p38 pathway kinases as anti-inflammatory drug targets.

Mitogen-activated protein kinases (MAPK) are intracellular signaling molecules involved in cytokine synthesis. Several classes of mammalian MAPK have been identified, including extracellular signal-regulated kinase, c-jun N-terminal kinase, and p38 MAP kinase. p38alpha is a key MAPK involved in tumor necrosis factor alpha and other cytokine production, as well as enzyme induction (cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloproteinases) and adhesion molecule expression. An understanding of the broad biologic and pathophysiological roles of p38 MAPK family members has grown significantly over the past decade, as has the complexity of the signaling network leading to their activation. Downstream substrates of MAPK include other kinases (e.g., mitogen-activated protein-kinase-activated protein kinase 2) and factors that regulate transcription, nuclear export, and mRNA stability and translation. The high-resolution crystal structure of p38alpha has led to the design of selective inhibitors that have good pharmacological activity. Despite the strong rationale for MAPK inhibitors in human disease, direct proof of concept in the clinic has yet to be demonstrated, with most compounds demonstrating dose-limiting adverse effects. The role of MAPK in inflammation makes them attractive targets for new therapies, and efforts are continuing to identify newer, more selective inhibitors for inflammatory diseases.

Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist.

Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.

Bivalent binding and signaling characteristics of Leridistim, a novel chimeric dual agonist of interleukin-3 and granulocyte colony-stimulating factor receptors.

Leridistim is a member of a novel family of engineered chimeric cytokines, myelopoietins, that contain agonists of both interleukin-3 (IL-3) receptors (IL-3R) and granulocyte colony-stimulating factor (G-CSF) receptors (G-CSFR). To more clearly understand Leridistim's function at the molecular level, binding to both IL-3R and G-CSFR and subsequent signaling characteristics have been delineated. The affinity of Leridistim for the human G-CSFR was found to be comparable to that of native G-CSF (IC(50) = 0.96 nM and 1.0 nM, respectively). Both Leridistim and G-CSF induced receptor tyrosine phosphorylation to a similar maximal level. Compared with native recombinant human IL-3 (rhIL-3), Leridistim was found to possess higher affinity for the IL-3R alpha chain (IL-3Ralpha) (IC(50) = 85 nM and 162 nM, respectively). However, the increase in Leridistim binding affinity to the functional, high-affinity heterodimeric IL-3Ralphabeta(c) receptor is lower than that observed with rhIL-3 (85 nM and 14 nM vs 162 nM and 3.5 nM, respectively). Leridistim induced tyrosine phosphorylation of beta(c) to a level comparable to native IL-3, and the level of JAK2 tyrosine phosphorylation in cells expressing both IL-3R and G-CSFR was comparable to that observed with IL-3 or G-CSF alone. The ability of Leridistim to interact with IL-3R and G-CSFR simultaneously was demonstrated using surface plasmon resonance analysis. These studies were extended to demonstrate that Leridistim exhibited a higher affinity for the IL-3R on cells that express both the IL-3Ralphabeta(c) and the G-CSFR (IC(50) = 2 nM) compared with cells that contain the IL-3Ralphabeta(c) alone (IC(50) = 14 nM). Leridistim binds to both IL-3R and G-CSFR simultaneously and has been shown to activate both receptors. The bivalent avidity may explain the unique biologic effects and unexpected potency of Leridistim in hematopoietic cells compared with rhIL-3 or G-CSF alone or in combination.

Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor.

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.

Synthesis, preliminary evaluation and molecular modeling studies of new, conformationally constrained analogues of the competitive NMDA receptor antagonist 4-(phosphonomethyl)-2-piperidinecarboxylic acid (CGS 19755).

Two new spirobicyclophosphonate isomers (19 and 20), conformationally constrained analogues of the potent competitive NMDA antagonist CGS 19755 (4), have been designed and synthetized with the aim of gaining insight into the conformational preference of the crucial distal phosphonate moiety at the antagonist NMDA binding site. The preliminary biological evaluation reveals that the activity as NMDA antagonist resides only in the (1R,5S,7R)-isomer (19), characterized by a (-)-gauche disposition around the C1-C5 bond, thus confirming previously reported pharmacophore models.

Characterization of a monoclonal antibody that neutralizes the activity of prostaglandin E2.

We describe the generation of an IgG1 mAb, 2B5, that neutralizes the biologic activity of PGE2 in vitro and in vivo. The Ab was derived from a BALB/c mouse that was immunized with a PGE2-thyroglobulin conjugate. 2B5 is one of the highest affinity and specific anti-PGE2 mAbs reported to date. The Kd for PGE2 was approximately 300 pM and crossreactivity toward eicosanoids other than PGE1 was less than 1%. The addition of 2B5 to [3H]PGE2 blocked the binding of radioligand to cell membranes transfected with the murine EP3 prostaglandin receptor. In functional studies, 2B5 neutralized the capacity of PGE2 to suppress T cell proliferation induced by a mitogenic anti-CD3 Ab in vitro. In contrast, immunosuppression by the phosphodiesterase inhibitor, isobutylmethylxanthine was not affected. In an in vivo model of nociception, 2B5 substantially reduced the dorsoflexion response of mice to phenylbenzoquinone. This response is associated with prostaglandin production and is blocked by inhibitors of prostanoid synthesis. Our findings demonstrate that this nociceptive response is largely due to PGE2. In the absence of antagonists that prevent PGE2 from activating a diverse family of receptor subtypes, neutralizing Abs to PGE2 should represent useful reagents to delineate the biologic properties of this eicosanoid in vivo.

Rapid and selective induction of blood eosinophilia in guinea pigs by recombinant human interleukin 5.

Interleukin 5 (IL-5) has been implicated as a causal mediator in the development of pulmonary eosinophilia and airway hyperreactivity in human asthma. Supportive evidence for a pathogenic role of IL-5 in this disease has been provided by guinea pig models in which antigen-induced lung eosinophilia and bronchial hyperresponsiveness have been specifically attenuated with a neutralizing antibody to IL-5. In the present report, we describe a rapid mechanism-based model of IL-5-induced eosinophilia in the guinea pig. Our results show that intravenous injection of human IL-5 induced a 5-10-fold increase in the percentage and number of eosinophils in blood within 1 hour. In contrast, neutrophils and mononuclear cells were not recruited during this time. The increase in eosinophils was blocked by pretreatment of animals with an anti-IL5 monoclonal antibody (TRFK5) in doses similar to those which inhibit eosinophilia in guinea pig asthma models. Furthermore, dexamethasone, a potent inhibitor of eosinophilia in guinea pig asthma, profoundly suppressed the eosinophilia induced by human IL-5. This rapid model will be useful for elucidating the eosinophil activating properties of IL-5 in vivo and may potentially facilitate the development of IL-5 receptor antagonists for the specific blockade of the eosinophilic inflammation observed in human asthma.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.

Indole-2-carboxylates, novel antagonists of the N-methyl-D-aspartate (NMDA)-associated glycine recognition sites: in vivo characterization.

Recent in vitro receptor binding studies have indicated that indole-2-carboxylates with halogen substitutions at the position 5 or 6 are potent competitive antagonists of the NMDA (N-methyl-D-aspartate)-associated strychnine-insensitive glycine receptor (Gray N. M., Dappen M. S., Cheng B. K., Cordi A. A., Biesterfeldt J. P., Hood W. F. and Monahan J. B. (1992) J. med. Chem. 34: 1283-1292; Hood W. F., Gray N. M., Dappen M. S., Watson G. B., Compton R. P., Cordi A. A., Larthorn T. H. and Monahan J. B. (1992) J. Pharmac. exp. Ther. 262: 654-660). In the present investigation, a series of indole-2-carboxylates and two putative antagonists of glycine receptor HA-966 (3-amino-l-hydroxypyrrolidin-2-one) and 7-chlorokynurenic acid were examined for their effects on cGMP responses, mediated by the NMDA receptor complex, in vivo. Both SC-49648 (6-chloro-2-carboxyindole-3-acetic acid, intracerebellar injection, i.c.b.) and HA-966 (i.c.b. or intraperitoneal, i.p.) antagonized increases in levels of cyclic GMP in the cerebellum of the mouse, induced by the intracerebellar administration of NMDA and D-serine, agonists of the NMDA and the NMDA-associated glycine recognition sites, respectively. The drugs SC-49648 and 7-chlorokynurenic acid (i.p.) did not affect cGMP responses, suggesting poor bioavailability in brain. Following direct intracerebellar injection, SC-49648 was eliminated with a half-life of 12 min from the brain. Following intraperitoneal administration, SC-50132, the 3-ethylester analog of SC-49648, was eliminated from the brain with a half-life of 35 min and was found to be metabolized to SC-49648, in vivo. Some lipophilic analogs of SC-49648, designed as its prodrugs, were minimally active as glycine antagonists, in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of indole-2-carboxylate derivatives as antagonists of N-methyl-D-aspartate receptor activity at the associated glycine recognition site.

We have synthesized a series of indole-2-carboxylate derivatives and, with the use of radioligand binding, electrophysiological techniques and an in vivo transient bilateral carotid occlusion model of ischemic damage known to be sensitive to NMDA antagonists, have evaluated the indole-2-carboxylate derivatives ability to inhibit N-methyl-D-aspartate (NMDA) receptor activity through the associated glycine modulatory site. By using [3H]glycine to label this modulatory site, we found that the compounds with the highest affinity (Ki less than 1 microM) contained a chloro group at position C-6 and a polar, hydrogen-bond-accepting group at position C-3 of the indole ring. When these compounds were tested for their ability to modulate [3H]MK-801 [(+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclophepten-5,10- imine maleate) binding, a functional assessment of NMDA receptor activation, binding was inhibited, indicative of NMDA receptor antagonist character. Schild regression analysis indicated that this antagonism was competitive with glycine. Next, several of these indole-2-carboxylate derivatives were analyzed electrophysiologically in rat cortex mRNA-injected Xenopus oocytes shown to express a functional NMDA receptor channel complex. These compounds inhibited NMDA receptor activity in a manner noncompetitive with NMDA. They also produced a parallel right-ward shift in the glycine dose response for potentiation of the NMDA responses in the oocytes and thus provided further evidence for a competitive interaction at the glycine site. Finally, in vivo transient bilateral carotid artery occlusion experiments revealed that these compounds were capable of reducing the damage typically associated with an ischemic insult in Mongolian gerbil hippocampal neurons.

Characterization of 3-carboxy-5-phosphono-1,2,3,4-tetrahydroisoquinoline (SC-48981), a potent competitive N-methy-D-aspartate (NMDA) receptor antagonist, in vitro and in vivo.

(+/-)-3-Carboxy-5-phosphono-1,2,3,4-tetrahydroisoquinoline (SC-48981), a conformationally restricted analog of the potent competitive N-methyl-D-aspartate (NMDA) antagonist, 2-amino-5-phosphonopentanoate (AP-5), potently inhibited the binding of [3H]glutamate to the N-methyl-D-aspartate (NMDA) receptors with a Ki of 1.6 mcM, but with minimal affinity for kaininate and quisqualate receptors (Ki greater than 50 mcM), in vitro. Consistent with its ability to antagonize the NMDA receptor, SC-48981 decreased the binding of [3H]glycine and [3H]TCP [1-(2-thienyl)cyclohexylpiperidine] to the NMDA-associated glycine and phencyclidine (PCP) recognition sites, in vitro. SC-48981 attenuated levels of basal cGMP and harmaline-induced increases in levels of cGMP in the mouse cerebellum, in vivo, in a competitive manner, with ED50 values of 5.5 and 8.7 mg/kg, i.p. Direct intracerebellar injection of SC-48981 (0.5 microgram) attenuated increases in levels of cGMP induced by central injection of the NMDA-associated glycine receptor agonist, D-serine and by NMDA itself. Parenteral administration of SC-48981 (25 mg/kg, s.c.) decreased basal levels of cGMP for up to 3 h. These results indicate that SC-48981 represents a novel bioavailable competitive NMDA antagonist with a long duration of action.

Synthesis, absolute configuration and activity at N-methyl-D-aspartic acid (NMDA) receptor of the four D-2-amino-4,5-methano-adipate diastereoisomers.

The four D-2-amino-4,5-methano-adipates 26, 27, 32, 33 were synthesized and their biological activity at the N-methyl-D-aspartate (NMDA) receptor was assessed. The synthesis involved as a key step a rhodium acetate dimer catalyzed addition of ethyl diazoacetate to the protected D-allylglycine (17). In vitro receptor binding using L-[3H]glutamate as the radioligand provided affinity data, while modulation of [3H]TCP binding was used as a functional assay. The analogues were also evaluated in [3H]kainate and [3H]AMPA binding to assess selectivity over non-NMDA glutamate receptors. Three of the four diastereoisomer, D-CAA B (27), C (32) and D (33) were shown to have agonist properties at the NMDA-site, while the fourth, (2R,4R,5R) D-CAA A (26) was characterized as an NMDA-site atypic antagonist.

Novel indole-2-carboxylates as ligands for the strychnine-insensitive N-methyl-D-aspartate-linked glycine receptor.

A series of indole-2-carboxylates were prepared and evaluated for their ability to inhibit the binding at the strychnine-insensitive glycine receptor that is associated with the NMDA-PCP-glycine receptor complex. All of the compounds were selective for the glycine site relative to other sites on the receptor macrocomplex and several of the compounds in this series were found to have submicromolar affinity for this receptor. The lead compound, 2-carboxy-6-chloro-3-indoleacetic acid (Ki = 1.6 microM vs [3H]glycine), was also found to noncompetitively inhibit the binding of MK-801, a ligand for the phencyclidine site on the receptor macrocomplex. These latter data suggest that the compound functions as an antagonist at the strychnine-insensitive glycine receptor. The structural activity relationships within this series of indole-2-carboxylates is discussed and several key pharmacophores are identified for this series of glycine ligands. In general, the most potent compounds were the C-3 acetamides, with N-propyl-2-carboxy-6-chloro-3-indoleacetamide having the highest receptor affinity.

Contrasting neurochemical interactions of tiletamine, a potent phencyclidine (PCP) receptor ligand, with the N-methyl-D-aspartate-coupled and -uncoupled PCP recognition sites.

Neurochemical interactions of tiletamine, a potent phencyclidine (PCP) receptor ligand, with the N-methyl-D-aspartate (NMDA)-coupled and -uncoupled PCP recognition sites were examined. Tiletamine potently displaced the binding of [3H]1-(2-thienyl)cyclohexylpiperidine with an IC50 of 79 nM without affecting sigma-, glycine, glutamate, kainate, quisqualate, or dopamine (DA) receptors. Like other PCP ligands acting via the NMDA-coupled PCP recognition sites, tiletamine decreased basal, harmaline-, and D-serine-mediated increases in cyclic cGMP levels and induced stereotypy and ataxia. Tiletamine was nearly five times more potent than PCP at inhibiting the binding of 3-hydroxy[3H]PCP to its high-affinity NMDA-uncoupled PCP recognition sites. However, following parenteral administration, dizocilpine maleate (MK-801), ketamine, PCP, dexoxadrol, and 1-(2-thienyl)cyclohexylpiperidine HCl, but not tiletamine, increased rat pyriform cortical DA metabolism and/or release, a response modulated by the NMDA-uncoupled PCP recognition sites. Pretreatment with tiletamine did not attenuate the MK-801-induced increases in rat pyriform cortical DA metabolism, a result suggesting that tiletamine is not a partial agonist of the NMDA-uncoupled PCP recognition sites in this region. However, following intracerebroventricular administration (100-500 micrograms/rat), tiletamine increased pyriform cortical DA metabolism with a bell-shaped dose-response curve. These data indicate a differential interaction of tiletamine with the NMDA-coupled and -uncoupled PCP recognition sites. The paradoxical effects of tiletamine suggest that tiletamine might activate receptor(s) or neuronal pathways of unknown pharmacology.

Synthesis and biological evaluation of cyclopropyl analogues of 2-amino-5-phosphonopentanoic acid.

A series of cyclopropyl analogues related to 2-amino-5-phosphonopentanoic acid (AP5) were synthesized and their biological activity was assessed as competitive antagonists for the N-methyl-D-aspartate (NMDA) receptor. In vitro receptor binding using [3H]-L-glutamate as the radioligand provided affinity data, while modulation of [3H]MK-801 binding was used as a functional assay. The analogues were also evaluated in [3H]kainate binding to assess selectivity over non-NMDA glutamate receptors. Of the compounds tested, 4,5-methano-AP5 analogue 26 was the most potent selective NMDA antagonist; however, potency was lower than that for [[(+/-)-2-carboxypiperidin-4-yl]methyl]phosphonic acid (CGS 19755, 5).

Identification of a novel structural class of positive modulators of the N-methyl-D-aspartate receptor, with actions mediated through the glycine recognition site.

This study describes a new structural class of compounds which interact at the N-methyl-D-aspartate (NMDA) receptor-associated glycine recognition site. These E-gamma-substituted vinylglycine derivatives were active in displacing [3H]glycine binding from the NMDA receptor-associated recognition site in rat forebrain synaptic plasma membranes, with Ki values in the range of 0.24-8.7 microM. Functional analyses of these compounds indicate that they positively modulate basal [3H](+)-5-methyl-10,11-dihydro-5H- [a,d]cyclohepaten-5,10-imine ([3H]MK-801) binding, consistent with their having agonist characteristics. Little stereospecificity is observed with the gamma-substituted methyl and propyl derivatives while the L-isomer of the hexyl analog is significantly more potent than the D-isomer. The D- and L-hydroxyethyl gamma-substituted vinylglycines were the most potent inhibitors of [3H]glycine binding with Ki values of 0.75 +/- 0.06 microM and 0.24 +/- 0.02 microM, respectively. The 3,4-double bond was necessary for activity in that the saturated hexyl derivative (2-aminodecanoate) was inactive. Based on the results reported herein, the hypothesis that there is a distinct size restriction for functional agonists which interact with the glycine recognition site, should be altered to include these larger extensions of vinylglycine.

Sigma receptors modulate both A9 and A10 dopaminergic neurons in the rat brain: functional interaction with NMDA receptors.

The sigma receptor ligands, (+)-pentazocine and (+)-SKF 10,047, were found to increase dopamine metabolism (DOPAC, HVA) and release (3-MT) in both the striatum and olfactory tubercle of the rat, in a dose-dependent manner, after central as well as peripheral administration. The effect of (+)-SKF 10,047 was stereospecific. The increase in dopamine metabolism was not blocked by naloxone pretreatment, excluding an action via opioid receptors. More interestingly, this modulation was blocked by pretreatment with the NMDA receptor antagonist, CPP. Neither sigma ligand exhibited any affinity for D1 or D2 dopamine receptors or for NMDA, PCP or NMDA-associated glycine receptors. Sigma receptors thus appear to modulate dopaminergic function in both A9 and A10 projections. This modulation appears to involve a functional interaction with NMDA receptors or an NMDA-utilizing synapse downstream to neurons modulated by sigma receptors.

Cis-2,4-methanoglutamate is a potent and selective N-methyl-D-aspartate receptor agonist.

Cis- and trans-2,4-methanoglutamate were compared with L-glutamate as acidic amino acid ligands. Cis-2,4-methanoglutamate had a Ki of 0.052 microM against N-methyl-D-aspartate (NMDA)-specific L-[3H]glutamate binding compared with 0.050 microM for L-glutamate. Cis-2,4-methanoglutamate exhibited no significant affinity against [3H]kainate or [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate ([3H]AMPA) binding. Trans-2,4-methanoglutamate had no significant affinity for any of these sites. Cis-2,4-methanoglutamate increased [3H]N-1[2-thienyl]cyclohexyl-3,4-piperidine [( 3H]TCP) binding with EC50 of 0.35 +/- 0.14 microM. It produced an inward current in rat brain mRNA-injected Xenopus oocytes which was blocked by the NMDA antagonist, D-2-amino-7-phosphonoheptanoate (D-AP7). Cis-2,4-methanoglutamate (EC50 = 15.9 microM) was 100-fold more potent than L-glutamate (EC50 = 1,584 microM) in reducing the excitatory postsynaptic potential in CA1 of hippocampal slices. Cis-2,4-methanoglutamate is the most potent, selective NMDA agonist known.

Differential modulation of the associated glycine recognition site by competitive N-methyl-D-aspartate receptor antagonists.

The competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate and two other five-atom linkage (C-5) omega-phosphono-alpha-amino acid analogs reduced [3H]glycine binding, in a dose-dependent manner, to a maximum of 45-55%, whereas seven-atom linkage (C-7) analogs had significantly less effect. The IC50 of the C-5 antagonists for the inhibition of [3H]glycine binding closely paralleled their potency both in displacing NMDA-selective L-[3H]glutamate binding and in negatively modulating (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) binding. Additionally, reduction of glycine binding by the C-5 antagonists was reversed by both NMDA receptor agonists and C-7 competitive NMDA antagonists, providing evidence that the site of action of these C-5 antagonists is the NMDA recognition site, resulting in indirect modulation of the glycine site. These data imply a functional coupling between the NMDA and associated glycine recognition sites and, furthermore, suggest a differential interaction of C-5 and C-7 competitive NMDA antagonists with the NMDA receptor complex.

Characterization of D-3,4-cyclopropylglutamates as N-methyl-D-aspartate receptor agonists.

The 4 configurational isomers of D-3,4-cyclopropylglutamate (D-CGA) have been synthesized and analyzed for their interactions as excitatory amino acid recognition sites. Additionally, functional assessment of the action of these compounds at the N-methyl-D-aspartate (NMDA) receptor was performed. All 4 analogs function as agonists at the NMDA receptor as evidenced by their ability to stimulate [3H]MK-801 binding to the coupled PCP recognition site. Furthermore, the rank order of potency of these compounds in stimulating [3H]MK-801 binding corresponds with their Ki values for the displacement of NMDA-selective L-[3H]glutamate and [3H]CGS-19755 binding (D-CGA-C greater than D-CGA-B greater than D-CGA-D greater than D-CGA-A). The D-CGA-C isomer has affinity and potency at the NMDA receptor similar to the endogenous agonist, L-glutamate. This high potency coupled with greater specificity than L-glutamate, makes D-CGA-C a potentially useful pharmacological tool for the study of this receptor.