterial Agents/pharmacology , Excitatory Amino Acid Transporter 2/biosynthesis , Excitatory Amino Acid Transporter 2/genetics , Gene Expression Regulation/drug effects , Neuroprotective Agents/pharmacology , beta-Lactams/pharmacology , Animals , Ceftriaxone/pharmacology , Cell Count , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/drug effects , Drug Evaluation, Preclinical , Genes, Reporter/genetics , In Vitro Techniques , Ischemic Preconditioning , Mice , Mice, Transgenic , Motor Neurons/cytology , Motor Neurons/drug effects , Penicillins/pharmacology , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptional Activation , United States , United States Food and Drug Administration
19.
Toxicol Appl Pharmacol ; 200(2): 83-92, 2004 Oct 15.
Article in English | MEDLINE | ID: mdl-15476861

ABSTRACT

Beta-N-oxalyl-L-alpha-beta-diaminopropionic acid (beta-L-ODAP) is an unusual amino acid present in seeds of plants from the Lathyrus genus that is generally accepted as the causative agent underlying the motor neuron degeneration and spastic paraparesis in human neurolathyrism. Much of the neuropathology produced by beta-L-ODAP appears to be a direct consequence of its structural similarities to the excitatory neurotransmitter L-glutamate and its ability to induce excitotoxicity as an agonist of non-NMDA receptors. Its actions within the CNS are, however, not limited to non-NMDA receptors, raising the likely possibility that the anatomical and cellular specificity of the neuronal damage observed in neurolathyrism may result from the cumulative activity of beta-L-ODAP at multiple sites. Accumulating evidence suggests that system xc-, a transporter that mediates the exchange of L-cystine and L-glutamate, is one such site. In the present work, two distinct approaches were used to define the interactions of beta-L-ODAP with system xc-: Traditional radiolabel-uptake assays were employed to quantify inhibitory activity, while fluorometrically coupled assays that follow the exchange-induced efflux of L-glutamate were used to assess substrate activity. In addition to confirming that beta-L-ODAP is an effective competitive inhibitor of system xc-, we report that the compound exhibits a substrate activity comparable to that of the endogenous substrate L-cystine. The ability of system xc- to transport and accumulate beta-L-ODAP identifies additional variables that could influence its toxicity within the CNS, including the ability to limit its access to EAA receptors by clearing the excitotoxin from the extracellular synaptic environment, as well as serving as a point of entry through which beta-L-ODAP could have increased access to intracellular targets.


Subject(s)
Amino Acid Transport System y+/metabolism , Amino Acids, Diamino/metabolism , Glycine/analogs & derivatives , Neurotoxins/metabolism , Quisqualic Acid/pharmacology , Amino Acid Transport System y+/antagonists & inhibitors , Amino Acids, Diamino/pharmacology , Amino Acids, Diamino/toxicity , Animals , Binding, Competitive , Cell Line, Tumor , Cyanobacteria Toxins , Cystine/metabolism , Fluorometry , Glutamic Acid/metabolism , Glycine/pharmacology , Humans , Kinetics , Neurotoxins/pharmacology , Neurotoxins/toxicity , Rats , Receptors, Glutamate/metabolism
20.
Neuropharmacology ; 46(2): 273-84, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14680765

ABSTRACT

In addition to the well-characterized sodium-dependent excitatory amino acid transporters (EAATs) present in the mammalian CNS, a chloride-dependent, sodium-independent transporter has also been identified that is capable of mediating the uptake of L-glutamate. Named system x(c)(-), this transporter is an obligate exchanger that normally couples the export of intracellular L-glutamate with the import of extracellular L-cystine. Two cell lines that express high levels of system x(c)(-) are used to delineate the pharmacology of the transporter and demonstrate that it is distinct from both the EAATs and EAA ionotropic receptors. Potent competitive inhibitors of system x(c)(-) include: L-homocysteate, ibotenate, L-serine-O-sulphate, (RS)-4-bromohomoibotenate, quisqualate, and (S)-4-carboxyphenylglycine. A fluorescent-based assay that allows system x(c)(-)-mediated exchange of L-glutamate and L-cystine to be followed in real time is used to assess substrate activity. Interestingly, those compounds that proved to be the most potent competitive inhibitors (e.g. L-quisqualate and 4-S-CPG) also proved to be the least active as substrates, suggesting that distinct structural features may control binding and translocation. Lastly, the finding that a number of system x(c)(-) inhibitors are also commonly used as probes of excitotoxic pathology (e.g., L-quisqualate, ibotenate and L-homocysteate) raises some interesting questions regarding the mechanisms through which these analogues produce CNS damage.


Subject(s)
Amino Acid Transport System y+ , Carrier Proteins/metabolism , Cystine/pharmacokinetics , Glutamic Acid/pharmacokinetics , Animals , Cell Line, Tumor , Cystine/chemistry , Glutamic Acid/chemistry , Humans , Rats , Substrate Specificity/physiology
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