Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.     

Quinoxalines interact with the glycine recognition site of NMDA receptors: studies in guinea-pig myenteric plexus and in rat cortical membranes.

1. The effects of several quinoxalines, including 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7,dinitroquinoxaline-2,3-dione (DNQX), and of two kynurenates, kynurenate (KYNA) and 7-Clkynurenate (7-Cl-KYNA), have been evaluated on the N-methyl-D-aspartate (NMDA) receptors present in the guinea-pig ileum myenteric plexus preparation and on the strychnine-insensitive [3H]-glycine binding sites of cortical membranes. 2. Quinoxalines and kynurenates antagonized in a non-competitive manner L-glutamate-induced contraction. Their IC50s were (in microM): 5 for 7-Cl-KYNA, 7.5 for 6,7-Cl-3-hydroxy-2-quinoxaline carboxylate (6,7-Cl-HQCA), 20 for DNQX, 50 for CNQX, 76 for KYNA and 125 for 3-hydroxy-2-quinoxaline carboxylate (HQCA). 3. Glycine (5-50 microM) completely reversed the antagonism displayed by both quinoxalines and kynurenates. The interaction between glycine and the tested compounds appeared to be competitive in nature. 4. Quinoxalines and kynurenates displaced, in a concentration-dependent manner, [3H]-glycine from its strychnine-insensitive binding sites present in rat cortical membranes. Their IC50s for this action were (in microM): 0.45 for 7-Cl-KYNA, 0.6 for 6,7-Cl-HQCA, 2.4 for DNQX, 3.5 for CNQX, 20 for KYNA and 40 for HQCA. 5. When the IC50s for the displacement effect of [3H]-glycine binding were plotted against the IC50s obtained in the myenteric plexus, a significant correlation was found. 6. These data show that quinoxalines and kynurenates may antagonize the responses to L-glutamate by interacting with the glycine recognition sites of the NMDA receptor ion channel complex.     

Glycine and kynurenate modulate the glutamate receptors in the myenteric plexus and in cortical membranes

The responses evoked by stimulation of the N-methyl-D-aspartate receptors in the guinea-pig myenteric plexus were potentiated by micromolar concentrations of glycine and were non-competitively antagonized by kynurenate (IC50: 60 microM). The effects of kynurenate were competitively prevented by glycine. Furthermore, kynurenate displaced [3H]glycine from its binding sites on rat cortical membranes (IC50: 20 microM). Kynurenate and glycine, therefore, probably act at the same site, evoking opposite effects on the function of the ion channel complex of the N-methyl-D-aspartate receptor.     

Kynurenic acid analogues with improved affinity and selectivity for the glycine site on the N-methyl-D-aspartate receptor from rat brain.

The glycine site on the N-methyl-D-aspartate (NMDA) subtype of receptors for the excitatory neurotransmitter glutamate is a potential target for the development of neuroprotective drugs. We report here two chemical series of glycine site antagonists derived from kynurenic acid (KYNA), with greatly improved potency and selectivity. Disubstitution with chlorine or bromine in the 5- and 7-positions of KYNA increased affinity for [3H]glycine binding sites in rat cortex/hippocampus P2 membranes, with a parallel increase of potency for antagonism of NMDA-evoked responses in the rat cortical wedge preparation. The optimal compound was 5-I,7-Cl-KYNA, with an IC50 for [3H]glycine binding of 29 nM and an apparent Kb in the cortical wedge preparation of 0.41 microM. Reduction of the right-hand ring of 5,7-diCl-KYNA reduced affinity by 10-fold, but this was restored by substitution in the 4-position with the trans-phenylamide and further improved in the trans-benzylamide. The optimal compound was the transphenylurea (L-689,560), with an IC50 of 7.4 nM and an apparent Kb of 0.13 microM. Both series of compounds displayed a high degree of selectivity for the glycine site, having IC50 values of greater than 10 microM versus radioligand binding to the glutamate recognition sites of NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate receptors and the strychnine-sensitive glycine receptor. Selectivity versus AMPA receptor-mediated responses was also apparent in the rat cortical wedge and in patch-clamp recordings of cortical neurons in culture. Experiments using [3H]dizocilpine (MK-801) binding indicated that 5,7-diBr-KYNA, 5,7-diCl-KYNA, 5-I,7-Cl-KYNA, and L-689,560 all behaved as full antagonists and were competitive with glycine. Patch-clamp recordings of cortical neurons in culture also indicated that NMDA-induced currents were antagonized by competition for the glycine site, and gave no evidence for partial agonist activity. pKi values for 5,7-diBr-KYNA and L-689,560 in these experiments were 7.2 and 7.98, respectively, similar to the affinities of these compounds in the glycine binding assay. The high affinity and selectivity of these new derivatives make them useful tools to investigate the function of the glycine site on the NMDA receptor.     

Synthesis and excitatory amino acid pharmacology of a series of heterocyclic-fused quinoxalinones and quinazolinones.

As part of our program aimed at the development of potent excitatory amino acid antagonists, we synthesized and evaluated a series of substituted 1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-ones, 4, tetrazolo[1,5-a]quinoxalin-4(5H)-ones, 5, and pyrazolo[1,5-c]quinazolin-5(6H)-ones, 6, and an imidazo[1,2-a]quinoxalin-4(5H)-one, 7. In general, the same heterocycles which demonstrated the best affinity for the AMPA receptor also demonstrated the best affinity for the glycine site on the NMDA receptor complex. 1-Propyl-7,8-dichloro-1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-one, 4d, was found to bind with the greatest affinity to the AMPA receptor with an IC50 of 0.83 microM and antagonized 40 microM AMPA-induced depolarization in the cortical slice preparation with an IC50 of 44 microM. 7,8-Dichloro-1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-one, 4a, and 7,8-dichloroimidazo[1,2-a]quinoxalin-4(5H)-one, 7, possessed the best affinity for the glycine site with IC50 values of 0.63 and 1.26 microM, respectively. It is noteworthy that the SAR for the heterocyclic compounds did not directly parallel that of known quinoxalinediones (e.g. DNQX, 2, and DCQX, 15) at the AMPA receptor nor that of the kynurenic acids at the glycine site on the NMDA receptor complex.     

Kynurenic acid inhibits the activation of kainic and N-methyl-D-aspartic acid-sensitive ionotropic receptors by a different mechanism

The action of kynurenic acid on currents elicited by the activation of amino acid receptors was investigated in primary cultures of cortical neurons prepared from neonatal rats. Kynurenic acid was tested on currents elicited by both N-methyl-D-aspartic acid (NMDA) and kainate, using patch-clamp recording techniques in "outside-out" and "whole-cell" configurations. The inhibition by kynurenic acid was compared with that elicited by amino-phosphono-valeric acid (APV). Whole-cell currents, elicited by increasing doses of NMDA, were antagonized competitively by APV and non-competitively by kynurenic acid (ID50 70 microM); in contrast, kynurenic acid inhibited competitively the whole-cell currents elicited by kainic acid (ID50 500 microM). The non-competitive inhibition by kynurenic acid of the whole cell currents elicited by NMDA was antagonized competitively by glycine, a specific positive allosteric modulator of NMDA receptors; on the other hand glycine failed to change the inhibition by APV of the NMDA-elicited responses. Thus, kynurenic acid inhibits NMDA receptors allosterically (non-competitively) and kainic acid receptors isosterically (competitively).     

Heterocyclic excitatory amino acids. Synthesis and biological activity of novel analogues of AMPA.

The novel acidic amino acids 6a-c, 7, and 8 have been synthesized via 1,3-dipolar cycloadditions, using nitrile oxides and alkynes. The prepared compounds are heterocyclic analogues of glutamic acid with differing chain lengths. One of these compounds, (RS)-2-amino-3-(3-carboxy-5-methyl-4- isoxazolyl)propionic acid (ACPA, 8), was shown in [3H]AMPA binding studies to be more active than AMPA itself (IC50 = 20 nM compared to IC50 = 79 nM for AMPA). No affinity for NMDA receptors (NMDA-sensitive [3H]glutamic acid binding) was found, and only weak affinity in [3H]kainic acid binding (IC50 = 6.3 microM) was detected. The excitatory activity in rat cortical wedge also showed that ACPA was more potent than AMPA (EC50 = 1.0 microM compared to EC50 = 3.5 microM for AMPA). The depolarizing effect of ACPA could be fully antagonized by the selective non-NMDA antagonist 6-cyano-7-nitro-quinoxazoline-2,3-dione (CNQX), but was unaffected by the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (AP5).     

Spermidine enhancement of [3H]MK-801 binding to the NMDA receptor complex in human cortical membranes

The effects of spermidine on the binding of [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor complex was studied in human cerebral cortical membranes. [3H]MK-801 binding was increased from 56 +/- 5 fmol/mg protein (mean +/- S.E.M., n = 7) to 319 +/- 71 fmol/mg protein in the presence of 200 microM spermidine. The ED50 for spermidine stimulation of [3H]MK-801 binding was 89 +/- 22 microM (mean +/- S.E.M., n = 6). In the presence of glutamate (1 microM) plus glycine (1 microM) the ED50 was reduced to 5.5 +/- 0.7 microM. The increase in binding in the presence of spermidine was characterised by an increase in the rate of association of [3H]MK-801. In the presence of spermidine. [3H]MK-801 was inhibited by AP5. 7-chlorokynurenic acid and ifenprodil with IC50 values of 0.5 +/- 0.3 24 +/- 19 and 91 +/- 28 microM, respectively. None of these antagonists was a competitive inhibitor of the spermidine stimulation of [3H]MK-801 binding. Thus spermidine modulates the NMDA receptor complex in human brain, providing further evidence that the complex is similar in rat and human cortex.     

D,L-(tetrazol-5-yl) glycine: a novel and highly potent NMDA receptor agonist.

This paper describes the pharmacological activity of D,L-(tetrazol-5-yl)glycine, a structurally novel and highly potent agonist at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor. D,L-(Tetrazol-5-yl)glycine potently displaced NMDA receptor binding to rat brain membranes as measured using [3H]CGS19755 (IC50 = 98 +/- 7 nM) and [3H]glutamate (IC50 = 36 +/- 18 nM) as ligands. D,L-(Tetrazol-5-yl)glycine did not appreciably inhibit the binding of D,L-alpha-[5-methyl-3H] amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, or [3H]glycine (IC50s greater than 30,000 nM). D,L-(Tetrazol-5-yl)glycine was more potent than NMDA or cis-methanoglutamate as a depolarizing agent in the rat cortical slice, and unlike these other agents induced rapid receptor-mediated neurotoxicity. Depolarization by D,L-(tetrazol-5-yl)glycine was antagonized by LY233053, a selective NMDA receptor antagonist. D,L-(Tetrazol-5-yl)glycine was a highly potent convulsant when given to neonatal rats (ED50 = 0.071 mg/kg i.p.). Convulsions in neonatal rats or lethality in mice induced by D,L-(tetrazol-5-yl)glycine were selectively antagonized by competitive and non-competitive NMDA receptor antagonists. D,L-(Tetrazol-5-yl)glycine is a structurally novel (tetrazole-substituted) compound that is a highly potent and selective NMDA receptor agonist. D,L-(Tetrazol-5-yl)glycine could be used to probe further NMDA receptor function in vitro and in vivo.     

Differential modulation of [3H]TCP binding to the NMDA receptor by L-glutamate and glycine

At equilibrium (4 h incubation), [3H]TCP (N-(1-[2-thienyl]-cyclohexyl)-3,4-[3H]piperidine) binding to well-washed rat forebrain membranes was enhanced in a concentration-dependent and 2-APV (2-amino-5-phosphonovaleric acid)-sensitive fashion by L-glutamate (EC50 = 0.2 microM; maximal effect +280%). L-glutamate (10 microM) increased the affinity of [3H]TCP from 78 to 28 nM, but was without effect on the maximal binding capacity. The enhancing effect of L-glutamate on [3H]TCP binding was potentiated by glycine in a concentration-dependent manner (EC50 = 50 nM, maximal effect +30% in the presence of 10 microM L-glutamate; EC50 = 2 microM, maximal effect +29% in the presence of 0.1 microM L-glutamate). This effect was strychnine-insensitive. Glycine failed to enhance [3H]TCP binding in the presence of 10 microM 2-APV. The glycine effect was due to an increase in affinity (Kd = 21 nM in the presence of 10 microM glycine and 10 microM L-glutamate); glycine did not affect the maximal binding capacity. The glycine enhancement of L-glutamate-stimulated [3H]TCP binding was not antagonised by 1 microM strychnine and was mimicked by L-serine and L-alanine but not by GABA, taurine or beta-alanine. Kinetic analysis of the glycine and L-glutamate enhancement of [3H]TCP binding indicated that the L-glutamate effect was related to a decrease in the [3H]TCP dissociation rate while the glycine effect was due to an increase in the rate of [3H]TCP association in the presence of L-glutamate.     

Effects of RPR 118723, a novel antagonist at the glycine site of the NMDA receptor, in vitro

RPR 118723 ((8-chloro-5-methyl-2,3-dioxo-1,4-dihydro-5H-indeno[1, 2-b]pyrazin-5-yl) acetic acid) was previously reported to exhibit potent affinity for the glycine site of the N-methyl-D-aspartate (NMDA) receptor-channel complex in the nanomolar range (K(i)=3.1+/-0. 8 nM). We now report on the effects of RPR 118723 in two functional tests reflecting the interaction between the glycine site and the NMDA receptor. First, RPR 118723 potently inhibited [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in the presence of NMDA (IC(50)=3.5+/-0.4 nM). Second, RPR 118723 antagonized the NMDA-induced increase in [3H]dopamine release in mouse striatal slices (IC(50)=8.0+/-1.1 nM). In both experimental models, an excess of glycine reversed the effect of RPR 118723. These results show that RPR 118723 interferes functionally in the nanomolar range with the glycine site coupled to the NMDA receptor in vitro. The blockade of the glycine site with RPR 118723 may be useful for the therapy of the disorders linked to excessive NMDA stimulation.     

A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties

5.7-Dinitro-quinoxaline-2.3-dione (MNQX) displaced [3H]glycine binding to cortical membranes but had no effect n [3H]3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid ([3H]CPP) binding. MNQX potently antagonized N-methyl-D-aspartate (NMDA)-evoked release of [3H]GABA from cultured cortical neurones, NMDA evoked spreading depression and NMDA depolarizations in the rat neo-cortex. All of these responses were reversed by addition of glycine to the perfusion media. These results suggested that MNQX is an antagonist at the strychnine-insensitive glycine receptor associated with the NMDA receptor/ionophore complex. Furthermore the compound was found to antagonise audiogenic seizures in DBA-2 mice indicating the potential of glycine antagonists of this type in anticonvulsant therapy.     

NMDA receptor antagonists that bind to the strychnine-insensitive glycine site and inhibit NMDA-induced Ca2+ fluxes and [3H]GABA release

We have examined the actions of putative antagonists of the strychnine-insensitive glycine-mediated modulation of the N-methyl-D-aspartate (NMDA) receptor using [3H]MK801 binding, Ca2+ influx and [3H]GABA release assays. Kynurenic acid and HA-966 inhibited [3H]MK801 binding, NMDA and glycine induced Ca2+ influx measured using fura-2 and NMDA and glycine simulated [3H]GABA release. The effects of kynurenic acid could be partially overcome by the addition of excess glutamate and glycine, indicating limited selectivity for the glycine binding site. In addition, a component of the action of kynurenic acid was insensitive to agonist concentration, indicating a third action of kynurenic acid at high concentrations. In contrast, HA-966 was 100-fold selective for the glycine compared to the NMDA site. HA-966 only partially inhibited [3H]MK801 binding (IC50 19.7 microM), NMDA-induced Ca2+ influx and neurotransmitter release. The failure of HA-966 to completely block NMDA responses, even at high concentrations, suggests that glycine may not be an absolute requirement for the activation of NMDA receptors under these experimental conditions.     

Competitive inhibition of magnesium-induced [3H]N-(1-[thienyl] cyclohexyl)piperidine binding by arcaine: evidence for a shared spermidine-magnesium binding site

The polyamine competitive antagonist arcaine (1,4-diguanidino-butane) produced complete inhibition of basal [3H]N-(1-[thienyl] cyclohexyl)piperidine ([3H]TCP) binding, with an IC50 value of 4.52 +/- 0.93 microM. Arcaine (5 and 10 microM) produced a decrease in the affinity without a significant change in the receptor density of [3H]TCP binding under equilibrium conditions. In addition, arcaine did not alter either N-methyl-D-aspartate-specific [3H] glutamate or strychnine-insensitive [3H]glycine binding. Furthermore, increasing concentrations of arcaine produced parallel rightward shifts in the concentration-response curves for both spermidine- and magnesium-induced [3H]TCP binding, suggesting that arcaine is a competitive inhibitor of both agonists. Similar rightward shifts were observed for barium- and strontium-induced [3H]TCP binding. In contrast, arcaine decreased the efficacy of glutamate- and glycine-induced [3H]TCP binding without changing their EC50 values, indicating a noncompetitive type of inhibition. These results imply that spermidine and certain divalent cations including magnesium share the same mechanism for enhancing [3H]TCP binding, whereas glutamate and glycine have different sites of action. This is further supported by the additive effect of spermidine when tested in the presence of maximal concentrations of glutamate and glycine. On the other hand, spermidine and magnesium were not additive and, in fact, magnesium was able to block the effects of spermidine under certain conditions. The possibility that magnesium is a partial agonist at the polyamine site is discussed.     

Excitatory amino acid receptor ligands. Synthesis and biological activity of 3-isoxazolol amino acids structurally related to homoibotenic acid.

The 3-isoxazolol amino acid (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 2) and the isomeric compound (RS)-2-amino-3-(3-hydroxy-4-methylisoxazol-5-yl)propionic acid (4-methylhomoibotenic acid, 4a) are potent agonists at the AMPA subtype of central excitatory amino acid receptors. Using 4a as a lead structure, the amino acids 4c-e, in which the 4-methyl group of 4a is replaced by substituents of different size and polarity, were synthesized. Attempts to synthesize 4-(bromomethyl)homoibotenic acid (4f), a potential receptor alkylating agent, were unsuccessful. 4-Butylhomoibotenic acid (4c) and 4-(2-hydroxyethyl)homoibotenic acid (4e) were equipotent as inhibitors of [3H]AMPA binding (IC50 = 2 microM) and showed similar excitatory activity in the rat cortical slice preparation. 4d did not show significant affinity for AMPA receptor sites, but turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist. However, like 4c,e, 4d did not significantly affect the binding of the competitive NMDA antagonist, [3H]CPP, or the noncompetitive NMDA antagonist, [3H]MK-801. None of the amino acids 4c-e showed detectable affinity for [3H]kainic acid binding sites. Like the parent compound 4a (IC50 = 0.18 microM), 4c (IC50 = 0.18 microM), 4e (IC50 = 0.14 microM), and in particular 4d (IC50 = 0.02 microM) were effective inhibitors of calcium chloride-dependent [3H]glutamic acid binding, whereas AMPA is inactive (IC50 greater than 100 microM) in this binding assay. Thus, 4d is an effective and highly selective inhibitor of calcium chloride-dependent [3H]glutamic acid binding and may be a useful tool for studies of the physiological relevance and pharmacological importance of this binding affinity.     

4-Amido-2-carboxytetrahydroquinolines. Structure-activity relationships for antagonism at the glycine site of the NMDA receptor.

trans-2-Carboxy-5,7-dichloro-4-amidotetrahydroquinolines, evolved from the lead 5,7-dichlorokynurenic acid, have been synthesized and tested for in vitro antagonist activity at the glycine site on the N-methyl-D-aspartate (NMDA) receptor. Optimization of the 4-substituent has provided antagonists having nanomolar affinity, including the urea trans-2-carboxy-5,7-dichloro-4[[(phenylamino)carbonyl]amino]-1,2,3, 4-tetrahydroquinoline (35; IC50 = 7.4 nM vs [3H]glycine binding; Kb = 130 nM for block of NMDA responses in the rat cortical slice), which is one of the most potent NMDA antagonists yet found. The absolute stereochemical requirements for binding were found to be 2S,4R, showing that, in common with other glycine-site NMDA receptor ligands, the unnatural configuration at the alpha-amino acid center is required. The preferred conformation of the trans-2,4-disubstituted tetrahydroquinoline system, as shown by X-ray crystallography and 1H NMR studies, places the 2-carboxyl pseudoequatorial and the 4-substituent pseudoaxial. Modifications of the 4-amide show that bulky substituents are tolerated and reveal the critical importance for activity of correct positioning of the carbonyl group. The high affinity of trans-2-carboxy-5,7-dichloro-4-[1-(3-phenyl-2-oxoimidazolidinyl)]- 1,2,3,4-tetrahydroquinoline (55; IC50 = 6 nM) suggests that the Z,Z conformer of the phenyl urea moiety in 35 is recognized by the receptor. Molecular modeling studies show that the 4-carbonyl groups of the kynurenic acids, the tetrahydroquinolines, and related antagonists based on N-(chlorophenyl)glycine, can interact with a single putative H-bond donor on the receptor. The results allow the establishment of a three-dimensional pharmacophore of the glycine receptor antagonist site, incorporating a newly defined bulk tolerance/hydrophobic region.     

Evidence for direct interactions between the NMDA and glycine recognition sites in brain

The interaction between glycine and competitive N-methyl-D-aspartate (NMDA) antagonists was investigated. Glycine (IC50 = 170 nM) partially (approximately 60%) inhibited [3H]CGS-19755 ((+/-)-4-phosphonomethyl-2-piperdine carboxylic acid), but not [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) binding. The action of glycine was mimicked by D-serine and antagonized by 7-chlorokynurenate. CGS-19755 (IC50 = 230 nM) partially inhibited [3H]glycine binding from strychnine-insensitive sites; this effect was antagonized by NMDA. CPP and NPC 12626 (2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid) inhibited [3H]glycine binding, but only at concentrations 100- to 1000-fold greater than required to displace [3H]CGS-19755 or [3H]CPP. These data provide the first evidence for bidirectional interactions between glycine and NMDA recognition sites and suggest pharmacological differences among competitive NMDA antagonists.     

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.     

Spermine enhances binding to the glycine site associated with the N-methyl-D-aspartate receptor complex

Spermine enhanced strychnine-insensitive [3H]glycine binding 3-fold with an EC50 of 27 +/- 3.1 microM. Spermidine and putrescine were without effect, whereas the ethylenediamine analog of spermine had an intermediate effect. Eadie-Hofstee analysis revealed that spermine increased the affinity of glycine for its receptor without a significant change in receptor density. This effect persisted in the presence of glycine or N-methyl-D-aspartate receptor antagonists. Furthermore, spermine produced a leftward shift in the IC50 of glycine agonists in displacing [3H]glycine binding, without altering the IC50 for glycine antagonists. These data indicate that spermine interacts with the glycine receptor through a novel binding site and, further, that spermine can be used to discriminate glycine agonist and antagonist binding.     

NMDA receptor heterogeneity in mammalian tissues: focus on two agonists, (2S,3R,4S) cyclopropylglutamate and the sulfate ester of 4-hydroxy-(S)-pipecolic acid

Several potent and selective agonists of the glutamate (L-GLU) receptors of N-methyl-D-aspartate (NMDA) type have been tested on the L-[³H]GLU binding to rat cortical membranes, on the depolarization of mouse cortical wedges and on the contraction of guinea pig longitudinal muscle myenteric plexus preparations with the aim of comparing the NMDA receptors present in the cortex and those present in the gut.When the depolarization of the cortical wedges was evaluated, the EC₅₀ values of the agonists were (M): (R,S)-(tetrazol-5-yl)-glycine (TG) 0.3; trans-4-hydroxy (S)-pipecolic acid-4-sulfate (t-HPIS) 0.7; 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACBD) 0.8; NMDA 8; (2S,3R,4S) cyclopropylglutamate (L-CGA C) 12; quinolinic acid (QUIN) 400. When the contraction of the longitudinal muscle myenteric plexus was evaluated, the EC₅₀ values were (M): L-CGA C 1; TG 8; ACBD 50; t-HPIS 100; QUIN 500 and NMDA 680. When the displacement of NMDA specific L-[³H]GLU binding from rat cortical membranes was evaluated, the IC₅₀ values were (NM): L-CGA C 0.003; TG 0.005; ACBD 0.044; t-HPIS 0.062; NMDA 0.31 and QUIN 15. No significant correlation was found when the EC₅₀ values obtained in the ileum were plotted against the EC₅₀ values obtained in the cortex (r = 0.47). In particular it was noted that L-CGA C was approximately three orders of magnitude more potent than NMDA when tested in the ileum but had a potency not significantly different from that of NMDA when tested in the cortex. On the contrary, t-HPIS was particularly potent in cortical wedges.The results of these experiments suggest that different populations of NMDA receptors are present in the tissues of the three mammalian species investigated. In particular, the receptors present in the mouse cortical wedges are selectively stimulated by t-HPIS while those present in the guinea pig myenteric plexus are preferentially stimulated by L-CGA C. Thus at least two functional subtypes of NMDA receptors may be identified in mammalian tissues by using the order of potency of selective agonists.