A structure-activity relationship study of the positive allosteric modulator LY2033298 at the M4 muscarinic acetylcholine receptor

Article abstract of DOI:10.1039/c5md00334b

Positive allosteric modulators (PAMs) targeting the M₄ muscarinic acetylcholine receptor (mAChR) offer greater sub-type selectivity and unique potential as central nervous system agents through their novel mode of action to traditional orthosteric ligands. In an attempt to elucidate the molecular determinants of allostery mediated by the exemplar thienopyridine M₄ mAChR PAM, LY2033298, we report herein a systematic structure-activity relationship (SAR) study investigating different linkage points, halogen replacements to examine size and electronic effects, and different substitution combinations on the thienopyridine scaffold. We applied an operational model of allosterism to determine values of functional affinity (K_B), cooperativity (αβ) and intrinsic agonism (τ_B) for all compounds.

Full text of DOI:10.1039/c5md00334b

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A Structure-Activity Relationship Study of the Positive Allosteric
Modulator LY2033298 at the M4 Muscarinic Acetylcholine Recep-
tor
Received 00th January 20xx,
Accepted 00th January 20xx
Monika Szabo,ab Tracey Huynh,a Celine Valant,b J. Robert Lane,b Patrick M. Sexton,b Arthur
Christopoulos*b and Ben Capuano*a
DOI: 10.1039/x0xx00000x
www.rsc.org/medchemcomm
Positive allosteric modulators (PAMs) targeting the M4 muscarinic acetylcholine receptor (mAChR) offer greater sub-type
selectivity and unique potential as central nervous system agents through their novel mode of action to traditional
orthosteric ligands. In an attempt to elucidate the molecular determinants of allostery mediated by the exemplar
thienopyridine M4 mAChR PAM, LY2033298, we report herein a systematic structure-activity relationship (SAR) study
investigating different linkage points, halogen replacements to examine size and electronic effects, and different sub-
stitution combinations on the thienopyridine scaffold. We applied an operational model of allosterism to determine values
of functional affinity (KB), cooperativity (αβ) and intrinsic agonism (τB) for all compounds.
and/or direct activation by the allosteric ligand itself.5 Earlier
studies have deduced parameters for allosterism via
Introduction
The muscarinic acetylcholine receptors (mAChRs) are a class of
protein-coupled receptors (GPCRs) consisting of five
implementing an operational model of allosterism. This model
quantifies the magnitude and direction of the parameters of
allosterism including modulator binding affinity, KB,
modulation of binding affinity of the orthosteric ligand, α,
modulation of downstream efficacy upon binding of the
orthosteric ligand, β, and intrinsic allosteric agonism, τB.6
G
subtypes (M1-M5) and are expressed in the central nervous
system (CNS) and the periphery. The M4 mAChR is of clinical
interest as it has demonstrated an involvement in CNS
disorders such as schizophrenia.1 Specifically, the M4 mAChR
has been shown to alleviate the positive symptoms
(hallucinations, delusions) and potentially benefit the cognitive
There has been considerable research conducted in the field of
M4 PAMs, all of which focus on the thienopyridine scaffold.7-9
Whilst significant progress has been made, none have matched
the pharmacological profile of LY2033298 (Figure 1; 8a). In
attempts to move away from the 5-chloro-6-methoxy-4-
methyl substitution pattern on 8a, researchers at Vanderbilt
University (VU) developed a number of analogues based on a
4,6-dimethyl substitution pattern on the thienopyridine motif,
such as VU0152100 (Figure 1; 1a) and VU10004 (1b). We have
also published work on this scaffold investigating the
pharmacological impact of incorporating modifications to the
substitution pattern of the arylmethyl motif of VU015210010
and replacement of the cyclopropyl moiety of VU10004 with
alternative primary cycloalkanamines and cyclic secondary
amines.11
deficits (memory, learning) associated with schizophrenia.2,3
significant problem associated with clinically prescribed
antipsychotics designed to target designated GPCR
A
a
orthosteric site is their promiscuity and therefore lack of
receptor selectivity. This phenomenon often results in an
extensive side effect profile. Allosteric ligands offer a potential
solution to this problem by targeting a topographically distinct
site to the orthosteric site. While orthosteric sites tend to be
conserved across receptor subtypes, allosteric sites can differ,
making subtype selectivity
a
more eminent possibility.4
Previous work on the M4 mAChR has focused on positive
allosteric modulators (PAMs), whereby they can exhibit several
possible modes of action: potentiation of the binding of the
endogenous ligand, acetylcholine (ACh); potentiation of the
down-stream efficacy upon binding of the endogenous ligand;
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Figure 2 General overview of structural modifications to LY2033298
8a) investigated in this study.
(
Synthesis
The synthesis of derivatives with alkyl chains extending from
the methoxy group at the 6-position (O-alkyl derivatives) are
represented in Scheme 1. We commenced the sequence with
the monocyclic pyridinone (
2
); the chemistry of which has
been previously described.15 In one step, we combined
2
with
Figure 1 Muscarinic M4 positive allosteric modulators LY2033298 (8a),
VU0152100 (1a) and VU10004 (1b) with numbering systems.
the required primary alkyl iodide under mild basic conditions
to effect O-alkylation then subsequently added a stronger base
(1 M aqueous KOH) to convert the monocyclic intermediates
to bicyclic analogues (3a-d).10 The primary aromatic amine of
all analogues was protected as the phthalimide (4a-d) in good
yield using phthalic anhydride prior to halogenating the 5-
position (5a-f) with the appropriate N-halosuccinimide. This
strategy was implemented to circumvent unwanted N-
halogenation. Subsequent phthalimide de-protection under
standard conditions of hydrazine monohydrate afforded 6a-f
in good to excellent yield (46-89%). Base-promoted ester
hydrolysis (following acidic work-up) furnished the carboxylic
acids 7a-f in similar yields. A BOP-mediated amide coupling
reaction was then employed between cyclopropanamine and
carboxylic acids (7a-f) to generate the final O-alkyl analogues
To explore the structure activity relationships (SAR) of 8a, we
initially synthesised a focused series of compounds that
encompassed alkyl chain extensions from several strategic
points on the chemical structure of 8a (Figure 2). The aim was
to determine suitable tethering points and to observe if the
incorporation of larger groups would be tolerated at these
positions; with the future aim of structurally integrating either
an orthosteric mAChR moiety to generate bitopic ligands12 or
generate dual acting ligands that incorporate
a
pharmacophore that is selective for another protein target.13
This information may also be useful in determining the size of
the allosteric binding pocket at the M4 mAChR and therefore
the scope for structural diversification. We also assessed the
importance of the halogen at the 5-position in relation to both
size and electronic effects by substituting with larger halogens
(Br and I). In addition, we investigated an isosteric
(
8a-f). The N-alkyl analogues (9a-c) were furnished by simply
coupling the intermediate carboxylic acid 7a with the required
acyclic alkanamines. Synthesis of the ‘amine modified’
analogues was carried out by direct acylation of LY2033298
replacement of the C-Cl moiety for
N to yield a
thienopyrimidine bicyclic system in place of the
thienopyridine. Although, the new scaffold is lacking a chlorine
atom, it is predicted that the incorporation of an additional
nitrogen atom will maintain a relatively electron-deficient six-
membered ring of the bicyclic system comparable to that of
LY2033298. We also explored different combinations of
substituents on the thienopyridine ring of 8a to ascertain the
optimal substitution pattern essential for a PAM profile at the
M4 mAChR. To do this we looked at the following substitution
patterns of the thienopyridine core, namely 6-methoxy-4-
methyl, 5-chloro-4,6-dimethyl and 4,6-dimethyl. Although
these substitutions have been previously reported,9,14 the
underlying operational parameters describing their allosteric
effects have not been determined. All compounds were
characterized in functional M4 mAChR-mediated ERK1/2
phosphorylation (pERK1/2) assays and compounds that
showed significant differences compared to 8a, were further
profiled in radioligand binding assays.
(
8a) with the required acid chlorides to furnish the target
carboxamide derivatives (10a-c) in moderate yield (45-48%).
Scheme
1 Synthesis of O-alkyl, N-alkyl, N-acyl and des-chloro
derivatives of LY2033298. Reagents and conditions: (a) (i) Alkyl-I,
K2CO3, DMF, 4 h, RT; (ii) 1 M KOH, 15 min, 58-94%; (b) Phthalic
anhydride, AcOH, reflux, 20 h, 48-86%; (c) N-halosuccinimide, conc.
HCl, EtOH, reflux, 1.5 h, 80-99%; (d) (NH2)2.H2O, EtOH, reflux, 3 h, 46-
2 | J. Name., 2012, 00, 1-3
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89%;(e) 2 M NaOH, EtOH, reflux, 1.5 h, 49-96%; (f) Alkanamine, BOP
reagent, DIPEA, DMF, RT., 1-12 h, 15-72%; (g) Acid chloride, Et3N,
DCM, N2, reflux, 2 days, 45-48%.
concentrations of ACh (10 µM to 10 nM) at a stimulation time
of 6 minutes. The results of these interaction studies are
summarised in Table 1. By applying an operational model of
allosterism, we derived values of functional affinity (pKB),
intrinsic efficacy (τB) and overall cooperativity (αβ) of the
allosteric ligand’s effect on ACh.
All ligands displayed no notable enhancement in functional
affinity when compared across their respective groups, i.e. O-
alkyl, N-alkyl or halogen derivatives. For the O-alkyl analogues,
there was
proceeding from methyl (8a) and ethyl (8b) to the butyl (8c
and hexyl 8d derivatives. The intrinsic efficacy was
a significant loss in intrinsic efficacy when
)
(
)
Scheme 2 Synthesis of LY2033298/VU hybrid and thienopyrimidine
analogues. Reagents and conditions: (a) SO2Cl2, 1,2- DCE, reflux 6 h,
42%; (b) POCl3, MW, 100 °C, 1h, 88%; (c) Thiourea, EtOH, relux, 24 h,
maintained between 8a and 8b confirming that the addition of
the extra carbon at-om has little effect and that anything
29%; (d) NaOMe, MeOH, relux, overnight, 30%; (e) Acetyl chloride, larger is detrimental to the compound’s agonism. The
NH4SCN, dioxane, 23%; (f) Na metal, EtOH, reflux 4 h,75%.
cooperativity of the O-alkyl derivatives with ACh was also
maintained, however again the larger butyl (8c) and hexyl (8d
substituents resulted in a substantial loss in the cooperativity
(96- and 120-fold, respectively) when compared with 8a
)
Synthesis of des-chloro-LY2033298 (12) was achieved in good
yield by firstly converting the bicyclic ethyl ester (3a) to the
free carboxylic acid (11) via base hydrolysis and subsequent
acidic work-up, followed by a BOP-mediated coupling reaction
with cyclopropanamine to install the cyclopropyl moiety.
We envisaged the synthesis of the LY2033298/VU hybrid
.
Replacement of the chlorine at the 5-position of 8a with
bromine (8e) or iodine (8f), afforded no significant gain or loss
in intrinsic efficacy and cooperativity compared with the
parent compound. As such, the thienopyridine scaffold
analogue (18) commencing from the pyridinone precursor (13
)
possessing
larger
halogen
atoms
with
reduced
as illustrated in Scheme 2. The first chlorine atom was
successfully installed at the 5-position of the substituted
electronegativity is well tolerated by the M4 mAChR. For the
series of N-alkyl analogues synthesized, only the N-hexyl
derivative (9c) showed a significant drop in intrinsic agonism
(22-fold) and cooperativity (185-fold) when compared to the
N-cyclopropyl compound (8a). There were no significant
differences between the N-butyl compound 9b compared to
8a, confirming that a butyl linker is tolerated but the additional
two carbon atoms significantly affects the agonistic and PAM
properties of the ligand. The N-ethyl compound 9a maintained
similar allosteric properties to 8a, as the ethyl group occupies
a similar chemical and physical space to that of the cyclopropyl
group.
pyridinone core using sulfuryl chloride to give intermediate 14
Subsequent chlorination to furnish the dichloro substituted
pyridine 15 was effected using phosphoryl chloride.
.
(
)
Treatment of 15 with thiourea in ethanol at reflux afforded the
versatile pyridinethione (16) in moderate yield. Reaction of 16
with pre-synthesised 2-chloro-N-cyclopropylacetamide
(
17)
under basic conditions produced the target thienopyridine (18
)
with the 5-chloro-4,6-dimethyl substitution pattern. Synthesis
of the thienopyrimidine ana-logue 21 was successfully
(
)
achieved by firstly reacting 3-aminocrotononitrile (19), acetyl
chloride and ammonium thiocyanate to generate the
substituted pyrimidine core (20), followed by treatment with
chloroacetamide 17 and sodium metal in ethanol at reflux to
furnish the product in very good yield (75%).
The general trend observed between the O-alkyl and N-alkyl
analogues was as we sequentially increased the alkyl chain
length, we observed a decrease in both the intrinsic efficacy
and the cooperativity of the compounds with ACh. This is
illustrated in Figure 3 with N-alkyl analogues 9a-c exhibiting a
smaller decrease in activity as a function of chain length. As
such the allosteric pocket on the M4 mAChR appears to have a
greater degree of space to accommodate larger groups on this
side (9a-c), which is in agreement with the VU compounds that
incorporate larger alkyl and substituted benzyl groups at this
position.9 However, recent evidence on the binding
mechanism of allosteric ligands to the M2 mAChR showed that
upon receptor activation and binding of an allosteric ligand,
the allosteric site encloses the modulator, creating a tight
fitting binding pocket.16 Therefore major structural changes to
PAMs may affect how they can fit in the allosteric site, and
Results and discussion
To characterize the biological activity of the synthesized
compounds incorporating O-alkyl (8b-d) and N-alkyl chains (9a-
c
) on the LY2033298 scaffold, halogen replacements (8e-f) and
modification to the aromatic amine functionality (10a-c), we
tested all analogues in a functional pERK1/2 assay using intact
FlpIn-CHO cells stably transfected with the human M4 mAChR.
Compounds were initially tested in time-course assays (data
not shown) to determine the optimal incubation time for
maximum ERK1/2 phosphorylation for each compound. As the
response to all compounds peaked at a similar time to ACh,
interaction studies were performed using varying
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Figure 3 Effect of increasing alkyl chain length on the ability of N-alkyl analogues 9a-c to allosterically modulate the activity of ACh in M4 mAChR-mediated
pERK1/2 assays. Data points represent the mean of three experiments performed in duplicate.
Table 1 Functional ERK1/2 phosphorylation and binding data for LY2033298 (8a), O-alkyl analogues (8b-d), halogen replacement analogues (8e-f) and N-
alkyl analogues (9a-c) at the M4 mAChR
Functional ERK1/2 phosphorylation dataa,b
Binding dataa,c
C#
8a
R1
R2
R3
H
X
Cl
Cl
pKB(KB, μM)
log τB (τB)
log αβ (αβ)
pKi (Ki, μM)
*log α (α)
Methyl
Ethyl
6.41 (0.4)*
0.93 ± 0.14 (8.5)
0.97 ± 0.32 (9.3)
2.38 ± 0.23 (240)
1.79 ± 0.44 (61.7)
6.41 ± 0.11 (0.4)
6.71 ± 0.16 (0.2)
3.24 ± 0.14 (1738)
1.98 ± 0.12 (95.5)
8b
8c
8d
H
6.10 ± 0.37 (0.8)
Butyl
H
H
H
Cl
Cl
6.81 ± 0.40 (0.2)
7.42 ± 0.32 (0.04)
-0.51 ± 0.20 (0.3)
-0.75 ± 0.18 (0.2)
1.25 ± 0.40 (17.8)
0.40 ± 0.16 (2.5)
0.30 ± 0.11 (2.0)
1.91 ± 0.52 (81.3)
5.57 ± 0.40 (2.7)
nt
1.35 ± 0.16 (22.4)
nt
Hexyl
Methyl
8e
8f
Br 6.52 ± 0.44 (0.3)
6.28 ± 0.07 (0.52)
2.54 ± 0.11 (347)
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
H
H
I
6.66 ± 0.33 (0.2)
6.73 ± 0.39 (0.2)
6.29 ± 0.46 (0.5)
6.70 ± 0.32 (0.2)
n/a
0.37 ± 0.18 (2.3)
0.94 ± 0.33 (8.7)
0.44 ± 0.30 (2.8)
-0.35 ± 0.12 (0.4)
n/a
0.97 ± 0.44 (9.3)
1.84 ± 0.53 (69.2)
1.19 ± 0.51 (15.5)
0.13 ± 0.15 (1.3)
n/a
5.78 ± 0.09 (1.7)
2.43 ± 0.12 (269)
9a
Ethyl
Butyl
Hexyl
Cl
5.37 ± 0.32 (4.3)
2.75 ± 0.36 (562)
9b
9c
H
Cl
Cl
Cl
Cl
Cl
5.50 ± 0.14 (3.2)
2.09 ± 0.18 (123)
H
nt
nt
nt
nt
nt
nt
nt
nt
10a
10b
10c
COCH3
COC3H7
COC5H11
n/a
n/a
n/a
n/a
n/a
n/a
aData represent the mean ± SEM of three-four separate experiments performed in duplicate. bValues are obtained from pERK1/2 assays via interaction
with varying concentrations of Ach. cValues are obtained via radioligand ([3H]NMS) binding assays through interaction with varying concentrations of
ACh.*Log α’ is calculated by fixing log α to -100. n/a= Compound not active. nt= Not tested in binding assays as cooperativity (αβ) in pERK1/2 assays was
very low or compound was not active.*= The pKB is fixed to the pKi obtained from binding experiments.
may result in a loss of their ability to exert effects upon the
ligand in the orthosteric site.
Due to many analogues showing very similar functional
affinity, allosteric modulation and/or intrinsic efficacy to 8a,
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Data represent the mean ± SEM of three separate experiments per-
formed in duplicate. Values are obtained via interaction with
varying concentrations of ACh.
we performed radioligand binding assays to gain greater
insight into any subtle differences between the compounds,
because the pERK1/2 experiments reflect functional
responses that are determined by both ligand binding to the
receptor (i.e., affinity) and activation of the receptor (i.e.,
efficacy), and thus cannot differentiate the individual
contributions of allosteric modulator effects on orthosteric
ligand affinity (α) versus efficacy (β) on the overall estimated
functional cooperativity (αβ). An advantage of performing
binding assays is that they provide direct measures of
agonist affinity only, and thus any allosteric modulation
observed in these assays can be used to derive a direct
estimate of the α parameter. Therefore this information can
be used in conjunction with the functional assay analysis to
deduce the individual contributions of both α and β
parameters in driving the PAM effect on ACh. All
compounds in Table 1 were tested, excluding 8d and 9c as
both were very weak PAMs in the pERK1/2 signalling
pathway. Compared to 8a, there was a significant loss in
affinity modulation for O-alkyl analogues 8b and 8c (18- and
77-fold, respectively). The halogen substituted analogues 8e
and 8f also had a small but significant loss in affinity
modulation (5- and 6-fold, respectively) to that of 8a. The N-
alkyl analogues showed that 9a was able to maintain a
similar affinity modulation to 8a, whereas extending to the
butyl analogue (9b) resulted in a 14-fold loss in receptor
affinity. This suggests that increasing the size of O-alkyl
substituent at the 6-position on the thienopyridine affects
modulation of ACh binding affinity. In comparison,
substitution of alkyl groups at the amide nitrogen bearing
the cyclopropyl group is tolerated, but diminishes with
There are examples of ligands in the literature whereby
substitution at the 6-position of the thienopyridine scaffold
with pyridinylmethyl or morpholine derivatives show
activity, however groups longer and more lipophilic tend to
decrease the efficacy of the compounds.8 While it has been
previously reported that the combination of the VU scaffold
(4,6-dimethyl thienopyridine) and substitution at the
primary aromatic amine is mostly tolerated but results in a
loss of activity9, we found that the scaffold of 8a (5-chloro-4-
methyl-6-methoxy thienopyridine) in combination with
acylation of the primary aromatic amine (10a-c) abolished
activity completely. The next part of our study was to
elucidate if structural changes to the pyridine ring of the
thienopyridine scaffold of 8a had any major influences on its
allosteric properties and to pinpoint what substitutions are
crucial for its activity. The results of compounds 12
and VU10004 are summarised in Table 2. The pyrimidine
derivative 21), which was used to mimic the
, 18, 21
(
electronegativity and ring deactivating effects of the
chlorine at that position whilst also reducing the chemical
and physical space, had no significant advantages as
compared to the chlorine at that same position. There was
no statistical significance for compounds 12, 18, 21 and
VU10004 when compared to 8a and compared to each
other in relation to the functional affinity, intrinsic agonism
and cooperativity of the compounds. As such these results
highlight that the thienopyridine is able to tolerate small
structural changes to its pyridine ring, however it is not clear
what substitutions are essential for its activity and allosteric
properties. Combined with the results from Table 1, the
pharmacology of thienopyridine scaffold appears to be more
affected when substituted with longer and more lipophilic
groups. Changes to the scaffold itself, neither enhance nor
diminish the pharmacological profile. The results do
however reveal that a simple change from a butyl to a hexyl
chain can significantly alter the pharmacological profile. It is
also possible that the O- and N-alkyl chains may be too
flexible and therefore more rigid functional groups should
greater than two carbon length alkyl chains compared to 8a
.
Table 1 highlights that changes to the thienopyridine
scaffold of 8a can be tolerated, however larger structural
changes such as hexyl chains on both the O-alkyl and N-alkyl
analogues are detrimental to the allosteric modulatory
properties of the ligand.
Table 2 LY2033298 core variants (12
, 18 and 21) in functional
ERK1/2 phosphorylation assays at the M4 mAChR
be employed at these relative positions of 8a
.
Compd
12
R1
X
pKB
(KB, μM)
5.55 ± 0.38
(2.8)
6.45 ± 0.28
(0.4)
4.90 ± 0.26
(12.5)
5.37 ± 0.14
(4.3)
log τB
(τB)
log αβ
(αβ)
Conclusions
We performed a comprehensive SAR study of the PAM 8a by
(i) substituting progressively longer O- or N-alkyl chains from
the scaffold through the ether linkage (8b-d), (ii) halogen
replacement (8e-f), (iii) amide derivatives via acylation of
the primary aromatic amine (9a-c) and (iv) different
OMe CH
0.66 ± 0.30
(4.6)
1.11 ± 0.26
(12.9)
0.49 ± 0.16
(3.1)
0.77 ± 0.11
(5.9)
1.46 ± 0.39
(28.8)
1.89 ± 0.36
(77.6)
1.15 ± 0.27
(14.1)
1.72 ± 0.17
(52.5)
18
Me
Me
C-Cl
21
N
VU1000411 Me
CH
substitution combinations around the pyridine ring (12, 18
and 21). The allosteric properties of the ligands
progressively decreased when substituting alkyl chains
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greater than two carbon atoms from either side of 8a (Table
1 and Figure 2). Additionally, halogen replacements were
tolerated, but significantly decreased the ability of the
compounds to positively modulate agonist affinity as
compared to 8a, confirming that compound 8a largely
modulates the affinity of ACh in the orthosteric pocket of
the M4 mAChR rather than modulating the efficacy.
Acylation of the primary aromatic amine (10a-c) completely
abolished activity. Due to the small molecule nature of
PAMs at the M4 mAChR and the recent advances into the
understanding of mechanisms of how they bind to
muscarinic receptors16, the possibility of tethering larger
functional groups for a dual acting or a potential bitopic
mode of action based on the scaffold of 8a is likely to prove
challenging. Furthermore, whilst we can identify subtle
differences between PAMs by utilizing the operational
model of allosterism, it is difficult to determine exactly what
functional groups on the thienopyridine scaffold of 8a are
important for its PAM mode of action (Table 2).
Nonetheless, these results show the benefits of profiling
ligands using operational models of allosterism to gain a
more comprehensive appraisal into the contribution of each
of the parameters for a PAM. Additionally the results
represent useful SAR towards the understanding and
development of the thienopyridine scaffold of PAMs
targeting the M4 mAChR.
6. Keov, P., Sexton, P. M., Christopoulos, A.,
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P. R., Yin, H., Jadhav, S. B., Shirey, J. K., Conn, P. J.,
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Acknowledgements
This research was supported by the project grant
APP1049564 and program grant APP1055134 of the
National Health and Medical Research Council (NHMRC).
A.C. is a principal research fellow (NHMRC). J.R.L. is a R.D.
W., Hopkins, C. R., Bioorg. Med. Chem. Lett., 2013, 23
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346-350.
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K., Hubner, H., Pardon, E., Valant, C., Sexton, P. M.,
Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J.,
Weis, W. I., Garcia, K. C., Wess, J., Kobilka, B. K., Nature,
2013, 504, 101-106.
Wright
Biomedical
Career
Development
Fellow
(APP1052304, NHMRC) and a Larkin’s Fellow (Monash
University, Australia). M.S. and T.H. acknowledge an
Australian Postgraduate award and M.S. acknowledges a
Monash Post Graduate Publication Award.
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5. Gregory, K. J., Sexton, P. M., Christopoulos, A., Curr.
Neuropharmacol., 2007, 5, 157-167.
6 | J. Name., 2012, 00, 1-3
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