Chemical modulation of mutant mGlu1 receptors derived from deleterious GRM1 mutations found in schizophrenics

Article abstract of DOI:10.1021/cb500560h

(Figure Presented) Schizophrenia is a complex and highly heterogeneous psychiatric disorder whose precise etiology remains elusive. While genome-wide association studies (GWAS) have identified risk genes, they have failed to determine if rare coding single nucleotide polymorphisms (nsSNPs) contribute in schizophrenia. Recently, two independent studies identified 12 rare, deleterious nsSNPS in the GRM1 gene, which encodes the metabotropic glutamate receptor subtype 1 (mGlu₁), in schizophrenic patients. Here, we generated stable cell lines expressing the mGlu₁ mutant receptors and assessed their pharmacology. Using both the endogenous agonist glutamate and the synthetic agonist DHPG, we found that several of the mutant mGlu₁ receptors displayed a loss of function that was not due to a loss in plasma membrane expression. Due to a lack of mGlu₁ positive allosteric modulators (PAM) tool compounds active at human mGlu₁, we optimized a known mGlu₄ PAM/mGlu₁ NAM chemotype into a series of potent and selective mGlu₁ PAMs by virtue of a double "molecular switch". Employing mGlu₁ PAMs from multiple chemotypes, we demonstrate that the mutant receptors can be potentiated by small molecules and in some cases efficacy restored to that comparable to wild type mGlu₁ receptors, suggesting deficits in patients with schizophrenia due to these mutations may be amenable to intervention with an mGlu₁ PAM. However, in wild type animals, mGlu₁ negative allosteric modulators (NAMs) are efficacious in classic models predictive of antipsychotic activity, whereas we show that mGlu₁ PAMs have no effect to slight potentiation in these models. These data further highlight the heterogeneity of schizophrenia and the critical role of patient selection strategies in psychiatric clinical trials to match genotype with therapeutic mechanism.

Full text of DOI:10.1021/cb500560h

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Chemical Modulation of Mutant mGlu1 Receptors Derived from
Deleterious GRM1 Mutations Found in Schizophrenics
Hyekyung P. Cho,∥,†,‡ Pedro M. Garcia-Barrantes,∥,† John T. Brogan,† Corey R. Hopkins,†,‡,§
Colleen M. Niswender,†,‡ Alice L. Rodriguez,†,‡ Daryl F. Venable,†,‡ Ryan D. Morrison,†,‡
Michael Bubser,†,‡ J. Scott Daniels,†,‡ Carrie K. Jones,†,‡ P. Jeffrey Conn,†,‡ and Craig W. Lindsley*,†,‡,§
‡
†Vanderbilt Center for Neuroscience Drug Discovery, Department of Pharmacology, Vanderbilt University Medical Center,
Nashville, Tennessee 37232 United States
§Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232 United States
S
* Supporting Information
ABSTRACT: Schizophrenia is a complex and highly heterogeneous psychiatric disorder whose precise etiology remains elusive.
While genome-wide association studies (GWAS) have identified risk genes, they have failed to determine if rare coding single
nucleotide polymorphisms (nsSNPs) contribute in schizophrenia. Recently, two independent studies identified 12 rare,
deleterious nsSNPS in the GRM1 gene, which encodes the metabotropic glutamate receptor subtype 1 (mGlu1), in schizophrenic
patients. Here, we generated stable cell lines expressing the mGlu1 mutant receptors and assessed their pharmacology. Using both
the endogenous agonist glutamate and the synthetic agonist DHPG, we found that several of the mutant mGlu1 receptors
displayed a loss of function that was not due to a loss in plasma membrane expression. Due to a lack of mGlu1 positive allosteric
modulators (PAM) tool compounds active at human mGlu1, we optimized a known mGlu4 PAM/mGlu1 NAM chemotype into a
series of potent and selective mGlu1 PAMs by virtue of a double “molecular switch”. Employing mGlu1 PAMs from multiple
chemotypes, we demonstrate that the mutant receptors can be potentiated by small molecules and in some cases efficacy restored
to that comparable to wild type mGlu1 receptors, suggesting deficits in patients with schizophrenia due to these mutations may
be amenable to intervention with an mGlu1 PAM. However, in wild type animals, mGlu1 negative allosteric modulators (NAMs)
are efficacious in classic models predictive of antipsychotic activity, whereas we show that mGlu1 PAMs have no effect to slight
potentiation in these models. These data further highlight the heterogeneity of schizophrenia and the critical role of patient
selection strategies in psychiatric clinical trials to match genotype with therapeutic mechanism.
chizophrenia, schizo-affective disorder, and bipolar disorder
genes (e.g., DISC1, COMT),14,15 single-nucleotide polymor-
Sare heterogeneous, heritable psychiatric diseases with phisms (SNPs), rare de novo copy number variations (CNVs),
and increased deletions and/or duplications at coding
regions.16−20 Recently, Frank and co-workers21 examined rare
coding single nucleotide polymorphisms (nsSNPs), not
detected by CNV scans and SNP arrays, as possible
contributors to psychiatric disease. DNA sequencing of 10
hub genes, from both schizophrenics and controls, identified a
cluster of disease only nsSNPs, with the most significant being
significant overlap in terms of genetic origins and clinical
presentation. The exact etiology of the disease is mired in
complex receptor and circuitry dysfunction arising from
multiple genetic and environmental factors.1−6 Two dominant
hypotheses have guided therapeutic development: the “dop-
amine hypothesis” and the “NMDA receptor hypofunction
hypothesis”;1−11 however, these receptors are also functionally
linked through sophisticated signaling complexes by scaffolding
proteins, thus connecting the two, seemingly distinct,
approaches.12,13 Moreover, genome-wide association studies
(GWAS) of schizophrenia have identified a number of risk
Received: July 14, 2014
Accepted: August 19, 2014
Published: August 19, 2014
© 2014 American Chemical Society
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GRM1, the gene encoding the metabotropic glutamate receptor
subtype 1 (mGlu1); importantly, this is the first study
genetically linking mGlu1 with schizophrenia.21 A subsequent
study by Ayoub et al.22 confirmed the presence of deleterious
GRM1 mutations in schizophrenia but also found that families
with these mutations were also affected by multiple neuro-
psychiatric conditions, such as depression, anxiety, drug abuse,
and epilepsy. The nsSNP mutations identified (Figure 1) were
“double molecular switch”27 of an mGlu4 PAM chemotype.28
The ligands developed show improvements over the one
existing mGlu1 PAM tool compound and enabled in vivo studies
in a preclinical antipsychotic model. Results from these studies
further emphasize the importance of patient selection for highly
heterogeneous diseases such as schizophrenia.
RESULTS AND DISCUSSION
■
Pharmacological Characterization of the Schizo-
phrenic mGlu1 Mutant Receptors. Earlier work had
employed transiently transfected cell lines21,22 and assessed
functional activity based on quisqualate-induced PI hydrolysis, a
pathway known to be subject to ligand bias.29 In order to fully
characterize the pharmacology of the schizophrenic (Sz) mGlu1
mutant receptors, we prepared stable cell lines carrying
tetracycline-inducible human mGlu1 wild-type (WT) and
each of the nine mutations identified in schizophrenics
(T548M, K653N, L575V, F122L, Y632H, A683E, P729T,
P1014S, and P1015A) (Figure 1) in T-REx HEK-293 cells. As
alluded to above, ligand bias and probe-dependence of allosteric
modulator effects at mGlu1 suggest different modes of receptor-
agonist interactions;29,30 therefore, to accurately assess the
pharmacology of the nine schizophrenia mutants, we employed
both the endogenous agonist glutamate, as well as the synthetic
orthosteric agonist (S)-3,5-dihydroxyphenylglycine (DHPG).
Figure 2 highlights the calcium response of the WT and nine
mutant human mGlu1 receptors upon stimulation with
increasing concentrations of either glutamate or DHPG.
When glutamate was used as the agonist (Figure 2A and B),
all nine of the mutant mGlu1 receptors displayed a loss of
function, as judged by diminished calcium response, relative to
mGlu1 WT (mutants typically induced 45−65% of the WT
response with the exception of K563N and P1015A, at ∼90%
of WT response). With the synthetic group I mGlu agonist
DHPG (Figure 2C and D), once again, the majority of mutant
mGlu1 receptors display a loss of function relative to mGlu1
WT (typically 55−75% of WT response); however, K563N
displays a slight gain of function (∼110% of WT), and P1015A
only exhibits a slight diminution in activity (∼90% of WT
response), suggesting some role for ligand bias across the
mutants. Combined with the quisqalate-induced PI hydrolysis
data, it is clear that the mutant mGlu1 receptors generally
display a loss of function across multiple agonists, and signaling
pathways. It is possible that this loss of functional response was
due to reduced receptor expression at the cell membrane. As
mentioned earlier, data in transiently transfected mutant COS-7
cell lines using an ELISA-based read-out with mGlu1 antibodies
revealed that the majority of mutants retained plasma
membrane expression comparable to WT mGlu1.21,22 We
employed an independent assessment for plasma membrane
expression of the nine mutants using our stable cell lines and a
complementary method. Here, we biotinylated the cell surface
of the nine mutant receptor-expressing and WT mGlu1 cell
lines, followed by capture with streptavidin resin and standard
Western blotting with an mGlu1-specific antibody. As shown in
Figure 3, none of the mutants displayed reduced cell surface
expression relative to WT mGlu1, and, in fact, several displayed
increased expression, despite a loss of calcium signaling. Again,
the combined data strongly indicate that the nine mutant
human mGlu1 receptors are loss-of-function mutations with no
loss of plasma membrane/cell surface expression.
Figure 1. Representative (9 of 12) non-synonymous single nucleotide
polymorphisms (nsSNPs) in mGlu1 found in schizophrenia patients
that are evaluated in this study. Schematic structure and snake plot of
mGlu1 are presented. nsSNPs are shown in red circles at their
approximate location. VFTD: venus flytrap domain. CRD: cysteine-
rich domain. TMD: transmembrane domain.
predicted to have deleterious effects on receptor function, and
preliminary data with a functional quisqualate phosphoinositide
hydrolysis (IP1) assay suggested that, in transiently transfected
COS-1 cells, receptors encoding these mutations displayed loss
of function with no to minimal loss in plasma membrane
expression using ELISA with an anti-mGlu1 antibody.21,22 Prior
to these studies, mGlu1 was not regarded as a major
schizophrenia target, as opposed to its associated family
member mGlu5;23 however, observations from post-mortem
schizophrenic brains (increased expression of mGlu1 due to
NMDA hypofunction), sensorimotor gating deficits (PPI) in
GRM1 knockout mice, and the known role in both potentiating
NMDA receptor function as well as synaptic plasticity have
raised the possibility that mGlu1 may also play a role.24−26
Based on our longstanding interest in schizophrenia, we
pursued a more detailed pharmacological characterization of
nine of the 12 mutant mGlu1 receptors derived from these
deleterious GRM1 mutations found in schizophrenics, and
demonstrated that the mutations are indeed loss of function
mutations that can be rescued with novel, highly selective
mGlu1 positive allosteric modulators (PAMs), derived from a
Development of Novel mGlu1 PAMs. Chemical modu-
lation of mGlu1, much like mGlu5, cannot be effectively
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Figure 2. Calcium signaling induced by mGlu1 receptors encoding mutations associated with schizophrenia is reduced compared to WT. The
functional activity of the mGlu1 schizophrenia (Sz) mutant receptors was examined in Ca mobilization assay. Stable cell lines carrying tetracycline-
inducible mGlu1 WT or Sz mutants in T-REx-293 cells were incubated with 20 ng/mL or 1 μg/mL TET for WT and all mutants, respectively,
overnight to induce mGlu1 expression. Cells were stimulated with the increasing amounts of pan mGlu receptor agonist, glutamate (A and B), or the
group I mGlu-selective agonist, DHPG (C and D). The concentration−response curves were generated by normalizing the calcium responses to the
% of the WT maximal response of each agonist. Data represent at least three independent experiments performed in duplicates.
mediated by orthosteric, “glutamate-like” agonists in vivo, due
to the liability of excitotoxicity and seizures as a side
effect.23,31−33 In the case of mGlu5, these concerns have been
shown to be mitigated by mimicking physiological conditions
and potentiating the receptor with a positive allosteric
modulator (PAM). A multitude of mGlu5 PAMs have been
reported across literally dozens of chemotypes targeting
multiple, distinct allosteric binding sites with robust in vivo
efficacy and safety margins.23 However, mGlu1 is far less
developed, and only five mGlu1 PAMs have been reported
(Figure 4).31−33 Knoflach and co-workers from Roche
pioneered the field in the early 2000s,34 developing the first
four mGlu1 PAMs, 1 (Ro 01-6128), 2 (Ro 67-4853), 3 (Ro 67-
7476), and 5 (Ro 07-11401)35,36 with EC50s in the 56 to 200
nM range; however, only 2 and 5 were active at both human
and rat mGlu1, highlighting a serious concern with species
differences that was only recently understood in the studies
characterizing the mGlu1 NAM tool compounds.30−36 In
addition, the drug metabolism and pharmacokinetic (DMPK)
profile of 5 was not optimal for in vivo studies (vide infra).
Conn and co-workers later identified (VU-71) 4, derived from
the prototypical mGlu5 PAM chemotype, as a weak rat mGlu1
PAM (EC50 = 2.4 μM) with no activity at the human
receptor.37 In contrast, a wealth of mGlu1 NAMs have been
reported (6−10, Figure 4), with a range of species/ligand bias
that can be used to explore the ability of NAMs to
noncompetitively inhibit the mutant receptors.31,32 Again, to
fully evaluate the ability of small molecules to modulate the
mutant mGlu1 receptors, we needed to develop mGlu1 PAMs
in alternative chemotypes, beyond 5, to ensure caveats with
species differences and ligand bias did not preclude accurate
interpretations and mask physiological responses.
In lieu of an mGlu1 PAM high throughput screen (HTS), we
pursued several avenues to identify novel mGlu1 PAMs. A
multidimensional, iterative parallel synthesis effort was
employed to synthesize and screen several hundred analogs
of 4, resulting in little tractable SAR and no improvement in rat
mGlu1 PAM potency, and with no gain of human mGlu1
activity; thus, this approach was discontinued. In parallel, we
considered the phenomenon of “molecular switches” within
allosteric ligands,27 wherein subtle structural modifications can
modulate modes of ligand pharmacology and/or subtype
selectivity. Historically, there was evidence for cross-talk
between the allosteric sites on the group III mGlu receptor,
mGlu4 and the group I mGlu receptor, mGlu1 (Figure 5).38
The first mGlu4 PAM, (−)-PHCCC (11, EC50 = 1.4 μM) was
highly selective against all the mGlu subtypes except mGlu1,
where it was an equipotent mGlu1 NAM (IC50 = 2.1 μM).
Incorporation of a “molecular switch” in the form of aza-
congener 12 (VU0359516) not only enhanced PAM activity at
mGlu4 (EC50 = 380 nM) but also abolished activity at mGlu1
(IC50 > 30 μM).38 This observation led us to mine mGlu
selectivity data from our mGlu4 PAM program, where we had
prepared hundreds of analogs around 13 (VU0400195,
ML182), a potent and highly selective mGlu4 PAM (EC50
=
291 nM).28 This exercise led to the development of a highly
selective mGlu1 NAM 14 (VU0465334) within this chemotype
(mGlu1 IC50 = 220 nM, >10 μM versus mGlu4) via a
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mGlu1 and mGlu4. Functionalized picolinamides 19g−19o
were uniformly active at both mGlu1 species, yet abolished
activity at mGlu4 (EC50s >10 μM). Subsequent libraries held
the 3-methyl picolinamide (19k) or 3-chloropicolinamide (19l)
constant and surveyed alternative phthalimide moieties,
producing analogs 23 and 24, respectively, as human mGlu1
PAMs (Table 2). Of these analogs, incorporation of a
chlorophthalimide proved optimal, providing 23c
(VU0483737) and 24c (VU0483605) (Figure 6). 23c
(VU0483737) is a potent mGlu1 PAM at both human (EC50
= 370 nM) and rat (EC50 = 367 nM, pEC50 = 6.43 0.13, 118
7% Glu Max) and a very low efficacy mGlu4 PAM (EC50
=
199 nM, 55% Glu Max). 24c (VU0483605) proved superior
with excellent mGlu1 PAM activity at both human (EC50 = 390
nM) and rat (EC50 = 356 nM, pEC50 = 6.45 0.11, 113 5%
Glu Max) and no activity as an mGlu4 PAM (EC50 >10 μM).
Thus, data mining, coupled with identification of a double
“molecular switch”, enabled the development of two novel
mGlu1 PAMs, equipotent against both human and rat mGlu1,
and selective against mGlu4, despite originating from a potent
and highly selective mGlu4 chemotype. These two new
compounds 23c and 24c, along with the Roche mGlu1 PAM
5 and a cadre of mGlu1 NAMs, afforded the requisite tools to
explore chemical modulation of the schizophrenic mGlu1
mutant receptors.
Small Molecule Modulation of the Schizophrenic
mGlu1 Mutant Receptors. As the mutant receptors displayed
a loss of function in our standard calcium mobilization assay
(Figure 2), we evaluated the ability of the mGlu1 PAMs 5, 23c,
and 24c to potentiate the response of an increasing
concentration of either glutamate (Figure 7) or the synthetic
agonist DHPG (Figure 8) in the stable mGlu1 WT and mutant
cell lines. Figure 7A shows that all three PAMs potentiate WT
mGlu1, inducing a parallel left-ward shift of the glutamate
concentration response curve (CRC), with 5 and 24c showing
7.4- and 6.3-fold shifts respectively at 10 μM, while 23c was
only ∼3-fold. Each of the nine mutants is profiled in Figure 7B-
J, and all three PAMs are able to potentiate the decreased
calcium response in the mGlu1 mutants, and in the case of
K563N and P1015A mutants, which show minimal decreases in
calcium signaling, efficacy (%Glu Max) was also restored to the
level of WT mGlu1. As expected based on the data in Figure 2,
the three PAMs exhibit a more robust potentiation of DHPG
across both WT and mutant mGlu1 receptors (Figure 8). Here,
5 displays a 16.6-fold shift at WT mGlu1, whereas 24c was 6.6-
fold and 23c was 3.4-fold. Once again, across all the mutant
mGlu1 receptors, the three PAMs potentiate the decreased
calcium response. Hence, it is clear that the mutant mGlu1
receptors can be chemically modulated with small molecule
mGlu1 PAMs to partially restore decreased calcium responses.
These data suggest that chemical intervention with mGlu1
PAMs in schizophrenic patients harboring these mutations
could potentially be beneficial.
Figure 3. Decreased calcium signaling found in the mGlu1 mutants
associated with schizophrenia is not due to a loss of plasma membrane
receptor expression. Biotinylation of cell surface membrane receptors
was performed to compare the expression levels of the mutant
receptors with the WT receptor. The day before biotinylation, cells
expressing mGlu1 WT and Sz mutants were treated in the same
manner as shown in the calcium mobilization assay. Biotin labeling of
the cell surface receptors was performed at 4 °C, and the biotin-labeled
proteins were captured by streptoavidin resins. The streptoavidin-
bound receptors were solubilized in a SDS sample buffer containing
150 mM dithiothreitol, and then, the proteins were resolved by 8%
SDS-PAGE. The mGlu1 expression was detected by Western blotting
using an mGlu1-specific antibody (clone 20, BD transduction
Laboratories). Note that none of the Sz mutant receptors expressed
at lower levels than the WT regardless of the calcium response shown
in Figure 2. Data are represented as % of WT cell surface expression
from four independent experiments (Mean
SEM), two of which
were concomitantly performed with the calcium assay.
“molecular switch” modification to the imide moiety.30 Further
mining identified 15 (VU0405623), possessing a substituted
phthalimide moiety with potent, dual PAM activity at both
mGlu4 (EC50 = 61 nM) and mGlu1 (EC50 = 75 nM), in essence
a double “molecular switch”, and a lead compound from which
to develop a new, selective mGlu1 PAM chemotype, distinct
from Roche’s 5,34−36 if mGlu4 PAM activity could be
eliminated.
Analogs of 15 were readily prepared in three synthetic steps
from commercial 16a (Scheme 1) following a divergent plan. A
diversity library was developed by first treating 16 with phthalic
anhydride to generate phthalimide 17 in 88% yield. Reduction
of the nitro group and acylation under standard HATU
conditions provided amide analogs 19 (structure−activity
relationship (SAR) presented in Table 1), exploring diverse
moieties, in good overall yields. Once the optimal amide
moieties were identified in 19, further analogs 22 were accessed
by acylation of 16b with picolinic acids under HATU
conditions to deliver 20. Subsequent nitro reduction and
condensation with different phthalic anhydrides afforded
functionalized phthalimide congeners 22 (SAR presented in
Table 2), in good overall yields for the three step sequence.
As shown in Table 1, new analogs of 19 were screened
against human and rat mGlu1 as well as hmGlu4, generating
significant, tractable SAR with ideal compounds showing
equipotent activity at both human and rat mGlu1 to support
future translational studies. The pyridyl isomers 19a−19c
demonstrated that the 2-position/picolinamides (19a) was
essential for activity, with the 3- (19b) and 4-position (19c)
congeners inactive at both mGlu1 and mGlu4. Interestingly,
pyridazine congeners 19e and 19f were inactive, whereas the
pyrazine derivative 19d retained activity across human and rat
Finally, to complete the pharmacological characterization of
the mutant mGlu1 receptors, we examined the effect of an
mGlu1 NAM in high efficacy (K653N) and moderate efficacy
(A683E) mGlu1 mutant lines and compared it then to WT
mGlu1 (Supporting Information Figure 1). Cells were pre-
treated with 1 μM of potent mGlu1 NAM 7, and immediately,
cells were stimulated by glutamate or DHPG. In all cases,
noncompetitive antagonism (decrease in % Glu Max) was
noted, highlighting the fact that the mutant receptors behave as
WT mGlu1 in the presence of an mGlu1 NAM. Thus, the
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Figure 4. Structures of the only known mGlu1 PAMs (1−5) and structures of representative mGlu1 NAMs (6−10). Due to the existence of limited
mGlu1 PAMs with appropriate species selectivity, ancillary pharmacology, and disposition profiles, the therapeutic potential of mGlu1 PAMs is vastly
unexplored. 1−3 were the first mGlu1 PAMs described, but only 2 and 5 are active at both human and rat mGlu1. The rat/human species disconnect
is a major issue within multiple mGlu1 allosteric modulator chemotypes.
mutant receptors found in schizophrenic patients are capable of
being modulated by both PAMs and NAMs, suggesting they
may be targets for therapeutic intervention with small
molecules.
versus CYP1A2, 2C9, 2D6, and 3A4). In contrast, 5 possessed
high hepatic clearance in both rat (CLhep 63.3 mL/min/kg) and
human (CLhep 19.4 mL/min/kg) and a mixed CYP profile
(IC50 > 30 μM versus CYP2C9 and 3A4, 1A2 IC50 = 13 μM,
2C9 IC50 = 0.6 μM). Moreover, in a single point plasma/brain
level study (100 mg/kg i.p. at t = 1.25 h), 24c was highly brain
penetrant with a brain/plasma ratio (Kp) of 0.84, while 5 was
modest with a brain/plasma ratio (Kp) of 0.19. Thus, these are
acceptable first generation mGlu1 PAM tool compounds for in
vivo studies. These findings highlight the advantage of
“molecular switches”27 in effectively scaffold hopping within
allosteric chemotypes to an advanced lead for another receptor
subtype and opposing mode of pharmacology.
Historically, mGlu1 NAMs have shown efficacy in multiple
preclinical models that predict antipsychotic efficacy in WT
mice and rats. We confirmed this in house, by evaluating the
mGlu1 NAM 7 in an amphetamine-induced hyperlocomotion
(AHL) model (Supporting Information Figure 2). At doses of
10 and 30 mg/kg i.p., 7 completely reversed AHL. After a
detailed perusal of the literature, it appeared that the activity of
mGlu1 PAMs had never been reported in this model.
Therefore, we evaluated 24c in WT rats at doses up to 100
Preparation for Translational Studies with Schizo-
phrenic mGlu1 Mutant Receptors. In preparation for future
translational studies beyond cells and into animal models, we
first needed to understand if the mutations in human mGlu1
aligned with rodent mGlu1. Fortunately, all of the human
mutation sites in mGlu1 are sequence aligned with rat mGlu1
with a single exception; K563 is R563 in both rat and mouse
mGlu1 (Supporting Information Table 1). Thus, a future effort
will focus on developing and characterizing mutant mGlu1
mice. Based on these favorable data, we then assessed the
DMPK profiles of the three mGlu1 PAMs that were capable of
modulating the mutant mGlu1 receptors. In general (Support-
ing Information Table 2), both 23c and 24c displayed
improved disposition profiles relative to 5 in terms of both
intrinsic clearance and cytochrome P450 (CYP) inhibition
profiles. For example, 24c possessed moderate hepatic
clearance in both rat (CLhep 25.4 mL/min/kg) and human
(CLhep 9.46 mL/min/kg) and clean CYP profile (IC50 > 30 μM
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Figure 5. “Molecular switches” modulate mGlu subtype selectivity and mode of pharmacology. The first mGlu4 PAM, (−)-PHCCC (11), possessed
mGlu1 NAM activity, suggesting cross-talk between the allosteric sites on mGlu1 and mGlu4. A subtle molecular switch in the form of an aza-analog,
12, abolished activity at mGlu1 and improved mGlu4 PAM activity. An unrelated, highly selective mGlu4 PAM chemotype, 13, was subject to a
“molecular switch” by virtue of modification of the succinimide moiety, yielding a selective mGlu1 NAM 14. Based on these observations, we
screened our mGlu4 PAM library in an effort to identify a new mGlu1 PAM scaffold, and identified the phthalimide congener 15 as a dual mGlu1/
mGlu4 PAM with comparable potency, via a “double” molecular switch. Extensive SAR within 13−14 demonstrated that it was possible to dial-out
mGlu4 activity; thus, 15 served as a new lead upon which to develop a novel and selective mGlu1 PAM to profile against the Sz mutants.
a
Scheme 1. Synthesis of N-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)amides 19 and 22
a
Reagents and conditions. (a) phthalic anhydride, AcOH, reflux, 88%; (b) SnCl2, HCl, dioxane, rt, 72−93%; (c) carboxylic acids, HATU, DIEA,
DCM, rt, 45−90%; (d) picolinic acids, HATU, DIEA, μW 120 °C DCM, 75−78%; (e) phthalic anhydrides, μW 200 °C, N-methyl-2-pyrrolidone,
55−78%.
mg/kg i.p. and observed no effect up to the highest dose; in
contrast, a slight potentiation of amphetamine was noted in the
presence of good CNS exposure. Both 5 and 23c behaved
similarly. A key question that remains involved the potential for
antipsychotic activity of an mGlu1 PAM in genetic animals
possessing the mutant mGlu1 receptors? This further under-
scores the challenges in developing novel, single mechanism
antipsychotic agents to be clinically tested for efficacy in a
highly heterogeneous patient population. From these data, an
mGlu1 PAM would be expected to have no effect on patients
with WT mGlu1, but might give a signal in patients carrying the
mutant mGlu1 receptors.
glutamate, and the synthetic agonist DHPG, we demonstrate
that the mutant mGlu1 receptors display reduced calcium
signaling that does not result from a loss in plasma membrane
expression. Based on a lack of mGlu1 positive allosteric
modulators (PAM) tool compounds, we optimized a known
dual mGlu4 PAM/mGlu1 NAM chemotype into a series of
potent and selective mGlu1 PAMs by virtue of a double
“molecular switch”. Employing mGlu1 PAMs from multiple
chemotypes, we demonstrate that the mutant receptors can be
potentiated by small molecules, and in some cases efficacy
restored to that comparable to WT mGlu1 receptors, suggesting
schizophrenic patients harboring these mutations may be
amenable to intervention with an mGlu1 PAM. However, data
generated in WT animals in AHL studies further highlight the
heterogeneity of schizophrenia and the critical role of patient
selection in psychiatric clinical trials to match genotype with
In summary, we expressed each of the nine mGlu1 mutant
receptors, identified as GRM1 mutations associated with
schizophrenia, into stable cell lines and assessed their
pharmacology. Employing both the endogenous agonist
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Table 1. Structures and Activities for Selected Picolinamide Analogs 19
a
Calcium mobilization mGlu1 and mGlu4 assays; values are average of n = 3 performed in triplicate.
Cell Culture and Mutagenesis. Tetracycline-inducible human
mGlu1 WT-T-REx-293 cells39 were cultured at 37 °C in Dulbecco’s
Modified Eagle Medium (DMEM) growth medium containing 10%
Tet-tested FBS, 2 mM L-glutamine, 20 mM HEPES, 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, antibiotic/
antimycotic, 100 μg/mL hygromycin, and 5 μg/mL blasticidin in the
presence of 5% CO2. To generate a collection of stable cell lines
carrying tetracycline-inducible hmGlu1 schizophrenic mutants,21,22
site-directed mutagenesis of human mGlu1 WT in pcDNA5/TO was
performed using Quikchange II XL kit (Agilent Technologies, Santa
Clara, CA), and all point-mutations were confirmed by sequencing.
The mutant stable cell lines were generated in the same manner as the
WT as previously described and cultured in the growth medium
described above.
therapeutic mechanism. Future efforts will examine mGlu1
mutants harboring multiple mutations, and assessing the ability
of mGlu1 PAMs to modulate pharmacological responses.
METHODS
■
Molecular Pharmacology. Tetracycline-tested fetal bovine serum
(FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA),
and all other tissue culture reagents and Fluo-4-acetoxymethylester
(Fluo-4-AM) were purchased from Life Technologies (Carlsbad, CA).
Tetracycline hydrochloride (Sigma), L-glutamic acid (Tocris, Minne-
apolis, MN), and (S)-3,5-dihydroxyphenylglycine (DHPG) (Abcam,
Cambridge, MA). EZ-Link Sulfo-NHS-SS-Biotin and NeutrAvidin
agarose beads (Pierce Biotechnology, Rockford, IL).
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La Jolla, CA). To determine the potency of mGlu1 PAMs in calcium
assays, all procedures are performed in a similar manner to the ones
described above with the following modifications. Briefly, rat and
human mGlu1 WT-T-REx-293 cells were plated in poly-D-lysine coated
96-well plates at 80 000 cells/100 μL assay medium containing 10 ng/
mL and 50 ng/mL TET, respectively. After incubating cells with the
Fluo-4-AM dye solution, calcium flux was measured using Flexstation
II (Molecular Devices, Sunnyvale, CA). Compounds serially diluted at
half log concentrations in DMSO were further diluted in assay buffer.
The compounds or DMSO vehicle were added to cells and incubated
for 2.5 min, and an EC20 concentration of glutamate was added and
incubated for 1 min. An ECmax concentration of glutamate was also
added to cells that were incubated with DMSO vehicle to accurately
calculate the EC20 calcium response. Data were normalized by
subtracting the basal florescent peak before EC20 agonist addition from
the maximal peak elicited by EC20 agonist and PAMs. Using GraphPad
Prism 5.0, the concentration response curves were generated and the
potencies of the mGlu1 PAMs were determined.
Biotinylation of Cell Surface Expression of mGlu1 Receptor.
To determine the receptor expression level on the cell surface among
the mGlu1 WT and schizophrenia (Sz) mutant cell lines, cells were
plated in a 10 cm dish at 8 × 106 cells/8 mL density in the assay
medium containing either 20 ng/mL (WT) or 1 μg/mL (mutants)
TET to induce the receptor expression. The next morning, dishes were
placed on ice and gently washed with ice cold PBS. Immediately, cells
were scraped in cold PBS into a 1.5 mL tube, and spun down at 500g
for 4 min at 4 °C. All steps were carried at 4 °C unless otherwise
noted. Cells were gently resuspended in 1 mL of cold PBS containing
2 mg of EZ-Link Sulfo-NHS-SS-Biotin and gently rocked for 30 min.
Biotinylation was quenched by adding 500 uL of 150 mM Tris, pH 8.0,
and cells were spun down and resuspended in 1 mL of 50 mM Tris,
pH 8.0 to completely quench the reaction. Cells were spun down and
resuspended in 1 mL of lysis buffer (25 mM Tris, pH 7.4, 150 mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate containing protease
inhibitors) and incubated for 30 min. The lysates were spun at 16 000g
for 15 min, and the resulting supernatants were gently transferred to a
new 1.5 mL tube to incubate with 60 μL of 50% slurry of NeutrAvidin
beads by nutating at room temperature (RT) for 1 h. The beads were
washed three times with lysis buffer and the biotinylated proteins
bound to the beads were extracted by heating the beads at 65 °C for 5
min in SDS sample buffer containing 150 mM DTT. The proteins
were resolved by 8% SDS-PAGE and the mGlu1 expression level was
determined by Western blotting using the mGlu1−specific antibody
(Clone 20, BD Transduction Lab). The density of the mGlu1 protein
bands was measured using Quantity One software (Bio-Rad, Hercules,
CA).
Table 2. Structures and Human mGlu1 Activity for Selected
Substituted Phthalimide Analogs 23 and 24
a
EC50
pEC50
( SEM)
% Glu max
( SEM)
compd.
Ri
R2
(μM)
23a
24a
23b
24b
23c
24c
23d
24d
23e
24e
23f
H
CH3
0.176
0.177
>10.0
0.832
0.372
0.391
2.02
6.75 0.14
6.75 0.19
<5.00
101
7
110 21
130 26
CH3
H
H
CI
H
F
6.08 0.07
6.43 0.01
6.41 0.08
5.69 0.05
5.73 0.15
6.38 0.05
5.94 0.09
<5.00
82
4
126 25
110 15
92 11
CI
H
1.84
86
6
0.418
1.14
90 15
117
128
9
5
F
H
>10.0
3.88
24f
5.41 0.12
a
Calcium mobilization mGlu1 assays; values are average of n = 3,
performed in triplicate.
Figure 6. Novel mGlu1 PAMs derived from a double “molecular
switch” of an mGlu4 PAM/mGlu1 NAM preferring chemotype.
Calcium Mobilization Assay. To examine the functional activity
of all hmGlu1 mutants compared to the WT, calcium flux was
measured using the Functional Drug Screening System (FDSS7000,
Hamamatsu, Japan), as previously described.39 Briefly, the day before
the assay, the mGlu1-T-REx-293 cells were plated in black-walled,
clear-bottomed, poly-D-lysine coated 384-well plates (BD Biosciences,
San Jose, CA) at 20 000 cells/well in 20 μL of assay medium (DMEM
supplemented with 10% dialyzed FBS, 20 mM HEPES, and 1 mM
sodium pyruvate) that contains tetracycline (TET) to induce mGlu1
expression; 20 ng/mL TET was used for WT and 1 μg/mL TET for
all mutants. The next day, cells were washed with assay buffer (Hank’s
balanced salt solution, 20 mM HEPES, and 2.5 mM probenecid) using
ELx microplate washer (BioTek Instruments, Winooski, VT) and
immediately incubated with 20 μL of 1.15 μM Fluo-4 AM dye solution
prepared in assay buffer for 45 min at 37 °C. The dye was removed
and washed with assay buffer before measuring the florescent calcium
traces in FDSS. Agonists, glutamate or DHPG, were 1:5 serially diluted
into 11 point concentrations, added to cells, and incubated for 2 min.
The agonist-mediated calcium response was calculated by subtracting
the basal florescent peak before agonist addition from the maximal
peak elicited by agonist. When testing the effect of the mGlu1 PAMs,
cells were first incubated with the PAMs (10 μM final concentration)
or DMSO vehicle diluted in assay buffer for 2.2 min and then
stimulated with glutamate or DHPG. Concentration response curves
were generated using GraphPad Prism 5.0 (GraphPad Software, Inc.,
DMPK Methods. In Vitro. The in vitro DMPK assays, including
those assessing plasma protein binding (PPB), brain homogenate
binding (BHB), hepatic microsomal intrinsic clearance (CLint), and
cytochrome P450 inhibition, were performed as described previ-
ously.40 A potassium phosphate-buffered reaction mixture (0.1 M, pH
7.4) of test article (1 μM) and microsomes (0.5 mg mL−1) was
preincubated (5 min) at 37 °C prior to the addition of NADPH (1
mM). The incubations, performed in 96-well plates, were continued at
37 °C under ambient oxygenation and aliquots (80 μL) were removed
at selected time intervals (0, 3, 7, 15, 25, and 45 min). Protein was
precipitated by the addition of chilled acetonitrile (160 μL), containing
glyburide as an internal standard (50 ng/mL), and centrifuged at 3000
rpm (4 °C) for 10 min. Resulting supernatants were transferred to new
96-well plates in preparation for LC/MS/MS analysis. The in vitro
half-life (t1/2, min, eq 1), intrinsic clearance (CLint, mL/min·kg, eq 2)
and subsequent predicted hepatic clearance (CLhep, mL/min·kg, eq 3)
was determined employing the following equations:
t1/2 = Ln(2)/k
(1)
where k represents the slope from linear regression analysis (% test
article remaining) vs time
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Figure 7. Treatment of mGlu1 positive allosteric modulators partially restores the reduction in the glutamate-mediated calcium signaling in the Sz
mutants. To examine if mGlu1 PAM can potentiate the reduced calcium signaling induced by the Sz mutants, stable cells expressing the mGlu1 WT
and Sz mutants were incubated with 10 μM of Ro 07-11401 (5), VU0483605 (24c), VU0483737 (23c), or DMSO matched vehicle for 2.2 min, and
immediately stimulated with the increasing concentration of glutamate. (A) mGlu1 PAMs induce a leftward fold shift of the glutamate
concentration−response curve in cells expressing the mGlu1 WT receptor. Compared to an 870 nM EC50 for in the DMSO vehicle condition, both
Ro 07-11401 (5) and VU0483605 (24c) shifted the glutamate EC50 to 118 nM and 139 nM (7.4- and 6.3-fold shift, respectively) and showed a slight
increase in maximal glutamate response. VU0483737 (23c) displayed a lesser fold shift, shifting the EC50 to 294 nM. (B−J) All three mGlu1 PAMs
were able to potentiate the decreased glutamate-induced calcium response in the Sz mutants tested. Both Ro 07-11401 (5) and VU0483605 (24c)
showed comparable fold shifts off all mutant responses; VU0483737 (23c) showed a lesser effect. Data shown was generated by normalizing the
calcium responses to the % maximal glutamate-elicited response at the WT receptor in the presence of DMSO vehicle. Data represent four
independent experiments performed in duplicate.
Single-Use RED Plates with inserts (ThermoFisher Scientific,
CLint = (0.693/t1/2) (rxn vol./mg microsomes)
Rochester, NY). Briefly, plasma (220 μL) was added to the 96 well
× (45 mg microsomes/g of liver) (20 g of liver/kg body weight)
with scale-up factors of 20 (human) and 45 (rat)
Q × CLint
(2)
plate containing test article (5 μL) and mixed thoroughly.
Subsequently, 200 μL of the plasma-test article mixture was transferred
to the cis chamber (red) of the RED plate, with an accompanying 350
μL of phosphate buffer (25 mM, pH 7.4) in the trans chamber. The
RED plate was sealed and incubated 4 h at 37 °C with shaking. At
completion, 50 μL aliquots from each chamber were diluted 1:1 (50
μL) with either plasma (cis) or buffer (trans) and transferred to a new
96 well plate, at which time ice-cold acetonitrile (2 volumes) was
added to extract the matrices. The plate was centrifuged (3000 rpm, 10
min) and supernatants transferred to a new 96 well plate. The sealed
plate was stored at −20 °C until LC/MS/MS analysis.
Liquid Chromatography/Mass Spectrometry Analysis. In
Vivo Experiments. mGlu1 PAMs were analyzed via electrospray
ionization (ESI) on an AB Sciex API-4000 (Foster City, CA) triple-
quadrupole instrument that was coupled with Shimadzu LC-10AD
pumps (Columbia, MD) and a Leap Technologies CTC PAL
autosampler (Carrboro, NC). Analytes were separated by gradient
CLhep
=
Q + CLint
(3)
In Vivo. Male Sprague−Dawley rats (n = 2) weighing around 300 g
were purchased from Harlon laboratories (Indianapolis, IN) and
implanted with catheters in the carotid artery and jugular vein. The
cannulated animals were acclimated to their surroundings for
approximately 1 week before dosing and provided food and water
ad libitum. Samples were collected into chilled, EDTA-fortified tubes,
centrifuged for 10 min at 3000 rpm (4 °C), and resulting plasma
aliquoted into 96-well plates for LC/MS/MS analysis. All
pharmacokinetic analysis was performed employing noncompartmen-
tal analysis.
Plasma Protein Binding. Protein binding of the mGlu1 PAMs
were determined in rat plasma via equilibrium dialysis employing
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Figure 8. Potentiating effect of mGlu1 positive allosteric modulators reveal a probe-dependency in the DHPG-mediated Ca signaling. Stable cells
expressing the mGlu1 WT and Sz mutants were incubated with 10 μM of Ro 07-11401 (5), VU0483605 (24c), VU0483737 (23c), or DMSO-
matched vehicle for 2.2 min, and then stimulated with increasing concentrations of DHPG, group I mGlu-specific agonist. (A) mGlu1 PAMs induce a
leftward fold shift of the DHPG concentration−response curve at WT mGlu1. Ro 07-11401 (5) exhibited a dramatic 16.6 fold shift, resulting in 159
nM of EC50 DHPG concentration compared to 2.5 μM when cells were treated with DMSO. Comparable to the fold shifts in glutamate response,
both VU0483605 (24c) and VU0483737 (23c) shifted the DHPG EC50 value to 402 nM and 783 nM (6.6 and 3.4 fold shift, respectively). (B−J) All
three mGlu1 PAMs were able to potentiate the decreased DHPG-mediated calcium response in the Sz mutants tested. Both VU0483605 (24c) and
VU0483737 (23c) showed comparable fold shifts of all mutant responses while Ro 07-11401 (5) showed a stronger effect. Data shown was
generated by normalizing the calcium responses to the % maximal DHPG-elicited response at the WT receptor in the presence of DMSO vehicle.
Data represent three independent experiments performed in duplicate.
elution using a Fortis C18 2.1 × 50 mm, 3.5 μm column (Fortis
Technologies Ltd., Cheshire, U.K.) thermostated at 40 °C. HPLC
mobile phase A was 0.1% NH4OH (pH unadjusted), mobile phase B
was acetonitrile. The gradient started at 30% B after a 0.2 min hold
and was linearly increased to 90% B over 0.8 min, held at 90% B for
0.5 min, and returned to 30% B in 0.1 min, followed by a re-
equilibration (0.9 min). The total run time was 2.5 min and the HPLC
flow rate was 0.5 mL/min. The source temperature was set at 500 °C
and mass spectral analyses were performed using multiple reaction
monitoring (MRM) utilizing a Turbo-Ionspray source in positive
ionization mode (5.0 kV spray voltage). All data were analyzed using
AB Sciex Analyst 1.4.2 software.
Amphetamine-Induced Hyperlocomotion. Animals. Adult
male Sprague−Dawley rats (Harlan, Indianapolis, IN) were used for
the behavioral studies. They were group-housed under a 12-h light/
dark cycle (lights on from 7 AM−7 PM) with food and water available
ad libidum. All animal procedures were performed in accord with the
guidelines set by the Guide for the Care and Use of Laboratory Animals
and were approved by the Vanderbilt University Animal Care and Use
Committee.
Drugs. All compounds were dissolved in 20% hydroxypropyl-β-
cyclodextrin (BCD), and if necessary, the solutions were adjusted to
pH 6−7 using 1 N NaOH; D-amphetamine hemisulfate (Sigma-
Aldrich, St. Louis, MO) was dissolved in sterile water.
In Vitro Experiments. The mGlu1 PAMs were analyzed similarly to
that described above (In vivo) with the following exceptions: LC/MS/
MS analysis was performed employing a TSQ QuantumULTRA that was
coupled to a ThermoSurveyor LC system (Thermoelectron Corp., San
Jose, CA) and a Leap Technologies CTC PAL autosampler (Carrboro,
NC). Chromatographic separation of analytes was achieved with an
Acquity BEH C18 2.1 × 50 mm, 1.7 μm column (Waters, Taunton,
MA).
Reversal of Amphetamine-Induced Hyperlocomotion. Locomotor
activity was assessed using SmartFrame open field activity chambers
(40.5 cm × 40.5 cm × 38 cm; Kinder Scientific, Poway, CA) that were
equipped with a 16 × 16 array of infrared photobeams.41 Rats were
habituated in the open field for 30 min and then pretreated with
vehicle (20% BCD in water) or VU0483605 (100 mg/kg). Thirty
minutes later, amphetamine (0.75 mg/kg free base) was administered
IP and locomotor activity was recorded for an additional hour. The
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time course of locomotor activity (number of beam breaks per 5 min
interval) over the whole 120 min testing session and total locomotor
activity (total beam breaks from the time of amphetamine
administration to the end of the study) are presented. Data are
powder. 1H NMR (400.1 MHz, CDCl3) δ (ppm): 10.18 (1H, s), 8.55
(1H, dd, J = 4.5 Hz, J = 1.4 Hz), 8.19 (1H, d, J = 2.3 Hz), 7.91 (2H,
m), 7.75 (3H, m), 7.48 (1H, dd, J = 8.2 Hz, J = 4.5 Hz), 7.33 (1H, d, J
= 8.6 Hz). 13CNMR (100.6 MHz, CDCl3) δ (ppm): 165.2, 164.2,
160.7, 145.8, 144.5, 141.1, 139.7, 136.1, 135.3, 133.9, 133.7, 132.9,
132.0, 130.8, 127.6, 127.2, 124.7, 122.4, 120.8, 118.4. HRMS (TOF,
ES+) C20H11Cl3N3O3 [M + H]+ calc. mass 445.9866, found 445.9869.
shown as means
SEM and statistical comparisons were made by
one- or two-way ANOVA using GraphPad Prism V5.04.
Chemistry Experimental. All commercial chemicals and solvents
were reagent grade and were used without further purification unless
otherwise specified. All reactions were carried out employing standard
chemical techniques under inert atmosphere. Analytical thin layer
chromatography was performed on 250 μm silica gel plates from
Sorbent Technologies was employed routinely to follow the course of
reactions. NMR spectra were recorded on a 400 MHz Bruker AV-400,
500 MHz Bruker DRX-500, and 600 MHz AV-II instruments. 1H
chemical shifts are reported as δ values in ppm relative to the residual
solvent peak (DMSO-d6 = 2.50, CDCl3 = 7.26). Data are reported as
follows: chemical shift, integration, multiplicity (s = singlet, d =
doublet, dd = double of doublet, t = triplet, q = quartet, m = multiplet)
and coupling constant (Hz). 13C chemical shifts are reported as δ
values in ppm relative to the residual solvent peak (DMSO-d6 = 39.52,
CDCl3 = 77.16). Analytical HPLC was performed on an Agilent 1200
LCMS with UV detection at 214 and 254 nm along with ELSD
detection. The purity of all tested compounds was greater than 98%
based on analytical HPLC. Preparative purification of library
compounds was performed on a Gilson 215 preparative LC system.
Low resolution mass spectra were obtained on an Agilent 1200 LCMS
with electrospray ionization. High resolution mass spectra were
recorded on a Waters QToF-API-US plus Acquity system with
electrospray ionization.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details of compound synthesis and character-
ization as well as supplemental figures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: 615-322-8700. Fax: 615-343-6532. Email: craig.lindsley@
vanderbilt.edu.
Author Contributions
∥H.P.C. and P.M.G. contributed equally. C.W.L. and C.R.H.
oversaw and designed the chemistry. P.M.G. and J.T.B.
performed synthetic chemistry work. H.P.C. and C.M.N
oversaw, designed, and interpreted the molecular pharmacology
experiments. H.P.C., P.M.G., and J.T.B. performed the
molecular pharmacology experiments. J.S.D. oversaw the drug
metabolism assays, and R.D.M. performed the DMPK assays
and bioanalytical work.
N-(2-chloro-4-nitrophenyl)-3-chloropicolinamide (20b). In a
microwave vial, 347 mg (2.20 mmol) of the 3-chloropicolinic acid were
added and dissolved in 5 mL of DCE:DIEA (9:1), then 1254 mg (3.30
mmol) of HATU were added. The mixture was stirred for 5 min, and
456 mg (2.64 mmol) of 3-chloro-4-nitroaniline dissolved in 5 mL of
DCE:DIEA (9:1) were added, followed by 3 drops of DMF. The
reaction was heated in the microwave at 120 °C for 30 min. The
reaction was cooled to RT and water was added, causing the
precipitation of the product. The crude was filtrated in vacuo and
carefully triturated with cold methanol to give a pale yellow powder,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the National Institutes of Health (NIH;
U54084679, NS032373, MH062646, and MH097056) and the
Warren Family and Foundation for support of our programs in
schizophrenia. P.M.G acknowledge support from the Vander-
bilt International Scholar Program (VISP),
1
517 mg (75%). H NMR (400.1 MHz, DMSO-d6) δ (ppm): 11.36
(1H, s, −NH), 8.69 (1H, dd, J = 4.6 Hz, J = 1.3 Hz), 8.23 (1H, d, J =
2.1 Hz), 8.17 (2H, m), 7.92 (1H, dd, J = 9.0 Hz, J = 2.1 Hz), 7.68 (1H,
1H, dd, J = 8.2 Hz, J = 4.6 Hz). 13C NMR (100.6 MHz, DMSO-d6) δ
(ppm): 164.1, 149.7, 147.8, 143.7, 142.5, 139.6, 129.4, 128.0, 127.6,
127.1, 121.5, 118.9.
ABBREVIATIONS
■
mGlu, metabotropic glutamate receptor; CNS, central nervous
system; PAM, positive allosteric modulator; NAM, negative
allosteric modulator; HEK, human embryonic kidney; CRC,
concentration−response curve; GPCR, G-protein-coupled
receptor
N-(4-amino-2-chlorophenyl)-3-chloropicolinamide (21b). In
a vial, 510 mg (1.635 mmol) of 20b were resuspended in dioxane. The
suspension was cold in an ice bath and purged with argon. A
previously prepared solution of tin(II) chloride (1395 mg) in
concentrated hydrochloric acid (5 M concentration of SnCl2) was
added dropwise to the suspension. After 2 h of stirring at RT, the
reaction was neutralized carefully with aqueous potassium carbonate
20%, filtered and extracted with diethyl ether. The organic phase was
dried with magnesium sulfate, filtered and the volatiles eliminated in
REFERENCES
■
(1) Weinberger, D. R., and Harrison, P. J., Eds. (2011) Schizophrenia,
3rd Ed. Wiley-Blackwell Publishing, Ltd., Oxford, U.K.
(2) Carlsson, A., and Lindquist, M. (1963) Effect of chlorpromazine
or haloperidol on formation of 3-methoxytyramine and normetha-
nephrine in mouse brain. Acta Pharmacol. Toxicol. 20, 140−144.
(3) Andreasen, N. C. (2000) Schizophrenia: The fundamental
questions. Brain Res. Rev. 31, 106−112.
1
vacuo to yield a pale yellow powder (428 mg, 93%). H NMR (400.1
MHz, DMSO-d6) δ (ppm): 10.42 (1H, s, −NH), 8.61 (1H, dd, J = 4.6
Hz, J = 1.2 Hz), 8.09 (1H, dd, J = 8.2 Hz, J = 1.2 Hz), 7.74 (1H, d, J =
2.1 Hz), 7.59 (1H, dd, J = 8.2 Hz, J = 4.7 Hz), 7.33 (1H, dd, J = 8.7
Hz, J = 2.2 Hz), 6.79 (1H, d, J = 8.7 Hz), 5.24 (2H, s, −NH2). 13C
NMR (100.6 MHz, DMSO-d6) δ (ppm): 162.9, 151.6, 147.6, 141.8,
139.0, 128.8, 126.7, 121.0, 120.4, 116.9, 115.7.
(4) Karayiorgou, M. (2001) Genetic aspects of schizophrenia. Clin.
Neurosci. Res. 1, 158−163.
(5) Seeman, P., Lee, T., Chau-Wong, M., and Wong, K. (1976)
Antipsychotic drug doses and neuroleptic/dopamine receptors. Nature
261, 717−719.
(6) Seeman, P., and Lee, T. (1975) Antipsychotic drugs: Direct
correlation between clinical potency and presynaptic action on
dopamine neurons. Science 188, 1217−1219.
(7) Olney, J. W., Newcomer, J. W., and Farber, N. B. (1999) NMDA
receptor hypofunction model of schizophrenia. J. Psychiatr. Res. 33,
523−533.
N-(3-chloro-4-(4-chloro-1,3-dioxoisoindolin-2-yl)phenyl)-3-
chloropicolinamide (24c). In a microwave vial, 20 mg (0.0709
mmol) of 21b and 19 mg (0.106 mmol) of 3-chlorophthalic anhydride
were added and dissolved in 1 mL of NMP. The vial was sealed and
the mixture was heated at 200 °C for 20 min. After this time, the
reaction was quenched with the addition of water and was worked up
by extraction with DCM (2 mL, thrice). The organic phased was
filtered through a phase separator, volatiles were evaporated; the crude
was dissolved in DMSO and purified by preparative HPLC. Cream
2344
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Articles
(8) Lindsley, C. W., Shipe, W. D., Wolkenberg, S. E., Theberge, C. R.,
Williams, D. L., Jr., Sur, C., and Kinney, G. G. (2006) Progress towards
validating the NMDA receptor hypofunction hypothesis of schizo-
phrenia. Curr. Top. Med. Chem. 8, 771−784.
(9) Menniti, F. S., Lindsley, C. W., Conn, P. J., Pandit, J., Zagouras,
P., and Volkmann, R. A. (2013) Allosteric modulation for the
treatment of schizophrenia: Targeting glutamatergic networks. Curr.
Top. Med. Chem. 13, 26−54.
(24) Gupta, D. S., McCullumsmith, R. E., Beneyto, M.,
Haraoutunian, V., Davis, K. L., et al. (2005) Metabotropic glutamate
receptor protein expression in the prefrontal cortex and striatum in
schizophrenia. Synapse 57, 123−131.
(25) Volk, D. W., Eggan, S. M., and Lewis, D. A. (2010) Alteration in
metabotropic glutamate receptor 1a and regulator G protein signaling
4 in the prefrontal cortex in schizophrenia. Am. J. Psychiatry 167,
1489−1498.
(10) Moghaddam, B., and Javitt, D. (2012) From revolution to
evolution: the glutamate hypothesis of schizophrenia and its
implications for treatment. Neuropsychopharmacology Rev. 37, 4−15.
(11) Conn, P. J., Tamminga, C., Schoepp, D. D., and Lindsley, C.
(2008) Schizophrenia: Moving beyond monoamine antagonists. Mol.
Intervent. 8, 99−105.
(12) Yao, W., Spealman, R., and Zhang, J. (2006) Dopaminergic
signaling in dendritic spines. Biochem. Pharmacol. 75, 2055−2069.
(13) Zhang, J., Vinuela, A., Neely, M. H., Hallett, P. J., Grant, S. G.
N., Miller, G. M., Isacson, O., Caron, M. G., and Yao, W.-D. (2007)
Inhibition of the dopamine D1 receptor signaling by PSD-95′. J. Biol.
Chem. 282, 15778−15789.
(14) Soares, D. C., Carlyle, B. C., Bradshaw, N. J., and Porteus, D. L.
(2011) DISC1: Structure, function, and therapeutic potential for
mental illness. ACS Chem. Neurosci. 2, 609−632.
(26) Brody, S. A., Conquet, F., and Geyer, M. A. (2003) Disruption
in prepulse inhibition in mice lacking mGluR1. Eur. J. Neurosci. 18,
2261−2266.
(27) Wood, M. R., Hopkins, C. R., Brogan, J. T., Conn, P. J., and
Lindsley, C. W. (2011) Molecular switches on allosteric ligands that
modulate modes of pharmacology. Biochemistry 50, 2403−2410.
(28) Jones, C. K., Engers, D. W., Thompson, A. D., Field, J. R.,
Blobaum, A. L., Lindsley, S. R., Zhou, Y., Gogliotti, R. D., Jadhav, S.,
Zamorano, R., Daniels, J. S., Morrison, R., Weaver, C. D., Conn, P. J.,
Lindsley, C. W., Niswender, C. M., and Hopkins, C. R. (2011)
Discovery, synthesis, SAR development of a series of N-4-(2,5-
dioxopyrrolidin-1-yl)-phenylpicolinamides: Characterization of
VU0195 (ML182) as a centrally active positive allosteric modulator
of metabotropic glutamate receptor 4 (mGlu4) with oral efficacy in an
anti-Parkinsonian animal model. J. Med. Chem. 54, 7639−7647.
(29) Emery, A. C., DiRaddo, J. O., Miller, E., Hathaway, H. A.,
Pshenichikin, S., Takoudjou, G. R., Grajkowska, E., Yasuda, R. P.,
Wolfe, B. B., and Wroblewski, J. T. (2012) Ligand bias at metabotropic
glutamate 1a receptors: Molecular determinants that distinguish β-
arrestin-mediated from G protein-mediate signaling. Mol. Pharmacol.
82, 291−301.
(30) Cho, H. P., Engers, D. W., Venable, D. F., Niswender, C. M.,
Lindsley, C. W., Conn, P. J., Emmitte, K. A., and Rodriguez, A. L.
(2014) A novel class of succinimide-derived negative allosteric
modulators of metabotropic glutamate receptor subtype 1 provides
insight into a disconnect in activity between rat and human receptors.
ACS Chem. Neurosci. 5, 597−610.
(31) Owen, D. R. (2011) Recent advances in the medicinal chemistry
of the metabotropic glutamate receptor 1 (mGlu1). ASC Chem.
Neurosci. 2, 394−401.
(32) Urwyler, S. (2011) Allosteric modulation of Family C G-
Protein-Coupled receptors: From molecular insights to therapeutic
perspectives. Pharmacol. Rev. 63, 59−126.
(33) Engers, D. W., and Lindsley, C. W. (2013) Allosteric
modulation of Class C GPCRs: A novel approach for the treatment
of CNS disorders. Drug Discovery Today: Technol. 10, e269−e276.
(34) Knoflach, F., Mutel, V., Jolidon, S., Kew, J. N., Malherbe, P.,
Vieira, E., Wichmann, J., and Kemp, J. A. (2001) Positive allosteric
modulators of metabotropic glutamate 1 receptor: Characterization,
mechanism of action, and binding site. Proc. Natl. Acad. Sci.U.S.A. 98,
13402−13407.
(35) Vieira, E., Huwyler, J., Jolidon, S., Knoflach, F., Mutel, V., and
Wichmann, J. (2005) 9H-Xanthene-9-carboxylic acid [1,2,4]oxadiazol-
3-yl and (2H-tertazol-5-yl)-amides as potent, orally available mGlu1
enhancers. Bioorg. Med., Chem. Lett. 15, 4628−4631.
(15) Li, T., Shan, P. C., Vallada, H., Xie, T., Murray, R. M., and
Collier, D. A. (1996) Preferential transmission of the high activity
allele of COMT in schizophrenia. Pyschiatr. Genet. 6, 131−134.
(16) Walsh, T., McClellan, J. M., McCarthy, S. E., Addington, A. M.,
Pierce, S. B., Cooper, G. M., Nord, A. S., Kusenda, M., Malhotra, D.,
Bhandari, A., Stray, S. M., Rippey, C. F., Roccanova, P., Makarov, V.,
Lakshmi, B., Findling, R. L., Sikich, L., Stromberg, T., Merriman, B.,
Gogtay, N., Butler, P., Eckstrand, K., Noory, L., Gochman, P., Long, R.,
Chen, Z., Davis, S., Baker, C., Eichler, E. E., Meltzer, P. S., Nelson, S.
F., Singelton, A. B., Lee, M. K., Rapoport, J. L., King, M.-C., and Sebat,
J. (2008) Rare structural variants disrupt multiple genes in
neurodevelopmental pathways in schizophrenia. Science 320, 539−543.
(17) Stone, J. L., O’Donovan, M. C., Gurling, H., Kirov, G. K.,
Blackwood, D. H. R., Corvin, A., Craddock, N. J., Gill, M., Hultman, C.
M., Lichtenstein, P., McQuillin, A., Pato, C. N., Ruderfer, D. M.,
Owen, M. J., St. Clair, D., Sullivan, P. F., Sklar, P., and Purcell, S. M.
(2008) Rare chromosomal deletions and duplications increase risk of
schizophrenia. Nature 455, 237−241.
(18) Stefansson, H., Rujescu, D., Cichon, S., Pietilainen, O. P. H.,
̈
Ingason, A., Steinberg, S., Fossdal, R., Sigurdsson, E., Sigmundsson, T.,
et al. (2008) Large recurrent microdeletions associated with
schizophrenia. Nature 455, 232−236.
(19) Need, A. C., Ge, D., Weale, M. E., Maia, J., Feng, S., Heinzen, E.
L., Shianna, K. V., et al. (2009) A genome-wide investigation of SNPs
and CNVs in schizophrenia. PloS Genet. 5, e1000373.
(20) Zeggini, E., Rayner, W., Morris, A. P., Hattersley, A. T., Walker,
M., Hitman, G. A., Deloukas, P., Cardon, L. R., and McCarthy, M. I.
(2005) An evaluation of HapMap sample size and tagging SNP
performance in large-scale empirical and simulated data sets. Nat.
Genet. 37, 1320−1322.
(21) Frank, R. A. W., McRae, A. F., Pocklington, A. J., van de
Lagemaat, L. N., Navarro, P., Croning, M. D. R., Komiyama, N. H.,
Bradley, S. J., Challiss, R. A. J., Armstrong, J. D., Finn, R. D., Malloy,
M. P., MacLean, A. W., Harris, S. E., Starr, J. M., Bhaskar, S. S.,
Howard, E. K., Hunt, S. E., Coffey, A. J., Raganath, V., Deloukas, P.,
Rogers, J., Muir, W. J., Deary, I. J., Blackwood, D. H., Visscher, P. M.,
and Grant, S. G. N. (2011) Clustered coding variants in the glutamate
receptor complexes of individuals with schizophrenia and bipolar
disorder. PLoS One 6, e19011.
(36) Vieira, E., Huwyler, J., Jolidon, S., Knoflach, F., Mutel, V., and
Wichmann, J. (2009) Fluorinated 9H-Xanthene-9-carboxylic acid
oxazol-2-yl amides as potent, orally available mGlu1 enhancers. Bioorg.
Med., Chem. Lett. 19, 1666−1669.
(37) Hemstapat, K., dePaulis, T., Chen, Y., Brady, A. E., Grover, V.
K., Alagille, D., Tamagnan, G. D., and Conn, P. J. (2006) A novel class
of positive allosteric modulators of metabotropic glutamate receptor
subtype 1 interact with a site distinct from that of negative allosteric
modulators. Mol. Pharmacol. 70, 616−626.
(22) Ayoub, M. A., Angelicheva, D., Vile, D., Chandler, D., Morar, B.,
Cavanaugh, J. A., Visscher, P. M., Jablensky, A., Pfleger, K. D. G., and
Kalaydijeva, L. (2012) Deleterious GRM1 mutations in schizophrenia.
PLoS One 7, e32849.
(23) Stauffer, S. R. (2011) Progress toward positive allosteric
modulators of the metabotropic glutamate receptor subtype 5
(mGlu5). ACS Chem. Neurosci. 2, 450−470.
(38) Williams, R., Zhou, Y., Niswender, C. M., Luo, Q., Conn, P. J.,
Lindsley, C. W., and Hopkins, C. R. (2011) Re-exploration of the
PHCCC scaffold: Discovery of improved positive allosteric modu-
lators of mGluR4. ACS Chem. Neurosci. 1, 411−419.
(39) Wu, H., Wang, C., Gregory, K. J., Han, G. W., Cho, H. P., Xia,
Y., Niswender, C. M., Katrich, V., Meiler, J., Chrezov, V., Conn, P. J.,
and Stevens, R. C. (2014) Structure of a class C GPCR metabotropic
2345
dx.doi.org/10.1021/cb500560h | ACS Chem. Biol. 2014, 9, 2334−2346

ACS Chemical Biology
Articles
glutamate receptor 1 bound an allosteric modulator. Science 344, 58−
64.
(40) Wenthur, C. J., Niswender, C. M., Morrison, R., Daniels, J. S.,
Conn, P. J., and Lindsley, C. W. (2013) Discovery of (R)-(2-fluoro-4-
((-4-methoxyphenyl)ethynyl)phenyl)(3-hydroxypiperidin-1-yl)-
methanone (ML337), an mGlu3 selective and CNS penetrant negative
allosteric modulator (NAM). J. Med. Chem. 56, 3713−3718.
(41) Jones, C. K., Brady, A. E., Davis, A. A., Xiang, Z., Bubser, M.,
Tantawy, M. N., Kane, A., Bridges, T. M., Kennedy, J. P., Bradley, S.
R., Peterson, T., Baldwin, R. M., Kessler, R., Deutch, A., Lah, J. L.,
Levey, A. I., Lindsley, C. W., and Conn, P. J. (2008) Novel selective
allosteric activator of the M1 muscarinic acetylcholine receptor reduces
amyloid processing and produces antipsychotic-like activity in rats. J.
Neurosci. 28 (41), 10422−10433.
2346
dx.doi.org/10.1021/cb500560h | ACS Chem. Biol. 2014, 9, 2334−2346