Chemoselective regulation of TREK2 channel: Activation by sulfonate chalcones and inhibition by sulfonamide chalcones

Article abstract of DOI:10.1016/j.bmcl.2010.05.033

Although it has not been extensively studied, a significant volume of literature suggests that TREK2 will probably turn out to be an important channel in charge of tuning neuronal transmitter and hormone levels. Thus, pharmacological tools which can manip

Full text of DOI:10.1016/j.bmcl.2010.05.033

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4237–4239
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemoselective regulation of TREK2 channel: Activation by sulfonate
chalcones and inhibition by sulfonamide chalcones
Eun-Jin Kim b, Hyung Won Ryu a, Marcus J. Curtis-Long c, Jaehee Han b, Jun Young Kim a, Jung Keun Cho a,
a,
Dawon Kang b, , Ki Hun Park
*
*
a Division of Applied Life Science (BK21 program), EB-NCRC, Institute of Agriculture & Life Science, Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea
b Medical Research Center for Neural Dysfunction, Department of Physiology, School of Medicine, Gyeongsang National University, Jinju 660-751, Republic of Korea
c 12 New Road, Nafferton, East Yorkshire, YO25 4JP, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Although it has not been extensively studied, a significant volume of literature suggests that TREK2 will
probably turn out to be an important channel in charge of tuning neuronal transmitter and hormone lev-
els. Thus, pharmacological tools which can manipulate this channel, such as selective agonists are essen-
tial both in drug design and to further our understanding of this system. Our investigations have shown
that sulfonate (‘O’) chalcone and sulfonamide (‘N’) chalcones regulate the TREK2 channel in remarkably
Received 30 January 2010
Revised 30 April 2010
Accepted 12 May 2010
Available online 19 May 2010
different ways: sulfonamide chalcone 5 behaved as an inhibitor with an IC50 of 62
fonate analogue 11 activated TREK2 with EC50 value of 167 M.
lM, whereas the sul-
Keywords:
l
TREK2 channel
Sulfonate chalcone
Sulfonamide chalcone
Ó 2010 Elsevier Ltd. All rights reserved.
Potassium channels constitute the most diverse group of ion
channels. Four families of potassium channels in neurons have
been defined: voltage-gated (Kv), calcium-activated (Kca), inward
rectifying (Kir) and two-pore (K2p) potassium channels.1 The salient
feature of K2p membrane topology is they have four transmem-
brane domains (TM1-4) and two-pore forming domains (P1 and
P2).2 Two-pore domain K+ (K2P) channels contribute to background
or leak K+ currents that help set the resting membrane potential
and regulate cell excitability.3
TREK2 activators are applicable to neuropathic pain, neuroprotec-
tion, and insulin secretion. Importantly, recent studies have re-
ported that noradrenergic and GABAergic depressions of neuronal
excitability are related to modulation of the TREK2 channel.9,10
TREK2 inhibitors can be anticipated to exert an effect on the path-
ophysiology of mood disorders because TREK2 is inhibited by anti-
psychotics.11 Therefore, TREK2 channels have been suggested to be
a new potential therapeutic target for the treatment of neuropathic
pain and mood disorders.
Among K2P channels, TREK1 and TREK2 (TREKs) are mainly ex-
pressed in the central nervous system and are activated by a vari-
ety of pathophysiological stimuli, such as heat, intracellular
acidosis, and increased membrane tension.4 Evidence gathered
from the electrophysiological properties of TREK2 and pathological
changes of neuropathic pain suggests that neuronal excitability of
DRG neurons can be decreased in the process of neuropathic pain
by activating TREK2.5,6 TREK2 is also probably important in neuro-
protection by tuning the level of the resting membrane potential.
In this way it can cause a decrease in brain cell excitability and also
lower release of stimulative neurotransmitters.7 TREK2 can be
clearly identified in insulin secreting MIN6 cells, and contributes
to the background K+ conductance in MIN6 cells and hence may
regulate depolarization-induced secretion of insulin.8 In sum,
Chalcone is a biosynthetic product in the shikimate pathway
and can be easily obtained through the Claisen–Schmidt condensa-
tion of benzaldehyde and acetophenone using either basic or acidic
catalysis.12 Previously, we reported that sulfonate chalcones can
act as voltage-dependent K+ channel blockers:13 whereas sulfon-
amide chalcones showed antitumor activities in hepatocytes.14
In this study, we found that two series of compounds with clo-
sely similar chemical structures, sulfonamide and sulfonate chal-
cones, were able to decrease or increase the activity of this
channel, respectively (Fig. 1). All chalcones were tested for their
ability to regulate channel activity in HEK-293 cells which had
been co-transfected with rat TREK2 DNA.15 Cells expressing GFP
were detected by epifluorescence with a microscope equipped
with a mercury lamp light source. Cells were used one to three
days after transfection.
All sulfonamide chalcones 4–9 and sulfonate chalcones 10–16
were obtained through sulfonation of 4-hydroxyacetophenone or
4-aminoacetophenone with the relevant sulfonyl chloride deriva-
tive and an ensuing condensation of the sulfonated acetophenone
* Corresponding authors. Tel.: +82 55 751 8744 (D.K.); tel.: +82 55 751 5472; fax:
+82 55 757 0178 (K.H.P.).
E-mail addresses: dawon@gnu.ac.kr (D. Kang), khpark@gnu.ac.kr (K.H. Park).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.033

4238
E.-J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4237–4239
O
1.5
*
*
R1
*
OH
1.0
O
S
O
R2
R2
1
1
*
Activator:
O
Inhibitor:
N
S
R =
R = H
O
O
*
*
Figure 1.
0.5
*
*
and benzaldehyde using a catalytic amount of H2SO4 in MeOH
(Fig. 2).
0.0
Control
As shown in Figure 3 chalcones 1–3 which do not possess the
sulfonyl moiety do not effect inhibition or activation of whole-cell
TREK2 current in transfected HEK-293 cells, recorded at 60 mV. On
the other hand, sulfonamide chalcones 4–9 were found to exert an
inhibitory effect on whole-cell current in TREK2 (Fig. 3).16 The
TREK2 current was inhibited dose-dependently by sulfonamide
1
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16
Figure 3. Summary of effects of chalcone species (1–16) on TREK2 currents in HEK-
293 cells. The ratios of current recorded in presence of each individual test
compound (100 lM each) to the current in absence of compound (control) are
shown as mean values SD. The currents were measured at +60 mV. Asterisk (Ã)
indicates a significant difference against the control without application of chalcone
compounds (p <0.05).
chalcone 5 with an IC50 of 62 lM (Fig. 4A). Figure 5A and C show
that compound 5 elicits a very significant, rapid, stable and revers-
ible (shown by the washed out experiment) inhibition of whole-
cell TREK2 current. The current-potential relationship shows that
sulfonamide (5) inhibition of TREK2 current was observed at all
potentials (Fig. 5C). Inhibition of TREK2 currents is maintained as
along as sulfonamide application is maintained. When the free hy-
droxyl group (in 5, for example) was deleted (9) or protected (8),
potency was decreased (IC50, 62 vs >100 lM). This implies that a
H-bond donor is important in this position. Our data further indi-
cate that a para-hydrophobic group (Me) may be preferred on
the arene ring of the sulfonate group. The identification of a new
O
2'
2
Figure 4. Dose–response curve for duel effect of sulfonated chalcones on TREK2
channel. (A) The inhibition of TREK2 current by increasing concentrations of
chalcone compound 5, (B) The activation of TREK2 current by increasing concen-
trations of chalcone compound 11. Values are means SD from the data of 3–5 cells
R1
R2
4
4'
1 R1 =H, R2 = H
and fitted with logistic function for calculating IC50 or EC50
.
2 R1 =OH, R2 = OH
3 R1 =NH2, R2 = OH
O
O
R1
S
N
R2
H
O
4 R1 =H, R2 = OH
5 R1 =CH3, R2 = OH
6 R1 =F, R2 = OH
7 R1 =NH2, R2 = OH
8 R1 =CH3, R2 = OCH3
9 R1 =CH3, R2 = H
O
O
R1
S O
O
R2
10 R1 = H, R2 = OH
11 R1 = CH3, R2 = OH
12 R1 = F, R2 = OH
Figure 5. Comparison of effect of chalcone compounds on TREK2 current in
whole-cell mode. (A and B) Reversible effect of chalcone compound 5 and 11 on
TREK2 current. Whole-cell currents were recorded from HEK-293A cells express-
ing TREK2 before and after application of chalcone compounds 5 and 11 and
washout (n = 5). (C and D) The current-potential relationship show 5, 11 effect
on the TREK2 current observed at all potentials. Cell membrane potential was
held at À80 mV, and ramp pulses were applied from À120 mV to +60 mV once
every 5 s. Pipette solution contained 150 mM KCl and bath solution contained
5 mM KCl and 135 mM NaCl.
13 R1 = NO2, R2 = OH
14 R1 = NH2, R2 = OH
15 R1 = CH3, R2 = OCH3
16 R1 = CH3, R2 = H
Figure 2. Dual effect of chalcones on TREK2 Channel.

E.-J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4237–4239
4239
class of inhibitor is highly important because TREK2 is insensitive
Supplementary data
to classic potassium blockers such as tetraethylammonium and
Ba2+. These results further compare favorably with the prototypical
TREK2 inhibitor, quinine, that inhibits currents with an IC50 of
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.033.
100 l
M.17
Thus we were able to show that sulfonamide analogues are new
leadstructures forinhibitor screensofthe TREK2 channel. Derivatives
may prove to be useful for the treatment of mood disorder because
TREK2 is an important channel in a variety of neurotransmitters.
Sulfonate chalcones 10–14, which we have reported recently to
be voltage-dependent K+ channel blockers,13 were next investi-
gated. Surprisingly this structural class emerged to be activators
of the TREK2 channel. Figure 4B illustrates the dose-dependent
stimulating effect of sulfonate chalcone 11 on TREK2 current with
References and notes
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an EC50 of 167 lM. The increase in TREK2 activity was rapidly re-
versed after washing out of the compound, consistent with a
reversible interaction (Fig. 5B). The current-potential relationship
shows that sulfonate stimulation of TREK2 current was observed
at all potentials (Fig. 5D).
Once again, a hydroxy group on the B-ring is essential for acti-
vation because protected compound 15 and compound 16 which is
unsubstituted at this position did not give a significant difference
in activation from the control. This structure–activity relationship
is similar to that found for the sulfonamide chalcone derivatives
above. The compounds (13, 14) having amino or nitro group in
R1 were also not activators (Fig. 3).
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15. Cell culture and transfection of HEK-293 cells: HEK-293 cells were seeded at a
density of 2 Â 105 cells per 35 mm dish 24 h prior to transfection in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS;
Invitrogen, Grand Island, NY, USA) and 50 U/ml penicillin and streptomycin
In summary, we have made a thorough study of two structurally
similar compounds and their effect on TREK2 currents in transfected
HEK cells. Quite surprisingly, we uncovered that subtle changes in
structure were able to change the compound from an inhibitor (sul-
fonamide) to an activator (sulfonate). The structural requirements
for activity in each series were similar: a 4-hydroxy group in the B-
ring was found to be optimal in each case. This work has unveiled
a new class of both activator and inhibitor of the TREK2 channel.
These compounds are easily synthesized and are highly amenable
to SAR. Importantly, as they share similar structural requirements,
which could streamline any further investigations. We believe that
they will be highly useful as lead compounds in drug design and
pharmacology in the future.
(Invitrogen). HEK-293 cells were co-transfected with
(GenBank accession No, NM 023096) in pcDNA3.1 and pcDNA3.1/green
fluorescent protein (GFP) using LipofectAMINE (Invitrogen) and Opti-MEMÒ
a rat TREK2 DNA
I
Reduced Serum Medium (Invitrogen). For electrophysiological experiments,
transfected cells were plated and grown on 12-mm microscope cover glasses,
which were coated with poly-L-lysine for optimal cell attachment, in 35-mm
culture dishes and maintained for 48 h at 37 °C in a humidified atmosphere of
95% air and 5% CO2. The cells expressing GFP were detected by epifluorescence
with
a microscope (Axiovert 135; Carl Zeiss Jena GmbH, Jena, Germany)
equipped with a mercury lamp light source. Cells were used one to three days
after transfection.
16. (a) Selected spectroscopic data 4: mp: 207–208 °C; 13C NMR (75 MHz; CD3OD)
d 115.5, 117.9, 118.5, 126.3, 126.8, 128.4, 129.7, 130.5, 132.8, 133.6, 139.7,
142.3, 145.2, 160.3 and 189.6. (b) 5: mp: 206–208 °C; 13C NMR (75 MHz;
CD3OD) d 115.5, 117.9, 118.5, 126.3, 126.8, 128.4, 129.7, 130.5, 132.8, 133.6,
139.7, 142.3, 145.2, 160.3 and 189.6; (c) 6: mp: 226–229 °C; 13C NMR (75 MHz,
DMSO-d6) d 148.4, 146.1, 145.0, 131.35, 127.3, 124.8, 122.5, 120.9, 119.1,
118.3, 113.8, 25.7, 18.1212, 25.7, 18.1 and 4.22 (d) 7: mp: 216–217 °C; 13C
NMR (75 MHz; acetone-d6) d 113.1, 115.9, 118.2, 118.6, 125.7, 126.9, 129.2,
129.8, 130.6, 133.4, 142.9, 143.9, 153.1, 159.9 and 187.6.
Acknowledgments
This work was supported by the National Research Foundation
of Korea (NRF) by the Korean government (Nos. 20090081751,
R13-2005012010020). H.W.R. and E.J.K. were supported by a schol-
arship from the BK21 program.
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