In vitro and in vivo identification of novel positive allosteric modulators of the human dopamine D2 and D3 receptor

Article abstract of DOI:10.1124/mol.115.100172

Agonists at dopamine D2 and D3 receptors are important therapeutic agents in the treatment of Parkinson's disease. Compared with the use of agonists, allosteric potentiators offer potential advantages such as temporal, regional, and phasic potentiation of

Full text of DOI:10.1124/mol.115.100172

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2015/12/21/mol.115.100172.DC1.html
1
521-0111/89/2/303–312$25.00
http://dx.doi.org/10.1124/mol.115.100172
Mol Pharmacol 89:303–312, February 2016
MOLECULAR PHARMACOLOGY
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
In Vitro and In Vivo Identification of Novel Positive Allosteric
s
Modulators of the Human Dopamine D2 and D3 Receptor
Martyn Wood, Ali Ates, Veronique Marie Andre, Anne Michel, Robert Barnaby,
and Michel Gillard
UCB Biopharma SPRL, Chemin du Foriest, B-1420, Braine-l’Alleud, Belgium
Received May 29, 2015; accepted December 10, 2015
ABSTRACT
3
Agonists at dopamine D2 and D3 receptors are important thera- the number of high-affinity sites for [ H]-dopamine without affecting
peutic agents in the treatment of Parkinson’s disease. Compared Kd. We went on to identify a more potent PAM for use in native
with the use of agonists, allosteric potentiators offer potential receptor systems. This compound potentiated the effects of D2/D3
advantages such as temporal, regional, and phasic potentiation of signaling in vitro in electrophysiologic studies on dissociated striatal
natural signaling, and that of receptor subtype selectivity. We report neurons and in vivo on the effects of L-dopa in the 6OHDA
the identification of a stereoselective interaction of a benzothiazol (6-hydroxydopamine) contralateral turning model. These PAMs
racemic compound that acts as a positive allosteric modulator lacked activity at a wide variety of receptors, lacked PAM activity
(
PAM) of the rat and human dopamine D2 and D3 receptors. The R at related Gi–coupled G protein-coupled receptors, and lacked
3
isomer did not directly stimulate the dopamine D2 receptor but activity at D1 receptors. However, the PAMs did potentiate [ H]-
potentiated the effects of dopamine. In contrast the S isomer dopamine binding at both D2 and D3 receptors. Together, these
attenuated the effects of the PAM and the effects of dopamine. In studies show that we have identified PAMs of the D2 and D3
radioligand binding studies, these compounds do not compete for receptors both in vitro and in vivo. Such compounds may have
binding of orthosteric ligands, but indeed the R isomer increased utility in the treatment of hypodopaminergic function.
PAM) or attenuate (negative allosteric modulator, NAM) the
Introduction
effects of the endogenous ligand by affecting affinity and/or
There has been much interest in the identification of
efficacy (Wootten et al., 2013). As well as subtype selectivity,
allosteric modulators of G protein-coupled receptors (GPCRs),
allosteric modulators can present other potential advantages
both as tools to understand receptor mechanisms and as
from a drug-discovery perspective such as lack of direct effect
potential therapeutic agents (see Keov et al., 2011; Conn
or intrinsic efficacy; only potentiating the effect of the native
et al., 2012). GPCRs represent the largest family of cell-
transmitter where and when it is released; and reduced
surface receptors, and a large number of marketed drugs
propensity for inducing desensitization arising from constant
directly activate or block signaling mediated via these recep-
exposure to an agonist.
tors. However, for some GPCRs (e.g., peptide receptors) it has
The monoamine dopamine acts via two families of GPCRs to
proven difficult to develop small molecules; for others, achiev-
modulate motor function, reward mechanisms, central pro-
ing sufficient selectivity has been challenging due to the high
cessing, and other physiologic functions. The two families are
degree of homology in the ligand-binding site between GPCR
D1-like, comprising the dopamine D1 and D5 receptors, which
subtypes (e.g., dopamine D2 and dopamine D3). Accordingly,
couple to the Gs and Golf G-proteins and stimulate cAMP
much drug research has shifted to the identification of small
molecules that target sites distinct from the orthosteric
natural agonist and that induce a conformational change in
the GPCR, thereby allosterically modulating the receptor
function.
production; and D2-like, comprising the D2, D3, and D4
receptors which predominantly couple to Gi/o G-proteins and
attenuate cAMP production (see Neve et al., 2004; Beaulieu
and Gainetdinov, 2011). The D2 receptors exhibit a high
degree of sequence homology with D3 receptors, and they
share a predicted binding site for dopamine and other ligands
Allosteric ligands have a diverse range of activities, in-
cluding the ability to potentiate (positive allosteric modulator,
(Shi and Javitch, 2002). Many therapeutic agents target the
dopamine D2 and D3 receptors, notably agonists such as
ropinirole, pramipexole, and rotigotine used in the treatment
of motors disorders such as Parkinson’s disease (Perez-Lloret
dx.doi.org/10.1124/mol.115.100172.
s
This article has supplemental material available at molpharm.
aspetjournals.org.
ABBREVIATIONS: ANOVA, analysis of variance; CHO, Chinese hamster ovary; DMSO, dimethylsulfoxide; GPCR, G protein-coupled receptor; Gpp
NH)p, 5ʹ-guanylylimidodiphosphate; GTPgS, guanosine 59-O-(3-thio)triphosphate; HTRF, homogenous time-resolved fluorescence; IBMX,
(
3-isobutyl-1-methylxanthine; L-741,626, 4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol; NMDA, N-methyl-D-aspartate; 6OHDA,
6-hydroxydopamine; PAM, positive allosteric modulator; PBS, phosphate-buffered saline; SB269652, N-[4-[2-(7-cyano-3,4-dihydro-1H-isoquinolin-
2-yl)ethyl]cyclohexyl]-1H-indole-2-carboxamide.
303

3
04
Wood et al.
and Rascol, 2010) and antagonists such as atypical and typical
antipsychotic drugs (Seeman et al., 1976; Meltzer, 1999).
We therefore performed a high-throughput screen (HTS) on
Cell Culture and Membrane Preparation. Chinese hamster
ovary cells (CHO-K1) stably expressing the human dopamine D2 long
receptor or the human D3 receptor were generated in house. Human
embryonic kidney cells (HEK293) transiently expressing the rat
dopamine D2 long receptor were also generated in house using
8
0,000 compounds to identify novel allosteric modulators of
the human dopamine D2 receptor. By determining changes in
intracellular cAMP via homogenous time-resolved fluores-
cence (HTRF; CisBio Bioassays, Codolet, France) we identified
three hits that exhibited PAM properties at the dopamine D2
293fectin (Life Technologies, Carlsbad, CA). CHO cells expressing
human a2C adrenergic receptors were obtained in collaboration with
Dr. N. Moguilevsky, Free University of Brussels, Department of
Applied Genetics (Brussels, Belgium). CHO cells expressing the
receptor. However, one compound was a racemic mixture (1,3- human histamine H3 receptor were purchased from Euroscreen S.A.
benzothiazol-2-yl(2-methyl-2,3-dihydro-indol-1-yl)
(Gosselies, Belgium). The cells were cultured at 37°C in a humidified
methanone; Fig. 1). On synthesis and chiral resolution of the atmosphere of 5% CO . The CHO cells were grown in Dulbecco’s
modified Eagle’s medium/Ham’s-F12 GlutaMAX-I medium
GIBCO/Invitrogen, Gent, Belgium) containing 10% fetal bovine
serum (BioWhittaker/Lonza, Vervier, Belgium), 400 mg/ml geneticin
GIBCO/Invitrogen), and 100 IU/ml penicillin and 100 IU/ml strepto-
2
1
two stereoisomers, one (the R stereoisomer) was shown to act
as a PAM whereas the other displayed negative allosteric
modulator properties. We report here the properties and
characterization of these compounds in recombinant systems
on both function and radioligand binding, in native tissue and
in vivo in hemiparkinsonian rats.
(
(
mycin (Pen-Strep solution; BioWhittaker/Lonza).
Membranes were prepared from confluent cells grown in 500 cm
culture dishes. The cells were rinsed with 30 ml of phosphate-buffered
saline (PBS, pH 7.4) and detached by 5 to 10 minutes of incubation in
2
3
0 ml of 1 mM EDTA in PBS at 37°C and washed with 20 ml of PBS at
4°C. The cell suspension was centrifuged at 1500g for 10 minutes at
°C. The pellet was homogenized in 50 mM Tris-HCl (pH 7.4), 1 mM
EGTA, 0.3 mM EDTA, and 2 mM MgCl buffer with complete
Materials and Methods
Materials. [ S]-guanosine 59-O-(3-thio)triphosphate (GTPgS) and
4
35
3
2
[
H]-dopamine were obtained from PerkinElmer (Zaventem, Belgium).
protease inhibitor, using a glass/Teflon homogenizer (10 strokes on
ice at 1100 rpm). The cell pellet was then subjected to two freeze/
thaw cycles, and the membranes were incubated for 10 minutes at
Complete protease inhibitor was obtained from Roche Diagnostics
Vilvoorde, Belgium); DNAse, 3-isobutyl-1-methylxanthine (IBMX),
(
forskolin, and apomorphine from Sigma Aldrich (Diegem, Belgium);
and the cAMP dynamic range kit from Cisbio Bioassay (Codolet,
France). Quinpirole and L-741,626 [4-(4-chlorophenyl)-1-(1H-indol-3-
ylmethyl)piperidin-4-ol] were obtained from Tocris (Abingdon, United
Kingdom).
Compounds were synthesized and prepared in house, dissolved in
dimethylsulfoxide (DMSO) and diluted in assay buffer such that the
final concentration of DMSO in the assay was 1%.
Animals and Ethics Statement. All animal experiments were
performed according to the Helsinki declaration and were conducted
in accordance with the guidelines of the European Community Council
directive 86/609/EEC and approved by the ethics committee from UCB
Biopharma SPRL (LA1220040 and LA2220363). Male Sprague-
Dawley rats (Janvier, France) were housed in cages (four rats per
cage) for 1 week before the experiments. They were kept on a 12-hour
light/dark cycle with lights on at 06:00 and at a temperature
maintained at 20–21°C and at humidity of approximately 40%. All
animals had free access to standard pellet food and water before
assignment to experimental groups. The animals weighed 250–275 g
at the time of surgery and 400–450 g at the time of drug testing.
Additional enrichment and welfare were provided (Enviro-Dri;
PharmaServ, Framingham, MA) before and after the surgery. Animal
health was monitored daily by the animal care staff. Surgeries were
performed under ketamine and xylazine or under isoflurane anesthe-
sia, and all efforts were made to minimize suffering. The animals were
2
4
5°C with DNase (1 ml/ml). The membranes were then centrifuged at
0,000g for 25 minutes at 4°C, and the final pellet was suspended in
a 20 mM Tris-HCl (pH 7.4) and 250 mM sucrose buffer and stored in
liquid nitrogen at a protein concentration of 2–8 mg/ml.
Radioligand Binding Assays. The CHO D2 membranes (50–
3
1
1
00 mg Pr per well) were incubated with [ H]-dopamine (0.05 nM to
00 nM in saturation studies; 0.6 nM in compound concentration-
effect studies) for 120 minutes in 50 mM Tris buffer (pH 7.4 at 25°C,
final volume 1 ml) containing (final) MgCl (1 mM), pargyline
10 mM), ascorbic acid (1 mM), and test compound or DMSO (1% to
2
(
define total binding) or apomorphine (10 mM to define nonspecific
binding; Burt et al., 1975). The reaction was terminated by rapid
filtration (glass fiber filters GF/B), the filters were washed 4 times
with ice-cold 50 mM Tris buffer, and the retained radioactivity was
determined by liquid scintillation spectroscopy. All incubations were
performed in duplicate or triplicate.
The same method was used for CHO D3 membranes except that the
protein concentration was 3 mg Pr per well due to higher receptor
3
expression. [ H]-raclopride binding to CHO D2 membranes was
performed according to Wood et al. (2015).
Dopamine D2–Stimulated [35S]-GTPgS Binding. Membranes
(
5 mg Pr) expressing human D2 dopamine receptors were incubated for
1
3
5 minutes at 25°C in buffer (50 mM Tris-HCl [pH 7.4] containing
mM MgCl , 50 mM NaCl, 1 mM GDP, 10 mg/ml saponin) that
2
35
contained the test compound in a final volume of 0.2 ml. [ S]-GTPgS
0.15–0.2 nM) was then added, and the reaction continued for
0 minutes. Membrane-bound radioligand was separated by rapid
2
killed with CO or when necessary by exsanguination.
(
6
filtration through glass fiber filters (GFB) and washed 4 times with
filtration buffer (ice-cold 50 mM Tris-HCl (pH7.4), and the retained
radioactivity was counted by liquid scintillation spectroscopy. All
incubations were performed in duplicate or triplicate.
cAMP Cellular Assays. The effect of compounds on the levels of
cAMP in the D2 CHO cells was assessed using cAMP dynamic kit 2
from CisBio Bioassays following the manufacturer’s instructions.
Using HTRF technology, the assay is based on competition between
native cAMP produced by cells and cAMP labeled with the dye d2. The
tracer binding is visualized by an anti-cAMP antibody labeled with
cryptate. The assays were performed in 384 wells with 5000 cells per
well in a final volume of 80 ml. The cells were incubated with IBMX
O
N
N
S
*
(
500 mM final), compound (varying concentrations), and forskolin
Fig. 1. Chemical structure of the dopamine D2 receptor allosteric
modulator showing the racemic center.
(10 mM final) in the presence and absence of varying concentrations of

Identification of Dopamine D2/D3 Receptor PAMs
305
dopamine with DMSO (1% final) for 60 minutes at ambient temper-
ature. The reaction was terminated and the cells lysed, cAMP-d2
reagent and anti-cAMP antibody was added, and then it was in-
cubated for 60 minutes at ambient temperature. The level of cAMP
was then determined by measuring the fluorescence ratio (665 nm/
NMDA (N-methyl-D-aspartate) and quinpirole were dissolved in
2
O whereas the D2 antagonist L-741,626 and the test compound
H
were dissolved in DMSO. All experiments contained the same final
DMSO concentration (1%). Drugs were applied through a pressure-
driven fast perfusion system (Warner Instruments, Hamden, CT)
using an array of application capillaries synchronized by pClamp.
NMDA currents (3-second duration every 15 seconds) were evoked
6
20 nm). All incubations were performed in duplicate.
The same procedure was used for Lmtk-1 cells expressing the
human dopamine D1 receptor except that forskolin was not included; while holding the cell at 270 mV. The D2 agonist and antagonist were
the incubation was for 1 hour at room temperature with 20,000 cells
per well.
both preapplied and coapplied. In separate cells, one concentration of
the D2 agonist was tested before and after application of the tested
Selectivity. To investigate the selectivity of any allosteric effect, compound or vehicle. The baseline for NMDA currents (100 mM) was
compounds were evaluated at the Gi-coupled a2C-adrenergic receptor established, and the modulation of NMDA currents by the D2 agonist
and histamine H3 receptors and the related D1, D3, and D4 receptors. quinpirole was tested in neurons at one concentration. Quinpirole was
3
5
Histamine-stimulated [ S]-GTPgS binding was performed as de-
scribed in Célanire et al. (2009). Membranes (5–10 mg proteins)
then washed out, the test compound (10 mM) was applied for 2 minutes
before establishing a new baseline for NMDA currents, and retesting
was performed with quinpirole.
expressing human H
3
receptor were incubated for 15 minutes at
5°C in 0.2 ml of a 50 mM Tris-HCl buffer (pH 7.4) containing 3 mM
2
6-Hydroxydopamine Lesion. To protect norepinephrinergic
MgCl
2
, 50 mM NaCl, 1 mM GDP, 2 mg saponin, 1% DMSO, and neurons, animals were administered imipramine HCl (Sigma Aldrich)
increasing concentrations of compounds for agonism/inverse ago-
nism determination. Histamine and test compound were coincu-
15 minutes before surgery. They were subsequently anesthetized with
ketamine (Ceva, 75 mg/kg) and xylazine (10 mg/kg; Bayer, Diegem,
Belgium) and placed in stereotaxic frame (David Kopf Instrument,
Tujunga, CA). 6-Hydroxydopamine (6OHDA) was injected into the
right ascending medial forebrain bundle at the following coordinates
(in mm) relative to bregma and surface of the dura: anteroposterior2
3.5, mediolateral 21.5, and dorsoventral 28.7. Each rat received
one injection of 6OHDA HBr (4 mg/ml) over a period of 5 minutes
(0.5 ml/min) for a total of 10 mg per rat. Animals were monitored for
3 weeks to ensure full recovery and habituation to the environment
and experimenters.
On day 21 after surgery, all rats were challenged with a small
dose of subcutaneously administered apomorphine (0.05 mg/kg;
Sigma Aldrich). Rats showing more than 90 contralateral rotations
(360°) over a 45-minute recording period were included in the study.
L-Dopa methyl ester (Sigma Aldrich) was dissolved in physiologic
saline solution at a volume of 5 ml/kg. UCB compound (30 mg/kg;
UCB) was intraperitoneally administered as a suspension at a
dose volume of 5 ml/kg in vehicle [0.1% (w/v) Polysorbate 80
(Merck, Kenilworth, NJ); 0.1% (w/v) 1510 silicone antifoam (VWR,
Lutterworth, United Kingdom) in 1.0% (w/v) methylcellulose
(Sigma-Aldrich, UK)]. The compound was prepared extemporaneously
and homogenized by using a ultrasonic homogenizer (Covaris,
Woburn, MA) and magnetic stirring.
3
5
bated for 60 minutes, 0.2 nM of [ S]-GTPgS was added to the
samples, and the incubation was continued for another 30 minutes.
Assays were terminated by the addition of ice-cold 50 mM Tris-HCl
buffer (pH 7.4) followed by rapid filtration and radioactivity de-
termined as described earlier.
3
Binding of [ H]-dopamine to the D3 receptor was performed as for
the D2 receptor except that 3–5 mg protein was used per well and the
3
concentration of [ H]-dopamine was 2–3 nM.
35
Dopamine-stimulated [ S]-GTPgS binding at the D4 receptor was
performed as for the D2 receptor except that membranes were
prepared from CHO cells expressing the human dopamine D4.4
receptor and that 10 mg of protein of the membranes was used per
well (Wood et al., 2015).
Dopamine-stimulated increases in cAMP at the dopamine D1
receptor in Lmtk-1 cells (Wood et al., 2015) was performed as for the
dopamine D2 receptor except that 20,000 cells per well were used and
that the assay did not include forskolin.
Noradrenaline-stimulated [35S]-GTPgS binding was performed as
described earlier using 10 mg of protein from CHO cells expressing the
human a2C-adrenergic receptor in Tris buffer (pH 7.4) containing
1
mM MgCl
Acutely Dissociated Striatal Neurons. Detailed procedures
have been published (Cepeda et al., 1998; Flores-Hernandez et al.,
002; Cepeda et al., 2008). Adult rats were anesthetized with a mix of
2
, 2 mg saponin, and 1 mM GDP.
Behavioral Recording. Vehicle or UCB compound were admin-
istered as “add-on treatment” to a subthreshold dose of L-dopa
2
isoflurane and oxygen and sacrificed. Their brains were dissected and (15 mg/kg) without any dopa decarboxylase inhibitor to avoid
placed in ice-cold artificial cerebrospinal fluid containing (in mM)
additional potential pharmacokinetic interaction. UCB compound or
NaCl, 130; NaH PO , 1.25; NaHCO , 26; MgCl , 5; CaCl
, 1; and vehicle were administered intraperitoneally (i.p.) 15 minutes before
glucose, 10. Coronal slices (350 mm) were transferred to a chamber the L-dopa dose (15 mg/kg, i.p.).
containing artificial cerebrospinal fluid (with 2 mM CaCl and 2 mM
Rotational behavior was recorded using a computerized system.
MgCl ) oxygenated with 95% O –5% CO
(pH 7.2–7.4, 290–310 mOsm, Rats were fixed in a harness and linked to mechanical sensors
5 6 2°C). The dorsal striatum was dissected and treated for
5 minutes with papain (0.5 mg/ml, Calbiochem, San Diego, CA) at
2
4
3
2
2
2
2
2
2
2
1
3
connected directly to a computer. Each 360° clockwise or counterclock-
wise turn was automatically recorded for up to 120 minutes at the
5°C in a N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] maximum. Throughout the experiments, rats were allocated to indi-
HEPES)–buffered Hank’s balanced salts solution (Sigma Aldrich) vidual test cages.
(
supplemented with (in mM) pyruvic acid, 1; glutathione, 0.005; NG-
nitro-L-arginine, 0.1; and kynurenic acid, 1 (pH 7.4, 300–310 mOsm).
Data Analysis. In all studies values are presented as mean 6S.E.M.
from n separate experiments. In the electrophysiologic studies, the
, 2; data analyses were performed with Clampfit 10.3 (Molecular Devices,
Wokingham, United Kingdom). The group means for all measures
were compared using Student’s t tests (for two-group comparisons)
The tissue was rinsed with (mM) Na isethionate, 140; KCl, 2; MgCl
CaCl , 0.1; glucose, 23; and HEPES, 15. Striatal slices were mechan-
ically dissociated and plated onto a stage of an inverted microscope.
2
2
Standard whole-cell patch clamp techniques were used to obtain and analysis of variance (ANOVA) followed by Bonferroni t tests
voltage clamp recordings. The internal solution consisted of (in mM)
N-methyl-D-glucamine, 175; HEPES, 40; MgCl , 2; EGTA, 10; phos-
phocreatine, 12; Na ATP, 2; Na GTP, 0.2; and leupeptin 0.1 (pH 7.25,
65–270 mOsm). The external solution consisted of (in mM) NaCl, 135;
(multiple-group comparisons) using SigmaStat software (SPSS,
Chicago, IL). P , 0.05 was considered statistically significant.
In all other in vitro assays, data analysis was performed in Prism
(GraphPad Software, La Jolla, CA) using the sigmoidal dose-response
equation for pharmacologic studies and one-site specific equation for
2
2
2
2
2 2
CsCl, 20; BaCl , 3; CaCl , 2; glucose, 10; HEPES, 10; and tetrodotoxin,
0
.0003 (pH 7.4, 300–310 mOsm). Negative pressure was used to obtain saturation studies.
tight seals (.1 GV), and the data were collected from neurons that had
access resistances below 20 MV.
The efficacy of the compound to modify the level of L-dopa-induced
contralateral rotations was assessed with two-way mixed ANOVA,

3
06
Wood et al.
combining the drug effect (two levels) as a between-group factor with
the time (12 levels) as a within-subjects factor. Statistical analyses
were performed using the Statistica software (StatSoft, Tulsa, OK).
For each test, statistical significance was assumed if P , 0.05, and
data were log-transformed before analysis.
Results
Effects on [35S]-GTPgS Binding. In membranes from
CHO cells expressing the human dopamine D2 receptor,
3
5
dopamine stimulated [ S]-GTPgS binding with a pEC of
5
0
6
.46 6 0.05 (mean 6 S.E.M., n 5 8; 350 nM) and a stimulation
of 91% 6 8% over basal. The initial compound identified at
concentrations up to 10 mM lacked any significant effect on
3
5
[
S]-GTPgS binding in the same cells on its own (6%
stimulation over basal 6 3 at 10 mM, n 5 6) (Fig. 2A).
However, in the presence of a low concentration of dopamine
3
5
(
30 nM) close to the EC20, the compound now stimulated [ S]-
GTPgS binding by 27% 6 6% (fitted Emax) over basal (basal
defined in the presence of dopamine) with a pEC50 of 5.99 6
0
.32 (n 5 3; 1000 nM) (Fig. 2B). Upon synthesis of the
3
5
stereoisomers, the R isomer stimulated [ S]-GTPgS binding
in the presence of a low concentration of dopamine by 45% 6
3
% (n 5 3) with the same pEC50 of 6.00 6 0.13 whereas the
S isomer lacked significant effects (Fig. 2B).
The effects of the stereoisomers on the concentration–
response curve to dopamine were then investigated (Fig.
2
C). At 10 mM, the R isomer significantly increased the
potency (pEC50) of dopamine from 6.60 6 0.1 to 7.19 6 0.1
250 nM to 65 nM; n 5 3, P , 0.001, F test; GraphPad Prism)
(
and increased the Emax by 14% (P , 0.01, F test). In contrast,
the S isomer had no effect on the potency of dopamine to 6.44
(370 nM, not statistically significant) but reduced the Emax of
dopamine by 20% (P , 0.01, F test).
We then examined whether the R isomer would potentiate
the effects of other dopamine D2 receptor agonists. At 10 mM,
the R isomer increased the potency of quinpirole (pEC )
5
0
from 6.21 6 0.08 to 6.69 6 0.11 (620 nM; mean 6 S.E.M., n 5 4,
P , 0.01 Student’s paired t test) (Fig. 2D). Quinpirole was a
full agonist compared with dopamine (Emax 99% 6 2%
compared with dopamine fitted Emax 5 100%, mean 6 S.E.M.,
n 5 4), and the presence of the R isomer at 10 mM increased
this Emax by 35% 6 2% (P , 0.01, F test; GraphPad Prism).
We next investigated the effects of the compounds in
membranes expressing the rat dopamine D2 receptor. At the
3
5
rat D2 receptor, dopamine stimulated [ S]-GTPgS binding
with a pEC50 of 6.83 6 0.16 (n 5 5; 150 nM). At the rat D2
3
5
receptor, the R isomer potentiated [ S]-GTPgS binding in the
presence of a low concentration of dopamine (10 nM) by 57% 6
6
% over basal (fitted Emax and basal with basal defined as
binding in the absence of compound but presence of 10 nM
dopamine) with a pEC50 of 5.84 6 0.06 (n 5 5; 140 nM). In the
absence of dopamine, there was no statistically significant
effect.
Effects on cAMP at the hD2L Receptor. In the pres-
Fig. 2. Effect of compounds on D2-mediated [ S]-GTPgS binding. The ence of forskolin, dopamine produced a concentration-
results are mean and S.E.M. from triplicate determinations in a single dependent decrease in cAMP levels with a pEC50 of 9.0 6 0.1
3
5
representative experiment that was repeated twice. (A) The R isomer lacks
direct effects. The response was basal in the absence of dopamine (DA). (B)
The R isomer caused a greater potentiation of D2 receptor signaling in the
presence of a low concentration of dopamine than the racemate in a
concentration-related manner. The S isomer lacked effects. Results are
normalized to fitted max DA response and minimum (basal) response in
presence of EC20 [DA]. (C) In the presence of 10 mM of the R isomer, the
potency and efficacy of dopamine was increased compared with dopamine
alone. The presence of the S isomer (10 mM) had no effect. (D) In the
presence of 10 mM of the R isomer, the potency and efficacy of quinpirole
was increased compared with quinpirole alone.

Identification of Dopamine D2/D3 Receptor PAMs
307
(
n 5 4; 1 nM). In the presence of 10 mM of the racemic initial compound on basal levels of cAMP, and there was no change in
compound, the potency (pEC50) of dopamine was increased to the maximum response to dopamine. We then tested the
9
.6 6 0.1 (0.25 nM) (Fig. 3A). There was no effect of the effects of the stereoisomers on the dopamine concentration–
response curve (changes in cAMP assessed using the HTRF
ratio; Fig. 3B).The R isomer (10 mM) produced a significant
shift in the potency of dopamine pEC50 (EC50) from 8.3
(4.6 nM) to 9.03 (0.9 nM), and the S isomer had a small but
nonsignificant reduction in dopamine potency to 8.1 (8 nM).
This change in apparent dopamine potency was due to the
correction of raw counts to nM cAMP using the standard
curve.
Effects on D2 Receptor Binding. At concentrations up
to 10 mM both the R and S isomers had no statistically
3
significant effect (,10%) on the binding of [ H]-raclopride to
the human D2 receptor (data not shown). The effects of the
potential allosteric modulators on the binding of the endoge-
3
nous agonist, [ H]-dopamine, to the hD2 receptor in CHO cell
membranes was then investigated. Previous literature has
1
1
shown the importance of including Mg
ions both in the
membrane preparation and in the assay buffer (Hamblin and
Creese, 1982), and in the present studies the best window
was obtained using 1 mM MgCl
2
. The concentration of
3
[
H]-dopamine used in competition studies was selected based
on signal size and also on window size (specific: nonspecific).
The specific binding window was increased in the presence
of pargyline and ascorbic acid compared with that in the
absence and no specific filter binding was seen. No specific
binding was seen in membranes prepared from cells lacking
the D2 receptor. Control compounds such as apomorphine,
chlorpromazine, and (1)butaclamol produced a concentration-
3
dependent full inhibition of [ H]-dopamine binding to the D2
receptor with a pEC50 of 9.16 6 0.04 (n 5 3; 0.25 nM), 9.00 6
0
.50 (n 5 4, 1 nM) and 8.58 6 0.10 (n 5 3; 2.6 nM), respectively.
In contrast, the R isomer produced a concentration-dependent
3
increase in [ H]-dopamine binding (Fig. 4A). This potentiation
was difficult to quantify as the response did not saturate at the
concentrations tested (we could not go to higher concentra-
tions of the compound due to solubility), but a marked 100%
increase in radioligand binding was seen at 10 mM of the
compound and a statistically significant effect was seen at
1
mM. The S isomer produced an inhibition of radioligand
binding. Again the effect was difficult to quantify, but a
statistically significant inhibition of 16% 6 3% (n 5 3) was
seen at 3 mM compound.
3
The effect of the isomers on the saturation binding of [ H]-
dopamine to the dopamine D2 receptor was then studied.
Analysis of the saturation binding revealed the presence of one
binding site (F-test, GraphPad Prism). Saturation studies
were performed in the presence and absence of 10 mM of the R
and S isomers (Table 1). There was no statistically significant
effect on the apparent affinity of dopamine (K 15 nM), but the
d
maximum number of binding sites (Bmax; 3.2 6 0.2 pmol/mg
Pr) was statistically significantly increased in the presence of
the R isomer (P , 0.5, Student’s paired t test) whereas there
Fig. 3. Effect of compounds on D2-mediated cAMP levels. The results are
mean and S.E.M. from triplicate determinations in a single representative was a small but nonsignificant reduction in the Bmax with the
experiment that was repeated at least twice. (A) The presence of 10 mM of the
racemate increases the potency of dopamine 4-fold to inhibit cAMP pro-
duction at the human dopamine D2 receptor compared with dopamine alone.
S isomer (Supplemental Data). The Bmax for the antagonist
3
radioligand [ H]-raclopride in the same membranes was
Data are shown for after conversion to [cAMP]. (B) In the presence of 10 mM 17 pmol/mg Pr (data not shown).
of the R isomer, the potency (5-fold) and efficacy of dopamine was increased
compared with dopamine alone. There was a small, nonsignificant reduction
in the potency of dopamine in the presence of the S isomer (10 mM). The raw
data are shown in HTRF fluorescent ratio. (C) The improved compound at 10
mM increased the potency of dopamine by 15-fold.
To further explore the activities of these PAM effects at the
dopamine receptors, we went on to identify [5-fluoro-
-(hydroxymethyl)-2-methoxyphenyl]-(4-fluoro-1H-indol-1-yl)
methanone (see the Supplemental Data for synthesis and
4

3
08
Wood et al.
TABLE 1
3
Effects of the stereoisomers on the saturation binding of [ H]-dopamine to
the human dopamine D2 receptor
Data are mean 6 standard deviation from three separate experiments.
Control
+ R Isomer (10 mM)
+ S Isomer (10 mM)
K
d
(nM)
max (% control)
15 6 3
100 6 6
13 6 3
137 6 10*
17 6 6
82 6 15
B
*P , 0.05, Student’s paired t test.
the G protein level, experiments were conducted using the
human a2C-noradrenergic receptor and the histamine H3
receptor. No statistically significant effects were seen at
concentrations up to 10 mM on basal or agonist-stimulated
3
5
[
S]-GTPgS binding at either receptor for any of the com-
pounds (data not shown, but examples shown in the Supple-
mental Data).
To examine selectivity further, the effects of the compounds
were tested as allosteric modulators at human dopamine D1
and D3 receptors. At the D1 receptor, dopamine stimulated
cAMP production with a pEC50 of 8.6 6 0.1 (n 5 4; 2.5 nM).
None of the three compounds had an effect on the D1 response
(data not shown), neither on their own nor in the presence of
dopamine (1.5 nM); nor did they have an effect on the dopamine
concentration–response curve (Supplemental Data). In contrast,
similar to their effects at the human dopamine D2 receptor, the R
3
isomer potentiated the binding of [ H]-dopamine to the D3
receptor and the S isomer attenuated binding (Fig. 4B). The
compounds also lacked activity either on their own or in the
presence of dopamine at the human dopamine D4 receptor (data
3
Fig. 4. Effect of compounds on [ H]-dopamine binding to the human
dopamine receptors. The results are mean and S.E.M. from triplicate not shown).
determinations in a single representative experiment that was repeated at
Effects in Electrophysiologic Studies in Rat Striatal
concentration-related increase in [ H]-dopamine binding whereas the S Neurons. At concentrations above 3 mM, quinpirole dose-
least twice. (A) At the human D2 receptor, the R isomer caused a
3
isomer decreased binding. (B) At the human D3 receptor, the R isomer
caused a concentration-related increase in [ H]-dopamine binding whereas
dependently decreased NMDA currents (P , 0.0001, ANOVA,
F 6,35 5 70.29) (Fig. 6A). The D2 antagonist L-741,626
3
the S isomer decreased binding.
(30 mM; Hattori et al., 2006) blocked the 10 mM quinpirole
effect (not shown), demonstrating that this effect is mediated
by D2 receptor activation. In the presence of the D2 antago-
structure) as a more potent and efficacious PAM suitable for in nist, we still observed a slight decrease of NMDA currents
3
5
vivo experimentation. On human D2 GTP[ S]-GTPgS bind- (213.4 6 3.4%, n 5 4). However, it was similar to the decrease
ing, this improved PAM lacked statistically significant effects observed with the lower concentrations of quinpirole, suggest-
on its own (,10% activation at 10 mM compared with maximal ing that the remaining decrease is induced in part by current
effect of dopamine) but potentiated the effects of a low rundown. For all further studies, the effects of compounds
concentration of dopamine with a pEC50 of 6.2 6 0.24 (n 5 5; were therefore compared with time-matched controls.
6
5
30 nM) and by an Emax of 89% over basal 6 12 (compared with
The R isomer tested alone at 10 mM did not have a
7% for the R isomer and 100% for dopamine). This higher statistically significant effect on NMDA currents (25.3 6
level of potentiation was evidenced by a greater shift or 1.3%, n 5 29). In the presence of the R isomer (10 mM), the
increase in the potency of dopamine of 15-fold (from 6.4 nM effect of quinpirole at 3, 10, 30, 100, and 300 mM (n 5 4–11)
to 0.4 nM) (Fig. 4C) compared with 4-fold for the R isomer. We was increased (P , 0.05, paired t test), and the potency of
therefore studied the effect of the improved PAM on the quinpirole pEC50 was increased from 4.3 (52.5 mM) to 4.8
3
saturation binding of [ H]-dopamine as earlier. This PAM (15.9 mM) (Fig. 6B). As a control, incubation in 1% DMSO
(
10 mM) markedly increased the number of high-affinity failed to potentiate the 3 mM quinpirole effect (n 5 5, Fig. 6C).
binding sites, giving a 3-fold increase which was markedly The PAM (R isomer) had no effect on the lowest concentration
greater than that seen with the R isomer and consistent with of quinpirole tested (1 mM), suggesting that any effect was not
its higher efficacy (Fig. 5; Table 2). The nonhydrolysable GTP due to current rundown, which would be present at all
analog 5ʹ-guanylylimidodiphosphate [Gpp(NH)p] (32 mM) re- quinpirole concentrations.
3
duced the affinity of [ H]-dopamine binding and increased the
In Vivo Effects on L-Dopa Turning Behavior. The
apparent number of sites. The effect of the PAM was not
improved PAM D2 compound statistically significantly in-
affected by the presence of Gpp(NH)p.
creased [F (1,14) 5 10.45, P , 0.01] and prolonged [F (11,154) 5
Selectivity Assays. To ensure that potentiation in dopa- 50.55, P , 0.0001] the L-dopa-induced contralateral rotations
mine mediated receptor signaling was not due to an effect at (Fig. 7). An effect of the treatment with time was also observed

Identification of Dopamine D2/D3 Receptor PAMs
TABLE 2
Effect of the improved PAM on the saturation binding of [ H]-dopamine
binding to the human dopamine D2 receptor
309
3
Data are mean 6 standard deviation from three to four separate experiments.
+
PAM
+ Gpp(NH)p
(32mM)
+ PAM
+Gpp(NH)p
Control
(
10 mM)
K
B
d
(nM)
18 6 12
11 6 4
87 6 32*
20 6 13
289 6 47*
max (% control) 100 6 26 278 6 51* 200 6 120
*P , 0.05, Student’s paired t test compared with control.
presence of the S isomer in the racemate attenuated the effects
of the R isomer.
We then compared the ability of the isomers to affect the
concentration–response curve with the endogenous ligand
dopamine. The R isomer increased both the potency and, to
a lesser extent, the efficacy of dopamine, whereas the S isomer
produced a small but significant reduction in the maximal
response to dopamine with no effect on dopamine potency.
Similar results were seen on the ability of dopamine to inhibit
cAMP levels, although a reduction in maximal response to
dopamine in the presence of the S isomer was not observed.
This may reflect the greater receptor reserve seen in the cAMP
pathway, as evidenced by the markedly higher potency of
3
5
dopamine in the cAMP assay (pEC50 9.0) than in the [ S]-
GTPgS binding assay (pEC50 6.6).
To check that this allosteric effect was mediated at the
receptor level (as opposed to an effect on the G protein), we
then sought to determine whether the compounds affect other
3
5
Gi-coupled receptors. Using [ S]-GTPgS binding, we showed
that neither compound had any significant affect as either an
agonist or as a modulator at the Gi-coupled a2C-adrenergic
3
and histaminergic H3 receptors. Using the antagonist [ H]-
raclopride, we found no significant effect at concentrations up
to 10 mM, indicating that the compounds did not compete at
3
Fig. 5. Effect of [5-fluoro-4-(hydroxymethyl)-2-methoxyphenyl](4-fluoro- the orthosteric site. Using [ H]-dopamine, we showed that the
3
1
H-indol-1-yl)methanone (10 mM) on saturation binding of [ H]-dopamine.
R isomer did not act as an orthosteric ligand to inhibit binding
to the dopamine D2 receptor; rather, it increased the binding,
3
(
A) The specific binding of [ H]-dopamine in the presence and absence of
the compound. Data from a single experiment with triplicate determina-
3
3
whereas the S isomer decreased binding. The increase in [ H]-
tion. (B) The effect of GppNHp on the saturation binding of [ H]-dopamine.
3
5
(C) The lack of effect of GppNHp on the effect of the PAM.
dopamine binding was consistent with its potency on [ S]-
GTPgS binding, although this was difficult to quantitate
because solubility of the compounded precluded testing at
concentrations greater than 10 mM. Similar effects on the
[
F (11,154) 5 7.60, P , 0.0001]. Additional statistical analysis
showed that the group treated with the UCB compound had a
statistically significantly higher level of contralateral rota-
tions than the vehicle-treated group for the first 60 minutes of
the test.
3
binding of [ H]-dopamine to the human dopamine D3 receptor
were observed with both the R and S isomers.
Novel allosteric binding sites on GPCRs have been sug-
gested to offer selectivity benefits compared with the orthos-
teric site, which is often highly conserved across receptor
subtypes (Conn et al., 2009; Leach et al., 2010). In this study,
we have shown selectivity for the D2 receptor over the D1
Discussion
We report the identification of novel allosteric modulators of receptor, but not over the D3 receptor. There is a high degree of
the dopamine D2 receptor and D3 receptors. We identified an sequence similarity between the D2 and D3 receptors—over
initial hit from an HTS that lacked agonist effects on its own 78% within the transmembrane domains and a near identity
(
at concentrations up to 10 mM) but potentiated the effect of of the residues inferred to form the dopamine-binding site. For
3
5
dopamine on the human D2 receptor in cAMP and on [ S]- the antagonist eticlopride, 17 of the 18 residues that form
GTPgS binding. However, this initial hit was a mix of two its binding site are identical across the D2 and D3 receptors
isomers. We therefore separated the isomers and confirmed (Shi and Javitch, 2002; Chien et al., 2010). Interestingly,
their conformation by X-ray—the R isomer with the methyl SB269652 (N-[4-[2-(7-cyano-3,4-dihydro-1H-isoquinolin-2-yl)
group up, and the S isomer with the methyl group down. ethyl]cyclohexyl]-1H-indole-2-carboxamide) was identified
Compared with the racemic mixture, the R isomer produced a as an allosteric antagonist at both the dopamine D3 and
3
5
greater enhancement of D2-stimulated [ S]-GTPgS binding D2 receptors (Silvano et al., 2010). Recent studies have
whereas the S isomer appeared inactive, suggesting that the suggested that this compound acts as a bitopic ligand with

3
10
Wood et al.
Fig. 6. (A) Traces of 100 mM NMDA-mediated calcium and barium currents before and after coapplication of 10 mM quinpirole, and before and after
incubation with the R isomer. (B) Graph shows the dose–response effect of quinpirole or quinpirole + R isomer on NMDA currents. The dose–response curve
was shifted to the left in the presence of PAM D2. (C) While incubation with improved PAM D2 compound potentiated the effect of 3 mM quinpirole compared
with vehicle control. Paired t test *P , 0.05; **P , 0.01; ***P , 0.001. The blue line indicates D2 agonist + R isomer. The green line indicates D2 agonist alone.
the indole-2-carboaxamide moiety interacting with an alloste- possibility that the allosteric pocket is conserved between
ric pocket (Lane et al., 2014) and has a close structural dopamine D2 and D3 receptors, but not across dopamine D4 or
similarity to the compounds identified here. This raises the D1 receptors.
Fig. 7. Effect of improved PAM com-
pound (30 mg/kg, i.p.) on the level of L-
dopa-induced contralateral rotations in
unilateral 6OHDA-lesioned rats (15 mg/kg,
i.p.). *P , 0.05: mean is significantly
different from that of the vehicle-treated
group (planned contrasts for every 10-
minute time interval).

Identification of Dopamine D2/D3 Receptor PAMs
311
We had good evidence that the compounds acted as
We have used the unilateral 6OHDA lesion model in rats
allosteric modulators at the dopamine D2 and D3 receptor, (Schwarting and Huston, 1996) to investigate the effects of the
so we investigated the mechanism of this interaction by PAM using a threshold dose of L-dopa that induced a low level
investigating their effects on the saturation binding of the of contralateral turns. This model was expected to be sensitive
3
orthosteric agonist [ H]-dopamine. Since the description of to the effects of a PAM. Indeed, we saw that the compound, at
this assay, there have been few reports on its application, 30 mg/kg, potentiated the effects of a threshold dose of L-dopa
probably due to the low level of specific binding (Burt et al., on contralateral turning in the 6OHDA model. We confirmed
1
975) compared with newer antagonist radioligands and the that this was not due to an effect on L-dopa metabolism as
11
observation that the inclusion of Mg
in the membrane there was no change in the plasma exposure of L-dopa
preparation is important (Hamblin and Creese, 1982). The (R. Barnaby, personal communication). Unfortunately we
3
affinity of [ H]-dopamine determined in the present study is did not have sufficient material to check the effect of the
in good agreement with that obtained in rat tissue (5– compound alone, but no overt effect of the compound was seen
1
2
0 nM, Burt et al., 1975) and in CHO cells (Torvinen et al., in the pharmacokinetic study, supporting the hypothesis that
004). Our data showed that the PAM did not affect the the potentiation was due to an allosteric mechanism. These
3
affinity of [ H]-dopamine for the D2 receptor, but instead data indicate that a positive allosteric modulator of the
increased the proportion of receptors in the high-affinity dopamine D2/D3 receptor can potentiate dopamine effects on
agonist state. This suggests that the PAM acts to enhance neuronal activity in rat brain.
signaling or efficacy at the receptor. The change in potency
In conclusion, we have shown a stereoselective interaction
of dopamine observed in the functional assays is probably on the allosteric modulation of the dopamine D2 and D3
due to a high receptor reserve leading to a ceiling in receptor. The R isomer acts as a PAM in that it lacks effects on
efficacy (see Kenakin, 2013). In support of this, (2)-[3H]- its own, but potentiates the effects of dopamine at the D2
(
3-hydroxyphenyl)-N-(1-propyl)-piperidine [(2)-3-PPP] has receptor on G protein activation and cAMP signaling, and
3
been reported as a partial agonist at the dopamine D2 potentiates [ H]-dopamine binding to the D2 receptor. The
receptor (Burris et al., 2002) but appeared as a full agonist potentiation in Emax seen in [ S]-GTPgS binding compared
3
5
in the present study (data not shown). GppNHp reduced the with the lack of effect on the downstream signaling cAMP
3
apparent affinity for [ H]-dopamine and increased the together with the potentiation in Bmax and the lack of change
3
apparent number of sites, suggesting a conversion to a in affinity for [ H]-dopamine, suggest that the R isomer acts as
low-affinity inactive state. In support of this, the number of an efficacy modulator by stabilizing the active state of the
3
high-affinity sites labeled by [ H]-dopamine was lower than receptor. In contrast, the S isomer attenuates the effects of the
3
3
that seen with [ H]-raclopride. The lack of effect of Gpp(NH) R isomer and inhibits [ H]-dopamine binding.
3
p on [ H]-dopamine binding in the presence of the PAM
Although the evidence for an allosteric action at the dopamine
suggests that this conformational change is independent of D3 receptor is not as robust as that at the dopamine D2 receptor,
3
the G protein.
the potentiation of [ H]-dopamine binding in the absence of an
As we have demonstrated robust allosteric effects at the effect on the orthosteric antagonist binding is consistent with
dopamine D2 receptor, it is important to show that these the profile demonstrated at the D2 receptor. Unfortunately, the
effects are maintained under physiologic expression and R isomer and S isomers both produced a nonspecific effect, which
signal transduction conditions. We show that the R isomer interfered with our D3 receptor label-free functional assay
potentiates the effect of a dopamine D2/D3 agonist on a D2/D3 (Wood et al., 2015) and precluded further testing.
receptor-mediated response in a native tissue system, rat
Importantly, we show that the PAM maintains its activity in
striatal neurons. Activation of the D2/D3 receptor induced native tissue and further that this potentiation was main-
decreases of NMDA currents in adult rat striatal neurons, and tained in vivo. In both these studies, an action at both
this was blocked by a dopamine D2/D3 receptor antagonist. In dopamine D2 and D3 receptors could be involved, and further
this assay, there was no effect of the compound alone, studies with selective D3 receptor antagonists are needed. The
demonstrating a lack of agonist-like properties under physi- identification of positive allosteric modulators at dopamine D2
ologic expression, but it increased the potency (EC50) of and D3 receptors could have important applications in
quinpirole from 52 to 16 mM. The potency of quinpirole pathologic conditions of hypodopaminergic function, such as
(
pEC50 4.28) was in agreement with other findings (Andre in Parkinson’s disease and restless leg syndrome.
et al., 2010; Jocoy et al., 2011) and was consistent with a lower
D2 receptor expression in the native tissue compared with the
recombinant systems expressing rat D2 receptors where the
pEC50 for quinpirole was 6.2.
To test the hypothesis that a D2 PAM should potentiate the
effects of released dopamine in vivo, we went on to identify
Acknowledgments
The authors thank David Urbain for technical assistance.
Authorship Contributions
Participated in research design: Wood, Andre, Ates, Gillard,
Michel, Barnaby.
Conducted experiments: Andre.
Performed data analysis: Wood, Andre, Michel, Barnaby, Ates.
Wrote or contributed to the writing of the manuscript: Wood, Ates,
Andre, Michel, Gillard.
[
5-fluoro-4-(hydroxymethyl)-2-methoxyphenyl](4-fluoro-1H-
indol-1-yl)methanone (Supplemental Data) as a more potent
and efficacious PAM D2/D3 compound. This compound was
slightly more potent, but was markedly more efficacious
(
4
almost 90% potentiation of GTPgS assay compared with
0% for the initial PAM and 3-fold shift in Bmax of [ H]-
3
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