Discovery of a negative allosteric modulator of GABAB receptors

Article abstract of DOI:10.1021/ml500162z

Initialized from the scaffold of CGP7930, an allosteric agonist of GABA_B receptors, a series of noncompetitive antagonists were discovered. Among these compounds, compounds 3, 6, and 14 decreased agonist GABA-induced maximal effect of IP3 production in HEK293 cells overexpressing GABA_B receptors and Gq_i9 proteins without changing the EC₅₀. Compounds 3, 6, and 14 not only inhibited agonist baclofen-induced ERK1/2 phosphorylation but also blocked CGP7930-induced ERK1/2 phosphorylation in HEK293 cells overexpressing GABA_B receptors. The results suggested that compounds 3, 6, and 14 are negative allosteric modulators of GABA_B receptors. The representative compound 14 decreased GABA-induced IP3 production with IC₅₀ of 37.9 μM and had no effect on other GPCR Class C members such as mGluR1, mGluR2, and mGluR5. Finally, we showed that compound 14 did not bind to the orthosteric binding sites of GABA_B receptors, demonstrating that compound 14 negatively modulated GABA_B receptors activity as a negative allosteric modulator.

Full text of DOI:10.1021/ml500162z

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Negative Allosteric Modulator of GABAB Receptors
Lin-Hai Chen,†,§ Bing Sun,‡,§ Yang Zhang,‡ Tong-Jie Xu,‡ Zhi-Xiong Xia,‡ Jian-Feng Liu,*,‡
and Fa-Jun Nan*,†
†National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai, China
‡Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and
Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China
S
* Supporting Information
ABSTRACT: Initialized from the scaffold of CGP7930, an
allosteric agonist of GABAB receptors, a series of non-
competitive antagonists were discovered. Among these
compounds, compounds 3, 6, and 14 decreased agonist
GABA-induced maximal effect of IP3 production in HEK293
cells overexpressing GABAB receptors and Gqi9 proteins
without changing the EC50. Compounds 3, 6, and 14 not
only inhibited agonist baclofen-induced ERK1/2 phosphor-
ylation but also blocked CGP7930-induced ERK1/2 phos-
phorylation in HEK293 cells overexpressing GABAB receptors.
The results suggested that compounds 3, 6, and 14 are negative allosteric modulators of GABAB receptors. The representative
compound 14 decreased GABA-induced IP3 production with IC50 of 37.9 μM and had no effect on other GPCR Class C
members such as mGluR1, mGluR2, and mGluR5. Finally, we showed that compound 14 did not bind to the orthosteric binding
sites of GABAB receptors, demonstrating that compound 14 negatively modulated GABAB receptors activity as a negative
allosteric modulator.
KEYWORDS: GABAB receptors, negative allosteric modulator, CGP7930, α-keto acid
and GS39783 (Figure 1) are pioneering PAMs of GABAB
receptors reported in 200126 and 2003.27 They have been
proven effective in enhancing the potency of GABA both in
vitro and in vivo,28 but their developments were restricted to
some extent due to their relative low potencies (in the μM
range) and genotoxicity.29 On their basis, more potent and
safer PAMs including NVP-BHF177,29 BHFF30 (Figure 1), and
γ-Aminobutyric acid (GABA) is the primary inhibitory
neurotransmitter in the mammalian central nervous system
(CNS).1 It activates two classes of receptors: ionotropic
GABAA and GABAC receptors and metabotropic GABAB
receptors. GABAB receptors are widely expressed in the brain
where they regulate synaptic activity both at the pre- and
postsynaptic levels.2 The GABAB receptors belong to the class
C G protein-coupled receptors (GPCRs) and are functional as
heterodimers composed by two subunits, GB1 and GB2.3−8
The GB1 subunits contain the orthosteric binding sites that
bind both agonists (e.g., GABA and baclofen) and competitive
antagonists (e.g., CGP54626), whereas GB2 subunits are
responsible for escorting GB1 subunits to the cell surface and
for activating the Gi/o-protein.7−11
GABAB receptors have been implicated in a variety of
neurological and psychiatric disorders including nociception,
cognitive impairment, epilepsy, spasticity, and drug addiction.12
The therapeutic effect of GABAB receptors agonist, Lioresal
(also known as baclofen), a drug used to treat spasticity in
multiple sclerosis patients, is muscle relaxation.12,13 Its usage is
limited for treatment of neurological and psychiatric disorders
because of this unwanted, adverse side effect and also its poor
ability to penetrate the blood−brain barrier (BBB).14,15 Positive
allosteric modulators (PAMs), which interact with GB2 subunit
transmembrane domain, can potentiate the activity of GABAB
receptors, with improved selectivity and safety.16−25 CGP7930
Figure 1. Representative positive allosteric modulators of GABAB
receptors.
Received: November 17, 2013
Accepted: May 27, 2014
© XXXX American Chemical Society
A
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ADX7144131 (structure undisclosed) have been developed, and
they showed efficacy in rodent models of nicotine32 or alcohol
addition,33 stress-induced hypothermia,30 and overactive
bladder,31 respectively. PAMs with novel scaffolds have also
been discovered,25 the representative compounds COR627 and
COR628 (Figure 1) both potentiated the sedative/hypnotic
effect of baclofen in DBA mice.34 Furthermore, we and other
groups have shown that CGP7930 alone may activate GABAB
receptors as an allosteric agonist.9,35,36 However, until now, no
negative allosteric modulators (NAMs) of GABAB receptors
have been reported.
In this context, we report the synthesis and characterization
of the compounds based on the scaffold of CGP7930. Among
these compounds, compounds 3, 6, and 14 decreased GABA-
induced maximal effect of IP3 production in HEK293 cells
overexpressing GABAB receptors and Gqi9 proteins without
changing the EC50, thus indicating that compounds 3, 6, and 14
may be noncompetitive antagonists. The term “noncompetitive
antagonism” can be used to describe two distinct phenomena:
one in which the antagonist binds to the active site of the
receptor, and one in which the antagonist binds to an allosteric
site of the receptor.37 We further showed that compounds 3, 6,
and 14 not only inhibited agonist baclofen-induced ERK1/2
phosphorylation but also blocked the allosteric agonist
CGP7930-induced ERK1/2 phosphorylation in HEK293 cells
overexpressing GABAB receptors. The antagonist, CGP54626,
blocked baclofen-induced ERK1/2 phosphorylation but failed
to block CGP7930’s effect. These results suggested that
compounds 3, 6, and 14 may be the antagonists that bind to
the allosteric sites of GABAB receptors as NAMs. Finally, we
showed that the representative compound 14 had no effect on
other GPCR Class C members such as mGluR1, mGluR2, and
mGluR5, demonstrating the specificity of compound 14 toward
GABAB receptors. Moreover, we showed that compound 14 did
not bind to the orthosteric binding sites of GABAB receptors,
thus demonstrating that compound 14 negatively modulated
GABAB receptors activity as a negative allosteric modulator.
In order to find PAMs of GABAB receptor with better
activity, we have been devoted to the structure optimization of
CGP7930 and obtained the unexpected compound 3
containing a rare hemiacetal group, when synthesizing
compound 2 (Figure 2A). We showed that compound 3
inhibited the GABA-induced IP3 (inositol trisphosphate)
production in HEK-293 cells overexpressing GABAB receptors
and Gqi9 chimeric proteins, while compound 2 acted as a PAM
to potentiate GABA-induced IP3 production (Figure 2B). As
compound 3 shares the same scaffold with CGP7930, we
hypothesized if compound 3 inhibited the GABA-induced IP3
production as a negative allosteric modulator.
Figure 2. Discovery and biological evaluation of compound 3. (A)
Synthesis of compounds 2 and 3. (B) Effect of CGP7930 and its
analogues (100 μM) on GABA (1 μM)-induced IP3 formation in
HEK293 cells overexpressing GABAB receptors (GB1 + GB2) and
Gqi9 chimeric proteins. (C) Dose−response curves for GABA-induced
IP3 formation in HEK293 cells overexpressing GABAB receptors (GB1
●
+ GB2) and Gqi9 chimeric proteins in the absence ( ) or presence
○
( ) of compound 3 (100 μM). (B,C) Data are the mean SEM from
three independent experiments. **p < 0.01, versus treated with GABA
in the absence of compounds. (D) Effect of compound 3 on either
baclofen (100 μM) or CGP7930 (100 μM)-stimulated ERK1/2
phosphorylation in HEK293 cells overexpressing GABAB receptors
(GB1 + GB2).
antagonist, CGP54626, failed to inhibit CGP7930 induced
phosphorylation of ERK1/29,35 suggests that compound 3
antagonizes the effect on GABAB receptors induced by the
allosteric agonist CGP7930 to act as a negative allosteric
modulator (NAM).
Before the mechanistic investigation of compound 3, we
found the inhibiting ability of compound 3 was fading during
the repeated activity confirmation lasted for several months,
which supported the following observation of partial decom-
position of compound 3 stock solution in DMSO utilizing the
liquid chromatography−mass spectrometry (LC−MS) analysis.
After the routine column chromatography isolation and
structure identification, four compounds 4−7 were acquired
(Figure 3A), with the recovery of compound 3. We further
compared the activities of compounds 4−7 with compound 3
(Figure 3B). On HEK293 cells expressing GABAB receptors
and Gqi9 chimeric proteins, compound 6 inhibited the GABA-
induced IP3 production to a similar extent of compound 3,
while the other three displayed little effect compared to the
control.
Compound 6 has a conjugated aldehyde group, which is
prone to react with nucleophilic reagents such as amines and
thiols in the cells. Does the activity of compound 6 result from
the nonspecific reaction with nucleophiles like GABA? To
answer this question, we assessed the activity of compound 6
analogues as compounds 8−12 (Figure 3C). The result showed
that only compound 8 displayed moderate activity, among
these three compounds possessing conjugated carbonyl group
(Figure 3D). Compound 12 also showed moderate activity,
which contains the same substituted phenyl group as
compound 6 but has the allylic alcohol group instead of
conjugated aldehyde. Compound 11 had little activity due to
the deletion of two tert-butyl groups on the phenyl group.
To better characterize the inhibition mechanism of
compound 3, we tested the effect of compound 3 (100 μM)
on the GABA-induced IP3 production maximum effect and
EC50. In HEK293 cells expressing GABAB receptors and Gqi9
chimeric proteins, compound 3 decreased the maximal
response of GABA-induced IP3 production without changing
the EC50 (Figure 2C). This result is consistent with a
noncompetitive mode of action. To evaluate the effect of
compound 3 on ERK1/2 phosphorylation, HEK293 cells
expressing GABAB receptors were treated by baclofen or
CGP7930 with or without compound 3, we found that
compound 3 blocked ERK1/2 phosphorylation either induced
by baclofen or CGP7930, without altering the protein
expression level (Figure 2D). The fact that GABABR
B
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Figure 4. Biological evaluation of compound 6. (A) Dose−response
curves for GABA (1 μM)-induced IP3 formation in HEK293 cells
overexpressing GABAB receptors and Gqi9 chimeric proteins in the
●
○
absence ( ) or presence ( ) of compound 6 (100 μM). (B) Dose−
response curves for compound 6-inhibited IP3 formation in HEK293
cells overexpressing GABAB receptors (GB1 + GB2) and Gqi9
chimeric proteins in the presence of GABA (1 μM). (A,B) Data are
the mean SEM from three independent experiments. (C) Effect of
compound 6 (100 μM) on baclofen (100 μM) or CGP7930 (100
μM)-stimulated ERK1/2 phosphorylation in HEK293 cells over-
expressing GABAB receptors (GB1 + GB2). **p < 0.01 versus treated
with baclofen in the absence of compounds, and #p < 0.05 versus
treated with CGP7930 in the absence of compounds.
Figure 3. Discovery and biological validation of compound 6. (A)
Structures of the four compounds isolated from decomposition
products of compound 3. (B) Effects of compounds 3−7 (100 μM) on
GABA (1 μM)-induced IP3 formation in HEK293 cells overexpressing
GABAB receptors (GB1 + GB2) and Gqi9 chimeric proteins. (C)
Structures of compound 6 analogues. (D) Effects of compound 6 and
its analogues (100 μM) on GABA (1 μM)-induced IP3 formation in
HEK293 cells overexpressing GABAB receptors (GB1 + GB2) and
Gqi9 chimeric proteins. (B,D) Data are the mean SEM from three
independent experiments. *p < 0.05 and ***p < 0.001 versus treated
with GABA in the absence of compounds.
Figure 5. Structure and biological evaluation of compounds 13a−17.
(A) Structures of CGP7930 analogues containing highly electrophilic
groups. (B) Effects of compounds 13a−17 (100 μM) on GABA (1
μM)-induced IP3 formation in HEK293 cells overexpressing GABAB
receptors and Gqi9 chimeric proteins. (C) Dose−response curves for
GABA-induced IP3 formation in HEK293 cells overexpressing GABAB
These results indicate that both the conjugated carbonyl group
and the substitutions on phenyl group are essential for the
inhibition activity of compound 6 and exclude the possibility
that the activity of compound 6 origins from the nonspecific
reaction with nucleophiles.
●
receptors (GB1 + GB2) and Gqi9 chimeric proteins in the absence ( )
○
or presence ( ) of compound 14 (100 μM). (B,C) Data are the mean
The further biological evaluation showed that compound 6
decreased the maximal response in the GABA-induced IP3
production without changing the EC50 of GABA (Figure 4A)
and inhibited the GABA-induced IP3 production in a
concentration-dependent manner with IC50 of 21.3 μM (Figure
4B). Compound 6 also blocked the phosphorylation of ERK1/
2 either induced by baclofen or CGP7930 in HEK293 cells
overexpressing GABAB receptors (Figure 4C). These results
suggest that compound 6 modulates GABAB receptors activity
as a NAM since it antagonized the phosphorylation of ERK1/2
induced by CGP7930. Although compound 6 displayed weaker
activity than compound 3, its stability and the preliminary
structure−activity relationship (SAR) provide us a fresh start
for the discovery of more potent NAMs of GABAB receptors.
The aforementioned activity difference between compounds
6 and 12 indicates that the highly electron-deficient carbonyl
group played an important role in the negative allosteric activity
of this scaffold. Increasing the electron-deficiency may enhance
the activity of these compounds. Thus, we designed and
synthesized two types of analogues as compounds 13a−17,
containing electrophilic trifluoromethyl ketone group or α-keto
acid (ester) group separately (Figure 5A). We showed that
SEM from three independent experiments. **p < 0.01 and ***p <
0.001 versus treated with GABA in the absence of compounds.
compounds 14 and 15 had the best effects to modulate GABAB
receptors activity, comparing to compounds 13a and 13b with
trifluoromethyl ketone group and their ester form, compounds
16 and 17 (Figure 5B). Since compound 14 is easy to
synthesize and its carboxylic acid group facilitates further
modification, we performed the functional analysis for this
compound. We showed that compound 14 decreased the
maximal response in the GABA-induced IP3 production
without changing the EC50 of GABA (Figure 5C) and inhibited
the GABA-induced IP3 production in a concentration-depend-
ent manner with IC50 of 37.9 μM (Figure SI, Supporting
Information).
To verify the selectivity of compound 14 on GABAB
receptors, we performed the experiment to verify if compound
14 blocked activation of other members of Class C GPCRs
such as mGluR1, mGluR2, and mGluR5. Among these Class C
GPCRs, mGluR1 and mGluR5 belong to Gq-protein coupled
receptors, whereas GABAB receptors and mGluR2 belong to
C
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Gi/o-protein coupled receptors. We compared GABAB
receptor-induced Ca2+ release in HEK293 cells overexpressing
GABAB receptors and Gqi9 chimeric proteins with different
members of mGluRs-induced Ca2+ release in HEK cells
overexpressing mGluR1, mGluR2 (with Gqi9), or mGluR5.
As shown in Figure 6A, 50 μM of compound 14 inhibited
to block allosteric agonist CGP7930-induced ERK1/2
phosphorylation in HEK293 cells overexpressing GABAB
receptors (Figure SII, Supporting Information). Compound
14 blocked the phosphorylation of ERK1/2 either induced by
baclofen or CGP7930 in HEK293 cells overexpressing GABAB
receptors (Figures 6C and SII, Supporting Information). These
results demonstrated that compound 14 modulated GABAB
receptor activity as a NAM for GABAB receptors.
In summary, we have developed a class of noncompetitive
antagonists of GABAB receptors derived from the scaffold of
CGP7930. Among them, compound 14 acts as a NAM since it
did not bind to the orthosteric binding sites of GABAB
receptors. Whether compound 14 interacts with a GB2
transmembrane domain as CGP7930 remains to be further
investigated. The common electrophilic groups shared by these
structures indicate a possible specific interaction between ligand
and receptor. The fine-tuning of GABAB receptor activity by a
NAM may provide a new strategy for developing novel
therapies. More detailed SAR studies focusing on this type of
compounds are in progress.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization of new chemical
entities. This material is available free of charge via the Internet
at http://pubs.acs.org.
Figure 6. Specificity of compound 14 toward GABAB receptors as a
negative allosteric modulator. (A) Effect of compound 14 (50 μM) on
agonist-induced Ca2+ release in HEK293 cells overexpressing GABAB
receptors (with Gqi9), mGluR1, mGluR2 (with Gqi9), or mGluR5,
respectively. ***p < 0.001 and ns, no effect, versus treated with
agonists (GABA (100 μM) for GABAB receptor and glutamate (10
μM) for mGluR1, 2, and 5 respectively) in the absence of compounds.
(B) Effect of compound 14 ( ) and GABA ( ) on the displacement
of the antagonist radioligand [3H]CGP54626 from CHO cells
membrane overexpressing GABAB receptors (GB1 + GB2). The
binding of [3H]CGP54626 to membranes from transfected CHO cells
was measured in a scintillation proximity assay. (C) Effects of
compound 14 (100 μM) on baclofen (100 μM) or CGP7930 (100
μM)-stimulated ERK1/2 phosphorylation in HEK293 cells over-
expressing GABAB receptors (GB1 + GB2). Data are the mean SEM
from three independent experiments. ***p < 0.001 versus treated with
baclofen in the absence of compounds, and ##p < 0.01 versus treated
with CGP7930 in the absence of compounds.
AUTHOR INFORMATION
Corresponding Authors
*(J.-F.L.) E-mail: jfliu@mail.hust.edu.cn.
*(F.-J.N.) E-mail: jnan@mail.shcnc.ac.cn.
■
Author Contributions
○
●
§(L.-H.C. and B.S.) These authors contributed equally to this
work.
Funding
This work was supported by the Ministry of Science and
Technology (grant number 2012CB518000), the National
Natural Science Foundation of China (NSFC) (grant numbers
31130028, 31225011, and 91213303), the Program of
Introducing Talents of Discipline to the Universities of the
Ministry of Education (grant number B08029), and the
́ ́
Merieux Research Grants Program of Institut-Merieux (to J.-
GABA-induced Ca2+ release but had no effect at all on
glutamate-induced Ca2+ release, demonstrating the specificity of
compound 14 on GABAB receptors. Furthermore, our data in
Figure 6A showed that compound 14 only inhibited GABAB
receptor-mediated Gi/o signaling but had no effect on
mGluR2-mediated Gi/o signaling, demonstrating that com-
pound 14 had no effect on GABAB receptors downstream of
Gi/o-protein signaling and interacted only with GABAB
receptors at the receptor level.
F.L.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Jean-Philippe Pin and Dr. Philippe Rondard
■
(Institut de Gen
for their helpful discussions.
́
omique Fonctionnelle, Montpellier, France)
To verify if compound 14 binds to orthosteric binding sites
in GB1, we thus performed radioactivity-labeled binding assay
through displacement of [3H]CGP54626 from membranes of
CHO cells overexpressing GABAB receptors.38 As shown in
Figure 6B, [3H]CGP54626 binding was displaced in a
concentration-dependent manner by the GABAB receptors
agonist, GABA. In contrast, compound 14 failed to displace
[3H]CGP54626 binding even at concentrations up to 100 μM,
demonstrating that compound 14 did not bind to orthosteric
binding sites in GB1 subunits. Furthermore, CGP54626
blocked baclofen-induced ERK1/2 phosphorylation but failed
ABBREVIATIONS USED
■
GABA, γ-aminobutyric acid; GPCR, G-protein coupled
receptor; PAM, positive allosteric modulator;; NAM, negative
allosteric modulator; IP3, inositol trisphosphate; ERK1/2,
extracellular-signal-regulated kinases 1/2; SAR, structure−
activity relationship
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