0022-3565/92/2623-0947$03.

00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 262, No. 3
Copyright C 1992 by The American Society for Pharmacologj, and Experimental Therapeutics Printd in U.S.A.

Potent Indole- and Quinoline-Containing N-Methyl-D-Aspartate

Antagonists Acting at the Strychnine-Insensitive Glycine Binding
Site

B. M. BARON, B. L. HARRISON, I. A. McDONALD, B. S. MELDRUM, M. G. PALFREYMAN, F. G. SALITURO,

B. W. SIEGEL, A. L. SLONE, J. P. TURNER1 and H. S. WHITE
Marion Merrell Dow Research Institute, 21 10 E. Galbraith Rd., Cincinnati, Ohio 45215 (B.M.B., B.L.H., 1A.M., M.G.P., F.G.S., B.W.S., A.L.S.);
Department of Pharmacology and Toxicology, Anticonvulsant Drug Development Program, 421 Wakara Way, University of Utah,
Saft Lake City, Utah 84108 (H.S.W.); and Department of Neurology, Institute of Psychiatry, De Crespigny Park,
Denmark Hill, London SE5 8AF, U.K. (B.S.M., J.P.T.)
Accepted for publication May 18, 1992

ABSTRACT
The N-methyl-D-aspartate (NMDA)-prefemng glutamate receptor inhibited the use-dependent binding of [3H]N-[1 -(2-thienyl)
subtype possesses, in addition to the recognition site for gluta- cyclohexyl]-piperidine and were noncompetitive, glycine-reversi-
mate, a binding site for glycine. We report here on the phar- ble inhibitors of both NMDA-induced biochemical and electro-
macological properties of 3-(4,6-dichloro-2-carboxyindol-3-yl)- physiological responses in brain slice preparations. A competitive
propionic acid (MDL 29,951) and 4-carboxymethylamino-5,7- interaction with the glycine binding site was also evident in that
dichloroquinoline-2-carboxylic acid (MDL 100,748), two novel MDL 29,951 and MDL 1 00,748 produced parallel rightward shifts
glycine antagonists of NMDA receptor activation in vitro and in in the glycine requirement for demonstration of NMDA-stimulated
vivo. We have measured in parallel the effects of two previously elevations in cytosolic calcium in cultured neuronal preparations.
described glycine antagonists, 7-chlorokynurenic acid and 5,7- The glycine antagonists were potent anticonvulsants after their
dichlorokynurenic acid. All were potent inhibitors of [3H]glycine i.c.v. administration to audiogenic seizure-susceptible DBA/2J
binding. K, values (SM) were 0.36 (7-chlorokynurenic acid), 0.08 mice. Because the compounds chosen encompass a variety of
(5,7-dichlorokynurenic acid), 0.07 (MDL 1 00,748) and 0.1 4 (MDL chemical structures, the results indicate that glycine is required
29,951). MDL 1 00,748 and MDL 29,951 were approximately for NMDA receptor activation and that bioavailable glycine an-
2000-fold selective for the glycine binding site relative to the tagonists may form the basis of a novel therapy for epilepsy.
glutamate recognition sites. All four compounds completely

The NMDA class of excitatory amino acid (EAA) receptors et at., 1990), or that it may control the rate of desensitization
consists of a glutamate recognition site linked to an ion channel of the receptor (Mayer et aL, 1989), or, indeed, both (Lerma et
permeable to monovalent cations and Ca. The activation of aL, 1990; for a review see Palfreyman and Baron, 1991). The
the receptor complex is modulated by a number of sites (for nonselective EAA antagonist, kynurenic acid, has been dem-
reviews see Watkins, 1989; Mayer, 1991). These sites include a onstrated to interact with the glycine site on the NMDA
strychnine-insensitive glycine binding site, first described in receptor with moderate affinity (Kessler et aL, 1989a). However,
cultured mouse brain neurons by Johnson and Ascher (1987), at similar concentrations, kynurenic acid also binds to the
through which glycine enhances NMDA receptor activation. glutamate/NMDA recognition site (Olverman et al., 1988),
Subsequent studies suggested that glycine is an obligatory making this molecule a poor tool to study the role of glycine in
coagonist of glutamate at the NMDA receptor (Kleckner and receptor activation. Other compounds such as 6-cyano-7-nitro-
Dingledine, 1988; Luzzi et aL, 1988; Shirasaki et aL, 1990; Baron quinoxaline-2,3-dione, 6,7-dinitro-quinoxaline-2,3-dione and
6,7-dichloro-3-hydroxyquinoxaline-2-carboxylic acid (Birch et
Received for publication March 31, 1992. at., 1988; Kessler et aL, 1989b; Kleckner and Dingledine, 1989),
1 Present address: University of Wales College of Cardiff, Department of 7-CKA (Kemp et al., 1988) and 5,7-DCKA (Baron et at., 1990,
Physiology, Cardiff CF1 1SS, U.K. 1991; McNamara et aL, 1990) have also been demonstrated to

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; MDL 29,951 , 3-(4,6-dichloro-2-carboxyindol-3-yl)propionic acid; MDL 1 00,748, 4-carboxymeth-
ylamino-5,7-dichloroquinoline-2-carboxylic acid; 7-CKA, 7-chlorokynurenic acid; 5,7-DCKA, 5,7-dichlorokynurenic acid; TCP, N-[1-(2-thienyl)cyclo-
hexyl]pipendine; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate; CPP, 3-(2-carboxypiperazin-4-yl)-propyl-1 -phosphonic acid; EAA, ex-
citatory amino acid.

947
948 Baron etal. Vol. 262

be antagonists at the glycine site, although they are also, in centrifuged at 4’C. This
resuspension and incubation procedure was
varying degrees, antagonists of other EAA receptor subtypes repeated two more Assays were performed
times. in glass tubes con-
(Honore et at., 1988). taming 2 nM [3H]TCP, 100 g of membrane protein and unlabeled
liganda as indicated in a final volume of 0.5 ml of 5 mM Tris-acetate
Recently we have described the chemical syntheses and prop-
(pH 7.4) buffer. Nonspecific binding was defined as that remaining in
erties of two new series of glycine antagonists, MDL: 100,748
the presence of 100 iM unlabeled phencyclidine. After a 2-h incubation
(Harrison et at., 1990; fig. 1) and MDL 29,951 (Salituro et a!.,
at 25#{176}C,
samples were filtered through glass fiber (GF/B) filter strips
1990; fig. 1). In this article, we present full details ofthe binding presoaked in 0.1% (v/v) polyethyleneimine and washed three times
properties of the compounds and show that they are potent, with 3 ml of ice-cold assay buffer. These assay parameters were chosen
selective glycine site liganda. We also show that there is a to maximize the magnitude of NMDA and glycine-induced increases
relationship between binding potency ([3H]glycine) and the in [3H]TCP binding and do not necessarily represent equilibrium
effects of the compounds in attenuating NMDA-elicited bio- conditions. Specific binding (n = 3 experiments), measured in the
chemical and electrophysiological responses. These results are presence of 10 nM glycine + 100 iM NMDA was 623 ± 71 fmol/mg

compared with those obtained using 7-CKA and 5,7-DCKA. protein and rose to 1102 ± 241 fmol/mg in the presence of 300 nM
glycine + 100 M NMDA.
Further evidence will be presented to demonstrate that MDL
[311]AMPA binding. [3HJAMPA (27.6 Ci/mmol) binding was per-
100,748 and MDL 29,951 are extremely effective antiseizure
formed as describedby Honore and Drejer (1988). Assay vials contained
agents in DBA/2J audiogenic seizure-susceptible mice.
0.5 mg ofmembrane protein, 5 nM [3HJAMPA, 100 mM KSCN, various
concentrations of test compounds, 2.5 mM CaC12 and 30 mM Tris-HC1
Materials and Methods (pH 7.4 at 4#{176}C) in a final volume of 0.5 ml. Nonspecific binding was
defined using 1 mM L-glutamate. Specific binding accounted for >85%
Synthesis of glycine antagonists. 7-CKA, 5,7-DCKA, MDL of total binding. After incubation on ice for 60 mm, bound ligand was
29,951 and MDL 100,748 were synthesized (vide at the Marion
supra) collected by centrifugation.
Merrell Dow Research Institute (Cincinnati, OH) and were >99% pure, NMDA-evoked elevation of intracellular calcium in cerebel-
as indicated from elemental analysis and spectroscopic data. lax granule cells. Cerebella from 12 to 16 8-day-old rats were removed,
[3H]Glycine binding. [3HjGlycine (43.5 Ci/mmol, DuPont NEN, fmely chopped using a razor blade and suspended in 30 ml of buffer A
Boston, MA) binding was measured as described by Baron et at. (1990). consisting of Krebs-Ringer bicarbonate buffer (mM: 121.0 NaC1, 4.8
Assays were conducted in minivials, incubated for 30 mm on ice in 0.5 KC1, 1.2 KH2PO4, 25.5 NaHCO3, 14.3 glucose, 0.15% phenol red, pH
ml of 50 mM HEPES-KOH (pH 7.4) containing 20 nM [3Hjglycine, 7.4) supplemented with 1.2 mM MgSO4 and 0.3% BSA. After centrif-
100 g of membrane protein, unlabeled ligands as indicated and 100 ugation for 15 sec at 150 x g, the pelleted tissue was resuspended in 20
M strychnine. Nonspecific binding was defined by inclusion of 100 ml of buffer A, 2 ml of 2.5 mg/mi trypsin (type III, Sigma Chemical
,M D-serine. Bound ligand was isolated by centrifugation. Specific Co., St. Louis, MO) was added and the mixture was incubated for 15
binding accounted for 75% of total binding, which was generally about mm at 37’C in a shaking water bath. Buffer A (30 ml), containing 10
19,000 dpm/assay vial. ig/ml DNAse (Sigma Chemical Co.) and 80 ig/ml soybean trypsin
[3H)CPP binding. [3HJCPP (30.7 Ci/mmol, DuPont NEN) binding inhibitor (Sigma Chemical Co.) was added, the tube was mixed and
assays were conducted as described by Baron
Assays were et at. (1990). centrifuged for 15 sec at 150 x g. The pellet was resuspended in 6 ml
conducted in minivials, incubated for 15 mm at 25’C in 1 ml of 50 mM of buffer A containing 80 g/ml DNAse, 0.5 mg/ml soybean trypsin
Tris-HC1 (pH 7.4) containing 10 nM [3HJCPP, 200 g of membrane inhibitor and an additional 2.7 mM MgSO4, and then triturated with a
protein and unlabeled ligands as indicated. Nonspecific binding was Pasteur pipet and allowed to settle for 10 mm. The supernatant was
defined using 1 mM L-glutamate. Bound ligand was separated by removed with a pipet, adjusted to 6 ml with buffer A and the cells were
centrifugation. Specific binding accounted for approximately 80% of dispersed by trituration. KRB (6-S ml) containing 0.3% BSA, 2.4 mM
total binding. MgSO4 and 0.1 mM CaCl2 was added and the cells remaining in the
[3HJTCP binding. [3H]TCP (47.6 Ci/mmol) binding was measured supernatant after 5 mm standing were removed, centrifuged (150 X g,
using a more extensively washed membrane preparation. On the day 10 mm), and the resulting pellet resuspended in culture medium (basal
of assay, the Triton-washed membranes were thawed at room temper- Eagle’s medium with Earle’s salts containing 10% fetal calf serum, 100
ature and resuspended in 30 ml of 25#{176}C
assay buffer. This suspension g/ml gentamicin, 25 mM KC1 and 2 mM glutamine) to give 1 x 106
was incubated for 15 mm at 25’C in a shaking water bath and then cells/ml. One ml of cell suspension was plated on poly-L-lysine-coated
cover slips. The media was replaced at 24 h and cytosine arabinoside
CI (10 ,M) was added.
NMDA-evoked changes in cytosolic free calcium concentrations
([Ca1]) were monitored in single, cultured granule cell neurons using
microspectrofluorimetry. Cover slips containing cells (grown 8 days in
CI CO2H
vitro) were placed in new plates and loaded with 2 M fura 2-AM for
H 30 mm at 37’C in assay buffer containing (mM: 137 NaCl, 5.4 KC1, 1.8
7-CKA 5,7-DCKA CaC12, 0.44 KH2PO4, 0.64 Na2HPO4, 3 NaHCO3, 5.5 glucose, 20 HEPES,
pH 7.35). Single cell [Ca,] was monitored as the ratio of emitted light
(>485 nm) at each of two excitation wavelengths (350 and 390 nm).
Ratio values were acquired at 10 Hz. Drugs were dissolved in assay
buffer and applied by superfusion (0.9 mi/mm) at room temperature.
CI NCO2H NMDA-stimulated cGMP accumulation in cerebellar slices.
NMDA-stimulated cGMP accumulation was measured in neonatal rat
cerebellar slices as described by Baron et at. (1990). Cerebella were
removed from 8-day-old rats and pooled in ice-cold saline. The tiasue
CI HCO2 was chopped into 400-zm slices using a Mcllwain tissue chopper. The
stage was then rotated 90’ and the tissue was chopped a second time.
Sliced tissue was removed with a Teflon-coated spatula and divided
MDL 100,748 MDL 29,951
between two vials, each containing 15 ml of oxygenated Krebs’ buffer
Fig. 1. Structures of glycine antagonists. (mM: 108 NaCl, 4.7 KC1, 2.5 CaCl2, 1.2 MgSO4, 1.0 KH2PO4, 25
1992 NMDA Receptor Glycine Antagonists 949

NaHCO3, 11.1
D-glucose, 10 HEPES-NaOH, pH 7.35, 0.001% phenol After 3 days, groups of six to eight mice were pretreated for 5 mm
red). The
vials were tightly capped, wrapped with Parafilm and vor- with either vehicle (0.9% saline, pH 7.2) or test compound. Both vehicle
texed for 5 sec. The vials were placed in a shaking water bath in a and antagonists were administered into the lateral ventricle of awake
horizontal orientation and incubated at 37#{176}C for 2 h. During this mice in a volume of 3 l with a 10-al Hamilton syringe. After 5 mm,
period, the vials were removed at 30-mm intervals, the slices allowed individual mice were placed into a round glass jar (diameter, 14.5 cm;
to settle and the medium replaced with 15 ml of fresh buffer. At the height, 30 cm). Each mouse was then exposed to a sound stimulus of
end of this period, the slices were washed four times with 20 ml of well- 110 dB (11 kHz) for 30 sec, during which time it was observed for the
oxygenated assay buffer (mM: 118 NaCl, 4.7 KC1, 2.5 CaCl2, 1.18 presence or absence of seizure activity. The severity of the seizure was
KH2PO4, 25 NaHCO3, ‘11.1 D-glucose, 10 HEPES-NaOH, pH 7.35). quantitated by assigning a numerical score to the observed response
Assays were
performed in stoppered polypropylene tubes in a final (e.g., no response = 0; wild running for <10 sec = 1; wild running for
volume of 500 l containing 25 of gravity-packed slices (200-400 g >10 sec = 2; clonic seizure = 3; forelimb extension/hindlimb flexion =
of membrane protein), various test compounds and, where indicated, 4; tonic extension = 5). The ED,o was calculated by log probit analysis
15 ,M NMDA. Studies measuring antagonism utilized a 10-mm prein- ofpercent protection vs. dose oftest compound. Animals not displaying
cubation with the antagonist before agonist challenge. Before NMDA tonic hindlimb extension were considered protected (seizure score 4).
addition, the assay tubes were cooled on ice, uncapped and the agonist Effects of the antagonists on motor behavior were assessed by moni-
was added. The tubes were gassed with 95% 02/5% CO2, recapped and toring the animals’ ability to remain on a rotating treadmill-like surface
incubated for 5 mm at 37#{176}C.
Assays were stopped
on ice using 50 Ml of (rotorod test).
44 mM EDTA. The samples were then homogenized, heated in a boiling
water bath for 4 mm and centrifuged. The supernatants containing the Results
cGMP were transferred to separate tubes. Pellets containing membrane
protein were dissolved in 2 ml of 0.1 N NaOH and assayed for protein Receptor binding. MDL 100,748, MDL 29,951, 7-CKA and
content using a dye binding method (BioRad, Richmond, CA). Levels 5,7-DCKA were all capable of inhibiting 100% of the specific
of cGMP were quantitated using a commercially available radio- binding of [3H]glycine (not shown). The rank order of potency
immunoas8ay kit (Amersham, Des Plaines, IL) according to the man- in the binding assay was MDL 100,748 5,7-DCKA > MDL
ufacturer’s instructions and were expressed as picomoles per milligram 29,951 > 7-CKA (see table 1). Slope factors were not signi.fi-
protein. cantly different from unity (P > .05, two-tailed t test) and were
NMDA-induced depolarization in cortical miniwedges. Ex-
0.89 (MDL 29,951), 0.94 (MDL 100,748), 0.82 (7-CKA) and
periments on the depolarizing responses to NMDA were performed on
0.85 (5,7-DCKA). All compounds showed a high degree of
a rat cingulate cortex preparation (Harrison and Simmonds, 1985).
selectivity for the glycine recognition site us. that for glutamate
Male Wistar rates (200-300 g) were sacrificed by decapitation and the
whole brain removed. Once submerged in chilled Krebs’ medium con- (table 1). Most selective were the dicarboxylic acid-containing
taming 1 mM Mg, serial transverse sections of forebrain (500 zm) compounds, MDL 100,748 and MDL 29,951, which were ap-
were made using a Vibroslice (Campden Instr. Ltd.). Sections at the proximately 2000-fold more potent against [3Hjglycine us. [3HJ
midline and lateral to the cingulate cortex resulted in wedge-shaped cPP. MDL 100,748 and MDL 29,951 displayed little interac-
pieces of tissue containing cingulate cortex and underlying white mat- tion with [3H]AMPA binding sites. ICo values were 273 ± 67
ter. The slices were then incubated in Krebs’ medium and gassed for and 46 ± 6 tiM, respectively (n = 3).
I .5 h before being placed in two compartment baths. The majority of The nonequilibrium binding ofthe radiolabeled open channel
tile cortical was in one compartment,
tissue with the remaining cortex
blocker, [3HJTCP, has been shown to be sensitive to a variety
and underlying white matter projecting through a greased slot in a two-
of NMDA receptor ligands. The four glycine antagonists were
piece barrier dividing the two compartments. Each compartment was
perfused independently with Krebs’ medium at 22 ± 1C. The Krebs’
tested for their ability to inhibit NMDA-induced [3HJTCP
medium contained (mM): 118 NaCl, 2.1 KC1, 1.2 KH2PO4, 2.5 CaCl2, binding under two conditions. low glycine (10 nM glycine +

25 NaHCO3, 1.0 MgSO4 and 11 glucose, and was gassed continuously 100 M NMDA) or high glycine (300 nM glycine + 100 M
with 95% 02 and 5% CO2. The Krebs’ buffer in the cortical compart- NMDA). As shown in figure 2, 7-CKA, 5,7-DCKA, MDL
ment was modified so that it lacked Mg to remove the voltage- 100,748 and MDL 29,951 were each capable of completely
dependent block of the NMDA receptor channel (Novak et aL, 1984). inhibiting [3H]TCP binding. In the presence of the low concen-
Tetrodotoxin (0.1 tiM) was also added to suppress the spontaneous tration of glycine, respective IC values (n = 3) were 7.4 ±
paroxysmal discharges, which
appear as a result of removing Mg. 4.7 (7-CKA), 1.7 ± 0.7 (5,7-DCKA), 1.3 ± 0.4 (MDL 100,748)
Drug solutions, diluted in Krebs’ buffer, were only passed through the
and 2.5 ± 1.1 iM (MDL 29,951). There is a near perfect
cortical compartment. The potential difference between the two corn-
relationship between the IC50 in the [3H]TCP assay and the
partments was recorded continuously, and negativity induced in the
glycine binding affinity (r = 0.998); however, there is an ap-
cortical compartment was interpreted as a depolarization ofthe cortical
tissue. proximately 20-fold difference in the compounds’ absolute po-
NMDA concentrations (5-20 M) at or below the EC concentration tency in the two types of assays. Increasing the glycine concen-
were selected and perfused for 2 mm. Responses were measured at peak tration present in the [3H]TCP assay decreased the potency of
amplitude. After a 30-mm exposure to the various glycine antagonists, each of the glycine antagonists 3- to 10-fold, as indicated both
NMDA responses were again measured. Increasing agonist concentra- by the parallel rightward shifts shown in figure 2 and by the
tions (up to 1 mM NMDA) were applied until control responses were
obtained. NMDA-evoked responses were in certain cases expressed as TABLE 1
“relative depolarization,” which is the fractional response observed in Binding affinity of glyclne antagonists
the presence ofthe antagonist relative to that achieved in control slices K, vs
in the presence of 20 iM NMDA. Com
(3H)GIy [3H)CPP
Sound-induced seizures in DBA/2 mice. Male DBA/2J audio-
genic-susceptible mice (18 days of age) were obtained from Jackson MM

Laboratory (Bar Harbour, ME). Upon arrival, they were housed in a MDL 100,748 0.07 ± 0.02 (8) 137 ± I (2) 1957
temperature- and humidity-controlled
environment and allowed free MDL 29,951 0.14 ± 0.02 (5) 358 ± 44 (4) 2557
7-CKA 0.36 ± 0.05 (6) 162 ± 57 (6) 450
access to Purina rat chow and water ad Ithitum. Lighting was main-
5,7-DCKA 0.08±0.02(4) 12.3±3.5(3) 154
tamed on a 12-h on/12-h off cycle (on 6:00 A.M., off 6:00 P.M.).
950 Baron et al. Vol. 262

7-CKA 5,7DCKA
100

C
0

0
-C
50
C

0
0.1 1 10 0.1 1

MDL 29,951 MDL 100,748

100 1
C
0
0
-C
C 50

0
0.01 0.1 1 10 100 0.01 0.1 1 10
[Antagonist], p.M [Antagonist], p.M
Fig. 2. Effect of glycine antagonists on [3H]TCP binding. Inhibition of the binding of the radiolabeled channel blocker [3HITCP was measured in the
presence of 100 zM NMDA plus either a low glycine (1 0 nM glycine, ) or a high glycine (300 nM glycine, 0) concentration. Results are means ±
S.E.M. of three independent experiments.

respective ICM values (n 3): 242 ± 8.4 (7-CKA), 16.5 ± 11.0 concentrations provided alone or in combination with either
(DCKA), 9.0 ± 4.2 (MDL 100,748) and 19.4 ± 10.6 iM (MDL 300 nM (n = 3 cells) or 1 iM (n = 5 cells) MDL 100,748. Only
29,951). one cell was selected from each cover slip. ECro values for
Effects on NMDA-elicited elevations in cytosolic cal.- glycine were calculated using pooled data and were 0.30, 0.77
cium. NMDA receptor activation leads to a glycine-dependent and 2.05 M, respectively. Curves were parallel with Hill slopes
elevation of cytosolic calcium which can be measured using the near unity (1.22, 1.57 and 1.10, respectively). As shown in the
fluorescent calcium indicator dye fura 2. Cerebellar granule inset, the data were transformed using the method of Arunlak-
cells were grown in monolayer culture on glasa cover slips shana and Schild (1959). The near unity slope of this plot (1.2)
loaded with fura 2-AM and placed in a superfusion chamber. is consistent with competitive inhibition at the glycine site and
Intracellular calcium concentrations were measured in single the value of the x-intercept (6.67) would imply a lCD for MDL
neurons, as described under “Materials and Methods.” Two 100,748 of 214 nM.
independent series of experiments were conducted, each focus- The second experiment (n = 7 cells) was carried out in a
ing on a single glycine antagonist, and designed to measure the similar fashion, but utilized MDL 29,951 (fig. 3). In the absence
antagonist’s affinity for the glycine binding site by examining of added glycine site ligands, NMDA elicited a response which
its ability to shift the concentration-response curve for glycine. was 7 ± 1% of the maximum response. Glycine ECro values
Data from each series of experiments were analyzed separately were increased in a concentration-dependent fashion by MDL
because, as shown below, we observed variations in the ECro of 29,951 and were 1.41 (control, n = 7 cells), 2.81 (300 nM MDL
glycine in different cell culture preparations. 29,951, n = 3 cells) and 9.98 M (1 jsM MDL 29,951, n 4
The first experiment (n = 8 cells) utilized MDL 100,748 as cells). Curves were, in each case, parallel with respective slope
the glycine antagonist. The response to NMDA (100 iM) was factors of 1.03, 1.09 and 1.08. Schild transformation (inset)
highly glycine dependent because in the absence of added gave a slope of 1.2 and x-intercept of 6.75, resulting in a
glycine, the response was 16 ± 2% of that observed in the calculated KD value of 178 nM.
presence of a maximally effective (30 tiM) glycine concentra- Effects on cGMP accumulation. All four compounds in-
tion. As shown in the representative traces provided in the hibited NMDA (15 M)-evoked increases in cGMP content of
upper panel of figure 3, each cell was tested with various glycine neonatal rat cerebellar slices (table 2). Inhibition by the antag-
1992 NMDA Receptor Glycine Antagonists 951

cc

Time, sec. Time, sec.

1
0.-
C/)

cc
C

0.1 1 10 100 0.1 1 10 100

[Gly], p.M [Gly], tM
Fig. 3. Effect of MDL 29,951 and MDL 1 00,748 on the glycine dependence of NMDA-evoked increases in cytosolic calcium. Agonists were applied
by superfusion to fura 2-loaded, cultured, cerebellar granule cell neurons, and cytosolic calcium was measured in single cells by microspectrofluori-
metry. The upper panels show the responses of representative cells obtained using 100 NMDA + various concentrations of glycine (in nM)
indicated by numbers above the traces. After the construction of a drug-free concentration-response curve, each cell was retested with various
glycine concentrations in the presence of either 300 nM or 1 M of the respective antagonists. In these representative traces, the period of drug
application (1 M) is indicated by the horizontal bar. Pooled data are shown as means ± S.E.M. in the lower panels. Concentrations of antagonist
utilized are indicated adjacent to each curve. The inset illustrates the same data transformed by the method of Schild.

TABLE 2 compounds antagonized depolarizing responses to NMDA (fig.

Inhibition of NMDA-stlmulated CGMP accumulation in rat cerebellar 5). In contrast to the cGMP experiments, in the cortical wedge
slices
preparation, low concentrations of the antagonists did not
Results are cx pressed as mean ± S.E.M with the
. number of experiments given in
parentheses. result in a flattening of the concentration-response curves for
NMDA. As in the cGMP experiments, in the presence of the
Compound lC,,,vS15MMNMDA
highest concentration of each antagonist tested, control levels
MM
of depolarization could not be elicited using up to 1 mM NMDA
MDL 100,748 2.7 ± 1 .1 (3)
(figs. 5 and 6), indicating a flattening of the concentration-
MDL 29,951 5.9 ± 0.8(4)
7-CKA 7.8±0.6(3) response curve for NMDA. This apparently insurmountable
5,7-DCKA 3.5 ± 0.8(4) antagonism of NMDA responses was, however, reversed when
100 M D-serine was applied together with the antagonists (fig.
6).
onists was fully reversible by inclusion of D-serine or glycine Anticonvulsant properties. The four glycine antagonists
(not shown). Studies of the nature of inhibition of the cGMP were potent anticonvulsants after i.c.v. administration to DBA/
response indicate that the four glycine antagonists show a 2J audiogenic seizure-susceptible mice (fig. 7). Anticonvulsant
common noncompetitive mechanism (fig. 4). In all cases, low EDro values (nmol) and 95% confidence intervals (in parenthe-
concentrations of the antagonist led to a flattening of the ses) were: MDL 100,748, 0.09 (0.05-0.19); MDL 29,951, 2.0
normally steep concentration-response curve to NMDA, which (1.6-2.6); 7-CKA, 6.9 (3.4-14.0); and 5,7-DCKA, 2.5 (2.1-3.0).
was accompanied by an increase in the ECro value of NMDA. Vehicle-injected animals showed normal seizure sensitivity (fig.
Higher concentrations of the antagonists led to a suppression 8), and at antagonist doses producing anticonvulsant effects,
of the maximal response to NMDA such that responses were there were no detectable behavioral disturbances either as
barely detectable, even at NMDA concentrations 100-fold determined by gross observation or as assessed using the roto-
greater than the ECro (approximately 10 tiM) for the agonist in rod test for motor impairment. The antagonists also produced
control slice preparations. a dose-dependent reduction in seizure score. With the exception
Effects on NMDA-mediated depolarization. All four of 7-CKA, the least potent of the four antagonists, maximally
952 Baron et al. Vol. 262

7-CKA 5,7-DCKA
200
U Control

Control

C 100
a)
0
0

.1.000 0.
E i 10 100

MDL 29,951 MDL 100,748

0
E 200
a-

a-
0
0 100 101

30MM

.1.000 0
01 10 100
NMDA, M
Fig. 4 Effect of glycine antagonists on NMDA-stimulated CGMP accumulation in neonatal rat cerebellar slices. NMDA concentration-response curves
(4 M to 1 mM NMDA) were constructed in the presence of fixed concentrations of the four glycine antagonists. Control responses are shown in
each case by squares. 5,7-DcKA, MDL 100,748 and MDL 29,951 were tested at 5 (#{149}) and 30 M (A), whereas 7-CKA was used at 20 (#{149}) and 100
zM (A). Results are expressed as picomoles of CGMP formed per milligram protein in the 5-mm agonist exposure period and are representative of
three to four independent experiments.

effective doses of antagonists completely suppressed clonic and [3H]TCP binding with a rank order of potency related to that
tonic seizure activity. Under these conditions, the anticonvul- observed in the glycine binding assay. Moreover, as expected,
sant potency of MDL 100,748 is approximately 12-fold greater the IC for each antagonist was shifted rightward in a parallel
than that displayed by MK-801 and about one-half that exhib- fashion by addition of excess glycine.
ited by CPP. ED,,o and 95% confidence limits are 1.1 (0.8-1.6) The initial consequence of NMDA receptor activation is an
and 0.043 (0.025-0.067) nmol for i.c.v.-administered MK-801 influx of calcium ions through the receptor-associated ion pore.
and CPP, respectively. We have previously used the technique of microspectrofluo-
rimetry to monitor agonist-induced changes in cytosolic cal-
Discussion cium (Ca1) concentrations in single, hippocampal neurons, and
have shown that 5,7-DCKA is a noncompetitive inhibitor of
MDL 100,748 and MDL 29,951 are highly potent, antagonist
the NMDA response (Baron et aL, 1990). Moreover, this effect
ligands for the strychnine-insensitive glycine binding site lo-
cated on the NMDA receptor complex. These compounds ex- of 5,7-DCKA was suggested to be a competitive action of the
hibit approximately 2000-fold greater affinity for the glycine antagonist on the glycine binding site, as it could be nullified
recognition site as compared to their affinity for the glutamate by adding excess glycine (Baron et at., 1990). In the present
binding site present on the same receptor complex. These studies, we used a more sophisticated approach to delineate the
properties make MDL 100,748 and MDL 29,951 the most mechanism of action of the glycine antagonists. In contrast to
selective and potent glycine site antagonists described to date. our previous work, we used cerebellar granule cell cultures
The nonequilibrium binding of the radiolabeled channel because these cells were found to be more robust in paradigms
blocker, [3H]TCP, previously has been proposed to provide a utilizing repetitive periods of stimulation. As in hippocampal
model of receptor activation, in that [3HJTCP binding is recip- neurons, NMDA produced a large increase in granule cell Ca1
rocally modulated by agents which are defined as agonists or which was nearly totally dependent upon added glycine. In this
antagonists using whole cell functional assays (e.g., Baron et manner, we were able to construct complete glycine concentra-
at., 1990 and references therein). Using this binding assay, we tion-response curves for each cell tested both in the absence
were able to demonstrate that MDL 100,748, MDL 29,951, 7- and the presence of a single concentration of glycine antagonist.
CKA and 5,7-DCKA were antagonist-like and that the site of MDL 100,748 and MDL 29,951 produced parallel rightward
action of each compound was the glycine binding site. Thus, shifts of the glycine concentration-response curve consistent
the four antagonists were each capable of completely inhibiting with a competitive interaction with the glycine binding site.
1992 NMDA Receptor Glycine Antagonists 953

2 7-CKA 2 5,7-DCKA
OM
30MM

Control Control

3OM
f+--t t1-;T;
10 100 10 100 1000

2 MDL 100,748 2 MDL 29,951

10,tM
Control
1 1

3OM
Control . . . . t I t 12MM40MM

01 #{149}1
b ibo 1#{149}o00 #{149}1
0 100 1000

Fig. 5. Effect of glycine
NMDA (5 M to 1 mM) in
square symbols. 5,7-DCKA
whereas MDL 29,951 was
depolarization achieved by
NMDA, M
antagonists on NMDA-mediated depolarization of a rat cortical wedge preparation. Dose-response lines are shown for
the presence of fixed concentrations of the four glycine antagonists. The control response is shown in each case by
and MDL 100,748 were tested at 3 (#{149}), 7-CKA was utilized at 10 (S), 30 (A) and 1 00 M (#{149}),
10 (A) and 30 MM (#{149}).
used at 4 (#{149}),
12 (A) and 40 zM (#{149}).
Each panel is a composite of four to six experiments. Results are normalized to the
20 MM NMDA in control slices for each individual experiment, which ranged from 1 .35 to 1 .88 mV.

This was corroborated using Schild transformations where There were, however, some qualitative differences in the two
slopes of the regression lines were very near unity. The antag- slice preparations which were particularly evident at low con-
onism was found to be rapidly reversible by superfusion with centrations of the glycine antagonists. Whereas all antagonist
antagonist-free buffer consistent with an action of the com- concentrations tested produced nonparallel rightward shifts in
pounds with an extracellularly located binding site. The KD the NMDA concentration-response curves in the cGMP assay,
values for MDL 100,748 and MDL 29,951 calculated from this nonparallelism was only apparent at high antagonist concen-
functional assay are very similar to those obtained in the glycine trations in the electrophysiological assay. It should be noted
binding assay. that the range of NMDA concentrations examined in the
Noncompetitive effects on NMDA-elicited events were also electrophysiological assay did not permit evaluation of the
evident in studies utilizing cortical or cerebellar slice prepara- effect of low concentrations of antagonists on the maximal
tions. NMDA evokes a large increase in cerebellar cGMP value of the response, and it is likely that more complete curves
content, and we have previously used this assay to monitor would reveal that the kinetic mechanisms of drug interaction
antagonism at the NMDA, glycine and polyamine recognition were similar in both preparations. High concentrations of the
sites (Baron et at., 1989, 1990; Reynolds et at., 1991). Similarly, glycine antagonists produced, in both assay types, a marked
NMDA elicits a depolarization of cortical slice preparations suppression in the maximal value of the NMDA response.
which is sensitive to both competitive and noncompetitive These results are indicative of a noncompetitive interaction
antagonists (Harrison and Simmonds, 1985; Lodge et aL, 1988). of these antagonists with respect to NMDA recognition site
However, unlike our experiments utilizing cultured cell prepa- and are entirely consistent with antagonism resulting from an
rations, we found that in both brain slice preparations, NMDA action at an obligatory second binding site. Additional evidence
is fully effective even in the absence of exogenous glycine, and, for the site of action is provided by the reversal of antagonism
thus, in these assays, the glycine site is most likely saturated by D-serine, an agonist ligand for the glycine site (Snell et at.,
by the endogenously released glycine. All four of the glycine 1988), in the cortical wedge and cerebellar slice cGMP experi-
antagonists were able to inhibit NMDA-stimulated cGMP ac- ments. Furthermore, the strong correlation between the antag-
cumulation and NMDA-induced depolarization in a glycine- onism of the two functional responses and [3H]glycine binding
reversible manner. ICro values were similar in both types of data would indicate that this noncompetitive interaction is,
functional assays, but were about 10-fold higher than those indeed, via the glycine binding site on the NMDA receptor
obtained in our measurements of Ca1 increases. complex.
954 Baron et al. Vol. 262

3 7-CKA 3 5 ,7 - DC KA

Control
j///
2
2

>1

./
1
E
C 30MM 5,7-DCKA
Control a- 100MM
+D-Serine #{149}
100MM 7-CKA
7-CKA a- C -#{149}

1
0 10 100 1000 10 100 #{149}‘‘ i#{246}oo
N
L.

MDL 100,748 2 MDL 29,951

0
a- Control
a)
Control

V.__Af//r 30MM
+1?- MSerineMDL
30 MDL 4OM MDL

0 +-+-4-H

10 100 1o.00
1000 1
NMDA, M
FIg. 6. Reversal of antagonism by o-senne. The dose-response relationship for NMDA-mediated depolarization of the cortical wedge Is shown for
individual experiments to illustrate the effect of the glycine antagonists measured in the presence (A) or absence (#{149})
of D-serine (100 M, applied
with the antagonists). COntrOl responses are shown in each case by U Antagonist concentrations were as follows; 7-CKA (100 zM), 5,7-DCKA (30
tM), MDL 100,748 (30 M) and MDL 29,951 (40 tiM).
MDL 29.951
100#{149}
MDL 100,748 MDL 100,748

C
0
-e-J
0 C
Fig. 7. Anticonvulsant effect of l.c.v.-administered gly-
a) one antagonists. AUdiOgeniC seizure-susceptible DBA/
0 2J mice recved various doses ofthe glycine antagonists
5 mm before presentation of a sound stimulus. The
0 absence of tonic extension was scored as protection
from sound-induced convulsions. Each point represents
C data from six to eight mace. Symbols are as follows: MDL
a) 100,748 (#{149}),
5,-7-DCKA (a), MDL 29,951 (A) and 7-CKA
0 (U).
a)

0.1 1 10 100
Dose, nmol Lc.v.

The potency in functional assays

was related to the affinity tionship between affinity measured in the functional assays
of the compounds for the glycine
binding site measured in and the ability of the compounds to inhibit NMDA-stimulated
receptor binding assays. Linear regression of relative potencies [3H]TCP binding. Ofparticular note are the approximately 20-
gave r values of0.92 for cGMP experiments and 0.97 for cortical fold greater antagonist concentrations which were required to
wedge experiments. Only two of the four compounds were inhibit function in the slice assays or to inhibit binding of the
evaluated in the calcium assay, so a comparable statistic was channel blocker, as opposed to that predicted based on occu-
not calculated. Similarly, there was a highly significant rela- pancy measurements ([3H]glycine binding). It should be re-
1992 NMDA Receptor Glycine Antagonists 955

7-CKA 5,7-DCKA
5 5
I
4 I I 4
I
3 3

2 2
I +
0
- ‘IL 1

C.) I I i I I 1
V)
CONT 3.35 6.7 13.4 CONT 1.5 2.2 2.9 3.6 4.4 5.8

N MDL 29,951 MDL 100,748

a) 5 5

4 .1. I 4 +
_+_
3

I 3

+
+__

I11
2 2

CONT SHAM 1.2 1.9 2.5 5

-

CONT
-

SHAM
-

0.019
-

0.029
-

0.039 0.078
I
0.16 0.63
Dose, nmol
Fig 8. Effect of glycine antagonists on the severity of audiogenic seizures. Data shown in figure 5 are repiotted as seizure score ± S.E.M. (no
response = 0, wild running < 10 sec = 1 , wild running >10 sec = 2, clonic seizure = 3, forelimb extension/hindlimb flexion = 4, hindlimb extension
= 5) vs. dose of each antagonist.

called that NMDA was able to elicit both depolarization and a potency to 5,7-DCKA at the glycine site, they are less potent
cGMP increase without the necessity for exogenous glycine at the NMDA recognition site, suggesting that the presence of
addition. Similarly, in the [3H]TCP assay, roughly equal the second carboxylic acid group improves binding site selectiv-
NMDA responses are observed in the absence of glycine (Baron ity. Moreover, the apparent requirement for glycine in receptor
et aL, 1990), the presence of low concentrations of glycine activation deduced from electrophysiological experiments is
(present work) or after addition of fully effective glycine con- confirmed by our pharmacological experiments. Because the
centrations (Baron et aL, 1990). In contrast, there was a clear four antagonists discussed here encompass considerable struc-
requirement for exogenous glycine for evocation of an NMDA- tural variability, their capability for complete inhibition of
induced rise in Ca1 in cultured neurons. In the latter prepara- NMDA responsiveness is likely due to shared characteristics
tion, the functionally derived lCD values for the glycine antag- ofglycine antagonists and is not limited to hypothesized inverse
onists agreed well with the respective affinity values calculated agonist qualities.
based on occupancy. It is likely that both the variations in The four glycine antagonists were potent antagonists of
glycine dependence of the control responses and the concentra- sound-induced convulsions in DBA/2J mice. As was the case
tions of competitive glycine antagonists required for their in- for the compounds’ ability to antagonize NMDA-mediated re-
hibition may be due to a relatively higher level of endogenous sponses, there was a significant correlation between anticon-
glycine in slice as compared to superfused cell monolayer prep- vulsant potency and glycine recognition site binding affinity (r
arations. There was less of a relationship between affinity for = 0.94). MDL 100,748 was particularly potent in this in vivo
the glutamate recognition site and functional antagonism (r = assay, with an EDro similar to that of CPP and superior to that
0.46 in the cGMP assays and r = 0.02 in the cortical wedge of MK-801. The completeness of the anticonvulsant effect was
experiments). In fact, all compounds inhibited the response to evident in that each of the glycine antagonists produced a dose-
NMDA at almost 40-fold lower concentrations than were re- dependent reduction in the severity of the seizure score. Thus,
quired to occupy the glutamate recognition site in binding in the presence of optimal doses of the antagonists, the auditory
assays. stimulus evoked only wild running without any evidence of
Structurally, MDL 29,951 and MDL 100,748 are interesting convulsive behavior. We have previously shown using 5,7-
in view of the second carboxylic acid group. Although of similar DCKA that as in the in vitro experiments, the dose-response
956 Baron et al Vol. 262

curve for the anticonvulsant effect can be shifted rightward by KESSLER, M., BAUDRY, M. AND LYNCH, G.: Quinoxaline derivatives are high
affinity antagonists of the NMDA receptor-associated glycine sites. Brain Res.
coinjection of the glycine agonist D-serine (Baron et at., 1990). 489: 377-382, 1989a.
Moreover, the anticonvulsant potency displayed by these corn- KE8SLER, M.,
TERRAMANI, T., LYNCH, G. AND BAUDRY, M.: A glycine site
with N-methyl-D-aspartic
associated acid receptors: characterization and iden-
pounds compares favorably with that achieved by the channel
of a new class of antagonists.
tification J. Neurochem. 52: 1319-1328, 1989b.
blocker MK-801 and the competitive glutamate antagonist KLECKNER, N. W. AND DINGLEDINE, R.: Requirement for glycine in activation
CPP. A variety of NMDA receptor antagonists, including chan- of NMDA receptors expressed in Xenopus oocytes. Science (Wash. DC) 241:
835-837, 1988.
nel blockers, glycine antagonists and glutamate antagonists, KLECKNER, N. W. AND R.: Selectivity
DINGLEDINE,of quinoxalines and kynu-
have been shown to possess anxiolytic activity; however, the renines of the glycine site on N-methyl-D-aspartate
as antagonists receptors.
glycine antagonists are distinctive in their apparent lack of Mol. Pharmacol. 36: 430-436, 1989.
KoEx, W. AND COLPAERT, F. C.: Selective blockade of N-methyl-D-aspartate
sedative or muscle-relaxant side effects (Kehne et at 1991; (NMDA)-induced convulsions by NMDA antagonists and putative glycine
Koek and Colpaert, 1990; Trullas et at., 1989). Although not antagonists: relationship
with phencyclidine-like behavioral effects. J. Phar-
quantified, a similar favorable therapeutic ratio was evident macol. Ezp. Ther. 252: 349-357, 1990.
LERMA, J., ZUKIN, R. S. AND BENNETT, M. V. L.: Glycine decreases desensiti-
both from our gross observations of behavior and from the zation of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus
apparent lack of impairment seen in rotorod testing of the oocytes and is required for NMDA responses. Proc. Natl. Aced. Sci. USA 87:
2354-2358, 1990.
DBA/2J mice receiving i.c.v.-administered glycine antagonists. LODGE, D., DAvis, S. N., JONES, M. G., MILLAR, J., MANALLACK, D. T.,
Thus, glycine antagonists might be particularly attractive in ORN5mIN, P. L., VERBERNE, A. J. M., YOUNG, N. AND Bwvr, P. M.: A
therapeutic situations where motor impairment would repre- comparison between the in uiuo and in vitro activity of five potent and
competitive NMDA antagonists. Br. J. Pharmacol. 95: 957-965, 1988.
sent an obstacle to successful treatment. LuzzI, S., ZILLETI, L, FRANCHI-MICHELI, S., G0RI, A. M., AND M0R0NI, F.:
Agonists, antagonists and modulators of excitatory amino acid receptors in the
Acknowledgments guinea pig myenteric plexus. Br. J. Pharmacol. 95: 1271-1277, 1988.
Financial support for some of the studies (J.P.T. and B.S.M.) was provided by MAYER, M. L., VYCKLICKY, L. AND CLEMENTS, J.: Regulation of NMDA receptor
the Medical Research Council. desensitization in mouse hippocampal neurons by glycine. Nature (Lond.) 338:
425-427, 1989.
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