Characterisation of the human NMDA receptor subunit NR3A glycine binding site

In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277-793nM)). Eight amino acids, which correspond to amino acids that are critical for ligand binding to other NMDA receptor subunits, situated within the S1S2 predicted ligand binding domain of hNR3A were mutated, which resulted in complete or near complete loss of [(3)H]-glycine binding to hNR3A. The NMDA NR1 glycine site agonist d-serine and partial agonist HA-966 (3-amino-1-hydroxypyrrolid-2-one), similarly to glycine displaced [(3)H]-glycine monophasically, suggesting a single common binding site. However, neither the partial agonist d-cycloserine nor the antagonist 7-chlorokynurenic acid displaced [(3)H]-glycine. Using homology modelling, a model of the NR3A binding pocket was generated which we suggest can be used to identify candidate agonists and antagonists. Our data show that glycine is a ligand, and most probably the endogenous ligand, for native NR3A at a binding site with unique pharmacological characteristics.     

Effect of 1-aminocyclopropanecarboxylic acid on N-methyl-D-aspartate-stimulated [3H]-noradrenaline release in rat hippocampal synaptosomes.

1. The effect of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant-like actions, was investigated in a functional assay for presynaptic NMDA receptors. 2. NMDA (100 microM) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]-noradrenaline ([3H]-NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 microM 7-chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor. 3. Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 +/- 11% and 1.82 +/- 0.04 microM, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 +/- 6 nM) and a lower maximal effect (Emax = 40 +/- 9%) than glycine. Furthermore, ACPC (0.1 microM) shifted the EC50 of glycine from 1.82 microM to > or = 3 mM. 4. These results show that ACPC can reduce the potentiation by glycine of NMDA-evoked [3H]-NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.     

Rat pup isolation calls are reduced by functional antagonists of the NMDA receptor complex

Compounds that reduce ion flux through N-methyl-D-aspartate (NMDA) coupled cation channels were evaluated for their effects on rat pup ultrasonic vocalizations (USV). Previous studies have demonstrated that rat pups emit ultrasonic calls during social isolation and that several classes of anxiolytics decrease, while putative anxiogenics increase, the number of these calls. The competitive NMDA antagonists 2-amino-7-phosphonoheptanoic acid (AP-7) and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [+/-)-CPP) as well as a partial agonist at the strychnine-insensitive glycine receptor, 1-aminocyclopropanecarboxylic acid (ACPC), reduced USV at doses that did not affect either motor activity or core temperature. A dose of glycine sufficient to elevate hippocampal glycine concentrations by 85% antagonized the effects of ACPC, but not AP-7. Glycine alone did not alter USV, but NMDA when given by itself increased USV by almost 50% at subconvulsant doses. Moreover, a dose of NMDA that did not affect USV antagonized the effects of AP-7 but not ACPC. Taken together, these findings are consistent with previous studies using conflict procedures which indicate that agents which reduce activity at NMDA receptor coupled cation channels may constitute a new class of anxiolytic agents.     

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.     

Putative partial agonist 1-aminocyclopropanecarboxylic acid acts concurrently as a glycine-site agonist and a glutamate-site antagonist at N-methyl-D-aspartate receptors

1-Aminocyclopropanecarboxylic acid (ACPC) has been shown to protect against neuronal cell death after ischemic insult in vivo. Such results can be correlated with in vitro assays in which ACPC protected neurons against glutamate-induced neurotoxicity by reducing the activity of N-methyl-D-aspartate (NMDA) channel activation. Electrophysiological studies have determined that ACPC inhibits NMDA receptor activity by acting as a glycine-binding site partial agonist. In this study, rapid drug perfusion combined with whole-cell voltage-clamp was used to elicit and measure the effects of ACPC on NMDA receptor-mediated responses from cultured hippocampal neurons and cerebellar granule cells. The ACPC steady-state dose-response curve had both stimulatory and inhibitory phases. Half-maximal activation by ACPC as a glycine-site agonist was 0.7 to 0.9 microM. Half-maximal inhibition by ACPC was dependent on NMDA concentration. Peak responses to a >100 microM ACPC pulse in the presence of 1 microM glutamate were similar to those of glycine but decayed to a steady-state amplitude below that of glycine. The removal of ACPC initially caused an increase in inward current followed by a subsequent decrease to baseline levels. This suggests that relief of low-affinity antagonism occurs before high-affinity agonist dissociation. Simulations of ACPC action by a two glutamate-binding site/two glycine-binding site model for NMDA channel activation in conjunction with the concurrent role of ACPC as a glycine-site full agonist and glutamate-site competitive antagonist were able to successfully approximate experimental results.     

NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones

1. NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR. 2. NMDA (1-100 microM, plus 10 microM glycine) induced a concentration-dependent increase in [Ca2+]i (EC50 = 5.7 microM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 microM) and nitrendipine (1 microM), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 microM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. 3. Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 microM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively. 4. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. 5. In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.     

Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling

We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N-methyl-D-aspartate (NMDA) receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, (R,S)-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by methanethiosulfonate ethylammonium potentiation of agonist-evoked currents. In addition, none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. The modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors, as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why "charge conserving" mutations of two residues in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA is not an agonist at AMPA receptors.     

Ethanol sensitivity of NMDA receptor function in developing cerebellar granule neurons

The mechanism by which ethanol inhibits the function of the NMDA subtype of glutamate receptor has not been elucidated. One possibility that has been suggested is that NMDA receptor subunit composition influences the sensitivity of the receptor to ethanol. We have taken advantage of developmental changes in subunit composition of the NMDA receptor in cultured neurons to examine possible changes in the effect of ethanol. We found an increase in expression of the NR2A subunit, and a decrease in expression of the NR2B subunit of the NMDA receptor in primary cultures of cerebellar granule neurons over time in culture, with no significant change in NR1 expression. This change in NR2 subunit expression was associated with the expected changes in functional properties of the NMDA receptor (measured as the NMDA-induced increase in intracellular Ca2+), i.e., ifenprodil sensitivity and glycine potency were higher when there was a relatively greater proportion of NR2B in the cultured neurons. However, the potency of ethanol to inhibit NMDA receptor function was lower when there was a greater proportion of NR2B subunits. Previous studies showed that ethanol inhibition of NMDA receptor function in cerebellar granule neurons resulted from an ethanol-induced decrease in potency of the co-agonist, glycine, and that this effect of ethanol was blocked by inhibitors of protein kinase C. Our current results suggest that the lower potency of ethanol to inhibit the response of NMDA receptors when cerebellar granule neurons are expressing a greater proportion of NR2B subunits is a result of the higher affinity of the NMDA receptors for endogenous levels of glycine at this point in time.     

Identification of a new site in the S1 ligand binding region of the NMDA receptor NR2A subunit involved in receptor activation by glutamate

Activation of N-methyl-d-aspartate (NMDA) receptors requires the binding of both glutamate and glycine to independent sites on the receptor. These ligands bind to NR2 and NR1 subunits respectively. Ligand binding residues are located in two non-contiguous domains, S1 and S2, which have been implicated in glutamate binding in other ionotropic glutamate receptor subunits. To further define the amino acids through which glutamate activates the receptor, we generated single-site mutations to the NR2A subunit, and expressed them with wild type NR1 in HEK 293 cells. Using calcium imaging and whole cell patch clamp we determined glutamate and glycine potencies. Of the eight residues mutated we identified five (E413, K484, A508, G685 and G688), whose mutation leads to a large reduction (from 4- to 1000-fold) in glutamate potency, consistent with a role for these residues in receptor activation by glutamate. The potency of glycine was largely unchanged by these mutations. Thus our results extend the knowledge base of residues involved in NMDA receptor function and identifies a new site in S1, in the region of A508, that has a role in receptor activation by glutamate.     

D-Cycloserine, a positive modulator of NMDA receptors, inhibits serotonergic function

The administration of the N-methyl-D-aspartate (NMDA)-associated glycine recognition site agonist D-cycloserine (DCS) to rats inhibited the head shakes and the forepaw treading induced by the serotonin (5HT) precursor, L-5-hydroxy-tryptophan [(-)5HTP], as well as the forepaw treading and motility elicited by the selective 5HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The head shakes typically induced by the 5HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), were unaffected by DCS pretreatment. The results are consistent with reduced serotonergic transmission produced by NMDA activation, as suggested by other authors. Due to the important role played in the pathogenesis of schizophrenia by glutamate deficiency/serotonin activation, the results support the view that positive modulators of NMDA receptors, activating glutamate receptors and reducing serotonergic tone, might be useful in the alleviation of psychotic symptoms. However, because of its partial agonist properties at the glycine recognition site, D-cycloserine shows some effects that might make it unsuitable for clinical use.     

Distinct effect of pregnenolone sulfate on NMDA receptor subtypes

Using rapid agonist applications to transfected HEK-293 cells, we investigated pregnenolone sulfate (PS) effects on deactivation and desensitization of recombinant NMDA receptors subtypes. PS prolonged the deactivation of responses produced by brief applications of L-glutamate with all subunit combinations tested. The action of PS was larger on NR1a/NR2A than on NR1a/NR2B channels. PS slowed the rate of macroscopic desensitization of the responses with all subunit combinations tested. In contrast, PS had little effect on current rise time and had much reduced action on responses with L-cysteate, a low affinity agonist. Our results suggest that PS decreases agonist unbinding. However, this action is counteracted by decreased desensitization. Since desensitization produces slow deactivating components, particularly with NR1a/NR2B receptors, this underlies the decreased PS effect with these subtypes. Indeed PS action was mainly observed on the fast component of deactivation. Furthermore, prolongation of NR1a/NR2A responses was similar to that of responses from NR1b/NR2B receptor, a subtype characterized by reduced desensitization. PS prolongation of evoked NMDA receptor mediated synaptic currents from cortical neuronal primary culture(s) was not significantly different from that of responses with NR1a/NR2B receptors indicating that native receptors in these neurons comprised at least some heteromeric combinations of these two subunits.     

NMDA receptor pharmacology: perspectives from molecular biology

The NMDA receptor is an important target for drug development, with agents from many different classes acting on this receptor. While the severe side effects associated with complete NMDA receptor blockade have limited clinical usefulness of most antagonists, the understanding of the multiple forms of NMDA receptors provides an opportunity for development of subtype specific agents with potentially fewer side effects. Different NMDA receptor subtypes are assembled from combinations of NR1 and NR2 subunits with each subunit conveying distinct properties. The NRI subunit is the glycine binding subunit and exists as 8 splice variants of a single gene. The glutamate binding subunit is the NR2 subunit, which is generated as the product of four distinct genes, and provides most of the structural basis for heterogeneity in NMDA receptors. Pharmacological heterogeneity results from differences in the structure of ligand binding regions, as well as structural differences between subtypes in a modulatory region called the LIVBP-like domain. This region in NR1 and NR2B controls the action of NR2B-selective drugs like ifenprodil, while this domain in receptors containing the NR2A subunit controls the action of NR2A-selective drugs such as zinc. This suggests that NMDA receptor subtype selective drugs can be created, and further understanding of subtype specific mechanisms ultimately may allow successful use of NMDA receptor antagonists as therapeutic agents.     

Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids

1. The neurosteroid pregnenolone sulphate (PS) potentiates N-methyl-D-aspartate (NMDA) receptor mediated responses in various neuronal preparations. The NR1 subunit can combine with NR2A, NR2B, NR2C, or NR2D subunits to form functional receptors. Differential NR2 subunit expression in brain and during development raises the question of how the NR2 subunit influences NMDA receptor modulation by neuroactive steroids. 2. We examined the effects of PS on the four diheteromeric NMDA receptor subtypes generated by co-expressing the NR1(100) subunit with each of the four NR2 subunits in Xenopus oocytes. Whereas PS potentiated NMDA-, glutamate-, and glycine-induced currents of NR1/NR2A and NR1/NR2B receptors, it was inhibitory at NR1/NR2C and NR1/NR2D receptors. 3. In contrast, pregnanolone sulphate (3alpha5betaS), a negative modulator of the NMDA receptor that acts at a distinct site from PS, inhibited all four subtypes, but was approximately 4 fold more potent at NR1/NR2C and NR1/NR2D than at NR1/NR2A and NR1/NR2B receptors. 4. These findings demonstrate that residues on the NR2 subunit are key determinants of modulation by PS and 3alpha5betaS. The modulatory effects of PS, but not 3alpha5betaS, on dose-response curves for NMDA, glutamate, and glycine are consistent with a two-state model in which PS either stabilizes or destabilizes the active state of the receptor, depending upon which NR2 subunit is present. 5. The selectivity of sulphated steroid modulators for NMDA receptors of specific subunit composition is consistent with a neuromodulatory role for endogenous sulphated steroids. The results indicate that it may be possible to develop therapeutic agents that target steroid modulatory sites of specific NMDA receptor subtypes.     

Structure-activity analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: 1-(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid

(2S*,3R*)-1-(biphenyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD) is a moderate affinity, competitive N-methyl-d-aspartate (NMDA) receptor antagonist with an atypical pattern of selectivity among NMDA receptor 2 subunit (NR2) subunits. We now describe the activity of several derivatives of PBPD tested at both rat brain NMDA receptors using l-[3H]-glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. Substituting various branched ring structures for the biphenyl group of PBPD reduced NMDA receptor activity. However, substituting linearly arranged ring structures - fluorenone or phenanthrene groups - retained or enhanced activity. Relative to PBPD, the phenanthrene derivative (2S*, 3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) displayed a 30- to 78-fold increase in affinity for native NMDA receptors. At recombinant receptors, PPDA displayed a 16-fold (NR2B) to 94-fold (NR2C) increase in affinity over PBPD. Replacement of the biphenyl group of PBPD with a 9-oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 2'-Bromo substitution on the biphenyl group of PBPD reduced antagonist affinity 3- to 5-fold at NR2A-, NR2B- and NR2D-containing receptors, but had little effect on NR2C-containing receptors. In contrast, 4'-fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. The aromatic rings of PBPD and PPDA increase antagonist affinity and appear to interact with a region of the NMDA receptor displaying subunit heterogeneity. PPDA is the most potent and selective NR2C/NR2D-preferring antagonist yet reported and thus may be useful in defining NR2C/NR2D function and developing related antagonists with improved NMDA receptor subtype selectivity.British Journal of Pharmacology (2004) 141, 508-516. doi:10.1038/sj.bjp.0705644     

Subunit-specific agonist activity at NR2A-, NR2B-, NR2C-, and NR2D-containing N-methyl-D-aspartate glutamate receptors

The four N-methyl-d-aspartate (NMDA) receptor NR2 subunits (NR2A-D) have different developmental, anatomical, and functional profiles that allow them to serve different roles in normal and neuropathological situations. Identification of subunit-selective NMDA receptor agonists, antagonists, or modulators could prove to be both valuable pharmacological tools as well as potential new therapeutic agents. We evaluated the potency and efficacy of a wide range of glutamate-like compounds at NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors. Twenty-five of 53 compounds examined exhibited agonist activity at the glutamate binding site of NMDA receptors. Concentration-response relationships were determined for these agonists at each NR2 subunit. We find consistently higher potency at the NR2D subunit for a wide range of dissimilar structures, with (2S,4R)-4-methylglutamate (SYM2081) showing the greatest differential potency between NR2A- and NR2D-containing receptors (46-fold). Analysis of chimeric NR2A/D receptors suggests that enhanced agonist potency for NR2D is controlled by residues in both of the domains (Domain1 and Domain2) that compose the bilobed agonist binding domain. Molecular dynamics (MD) simulations comparing a crystallography-based hydrated NR1/NR2A model with a homology-based NR1/NR2D hydrated model of the agonist binding domains suggest that glutamate exhibits a different binding mode in NR2D compared with NR2A that accommodates a 4-methyl substitution in SYM2081. Mutagenesis of functionally divergent residues supports the conclusions drawn based on the modeling studies. Despite high homology and conserved atomic contact residues within the agonist binding pocket of NR2A and NR2D, glutamate adopts a different binding orientation that could be exploited for the development of subunit selective agonists and competitive antagonists.     

Probing N-methyl-D-aspartate receptor desensitization with the substituted-cysteine accessibility method

Several forms of macroscopic N-methyl-D-aspartate (NMDA) receptor desensitization affect the amplitude and duration of postsynaptic responses. In addition to its functional significance, desensitization provides one means to examine the conformational coupling of ligand binding to channel gating. Segments flanking the ligand binding domain in the extracellular N terminus of the NMDA receptor NR2 subunit influence the glycine-independent form of desensitization. The NR2A pre-M1 region, the linker between the glutamate binding domain and the channel pore, plays a critical role in desensitization. Thus, we used the substituted-cysteine accessibility method to scan the accessibility of residues in the pre-M1 region and the first transmembrane domain (M1) of NR2A. Cysteine mutants were expressed with NR1 in human embryonic kidney 293 cells and were assayed by whole-cell recording. With activation of the receptor by glutamate and glycine, only a single mutant, V557C, which is located at the beginning of M1, led to irreversible inhibition by the methanethiosulfonate derivative methanethiosulfonate ethyltrimethylammonium (MTSET). The NR2 ligand glutamate was insufficient on its own to induce modification of V557C by MTSET, suggesting that the change in accessibility required channel gating. The rate of MTSET modification of the homologous residue on NR1 (NR1-1a(L562C)/NR2A) was much slower than V557C. We also substituted cysteine in the V557 site of mutant subunits that exhibit either enhanced or reduced desensitization. Modification by MTSET correlated with the degree of desensitization for these subunits, suggesting that V557C is a sensitive detector of desensitization gating.     

Riluzole, a glutamate release inhibitor, and motor behavior

Riluzole (2-amino-6-trigluoromethoxy benzothiazole) has neuroprotective, anticonvulsant, anxiolytic and anesthetic qualities. These effects are mediated by blockade of glutamate transmission, stabilizing of sodium channels and blockade of γ-aminobutyric acid (GABA) reuptake. The action profile of riluzole is dominated by its effects on glutamate transmission which are predominately mediated by N-methyl-D-aspartate (NMDA) receptor-linked processes in vitro. In vivo studies show that blockade and stimulation of the different NMDA receptor complex binding sites or AMPA receptors modulate motor behavior in a characteristic manner. It was therefore interesting to examine if blockade of glutamatergic transmission by riluzole induced similar behavioral effects as direct NMDA/AMPA receptor antagonists and if these effects are mediated by a specific receptor. The effects of riluzole alone and in combination with several other neuroactive compounds on the central nervous system was assessed by behavioral paradigms to evaluate sniffing behavior, locomotion, ataxia and rigidity. Accompanying compounds included the NMDA receptor agonist NMDA, the partial glycine site agonist D-cycloserine (DCS), and the α-amino-3-hydroxy-5-phenyl-4-isoxazolepropionic acid (AMPA) receptor antagonist GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzo-diazepine HCl]. Riluzole influenced neither stereotyped sniffing behavior nor locomotion but impaired motor coordination and attenuated rigidity induced by blockade of dopamine D1 and D2 receptor antagonists when given alone. At higher doses spontaneous behavioral activity decreased and motor coordination was more impaired. Augmentation of the riluzole effects were observed when NMDA, but not GYKI 52466, was coadministered. The glycine site agonist DCS increased the anticataleptic properties of riluzole. The results indicate that when given alone, riluzole has a behavioral profile resembling that of competitive NMDA receptor antagonists. However, coadministration of riluzole with NMDA/AMPA receptor ligands suggests that this assumption is incorrect, and that riluzole affects glutamatergic transmission by a more indirect mechanism. Nevertheless, the profile of riluzole together with its pre- and postsynaptic blockade of glutamatergic transmission implies beneficial properties in diseases where an overactive glutamate system induces chronic neurotoxicity and/or acute behavioral effects. Received: 15 December 1997 / Accepted: 12 May 1998     

Characterization of the binding of two novel glycine site antagonists to cloned NMDA receptors: evidence for two pharmacological classes of antagonists

The potency of two novel glycine site antagonists, GV150,526A and GV196,771A, was assessed by their ability to inhibit the binding of [(3)H]-MDL105,519 to cell homogenates prepared from mammalian cells transfected with either NR1-1a, NR1-2a, NR1-1a/NR2A, NR1-1a/NR2B, NR1-1a/NR2C or NR1-1a/NR2D NMDA receptor clones. The inhibition constants (K(i)s) for GV150,526A displacement of [(3)H]-MDL105,519 binding to either NR1-1a or NR1-2a expressed alone were not significantly different and were best fit by a one-site binding model. GV150,526A inhibition to NR1-1a/NR2 combinations was best fit by a two-site model with the NR1-1a/NR2C having an approximate 2 - 4 fold lower affinity compared to other NR1-1a/NR2 receptors. The K(i)s for GV196,771A displacement of [(3)H]-MDL105,519 binding to NR1-1a, NR1-2a and all NR1-1a/NR2 combinations was best fit by a two-site binding model. There was no significant difference between the K(i)s for the binding to NR1-1a and NR1-2a; NR1-1a/NR2A receptors had an approximate 4 fold lower affinity for GV196,771A compared to other NR1-1a/NR2 combinations. The K(i)s for both GV150, 526A and GV196,771A for the inhibition of [(3)H]-MDL105,519 binding to membranes prepared from adult rat forebrain were determined and compared to the values obtained for binding to cloned NMDA receptors. The K(i)s for a series of glycine site ligands with diverse chemical structures were also determined for the inhibition of [(3)H]-MDL105,519 binding to NR1-1a/NR2A receptors. L689,560 displayed similar binding characteristics to GV150,526A. It is suggested that glycine site antagonists may be divided into two classes based on their ability to distinguish between NR1 and NR1/NR2 receptors with respect to binding curve characteristics.     

Agonist-like character of the (R)-enantiomer of 1-hydroxy-3-amino-pyrrolid-2-one (HA-966).

HA-966 (1-hydroxy-3-amino-pyrrolid-2-one), an antagonist at the strychnine-insensitive glycine site on the N-methyl-D-aspartate (NMDA) receptor complex, only partially inhibits the binding of noncompetitive antagonists to the NMDA receptor but enhances the binding of the NMDA competitive antagonist CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid). Here we report that the IC50 of the active (R)-enantiomer of HA-966 for displacement of [3H]glycine binding is decreased in the presence of spermine, suggesting that spermine increases the affinity of (R)-HA-966 at the [3H]glycine binding site. The IC50 values of the agonist glycine and the partial agonist 1-aminocyclopropane-1-carboxylate are also decreased. The IC50 values of glycine antagonists 6,7-dinitroquinoxalin-2,3-dione and 7-chlorokynurenic acid are not significantly altered. The spermine shift represents the first demonstration of the agonist-like character of the (R)-enantiomer of HA-966 at the glycine site.     

N-methyl-D-aspartate antagonists for stroke and head trauma

The N-methyl-D-aspartate (NMDA) receptor is a ligand-gated ion channel which is widely distributed in the central nervous system (CNS), and which mediates most of the fast excitatory neuronal transmission in the CNS. As with other ligand-gated ion channels, the NMDA receptor is a macromolecular complex which possesses a number of intricate regulatory sites within and around a central ion channel. The key regulatory components for which prototypic antagonists have been developed are the competitive NMDA antagonist binding site, the non-competitive NMDA antagonist binding site within the ion channel, and the NMDA receptor-associated glycine antagonist site. The binding domains for each of these binding sites possess discrete and non-overlapping SAR with regard to the chemical series developed to date. The potential utility of NMDA antagonists in the treatment of stroke and traumatic brain injury was investigated soon after the synthesis of the first bioavailable NMDA antagonists. Efficacy in preclinical models was demonstrated with both competitive and non-competitive NMDA antagonists. However, preclinical testing also revealed potentially clinically-limiting side-effects which included phencyclidine (PCP)-like actions indicative of possible psychotomimetic activity, cerebral vacuolisation of limbic cortical neurones, low therapeutic indices relative to incapacitating motor side-effects and, in the case of non-competitive antagonists, hypertension. These limitations have led to the design of clinical trials that should define the therapeutic index for this type of compound in humans. Currently, the first competitive antagonist to enter clinical trials, selfotel, is on hold, while D-CPPene is still in development. The non-competitive antagonist, aptiganel, is currently in Phase III clinical trials and its therapeutic efficacy and index should be defined in 1997 and 1998. The well-defined limitations of competitive and non-competitive NMDA antagonists have been a key impetus in the investigation of alternative approaches to modulating the NMDA receptor complex. In the case of glycine site antagonists, these compounds have been shown in preclinical studies to be devoid of PCP-like actions and the neuronal vacuolisation associated with the competitive and non-competitive NMDA antagonists. This has induced the development of a number of chemical series with at least three compounds currently in Phase I and II clinical trials. These include ACEA 1021, GV150526A and ZD9379. Clinical efficacies and therapeutic indices of these compounds should be defined in 1998 and 1999. An alternative approach using a partial agonist of the glycine site (1-aminocyclopropane-carboxylic acid, ACPC) has been halted in Phase I. Another approach which has led to the development of NMDA receptor antagonists, selective for the NMDA receptor subunits 1A/2B (NR1A/2B subtype), was the discovery in early studies of the neuroprotective actions of ifenprodil. Structural analogues include eliprodil, CP-101,606 and lubeluzole. In the cases of eliprodil and lubeluzole, these compounds have demonstrated neuroprotection in preclinical models, but they possess the extremely dangerous side-effect of increasing cardiac repolarisation time (i.e., increased QTc interval). The therapeutic index for these compounds is low. This has led to the termination of eliprodil's development and has limited the current dosing strategy with lubeluzole. It has not been disclosed if CP-101,606 possesses this dose-limiting side-effect. In summary, strategies for drug design and development based on our knowledge of the NMDA receptor complex have led to the development of a new generation of compounds for the treatment of stroke and traumatic brain injury, which remain to be evaluated in the clinic. The success of this approach will be defined in the next two to three years.