Design, synthesis, and pharmacological evaluation of fluorescent and biotinylated antagonists of ρ1 GABAC receptors

Article abstract of DOI:10.1021/ml300476v

The ρ₁ GABA_C receptor is a ligand-gated chloride ion channel that shows promise as a therapeutic target for myopia, sleep disorders, memory and learning facilitation, and anxiety-related disorders. As such, there is a need for molecular probes to understand the role GABA _C receptors play in physiological and pathological processes. To date, no labeled (either radioactive or fluorescent) GABA_C selective ligand has been developed that can act as a marker for GABA_C receptor visualization and localization studies. Herein, we report a series of fluorescent ligands containing different-sized linkers and fluorophores based around (S)-4-ACPBPA [(4-aminocyclopenten-1-yl)-butylphosphinic acid], a selective GABA_C antagonist. One of these conjugates, (S)-4-ACPBPA-C5-BODIPY (13), displayed moderate potency (IC₅₀ = 58.61 μM) and selectivity (>100 times) for ρ₁ over α₁β₂γ_2L GABA_A receptors. These conjugates are novel lead agents for the development of more potent and selective fluorescent probes for studying the localization and function of GABA_C receptors in living cells.

Full text of DOI:10.1021/ml300476v

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Pharmacological Evaluation of Fluorescent
and Biotinylated Antagonists of ρ1 GABAC Receptors
Navnath Gavande,† Hye-Lim Kim,† Munikumar R. Doddareddy,† Graham A. R. Johnston,‡ Mary Chebib,†
and Jane R. Hanrahan*,†
†Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia
‡Adrien Albert Laboratory, Department of Pharmacology, The University of Sydney, Sydney, NSW 2006, Australia
S
* Supporting Information
ABSTRACT: The ρ1 GABAC receptor is a ligand-gated chloride
ion channel that shows promise as a therapeutic target for myopia,
sleep disorders, memory and learning facilitation, and anxiety-
related disorders. As such, there is a need for molecular probes to
understand the role GABAC receptors play in physiological and
pathological processes. To date, no labeled (either radioactive or
fluorescent) GABAC selective ligand has been developed that can
act as a marker for GABAC receptor visualization and localization
studies. Herein, we report a series of fluorescent ligands containing
different-sized linkers and fluorophores based around (S)-4-ACPBPA [(4-aminocyclopenten-1-yl)-butylphosphinic acid], a
selective GABAC antagonist. One of these conjugates, (S)-4-ACPBPA-C5-BODIPY (13), displayed moderate potency (IC50
=
58.61 μM) and selectivity (>100 times) for ρ1 over α1β2γ2L GABAA receptors. These conjugates are novel lead agents for the
development of more potent and selective fluorescent probes for studying the localization and function of GABAC receptors in
living cells.
KEYWORDS: Human ρ1 GABAC receptors, CNS-related disorders, GABAC antagonists, fluorescent and biotinylated probes,
homology modeling and docking
γ-Aminobutyric acid 1 [GABA (Figure 1)] is the most
abundant inhibitory neurotransmitter in the mammalian central
nervous system (CNS) and is essential for the overall balance
between neuronal excitation and inhibition.1,2 GABA released
from GABAergic axon terminals influences neurons via GABAA,
GABAB, and GABAC (GABAρ) receptors. These receptors are
grouped on the basis of their subunit composition, gating
properties, and pharmacological profiles. GABAA and GABAC
receptors are ligand-gated chloride ion channels that mediate
fast synaptic inhibition when activated by GABA,1 whereas
GABAB receptors are G-protein-coupled receptors that mediate
slow, longer-lasting synaptic inhibition by increasing potassium
and decreasing calcium conductances when activated by
GABA.3
Ionotropic GABAA receptors are transmembrane protein
complexes composed of five heteropentameric subunits. To
date, 16 human GABAA receptor subunits have been identified
and classified as α (α1−α6), β (β1−β3), γ (γ1−γ3), δ, ε, π, and
θ.4 In contrast, ionotropic GABAC receptors have pharmacol-
ogy, physiology, and subunit compositions distinct from those
of GABAA.5,6 In mammals, GABAC receptors are composed of
ρ subunits (ρ1−ρ3), forming homopentameric assemblies of
five ρ subunits (ρ1, ρ2, or ρ3 subunits) or pseudoheteropenta-
meric complexes comprising different ρ subunits (a combina-
tion of ρ1 and ρ2 or ρ2 and ρ3 subunits).5,7,8 GABAC receptors
are insensitive to bicuculline and (−)-baclofen, selectively
activated by (+)-CAMP 3 [(+)-cis-2-(aminomethyl)-
cyclopropanecarboxylic acid (Figure 1)] and 5-Me-I-4AA 4
(5-methyl-1H-imidazole-4-acetic acid), and inhibited by
TPMPA 5 (1,2,5,6-tetrahydropyridine-4-yl-methyl-phosphinic
acid), (S)-4-ACPBPA 6 {[(S)-4-aminocyclopenten-1-yl]-butyl-
phosphinic acid}9 and 3-GOHP 8 [3-(guanido)-1-oxo-1-
hydroxy-phospholane].10 This clearly indicates that the GABA
binding sites of GABAC and GABAA receptors are not identical.
GABAA receptors are widely distributed in the CNS,1
whereas GABAC receptors are mainly expressed in the superior
colliculus,11 cerebellum,12 lateral amyglada,13 hippocampus
(strong ρ2 subunit expression),14 and, most prominently, the
retina (strong ρ1 subunit expression).7 Various studies show
that GABAC receptors are potential therapeutic targets for
myopia, sleep disorders, managing memory-related disorders,
and peripheral antinociception.5,13 However, understanding the
role of GABAC receptors and information about the processes
triggered by ligand−receptor interactions in living cells are still
limited because of the lack of pharmacological tools.
Fluorescent ligands have proven to be useful tools offering a
wealth of information about the mapping or identification of
ligand binding sites,15,16 the mechanism of ligand binding,17 the
physical nature of the binding pocket, the movement and
Received: December 24, 2012
Accepted: February 18, 2013
Published: February 18, 2013
© 2013 American Chemical Society
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Figure 1. Structures of GABA (1), the GABAA agonist and GABAC partial agonist muscimol (2), the GABAC selective agonists (+)-CAMP (3) and
5-Me-I-4AA (4), and GABAC selective antagonists TPMPA (5), (S)-4-ACPBPA (6), (R)-4-ACPBPA (7), and 3-GOHP (8).
a
Scheme 1. Synthesis of Fluorescent ρ1 GABAC Antagonists (S)-11a−g and (R)-11d
a
Reagents and conditions: (a) (S)-4-ACPBPA 6 or (R)-4-ACPBPA 7 [for (R)-10d], NaHCO3, water/DME/THF (1:1:0.3), room temperature for
16 h, 81−89%; (b) TFA/DCM (1:1), room temperature for 1−2 h, 92−98%; (c) NMA N-succinimidyl ester, NaHCO3, water/DME/THF
(1:1:0.3), room temperature for 16 h, 75−84%.
internalization of receptors in living cells,18 and the localization
as well as visualization of the labeled receptors.19 Biotinylated
probes are useful for the isolation and purification of target
proteins of the receptor fragment for crystallization and X-ray
studies of the particular receptor-binding site,20 the localization
and visualization of the receptor by using antibodies,21 and the
study of ligand−protein interactions in living cells.22
In addition, fluorescent and biotinylated probes represent a
faster, safer, and less expensive alternative to radioligands in
receptor studies, circumventing several drawbacks associated
with radioligand studies such as health and safety concerns and
the need for a large number of cells with strong receptor
expression.23 Thus, the identification of fluorescent or
biotinylated ligands for GABAC receptors is of great interest.
The development of these tagged ligands usually starts with the
selection of a potent and selective pharmacophore, which is
then conjugated with the desired tag through an appropriate
linker.
To date, only a few fluorescent and biotinylated GABAB
receptor antagonist ligands have been reported,24 and N-
acylation of the amino moiety of muscimol (2), a widely used
GABAA agonist that it is also a potent partial agonist at human
ρ1 GABAC receptors with fluorophores, has been shown to
result in ligands that bind to the GABAA receptors.25 Vu et al.
presented evidence that muscimol linked through a 6-
aminohexanoyl chain to biotin (muscimol-biotin; EC50 = 20
μM) and BODIPY (muscimol-BODIPY) retained substantial
agonist activity compared to muscimol (2; EC50 = 2 μM) at
GABAC receptors,26 indicating a steric tolerance in the region
around the ligand’s amine binding site of the GABAC receptor.
To the best of our knowledge, no selective fluorescent ligand
for GABAC receptors has been described in the literature.
Therefore, we envisioned that incorporating fluorophores or
the versatile biotin moiety into selective GABAC ligands could
lead to selective probes. Herein, we report the design, synthesis,
and pharmacological evaluation of the selective fluorescent and
biotinylated probes for ρ1 GABAC receptors.
We selected two of our previously reported selective and
potent GABAC antagonists, (S)-4-ACPBPA 6 and (R)-4-
ACPBPA 7 (Figure 1), for the design of the fluorescent
GABAC antagonists.9 Both were synthesized according to our
previously published protocol.9 The length of the spacer chain
is known to significantly impact the affinity of ligand−
fluorophore conjugates for the receptors; therefore, we chose
a linker size that varied from 0 to 10 carbons to introduce
molecular flexibility into the conjugated fluorescent ligand. We
incorporated fluorescent groups with relatively small to large
molecular volumes to study the effect of perturbation on
receptor affinity. We used the fluorophores NMA (N-
methylanthranilic acid), BODIPY (4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene), and NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)
chloride, which are known to have different excitation
wavelengths and distinctive properties, to diversify fluorescence
experiments. BODIPY has been extensively utilized for the
development of fluorescent probes because of its distinctive and
useful features, including hydrophobicity, photochemical
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a
Scheme 2. Synthesis of Fluorescent ρ1 GABAC Antagonists 12 and 13
a
Reagents and conditions: (a) 7-nitrobenz-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), K2CO3, DMF, room temperature for 24 h, 76%; (b) BODIPY
N-succinimidyl ester, DIPEA, DMF, room temperature for 24 h, 72%.
a
Scheme 3. Synthesis of Biotinylated ρ1 GABAC Antagonists 15 and 16
a
Reagents and conditions: (a) (S)-4-ACPBPA 6, DIPEA, DMF, room temperature for 24 h, 75%; (b) (S)-10d, DIPEA, DMF, room temperature for
24 h, 81%.
a
Table 1. Pharmacological Evaluation of Fluorescent and Biotinylated Probes
human ρ1 GABAC receptor % inhibition and IC50
μM (95% confidence interval)
d
compound
(S)-4-ACPBPA (6)
spacer
300 μM
600 μM
human α1β2γ2L GABAA receptor % inhibition at 300 μM
b
g
97 3%
inactive
c
IC50 = 9.76, KB = 4.97 μM
b
g
(R)-4-ACPBPA (7)
92 7%
inactive
c
KB = 59.3 μM
(S)-4-ACPBPA-C1-NMA [(S)-11a]
(S)-4-ACPBPA-C3-NMA [(S)-11b]
(S)-4-ACPBPA-C4-NMA [(S)-11c]
(S)-4-ACPBPA-C5-NMA [(S)-11d]
(R)-4-ACPBPA-C5-NMA [(R)-11d]
(S)-4-ACPBPA-C6-NMA [(S)-11e]
(S)-4-ACPBPA-C7-NMA [(S)-11f]
(S)-4-ACPBPA-C10-NMA [(S)-11g]
(S)-4-ACPBPA-C5-NBD (12)
1
3
8
2%
23 4%
31 2%
62 7%
56 9%
55 3%
24 8%
4
g
5
inactive
g
5
inactive
6
7
10
5
8
3%
e
f
g
IC50 = 281.42 (274.96−332.11)
IC50 = 193.27 (181.67−224.81)
inactive
e
g
(S)-4-ACPBPA-C5-BODIPY (13)
5
IC50 = 103.14 (95.36−114.87)
inactive
f
IC50 = 58.61 (45.37−71.63)
inactive
b
(S)-4-ACPBPA-C0-biotin (15)
(S)-4-ACPBPA-C5-biotin (16)
0
5
e
g
IC50 = 147.92 (139.45−174.13)
9
7%
f
IC50 = 76.54 (68.33−102.96)
a
Determined electrophysiologically in Xenopus laevis oocytes expressing the human ρ1 GABAC receptor or human α1β2γ2L GABAA receptors as
previously described.9,10 Activity or percent inhibition by 300 μM compound of the current produced by a submaximal concentration of GABA (1
μM, EC50). Data from ref 9. Percent inhibition by 600 μM compound of the current produced by a submaximal concentration of GABA (1 μM,
b
c
d
e
f
EC50), with a 5 min preincubation. IC50 values for compounds without preincubation. IC50 values for compounds with a 5 min preincubation.
g
Activity or percent inhibition by 300 μM compound of the current produced by a submaximal concentration of GABA (30 μM, EC50) without and
with a 5 min preincubation. All data are means the standard error of the mean (n = 3 oocytes).
stability, insensitivity to changes in experimental conditions,
efficient uptake into cell membranes, and high extinction
coefficient and fluorescence quantum yield.27 Initially, we
synthesized fluorescent probes using the potent and selective
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ACS Medicinal Chemistry Letters
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Figure 2. View of the ρ1 GABAC ligand-binding site with predicted binding modes. (A) Close-up of (S)-4-ACPBPA 6 (green carbons). (B) Close-up
of (S)-4-ACPBPA-C5-NMA (S)-11d (brown carbons), (S)-4-ACPBPA-C5-NBD 12 (red carbons), (S)-4-ACPBPA-C5-BODIPY 13 (aqua blue
carbons), and (S)-4-ACPBPA-C5-biotin 16 (magenta carbons) that are overlaid on (S)-4-ACPBPA 6 in the GABAC binding site, with the linker and
fluorophores external to the orthosteric site.
GABAC antagonist (S)-4-ACPBPA 6 by linking the small
fluorophore NMA through linkers of various sizes.
(R)-11d, 12, 13, 15, and 16] were evaluated for activity alone
and in the presence of GABA on GABAA (α1β2γ2L), and ρ1
GABAC receptors to determine whether they behave as
agonists, antagonists, or modulators.
The synthesis of (S)-4-ACPBPA-NMA- and (R)-4-ACPBPA-
C5-NMA-conjugated target compounds (S)-11a−g and (R)-
11d is depicted in Scheme 1. N-Succinimidyl esters 9a−g were
prepared by protection of the amine function of the amino
acids with a tert-butyloxycarbonyl group (Boc), followed by
activation of the acid function of the amino acids with an N-
hydroxysuccinimide (NHS) (see the Supporting Information
for the experimental details). The activated N-succinimidyl
esters 9a−g were further reacted with the GABAC antagonist
(S)-4-ACPBPA 6 in an aqueous NaHCO3 solution at room
temperature, and subsequent removal of the Boc group using
TFA in CH2Cl2 to afford compounds 10a−g in moderate to
good yields. Target compounds (S)-11a−g were obtained by
amide coupling of compounds (S)-10a−g with NHS-activated
fluorophore NMA (NMA N-succinimidyl ester) in an aqueous
NaHCO3 solution at room temperature. (R)-10d was obtained
from 9d as described above for the preparation of (S)-10d. (R)-
11d was synthesized by reaction of (R)-4-ACPBPA 7 with the
previously prepared ester (R)-10d under basic conditions at
room temperature.
Fluorescent compounds 12 and 13 containing the NBD and
BODIPY moieties were prepared as depicted in Scheme 2.
Condensation of 7-nitrobenz-2-oxa-1,3-diazol-4-yl chloride
(NBD chloride) with (S)-10d in a K2CO3/DMF mixture at
room temperature afforded target compound 12 in moderate
yield. The synthesis of BODIPY N-succinimidyl ester (see the
Supporting Information for the experimental details) was
achieved in two steps according to a previously described
method.28 Compound 13 was obtained in moderate yield by
reaction of BODIPY succinimidyl ester and the primary amine
of (S)-10d in a DIPEA/DMF mixture at room temperature.
Biotinylated conjugates 15 and 16 were prepared according
to Scheme 3. Formation of an amide bond between biotin N-
succinimidyl ester 14 and the corresponding primary amine of
(S)-4-ACPBPA 6 and compound (S)-10d in a DIPEA/DMF
mixture at room temperature afforded biotinylated conjugates
15 and 16, respectively.
During the initial pharmacological screening, we found that
stable and reproducible levels of inhibition of GABA (1 μM) at
ρ1 GABAC receptors were reached only after a 5 min
preincubation with compounds (S)-11a−g and (R)-11d (see
the Supporting Information for details). The preincubation
needed for some of the compounds for reaching stable and
reproducible levels of inhibition of GABA may be due to a
molecule’s size and lipophilicity needing additional time to
diffuse into the binding site. In the (S)-4-ACPBPA-NMA series
where the NMA group was attached to the primary amine of
the ligand through an aminoalkanoyl chain, we observed that
compounds with a chain length of fewer than five carbons
[compounds (S)-11a−c] exhibited weak antagonist activity,
compared to compound (S)-11d with a five-carbon chain. The
activity for compounds (S)-11e−g decreased with an increasing
chain length. These compounds had a chain length of 6−10
carbons, indicating that a linker of five carbons is optimal. The
decrease in activity for compounds (S)-11a and (S)-11b
(spacer size of fewer than four carbons) could be explained by
the relative rigidity caused by the amido-containing chain with
the ligand moiety at the binding site. With spacer sizes of more
than six carbons, the fluorescent tag may occupy a less favorable
area, resulting in a decrease in inhibitory activity.
The optimal chain length of the linker is important for the
introduction of molecular flexibility for more convenient
positioning of the ligand into the receptor. After optimization
of the chain length for fluorescent probes, we linked (R)-4-
ACPBPA through a 6-aminohexanoyl chain to NMA [(R)-11d]
and evaluated its activity on ρ1 GABAC receptors. (R)-4-
ACPBPA 7 is 12-fold less potent than (S)-4-ACPBPA 6 at the
ρ1 GABAC receptor (Figure 1).9 However, (R)-4-ACPBPA-C5-
NMA (R)-11d exhibited activity almost equal to that of (S)-4-
ACPBPA-C5-NMA (S)-11d, indicating N-acylation of the
amine removed the differential activity of enantiomers.
We also evaluated NBD and BODIPY (12 and 13,
respectively) containing probes on both ρ1 GABAC and
α1β2γ2L GABAA receptors with and without preincubation
(Table 1). (S)-4-ACPBPA-C5-NBD 12 exhibited poor potency
(IC50 = 281.42 μM) when tested without preincubation, but its
We examined the functional characterization of fluorescent
and biotinylated probes on human recombinant GABA
receptors expressed in Xenopus oocytes using the two-electrode
voltage clamp method (Table 1).9,10 All 12 probes [(S)-11a−g,
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ACS Medicinal Chemistry Letters
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activity was slightly improved (IC50 = 193.27 μM) with a 5 min
preincubation at ρ1 GABAC receptors. (S)-4-ACPBPA-C5-
BODIPY 13 displayed moderate and improved potency (IC50 =
58.61 μM with preincubation, and IC50 = 103.14 μM without
preincubation) at ρ1 GABAC receptors. The increase in activity
for (S)-4-ACPBPA-C5-BODIPY 13 but not for (S)-4-
ACPBPA-C5-NMA (S)-11d and (S)-4-ACPBPA-C5-NBD 12
indicates that bulkier fluorophores linked by a 6-aminohexanoyl
chain are more favorable for activity. These results confirmed
the prediction of molecular modeling studies that the large
hydrophobic region of the receptor is located adjacent to the
binding site of the basic amino group of (S)-4-ACPBPA. As
expected, probes (S)-11d, (R)-11d, 12, and 13 were found to
be inactive (at 300 μM) at α1β2γ2L GABAA receptors.
We also evaluated biotinylated probes of (S)-4-ACPBPA
(compounds 15 and 16) on the ρ1 GABAC receptor. Biotin-
coupled probes are suitable for subcellular localization of
receptors because biotin forms a tight complex with
strept(avidin),21 which can be visualized by confocal micros-
copy using antibodies. Direct attachment of the biotin group to
(S)-4-ACPBPA was deleterious for activity, (compound 15 was
inactive at 300 μM). However, a linker length of five carbons
(compound 16) is sufficient to restore moderate activity (IC50
= 76.54 μM with preincubation, and IC50 = 147.92 μM without
preincubation) at ρ1 GABAC receptors. These findings
represent the first demonstration of electrophysiological activity
by N-acyl derivatives of selective antagonists (S)-4-ACPBPA 6
and (R)-4-ACPBPA 7.
To identify structural determinants for the observed activity
for the ρ1 GABAC receptor, we flexibly docked the structures of
(S)-4-ACPBPA 6 and fluorescent and biotinylated probes (S)-
4-ACPBPA-C5-NMA (S)-11d, (S)-4-ACPBPA-C5-NBD 12,
(S)-4-ACPBPA-C5-BODIPY 13, and (S)-4-ACPBPA-C5-biotin
16 into the ligand-binding site of a ρ1 GABAC homology
model.29 Figure 2B shows predicted binding modes for (S)-4-
ACPBPA-C5-NMA (S)-11d, (S)-4-ACPBPA-C5-NBD 12, (S)-
4-ACPBPA-C5-BODIPY 13, and (S)-4-ACPBPA-C5-biotin 16,
which show modes of binding for probe-linked (S)-4-ACPBPA
similar to that of parent (S)-4-ACPBPA 6 (Figure 2A). The
binding affinity is largely ascribed to various electrostatic
interactions,29−31 including (i) a salt bridge interaction between
the phosphinic acid and Arg104 and Arg158,29,30 (ii) hydrogen
bond contacts of the amine of parent ligand or amide of probes
with Glu196,9 and (iii) hydrogen bond contacts with Thr244.31
The docked model of compounds 6, (S)-11d, 12, 13, and 16
(Figure 3) orients the (S)-4-ACPBPA moiety in the active site
cavity of the ρ1 GABAC receptor (shown with wire mesh) and a
flexible spacer between the (S)-4-ACPBPA core and probe
where the probe is pointing out of or residing in the
noninteractive binding site of the receptors, which cannot be
ruled out at this stage. The fluorophores or biotin tags are
shown by molecular modeling studies to reside in a pocket
away from the orthosteric site or extend into the extracellular
space.
Figure 3. View of the (S)-4-ACPBPA moiety (wire mesh) in the active
site cavity of the ρ1 GABAC receptor model and flexible spacer
between the (S)-4-ACPBPA core and probes pointing away from the
orthosteric site.
visualizing, and studying the physiopathological processes of
GABAC receptors. Further optimization of probes at amino and
phosphinic acid moieties of (S)-4-ACPBPA, in vitro pharmaco-
logical evaluation, and fluorescent spectroscopic character-
izations of all the probes are underway.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic method, characterization of compounds, pharmacol-
ogy, and molecular modeling. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +61-2-93512078. E-mail: jane.hanrahan@sydney.
edu.au.
■
Funding
N.G. and H.-L.K. acknowledge support from an Endeavour
International Postgraduate Research Scholarship (EIPRS) and a
Faculty of Pharmacy Postgraduate Award, respectively. Both
N.G. and H.-L.K. also acknowledge the John A. Lamberton
Scholarship.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are thankful to Bruce Tattam and Dr. Keith Fischer for
technical assistance with mass spectrometry. We are also
thankful to Dr. Izumi Yamamoto, Dr. Nasiara Karim for
injecting human ρ1, α1, β2, and γ2L mRNA into Xenopus oocytes
for this study, and Dr. Heba Abdel-Halim for performing initial
docking studies.
In conclusion, we have designed and synthesized for the first
time fluorescent and biotinylated probes of selective antagonists
for ρ1 GABAC receptors. Pharmacological studies show that 12,
13, and 16 exhibit activity similar to that of the previously
reported muscimol-based probes,25,26 with the added advantage
that they are selective GABAC receptors. These results offer
new knowledge regarding the binding site and receptor
flexibility of GABAC receptors. The described fluorescent and
biotinylated probes will be useful tools for localizing,
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