Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.

PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.

A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.

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.

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.

Abstract: In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1‐[1‐(2‐thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine‐insensitive [3H]glycine binding are altered in the presence of N‐methyl‐D‐aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis‐4‐phosphonomethyl‐2‐piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L‐glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 γM vs. 0.129 γM). Similar decreases in affinity are observed for the glycine site agonists, D‐serine and 1‐aminocyclopropane‐1‐carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1‐hydroxy‐3‐amino‐2‐pyrrolidone and 1‐aminocyclobutane‐1‐carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L‐glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

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.

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.

Activation of the N-methyl-D-aspartate (NMDA) receptor complex is subject to modulation via interactions at a coupled [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM). We examined the effect of the potent and specific glycine site antagonists, 1-hydroxy-3-amino-2-pyrrolidone (HA-966) and 1-aminocyclobutane-1-carboxylate (ACBC), on the NMDA recognition site. These glycine analogs were found to significantly stimulate the binding of the competitive NMDA antagonist, [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate ([3H]CPP) in a dose-dependent fashion, whereas both compounds inhibited NMDA-specific L-[3H]glutamate (agonist) binding. Additionally, both glycine antagonists reduced the binding of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to SPM, a functional assessment of activation of the NMDA receptor-channel complex. The glycine site agonists, glycine and serine reversed these effects in a dose-dependent manner, with the serine reversal being stereospecific for D-serine. The relative potencies of these compounds in reversing the glycine antagonist effects on the NMDA recognition site corresponded with their ability to competitively displace strychnine-insensitive [3H]glycine binding. These results provide evidence for a functional coupling between the glycine and NMDA recognition sites and further, may provide a mechanism by which compounds interacting at the glycine recognition site may modulate NMDA receptor activity.

Following intravenous administration, 1-aminocyclobutane-1-carboxylate (ACBC, 100 mg/kg), a N-methyl-D-aspartate (NMDA)-associated glycine receptor antagonist, was eliminated with a T1/2 of 5 min in mouse brain and 4 min in rat cerebrospinal fluid (CSF). 1-Aminocyclopropane-1-carboxylate (ACC), a NMDA-associated glycine receptor agonist, was found to have a T1/2 of less than 5 min in mouse brain. ACC and ACBC did not alter basal cerebellar cGMP. Glycine and D-serine increased cGMP, and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), a glycine antagonist, reversed the D-serine-induced increases in cGMP. In contrast, ACBC did not reverse the D-serine-induced increases in cGMP. These data suggest that despite their brain bioavailability and marked potency at the glycine receptor in vitro, ACC and ACBC are rapidly inactivated and thus have limited in vivo utility.

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.

Binding of the glycine site antagonist 3-[2-(Phenylamino-carbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt ([3H]GV150526A) was characterised in rat cerebral cortical membranes. Saturation experiments indicated the existence of a high affinity binding site, with a pKd value of 9.08 (Kd=0.8 nM) and a Bmax of 3.4 pmol/mg of protein. A strong linear correlation was observed between the displacement potencies for [3H]GV150526A and [3H]glycine of 13 glycine site ligands (r=0.991). The association kinetics of [3H]GV150526A binding was monophasic, with a kon value of 0.047 (nM)−1 min−1. Dissociation was induced by the addition of an excess of glycine, GV150526A, or 5,7-dichlorokynurenic acid (DCKA), another glycine antagonist. With GV150526A and DCKA, the dissociation curves presented similar koff values (0.068 and 0.069 min−1, respectively), as expected from ligands binding to the same site. Conversely, a significantly lower koff value (0.027 min−1) was found with glycine. Although these data may suggest that glycine agonists and antagonists bind to discrete sites with an allosteric linkage (rather than interacting competitively), the reason for this difference remains to be elucidated. It is concluded that [3H]GV150526A can be considered a new valuable tool to further investigate the properties of the glycine site of the NMDA receptor.

Guanine nucleotides have been examined for their effect on [3H]1-[1-(2-thienyl)-cyclohexyl]-piperidine ([3H]TCP) binding to rat forebrain synaptic plasma membranes (SPM). We report that of the series of guanine nucleotides tested, GTP, GDP, 5'-guanylylimidodiphosphate (Gpp(NH)p) and 5'-guanylylmethylenediphosphate (Gpp(CH2)p) are significantly more potent at decreasing [3H]TCP binding than GMP, cyclic GMP, and guanosine. GTP, the most potent compound tested, inhibited basal [3H]TCP binding with an IC50 of 38.7 microM. Stimulation of [3H]TCP binding with either the N-methyl-D-aspartate (NMDA) agonist, L-glutamate, or Mg2+ was also inhibited by GTP. Addition of GTP resulted in a rightward shift in the glutamate dose-response curve and a decrease in the maximum level of stimulation. The Mg2+ stimulation of [3H]TCP binding was completely blocked by the addition of GTP. These results, coupled with the previous findings that guanine nucleotides inhibit the binding of L-[3H]glutamate to the NMDA recognition site (Monahan et al., 1988), indicate that guanine nucleotides antagonize NMDA receptor-mediated neurotransmission, at least in part, through their action (direct or indirect) on the NMDA recognition site and thus may be endogenous negative modulators of the NMDA receptor.

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.

NMDA receptors (NMDARs) play an important role in neural plasticity including long-term potentiation and long-term depression, which are likely to explain their importance for learning and memory. Cognitive decline is a major problem facing an ageing human population, so much so that its reversal has become an important goal for scientific research and pharmaceutical development. Enhancement of NMDAR function is a core strategy toward this goal. In this review we indicate some of the major ways of potentiating NMDAR function by both direct and indirect modulation. There is good evidence that both positive and negative modulation can enhance function suggesting that a subtle approach correcting imbalances in particular clinical situations will be required. Excessive activation and the resultant deleterious effects will need to be carefully avoided. Finally we describe some novel positive allosteric modulators of NMDARs, with some subunit selectivity, and show initial evidence of their ability to affect NMDAR mediated events.This article is part of a Special Issue entitled 'Cognitive Enhancers'.

We have previously shown that D-cycloserine displaces [3H]glycine binding to a recognition site with properties consistent with an N-methyl-D-aspartate (NMDA) receptor modulatory site. Additionally, D-cycloserine positively modulates the NMDA receptor as evidenced by its dose-dependent enhancement of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the NMDA receptor-coupled ionophore. Further evaluation of this compound indicates that the maximal stimulation of [3H]TCP binding induced by D-cycloserine is lower than that produced by other compounds acting at the NMDA receptor associated glycine modulatory site (glycine and D-serine). Moreover, the stimulation of [3H]TCP binding induced by D-cycloserine in the presence of various fixed concentrations of glycine results in a family of dose-response curves which asymptotically converge to 40-50% of the maximal stimulation induced by glycine alone. These results are consistent with D-cycloserine acting as a partial agonist of the NMDA receptor via its interaction with the coupled glycine modulatory site.

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.

The pharmacokinetics of 2-amino-7-phosphonoheptanoate (AP7) in rat plasma and appearance in cerebrospinal fluid (CSF) are reported. Using high-performance liquid chromatography (HPLC) with fluorescence detection, concentrations of AP7 can be detected as low as 1.0 μM. Peak CSF concentrations (12 15 μM) for both the d -AP7 and d,l -AP7 are observed 10 15 min after i.v. administration and amount to approximately 0.1% of a 1 mmol/kg dose. Significant quantities (3 μM) are present in CSF at 2 h and no AP7 is detectable at 4 h. Following i.v. administration, a monoexponential clearance was observed for d -AP7 clearance from plasma, 15.4 ± 0.8 S.E.M. ml/min/kg with a t 1 2 of 38.9 ± 0.8 S.E.M. min. However, a biexponential clearance from plasma was observed for d,l -AP7.