Pharmacological characterization of 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242), a high-affinity antagonist selective for κ-opioid receptors.

2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242) is a novel κ-opioid receptor (KOR) antagonist with high affinity for human (3 nM), rat (21 nM), and mouse (22 nM) KOR, a ∼ 20-fold reduced affinity for human µ-opioid receptors (MORs; K(i) = 64 nM), and negligible affinity for δ-opioid receptors (K(i) > 4 µM). PF-04455242 also showed selectivity for KORs in vivo. In rats, PF-04455242 blocked KOR and MOR agonist-induced analgesia with ID(50) values of 1.5 and 9.8 mg/kg, respectively, and inhibited ex vivo [(3)H](2-(benzofuran-4-yl)-N-methyl-N-((5S,7R,8R)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-yl)acetamide ([(3)H]CI977) and [(3)H](2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl) propanoyl]amino]propanoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide ([(3)H]DAMGO) binding to KOR and MOR receptors with ID(50) values of 2.0 and 8.6 mg/kg, respectively. An in vivo binding assay was developed using (-)-4-[(3)H]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(3)H]PF-04767135), a tritiated version of the KOR positron emission tomography ligand (-)-4-[(11)C]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(11)C]GR103545) in which PF-04455242 had an ID(50) of 5.2 mg/kg. PF-04455242 demonstrated antidepressant-like efficacy (mouse forced-swim test), attenuated the behavioral effects of stress (mouse social defeat stress assay), and showed therapeutic potential in treating reinstatement of extinguished cocaine-seeking behavior (mouse conditioned place preference). KOR agonist-induced plasma prolactin was investigated as a translatable mechanism biomarker. Spiradoline (0.32 mg/kg) significantly increased rat plasma prolactin levels from 1.9 ± 0.4 to 41.9 ± 4.9 ng/ml. PF-04455242 dose-dependently reduced the elevation of spiradoline-induced plasma prolactin with an ID(50) of 2.3 ± 0.1 mg/kg, which aligned well with the ED(50) values obtained from the rat in vivo binding and efficacy assays. These data provide further evidence that KOR antagonists have potential for the treatment of depression and addiction disorders.

Determination of guinea-pig cortical gamma-secretase activity ex vivo following the systemic administration of a gamma-secretase inhibitor.

(2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide (compound E) is a gamma-secretase inhibitor capable of reducing amyloid beta-peptide (1-40) and amyloid beta-peptide (1-42) levels. In this study we investigated the effect of in vivo administration of compound E on guinea-pig plasma, CSF and cortical amyloid beta-peptide (1-40) concentration. Using repeated sampling of CSF, compound E (30 mg/kg p.o.) was shown to cause a time-dependent decrease in CSF amyloid beta-peptide (1-40) levels, which was maximal at 3 h (70% inhibition), compared to baseline controls. After 3 h administration, compound E (3, 10 and 30 mg/kg p.o.), reduced plasma, CSF and DEA-extracted cortical amyloid beta-peptide (1-40) levels by 95, 97 and 99%; 26, 48 and 78%; 32, 33, and 47%, respectively, compared to vehicle control values. In the same animals, compound E (3, 10 and 30 mg/kg p.o.) inhibited cortical gamma-secretase activity, determined ex vivo using the recombinant substrate C100Flag, by 40, 71 and 79% of controls, respectively. These data demonstrate the value of determining not only the extent by which systemic administration of a gamma-secretase inhibitor reduces amyloid beta-peptide, but also the inhibition of brain gamma-secretase activity, as a more direct estimate of enzyme occupancy.

2-Carboxytetrahydroquinolines. Conformational and stereochemical requirements for antagonism of the glycine site on the NMDA receptor.

2-Carboxy-1,2,3,4-tetrahydroquinoline derivatives, derived from kynurenic acid, have been synthesized and evaluated for in vitro antagonist activity at the glycine site on the NMDA receptor. 2,3-Dihydrokynurenic acids show reduced potency relative to the parent lead compounds (Table I) possibly as a result of conformational effects. Removal of the 4-oxo group results in further reduced potency, but introduction of a cis-carboxymethyl group to the 4-position restores antagonist activity (Tables III and IV). Replacement of the keto group of 5,7-dichloro-2,3-dihydrokynurenic acid with other alternative H-bonding groups, for example cis- and trans-benzyloxycarbonyl and cis- and trans-carboxamido (Table V), gives comparable activity, but there is negligible stereoselectivity. A significant increase in potency and stereoselectivity is seen within the 4-acetate series (Table VI). The trans-4-acetic acid is significantly more potent than the corresponding lead kynurenic acid and has 100-fold greater affinity than the cis isomer. The results are consistent with a requirement in binding for a pseudoequatorially placed 2-carboxylate and clearly demonstrate the importance for binding of a correctly positioned hydrogen-bond-accepting group at the 4-position. The high-affinity binding of an anionic group in the 4-substituent binding pocket suggests that the glycine site and the neurotransmitter recognition (NMDA) site may have some features in common.

Identification of 3,5-dihydro-2-aryl-1H-pyrazolo[3,4-c]quinoline-1,4(2H)-diones as novel high-affinity glycine site N-methyl-D-aspartate antagonists.

Almost all of the existing known antagonists at the glycine site of the N-methyl-D-aspartate (NMDA) receptor have a low propensity for crossing the blood-brain barrier. It has been suggested that in many cases this may be due to the presence of a carboxylic acid which is a common feature of most of the potent full antagonists at this receptor. In this study, 2-aryl-1H-pyrazolo[3,4-c]quinoline-1,4(2H)-diones were found to have high-affinity binding at the glycine receptor. In particular, structure-activity studies identified 7-chloro-3,5-dihydro-2-(4-methoxyphenyl)-1H-pyrazolo[3,4-c]quinoline- 1,4(2H)-dione as the most potent of a series of analogues with an IC50 of 3.3 nM. The measured pKa values in this class of compounds (typically 4.0) indicate they are of equivalent acidity to carboxylic acids. Functional antagonism was demonstrated by inhibition of NMDA-evoked responses in rat cortical slices. Anticonvulsant activity in DBA/2 mice was achieved after dosing by direct injection into the cerebral ventricles, but no activity was seen after systemic administration, suggesting low brain penetration with this class of antagonists.

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

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

4-substituted-3-phenylquinolin-2(1H)-ones: acidic and nonacidic glycine site N-methyl-D-aspartate antagonists with in vivo activity.

4-Substituted-3-phenylquinolin-2(1H)-ones have been synthesized and evaluated in vitro for antagonist activity at the glycine site on the NMDA (N-methyl-D-aspartate) receptor and in vivo for anticonvulsant activity in the DBA/2 strain of mouse in an audiogenic seizure model. 4-Amino-3-phenylquinolin-2(1H)-one (3) is 40-fold lower in binding affinity but only 4-fold weaker as an anticonvulsant than the acidic 4-hydroxy compound 1. Methylsulfonylation at the 4-position of 3 gives an acidic compound (6, pKa = 6.0) where affinity is fully restored but in vivo potency is significantly reduced (Table 1). Methylation at the 4-position of 1 to give 18 results in the abolition of measurable affinity, but the attachment of neutral hydrogen bond-accepting groups to the methyl group of 18 produces compounds with comparable in vitro and in vivo activity to 1 (e.g., 23 and 28, Table 2). Replacement of the 4-hydroxy group of 1 with an ethyl group abolishes activity (42), but again, incorporation of neutral hydrogen bond acceptors to the terminal carbon atom restores affinity (e.g., 36, 39, and 40, Table 3). Replacement of the 4-hydroxy group of the high-affinity compound 2 with an amino group produces a compound with 200-fold reduced affinity (43; IC50 = 0.42 microM, Table 4) which is nevertheless still 10-fold higher in affinity than 3. The results in this paper indicate that anionic functionality is not an absolute requirement for good affinity at the glycine/NMDA site and provide compelling evidence for the existence of a ligand/receptor hydrogen bond interaction between an acceptor attached to the 4-position of the ligand and a hydrogen bond donor attached to the receptor.

Effect of plasma protein binding on in vivo activity and brain penetration of glycine/NMDA receptor antagonists.

A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity increases with lipophilicity of the 3-substituent up to a maximum at a log P around 3, then does not improve further. When combined with increasing protein binding, this gives a parabolic relationship between predicted in vivo activity and log P, with a maximum log P value of 2.39. Finally, the plasma protein binding studies have been extended to other series of glycine site antagonists, and its is shown that for a given log P these have similar protein binding to the 4-hydroxyquinolones, except for compounds that are not acidic. The results have implications for the design of novel glycine site antagonists, and it is suggested that it is necessary to either keep log P low or pKa high to obtain good central nervous system activity.

The pharmacological specificity of N-methyl-D-aspartate receptors in rat cerebral cortex: correspondence between radioligand binding and electrophysiological measurements.

1. The pharmacological specificity of N-methyl-D-aspartate (NMDA) receptors has been investigated in the rat cerebral cortex by use of radioligand binding and electrophysiological techniques. 2. A comparison was made between a functional assay (NMDA-induced depolarizations in a rat cortical slice preparation) and NMDA-sensitive L-[3H]-glutamate binding in the same brain region and species, to provide accurate affinity values for agonists and antagonists at the NMDA recognition site. 3. In a preparation of crude postsynaptic densities (PSD) from rat cortex, L-[3H]-glutamate bound with high affinity to an NMDA-sensitive population of sites with KD (geometric mean (-s.e.mean. + s.e. mean) = 120 (114, 126) nM, Bmax (mean +/- s.e.mean) = 11.4 +/- 0.8 pmol mg-1 protein and Hill coefficient (mean +/- s.e.mean) = 1.2 +/- 0.17 (n = 3). 4. There was a good agreement between the relative affinities in radioligand binding and electrophysiological assays for the receptor agonists NMDA, N-methyl-L-aspartate, quinolinate and trans-2,3-piperidine dicarboxylate, which are poor substrates of acidic amino acid transport systems. However, agonists which are good substrates for high affinity uptake systems (L- and D-glutamate, L- and D-aspartate, D-aspartate-beta-hydroxamate and L-glutamate-gamma-hydroxamate) were much weaker in the electrophysiological experiments. 5 Schild analysis of the antagonism of NMDA responses in the rat cortical slice by DL-3(2- carboxypiperazin-4-yl)propyl-1-phosphonate, D- and DL-2-amino-5-phosphonovalerate, D- and DL-2- amino- 7-phosphonoheptanoate, D-beta-aspartylaminomethylphosphonate, D-gamma-glutamylglycine and D-Ofaminoadipate (D-AA) indicated a competitive interaction with respective pA2 values of 6.17, 5.62, 5.24, 5.28, 5.20, 5.00, 4.43 and 3.97. 6 In the radioligand binding experiments the same antagonists inhibited only the NMDA-sensitive component of L-[3H]-glutamate binding. IC50 values showed a good correlation with the pA2 values (correlation coefficient = 0.96), with the exception of D-AA which was more potent than anticipated in the binding experiments (IC50 = 9.8 microM).7 These results confirm that NMDA-sensitive L-[3H]-glutamate binding sites represent the NMDA recognition site of the NMDA receptor and provide affinity values for both agonists and antagonists in the rat cerebral cortex, agreeing well with previous estimates in this and other tissues.

Comparison of the effects of the K(+)-channel openers cromakalim and minoxidil sulphate on vascular smooth muscle.

1 The actions of the potassium channel openers, cromakalim and minoxidil sulphate, were compared in a range of isolated blood vessel preparations. 2 Cromakalim and minoxidil sulphate inhibited spontaneous mechanical activity of the guinea-pig portal vein and relaxed the noradrenaline precontracted rat aorta with similar potency. In contrast, minoxidil sulphate was less potent than cromakalim in inhibiting spontaneous activity in the rat portal vein and was essentially inactive in the noradrenaline precontracted rat mesenteric artery and rabbit aorta. 3 Minoxidil sulphate did not antagonize the effects of cromakalim in the rabbit aorta indicating it was not acting as a partial 'agonist'. 4 Charybdotoxin, noxiustoxin and rubidium failed to discriminate between cromakalim and minoxidil sulphate indicating that the apparently selective effects of minoxidil sulphate were not mediated by either Ca(2+)-activated potassium channels, delayed rectifiers or rubidium impermeable potassium channels. 5 Glibenclamide antagonized the effects of cromakalim in an apparently competitive manner whereas the effects of minoxidil sulphate were antagonized in a non-competitive manner. The involvement of subtypes of ATP-sensitive potassium channels is discussed.

NR2B-containing NMDA receptors are up-regulated in temporal cortex in schizophrenia.

Saturation analyses of [3H]L-689,560, [3H]CGP 39653 and NMDA-specific [3H]ifenprodil binding revealed an equivalent increase (0.7 pmol/mg) in the number of [3H]L-689,560 and [3H]ifenprodil binding sites in superior temporal cortex (BA22) from drug-treated chronic schizophrenic patients and control subjects. No differences were observed between control and schizophrenic subjects for [3H]CGP 39653 binding in BA22, or for any of the radioligands binding to pre-motor cortex (BA6). Since [3H]L-689,560, [3H]CGP 39653 and [3H]ifenprodil label the glycine, glutamate and ifenprodil sites of the NMDA receptor complex, which are associated with NR1, NR1/NR2A and NR1/NR2B subunits respectively, our findings suggest that NR2B-containing receptors are selectively up-regulated in superior temporal cortex in schizophrenia.

Polyamines modulate [3H]L-689,560 binding to the glycine site of the NMDA receptor from rat brain.

The N-methyl-D-aspartate (NMDA) receptor complex possesses distinct recognition sites for glutamate, glycine and polyamines, which appear to be allosterically linked. We have investigated the effects of polyamines on the binding of the glycine site antagonist [3H](+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino - 1,2,3,4-tetrahydroquinoline ([3H]L-689,560), using rat cortex/hippocampus P2 membranes. Spermine and spermidine partially inhibited [3H]L-689,560 binding under non-equilibrium conditions, with IC50 values of 25.9 and 106 microM, respectively. The putative polyamine site antagonists arcaine, 1,10-diaminodecane, diethylenetriamine and putrescine had no effect on [3H]L-689,560 binding per se at 1 mM. The inhibition of [3H]L-689,560 binding by spermine was antagonised by arcaine in a competitive manner, but not by 1,10-diaminodecane, diethylenetriamine or putrescine. Kinetic analysis revealed that spermine (100 microM) decreased the association and dissociation rates of [3H]L-689,560 binding. In saturation experiments 100 microM spermine increased the KD for [3H]L-689,560 binding from 1.99 nM to 4.03 nM, with no effect on the number of binding sites. Spermine increased the affinity of glycine site agonists in displacing [3H]L-689,560 binding, with no effect on inhibition by partial agonists or antagonists, suggesting that spermine promotes an 'agonist-preferring' state. Modulation of [3H]L-689,560 binding by agonists for the polyamine and glutamate sites on the NMDA receptor did not appear to be additive in nature.

Allosteric modulation of the glutamate site on the NMDA receptor by four novel glycine site antagonists.

Using radioligand binding studies, we have investigated the binding properties of four 4-hydroxy-2-quinolones, a novel series of selective antagonists for the glycine site on the N-methyl-D-aspartate (NMDA) receptor. L-701,324, L-703,717, L-698,532 and L-695,902 inhibited [3H]L-689,560 (glycine site antagonist) binding to rat cortex/hippocampus P2 membranes with IC50 values of 1.97, 4.47, 209 and 6448 nM, respectively, whilst also inhibiting non-equilibrium [3H]dizocilpine binding to the NMDA receptor ion-channel. All four compounds partially inhibited L-[3H]glutamate (approximately 50% inhibition; agonist) binding and enhanced [3H]cis-4-phosphonomethyl-2-piperidine carboxylate ([3H]CGS-19755; 41-81% enhancement; 'C-5' antagonist) and [3H]3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate ([3H]CPP; 28-66% enhancement; 'C-7' antagonist) binding to the glutamate recognition site of the NMDA receptor with EC50 values similar to those observed for [3H]L-689,560 binding. These results provide further evidence for allosteric interactions between the glutamate and glycine recognition sites of the NMDA receptor complex, and as the 4-hydroxy-2-quinolones are 'full' antagonists at the glycine site, indicate that these interactions are not caused by the intrinsic activity of a compound.

Modulation of [3H]MK-801 binding to NMDA receptors in vivo and in vitro.

[3H]MK-801 binding in vivo was used to determine the occupancy of NMDA receptor ligands shown to allosterically modulate binding in vitro. ED(50) values (mg/kg) were obtained for the channel blockers (+)-5-methyl-10,11-dihydro-5,4-dibenzo[a,d]cyclohepten-5,10-imine maleate ((+)-MK-801, 0.2), 1-(1-phenylcyclohexyl)piperidine (phencyclidine, PCP, 1.7) and ketamine (4.4). Antagonists at the glutamate (DL-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (DL-CPP, 5.7)) and glycine site (7-Chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2(H)quinolinone (L-701,324, 14.1), 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414, 15.1)) inhibited [3H]MK-801 binding in vivo to varying maximum levels (69%, 103% and 45%, respectively). NR2B subunit-selective compounds acting at the ifenprodil site inhibited [3H]MK-801 in vivo by a maximum of 52-72% and gave ED(50) values (mg/kg) of: (+/-)-(1S*, 2S*)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol ((+/-)CP-101,606), 1.9; (+/-)-(3R, 4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol ((+/-)CP-283,097), 1.8; (+/-)-(R*, S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propanol ((+/-)Ro 25-6981), 1.0; ifenprodil, 6.0. The glycine site agonist D-serine stimulated binding to 151% of control with an ED(50) of 1.7 mg/kg. Results show that [3H]MK-801 binding in vivo may be used to measure receptor occupancy of ligands acting not only within the ion channel but also at modulatory sites on the NMDA receptor complex.

Neurophysiological signals as potential translatable biomarkers for modulation of metabotropic glutamate 5 receptors.

The Group I metabotropic glutamate receptor subtype 5 (mGluR5) is widely distributed in the brain with dense expression in the cerebral cortex, hippocampus, and basal ganglia. These receptors have been implicated in psychiatric and neurological disorders such as schizophrenia, Fragile X syndrome, addiction, anxiety/depression, Parkinson's disease and neuropathic pain. The present study evaluated the effects of the mGluR5 negative allosteric modulators (NAMs) 4-difluoromethoxy-3-(pyridine-2-ylethynyl)phenyl)5H-pyrrolo[3,4-b]pyridine-6(7H)-yl methanone (GRN-529) and methyl (3aR,4S,7aR)-4-hydroxy-4-[(3-methylphenyl)ethynyl]octahydro-1H-indole-1-carboxylate (AFQ056) on polysomnographic (PSG) and quantitative electroencephalographic (qEEG) measures in freely moving rats. Furthermore, the anxiolytic profile of GRN-529 was characterized in anesthetized rats by measuring stimulation-induced hippocampal theta oscillation. The present findings demonstrate that inhibition of mGluR5 via its allosteric site profoundly modulates high-level neuronal network activities as indicated by changes in sleep-wake activity and power distribution of qEEG. Both GRN-529 and AFQ056 reduced the total time spent in rapid-eye movement with AFQ056 producing a significant increase in wakefulness at the highest dose tested. Additionally, qEEG revealed significant compound-induced increases in delta power concomitant with more subtle decreases in theta and alpha band power. Receptor occupancy (RO) studies revealed that GRN-529 and AFQ056 at all doses resulted in over 45% mGluR5 occupancy. Furthermore, GRN-529 dose-dependently decreased elicited hippocampal theta frequency, consistent with previous findings using clinically active anxiolytic compounds. The described changes in neurophysiological signals identified in freely moving rats may be considered suitable translational biomarkers for the clinical evaluation of mGluR5 NAMs.

Interactions between the glutamate and glycine recognition sites of the N-methyl-D-aspartate receptor from rat brain, as revealed from radioligand binding studies.

The N-methyl-D-aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P2 membranes we have investigated the effect of glutamate recognition site ligands on [3H]glycine (agonist) and (+-)-4-trans-2-carboxy-5,7-dichloro-4- [3H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([3H]L-689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on L-[3H]glutamate (agonist), DL-3-(2-carboxypiperazin-4-yl)-[3H]propyl-1-phosphonate ([3H]CPP; "C-7" antagonist), and cis-4-phosphonomethyl-2-[3H]piperidine carboxylate ([3H]CGS-19755; "C-5" antagonist) binding to the glutamate site. "C-7" glutamate site antagonists partially inhibited [3H]L-689,560 binding but had no effect on [3H]glycine binding, whereas "C-5" antagonists partially inhibited the binding of both radioligands. Glycine, D-serine, and D-cycloserine partially inhibited [3H]CGS-19755 binding but had little effect on L-[3H]glutamate or [3H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (L-687,414), and 1-amino-1-carboxycyclobutane all enhanced [3H]CPP binding but had no effect on [3H]CGS-19755 binding, and (+)-HA-966 and L-687,414 inhibited L-[3H]glutamate binding. The association and dissociation rates of [3H]L-689,560 binding were decreased by CPP and D-2-amino-5-phosphonopentanoic acid ("C-5"). Saturation analysis of [3H]L-689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [3H]L-689,560 is discussed.

Recombinant human NMDA homomeric NR1 receptors expressed in mammalian cells form a high-affinity glycine antagonist binding site.

The cDNA NYMDAR1 (NR1) encodes a single polypeptide that forms a receptor-channel complex with electrophsiological and pharmacological properties characteristic of the N-methyl-D-aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-[3H]phenylaminocarbonylamino - 1,2,3,4-tetrahydroquinoline ([3H]L-689,560) bound to NR1a- and NR1e-transfected cells with high affinity (KD = 3.29 and 1.61 nM, respectively). Bmax values for NR1a- and NR1e-transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. Ki values for glycine site antagonists inhibiting [3H]L-689,560 binding to NR1e-transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four-to 16-fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist-preferring state. Ki values obtained with NR1a-transfected cells were approximately twofold lower than those obtained with NR1e-transfected cells. High-affinity binding to NR1-transfected cells was not observed with the transmitter recognition site radioligands L-[3H]glutamate and D,L-(epsilon)-2-[3H]amino-4-propyl-5-phosphono-3-pentanoic acid ([3H]CGP-39653) or the ion-channel radioligand [3H]dizocilpine ([3H]MK-801).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes.

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.

Characterization of the binding of [3H]L-689,560, an antagonist for the glycine site on the N-methyl-D-aspartate receptor, to rat brain membranes.

The binding characteristics of [3H]L-689,560 [(+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline], a selective antagonist for the glycine site on the N-methyl-D-aspartate receptor, have been evaluated using rat cortex/hippocampus P2 membranes. Specific [3H]L-689,560 binding was saturable, having a Kd of 2.97 nM and a Bmax of 4.15 pmol/mg of protein. The Bmax value was not significantly different from that obtained for [3H]glycine in the same membrane preparation, and L-689,560 and glycine were found to be mutually competitive. The specific binding of [3H]L-689,560 (1 nM) represented 96 +/- 0.02% (four experiments) of total binding. Association experiments at 4 degrees revealed that [3H]L-689,560 reached equilibrium in 120 min, with a t1/2 of 40 min. The dissociation of [3H]L-689,560 was slow at 4 degrees (t1/2 = 118 min), allowing the use of filtration to separate free from bound radioactivity. Both association and dissociation curves were best fitted by a double-exponential function, suggesting the presence of two components. Comparison of IC50 values obtained using [3H]glycine and [3H]L-689,560 binding for 21 glycine site ligands (including agonists, partial agonists, and antagonists, with affinities spanning 5 orders of magnitude) showed a 1:1 correlation, with a correlation coefficient of 0.97. This suggests that efficacy does not have a large influence on the affinity of glycine site ligands when an agonist or antagonist radioligand is used. Ligands for other amino acid recognition sites did not directly inhibit [3H]L-689,560 binding. [3H]L-689,560 is an improved radioligand for the glycine site that will facilitate further investigations of its properties.

Modulation of 45Ca2+ influx into cells stably expressing recombinant human NMDA receptors by ligands acting at distinct recognition sites.

A 45Ca2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45Ca2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45Ca2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45Ca2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45Ca2+ influx into cells expressing NR1a/NR2A receptors (IC50 = 408 microM) and NR1a/NR2B receptors (EC50 = 37.3 microM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC50 = 0.099 microM) compared with NR1a/NR2A receptors (IC50 = 164 microM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg2+ and Zn2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45Ca2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.

Delineation of the structural determinants of the N-methyl-D-aspartate receptor glycine binding site.

In this study, we have further delineated the domains of the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine co-agonist binding site. Taking an iterative approach, we have constructed truncation mutants of the NR1 subunit, transiently expressed them in HEK-293 cells, and determined the binding of the glycine site antagonist [3H]L-689,560. Amino acids 380-811 were sufficient to form a glycine binding site with affinities for [3H]L-689,560 and glycine that were not significantly different from wild-type NR1. More extensive deletions, from either the amino- or the carboxy-terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380-843 of NR1, replacing the transmembrane (TMI-TMIII) domain with intervening linker sequences while retaining the TMIV domain so as to anchor the polypeptide to the membrane. Although robust amounts of polypeptides were synthesised by transfected cells, only low levels of [3H]L-689,560 binding sites could be detected. This suggests that only a small proportion of the synthesised polypeptide folds in the appropriate manner so as to form a ligand binding site. These data indicate that although it is possible to reduce the glycine binding site to minimal so-called S1 and S2 domains, efficient folding of the polypeptide so as to form a ligand binding site may require sequences within the TMI-TMIII domain.