Pharmacological characterization of JNJ-40068782, a new...

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Pharmacological characterization of JNJ-40068782, a new potent, selective

and systemically active positive allosteric modulator of the mGlu2 receptor

and its radioligand [3H]JNJ-40068782

Hilde Lavreysen, Xavier Langlois, Abdel Ahnaou, Wilhelmus Drinkenburg, Paula te Riele,

Ilse Biesmans, Ilse Van der Linden, Luc Peeters, Anton Megens, Cindy Wintmolders, José

Cid, Andrés A. Trabanco, Ignacio Andrés, Frank M. Dautzenberg, Robert Lütjens, Gregor

Macdonald, John Atack

Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium (HL, XL, AA,

WD, PtR, IB, IVdL, LP, AM, CW, FD, GM, JA)

Janssen Research and Development, Jarama 75, 45007 Toledo, Spain (JC, AT, IA)

Addex Therapeutics, Chemin des Aulx 12, 1228 Plan-les-Ouates, Geneva, Switzerland (RL)

JPET Fast Forward. Published on June 13, 2013 as DOI:10.1124/jpet.113.204990

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 13, 2013 as DOI: 10.1124/jpet.113.204990

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Running title: Pharmacological characterization of JNJ-40068782, a mGlu2 receptor PAM

Address correspondence, proofs and reprint requests to Dr. Hilde Lavreysen at: Neuroscience,

Janssen, Turnhoutseweg 30, B-2340 Beerse, Belgium

Tel: +32 (0)14 607680

Fax: +32 (0)14 606121

E-mail: [email protected]

Number of text pages: 43

Number of tables: 2

Number of figures: 10

Number of references: 53

Words in abstract: 260

Words in Introduction: 783

Words in Discussion: 1771

Abbreviations:

JNJ-40068782, 3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-

one; JNJ-40264796, 1-Butyl-4-[4-(2-methylpyridin-4-yloxy)phenyl]-2-oxo-1,2-

dihydropyridine-3-carbonitrile; DCG-IV, (2S,2′R,3′R)-2-(2′,3′-

dicarboxylcyclopropyl)glycine; BINA, biphenyl-indanone A; LY487379, 2,2,2-Trifluoro-N-

[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide; LY341495, 2S-2-

amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl) propanoic acid; LY404039, (-)-

(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid;

THIIC/LY2607540, N-(4-((2-(trifluoromethyl)-3-hydroxy-4-


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(isobutyryl)phenxoy)methylbenzyl)-1-methyl-1H-imidazo-4-carboxamide; GDP, guanosin 5’-

diphosphate sodium salt; EGTA, ethylene glycol-bis(2-amino-ethylether)-N,N,N’,N’-tetra-

acetic acid; BSA, albumin from bovine serum; Tris, Tris(hydroxymethyl)-aminomethane;

PMSF, phenylmethanesulfonyl fluoride; HBSS, Hank’s Buffered Salt Solution; REM, rapid

eye movement

Recommended Section Assignment: Neuropharmacology


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Abstract

Modulation of the metabotropic glutamate type 2 (mGlu2) receptor is considered a promising

target for the treatment of CNS diseases like schizophrenia. Here, we describe the

pharmacological properties of the novel mGlu2 receptor positive allosteric modulator (PAM)

3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-one (JNJ-

40068782) and its radioligand [3H]JNJ-40068782. In [35S]GTPγS binding, JNJ-40068782

produced a left- and up-ward shift in the glutamate concentration-effect curve at human

recombinant mGlu2 receptors. The EC50 of JNJ-40068782 for potentiation of an EC20-

equivalent concentration of glutamate was 143 nM. While JNJ-40068782 did not affect

binding of the orthosteric antagonist [3H] 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-

3-(xanth-9-yl) propanoic acid (LY-341495), it did potentiate the binding of the agonist [3H]

(2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine (DCG-IV), demonstrating that it can

allosterically affect binding at the agonist recognition site. The binding of [3H]JNJ-40068782

to human recombinant mGlu2 receptors in CHO cells and rat brain receptors was saturable

with a KD of ∼10 nM. In rat brain, the anatomical distribution of [3H]JNJ-40068782 was

consistent with mGlu2 expression previously described and was most abundant in cortex and

hippocampus. The ability of structurally unrelated PAMs to displace [3H]JNJ-40068782

suggests that PAMs may bind to common determinants within the same site. Interestingly,

agonists also increased the binding affinity of [3H]JNJ-40068782. JNJ-40068782 influenced

rat sleep-wake organization by decreasing REM sleep with a lowest active dose of 3 mg/kg

p.o. In mice, JNJ-40068782 reversed PCP-induced hyperlocomotion with an ED50 of 5.7

mg/kg s.c. Collectively, the present data demonstrate that JNJ-40068782 has utility in

investigating the potential of mGlu2 modulation for the treatment of diseases characterized by

disturbed glutamatergic signalling, and highlight the value of [3H]JNJ-40068782 in exploring

allosteric binding.


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Introduction

Glutamate is the major excitatory neurotransmitter in the central nervous system of

vertebrates and elicits and modulates synaptic responses by activating ionotropic or

metabotropic glutamate (mGlu) receptors (Kew and Kemp, 2005). To date, eight mGlu

receptor subtypes have been cloned and classified into three groups based upon sequence

homology, pharmacological profile and preferential signal transduction pathway (Niswender

and Conn, 2010).

The drug discovery efforts related to group III mGlu receptors have lagged behind

(Lavreysen and Dautzenberg, 2008), and various research groups have focussed on the

development of ligands acting at group I and II receptors. For example, mGlu1 receptor

blockade is thought to have anxiolytic-like properties and to relieve pain (Tatarczynska et al.,

2001; Steckler et al., 2005; Varney and Gereau, 2002), whereas inhibitors of the mGlu5

receptor are believed to be alternative treatments for anxiety, depression and Fragile X, while

mGlu5 activators may have beneficial effects in schizophrenia (Spooren et al., 2003,

Auerbach et al., 2011). Since group II mGlu receptors reduce transmission at glutamatergic

synapses in brain regions where excessive glutamatergic transmission may be implicated in

the pathophysiology of anxiety and schizophrenia, it is hypothesized that activation of group

II mGlu receptors may provide anxiolytic and/or antipsychotic effects (Swanson et al., 2005).

Accordingly, mGlu2/3 receptor agonists, such as (-)-(1R,4S,5S,6S)-4-amino-2-

sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) are effective in a number of

animal models of schizophrenia (Rorick-Kehn et al., 2007) and anxiety (Helton et al, 1998;

Tizzano et al., 2002; Swanson et al., 2005; Witkin et al., 2007). These observations have been

extended into the clinic where LY2140023, a methionine amide prodrug of LY404039, has

been shown to demonstrate efficacy in a Phase II study in schizophrenia patients (Patil et al.,

2007; Mezler et al., 2010), though these observations could not be confirmed in a follow-up


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trial (Kinon et al., 2011). The structurally-related analogue LY354740 was able to reduce

anxiety in healthy volunteers in a fear-potentiated startle model (Grillon et al., 2003) and

attenuated CO2-induced panic in subjects with panic disorder (Schoepp et al., 2003). These

anxiolytic-like properties were reproduced in patients with generalised anxiety disorder using

LY354740 (Michelson et al., 2005) as well as its prodrug LY544344 (Dunayevich et al.,

2008).

The use of knockout mice lacking either mGlu2 or mGlu3 receptors has further refined

our understanding of the mechanism of action of mGlu2/3 receptor agonists. For example, the

anxiolytic actions of LY354740 in the elevated plus maze test require the expression of one or

both of these receptors (Linden et al., 2005). On the other hand, mGlu2 rather than mGlu3

receptors appear to be responsible for the antipsychotic-like properties of LY404039 (Fell et

al, 2008), LY379268 (Wooley et al., 2008) and LY314882, which is the racemate of

LY354740 (Spooren et al., 2000). Based upon the collective data using mGlu2/3 selective

agonists as well as mice lacking either the mGlu2 or mGlu3 receptor, the mGlu2 receptor

would appear to be an attractive potential therapeutic target (Niswender and Conn, 2010).

mGlu2 positive allosteric modulators (PAMs) that bind at a receptor site that is different

(i.e. allosteric) from glutamate offer an alternative approach for enhancing mGlu2 activity.

mGlu2 PAMs have minimal effects on their own but increase the response of glutamate.

Consequently, mGlu2 receptor PAMs offer an opportunity to enhance mGlu2 receptor

signalling by maintaining activity based on transient release of glutamate, but have a lower

risk of tolerance development (Conn et al, 2008 and 2009). 2,2,2-Trifluoro-N-[4-(2-

methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide (LY487379) was

identified as the first mGlu2 receptor-specific PAM (Johnson et al., 2003) and was active in

animal models predictive of anxiolytic or antipsychotic activity (Johnson et al., 2003, 2005;

Galici et al., 2005), providing preclinical proof-of-concept that PAMs can mimic aspects of


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the pharmacology of agonists. Minimal efficacy at sufficiently low doses as well as short

duration of action however limited its use for further development. Pinkerton et al. (2005)

reported an alternative series of mGlu2 PAMs, of which the prototypic example biphenyl-

indanone A (BINA) demonstrates long-lasting activity in some animal models used to predict

potential antipsychotic and anxiolytic activity (Galici et al., 2006). Recently, the structurally

novel allosteric potentiator N-(4-((2-(trifluoromethyl)-3-hydroxy-4-

(isobutyryl)phenxoy)methylbenzyl)-1-methyl-1H-imidazo-4-carboxamide (THIIC, also

known as LY2607540), which seems to have activity in models predicting either anxiolytic or

antidepressant effects, was reported (Fell et al., 2010). While preclinical data hold promise

for mGlu2 PAMs as novel therapeutics, clinical proof-of-concept is awaited with more drug-

like compounds compared to the first generation PAMs.

Here, we describe the in vitro and in vivo properties of the novel PAM 3-cyano-1-

cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-one (JNJ-40068782) (Cid et

al., Patent WO 2008/107481 A1 2008 and Imogai et al. Patent WO 2007/104783 A2 2007].

We furthermore describe [3H]JNJ-40068782 as the first high affinity, selective mGlu2 PAM

radioligand and show that this is an excellent tool to study allosteric mGlu2 interactions.


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Methods

Materials.

BINA, LY487379, THIIC (LY2607540), 1-Butyl-4-[4-(2-methylpyridin-4-yloxy)phenyl]-2-

oxo-1,2-dihydropyridine-3-carbonitrile (JNJ-40264796) and JNJ-40068782 and [3H]JNJ-

40068782 (Figure 1) were synthesized at Janssen Research and Development. Both [3H]-

DCG-IV and [3H]-LY341495 were obtained from American Radiolabeled Chemicals. [35S]-

GTPγS (specific activity 1250 Ci/mmol; guanosine 5’-(γ-thio)triphosphate was obtained from

PerkinElmer (Boston MA). GDP, HEPES, EGTA, BSA, probenecid and glutamate were

purchased from Sigma (St-Louis, MO); MgCl2 was purchased from VWR Prolabo (Belgium),

saponin from Calbiochem (US & Canada), HBSS and fluo-4-AM from Invitrogen (Oregon,

USA), and PMSF was obtained from Fluka (Germany). All other reagents were obtained

from Merck (Germany). For in vitro experiments, concentration-response curves of JNJ-

40068782 were prepared in 100% DMSO and further diluted in assay buffer with final assay

concentrations reaching 1% DMSO. For in vivo studies, JNJ-40068782 solutions were

prepared in 20% hydroxypropyl-β-cyclodextrine.

Tritiation of JNJ-40068782

The brominated precursor 4-(4-(4-bromophenyl)piperidin-1-yl)-1-(cyclopropylmethyl)-2-oxo-

1,2-dihydropyridine-3-carbonitrile was dissolved in tetrahydrofuran. Next N,N-

Diisopropylethylamine and Pd/C were added. The reaction mixture was attached to a tritium

manifold (RC Tritec) and 183 mbar of tritium gas was placed on top of the reaction mixture

and was stirred for 1 h at room temperature. The remaining tritium gas was trapped on a

Uranium bed and all volatile compounds were lyophilized to a waste ampoule. The crude

reaction mixture was rinsed with methanol, dissolved in ethanol (and filtered over an

Acrodisk filter, yielding 16.4 GBq. A small portion of this material was further purified and


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analyzed by HPLC to obtain the tritiated compound 4-(4-(4-tritiumphenyl)piperidin-1-yl)-

1(cyclopropylmethyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (radiochemical purity of

>98%, 865 GBq/mmol).

Functional mGlu2 assays.

Ca2+ assay. Intracellular Ca2+ levels were measured using the Functional Drug

Screening System (FDSS 6000, Hamamatsu). Human mGlu2 receptor expressing Gα16-HEK

293 cells were seeded at 10,000 cells/well in 384-well black/clear bottom poly-D-lysine

coated plates 24 h before the experiment. On the day of the experiment, medium was

removed by brief centrifugation and 20 μl of a fluo-4-AM solution (HBSS buffer containing

20 mM HEPES, 2.5 mM probenecid and ∼2 μM fluo-4-AM, pH 7.4) was added. After an

incubation period of 1 h at room temperature in the dark, 40 μl buffer was added and cells

were kept at room temperature for another 10 min before measurement. Fluorescence was

recorded upon the addition of compound (20 μl), and the ratio of the peak over basal

fluorescence was exported and used for further calculation. For the measurement of intrinsic

agonism, compound was added alone (i.e. in the absence of glutamate); for measurement of

PAM activity, compound was added together with an EC20 equivalent concentration of

glutamate (0.3 μM).

[35S]GTPγS binding. CHO cells expressing the rat or human mGlu2 receptor were

grown until 80% confluence, washed in ice-cold phosphate-buffered saline and stored at -

20°C until membrane preparation. After thawing, cells were suspended in 50 mM Tris-HCl,

pH 7.4 and collected through centrifugation for 10 min at 23,500 g at 4°C. Cells were lysed

in 5 mM hypotonic Tris-HCl, pH 7.4 and, after recentrifugation for 20 min at 30,000 g at 4°C,

the pellet was homogenized with an Ultra Turrax homogenizer in 50 mM Tris-HCl, pH 7.4.


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Protein concentrations were measured by the Bio-Rad protein assay using bovine serum

albumin as standard.

For [35S]GTPγS measurements, compound and glutamate were diluted in buffer

containing 10 mM HEPES acid, 10 mM HEPES salt, pH 7.4, containing 100 mM NaCl, 3

mM MgCl2 and 10 μM GDP. Membranes were thawed on ice and diluted in the same buffer,

supplemented with 14 μg/ml saponin (final assay concentration of 2 μg/ml saponin). Final

assay mixtures contained 7 (human mGlu2) or 10 (rat mGlu2) μg of membrane protein and

were pre-incubated with compound alone (determination of agonist effects) or together with

an EC20 concentration (4 μM) of glutamate (determination of PAM effects) for 30 min at

30°C. [35S]GTPγS was added at a final concentration of 0.1 nM and incubated for another 30

min at 30°C. Reactions were terminated by rapid filtration through Unifilter-96 GF/B filter

plates (PerkinElmer, Meriden, CT) using a Unifilter-96 harvester (PerkinElmer). Filters were

washed 3 times with ice-cold 10 mM NaH2PO4/10 mM Na2HPO4, pH 7.4 and filter-bound

radioactivity was counted in a Topcount NXT Microplate Scintillation and Luminescence

Counter from PerkinElmer. Typically, [35S]GTPγS binding in the absence of agonist was in

the region of 400 cpm whereas a maximal glutamate response corresponded to ∼2500 cpm.

[35S]GTPγS binding to rat brain sections. Autoradiography of agonist-stimulated

[35S]GTPγS binding in brain sections was performed as previously described (Happe et al.,

2001). Hence, 20 μm thick sagital sections were incubated twice in assay buffer pH 7.5 (50

mM Tris-HCl, 10 mM MgCl2, 0,2 mM EGTA, 100 mM NaCl, 0,1 mM PMSF and 1% BSA)

for 10 min at 25°C and once in assay buffer with 2 mM GDP for 15 min at 25°C. Slides were

then incubated in assay buffer containing 0.04 nM [35S]GTPγS, in presence or absence of 10

µM glutamate and increasing concentrations of JNJ-40068782 for 2 h at 25°C. The incubation

was stopped by washing twice for 3 min in ice-cold 50 mM Tris-HCl (pH 7.4) and a quick dip

in ice-cold distilled water.


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Sections were then dried under a cold stream of air and placed in a light-tight cassette

together with commercially available radioactive polymer standards and exposed to Kodak

Biomax MR film (Kodak, PerkinElmer, Cambridge, UK) for 4 days. Films were developed by

standard techniques, digitized and analyzed using the MCID basic 7.0 system (Imaging

Research, Canada).

Agonist-stimulated activity in brain sections was calculated by subtracting the optical

density in basal sections (incubated with GDP alone) from that of agonist-stimulated sections;

results were expressed as percentage basal activity.

Radioligand binding to human mGlu2-CHO membranes or rat cortical membranes.

Membranes from human mGlu2 receptor stably transfected CHO cells were prepared

as above (see [35S]GTPγS binding). For cortical membranes, male Wistar rats (150 g) were

sacrificed and decapitated. Brains were removed and cortex tissue was dissected and kept on

ice. Tissue was homogenized with an Ultra-Turrax homogenizer in 50 mM Tris-HCl, pH 7.4

and the homogenate was centrifuged for 10 min at 16000 RPM. The pellet was resuspended

and homogenized again with a glass-teflon homogenizer (Dual). After 2

washing/centrifugation steps, the final pellet was resuspended in 50 mM Tris-HCl, pH 7.4.

Aliquots were made and frozen at -80°C until further use. Protein concentration was

determined with the Bio-Rad Protein Assay method using bovine serum albumin as standard.

After thawing, membranes were homogenized using an Ultra Turrax homogenizer and

suspended in ice-cold binding buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2,

and 2 mM CaCl2.

[3H]LY341495 binding. For competition binding studies, assay mixtures were

incubated for 60 min at room temperature in a volume of 0.5 ml containing 10 μg membrane

protein (hmGlu2 CHO), appropriate concentrations of test compound and 2 nM


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[3H]LY341495. Non-specific binding was determined in the presence of 1000 μM glutamate

and was about 10% of total binding. The incubation was stopped by rapid filtration using

Unifilter-96 GF/B filter plates and a Unifilter-96 harvester (PerkinElmer).

[3H]DCG-IV binding. For saturation experiments, 50-75 μg membrane protein

(hmGlu2 CHO), test compound and increasing concentrations ranging from 2 to 800 nM of

[3H]DCG-IV were incubated for 60 min at room temperature (in total, 11 or 12 concentrations

were tested). Non-specific binding (about 30% of total binding) was determined in the

presence of 10 μM LY341495. Displacement studies were done with 50 nM of [3H]DCG-IV.

Filtration was performed using Unifilter-96 GF/C filters pre-soaked in 0.1% PEI and a

Unifilter-96 harvester (PerkinElmer).

[3H]JNJ-40068782 binding. For saturation experiments, 75 μg (hmGlu2 CHO) or 75-

125 μg (cortex) membrane protein, test compound and increasing concentrations

(concentrations in assay ranged from 0.5 to 200 nM and a total of 12 concentrations were

tested) of [3H]JNJ-40068782 were incubated for 60 min at room temperature, in a total

volume of 0.5 ml. Non-specific binding (about 30% of total binding) was determined in the

presence of 10 μM 1-butyl-4-[4-(2-methylpyridin-4-yloxy)phenyl]-2-oxo-1,2-

dihydropyridine-3-carbonitrile JNJ-40264796, a structurally-related molecule. Displacement

studies were performed using 10 nM of radioligand. Filtration was performed using Unifilter-

96 GF/C filters pre-soaked in 0.1% PEI and a 40-well manifold or 96-well Brandell harvester

96.

For all radioligands, [3H]LY341495, [3H]DCG-IV, and [3H]JNJ-40068782, assay

conditions including radioligand concentrations were chosen such that <10% of the free

radioligand concentration was receptor-bound. After the addition of scintillation liquid,


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radioactivity on the filters was counted in a Microplate Scintillation and Luminescence

Counter or Liquid Scintillation Analyzer from Perkin Elmer.

[3H]JNJ-40068782 rat brain autoradiography.

Tissue Preparation. Male Wistar rats (200 g) were sacrificed by decapitation. Brains

were immediately removed from the skull and then rapidly frozen in dry-ice-cooled 2-

methylbutane (-40°C). Brains were then stored at -20°C until sectioning. Sagittal sections (20

μm) were cut using a Leica C3050 cryostat microtome (Leica Microsystems, Wetzlar,

Germany) and thaw-mounted on SuperFrost Plus microscope slides (Menzel-Glaser GmbH,

Braunschweig, Germany). The sections were then kept at -20°C until use. Male wild type

C57BL/6 and mGlu2 knock-out mice (Deltagen, Inc.) weighing 25-30 g were sacrificed by

decapitation and their brain prepared as stated above.

Receptor Autoradiography. Sections were thawed and dried under a stream of cold air,

and preincubated (2 times 10 min) at room temperature in incubation buffer (50 mM Tris-

HCl, 2 mM MgCl2, 2 mM CaCl2, pH 7.4). Sections were then incubated for 60 min at room

temperature in buffer containing 10 nM [3H]JNJ-40068782 with nonspecific binding

determined by addition of 10 μM JNJ-40068782. After incubation, the excess of radioligand

was washed off (2 times 10 min) in ice-cold buffer, followed by a rapid dip in cold distilled

water. The sections were dried under a stream of cold air. Rat brain sections were exposed to

[3H]Hyperfilm (Amersham Biosciences, Buckinghamshire, UK) for 6 weeks at room

temperature. The films were developed manually in Kodak D19 and fixed with Kodak

Readymatic (Eastman Kodak, Rochester, NY). Mouse brain sections were exposed for 6 days

to a Phosphorimager screen. Digital autoradiograms were obtained using a PhosphorImager

FLA7000 (Fuji).


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Selectivity panel.

Ca2+ assays with human mGlu1, 3, 5, 7 or 8 receptor-expressing HEK 293 cells.

Measurement of intracellular Ca2+ mobilization in HEK293 cells stably transfected with the

human mGlu1a receptor proceeded as follows. Cells were seeded at 10,000 cells/well in a

384-well plate. Twenty-four h after seeding, FLIPR Ca2+ assay kit (Molecular Devices)

dissolved in phosphate-buffered saline supplemented with 5 mM probenecid, pH 7.4 (f.c. 2.5

mM probenecid as loading buffer was added on the cell layer with no removal of medium)

was added and measurements were initiated 90 min thereafter.

Ca2+ assays with human mGlu3 receptor expressing Gα16-HEK 293 cells were

performed similarly as for human mGlu2 expressing cells, with these exceptions: cells were

seeded at a density of 10,000 cells/well and cell incubation with fluo-4-AM took place at

37°C instead of room temperature.

For the mGlu5 receptor, cells were seeded at 40,000 cells/well in PDL-coated 384-

well plates, and a day later, medium was removed before addition of a fluo-4-AM solution

(HBSS, 2.5 mM probenecid and ∼2 μM fluo-4-AM, pH 7.4). Measurements were performed

90 min later (while incubation at 37°C and 5% CO2).

For the human mGlu7 and 8 receptor, stably expressing Gα15-HEK cells were seeded

at a density of 15,000 cells/well (PDL-coated 384-well plates) and incubated with FLIPR

Calcium 4 assay kit (dissolved in HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) for 90

min at 25°C before measurement.

The ratio of peak versus basal fluorescent signals was used for data analysis.

[35S]GTPγS binding to membranes from CHO cells expressing the rat mGlu6 receptor

and to membranes from L929sA cells expressing the human mGlu4 receptor.

Membrane preparations were performed as described above (see [35S]GTPγS binding).

Membranes, compound and glutamate were diluted in assay buffer (10 mM HEPES acid, 10


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mM HEPES salt, pH 7.4, containing 100 mM NaCl, 0.3 mM MgCl2 and 10 μM GDP). Assay

mixtures included 10 μg of membrane protein and were pre-incubated with compound alone

or together with glutamate for 30 min at 37°C. [35S]GTPγS was added at a final concentration

of 0.1 nM and incubated for another 30 min at 37°C. Reactions were terminated by rapid

filtration through Unifilter-96 GF/B filter plates (Packard, Meriden, CT) using a 96-well

Packard filtermate harvester.

Selectivity of JNJ-40068782 in additional radioligand binding assays. JNJ-40068782

was tested at a concentration of 10 μM by CEREP (Celle L’Evescault, France) for its

inhibition of radioligand binding to a battery of neurotransmitter and peptide receptors, ion

channels and transporters.

Data analysis.

For both [35S]GTPγS and Ca2+ mobilization assays, data were processed using either

the Lexis software interface developed at Janssen Research & Development or GraphPad

Prism (version 4.02) and were calculated as percentage of the control agonist challenge.

Sigmoid concentration-response curves plotting percentages of effect versus the log

concentration of the test molecule were analysed using nonlinear regression analysis.

To further describe the signalling of glutamate in the presence of a PAM, the fold shift

of the glutamate concentration-response was plotted in function of the concentration of

modulator in GraphPad Prism (version 4.02) using nonlinear regression, interpolation was

then used to allow estimates of concentrations needed for a 5-fold shift.

Inhibition [3H]JNJ-40068782 binding data were fitted to a one-site binding model

(GraphPad Prism version 4.02) and estimates of dissociation constants (Ki) were derived

using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). Saturation binding experiments


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were analysed using nonlinear regression (one-site binding hyperbola; GraphPad Prism

version 4.02).

In vivo studies.

All animal experiments were carried out in strict accordance with the Declaration of Helsinki

and the European Communities Council Directive of 24th November 1986 (86/609/EEC) ,

and were approved by the animal care and use committee of Janssen Research and

Development as well as the local ethical committee.

Pharmacokinetic analysis. Animals were given free access to food and water overnight prior

to dosing, and throughout the study. Male Sprague-Dawley rats were dosed with a single

oral dose of 10 mg/kg. Blood samples were taken at 0.5, 1, 2, 4, 8 and 24 h from the tail vein

using EDTA as the anti-coagulant. Male Swiss mouse were dosed subcutaneously with a

dose of 10 mg/kg, and blood samples were taken at 0.5, 1 and 1.5h after dosing. Plasma was

obtained following centrifugation, extracted using protein precipitation, and subsequently

analysed for JNJ-40068782 using a liquid chromatography/tandem mass spectrometry

qualified research method on an API4000 instrument (Applied Biosystems)

Sleep-wake architecture study in rat. Male Sprague Dawley rats (Charles River, France)

weighing 250–300 g at the time of surgery were used. The effect of oral administration of

JNJ-40068782 (administered p.o. 2 h after light onset using a dose volume of 10 ml/kg) on

sleep-wake organization in rats was determined as described in Ahnaou et al., 2009. In brief,

sleep polysomnographic variables were determined in 32 adult, male Sprague Dawley rats

(n=8 each group), which were chronically implanted with electrodes for recording the

cortical electroencephalogram (EEG), electrical neck muscle activity (EMG), and ocular

movements (EOG). Polysomnographic data was scored with an automated sleep stages

analyzer, scoring each 2-second epoch before averaging stages over 30-minutes periods.


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Sleep-wake state classifications were assigned based upon the combination of dynamics of

five EEG frequency domains, integrated EMG, EOG, and body activity level: Active wake;

Passive wake; Rapid Eye Movement sleep (REM); Intermediate Stage (pre-REM transients);

light non-REM sleep; and deep non-REM sleep. Different sleep-wake parameters like

amount of time spent in each state were investigated over 20 post-administration hours.

Time spent in each sleep-wake state was expressed as a percentage of the recording

period. A statistical analysis of the obtained data was carried out by a non-parametric analysis

of variance of each 30-min period, followed by a Wilcoxon-Mann-Whitney rank sum test of

comparisons with the control group.

PCP-induced hyperlocomotion in mice. Test compound or solvent was administered

alone (to monitor effects on spontaneous locomotion) or together with phencyclidine (PCP; 5

mg/kg) to male NMRI mice (22 ± 4 g and fasted overnight; Charles River Germany) 30 min

before measurement. Both JNJ-40068782 and PCP were injected subcutaneously (s.c.) and

locomotor activity was measured for a period of 30 min. ED50 values (the doses inducing

50% responders to criterion) were determined as follows.

All-or-nothing criteria for significant (p < 0.05) effects were defined by analyzing a

frequency distribution of a large series of historical control data obtained in solvent-treated

animals. Based on the criteria obtained in this way, the ED50 and corresponding 95%

confidence limits were determined according to the modified Spearman-Kaerber estimate,

using theoretical probabilities instead of empirical ones. This modification allows

determination of the ED50 and its confidence interval as a function of the slope of the log

dose-response curve. Criterion for drug-induced decrease of spontaneous locomotion: total

distance < 2,500 counts (4.1% false positives in controls; n = 410). Criterion for drug-

induced inhibition of PCP-induced hyperlocomotion: total distance < 5,500 counts (3.9%

false positives in controls; n = 154).


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Results

JNJ-40068782 acts as a selective positive allosteric modulator at the mGlu2 receptor.

Figure 2 shows the concentration-response curves for the glutamate-stimulated

increase in [35S]GTPγS binding in absence and presence of JNJ-40068782, BINA, LY487379

or LY2607540. JNJ-40068782 like the other PAMs enhances the glutamate-stimulated

increase in [35S]GTPγS. Interestingly, all compounds magnified the response produced by

glutamate, shifting the glutamate concentration-effect curve both up-ward and left-ward.

While a concentration of 429 nM of BINA, 568 nM of JNJ-40068782 or 305 nM of

LY2607540 increases the glutamate potency (i.e. lower the glutamate EC50) by 5-fold,

LY487379 causes only a 2-3 fold shift in the glutamate concentration-response curve at the

highest tested concentration of 1 μM. Maximal decreases in glutamate EC50 of 10-12 fold are

observed for the other compounds.

By quantifying the increase in response at a fixed glutamate concentration, for

example the glutamate EC20, the potency (EC50) of JNJ-40068782 at the human mGlu2

receptor was estimated to be 143 nM (Table 1 and Figure 2B). This value is similar

(overlapping confidence intervals) to the potencies of BINA and LY487379 and LY2607540

(respective EC50 values of 89, 256 and 95 nM). The maximal response to glutamate was

increased to 309 ± 23% upon addition of JNJ-40068782, which is similar to the potentiation

seen with BINA and LY2607540 (221 ± 9 and 215 ± 7%, respectively) (Table 1). However,

LY487379 showed a more modest, 147 ± 9%, increase in glutamate Emax. Though both EC50

and Emax values slightly increased at the rat compared to the human mGlu2 receptor, we found

a good correspondence between compound potencies obtained in the rat and human assays

(Table 1).


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At higher concentrations, JNJ-40068782 also increased [35S]GTPγS binding in the

absence of glutamate, suggesting that it acts as an ‘ago-potentiator’. Similar profiles were

observed for BINA and LY2607540 but not LY487379. Interestingly, JNJ-40068782 along

with the reference compounds acted as a pure mGlu2 receptor PAM at recombinant rat mGlu2

receptors (i.e. did not have any intrinsic activity in the absence of glutamate; Table 1).

Further profiling of JNJ-40068782 showed that it also facilitated glutamate-induced

Ca2+ mobilization at human recombinant mGlu2 receptors. In HEK293 cells co-expressing

Gα16, JNJ-40068782 potentiated the EC20 glutamate response with an EC50 value of 15 nM

(95% confidence interval 12 - 19 nM; n=30). Interestingly, in this assay JNJ-40068782 did

not increase the maximal response of glutamate (Emax = 119 ± 2%; also see Figure 2C). JNJ-

40068782 induced intracellular Ca2+ mobilization on its own (allosteric agonism) with an

EC50 of 618 nM (95% confidence interval 497 - 768 nM) and Emax of 81 ± 3% (n=25).

JNJ-40068782 did not show agonist, antagonist or PAM activity at the human mGlu3

receptor or any of the group I or group III mGlu receptors up to 10 μM (data not shown).

Further profiling (CEREP) revealed 59% and 85% inhibition of h5HT2A (determined with

[3H]ketanserin) and hCCKA (determined with [125I]CCK-8s) receptor binding at 10 μM. The

complete selectivity profile based on CEREP data is shown in Supplementary Table 1.

Modulation of [35S]GTPγS binding by JNJ-40068782 at native rat mGlu2 receptor.

Despite extensive washing of the rat brain sections, in the absence of exogenous

glutamate, there remained a basal level of [35S]GTPγS binding that could be reduced by

mGlu2/3 receptor antagonists (data not shown), suggesting that there was sufficient residual

endogenous glutamate to produce a degree of mGlu2 and/or mGlu3 receptor activation. As a

result, in rat brain sections glutamate produced much smaller responses than observed in the


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recombinant human mGlu2 cell line, with, for example, 10 μM glutamate producing a

maximal response of about 25% over basal.

The [35S]GTPγS binding to rat brain sections nevertheless does give a clear illustration

that JNJ-40068782 can modulate the effects of glutamate at native rat brain mGlu2 receptors.

Hence, JNJ-40068782 was able to increase the [35S]GTPγS binding to rat brain slices in the

absence of exogenous glutamate (Figure 3). Although this may imply that JNJ-40068782 has

intrinsic direct agonist effects at native rat mGlu2 receptors (i.e., can activate mGlu2

receptors in the absence of glutamate), the possibility that this may be a modulation of

endogenous glutamate that was not removed during washing of the slices cannot be excluded.

In the presence of 10 μM glutamate, JNJ-40068782 produced a robust increase in [35S]GTPγS

binding, particularly in the cortex, striatum and hippocampus (Figure 3). Potentiation of

glutamate by JNJ-40068782 was evident at the lowest concentration of the allosteric

modulator, and quantification of these data showed that whereas 10 μM glutamate or 1 μM

JNJ-40068782 alone increased [35S]GTPγS binding in the cortex by ∼10% and ∼20%,

respectively, the combination led to a stimulation of ∼80%. In combination with glutamate, a

maximal stimulation of ∼175% was detected after the addition of 10 or 100 μM JNJ-

40068782; in the absence of glutamate, 100 μM JNJ-40068782 increased the response to

125%. Hence, although the potency of JNJ-40068782 on [35S]GTPγS binding at native rat

receptors is reduced relative to recombinant systems, presumably as a result of the

complication of endogenous glutamate, the anatomical distribution of the responses (i.e.,

preferential stimulation of the binding in the cortex, striatum and hippocampus ) is consistent

with an mGlu2 receptor-mediated response.


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JNJ-40068782 does not affect [3H]LY341495 binding but increases [3H]DCG-IV binding.

To ascertain its allosteric character, JNJ-40068782 was tested for displacement of the

prototypic orthosteric (i.e. glutamate site) mGlu2/3 receptor antagonist [3H]LY341495

binding to human mGlu2 receptor-expressing membranes. [3H]LY341495 binding to the

human mGlu2 receptor was not inhibited upon the addition of JNJ-40068782 10 μM (Figure

4A). In contrast to the lack of effect of JNJ-40068782 on the binding of the antagonist

radioligand, binding of the radiolabeled orthosteric agonist [3H]DCG-IV was increased up to

2.5-fold upon the addition of JNJ-40068782 at 3 μM (Figure 4B).

In order to assess whether JNJ-400068782 alters the apparent affinity of an agonist for

the orthosteric binding site, saturation binding of the agonist [3H]DCG-IV was performed in

the absence and presence of 3 μM JNJ-40068782. Figure 5A clearly demonstrates that there

was a left-ward shift in the [3H]DCG-IV binding curve in the presence of JNJ-40068782.

Quantification of these data shows that in the absence of JNJ-40068782, the affinity of

[3H]DCG-IV for the orthosteric binding site was 240 ± 39 nM (average ± S.E.M. of 6

experiments) whereas with the PAM present, KD decreased about 8-fold, reflecting increased

affinity (Table 2). Similar results were seen with other mGlu2 receptor PAMs. The number

of binding sites, represented by the Bmax value, was unchanged. The linearity of the Scatchard

analysis of the saturation binding data, illustrates that both in the absence and presence of

JNJ-40068782, the binding of [3H]DCG-IV is best described by a single binding site. In order

to see whether the same effects were seen for the natural agonist glutamate, glutamate-

mediated inhibition of [3H]DCG-IV binding was assessed in the absence and presence of 3

μM JNJ-40068782. In line with previous data, JNJ-40068782 not only increased the maximal

binding, but also enhanced the affinity of glutamate, i.e. lowered the IC50 of glutamate by ∼5-

fold (4.5- and 6.7-fold found in 2 independent experiments; Figure 5B).


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Binding of [3H]JNJ-40068782 to recombinant human mGlu2 receptors.

JNJ-40068782 was tritiated and used to characterize the binding to human recombinant

mGlu2 receptors (Figure 6A). Binding was saturable and was best-fit to a single-site model

defined by a KD of 12 ± 4 nM with a Bmax of 4.5 ± 1.9 pmol/mg protein (n=12). At a

concentration of 10 nM, the level of specific binding was in the region of 70%. Interestingly,

in the presence of agonist (either glutamate, DCG-IV or LY354740), the apparent affinity of

[3H]JNJ-40068782 was increased (average KD in the presence of 100 μM glutamate was 4.0 ±

1.2 nM; n=4), with the saturation curve being left-ward shifted (Figure 6B). In each of the 4

experiments where binding was measured in the absence or presence of glutamate, the Bmax

value of [3H]JNJ-40068782 was increased upon addition of the agonist, with an average

increase of 2-fold.

The binding of [3H]JNJ-40068782 to human recombinant mGlu2 receptors was

inhibited by JNJ-40068782 with a mean IC50 of 38 nM (95% confidence interval 27-49 nM,

n=9, Figure 7A). In addition, BINA, LY483739 and LY2607540 inhibited [3H]JNJ-40068782

with respective IC50 values of 385 nM (95% confidence interval 217-681 nM, n=8), 116 nM

(95% confidence interval 78-171 nM, n=6) and 98 nM (95% confidence interval 44-216 nM,

n=4). Though it is not clear whether the interaction between these molecules and JNJ-

40068782 is completely competitive, hill slopes of the inhibition curves are close to one and

binding of [3H]JNJ-40068782 was fully displaced upon addition of compound (i.e. the same

level of non-specific binding was observed for all – structurally unrelated – modulators),

suggesting a competitive interaction. Hence, IC50 values were also converted to Ki values

according to the Cheng-Prusoff equation (Ki was 20 nM for JNJ-40068782, 215 nM for

BINA, 65 nM for LY483739 and 55 nM for LY2607540). Additional studies showed that the


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negative allosteric modulator RO4491533 also fully displaced [3H]JNJ-40068782 (IC50 of 16

nM (95% confidence interval 7-39 nM, n=4; Ki of 9 nM; data not shown).

Binding of [3H]JNJ-40068782 to rodent brain tissue.

[3H]JNJ-40068782 bound with a similar affinity to receptors expressed in rat cortex; KD

and Bmax were 10 ± 4 nM (c.f. 12 ± 4 nM at human recombinant mGlu2 receptors) and 14.7 ±

5.7 pmol/mg protein (n=6), respectively. IC50 values for displacing cortical mGlu2 receptors

were 52 nM (95% confidence interval 34-78 nM, n=7), 617 nM (95% confidence interval

242-1571 nM, n=5), 102 nM (95% confidence interval 55-190 nM, n=5) and 79 nM (95%

confidence interval 43-143 nM, n=3) for JNJ-40068782, BINA, LY483739 and LY2607540

respectively (assuming a competitive interaction, Ki values are 26, 305, 51 and 39 nM,

respectively), confirming that these PAMs also bind to native receptors (Figure 7B).

Using radioligand autoradiography, we examined [3H]JNJ-40068782 binding distributions in

rat brain sections in further detail (Figure 8A). High specific binding was observed in the

hippocampal formation, granular layer of the cerebellum, striatum and cortex, corresponding

very well with the expression pattern demonstrated using the mGlu2/3 receptor antagonist

[3H]LY341495. [3H]JNJ-40068782 binding site distribution was similar in wild type mice

brain. Importantly, binding was lost in mGlu2 knock-out mice underlining the mGlu2-

specificity of the radioligand (Figure 8B).

Effects of JNJ-40068782 on sleep-wake organization in the rat.

The preceding data collectively demonstrate that JNJ-40068782 not only binds to

native mGlu2 receptors but also selectively potentiates the functional response at rat brain

mGlu2 receptors. As JNJ-40068782 was shown to be brain-penetrant (with Cmax of ∼1400


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ng/ml, and brain : plasma ratio of ∼0.4 after oral dosing of 10 mg/kg in rats), we set out to

evaluate some of the in vivo functional effects of this novel mGlu2 receptor PAM.

We have previously shown that selective activation or positive allosteric modulation

of the mGlu2 receptor in vivo affects the sleep-wake architecture of rats (Ahnaou et al.,

2009). To assess whether JNJ-40068782 has in vivo central activity, we evaluated sleep-

wake organization of rats after oral dosing of JNJ-40068782.

The time course of vigilance states over 20 h following administration of vehicle and

JNJ-40068782 is illustrated in Figure 9. Acute administration of JNJ-40068782 (3, 10 and 30

mg/kg, p.o.) significantly suppressed the time spent in REM sleep and intermediate stage,

whereas no major effects on the other sleep-wake stages were observed. Quantification of

effect over a 4-h time period following compound administration revealed a dose-related

reduction in time spent in REM sleep with a lowest active dose of 3 mg/kg p.o.,

corresponding to a plasma drug concentration of ∼420 ng/ml. REM sleep reduction was

accompanied by consistent lengthening in REM sleep onset latency (Figure 8, upper small bar

graph panel in REM sleep stage).

Effects of JNJ-40068782 on PCP-induced hyperactivity in mice.

The effects of JNJ-40068782 on spontaneous locomotor activity was measured at

doses ranging from 2.5-10 mg/kg s.c. (Figure 10A). The compound produced a modest

reduction on spontaneous activity, with the total distance travelled dropping by 20% from

2974 cm in vehicle-treated mice to 2365 cm in animals receiving 20 mg/kg JNJ-40068782.

We subsequently evaluated whether JNJ-40068782 was able to mimic some of the

effects of mGlu2/3 agonists in an animal model that is used to predict potential anti-psychotic

activity. At a dose of 5 mg/kg (s.c.), PCP induces a profound increase in the total distance

travelled by mice. Subcutaneous injection of JNJ-40068782 significantly attenuated the


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increase in locomotor activity evoked by PCP, with an ED50 of 5.7 mg/kg s.c., corresponding

to a plasma drug concentration of ∼1700 ng/ml (Figure 10B).


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Discussion

The pioneering work emanating from Eli Lilly using mGlu2/3 orthosteric (glutamate) binding

site agonists such as LY404039 and LY354740 have defined the group II mGlu receptor

subfamily as an attractive potential therapeutic target for anxiety disorders and schizophrenia

(Swanson et al., 2005; Fell et al., 2012). A key role of the mGlu2 receptor in mediating the

pharmacological effects observed with mGlu2/3 receptor agonists is emphasized by data

demonstrating that compounds which selectively potentiate the effects of glutamate at mGlu2

receptors (mGlu2 PAMs) can reproduce some of the effects seen with the non-selective

mGlu2/3 receptor agonists. Such compounds include CBiPES (Johnson et al., 2005), BINA

(Galici et al., 2005, 2006) and LY487379 (Johnson et al., 2003; Schaffhauser et al., 2003;

Galici et al., 2005).

Our studies have provided new pharmacological tools to study the mGlu2 receptor PAM

concept, including JNJ-40068782 and [3H]JNJ-40068782. While the in vitro potencies for the

reference mGlu2 receptor PAMs vary from lab to lab, presumably as a result of difference in

receptor expression systems and/or assay methodologies, it nevertheless seems that

LY487379 is consistently less potent (with in vitro potency for the human mGlu2 receptor

ranging from ∼250 nM to ∼1.5 μM) than BINA, LY2607540 and JNJ-40068782, all of which

may be considered equipotent (with potencies ranging from ∼20 nM to 1 μM depending on

the assay).

Using a [35S]GTPγS assay for mGlu2 receptors expressed in CHO cells, there does not seem

to be any marked rat versus human species differences for the four above-mentioned PAMs

although EC50 values were generally 2-fold lower and Emax 1.5–fold higher between rat and

human mGlu2 data. Screening of over 100 additional compounds furthermore confirmed this

pattern, with no examples of compounds being active in one species but not the other (data


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not shown; correlation plots can be found in Supplementary Figure 1). JNJ-40068782 induced

a receptor response even in the absence of agonist in some but not all in vitro assays,

indicating that the functional effects of this molecule can be different depending on the

functional read-out as well as the level of receptor expression and hence seem context-

dependent. This is in line with the thinking that ligand ‘behaviours’ rather than inherent

‘properties’ can be described and that detection of agonist ‘behaviour’ depends on the ligand

and sensitivity of the assay used (Langmead, 2012). It can also not be ruled out that the

response can, at least in part, be attributed to the presence of endogenous glutamate.

It is not yet clear why JNJ-40068782 seems to be more potent in the Ca2+ assay compared to

the [35S]GTPγS binding assay. Moreover, the maximal response to glutamate was not

increased when measuring changes in intracellular Ca2+ concentrations, in contrast to the

dramatic increase in response when measuring [35S]GTPγS accumulation. Several reasons

may underlie these differences including the use of different cellular backgrounds, potential

differences in receptor and/or G protein expression levels, the transient (Ca2+) versus end

point ([35S]GTPγS) nature of the assay, potential fluorescent dye saturation, differences in

equilibrium binding conditions, or compound solubility during the assay procedure. It can

furthermore not be excluded that forced coupling of the mGlu2 receptor to the Ca2+ pathway

affects ligand potency compared to using an assay where the receptor signals via a natural,

endogenous protein.

JNJ-40068782 also enhanced native mGlu2 receptor signalling as shown using GTPγS

autoradiography on rat brain slices.

Allosteric modulators may not only enhance the efficacy of the endogenous agonist by

“magnifying” the intracellular responses produced by the agonist but also by altering the

apparent affinity of the agonist, or a mixture of the two (Conn et al., 2009). In the present


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study, JNJ-40068782, along with BINA and LY487379, was able to increase the affinity of

agonists, with the KD for [3H]DCG-IV binding to recombinant human mGlu2 receptors

decreasing by around 5-10-fold in the presence of mGlu2 PAMs. Moreover, and as expected,

although JNJ-40068782 increased the affinity of agonists at the orthosteric binding site, it did

not alter the binding of the orthosteric antagonist [3H]LY341495.

While our data thus far enabled to get an understanding of how PAMs can affect agonist

signalling and binding, we set out to study effects on allosteric interaction by agonists with

the use of selective mGlu2 receptor PAM radioligand binding. Radioligands that selectively

recognize an allosteric modulatory site have been described for the mGlu1 ([3H]R214127;

Lavreysen et al., 2003) and mGlu5 receptor ([3H]M-MPEP, Gasparini et al., 2002;

[3H]mPEPy, Cosford et al., 2003). Recently, a novel mGlu2/3 receptor agonist radioligand

was reported (Wright et al., 2012). However, [3H]JNJ-40068782 is the first selective mGlu2

radioligand that is described extensively. JNJ-40068782 was selective for mGlu2 receptors as

defined by CEREP profiling, where only a significant interaction at CCKA receptors, which

are known to be only expressed in very discrete brain regions such as the interpeduncular

nucleus and nucleus tractus solitarius (Hill et al., 1987), was observed at 10 μM of JNJ-

40068782. At relatively high concentrations, JNJ-40068782 also acted on the 5HT2A receptor

(IC50 ∼ 10 μM); it is, however, unlikely that it reaches sufficient amounts in vivo to trigger a

functional 5HT2A-related effect. Using [3H]ketanserin, we did not observe in vivo binding up

to 30 mg/kg p.o. (data not shown); also, JNJ-40068782 did not affect sleep-wake behaviour in

rats, while ample evidence exists for modulation of deep non-REM sleep via the 5HT2A

receptor (Al-Shamma et al., 2010). Moreover, in rat brain [35S]GTPγS binding experiments,

the preferential stimulation of binding in the cortex and striatum is again consistent with JNJ-

40068782 selectively modulating the function of mGlu2 receptors. Finally, the selectivity of


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the radioligand for mGlu2 versus mGlu3 receptors was confirmed by the fact that binding was

lost in mGlu2 but retained in mGlu3 receptor knockout mice brains.

[3H]JNJ-40068782 binding met all the requirements for a ligand well suited to study binding

properties, pharmacology, and distribution of mGlu2 receptors. [3H]JNJ-40068782 showed

saturable one-site binding, with its affinity (∼10 nM) being similar between cloned human

receptors and native mGlu2 receptors in rat cortex. The structurally diverse PAMs BINA,

LY487379 and LY2607540 seem to share common determinants in the allosteric binding site

since, both in recombinant cells and in brain tissue, they could all displace [3H]JNJ-40068782.

Interestingly, the mGlu2 receptor negative allosteric modulator RO4491533 was also able to

inhibit the binding of JNJ-40068782 indicating that both negative and positive allosteric

modulators share common determinants of binding within the allosteric modulatory site.

[3H]JNJ-40068782 binding was used to get more insight in how orthosteric binding would

influence binding to the allosteric 7TM domain. In line with the effect of JNJ-40068782 on

agonist binding, demonstrated with both [3H]DCG-IV and glutamate, there was a reciprocal

effect of orthosteric agonists (glutamate, DCG-IV and LY354740) enhancing binding affinity

of [3H]JNJ-40068782. While agonists predominantly label the high affinity or G protein-

coupled receptor state, an antagonist shows equal affinity for coupled and uncoupled

receptors, or for both the high- and low-affinity states of the receptor. Our finding that the

Bmax for [3H]JNJ-40068782 was only about half of that of [3H]DCG-IV is interesting and may

suggest that PAMs preferentially bind to certain receptor states that are not necessarily similar

to those preferentially recognized by DCG-IV. Alternatively, JNJ-40068782 may bind to

only one of the subunits within the mGlu2 homodimer, whereas DCG-IV likely binds to both,

which might be consistent with previous reports that binding and closure of both extracellular

domains within the mGlu receptor dimer is needed for full activity, whereas only a single


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heptahelical domain is turned on upon receptor activation (Kniazeff et al., 2004; Hlavackova

et al., 2005). Even more intriguing is the finding that in the presence of agonists (glutamate

or LY354740), the Bmax of [3H]JNJ-40068782 was consistently increased, opening the

possibility of a changed receptor conformation allowing binding to both receptor subunits in

the mGlu2 receptor dimer, upon which a ‘super-activated’ state may be imposed.

Because of its specificity, [3H]JNJ-40068782 proved to be suitable for investigation of mGlu2

receptor distribution in brain sections using radioligand autoradiography. These studies

showed that the highest level of mGlu2 specific binding was present in the hippocampus,

striatum, cortex and granular layer of the cerebellum. These results correspond to the binding

of [3H]LY341495, and to the binding of [3H]LY354740 reported by Richards et al. (2005),

where [3H]LY354740 was used under such conditions that it bound selectively to mGlu2

receptors. The distribution of [3H]JNJ-40068782 binding was also comparable to the

anatomical distribution of mGlu2 receptor mRNA and protein (Ohishi et al., 1993, 1998; Gu

et al., 2008).

JNJ-40068782 proved to have appropriate pharmacological and pharmacokinetic properties to

evaluate its effects in vivo. It is well-established that mGlu2 receptor modulation affects

sleep-wake architecture in rats and mice (Feinberg et al., 2002; Ahnaou et al., 2009; Fell et

al., 2010). Consistent with these data, we found that JNJ-40068782 dose-dependently

decreased the amount of REM sleep in rats when dosed early in the light phase. The effects

on sleep-wake organization in rats may well reflect the modulation of physiological

glutamatergic signalling since it has been shown that glutamate levels are increased during

REM sleep in rat orbitofrontal and cerebral cortex (Lopez-Rodriguez et al., 2007 and Dash et

al., 2009). Moreover, because of the translational nature of the sleep-wake EEG paradigm –

polysomnography measurements in man may be used as a reproducible and sensitive


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biomarker. Nevertheless, it remains to be determined to what extent the changes in REM

sleep, although a reliable pharmacodynamic marker, have relevance for potential mGlu2-

mediated therapeutic efficacy.

As regards to potential therapeutic indication for mGlu2 receptor modulation, JNJ-40068782

decreased PCP-induced hyperlocomotion, which is a model in which dopamine D2-related

antipsychotics demonstrate robust efficacy. However, the doses at which JNJ-40068782 is

active in this model correspond to plasma drug concentrations somewhat higher than those in

the sleep-wake organization paradigm, although this may merely reflect the fact that greater

drug concentrations may be required to demonstrate efficacy in a challenge model (PCP-

induced hyperactivity) compared to a more physiological situation (sleep-wake). While these

findings provide more evidence that mGlu2 PAMs can mimic aspects of the pharmacology of

mGlu2/3 receptor agonists, and hence have the potential to exhibit antipsychotic behavior,

studies are ongoing to provide more insight in the translation of compound activities in

preclinical animal models into relevant dose levels in a clinical setting.

Collectively, our data indicate that JNJ-40068782 is a selective PAM of native and

recombinant rat and recombinant human mGlu2 receptors. It not only enhances the affinity of

agonists, but also increases the maximal response. In the absence of exogenous glutamate,

JNJ-40068782 has a modest degree of intrinsic agonist efficacy. [3H]JNJ-40068782 represents

an excellent novel radiotracer to study mGlu2 allosteric interaction, expression and

distribution. With [3H]JNJ-40068782, we show for the first time that also agonists influence

the affinity of PAMs. Finally, since JNJ-40068782 is active in vivo, it is a suitable

pharmacological tool to increase our understanding how mGlu2 receptor modulation may

affect diseases where glutamatergic transmission is disrupted.


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Acknowledgements

The authors wish to thank Stefan Pype and Claire Mackie for their continuous support;

Michel Mahieu and Heidi Huysmans for technical assistance; and Addex Therapeutics for

their collaboration on the mGlu2 PAM discovery program, with special thanks to Francoise

Girard, Emmanuel Le Poul and Guillaume Duvey.


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Authorship Contributions

Participated in research design: Lavreysen, Langlois, Ahnaou, Drinkenburg, te Riele,

Biesmans, Van der Linden, Peeters, Megens, Wintmolders, Macdonald, Lütjens,

Dautzenberg, Atack

Conducted experiments: Lavreysen, te Riele, Biesmans, Van der Linden, Peeters,

Wintmolders

Contributed new reagents or analytic tools: Cid, Trabanco, Andrés

Performed data analysis: Lavreysen, Ahnaou, te Riele, Biesmans, Van der Linden, Peeters,

Megens, Wintmolders

Wrote or contributed to the writing of the manuscript: Lavreysen, Langlois, Ahnaou,

Drinkenburg, te Riele, Biesmans, Van der Linden, Peeters, Megens, Wintmolders, Cid,

Trabanco, Andrés, Macdonald, Lütjens, Dautzenberg, Atack


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Figure Legends

Figure 1. Structure of JNJ-40068782. The asterix denotes the position of the tritium in [3H]JNJ-40068782.

Figure 2. [35S]GTPγS binding to hmGlu2 receptors expressed in CHO cells. A. Stimulation of [35S]GTPγS

binding produced by glutamate in the absence and presence of various concentrations of JNJ-40068782; BINA;

LY487379; and LY2607540. Data are from a representative experiment. The experiment was repeated at least

twice giving similar results. B. The concentration-effect curves for JNJ-40068782, BINA, LY487379 and

LY2607540 on the binding of [35S]GTPγS at an EC20-equivalent concentration of glutamate. Data are expressed

as percentage of the maximal response to glutamate and are mean ± S.E.M of 6-13 experiments. C. The

concentration-effect curves for JNJ-40068782 on the binding of [35S]GTPγS versus mobilization of Ca2+ at an

EC20-equivalent concentration of glutamate (Glu) . Data are expressed as percentage of the maximal response to

glutamate and are mean ± S.E.M of 6 ([35S]GTPγS) or 30 (Ca2+) experiments.

Figure 3. Binding of [35S]GTPγS to rat brain sections revealed by quantitative autoradiography. A. Sagital rat

brain sections were incubated with various concentration of JNJ-40068782 in the absence or presence of 10 μM

glutamate. The top left brain section represents, in absence of glutamate and JNJ-40068782, the basal

[35S]GTPγS binding. B. Quantification of the data represented in the images. Bar graphs represent the average

(n = 3 rats) percent stimulation by glutamate and/or PAM over basal binding.

Figure 4. [3H]LY341495 (A) and [3H]DCG-IV (B) binding in the presence of JNJ-40068782 and other mGlu2

receptor PAMs.

Figure 5. A. Saturation binding of the agonist [3H]DCG-IV to human recombinant mGlu2 receptors in the

absence and presence of 3 μM JNJ-40068782. In the inset, the Scatchard analysis is shown. B. Displacement of

[3H]DCG-IV from the human mGlu2 receptor by glutamate in the absence and presence of JNJ-40068782. Data

are from one representative experiment.

Figure 6. A. Binding of [3H]JNJ-40068782 to human recombinant mGlu2 receptors. A. Representative saturation

curve showing the total and specific binding of [3H]JNJ-40068782, with specific binding being calculated as


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total binding minus nonspecific binding. B. Saturation binding in the absence and presence of the orthosteric

(glutamate) binding site agonists glutamate (100 μM), DCG-IV (100 μM) and LY354740 (1 μM).

Figure 7. JNJ-40068782, BINA, LY487379 and LY2607540 displacement of [3H]JNJ-40068782 binding to

human recombinant (A) and rat cortical (B) mGlu2 receptors. Data are expressed as % specific binding ± S.E.M.

and are from 3-9 experiments.

Figure 8. A. Binding of 10 nM [3H]JNJ-40068782 to rat brain sections and comparison with the labelling of

group II mGlu receptors with the mGlu2/3 receptor antagonist [3H]LY341495. B. Binding of 10 nM [3H]JNJ-

40068782 to wild-type versus mGlu2 knock-out mouse brain sections.

Figure 9. Percentage distribution of sleep-wake states for each h period of the 20 h recording session following

oral administration of JNJ-40068782 (3, 10, 30 mg/kg) or vehicle. Open and dark areas in the abscissa axis

indicate light and dark phase of the circadian time, respectively. Small bar charts indicate amounts of vigilance

states in minutes (plus S.E.M.) during the first 2 hours post-administration (n=8 for each group). Small bottom

right upper panel indicates REM sleep onset latency (ROL). Values are presented as means ± S.E.M. for each

condition. Each symbol on curve plots indicate statistically significant difference (P<0.05) between vehicle and

drug-dose injected groups. *indicates p<0.05: Wilcoxon-Mann-Whitney rank sum tests as compared to vehicle

values.

Figure 10. Effect of JNJ-40068782 (0, 2.5, 5, 10 and 20 mg/kg, s.c.) on spontaneous locomotion (A) and PCP (B)

(5 mg/kg, s.c.; -0.5 h)-induced hyperlocomotion in mice. The dotted lines at 2500 and 5500 cm indicate the critical

level for hypolocomotion and hyperlocomotion, respectively.


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Tables

Table 1. Functional activity of JNJ-40068782, BINA, LY487379 and LY2607540 at human and rat recombinant

mGlu2 receptors, in absence or presence of an EC20 concentration of glutamate. Values are an average of 6-13

experiments, %Emax values are indicated as mean ± S.E.M., EC50 values are geometric means with 95%

confidence intervals indicated in brackets.

absence of glutamate presence of EC20 glutamate

% Emax EC50 (nM) % Emax EC50 (nM)

human mGlu2

JNJ-40068782 69 ± 9 925 (573 - 1493) 309 ± 23 143 (108-190)

BINA 60 ± 3 572 (141 – 2324) 221 ± 9 89 (48-162)

LY487379 20 ± 3 >50.000 147 ± 9 256 (193-337)

LY2607540 28 ± 2 720 (631 – 821) 215 ± 7 95 (77-117)

rat mGlu2

JNJ-40068782 31 ± 3.7 >30.000 444 ± 86 307 (287 – 327)

BINA 35 ± 4.9 >30.000 390 ± 63 138 (57-336)

LY487379 7.4 ± 3.9 >30.000 166 ± 14 501 (417-602)

LY2607540 13 ± 1.5 >30.000 273 ± 10 314 (251 – 392)


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Table 2: Characteristics of [3H]DCG-IV binding to human recombinant mGlu2 receptors in the absence and

presence of JNJ-40068782, BINA and LY487379. The KD and Bmax of [3H]DCG-IV in the absence of

compound is an average ± S.E.M. of 6 experiments. The same parameters measured in the presence of

compound come from 2 independent experiments (values from both experiments are given).

[3H]DCG-IV binding

KD (nM) Bmax (pmol/mg protein)

Control 240 ± 39 14.5 ± 2.8

JNJ-40068782 (3 μM) 22 / 39 16.6 / 7.6

BINA (3 μM) 47 / 81 15.5 / 9.0

LY487379 (3 μM) 35 / 53 17.3 / 7.0


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N

O

N

N

*

Figure 1


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C

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+ 10 µM

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+ 1 µM+ 300 nM+ 100 nM+ 50 nM+ 10 nM+ 5 nM

+ 10 µM

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control

+ 1 µM+ 300 nM+ 100 nM+ 50 nM+ 10 nM+ 5 nM

+ 10 µM

Log [Glutamate], M

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JNJ-40068782 BINA

LY487379 LY2607540

-11 -10 -9 -8 -7 -6 -5 -4

0

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350 JNJ-40068782

BINALY487379

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Log [compound], M

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-12 -11 -10 -9 -8 -7 -6 -5 -4

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Figure 2


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no JNJ-40068782

1 µM JNJ-40068782

10 µM JNJ-40068782

100 µM JNJ-40068782

0 µM 1 µM 10 µM 100 µM0

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250 no glutamate10 µM glutamate

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[35S

]GTP

γS b

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Figure 3


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[3H]DCG-IVorthosteric mGlu2/3 agonist

[3H]LY341495orthosteric mGlu2/3 antagonist

A B

control

100 µM Glu

1 µM BINA

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10 µM JNJ-40068782 0

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[3H]H]JNJ-40068782, nM

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60

80

100

120

140 JNJ-40068782BINALY487379LY2607540

Log [compound], M

% s

peci

fic b

indi

ng

-11 -10 -9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120

140 JNJ-40068782BINALY487379LY2607540

Log [compound], M

% s

peci

fic b

indi

ngFigure 7


at ASPE



Dow

nloaded from


Cer

HipCx

Str

[3H]LY341495[3H]JNJ-40068782non-specific binding

A

B wild-type mGlu2 KO

Cer

Hip

Str

Figure 8


at ASPE



Dow

nloaded from


0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 9 1011121314151617181920

Lightsleep

0

20

40

60

80

0 1 2 3 4 5 6 7 8 9 1011121314151617181920

Deep sleep

0

4

8

12

16

20

0 1 2 3 4 5 6 7 8 9 1011121314151617181920

REMsleep

0

1

2

3

4

0 1 2 3 4 5 6 7 8 9 1011121314151617181920

Inter-mediate

state

0

4

8

12

16

0 1 2 3 4 5 6 7 8 9 1011121314151617181920

Passivewake

010203040

05

101520

0

10

20

30

020406080

0.00.51.01.52.0

0

5

10

15

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 1011121314151617181920

Active wake

Sta

te a

mou

nt (%

reco

rdin

g tim

e)

Time (# hours post-administration)

* ***

JNJ-40068782 (mg/kg)

3 10 30Vehicle

*

04080

120160200

*ROL

Figure 9


at ASPE



Dow

nloaded from


B PCP-induced hyperlocomotion

veh/v

eh

0 JNJ-4

0068

782

2.5 JN

J-400

6878

2

5 JNJ-4

0068

782

10 JN

J-400

6878

2

20 JN

J-400

6878

20

5000

10000

15000

20000

PCP (5 mg/kg, s.c.)

tota

l dis

tanc

e (c

m)

0 JNJ-4

0068

782

2.5 JN

J-400

6878

2

5 JNJ-4

0068

782

10 JN

J-400

6878

2

20 JN

J-400

6878

20

5000

10000

15000

20000

tota

l dis

tanc

e (c

m)

A Spontaneous locomotionFigure 10


at ASPE



Dow

nloaded from


Pharmacological characterization of JNJ-40068782, a new...

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