Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides.

The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal gamma-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[gamma10K/gamma14K] and con-R[gamma11A/gamma15A] displayed 5.6- and 8.4-fold decreases in tau(off), respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the tau(off) was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 microM spermine enhanced the onset rate constant from 3.1x10(3)M(-1) x s(-1) to 12.6x10(3)M(-1) x s(-1). From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K(i) value of 1.1 microM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 microM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.     

The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B

The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E⁶⁵⁷-I⁸¹⁴) were replaced by the corresponding S2 region of NR2B (E⁶⁵⁸-I⁸¹⁵), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M⁷³⁹ of NR2B for the equivalent K⁷³⁸ of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K⁷⁵⁵ of NR2B for Y⁷⁵⁴ of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes.     

Opposing action of conantokin-G on synaptically and extrasynaptically-activated NMDA receptors

Synaptic and extrasynaptic activation of the N-methyl-D-aspartate receptor (NMDAR) has distinct consequences on cell signaling and neuronal survival. Since conantokin (con)-G antagonism is NR2B-selective, which is the key subunit involved in extrasynaptic activation of the receptor, its ability to specifically elicit distinct signaling outcomes in neurons with synaptically or extrasynaptically-activated NMDARs was evaluated. Inhibition of Ca(2+) influx through extrasynaptic NMDAR ion channels was neuroprotective, as it effectively enhanced levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), activated cAMP response element binding protein (CREB), enhanced mitochondrial viability, and attenuated the actin disorganization observed by extrasynaptic activation of NMDARs. Conversely, the pro-signaling pathways stimulated by synaptically-induced Ca(2+) influx were abolished by con-G. Furthermore, subunit non-selective con-T was unable to successfully redress the impairments in neurons caused by extrasynaptically-activated NMDARs, thus indicating that NR2B-specific antagonists are beneficial for neuron survival. Neurons ablated for the NR2B subunit showed weak synaptic Ca(2+) influx, reduced sensitivity to MK-801 blockage, and diminished extrasynaptic current compared to WT and NR2A(-/-) neurons. This indicates that the NR2B subunit is an integral component of both synaptic and extrasynaptic NMDAR channels. Altogether, these data suggest that con-G specifically targets the NR2B subunit in the synaptic and extrasynaptic locations, resulting in the opposing action of con-G on differentially activated pools of NMDARs.     

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors

The N-methyl-D-aspartate receptor (NMDAR) antagonists: MK-801, phencyclidine and ketamine are open-channel blockers with limited clinical value due to psychotomimetic effects. Similarly, the psychotomimetic effects of the dextrorotatory opioids, dextromethorphan and its metabolite dextrorphan, derive from their NMDAR antagonist actions. Differences in the use dependency of blockade, however, suggest that the binding sites for MK-801 and dextrorphan are distinct. In the absence of exogenous glutamate and glycine, the rate of association of [3H]MK-801 with wild-type NR1-1a/NR2A receptors was considerably slower than that for [3H]dextrorphan. Glutamate individually, and in the presence of the co-agonist glycine, had substantial effects on the specific binding of [3H]MK-801, while the binding of [3H]dextrorphan was not affected. Mutation of residues N616 and A627 in the NR1 subunit had a profound effect on [3H]MK-801 binding affinity, while that of [3H]dextrorphan was unaltered. In contrast, NR1 residues, W611 and N812, were critical for specific binding of [3H]dextrorphan to NR1-1a/NR2A complexes with no corresponding influence on that of [3H]MK-801. Thus, [3H]dextrorphan and [3H]MK-801 have distinct molecular determinants for high-affinity binding. The ability of [3H]dextrorphan to bind to a closed channel, moreover, indicates that its recognition site is shallower in the ion channel domain than that of MK-801 and may be associated with the extracellular vestibule of the NMDAR.     

Design, synthesis, and biological evaluation of new 8-heterocyclic xanthine derivatives as highly potent and selective human A2B adenosine receptor antagonists

Here we report the synthesis of 8-heterocycle-substituted xanthines as potent and selective A(2B) adenosine receptor antagonists. The structure-activity relationships (SAR) of the xanthines synthesized in binding to recombinant human A(2B) adenosine receptors (ARs) in HEK-293 cells (HEK-A(2B)) and at other AR subtypes were explored. The synthesized compounds showed A(2B) adenosine receptor affinity in the nanomolar range and good levels of selectivity evaluated in radioligand binding assays at human (h) A(1), A(2A), A(2B), and A(3) ARs. We introduced several heterocycles, such as pyrazole, isoxazole, pyridine, and pyridazine, at the 8-position of the xanthine nucleus and we have also investigated different spacers (substituted acetamide, oxyacetamide, and urea moieties) on the heterocycle introduced. Various groups at the 3- and 4-positions of phenylacetamide moiety were studied. This study allowed us to identify the derivatives 2-(3,4-dimethoxyphenyl)-N-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl]acetamide (29b, MRE2028F20) [K(i)(hA(2B)) = 38 nM, K(i)(hA(1),hA(2A),hA(3)) >1000 nM], N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide (62b, MRE2029F20) [K(i)(hA(2B)) = 5.5 nM, K(i)(hA(1),hA(2A),hA(3)) > 1000 nM], and N-(3,4-dimethoxyphenyl)-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide (72b, MRE2030F20) [K(i)(hA(2B) = 12 nM, K(i)(hA(1),hA(2A), hA(3)) > 1000 nM], which showed high affinity at the A(2B) receptor subtype and very good selectivity vs the other ARs. Substitution of the acetamide with an urea moiety afforded bioisosteric xanthines with good affinity and selectivity comparable to the acetamide derivatives. Substitution at the para-position of a 4-benzyloxy group of the phenylacetamido chain enhanced affinity at the A(2B) receptor [compound 30b (K(i)(hA(2B)) = 13 nM) vs compound 21b (K(i)(hA(2B) = 56 nM)] but did not favor selectivity. The derivatives with higher affinity at human A(2B) AR proved to be antagonists, in the cyclic AMP assay, capable of inhibiting the stimulatory effect of NECA (100 nM) with IC(50) values in the nanomolar range, a trend similar to that observed in the binding assay (62b, IC(50) = 38 nM; 72b, IC(50) = 46 nM). In conclusion, the 8-pyrazolo-1,3-dipropyl-1H-purine-2,6-dione derivatives described herein represent a new family of selective antagonists for the adenosine A(2B) receptor.     

Inhibition by 2-Methoxy-4-ethylphenol of Ca2+ Influx Through Acquired and Native N-Methyl-d-aspartate–Receptor Channels

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-d-aspartate (NMDA), to modulate Ca²⁺ influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca²⁺ levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca²⁺ levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit–selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits. 2M4EP significantly prevented the increased intracellular Ca²⁺ levels by NMDA in cultured rat hippocampal neurons. Brief exposure to NMDA led to a drastic decrease in cellular viability 24 h later in cultured hippocampal neurons, while 2M4EP significantly prevented the loss of cellular vitality by NMDA. Similarly, 2M4EP more efficiently protected HEK293 cells with NR1/NR2B subunits than those with NR1/NR2A subunits. These results suggest that 2M4EP may protect neurons from excitotoxicity through inhibition of Ca²⁺ influx across NMDAR channels composed of NR1/ NR2B, rather than NR1/NR2A, subunits.     

The NMDA receptor NR2B subunit: a valid therapeutic target for multiple CNS pathologies

The NMDAR2B subunit is the focus of increasing interest as a therapeutic target in a wide range of CNS pathologies, including acute and chronic pain, stroke and head trauma, drug-induced dyskinesias, and dementias. Due to significant pharmaceutical endeavor, an impressive collection of chemical leads has been developed which target the NR2B subunit, some of which appear to discriminate between closely related subtypes. We now have the benefit of a structural template for the ifenprodil binding site which should further improve future structure activity relationships. A growing appreciation of the likely extrasynaptic localisation of the NR2B receptor subtype and importance of NR2B protein modification, notably tyrosine phosphorylation, may explain its therapeutic importance. The apparent superior preclinical and clinical data for the second and third generation NR2B compounds is likely to reflect subtype selectivity, a unique mode of action and cellular location of the NR2B receptors in the CNS.     

Subtype-selective N-methyl-D-aspartate receptor antagonists: synthesis and biological evaluation of 1-(heteroarylalkynyl)-4-benzylpiperidines

4-[4-(4-Benzylpiperidin-1-yl)but-1-ynyl]phenol (8) and 4-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]phenol (9) are potent NR1A/2B receptor antagonists (IC(50) values 0.17 and 0.10 microM, respectively). Administered intraperitoneally, they both potentiated the activity of L-DOPA in the unilaterally 6-hydroxydopamine-lesioned (6-OHDA) rat, a model of Parkinson's disease. However, compound 9 was not active orally, likely due to rapid first-pass metabolism of the phenol moiety. The phenol was replaced by several bicyclic heterocyclic systems containing an NH group to function as a H-bond donor in the hope that these would be less likely to undergo rapid metabolism. In general, indoles, indazoles, benzotriazoles, indolones, and isatins gave analogues with weaker NR1A/2B activity than the parent phenols, while benzimidazolones and benzimidazolinones gave equipotent or more potent analogues. The preference for a para arrangement between the H-bond donor and the linking acetylene moiety was confirmed, and a propyne link was preferred over a butyne link. Substitution on the benzyl group or a 4-hydroxyl group on the piperidine had little effect on NR1A/2B potency; however, 4-hydroxypiperidines demonstrated slightly improved selectivity for NR1A/2B receptors versus alpha-1 adrenergic and dopamine D2 receptor affinity. From this study, 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1, 3-dihydrobenzoimidazol-2-one (46b) was identified as a very potent, selective NR1A/2B receptor antagonist (IC(50) value 0.0053 microM). After oral administration at 10 and 30 mg/kg, 46b potentiated the effects of L-DOPA in the 6-OHDA-lesioned rat and seemed to have improved oral bioavailability but lower brain penetration compared to phenol 9.     

Effects of volatile solvents on recombinant N-methyl-D-aspartate receptors expressed in Xenopus oocytes

1. We have previously shown that toluene dose-dependently inhibits recombinant N-methyl-D-aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC(50) value for toluene of 0.17 mM. 2. We now report on the effects of other commonly abused solvents (benzene, m-xylene, ethylbenzene, propylbenzene, 1,1,1-trichlorethane (TCE) and those of a convulsive solvent, 2,2,2-trifluoroethyl ether (flurothyl), on NMDA-induced currents measured in XENOPUS oocytes expressing NR1/2A or NR1/2B receptor subtypes. 3. All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA-induced currents that was dose- and subunit-dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype. 4. The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes. 5. Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain.     

Site within N-Methyl-D-aspartate receptor pore modulates channel gating

N-methyl-d-aspartate-type glutamate receptors (NMDARs) are ligand-gated ion channels activated by coagonists glutamate and glycine. NMDARs play a critical role in synaptic plasticity and excitotoxicity, largely because of their high calcium permeability and slow deactivation and desensitization kinetics. NR1 is an obligate subunit in all NMDAR complexes, where it combines with NR2A, 2B, 2C, and/or 2D. NR1 binds glycine, and residue Asn598 in the re-entrant membrane loop M2 largely determines NMDAR calcium permeability. In contrast, NR2 subunits bind glutamate and contain regions that regulate receptor desensitization and deactivation. Here, we report that mutations of NR1(Asn598) in combination with wild-type NR2A, expressed in human embryonic kidney 293 cells, exhibit altered glycine-independent desensitization. In the absence of extracellular calcium, substitution of Arg for Asn598 (NR1R) slowed desensitization by 2- to 3-fold compared with wild-type NR1/NR2A, and glutamate-evoked peak current EC50 and deactivation rate were also affected. Replacement of Asn by Gln (NR1Q) produced two distinct rates of calcium- and glycine-independent desensitization. Moreover, in the presence of extracellular calcium, the voltage-dependent pore block by calcium for the NR1Q mutant mimicked the effects of the positively charged Arg at this site in NR1R on slowing desensitization and deactivation. A kinetic model of the NMDA receptor-channel suggests that these results can be explained by altered gating and not ligand binding. Our data increase understanding of the role that amino acids within the NMDAR pore play in channel gating.     

Synthesis and in vitro characterization of novel amino terminally modified oxotremorine derivatives for brain muscarinic receptors.

A series of novel 2-substituted acetylenic pyrrolidines and piperidines related to oxotremorine (1) were prepared and evaluated in vitro as muscarinic cholinergic agents at brain M1 and M2 receptors. One analogue, 3-(2-oxo-1-pyrrolidinyl)-1-[2(R)-pyrrolidinyl]-1-propyne hydrogen oxalate (6a), was found to be a partial agonist producing a PI hydrolysis response at cortical M1 receptors approximately 3-fold larger than that produced by 1. The intrinsic activity profile of 6a at brain muscarinic receptors is similar to those of azetidine oxo analogue 2 and dimethylamino oxo analogue. All three compounds are partial M1 agonists and full M2 agonists; however, the profile of 6a in binding studies is significantly different. While 2 and 3 exhibit large M2 selectivities ranging between 8-fold to several hundred-fold, the binding profile of 6a shows almost no subtype selectivity.     

HIV-1 Glycoprotein 120 Enhancement of N-Methyl-D-Aspartate NMDA Receptor-Mediated Excitatory Postsynaptic Currents: Implications for HIV-1-Associated Neural Injury

It is widely accepted that human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) plays an important role in HIV-1-induced neural injury and pathogenesis of HIV-1-associated dementia (HAND). Multiple pathways have been proposed for gp120-induced neurotoxicity, amongst is the activation of N-Methyl-D-Aspartate receptors (NMDARs). It has been shown that gp120 causes neuronal injury or death and gp120 transgenic mice exhibit neurological similarity to that of HAND, all of which can be blocked or attenuated by NMDAR antagonists. Several lines of evidence indicate the subtype and location of activated NMDARs are key determinants of the nature of NMDAR physiology. To examine the subtype and the location of NMDARs affected by gp120, we studied gp120 on subtype NMDAR-mediated EPSCs in the CA1 region of rat hippocampal slices through “blind” whole-cell patch recordings. Our results showed bath application of gp120 increased both NR2A- and NR2B-mediated EPSCs possibly via a presynaptic mechanism, with much stronger effect on NR2B-mediated EPSCs. In contrast, gp120 failed on enhancing AMPA receptor-mediated EPSCs. Ca²⁺ imaging studies revealed that gp120 potentiated glutamate-induced increase of intracellular Ca²⁺ concentration in rat hippocampal neuronal cultures which were blocked by a NMDAR antagonist, but not by an AMPA receptor antagonist, indicating gp120 induces Ca²⁺ influx through NMDARs. Further investigations demonstrated that gp120 increased the EPSCs mediated by extrasynaptic NR2BRs. Taken together, these results demonstrate that gp120 interacts with both NR2A and NR2B subtypes of NMDARs with a predominant action on the extrasynaptic NR2B, implicating a role NR2B may play in HIV-1-associated neuropathology.     

Pharmacological characterization of recombinant NR1/NR2A NMDA receptors with truncated and deleted carboxy termini expressed in Xenopus laevis oocytes

Background and purpose:

The carboxy terminal domain (CTD) of NR2 N-methyl-d-aspartate receptor (NMDAR) subunits interacts with numerous scaffolding and signal transduction proteins. Mutations of this region affect trafficking and downstream signalling of NMDARs. This study determines to what extent characteristic pharmacological properties of NR2A-containing NMDARs are influenced by this key functional domain.

Experimental approach:

Using recombinant receptor expression in Xenopus laevis oocytes and two electrode voltage clamp recordings we characterized pharmacological properties of rat NR1/NR2A NMDARs with altered CTDs. We assessed the effects of truncating [at residue Iso1098; NR2A(trunC)] and deleting [from residue Phe822; NR2A(delC)] the CTD of NR2A NMDAR subunits on agonist potencies, channel block by Mg²⁺ and memantine and potentiation of NMDAR-mediated responses by chelating contaminating divalent cations.

Key results:

Truncation or deletion of the CTD of NR2A NMDAR subunits did not affect glutamate potency [EC₅₀ = 2.2 µmol·L⁻¹, NR2A(trunC); 2.7 µmol·L⁻¹, NR2A(delC) compared with 3.3 µmol·L⁻¹, NR2A(WT)] but did significantly increase glycine potency [EC₅₀ = 500 nmol·L⁻¹, NR2A(trunC); 900 nmol·L⁻¹, NR2A(delC) compared with 1.3 µmol·L⁻¹, NR2A(WT)]. Voltage-dependent Mg²⁺ block of NR2A(WT)- and NR2A(trunC)-containing NMDARs was similar but low concentrations of Mg²⁺ (1 µmol·L⁻¹) potentiated NR1/NR2A(delC) NMDARs. Memantine block was not affected by changes to the structure of the NR2A CTD. EDTA-induced potentiation was similar at each of the three NMDAR constructs.

Conclusions and implications:

Of the parameters studied only minor influences of the CTD were observed; these are unlikely to compromise interpretation of studies that make use of CTD-mutated recombinant receptors or transgenic mice in investigations of the role of the CTD in NMDAR signalling.     

Synthesis and pharmacology of N1-substituted piperazine-2,3-dicarboxylic acid derivatives acting as NMDA receptor antagonists

The binding site for competitive NMDA receptor antagonists is on the NR2 subunit, of which there are four types (NR2A-D). Typical antagonists such as (R)-AP5 have a subunit selectivity of NR2A > NR2B > NR2C > NR2D. The competitive NMDA receptor antagonist (2R,3S)-(1-biphenylyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD, 16b) displays an unusual selectivity with improved relative affinity for NR2C and NR2D vs NR2A and NR2B. Analogues of 16b bearing aroyl or aryl substituents attached to the N(1) position of piperazine-2,3-dicarboxylic acid have been synthesized to probe the structural requirements for NR2C/NR2D selectivity. A phenanthrenyl-2-carbonyl analogue, 16e, had >60-fold higher affinity for NR2C and NR2D and showed 3-5-fold selectivity for NR2C/NR2D vs NR2A/NR2B. The phenanthrenyl-3-carbonyl analogue (16f) was less potent but more selective, having 5- and 7-fold selectivity for NR2D vs NR2A and NR2B, respectively. Thus, antagonists bearing bulky hydrophobic residues have a different NR2 subunit selectivity than that of typical antagonists.     

The role of NMDAR subtypes and charge transfer during hippocampal LTP induction

Activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations, such as long-term potentiation of synaptic transmission (LTP). NMDARs are composed of NR1 and NR2 subunits, and NR2 subunit-dependent gating properties of NMDAR subtypes cause dramatic differences in the timing of charge transfer. These postsynaptic temporal profiles are further influenced by the frequency of synaptic activation. Here, we investigated in the CA1 region of hippocampal slices from P28 mice, whether particular NMDAR subtypes are recruited based on NR2 subunit-specific gating following different induction protocols. For high frequency afferent stimulation (HFS), we found that genetic impairment of NR2A or pharmacological block of NR2A- or NR2B-type NMDARs can reduce field LTP. In contrast, when pairing low frequency synaptic stimulation with postsynaptic depolarization (LFS pairing) in single CA1 neurons, pharmacological antagonism of either subtype modestly reduced the charge transfer during LFS pairing without reducing the LTP magnitude. These results indicate that HFS-triggered LTP is induced by more than one NMDAR subtype, whereas a single subtype is sufficient during LFS pairing. Analysis of charge transfer during LFS pairing in 13 different conditions revealed a threshold for LTP induction, which was independent of the NR2 antagonist tested. Thus, at least for LFS pairing, the amount of charge transfer, and thus Ca2+ influx, during LTP induction is a factor more critical than the participation of a particular NMDAR subtype.     

N-methyl-D-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein

The effects of a synthetic apoE peptide, viz., residues 133-149 (apoE[133-149]), a mimetic that comprises the apoE receptor binding domain, on N-methyl-D-aspartate (NMDA)/glycine-induced ion flow through NMDA receptor (NMDAR) channels, have been investigated. The activity of apoE[133-149] was found to depend on the low-density lipoprotein receptor-related protein (LRP). Competition experiments with receptor-associated protein (RAP) and activated alpha(2)-macroglobulin (alpha(2)M*), two proteins that compete for apoE binding to LRP, demonstrate that apoE[133-149] inhibition of NMDAR function is mediated at a locus in LRP that overlaps with the binding sites of RAP and alpha(2)M*. A coreceptor of LRP, cell surface heparin sulfate proteoglycan, did not function in this system. Additional electrophysiology experiments demonstrated that the inhibitory potency of apoE[133-149] was threefold greater for NMDAR-transfected wild-type Chinese hamster ovary (CHO) cells compared with NMDAR-transfected CHO cells deficient in LRP. Studies with truncation and replacement variants of the apoE peptide demonstrated that the NMDAR inhibitory properties of these peptides correlate with their binding affinities for LRP. These novel results indicate that apoE functions as an inhibitor of NMDAR ion channels indirectly via LRP, and are suggestive of a participatory role for LRP in NMDAR-based neuropathies.     

Loss of synaptic D1 dopamine/N-methyl-D-aspartate glutamate receptor complexes in L-DOPA-induced dyskinesia in the rat

Glutamate-mediated mechanisms are related to the motor complications of L-DOPA therapy in Parkinson's disease (PD). In striatal postsynaptic densities (PSD), the dopamine D1 receptor (D1R) is part of an oligomeric complex with the glutamate N-methyl-D-aspartate receptor (NMDAR), determining the strength of corticostriatal transmission. We studied D1R/NMDAR complex alterations induced by L-DOPA in the 6-hydroxydopamine-lesioned rat model of PD. L-DOPA-treated hemiparkinsonian rats were determined to be dyskinetic or nondyskinetic based on behavioral testing. D1R/NMDAR assemblies containing NR1-C2 and NR2B subunits were decreased in the PSD of lesioned striatum. Short-term L-DOPA administration improved akinesia and restored the synaptic abundance of D1R, NR1-C2 and NR2B. Prolonged L-DOPA treatment also normalized synaptic D1R/NMDAR complexes in nondyskinetic rats, but remarkably reduced them in the dyskinetic group without changing their interaction. This decrease involved NR1-C2, NR1-C2', NR2A, and NR2B subunits. The composition of residual synaptic D1R/NMDAR complexes in dyskinetic rats may thus be different from that observed in lesioned rats, suggesting that expression of different motor dysfunctions might be related to the receptor profile at corticostriatal synapses. The levels of D1R/NMDAR complexes were unchanged in total striatal membrane proteins, suggesting that the decrease of these species in the PSD is likely to reflect an altered receptor trafficking. In human embryonic kidney 293 cells expressing the D1R/NMDAR, complex costimulation of both D1R and NMDAR, but not individual receptor activation, promoted internalization, suggesting that development of dyskinesias might be related to agonist-mediated down-regulation of the D1R/NMDAR complex at corticostriatal synapses.     

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     

Subtype-selective N-methyl-D-aspartate receptor antagonists: synthesis and biological evaluation of 1-(arylalkynyl)-4-benzylpiperidines.

A search of our compound library for compounds with structural similarity to ifenprodil (5) and haloperidol (7) followed by in vitro screening revealed that 4-benzyl-1-(4-phenyl-3-butynyl)piperidine (8) was a moderately potent and selective antagonist of the NR1A/2B subtype of NMDA receptors. Substitution on the benzyl group of 8 did not significantly affect NR1A/2B potency, while addition of hydrogen bond donors in the para position of the phenyl group enhanced NR1A/2B potency. Addition of a hydroxyl moiety to the 4-position of the piperidine group slightly reduced NR1A/2B potency while reducing alpha-1 adrenergic and dopamine D2 receptor binding affinities substantially, resulting in improved overall selectivity for NR1A/2B receptors. Finally, the butynyl linker was replaced with propynyl or pentynyl. When the phenyl was para substituted with amine or acetamide groups, the NR1A/2B potency order was butynyl > pentynyl > propynyl. For the para methanesulfonamide or hydroxyl groups, the order was butynyl approximately propynyl > pentynyl. The hydroxyl propyne (48) and butyne (23) were among the most potent NR1A/2B antagonists from this study. They both potentiated the effects of L-DOPA in the 6-hydroxydopamine-lesioned rat, a model of Parkinson's disease, dosed at 10 mg/kg ip, but 48 was not active at 30 mg/kg po.