A novel family of potent negative allosteric modulators of group II metabotropic glutamate receptors

Article abstract of DOI:10.1124/jpet.106.117093

Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a n

Full text of DOI:10.1124/jpet.106.117093

0022-3565/07/3221-254–264$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 322:254–264, 2007
Vol. 322, No. 1
117093/3217936
Printed in U.S.A.
A Novel Family of Potent Negative Allosteric Modulators of
Group II Metabotropic Glutamate Receptors
Kamondanai Hemstapat, Herve Da Costa, Yi Nong, Ashley E. Brady, Qingwei Luo,
Colleen M. Niswender, Gilles D. Tamagnan, and P. Jeffrey Conn
Department of Pharmacology and Vanderbilt Institute of Chemical Biology Program in Drug Discovery, Vanderbilt University
Medical Center, Nashville, Tennessee (K.H., Y.N., A.E.B., Q.L., C.M.N., P.J.C.); and Institute for Neurodegenerative Disorders,
New Haven, Connecticut (H.D.C., G.D.T.)
Received November 14, 2006; accepted April 5, 2007
ABSTRACT
Group II metabotropic glutamate receptors (mGluRs), mGluR2 onist [3H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopro-
and mGluR3, play a number of important roles in mammalian pan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagen-
brain and represent exciting new targets for certain central esis revealed that a single point mutation in transmembrane V
nervous system disorders. We now report synthesis and char- (N735D), previously shown to be an important residue for
acterization of a novel family of derivatives of dihydro- potentiation activity of the mGluR2 allosteric potentiator
benzo[1,4]diazepin-2-one that are selective negative allosteric LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoro-
modulators for group II mGluRs. These compounds inhibit both ethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhib-
mGluR2 and mGluR3 but have no activity at group I and III itory activity of negative allosteric modulators of group II
mGluRs. The novel mGluR2/3 antagonists also potently block mGluRs. However, this single mutation in human GluR2 almost
mGluR2/3-mediated inhibition of the field excitatory postsyn- completely blocked the enhancing activity of biphenyl-in-
aptic potentials at the perforant path synapse in hippocampal danone A, a novel allosteric potentiator of mGluR2. Our data
slices. These compounds induce a rightward shift and de- suggest that these two positive allosteric modulators of
crease the maximal response in the glutamate concentration- mGluR2 may share a common binding site and that this site
response relationship, consistent with a noncompetitive antag- may be distinct from the binding site for the new negative
onist mechanism of action. Furthermore, radioligand binding allosteric modulators of group II mGluRs.
studies revealed no effect on binding of the orthosteric antag-
The eight known subtypes of metabotropic glutamate re- clude group I (mGluR1 and 5), group II (mGluR2 and 3), and
ceptors (mGluRs) have been classified based on sequence
homology, pharmacology, and signal transduction. These in-
group III receptors (mGluR4, 6, 7, and 8). The group I recep-
tors couple to G␣q and phospholipase C, whereas group II and
group III mGluRs couple to G␣i (Conn and Pin, 1997; Schoepp
This work was supported by grants from National Institute of Mental et al., 1999). A large body of in vitro and in vivo preclinical
Health; NINDS, National Institutes of Health; The National Alliance for
Research on Schizophrenia and Depression; and the Stanley Foundation.
studies suggest that specific mGluR subtypes play a broad
range of neuromodulatory roles in different central nervous
system circuits and that specific subtypes may provide viable
targets for novel treatment strategies for a range of neuro-
logical and psychiatric disorders, including anxiety (Linden
Vanderbilt University is a site in the National Institutes of Health-supported
Molecular Libraries Screening Center Network.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.117093.
ABBREVIATIONS: mGluR, metabotropic glutamate receptor; CHO, Chinese hamster ovary; HEK, human embryonic kidney; TM, transmembrane;
DCG-IV, (2S,2ЈR,3ЈR)-2-(2Ј,3Ј-dicarboxycyclopropyl)glycine; LY341495, 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propi-
onic acid; LY487379, N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroetylsulfonyl)-pyrid-3-ylmethylamine; BINA, biphenyl-indanone A or 3Ј-
(((2-cyclopropyl-6,7-dimethl-1-oxo2,3-dihydro-1H-inden-5-yl)oxy)methyl)biphenyl-4-carboxylic acid; DMSO, dimethyl sulfoxide; [35S]GTP␥S,
guanosine 5Ј-O-(3-[35S]thio)triphosphate; ACSF, artificial cerebrospinal fluid; L-AP4, L(ϩ)-2-amino-4-phosphonobutyric acid; FBS, fetal bovine
serum; DMEM, Dulbecco’s modified Eagle’s medium; MPP, medial perforant path; fEPSP, field excitatory postsynaptic potential; LY379268,
(Ϫ)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate;
LY354740,
(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid;
MGS0039, (1R,2R,3R,5R,6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; MNI-135, 3-(7-iodo-4-oxo-
4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-benzonitrile; MNI-136, 7-bromo-4-(3-pyridin-3-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
MNI-137, 4-(7-bromo-4-oxo-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-pyridine-2-carbonitrile; HBSS, Hanks’ balanced salt solution; DG, den-
tate granule; hmGluR, human GluR; rmGluR, rat GluR.
254

Novel Negative Allosteric Modulators of Group II mGluRs
255
et al., 2005; Swanson et al., 2005), pain (Fisher et al., 2002; modulators that are active at both group II mGluR sub-
Adwanikar et al., 2004), Parkinson’s disease (Conn et al., types but are without effects on group I and group III
2005), schizophrenia (Moghaddam, 2004; Kinney et al., 2005; mGluRs. These compounds are useful in both cell lines and
Shipe et al., 2005), and cognitive disorders (Campbell et al., in blocking electrophysiological effects of group II mGluRs
2004; Homayoun et al., 2004; Moghaddam, 2004). Thus, com- in brain slices. Interestingly, these compounds do not alter
pounds that are selective for a specific subtype of mGluR binding to the orthosteric site and may act at an allosteric
could provide a valuable tool to further investigate the in- site that is distinct from that of recently described positive
volvement of these receptors in various diseases.
allosteric modulators that are selective for mGluR2.
Group II mGluRs (mGluR2/3) are abundantly expressed in
forebrain regions, such as cortex, hippocampus, striatum,
and amygdala (Ohishi et al., 1998). Activation of group II
mGluRs by group-selective agonists, including (Ϫ)-2-oxa-4-
Materials and Methods
Materials. All tissue culture reagents were obtained from Invitro-
gen (Carlsbad, CA). G418 sulfate was obtained from Mediatech, Inc.,
(Herndon, VA). [3H]LY341495 (28.28 Ci/mmol) was obtained from
American Radiolabeled Chemicals, Inc. (St Louis, MO). L-Glutamate,
DCG-IV, L-AP4 [L-(ϩ)-2-amino-4-phosphonobutyric acid], and
LY341495 were obtained from Tocris (Ellisville, MO). Methotrexate
was purchased from Calbiochem (La Jolla, CA). BINA was synthe-
sized as described previously (Galici et al., 2006). The Calcium 3
Assay Kit was obtained from Molecular Devices (Sunnyvale, CA).
The indicator dye fluo-4 was obtained from Invitrogen. Probenecid,
dimethyl sulfoxide (DMSO), puromycin dihydrochloride, GDP, and
aminobicyclo[3.1.0]hexane-4,6-dicarboxylate
(LY379268)
and methyl substitution of 2-aminobicyclo[3.1.0]hexane 2,6-
dicarboxylate (LY354740), leads to robust anxiolytic-like ef-
fects and antipsychotic-like activity in rodents (Carter et al.,
2004; Linden et al., 2005) and has also shown anxiolytic
activity in humans (Grillon et al., 2003). Whereas the ther-
apeutic significance of group II mGluR antagonists has not
been widely investigated, recent studies of selective compet-
itive group II mGluR antagonists, including 2S-2-amino-2-
(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic guanosine 5Ј-3-O-(thio)triphosphate were purchased from Sigma-
Aldrich, Inc., (St. Louis, MO). Unifilter-96 GF/B plates and Mi-
croScint-20 were obtained from PerkinElmer Life and Analytical
Sciences (Boston, MA). BioCoat poly-D-lysine 96-well culture plates
were obtained from BD Biosciences Discovery Labware (Bedford,
MA). QuikChange site-directed mutagenesis kit and Pfu Ultra high
fidelity DNA polymerase were obtained from Stratagene (La Jolla,
CA). Complimentary oligonucleotides were obtained from Operon
Biotechnologies (Huntsville, AL). All test compounds were synthe-
sized as described previously (Adam et al., 2003); the synthesis of
three representative compounds is described here.
MNI-135. [3-(7-iodo-4-oxo-4,5-dihydro-3H-benzo[1,4]diazepin-2-
yl)-benzonitrile]. Under N2, trifluoroacetic acid (30 mmol) was slowly
added at 0°C to a solution of [4-iodo-2-[3-(2-cyano-pyridin-4-yl)-3-
oxo-propionylamino]-phenyl]-carbamic acid tert-butyl ester (1.9
mmol) in dichloromethane (20 ml). The reaction was stirred 7 h at
room temperature. The mixture was washed with 10% aqueous
NaHCO3, the organic layer was dried over Na2SO4, and the solvent
was removed on the rotary evaporator. The residue was purified by
flash chromatography on silica gel using 60:40 hexane-ethyl acetate
to yield 610 mg (80%) of MNI-135 as a white solid. Mp: 226°C; 1H
NMR (DMSO), ␦ ppm: 3.60 (2H, s), 7.21 (1H, d, J ϭ 9.2 Hz), 7.55–7.57
acid (LY341495) and (1R,2R,3R,5R,6R)-2-amino-3-(3,
4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0] hexane-2,6-
dicarboxylic acid (MGS0039), have suggested that these com-
pounds exhibit antidepressant-like activity and anti-
obsessive-compulsive disorder-like effects in animal models
(Chaki et al., 2004; Shimazaki et al., 2004; Palucha and Pilc,
2005).
Due to the high level of conservation of the orthosteric
binding site of mGluRs, it has proven difficult to develop
subtype-specific ligands for these receptors. As such, the
most widely used orthosteric antagonists for group II
mGluRs show some level of activity at all mGluR subtypes
(Kingston et al., 1998). However, major advances have been
made in developing highly selective antagonists of group I
mGluRs by targeting allosteric sites on the receptor to non-
competitively block receptor function (Gasparini et al., 1999;
Lavreysen et al., 2003). The ability to achieve higher selec-
tivity with these compounds is probably due to the fact that
they bind within the seven-transmembrane (TM)-spanning
domain of the mGluR, which is less highly conserved than (2H, m), 7.74 (1H, t, J ϭ 7.6 Hz), 8.03 (1H, d, J ϭ 7.6 Hz), 8.35 (1H,
d, J ϭ 8 Hz), 8.46 (1H, s), 10.62 (1H, s); 13C NMR (DMSO) ␦ ppm:
the glutamate binding pocket. Moreover, allosteric antago-
40.0, 91.4, 112.1, 118.3, 129.8, 130.0, 130.2, 131.2, 131.6, 132.1,
nists may provide other advantages in that their activity is
132.7, 134.5, 138.0, 138.6, 157.2, 165.9; Anal. (C16H10IN3O) C, H, N,
not altered by the presence of competing orthosteric agonists.
Recently, the discovery of two highly selective allo-
steric potentiators of mGluR2, N-(4-(2-methoxyphen-
oxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmeth-
ylamine (LY487379) (Johnson et al., 2003) and biphenyl-
indanone A (BINA) (Galici et al., 2006), has provided
excellent tools to selectively activate this group II mGluR
MS: 388.3 (M ϩ 1).
MNI-136. [7-bromo-4-(3-pyridin-3-yl-phenyl)-1,3-dihydro-benzo
[1,4] diazepin-2-one]. Under N2, N-(4-bromo-2-nitro-phenyl)-3-oxo-3-
(3-pyridin-3-yl-phenyl)-propionamide (1.9 mmol) was dissolved in
ethanol, and tin chloride (5.7 mmol) was added. The mixture was
stirred 7 h at reflux. The solvent was evaporated, and the residue
was basified with 1 N NaOH (pH 7–8) and extracted with ethyl
subtype. Less progress has been achieved with the discov- acetate. The organic layer was dried over Na2SO4, and the solvent
was removed on the rotary evaporator. The residue was purified by
chromatography on silica gel using 70:30 hexane-ethyl acetate to
yield 650 mg (86%) of MNI-136 as a white solid. Mp: 218°C; 1H NMR
(DMSO) ␦ ppm 3.66 (2H, s), 7.16 (1H, d, J ϭ 8.8 Hz), 7.46 (1H, dd, J ϭ
6 Hz, J ϭ 9.2 Hz), 7.53–7.56 (1H, m), 7.64 (1H, d, J ϭ 2.3 Hz), 7.69
(1H, t, J ϭ 7.6 Hz), 7.95 (1H, d, 8.8 Hz), 8.12 (1H, d, J ϭ 7.6 Hz), 8.18
(1H, d, J ϭ 8 Hz), 8.36 (1H, s), 8.62–8.64 (1H, m), 8.97–8.99 (1H, m),
10.71 (1H, s); 13C NMR (DMSO) ␦ ppm: 40.1, 115.8, 123.9, 124.0,
126.2, 127.3, 128.9, 129.6, 129.7, 129.8, 129.9, 134.3, 135.0, 137.6,
137.8, 140.7, 147.8, 148.9, 159.5, 166.0; Anal. (C20H14BrN3O) C, H,
ery of negative allosteric modulators (allosteric antago-
nists) of group II mGluRs. However, preliminary report
presented in abstract form suggested that a series of dihy-
dro-benzo[1,4]diazepin-2-one derivatives may have alloste-
ric activity at the group II mGluRs (Gatti et al., 2001).
Based on this finding, we synthesized a series of com-
pounds based on the scaffold described by Adam et al.
(2003) and determined the activity of these molecules at
group II mGluRs. Here we report that these compounds
provide a novel family of potent and negative allosteric N, MS: 393.6 (M ϩ 1).

256
Hemstapat et al.
MNI-137. [4-(7-bromo-4-oxo-4,5-dihydro-3H-benzo[1,4]diazepin-2- glutamate (EC80) or as a percentage of the maximal response to
yl)-pyridine-2-carbonitrile]. Under N2, trifluoroacetic acid (30 mmol) glutamate (10 ␮M).
was slowly added at 0°C to a solution of [4-bromo-2-[3-(2-cyano-
Functional Calcium Mobilization Assay for mGluR8. The rat
pyridin-4-yl)-3-oxo-propionylamino]-phenyl]-carbamic acid tert-butyl mGluR8/Gqi9/CHO cell lines were plated at a seeding density of 4 ϫ
ester (3 mmol) in dichloromethane (20 ml). The reaction was stirred 105 cells/well in clear-bottomed, poly-D-lysine-coated 384-well plates
7 h at room temperature. The mixture was washed with 10%
(black-walled) in DMEM containing 10% FBS, 100 U/ml penicillin/
NaHCO3, the organic layer was dried over Na2SO4, and the solvent streptomycin and 20 mM HEPES. Cells were plated in the morning
and incubated at 42°C for 2 h in the afternoon before analysis (“heat
shock”) and then incubated overnight at 37°C in the presence of 5%
CO2. On the day of the assay, the medium was removed, and 0.9 ␮M
indicator dye fluo-4 was added in HBSS containing 20 mM HEPES,
pH 7.3 (20 ␮l/well). Cells were incubated for 1 h at room tempera-
ture, and the dye was replaced with 20 ␮l/well HBSS. Test com-
pounds were dissolved as a 10 mM stock in 100% DMSO and serially
diluted in DMSO. Test compound plates were prepared in HBSS at
2.5ϫ their final concentration in 0.25% DMSO; L-AP4 was at 5ϫ the
final concentration to be assayed in HBSS. Cell plates and compound
plates were loaded onto a Hamamatsu FDSS 6000 kinetic imaging
plate reader (Hamamatsu Corporation, Bridgewater, NJ). The assay
was initiated by collecting 10 images at 1 Hz before the addition of
the test compound. Test compound (20 ␮l/well) was then added, and
was removed on the rotary evaporator. The residue was purified by
chromatography on silica gel using 60:40 hexane-ethyl acetate to
yield 610 mg (80%) of MNI-137 as a white solid. Mp: 238°C; 1H NMR
(DMSO) ␦ ppm: 3.65 (2H, s), 7.42 (3H, m), 8.25 (1H, dd, J ϭ 1 Hz, J ϭ
5 Hz), 8.54 (1H, s), 8.92 (1H, d, J ϭ 5 Hz), 10.79 (1H, s); 13C NMR
(DMSO), ␦ ppm 40.2, 117.3, 119.5, 124.2, 125.1, 126.6, 127.1, 130.1,
131.7, 133.6, 137.8, 145.2, 152.3, 155.7, 165.7; Anal. (C15H9BrN4O)
C, H, N, MS: 342.3 (M ϩ 1).
Cell Culture and Transfections. Baby hamster kidney cells
stably expressing the rat mGluR1a (rmGluR1a) were generously
provided by Dr. Betty Haldeman (Zymogenetics, Seattle, WA). Cells
were grown in Dulbecco’s modified Eagle’s medium (DMEM) contain-
ing 5% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine
(GlutaMAX I; Invitrogen), antibiotic-antimycotic (100 U of penicillin,
100 ␮g of streptomycin, and 0.25 ␮g of amphotericin B), 1 mM
sodium pyruvate, 20 mM HEPES, and 250 nM methotrexate. Rat
mGluR2 and the promiscuous G protein, Gqi5, were cotransfected
into HEK293A cells using Lipofectamine 2000 (Invitrogen) and were
grown in DMEM containing 10% heat-inactivated FBS, 2 mM L-
glutamine, antibiotic-antimycotic, 0.1 mM nonessential amino acids,
and 20 mM HEPES. Chinese hamster ovary (CHO) cells stably
expressing human mGluR2 (hmGluR2), which were transiently
transfected with Gqi5, and CHO cells stably expressing both the
hmGluR4 and Gqi5 were grown in DMEM containing 10% heat-
inactivated dialyzed FBS, 2 mM L-glutamine, antibiotic-antimycotic,
1 mM sodium pyruvate, 20 mM HEPES, 5 nM methotrexate, and 20
␮g/ml L-proline. CHO cells stably expressing the rmGluR3 were
grown in DMEM containing 10% heat-inactivated FBS, 2 mM L-
glutamine, 100 U of penicillin, 100 ␮g of streptomycin, 1 mM sodium
pyruvate, 0.1 mM nonessential amino acids, and 20 mM HEPES. The
rat mGluR8/Gqi9/CHO cell line was a generous gift of Jarda Wrob-
lewska (Georgetown University, Washington, DC) and was main-
tained in 90% DMEM, 10% FBS, 100 U/ml penicillin/streptomycin,
20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, and 2 mM glu-
tamine supplemented with 800 ␮g/ml G418. HEK cells stably ex-
pressing rat mGluR7a were grown in 45% DMEM, 45% Ham’s F12,
10% FBS, 100 U/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3),
1 mM sodium pyruvate, 2 mM glutamine, 600 ng/ml puromycin
dihydrochloride, and 700 ␮g/ml G418 at 37°C in the presence of 5%
CO2.
Functional Calcium Mobilization Assay for mGluRs 1, 2, 4,
and 5. Recombinant cell lines, including mGluR1, 2, 4, and 5, were
plated at a seeding density of 7 to 8 ϫ 105 cells/well in clear-
bottomed, poly-D-lysine-coated 96-well plates. Cells were then incu-
bated in glutamate/glutamine-free medium overnight at 37°C in an
atmosphere of 95% O2/ 5% CO2, with the exception of baby hamster
kidney cells stably expressing rmGluR1a, which were maintained in
regular medium. Cells were loaded with calcium indicator dye (Cal-
cium 3 Assay Kit) at 37°C for 1 h. Dye was removed and replaced
with the appropriate volume of assay buffer containing 1ϫ Hanks’
balanced salt solution (HBSS), 20 mM HEPES, and 2.5 mM probe-
necid, pH 7.4. At this stage, cells were used for the calcium mobili-
zation assay.
A
rmGluR2
120
MNI-134
MNI-135
MNI-136
MNI-137
MNI-139
MNI-140
MNI-141
MNI-142
MNI-144
100
80
60
40
20
0
-10
-9
-8
-7
-6
-5
log[Compound], M
B
rmGluR3
120
100
80
MNI-134
MNI-135
MNI-136
MNI-137
MNI-139
MNI-140
MNI-141
MNI-142
MNI-144
60
40
20
0
-20
-40
0
-9
-8
-7
-6
-5
log[Compound], M
Fig. 1. All test compounds produced a concentration-dependent inhibi-
tion of glutamate-induced responses in HEK293A cells expressing rm-
GluR2, as assessed by calcium mobilization assay (A) or in membranes
expressing rmGluR3, as assessed by [35S]GTP␥S binding assay (B). Cells
were preincubated for 5 min with various concentrations of test com-
pounds before the addition of a nearly maximal concentration (EC80) of
glutamate. Changes in calcium mobilization were measured using the
FLEXstation II. For the [35S]GTP␥S assay, membranes were prepared
from CHO cells stably expressing rmGluR3. The assay mixtures con-
tained 10 ␮g of membrane protein, test compound, glutamate, 0.1 nM
All test compounds were dissolved in 100% DMSO and then seri-
ally diluted into assay buffer containing 0.1% bovine serum albumin
for a 5ϫ stock. The stock solution was added to the assay plate to a
final DMSO concentration of 0.1%. Glutamate and L-AP4 were pre-
pared as a 10ϫ stock solution in assay buffer before addition to assay
plates. Calcium mobilization was measured using the FLEXstation
II (Molecular Devices). The signal amplitude was normalized as a
percentage of the response to the nearly maximal concentration of
[
35S]GTP␥S, and assay buffer. The mixtures were incubated at 30°C for
60 min, and the binding equilibrium was terminated by rapid filtration
through Unifilter-96 GF/B filter plates. The radioactivity was determined
by TopCount NXT. Data were normalized as a percentage of the maximal
response to 31.6 or 10 ␮M glutamate for [35S]GTP␥S binding and calcium
mobilization assays, respectively. Data are presented as the mean of
three individual experiments performed in triplicate, with error bars
representing mean Ϯ S.E.M.

Novel Negative Allosteric Modulators of Group II mGluRs
257
data were collected for 5 min. After this 5-min period, 10 ␮l of vehicle
Membrane Preparation. Membranes from cell lines expressing
or agonist was added, and data were collected for an additional 2 hmGluR2 or rmGluR3 were prepared as described previously (John-
min. Functional response was quantified using Microsoft Excel by son et al., 1999; Schaffhauser et al., 2003). In brief, confluent cells of
calculating the maximal fluorescence generated during the time both cell lines were washed once with ice-cold phosphate-buffered
window after agonist addition and subtracting the fluorescence ob- saline. hmGluR2-expressing cell lines were harvested and resus-
tained in the vehicle control wells. The signal amplitude was then pended in ice-cold binding buffer containing 10 mM potassium phos-
normalized as the percentage of the response to the EC80 concentra- phate monobasic and 100 mM potassium bromide, pH 7.6, whereas
tion of L-AP4.
rmGluR3-expressing cell lines were harvested and resuspended in
Functional Cyclase Inhibition Assay-mGluR7. HEK cells sta- ice-cold binding buffer 1 (20 mM HEPES and 10 mM EDTA, pH 7.4).
bly expressing rat mGluR7a were plated in clear-bottomed, poly-D- Once harvested, cells were homogenized on ice using a Polytron
lysine-coated 96-well plates 1 day before assay. After serum starving homogenizer (Tekmar, Inc., Cincinnati, OH) for 2 s. The homogenate
for 2 h, cells were equilibrated to assay buffer (DMEM, 20 mM was centrifuged at 40,000g for 20 min at 4°C. This last step was
HEPES, and 0.025% ascorbic acid) for 10 min at room temperature.
Compounds were serially diluted in 100% DMSO. Cells were then
repeated two additional times, homogenizing between centrifuga-
tions for 10 and 5 s, respectively, with the exception of rmGluR3
incubated with 500 ␮M 3-isobutyl-1-methylxanthine and test com- pellets, which were resuspended in ice-cold binding buffer 2 (20 mM
pounds or appropriate vehicle for 30 min at 37°C. Forskolin (15 ␮M) HEPES and 0.1 mM EDTA, pH 7.4) in the final centrifugation. The
and an EC80 concentration, L-AP4, were then added to cells and
incubated at 37°C for an additional 20 min. Drug solutions were
final pellets were resuspended and homogenized on ice using a
Dounce glass homogenizer (Fisher Scientific Co., Pittsburgh, PA)
decanted from cells, and 3% trichloroacetic acid was added for Ͼ2 h and were stored in aliquots at Ϫ80°C until use. Protein concentra-
at 4°C to extract intracellular cAMP. Samples were then incubated tions were measured using the Bio-Rad protein assay kit (Bio-Rad
with bovine protein kinase A and [3H]cAMP (1 nM) for 2 h on ice. Laboratories, Hercules, CA) using serum albumin as the standard
After harvesting samples onto a GF/B filter plate (PerkinElmer Life
and Analytical Sciences), samples were counted in a Packard Top-
(Pierce, Rockford, IL).
Radioligand Binding Studies. After thawing, the hmGluR2
Count (PerkinElmer Life and Analytical Sciences, Waltham, MA), membranes were resuspended and homogenized in ice-cold binding
and [cAMP] in each sample was determined based on a standard buffer as described above using a Dounce glass homogenizer. Bind-
curve of unlabeled cAMP. The effect of the test compounds was then
ing experiments were prepared as described previously (Johnson et
normalized as a percentage of the response to the EC80 concentration al., 1999). Test compounds were dissolved in 100% DMSO and then
of L-AP4.
serially diluted in binding buffer to yield a 5ϫ stock. The stock
solution was added to the assay plate to a final DMSO concentration
A
hmGluR2
hmGluR2
120
100
80
60
40
20
0
MNI-135, IC50= 118.2 23.6 nM
A
MNI-136, IC50= 46.6 2.5 nM
MNI-137, IC50= 72.7 5.0 nM
140
MNI-134
MNI-135
120
100
80
60
40
20
0
MNI-136
MNI-137
MNI-139
MNI-140
MNI-141
MNI-142
MNI-144
-10
-9
-8
-7
-6
-5
0
-9
-8
-7
-6
-5
log[Compound], M
log[Compound], M
rmGluR3
B -6.5
Rat v. Human
B
MNI-135, IC50= 30.2 3.6 nM
MNI-136, IC50= 21.3 1.6 nM
MNI-137, IC50= 20.3 4.0 nM
r2 = 0.958; p < 0.0001
140
120
100
80
-7.0
-7.5
-8.0
-8.5
60
40
20
0
-8.5
-8.0
-7.5
-7.0
-6.5
0
-9
-8
-7
-6
-5
Rat (IC50 Log, M)
log[Compound], M
Fig. 2. A, similar to results observed with rmGluR2, test compounds
exhibited inhibitory effects in a calcium mobilization assay in cells ex-
pressing hmGluR2. B, a statistically significant (p Ͻ 0.0001) correlation
was observed between the IC50 values obtained from cells expressing
rmGluR2 and hmGluR2 for all test compounds evaluated in a glutamate-
induced calcium mobilization assay. Data were analyzed using linear
regression analysis and are the mean of three independent experiments
performed in triplicate. Error bars represent mean Ϯ S.E.M.
Fig. 3. Three representative antagonists inhibited an EC80 concentration
of glutamate-induced [35S]GTP␥S binding in membranes from cell lines
stably expressing hmGluR2 or rmGluR3 in a concentration-dependent
manner. Results are expressed as the percentage of the maximal stimu-
lation induced by 1 mM or 31.6 ␮M glutamate for hmGluR2 and rm-
GluR3, respectively. Data are presented as the mean of three individual
experiments performed in triplicate with error bars representing mean Ϯ
S.E.M.

258
Hemstapat et al.
of 0.1%. All binding reactions were performed in 96-well deep plates
Site-Directed Mutagenesis. The cDNA encoding human
in a final volume of 100 ␮l. [3H]LY341495 binding assay mixtures mGluR2 in the pGTh backbone (Grinnell et al., 1991) was generously
contained 15 ␮g of membrane protein, 1 nM [3H]LY341495, and provided by Dr. M. Baez (Eli Lilly & Co., Indianapolis, IN). Point
various concentrations of test compounds. The assay mixtures were mutations were generated using the QuikChange site-directed mu-
incubated on ice for 30 min. Nonspecific binding was determined in
the presence of 1 mM glutamate.
tagenesis kit (Stratagene) according to the manufacturer’s instruc-
tions. Complimentary oligonucleotides were designed to contain the
The binding equilibrium was terminated by rapid filtration desired mutations and a novel restriction site, which does not alter
through Unifilter-96 GF/B filter plates (presoaked with ice-cold bind- the amino acid sequence, to be used for screening purposes. Sense
ing buffer), and the filter plates were washed three times with and antisense oligonucleotides were based on the following se-
ice-cold binding buffer using a 96-well Brandel harvester (Brandel quences and were used to introduce three point mutations into the
Inc., Gaithersburg, MD). Filter plates were dried and filled with 30 human mGluR2 sequence. 5Ј-CTGCCTGGCACTTATACTAGTC-
␮l of MicroScint-20, and the radioactivity was determined by Top- CAGCTGCTCATCGTG-3Ј was used for generation of two consecu-
Count NXT microplate scintillation and luminescence counter tive point mutations (S688L, G689V) along with a novel SpeI site,
(PerkinElmer Life and Analytical Sciences).
[
and 5Ј-GGCTCGCTGGCCTACGACGTCCTCCTCATCGCGCTC-3Ј
35S]GTP␥S Binding Studies. rmGluR3 membranes were was used to introduce a third single mutation (N735D) in combina-
thawed and homogenized using a glass homogenizer in ice-cold bind-
tion with a novel AatII site. PCR amplification was performed using
ing buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 150 mM Pfu Ultra high-fidelity DNA polymerase. The mutated hmGluR2
NaCl, 1 mM EDTA, 10 ␮g/ml saponin, and 1 ␮M GDP. Assay mix- sequence was dropped out of the pGTh backbone using flanking SalI
tures contained 10 ␮g of membrane protein, test compound, gluta- sites and subcloned into the pcDNA 3.1ϩ vector from Invitrogen at
mate, 0.1 nM [35S]GTP␥S, and assay buffer to yield a total volume of the complimentary XhoI site. Final constructs were verified by se-
100 ␮l. Nonspecific binding was determined in the presence of 10 ␮M quencing using an Applied Biosciences DNA analysis system at the
unlabeled guanosine 5Ј-3-O-(thio)triphosphate. The assay mixtures Vanderbilt University DNA sequencing facility.
were incubated at 30°C for 60 min. The reaction was terminated in
Field Potential Recording in Hippocampus Slice. Hippocam-
a manner similar to that described above in radioligand binding pal brain slices were prepared from 3 to 4-week-old male Sprague-
studies.
Dawley rats as described previously with minor modifications
B
A
140
120
100
80
rmGluR1
rmGluR5
140
120
100
80
60
60
MNI-135
MNI-136
MNI-137
MNI-135
MNI-136
MNI-137
40
40
20
20
0
0
-11
-10
-9
-8
-7
-6
-5
-11
-10
-9
-8
-7
-6
-5
log[Compound],M
log[Compound], M
Fig. 4. Three representative antagonists exhibited
no effect toward rmGluR1, rmGluR5, hmGluR4,
rmGluR7, or rmGluR8 in functional assays. In-
creasing concentrations of antagonists were unable
to inhibit an EC80 concentration of glutamate- or
L-AP4-induced function in cells lines expressing
rmGluR1 (A), rmGluR5 (B), and hmGluR4 (C), or
rmGluR8 (D) using calcium mobilization assay or
rmGluR7 (E) using a cyclase inhibition assay. Data
were analyzed using nonlinear regression analysis
and are the mean of three separate experiments
performed with a number of three (A-C), four (E), or
eight (D) independent replicates per experiment.
Error bars represent mean Ϯ S.E.M.
C
D
rmGluR7
hmGluR4
140
120
100
80
140
120
100
80
60
60
MNI-135
MNI-136
MNI-137
MNI-135
MNI-136
MNI-137
40
40
20
20
0
0
-11
-10
-9
-8
-7
-6
-5
-11
-10
-9
-8
-7
-6
-5
log[Compound],M
log[Compound],M
E
140
120
100
80
rmGluR8
60
MNI-135
40
20
0
MNI-136
MNI-137
-11
-10
-9
-8
-7
-6
-5
log[Compound],M

Novel Negative Allosteric Modulators of Group II mGluRs
259
(Macek et al., 1996, 1998). Brains were rapidly removed and sub-
the desired concentration. All compounds were added to the record-
merged in an ice-cold sucrose replacement solution containing 200 ing chamber via addition to the perfusion solution.
mM sucrose, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.3 mM MgCl2, 8 mM
Data Analysis. For calcium mobilization, radioligand binding
MgSO4, 20 mM glucose, and 46 mM NaHCO3, equilibrated with 95% and [35S]GTP␥S binding studies, data analysis was performed using
O2/5% CO2. Coronal hippocampal slices (400-␮m thick) were made GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA). Con-
using a microtome (Leica Microsystems Inc., Bannockburn, IL). The centration-response curves were analyzed using a nonlinear regres-
slices were placed in a holding chamber containing artificial cerebro- sion analysis and were generated from the means of three separate
spinal fluid (ACSF) comprising 124 mM NaCl, 2.5 mM KCl, 1.3 mM experiments. Inhibition curves were fitted by nonlinear regression
MgSO4, 1.0 mM NaH2PO4, 2 mM CaCl2, 20 mM glucose, and 26 mM analysis using the one-site competition equation. Error bars repre-
NaHCO3, equilibrated with 95% O2/5% CO2, which was maintained sent the mean Ϯ S.E.M. For the electrophysiology experiments, data
at room temperature. To increase slice viability, 5 ␮M glutathione, were analyzed using pClampfit 9.2. All results are expressed as
500 ␮M pyruvate, and 250 ␮M kynurenic acid were added to the mean ϮS.E.M., and statistical significance was determined using
sucrose replacement buffer, maintaining ACSF buffer in all experi- Student’s t test.
ments.
After a recovery period of at least 1 h, a slice was transferred to a
submerged brain slice recording chamber, where it was perfused
Results
Negative Allosteric Modulators Have Inhibitory Ac-
tivity for Group II mGluRs. We synthesized a range of
novel molecules based on the structure reported by Adam et
al. (2003). We first examined their inhibitory activity on cells
expressing rmGluR2 by employing a calcium mobilization
continuously with oxygenated ACSF at 2 to 3 ml/min at room tem-
perature. Recording electrodes were pulled on a Narishige (East
Meadow, NY) vertical patch pipette puller from a borosilicate glass
and filled with 2.5 mM NaCl, in which the electrode resistance
ranged from 0.3 to 1.0 M⍀. A bipolar tungsten electrode (FHC,
Bowdoinham, ME) and recording electrode were placed in the middle
third layer of the molecular layer at the dentate gyrus for stimula-
tion of the medial perforant path (MPP) fibers. Stimuli (0.1 ms in
duration) were delivered at 0.05 Hz using Grass S48 stimulator and
Grass isolator (Grass Technologies, West Warrick, RI). In each ex-
periment, an input-output curve was performed, and the stimulus
intensity was adjusted to produce a field excitatory postsynaptic
potential (fEPSP) approximately 50 to 70% of the maximal response.
The fEPSPs were recorded by an Axon MultiClamp 700B amplifier
(Molecular Devices) in current clamp mode. Data were filtered at 2
kHz, digitized with DigiData 1322A (Molecular Devices), and ac-
quired by the pClampex 9.2 program (Molecular Devices). The fEPSP
slope was measured by pClampfit 9.2 program (Molecular Devices).
The starting six consecutive slopes were averaged to define the
baseline fEPSP slope value for a given experiment. The subsequent
fEPSP slopes in the experiment were normalized to the baseline
fEPSP slope value and expressed as a percentage. Six consecutive
fEPSP slopes after 12 min of application of DCG-IV were averaged
and defined as the value of applying DCG-IV. All normalized fEPSP
slopes for each concentration of DCG-IV were then expressed as the
mean Ϯ S.E.M. All compounds were dissolved in double deionized
water with 1 N sodium hydroxide, with the exception of MNI-137,
which was dissolved in DMSO, and was then diluted with ACSF to
A
120
100
80
60
LY341495
MNI-135
40
MNI-136
MNI-137
20
0
-10
-9
-8
-7
-6
-5
log[Compound], M
B
120
100
80
60
40
20
0
rmGluR2
Vehicle
100
Vehicle
+ MNI-135
+ MNI-136
+ MNI-137
+ 31.6 nM MNI-135
+ 31.6 nM MNI-136
+ 31.6 nM MNI-137
90
80
70
60
50
40
30
20
10
0
-7
-6
-5
-4
-3
log[Glutamate], M
Fig. 6. Three representative antagonists had no effect on [3H]LY341495
binding. Membranes were prepared from CHO cells stably expressing hm-
GluR2 and were incubated with 1 nM [3H]LY341495 in a final volume of 100
␮l for 30 min at 4°C in the presence of varying concentrations of LY341495
or three representative antagonists. Bound and free radioligand were sepa-
rated by vacuum filtration through Unifilter-96 GF/B filter plates. Nonspe-
cific binding was determined in the presence of 1 mM glutamate. The
radioactivity was determined by TopCount NXT. A, binding of 1 nM
[3H]LY341495 was displaced by cold LY341495 but not by the three repre-
sentative compounds tested. B, the presence of three representative com-
pounds (1 ␮M) did not affect the displacement of [3H]LY341495 by gluta-
mate. The graphs summarize data from three independent experiments
performed in triplicate. Error bars represent mean Ϯ S.E.M.
0
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0
log[Glutamate], M
Fig. 5. Concentration-response curves of glutamate-induced calcium mo-
bilization were inhibited in a noncompetitive manner by three represen-
tative antagonists (31.6 nM) in HEK293A cells expressing rmGluR2.
Data are expressed as a percentage of the maximal response to glutamate
(10 ␮M) and are the mean of three individual experiments performed in
triplicate. Error bars represent mean Ϯ S.E.M.

260
Hemstapat et al.
assay. Consistent with action as antagonists of group II
MNI-135, MNI-136, and MNI-137 Are Noncompetitive
mGluRs, all test compounds blocked a glutamate-induced Group II mGluR Antagonists. To characterize the mech-
increase in calcium (Fig. 1A). A similar effect was observed anism of inhibition of the novel negative allosteric modula-
using [35S]GTP␥S binding studies to measure activation of tors, the effect of one single concentration (31.6 nM) of each
rmGluR3 overexpressed in CHO cells, as [35S]GTP␥S binding of these compounds on the glutamate concentration-response
was attenuated in these cells following an application of test curves was assessed. In cells expressing rmGluR2, all three
compounds (Fig. 1B). Inhibitory effects of these compounds compounds shifted glutamate concentration-response curves
on calcium mobilization were also observed in CHO cells in a calcium mobilization assay to the right with a concomi-
stably expressing hmGluR2 (Fig. 2A). For the calcium mobi- tant decrease in the maximal response (Fig. 5). These results
lization assay, a highly significant positive correlation (r2 ϭ are consistent with a noncompetitive mode of action. To con-
0.958, p Ͻ 0.0001) was observed between the IC50 values firm that these compounds act as noncompetitive antago-
obtained from cells expressing rmGluR2 and hmGluR2 (Fig. nists, we determined their ability to bind to the orthosteric
2B), suggesting that there is no species specificity exhibited site by measuring their ability to displace the binding of
within this series.
[3H]LY341495, a potent orthosteric antagonist of group II
Negative Allosteric Modulators Are Selective for mGluRs. [3H]LY341495 binding was measured in CHO cell
Group II Relative to Group I and III mGluRs. Three membranes stably expressing hmGluR2. As shown in Fig.
compounds (MNI-135, MNI-136, and MNI-137) that exhib- 6A, the three compounds tested had no effect on binding of 1
ited the most potent inhibitory activity in the calcium mobi- nM [3H]LY341495 at concentrations up to 10 ␮M, whereas
lization assay at rmGluR2 were selected for further studies radioactive LY341495 potently displaced binding of the ra-
aimed at determining their selectivity for specific mGluR dioligand. The ability of glutamate to displace the binding of
subtypes. Similar to their activity observed in the calcium 1 nM [3H]LY341495 was not altered in the presence of these
mobilization assay, these three representative compounds compounds (Fig. 6B). For comparison, the potency of inhibi-
inhibited glutamate-induced [35S]GTP␥S activation in cells tion of all test compounds on glutamate-induced responses
expressing hmGluR2 (Fig. 3A). Comparison of these data for both calcium mobilization and [35S]GTP␥S binding assay
with the effects of these compounds on rmGluR3 (Fig. 3B), as are summarized in Table 1.
assessed by [35S]GTP␥S binding, revealed that these com-
MNI-137 Inhibits Group II mGluR Agonist-Mediated
pounds were slightly more potent at inhibiting glutamate- Responses at Medial Perforant Path-Dentate Gyrus
induced activation of mGluR3 relative to mGluR2. However, Synapses in Rat Hippocampal Slices. Previous findings
none of the compounds provided sufficient selectivity be- revealed that group II mGluRs, the predominant subtype at
tween the group II mGluRs to be useful for differentiating the medial perforant path-dentate granule cell synapse
between the group II mGluR subtypes. To further character- (MPP-DG) inhibits excitatory synaptic transmission by re-
ize the selectivity of these compounds, we used the calcium ducing glutamate release from presynaptic terminals (Macek
mobilization assay with cells expressing mGluR1, 4, 5, or 8, et al., 1996, 1998). Thus, the MPP-DG synapses of the rat
as well as a cyclase inhibition assay with cells expressing hippocampus were employed as a native system for deter-
mGluR7. None of these compounds exhibited inhibitory ac- mining the effects of MNI-137, a representative novel nega-
tivity induced by activation of any of these receptor subtypes tive allosteric modulator identified in this study, on group II
at concentrations up to 10 ␮M (Fig. 4, A–E).
mGluR-mediated responses.
TABLE 1
IC50 values for the inhibition of an EC80 concentration of glutamate-induced response in both calcium mobilization and ͓35S͔GTP␥S binding assay
on cells expressing rmGluR2, hmGluR2 or rmGluR3. Error expressed as S.E.M.
Calcium Assay Potency
mGluR2
͓
35S͔GTP␥S Binding Potency
Compounds
R1
R2
R3
Y
mGluR2
mGluR3
IC50 (Rat)
IC50 (Human)
IC50 (Human)
IC50 (Rat)
nM
nM
MNI-134
MNI-135
MNI-136
MNI-137
MNI-139
MNI-140
MNI-141
MNI-142
MNI-144
Cl
H
Br
H
CH3
H
Br
Cl
I
CH
CH
CH
N
CH
CH
N
141.7 Ϯ 4.0
19.6 Ϯ 0.7
19.7 Ϯ 0.7
12.6 Ϯ 1.4
164.9 Ϯ 16.6
22.5 Ϯ 1.4
47.9 Ϯ 5
73.9 Ϯ 33.8
10.51 Ϯ 2.3
8.8 Ϯ 1.7
NT
118.2 Ϯ 23.6
46.6 Ϯ 2.5
72.7 Ϯ 5.0
NT
73.8 Ϯ 16.3
30.2 Ϯ 3.6
21.3 Ϯ 1.6
20.3 Ϯ 4.0
57.7 Ϯ 9.6
17.4 Ϯ 2.1
182.7 Ϯ 25
378.8 Ϯ 84
183.9 Ϯ 37.6
I
CN
H
3-C5H4N
CN
I
3-C5H4N
CN
CN
CN
Br
CH3
Br
H
8.3 Ϯ 1.4
158.1 Ϯ 10.2
19.3 Ϯ 1.7
33.0 Ϯ 2.8
151.4 Ϯ 8.6
99.2 Ϯ 14.6
NT
NT
CH3NH
CH3CO
Cl
Cl
N
N
170.0 Ϯ 33.0
167.7 Ϯ 15.2
NT
NT
NT, not tested.

Novel Negative Allosteric Modulators of Group II mGluRs
261
To assess the effects of activation of group II mGluRs on present functional and radioligand binding studies suggest
synaptic transmission at the MPP-DG synapse, extracellular that these negative allosteric modulators do not bind to the
field potentials were recorded from the middle third of the orthosteric binding site (i.e., glutamate binding site). A pre-
molecular layer in the dentate gyrus. Consistent with our vious study reported that the amino acid residues forming
previous results, bath application of the group II agonist the binding pocket for a novel positive allosteric modulator
DCG-IV induced a concentration-dependent decrease in fEP- for mGluR2, LY487379, are Ser688, Gly689, and Asn735
SPs at this synapse (data not shown) (Galici et al., 2006). 1 (Schaffhauser et al., 2003). We next sought to investigate
␮M DCG-IV inhibited the fEPSP slope by 60.31 Ϯ 2.58% (n ϭ whether these residues are also critical for the inhibitory
12) (Fig. 7, A and C). In addition, the effect of 1 ␮M DCG-IV activity of our novel negative allosteric modulators.
was blocked by the orthosteric mGlu2/3 receptor antagonist
We first examined the enhancing property of LY487379 in
LY341495 (1 ␮M), (data not shown). Consistent with the HEK293A cells transiently expressing wild-type hmGluR2 or
finding that MNI-137 inhibited glutamate-induced increases mutant hmGluR2 containing a single (N735D in TM V),
in [35S]GTP␥S binding in cells expressing hmGluR2, with a double (S688L/G689V in TM IV), or triple (S688L/G689V/
maximal effect in the low micromolar range, 3 ␮M MNI-137 N735D) point mutation in the presence of single concentra-
significantly blocked the effect of 1 ␮M DCG-IV on fEPSP tion of LY487379 (1 ␮M) as a control experiment. Consistent
slope (16.84 Ϯ 3.01%; n ϭ 8; ء, p Ͻ 0.01) (Fig. 7, B and C).
with previous studies, LY487379 induced parallel shifts of
Point Mutation of Residue Asn735 Located in Trans- the glutamate concentration-response curve approximately 6
membrane V Abolishes Potentiation Activities of Pos- to 7-fold to the left in HEK293A cells transiently expressing
itive Allosteric Modulators of mGluR2 but Not Nega- wild-type hmGluR2 (Fig. 8A, left). The single point mutation,
tive Allosteric Modulators of Group II mGluRs. The N735D, in TM V of hmGluR2 markedly attenuated the po-
tentiation activity of LY487379 compared with wild type
(Fig. 8A, right). In addition, approximately 30 and 85% re-
duction of the potentiation by LY487379 of glutamate-in-
duced calcium mobilization was observed in mutant hm-
DCG- IV 1 µM
A
GluR2 containing the double mutation (S688L/G689V) or
triple mutation (S688L/G689V/N735D), respectively (data
not shown). These results suggest that residue Asn735 plays
0.2mv
5ms
Control
a critical role for the enhancing activity of this modulator.
Based on this finding, we sought to examine whether this
residue was also critical for potentiation activity of a struc-
turally distinct newly reported positive allosteric modulator
of mGluR2, BINA. Similar to LY487379, BINA induced par-
allel shifts of the glutamate concentration-response curve
approximately 11-fold to the left in HEK293A cells tran-
siently expressing wild-type hmGluR2 (Fig. 8B, left) (data
were from Galici et al. (2006). Potentiation activity of BINA
was almost completely abolished in mutant hmGluR2 con-
taining N735D (Fig. 8B, right).
To determine whether residues Ser688, Gly689, and
Asn735 contribute to the inhibitory activity of our novel
negative allosteric modulators of group II mGluRs, we used
calcium mobilization and mutagenesis approaches similar to
those performed with LY487379 and BINA. We found that
the ability of MNI-135, MNI-136, and MNI-137 to block an
EC80 glutamate-induced response was not altered in
HEK293A cells transiently expressing mutant hmGluR2 con-
taining N735D (Fig. 9, right) compared with wild-type (Fig.
9, left). Similar results were also observed with the double
mutant (S688L/G689V) or triple mutant (S688L/G689V/
N735D) (data not shown), suggesting that these residues are
not critical for the inhibitory activity of our novel negative
allosteric modulators.
DCG-IV 1 µM
+ 3 µM MNI-137
B
C
0.2mv
5ms
Control
(n=12)
80
60
40
20
0
(n=8)
*
DCG-IV 1 µM DCG-IV 1 µM
+ 3 µM MNI-137
Fig. 7. MNI-137 blocked DCG-IV-induced inhibition of the fEPSP slope at
MPP-DG synapses in rat hippocampal slices. A, representative fEPSP
traces depicting the effect of a 12-min application of 1 ␮M DCG-IV alone
on the synaptic transmission at MPP-DG synapses. B, representative
fEPSP traces depicting the effect of 1 ␮M DCG-IV following a 10-min
preincubation with 3 ␮M MNI-137. Each representative fEPSP trace is
an average of six consecutive evoked responses at this synapse. C, the bar
graph illustrates the effects of application of 1 ␮M DCG-IV (n ϭ 12) in the
absence or presence of 3 ␮M MNI-137 (n ϭ 8) on the fEPSP slope at
MPP-DG synapses. The average of the fEPSP slope obtained from six
traces immediately before and after compound application was compared
to determine the effects of test compounds. All values are expressed as
Discussion
In the present study, we synthesized and rigorously char-
acterized a novel family of noncompetitive antagonists (neg-
ative allosteric modulators) of group II mGluRs based on the
general structure reported by Adam et al. (2003). These stud-
ies confirm a preliminary report suggesting that compounds
in this structural class have allosteric antagonist activity at
mean Ϯ S.E.M, Calibration bar: 0.2 mV/5 ms. ء, p Ͻ 0.01; Student’s t test. group II mGluRs (Gatti et al., 2001). These compounds are

262
Hemstapat et al.
LY487379
A
Wild-type
N735D
Vehicle, EC50= 814.4 13.4 nM
Vehicle, EC50= 1058.0 124.5 nM
1 µM LY487379, EC50= 122.2 13.3 nM
+1 uM LY487379, EC50= 750.4 61.2 nM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Fig. 8. Potentiator activity of both LY487379 (A) and BINA
(B), novel selective positive allosteric modulators of
mGluR2, on glutamate-induced calcium mobilization in
HEK293A cells is dependent on residue Asn735 in TM V of
mGluR2. HEK293A cells were transiently transfected with
the wild-type mGluR2 or a mutant mGluR2 containing a
single point mutation N735D. LY487379 and BINA caused
a parallel leftward shift of the glutamate concentration-
response curve in cells expressing wild-type mGluR2 (A
and B, left). However, the parallel leftward shift of the
glutamate concentration curve was markedly reduced with
the N735D mutant mGluR2, indicating a loss of potentia-
tion activity at this mutant (A and B, right). The fluores-
cence responses were normalized as a percentage of the
maximal response to glutamate (10 ␮M) and are the mean
of four independent experiments performed in triplicate.
Error bars are mean Ϯ S.E.M.
0
-8
-7
-6
-5
0
-8
-7
-6
-5
log[Glutamate], M
log[Glutamate], M
BINA
B
Wild-type
Vehicle, EC50= 1182 109.1 nM
300 nM BINA, EC50= 108 15.17 nM
N735D
Vehicle, EC50= 913.3 44.9 nM
300nM BINA, EC50= 865.6 12.3 nM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
-8
-7
-6
-5
0
-8
-7
-6
-5
Log[Glutamate], M
log[Glutamate], M
chemically and structurally unrelated to the competitive
These compounds were found to be noncompetitive antag-
group II antagonist, LY341495 or MGS0039, or to the allo- onists of glutamate action on group II mGluRs, and they did
steric mGluR2 potentiator, LY487379. These novel com- not displace [3H]LY341495 from its binding site. Interest-
pounds exhibited nanomolar potency for both rat and human ingly, the affinity of glutamate to displace the binding of
mGluR2 and rat mGluR3 expressed in cell lines and also [3H]LY341495 was not affected in the presence of these com-
blocked activation of group II mGluRs at the MPP-DG syn- pounds, suggesting that they do not act by allosterically
apse in rat hippocampal slices in a manner similar to that regulating binding of agonists to the orthosteric site. This is
observed in the recombinant systems.
similar to the effects of previously identified allosteric antag-
Wild-type
N735D
MNI-135, IC50= 24.4 1.4 nM
MNI-136, IC50= 18 3.2 nM
MNI-135, IC50= 21.4 0.9 nM
MNI-136, IC50= 17.5 0.3 nM
MNI-137, IC50= 11.4 1.1 nM
120
120
100
80
60
40
20
0
MNI-137, IC50= 17.0 0.1 nM
100
80
60
40
20
0
0
-9
-8
-7
-6
-5
0
-9
-8
-7
-6
-5
log[Compound], M
log[Compound], M
Fig. 9. The effect of novel negative allosteric group II mGluRs (i.e., MNI-135, MNI-136, and MNI-137) on glutamate-induced calcium mobilization in
HEK293A cells is independent of N735D in TM V of mGluR2. HEK293A cells were transiently transfected with the wild-type hmGluR2 or a mutant
mGluR2 containing a single point mutation, N735D. These compounds produced a concentration-dependent inhibition of glutamate-induced responses
in cells expressing wild-type mGluR2 (left). Single point mutation of residue Asn735 in hmGluR2 did not alter the inhibitory activity of these novel
compounds (right). The fluorescence responses were normalized as a percentage of the maximal response to glutamate (10 ␮M) and are the mean of
four independent experiments performed in triplicate. Error bars are mean Ϯ S.E.M.

Novel Negative Allosteric Modulators of Group II mGluRs
263
onists of group I mGluRs, which do not alter glutamate BINA. These data suggest that these two chemically unre-
binding but may act by altering conformational changes of lated allosteric potentiators of mGluR2, LY487379, and
the seven-transmembrane domain that in turn can inhibit BINA may share a common binding site. In contrast, the
coupling to G proteins (Kuhn et al., 2002).
single mutation N735D failed to block the inhibitory effect of
Importantly, these novel compounds did not inhibit gluta- our novel negative allosteric modulators of group II mGluRs.
mate- or L-AP4-induced function in cell lines expressing These studies are consistent with growing evidence that
mGluR1, 4, 5, or 8 by using calcium mobilization assay or other mGluR subtypes can contain multiple allosteric regu-
mGluR7 by using a cyclase inhibition assay, suggesting that latory sites. For instance, we recently reported that two fam-
they are selective group II mGluR antagonists. However, it is ilies of allosteric potentiators of mGluR1 share a common
important to note that the calcium fluorescence assay used binding site and that this site is distinct from the binding site
for measuring mGluR4 and mGluR8 activity relies on cou- that is common to multiple allosteric antagonists of mGluR1
pling to a promiscuous G protein that is not the native G (Hemstapat et al., 2006). Furthermore, we have provided
protein to which these receptors normally couple. Although evidence that N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoin-
unlikely, it is conceivable that this could affect the response dol-2-yl)methyl]phenyl}-2-hydroxybenzamide, an allosteric
of these receptors to antagonists
potentiator of mGluR5, acts at a site that is distinct from that
These novel compounds identified in the present study the of the site for the allosteric antagonist 2-methyl-6-(phenyl-
most selective noncompetitive antagonists of group II ethynyl)-pyridine (O’Brien et al., 2004). However, it is also
mGluRs available to date. The most commonly used antago- clear that other classes of compounds can bind to the 2-meth-
nist of these receptors is LY341495, which acts as a compet- yl-6-(phenylethynyl)-pyridine site with a range of activities,
itive antagonist at the orthosteric glutamate binding site (see including positive and negative allosteric modulators as well
Schoepp et al., 1999 for review). Although selective for group as neutral ligands (O’Brien et al., 2003; Kinney et al., 2005;
II mGluRs relative to other mGluR subtypes, this compound Rodriguez et al., 2005).
has antagonist activity at all known mGluRs and blocks
In summary, we have thoroughly characterized a novel
mGluR8 with a potency similar to its potency at mGluR2 and family of negative allosteric modulators of group II mGluRs.
mGluR3 (Kingston et al., 1998). The present findings are Although few reports have described the therapeutic signif-
consistent with a growing body of literature suggesting that icance of group II mGluR antagonists, recent studies have
allosteric antagonists provide a novel approach for develop- demonstrated that they may exhibit antidepressant-like
ing both antagonists and potentiators for mGluRs that have activity and antiobsessive-compulsive disorder-like effects
higher subtype selectivity than traditional orthosteric li- (Chaki et al., 2004; Shimazaki et al., 2004). Our current
gands. With the group I and group III mGluRs, this has finding of negative allosteric modulators of group II mGluRs
extended to the discovery of ligands that differentiate be- suggests that targeting of allosteric sites provides a viable
tween members within a group (Kuhn et al., 2002; Marino et approach for developing highly selective antagonists of these
al., 2003; Mitsukawa et al., 2005). Unfortunately, the present receptors.
compounds did not exhibit selectivity that would distinguish
between mGluR2 and mGluR3. Furthermore, there was no Acknowledgments
indication from the present studies that the structure-activ-
We thank Kari Myers for the construction of the mGluR7/HEK cell
ity relationship within this series could lead to subtype- lines and Douglas Sheffler for help with the cyclase assays.
specific compounds that differentiate among these closely
related subtypes.
An especially interesting finding in the current studies was
preliminary evidence suggesting that two structurally dis-
tinct allosteric potentiators of mGluR2 may share a common
binding site and that mutations that disrupt activity of these
potentiators are without effect on the response to the new
allosteric antagonists. Recent findings by Schaffhauser et al.
References
Adam G, Goetschi E, Mutel V, Wichmann J and Woltering TJ (2003) inventors;
Hoffmann-La Roche Inc., assignee. Dihydro-benzo[b][1,4]diazepin-2-one deriva-
tives. World Patent WO01/29012. 2003 April 15.
Adwanikar H, Karim F, and Gereau RW 4th (2004) Inflammation persistently
enhances nocifensive behaviors mediated by spinal group I mGluRs through sus-
tained ERK activation. Pain 111:125–135.
Campbell UC, Lalwani K, Hernandez L, Kinney GG, Conn PJ, and Bristow LJ (2004)
The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) potentiates
PCP-induced cognitive deficits in rats. Psychopharmacology (Berl) 175:310–318.
Carter K, Dickerson J, Schoepp DD, Reilly M, Herring N, Williams J, Sallee FR,
(2003) suggest that residues Ser688, Gly689, and Asn735 are
Sharp JW, and Sharp FR (2004) The mGlu2/3 receptor agonist LY379268 injected
part of the binding pocket for a novel positive allosteric
modulator for mGluR2, LY487379, and are important for its
potentiation activity. Therefore, we next sought to investi-
gate whether these residues are also critical for the inhibi-
tory activity of our novel negative allosteric modulators. Con-
sistent with this previous report, the enhancing effect of
LY487379 on glutamate-induced calcium mobilization was
markedly abolished by the single point mutation N735D,
whereas the double mutation S688L/G689V induced a slight
reduction. Moreover, the triple mutation, S688L/G689V/
N735D, further abolished LY487379-induced responses com-
pared with the single N735D mutant, suggesting that resi-
due Asn735 plays a critical role for the enhancing activity of
this allosteric modulator. Interestingly, we also found that
residue Asn735 is also critical for potentiation activity of a
newly reported selective allosteric potentiator of mGluR2,
into cortex or thalamus decreases neuronal injury in retrosplenial cortex produced
by NMDA receptor antagonist MK-801: possible implications for psychosis. Neu-
ropharmacology 47:1135–1145.
Chaki S, Yoshikawa R, Hirota S, Shimazaki T, Maeda M, Kawashima N, Yoshimizu
T, Yasuhara A, Sakagami K, Okuyama S, et al. (2004) MGS0039: a potent and
selective group II metabotropic glutamate receptor antagonist with antidepres-
sant-like activity. Neuropharmacology 46:457–467.
Conn PJ, Battaglia G, Marino MJ, and Nicoletti F (2005) Metabotropic glutamate
receptors in the basal ganglia motor circuit. Nat Rev Neurosci 6:787–798.
Conn PJ and Pin JP (1997) Pharmacology and functions of metabotropic glutamate
receptors. Annu Rev Pharmacol Toxicol 37:205–237.
Fisher K, Lefebvre C, and Coderre TJ (2002) Antinociceptive effects following intra-
thecal pretreatment with selective metabotropic glutamate receptor compounds in
a rat model of neuropathic pain. Pharmacol Biochem Behav 73:411–418.
Galici R, Jones CK, Hemstapat K, Nong Y, Echemendia NG, Williams LC, de Paulis
T, and Conn PJ (2006) Biphenyl-indanone A, a positive allosteric modulator of the
metabotropic glutamate receptor subtype 2, has antipsychotic- and anxiolytic-like
effects in mice. J Pharmacol Exp Ther 318:173–185.
Gasparini F, Lingenhohl K, Stoehr N, Flor PJ, Heinrich M, Vranesic I, Biollaz M,
Allgeier H, Heckendorn R, Urwyler S, et al. (1999) 2-Methyl-6-(phenylethynyl)-
pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor
antagonist. Neuropharmacology 38:1493–1503.
Gatti S, Knoflach F, Kew J, Adam G, Goetschi E, Wichmann J, Woltering T, Kemp

264
Hemstapat et al.
J, and Mutel
V
(2001) 8-Arylethynyl-1,3-dihydro-benzo1,4]diazepin-2-one-
Macek TA, Winder DG, Gereau RW 4th, Ladd CO, and Conn PJ (1996) Differential
involvement of group II and group III mGluRs as autoreceptors at lateral and
medial perforant path synapses. J Neurophysiol 76:3798–3806.
derivatives are potent non-competitive metabotropic glutamate receptor 2/3 an-
tagonists. Soc Neurosci Abstr 21:705.10.
Grillon C, Cordova J, Levine LR, and Morgan CA 3rd (2003) Anxiolytic effects of a
novel group II metabotropic glutamate receptor agonist (LY354740) in the fear-
potentiated startle paradigm in humans. Psychopharmacology (Berl) 168:446–
454.
Grinnell BW, Walls JD, and Gerlitz B (1991) Glycosylation of human protein C
affects its secretion, processing, functional activities, and activation by thrombin.
J Biol Chem 266:9778–9785.
Hemstapat K, de Paulis T, Chen Y, Brady AE, Grover VK, Alagille D, Tamagnan GD,
and Conn PJ (2006) A novel class of positive allosteric modulators of metabotropic
glutamate receptor subtype 1 interact with a site distinct from that of negative
allosteric modulators. Mol Pharmacol 70:616–626.
Homayoun H, Stefani MR, Adams BW, Tamagnan GD, and Moghaddam B (2004)
Functional interaction between NMDA and mGlu5 receptors: effects on working
memory, instrumental learning, motor behaviors, and dopamine release. Neuro-
psychopharmacology 29:1259–1269.
Johnson BG, Wright RA, Arnold MB, Wheeler WJ, Ornstein PL, and Schoepp DD
(1999) [3H]-LY341495 as a novel antagonist radioligand for group II metabotropic
glutamate (mGlu) receptors: characterization of binding to membranes of mGlu
receptor subtype expressing cells. Neuropharmacology 38:1519–1529.
Johnson MP, Baez M, Jagdmann GE Jr., Britton TC, Large TH, Callagaro DO,
Tizzano JP, Monn JA, and Schoepp DD (2003) Discovery of allosteric potentiators
for the metabotropic glutamate 2 receptor: synthesis and subtype selectivity of
N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmeth-
ylamine. J Med Chem 46:3189–3192.
Kingston AE, Ornstein PL, Wright RA, Johnson BG, Mayne NG, Burnett JP, Bel-
agaje R, Wu S, and Schoepp DD (1998) LY341495 is a nanomolar potent and
selective antagonist of group II metabotropic glutamate receptors. Neuropharma-
cology 37:1–12.
Kinney GG, O’Brien JA, Lemaire W, Burno M, Bickel DJ, Clements MK, Chen TB,
Wisnoski DD, Lindsley CW, Tiller PR, et al. (2005) A novel selective positive
allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo
activity and antipsychotic-like effects in rat behavioral models. J Pharmacol Exp
Ther 313:199–206.
Kuhn R, Pagano A, Stoehr N, Vranesic I, Flor PJ, Lingenhohl K, Spooren W, Gentsch
C, Vassout A, Pilc A, et al. (2002) In vitro and in vivo characterization of MPEP,
an allosteric modulator of the metabotropic glutamate receptor subtype 5: review
article. Amino Acids 23:207–211.
Marino MJ, Williams DL Jr., O’Brien JA, Valenti O, McDonald TP, Clements MK,
Wang R, DiLella AG, Hess JF, Kinney GG, et al. (2003) Allosteric modulation of
group III metabotropic glutamate receptor 4: a potential approach to Parkinson’s
disease treatment. Proc Natl Acad Sci U S A 100:13668–13673.
Mitsukawa K, Yamamoto R, Ofner S, Nozulak J, Pescott O, Lukic S, Stoehr N,
Mombereau C, Kuhn R, McAllister KH, et al. (2005) A selective metabotropic
glutamate receptor 7 agonist: activation of receptor signaling via an allosteric site
modulates stress parameters in vivo. Proc Natl Acad Sci U S A 102:18712–18717.
Moghaddam B (2004) Targeting metabotropic glutamate receptors for treatment of
the cognitive symptoms of schizophrenia. Psychopharmacology (Berl) 174:39–44.
O’Brien JA, Lemaire W, Chen TB, Chang RSL, Jacobson MA, Ha SN, Lindsley CW,
Schaffhauser HJ, Sur C, Pettibone DJ, et al. (2003) A family of highly selective
allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol Phar-
macol 64:731–740.
O’Brien JA, Lemaire W, Wittmann M, Jacobson MA, Ha SN, Wisnoski DD, Lindsley
CW, Schaffhauser HJ, Rowe B, Sur C, et al. (2004) A novel selective allosteric
modulator potentiates the activity of native metabotropic glutamate receptor
subtype 5 in rat forebrain. J Pharmacol Exp Ther 309:568–577.
Ohishi H, Neki A, and Mizuno N (1998) Distribution of a metabotropic glutamate
receptor, mGluR2, in the central nervous system of the rat and mouse: an immu-
nohistochemical study with a monoclonal antibody. Neurosci Res 30:65–82.
Palucha A and Pilc A (2005) The involvement of glutamate in the pathophysiology of
depression. Drug News Perspect 18:262–268.
Rodriguez AL, Nong Y, Sekaran NK, Alagille D, Tamagnan GD, and Conn PJ (2005)
A close structural analog of MPEP acts as a neutral allosteric site ligand on
metabotropic glutamate receptor subtype 5 and blocks the effects of multiple
allosteric modulators. Mol Pharmacol 68:1793–1802.
Schaffhauser H, Rowe BA, Morales S, Chavez-Noriega LE, Yin R, Jachec C, Rao SP,
Bain G, Pinkerton AB, Vernier JM, et al. (2003) Pharmacological characterization
and identification of amino acids involved in the positive modulation of metabo-
tropic glutamate receptor subtype 2. Mol Pharmacol 64:798–810.
Schoepp DD, Jane DE, and Monn JA (1999) Pharmacological agents acting at
subtypes of metabotropic glutamate receptors. Neuropharmacology 38:1431–1476.
Shimazaki T, Iijima M, and Chaki S (2004) Anxiolytic-like activity of MGS0039, a
potent group II metabotropic glutamate receptor antagonist, in a marble-burying
behavior test. Eur J Pharmacol 501:121–125.
Shipe WD, Wolkenberg SE, Williams DL Jr, and Lindsley CW (2005) Recent ad-
vances in positive allosteric modulators of metabotropic glutamate receptors. Curr
Opin Drug Discov Devel 8:449–457.
Lavreysen H, Janssen C, Bischoff F, Langlois X, Leysen JE, and Lesage ASJ (2003)
[3H]R214127: a novel high-affinity radioligand for the mGlu1 receptor reveals a
common binding site shared by multiple allosteric antagonists. Mol Pharmacol
63:1082–1093.
Linden AM, Shannon H, Baez M, Yu JL, Koester A, and Schoepp DD (2005) Anxio-
lytic-like activity of the mGLU2/3 receptor agonist LY354740 in the elevated plus
maze test is disrupted in metabotropic glutamate receptor 2 and 3 knock-out mice.
Psychopharmacology (Berl) 179:284–291.
Macek TA, Schaffhauser H, and Conn PJ (1998) Protein kinase C and A3 adenosine
receptor activation inhibit presynaptic metabotropic glutamate receptor (mGluR)
function and uncouple mGluRs from GTP-binding proteins. J Neurosci 18:6138–
6146.
Swanson CJ, Bures M, Johnson MP, Linden AM, Monn JA, and Schoepp DD (2005)
Metabotropic glutamate receptors as novel targets for anxiety and stress disor-
ders. Nat Rev Drug Discov 4:131–144.
Address correspondence to: Dr. P. Jeffrey Conn, Department of Pharma-
cology, Vanderbilt University Medical Center, 23rd Avenue South at Pierce,
417-D Preston Research Building, Nashville, TN 37232-6600. E-mail:
jeff.conn@vanderbilt.edu