A photoreactive probe that differentiates the binding sites of noncompetitive GABA receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.01.118

γ-Aminobutyric acid (GABA) receptors are postsynaptic membrane protein complexes that are important not only in the regulation of the nervous system but also as targets of drugs and insecticides. We synthesized a photoreactive straight-chain noncompetitiv

Full text of DOI:10.1016/j.bmcl.2011.01.118

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1598–1600
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A photoreactive probe that differentiates the binding sites of noncompetitive
GABA receptor antagonists
⇑
Hiroshi Shimotahira, Sayaka Fusazaki, Izumi Ikeda, Yoshihisa Ozoe
Department of Life Science and Biotechnology, Shimane University, Matsue, Shimane 690-8504, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
c
-Aminobutyric acid (GABA) receptors are postsynaptic membrane protein complexes that are important
Received 13 November 2010
Revised 16 January 2011
Accepted 26 January 2011
Available online 2 February 2011
not only in the regulation of the nervous system but also as targets of drugs and insecticides. We synthe-
sized a photoreactive straight-chain noncompetitive antagonist (NCA), 2-nitro-4-[3-(trifluoromethyl)-
3H-diazirin-3-yl]phenyl 4-(4-methoxycarbonyl-1-butynyl)benzoate (NMB), to probe the NCA binding
site. Our data show that this probe labels the NCA site and demonstrate that the NCA insecticide fipronil
binds at a site distinct from that of other NCAs, such as picrotoxinin and 40-ethynyl-4-n-propyl-
bicycloorthobenzoate. The unique molecule NMB will be useful in identifying the cross-linking site of
straight-chain NCAs in GABA receptors and mapping allosteric binding sites. Such studies should provide
invaluable information in designing novel NCAs.
Keywords:
Photoreactive probe
Noncompetitive antagonist
GABA
Ó 2011 Elsevier Ltd. All rights reserved.
Picrotoxinin
Fipronil
c
-Aminobutyric acid (GABA) is the major inhibitory neurotrans-
We previously found that the straight-chain compound 5-[4-
(3,3-dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP)
acts as an NCA at GABA receptors,13 and we suggested that this un-
ique NCA might serve as a tool to probe the NCA binding site. To
show the utility of this class of NCAs, we synthesized a photoreac-
tive analogue of DBCPP and examined its competition with two
known antagonists for the NCA binding site of rat GABA receptors.
To examine the reactivity of ligands with the NCA site, compe-
tition assays were performed using [3H]40-ethynyl-4-n-propyl-
bicycloorthobenzoate ([3H]EBOB), a high affinity NCA.14 DBCPP is
the most potent competitive inhibitor of [3H]EBOB binding in the
mitter in the nervous system of vertebrates and invertebrates. The
receptors mediating its inhibitory effects are pentameric ligand-
gated ion channels, and each subunit is a four-transmembrane do-
main (4-TM) protein. The receptor’s integral channels open upon
GABA binding and enhance chloride permeability into neurons,
thereby suppressing the depolarization induced by excitatory neu-
rotransmitters. GABA receptors are important not only physiologi-
cally but also as targets of a variety of anxiolytic/anticonvulsant
drugs, insecticides, and toxic substances.1,2
Noncompetitive antagonists (NCAs) inhibit the channel func-
tion by binding to a site(s) distinct from the orthosteric binding
site. The best studied NCA is picrotoxinin (PTX), a sesquiterpene
isolated from poisonous Menispermaceae plants (Fig. 1A).3 Since
the discovery of PTX as an NCA, many structurally diverse natural
and synthetic compounds have been reported to exhibit antagonist
activity.2 Site-directed mutagenesis and homology modeling stud-
ies have shown that the pore-lining amino acids at the 20-, 60-, and
90-positions of the second TM are involved in the interaction of the
receptor with a wide array of NCAs.4–9 However, the binding
modes of NCAs remain largely unclear, and several lines of evi-
dence suggest the existence of distinct binding sites for different
NCAs.6,10–12 Chemical approaches to clarify the binding mode(s)
and site(s) of NCAs are needed to facilitate rational design and
development of chemicals targeting these important receptors.
Figure 1. (A) Structures of NMB and two representative NCAs of GABA receptors.
(B) Concentration-inhibition curves of NMB on specific [3H]EBOB binding to rat
brain and housefly head membranes. Error bars represent SD (n = 3).
⇑
Corresponding author. Tel.: +81 852 32 6573; fax: +81 852 32 6092.
E-mail address: ozoe-y@life.shimane-u.ac.jp (Y. Ozoe).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.118

H. Shimotahira et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1598–1600
1599
class of straight-chain antagonists, with IC50 values of 88 nM and
3.41
M for rat and housefly GABA receptors, respectively.13 2-Ni-
l
tro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl 4-(4-methoxy-
carbonyl-1-butynyl)benzoate (NMB) (Fig. 1A) was synthesized as
a carbene-generating photoreactive analogue of DBCPP in 5.3%
yield over 8 steps from p-bromoanisole (Supplementary data). This
analogue inhibited [3H]EBOB binding to rat and housefly GABA
receptors in a concentration-dependent manner, with IC50 values
of 1.49 (1.07–2.08) lM and 0.24 (0.16–0.37) lM, respectively
(95% confidence intervals in parentheses) (Fig. 1B). Although the
affinity of NMB for housefly GABA receptors was found to be higher
than that for rat GABA receptors, membranes from housefly heads
were not used in photoaffinity labeling (PAL) experiments due to
their liability under the experimental conditions. Rat brain mem-
branes were used as a rich source of GABA receptors.
PAL experiments were conducted as shown in Figure 2. Stirred
suspensions containing rat brain P2 membranes and NMB were
irradiated at 365 nm in the absence and presence of PTX or fipronil
at 4 °C for 10 h after preincubation for 90 min at 37 °C in a dark
room. The optimum photolabeling condition was determined after
preliminary experiments performed in terms of UV intensity, irra-
diation time, incubation temperature, protein concentration, probe
concentration, ligand concentration, etc. The photolabeled product
was also identified in a model reaction with methanol (data not
shown). The preincubation before irradiation was necessary as a
process to establish an equilibrium. After washing out mem-
brane-bound ligands, the membranes were examined for the abil-
ity to bind [3H]EBOB (Supplementary data).
Figure 3. Photoaffinity labeling of rat GABA receptors with 10
l
M NMB (6.7 Ki) in
the absence and presence of 10 l
M PTX (19.6 Ki).24 Treated membranes were
examined for their ability to specifically bind [3H]EBOB. Error bars represent SD
(n = 3). ⁄P <0.01 (unpaired t-test).
Figure 3 compares the results of PAL of membranes with NMB
in the absence and presence of PTX. Without irradiation, [3H]EBOB
specifically bound to rat brain membranes at 82.2% of the control
level after incubation with NMB followed by washing. The mem-
branes also specifically bound [3H]EBOB after incubation in the
presence of both NMB and PTX, although the levels were slightly
lower than those found in the presence of NMB alone. With irradi-
ation, specific binding of [3H]EBOB was significantly reduced after
incubation with NMB in the absence of PTX. This result indicates
that [3H]EBOB binding was hindered by PAL of the EBOB binding
site with NMB. In contrast, [3H]EBOB binding was not significantly
reduced when similar experiments were performed in the pres-
ence of PTX. This result indicates that noncovalent binding of
PTX protected the EBOB binding site from covalent binding with
NMB.
Figure 4. Photoaffinity labeling of rat GABA receptors with 10 lM NMB (6.7 Ki) in
the absence and presence of 10 l
M fipronil (16.4 Ki).24 Treated membranes were
examined for their ability to specifically bind [3H]EBOB. Error bars represent SD
(n = 6). ⁄P <0.01 (unpaired t-test). ns, not significantly different (P >0.05).
tion of membranes in the presence of both NMB and fipronil re-
sulted in slightly reduced levels of [3H]EBOB binding compared
with those in the presence of NMB alone, most likely because fipro-
nil was not completely washed away from membranes due to its
hydrophobicity. With irradiation, incubation of membranes with
NMB resulted in reduced levels of [3H]EBOB binding due to PAL,
and, in contrast to the results with PTX, these levels were un-
changed in the presence of both NMB and fipronil. This result indi-
cates that fipronil did not protect the EBOB site from access by
NMB and that NMB was capable of forming a covalent bond with
the EBOB binding site even in the presence of fipronil.
Similar PAL experiments were conducted with fipronil, an
insecticide that acts as an NCA (Fig. 4). Without irradiation, incuba-
Despite efforts by several groups, the location of the fipronil
binding site on GABA receptors remains elusive, whereas numer-
ous reports indicate that PTX interacts with the 20 and 60 amino
acid residues of the second TM of each subunit.4,5,15–20 Because
GABA receptors with a mutation at the 20-position were less sensi-
tive to fipronil, fipronil is thought to interact with the 20 residue of
fruit flies (Drosophila melanogaster) and whitebacked planthoppers
(Sogatella furcifera).21,22 Housefly GABA receptors bearing a muta-
tion at the same position, however, bound fipronil with an affinity
similar to that of the wild-type receptor.23 Furthermore, Le Goff
et al. reported that a mutation in the third TM of GABA receptors
of fipronil-resistant fruit flies also reduced the sensitivity of GABA
receptors to fipronil, suggesting a different mode of binding for
fipronil.11 From the results of experiments using chemically reac-
tive fipronil analogues and cysteine mutants of rat GABA receptors,
it was proposed that fipronil interacts not only with the 20 residue
but also with the 170 residue, both of which are located within the
Figure 2. Protocol for photoaffinity labeling with NMB.

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H. Shimotahira et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1598–1600
channel.6 The presence of these two binding sites was supported
by recent homology modeling and docking studies.12 Our present
data indicate that fipronil has a binding site distinct from that of
EBOB and NMB in rat GABA receptors. However, as it is impossible
to readily extrapolate the results obtained with rats to insects in
view of the differences between insect and vertebrate GABA recep-
tors,2 similar studies using insect preparations need to be per-
formed when the sites of action of insecticides are considered.
In conclusion, the unique molecule NMB differentiates the bind-
ing sites of the insecticide fipronil and other NCAs in rat GABA
receptors. This probe will be useful in identifying the cross-linking
site of straight-chain NCAs in GABA receptors and mapping alloste-
ric binding sites. Such studies should provide invaluable informa-
tion for the design of novel NCAs.
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Supplementary data
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Supplementary data associated with this article can be found, in
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References and notes
24. The inhibition constants (Kis) were calculated using the Kd of EBOB = 2.45 nM,
1. The GABA Receptors; Enna, S. J., Möhler, H., Eds., 3rd ed.; Humana Press: Totowa,
2007.
2. Ozoe, Y.; Takaeda, M.; Matsuda, K. In Biorational Control of Arthropod Pests—
Application and Resistance Management; Ishaaya, I., Horowitz, A. R., Eds.;
Springer: Heidelberg, 2009; pp 131–162.
the IC50 of PTX = 0.62 lM, the IC50 of fipronil = 0.728 nM, and the concentration
of [3H]EBOB = 0.5 nM. For calculations see Cheng, Y.; Prusoff, W. H. Biochem.
Pharmacol. 1973, 22, 3099. For experimental details on binding assays see
Supplementary data.