Controlling Ca2+ Permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Photochromic Ion Channel Blockers

Article abstract of DOI:10.1021/acs.jmedchem.8b00756

Ionotropic glutamate receptors (iGluRs) play a critical role in normal brain function and neurodegenerative diseases. Development of light-dependent compounds would enable studies of iGluRs within intact mammalian neural tissue, as light is noninvasive and can be applied with high spatiotemporal precision. Here we develop a potent photochromic antagonist that selectively targets the Ca²⁺ permeable AMPA-type of iGuRs, thus providing an important tool to study the contribution of AMPA-type iGluRs on neuronal activity.

Full text of DOI:10.1021/acs.jmedchem.8b00756

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Brief Article
Controlling Ca2+ permeable #-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) Receptors with Photochromic Ion Channel Blockers
Niels Grøn Nørager, Mette Homann Poulsen, and Kristian Strømgaard
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00756 • Publication Date (Web): 20 Aug 2018
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Journal of Medicinal Chemistry
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Controlling
Ca2+
permeable
α-amino-3-hydroxy-5-methyl-4-
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isoxazolepropionic acid (AMPA) Receptors with Photochromic Ion
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Channel Blockers
Niels G. Nørager†, Mette H. Poulsen† and Kristian Strømgaard*
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copen-
hagen, Denmark.
ABSTRACT: Ionotropic glutamate receptors (iGluRs) play a critical role in normal brain function and neurodegenerative
diseases. Development of light-dependent compounds would enable studies of iGluRs within intact mammalian neural
tissue, as light is non-invasive and can be applied with high spatiotemporal precision. Here we develop a potent photo-
chromic antagonist that selectively targets the Ca2+ permeable AMPA-type of iGuRs, thus providing an important tool
study the contribution of AMPA-type iGluRs on neuronal activity.
agonists8 and recently a photoswitchable tethered antag-
INTRODUCTION
onist7a as well as a photochromic antagonist9. Such antag-
Optical control of neuronal activity has been achieved
onists are highly useful for studying the role of iGluRs in
through coupling of exogenous photosensitive receptors
neuronal activity. In particular, selectively targeting Ca2+-
from bacteria or protozoa into neurons as well as through
permeable AMPARs with photochromic antagonist is of
photosensitization of endogenous receptors using syn-
thetic photoswitchable small molecules.1 Of these endog-
great interest, as these receptors play key roles in brain
development, synaptic plasticity as well as in excitotoxici-
enous receptors, the family of ionotropic glutamate recep-
ty, which is believed to be associated with excessive re-
tors (iGluRs) is a promising target for integrating photo-
10
ceptor activity.2,
Ideally, photochromic antagonists
sensitivity to neuronal activity, due to its importance in
neuronal activity in the vertebrate brain, where it plays an
essential role in mediation of the excitatory synaptic
communication.2 Furthermore, the iGluRs are considered
an attractive drug target, as they are essential to normal
brain function and dysfunction is believed to play an
important role in several neurological and psychiatric
disorders.2-3 The iGluR family comprises the three sub-
families of cation selective ligand-gated ion channels, the
N-methyl-D-aspartate receptors (NMDARs), α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid receptors
(AMPARs) and kainate receptors (KARs). The iGluRs
assemble as hetero- and homo-tetrameric receptor sub-
types that are Ca2+-permeable or -impermeable.2 In most
glutamatergic synapses, the AMPARs and NMDARs are
co-localized in the postsynaptic membrane, where they
generate a dual-component response to glutamate, medi-
ating a fast and slow response component, respectively.4
The role of the KARs is more complex, as they modulate
release of both inhibitory and excitatory neurotransmit-
ters and forms post-synaptic ion channels.5
should enable receptor control, while preserving respon-
siveness to the native input. The existing tethered pho-
toswitchable and photochromic antagonists inhibit the
NMDAR or AMPARs and KARs, respectively, by binding
to the orthosteric binding site in the ligand binding do-
9
main.7a, Thus, acting as competitive antagonists, that
interferes with glutamate binding and activation of the
receptor. Development of photochromic antagonists that
target the receptor beyond the orthosteric binding site is
therefore of great interest. To this end, selective targeting
of Ca2+-permeable iGluRs would further provide a tool to
decipher the role of individual iGluR subtypes in neuronal
excitability and excitotoxicity.
Polyamine toxins are a class of small molecules isolat-
ed from spiders and wasps that are potent use- and volt-
age dependent open-channel blockers of iGluRs.11 They
are important pharmacological tools for examining the
iGluRs, owing to their high potency,11-12 and importantly,
polyamine toxins are capable of distinguishing between
Ca2+-permeable and -impermeable iGluR subtypes, with
selectivity for the former subtype.13
The iGluRs have been photosensitized using caged lig-
ands,6 photoswitchable tethered agonists7, photochromic
Argiotoxin-636 (ArgTX-636) is a polyamine toxin iso-
lated from the Argiope lobata spider, and is a potent, but
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non-selective inhibitor of iGluRs. We have previously
Page 2 of 8
shown to improve potencies in an earlier study (Scheme
1).15 For each of the three templates, six different azoben-
zene moieties were selected. For initial screening of the
ability of the azobenzene scaffold to be accommodated in
the ion channel vestibule, we selected the smallest possi-
ble, unsubstituted azobenzenes. To test differences be-
tween the orientations of the terminal phenyl ring, both
the para and meta substituted azobenzenes were tested.
To improve the spectroscopic properties, four azoben-
zenes were selected that carried electron-donating
groups, dimethylamine or diethylamine, in the 4’-
position. Furthermore, the methylene group connecting
the azobenzene to the rest of the polyamine toxin was
removed, thus attaching the azobenzene directly to the
electron withdrawing amide moiety, which ensured an
asymmetric push-pull electron distribution leading to
redshifted absorption.16 Redshifted absorption was desir-
able, as lower energy light has deeper tissue penetration
and lower risk of tissue damage when applied in living
systems.17 Substitution at the 4’ position had the addition-
al effect of increasing the size of the photoswitchable
moiety, thus potentially increasing the difference between
the conformational space mapped by the cis and trans
isomers.
The photoswitchable compounds were synthesized us-
ing a solid-phase organic procedure inspired by a previ-
ously reported argiotoxin synthesis (Scheme 2).15 The
synthesis began with loading of mono nosyl protected
diamines (4) on a polystyrene resin by reductive amina-
tion to a backbone amide linker (BAL).18 An arginine ami-
no acid was subsequently coupled to the resin-bound
secondary amine (5) providing intermediate 6, which was
extended with N-Teoc protected amino alcohols via a
Fukuyama-Mitsunobu alkylation reaction.19 The Teoc
protection group could subsequently be removed with
TBAF, resulting in intermediate 7, an intermediate for all
synthesized compounds. For synthesis of analogues of 1
and 2, the intermediate 7 was coupled to an asparagine
amino acid, followed by deprotection of the Fmoc group,
to give resin-bound 8. Six carboxyl azobenzenes were
then coupled to 8, and subsequent deprotection of the Ns
group, cleavage from the resin and simultaneous depro-
tection of acid labile protection groups provided the de-
sired photochromic analogues 10a–b and 15a–b in good
yields (8–25%, 9 steps, 76–87% average yields per step).
For the synthesis of analogues of template 3, six carboxy
azobenzenes were coupled directly to 7, followed by
deprotection of the Ns group, cleavage from the resin and
simultaneous deprotection of acid labile protection
groups (17–22, 11–28%, 7 steps, 73–83% average yields per
step).
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2
3
4
5
6
7
8
developed two analogues of ArgTX-636, denoted ArgTX-
93 (1) and ArgTX-48 (2), which are highly potent and
subfamily selective antagonists of the NMDARs and AM-
PARs, respectively.14
Furthermore, we have observed that these compounds
tolerate substitution of the terminal resorcinol moiety
with larger fluorophores with a clear correlation between
size of the fluorophore and retention of potency.15 Hence,
smaller fluorophores such as coumarins and boron-
dipyrromethene fluorophores could be accommodated in
the iGluR ion channel vestibule, while larger flourophores
rendered the compounds inactive. Based on this and the
comparable size of the photoswitchable azobenzene scaf-
fold to the smaller size fluorophores, we designed and
developed polyamine toxins carrying an azobenzene moi-
ety in place of the resorcinol group to incorporate photo-
sensitivity (Scheme 1). One compound, 14b, showed a
significant difference in potency between the cis and
trans isomers, and selectively targets the Ca2+-permeable
AMPARs.
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Scheme 1. Design of photochromic argiotoxin analogues
RESULTS AND DISCUSSION
The class of azobenzenes was chosen as photoswitch
due to its favorable geometric and photochemical proper-
16
ties.1a,
Azobenzenes have well-defined cis and trans
states with little overlap between the conformational
space mapped by the two isomers, thus leading to signifi-
cant geometric change upon isomerization.16 Further-
more, azobenzenes are photostable, have high isomeriza-
tion rates, molar extinction coefficients and quantum
yields, and their spectroscopic properties are highly tuna-
ble through ring substitution.16
The azobenzenes were incorporated in the polyamine
toxin templates of 1 and 2, as well as an analogue of 1
without the asparagine moiety (3), as this excision has
The substituted azobenzenes had been designed to
tune their spectroscopic properties towards redshifted
absorption. To evaluate this, representative absorption
spectra were recorded for the six compounds carrying
azobenzenes coupling to template 2 (Figure S1 in Support-
ing Information), showing redshift of absorption maxi-
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mum from 323–329 nm (10b–11b) to 460–487 nm (12b–
1
15b).
2
3
4
5
6
7
8
O
O
NH
b
Pbf c,d
a
HN
Ns
4
NH2
HN
Ns
NH
m
HN
Ns
N
N
H
6
N
H
m
m
Et2N
Me2N
NHBoc
N
R =
5
N
N
N
N
N
N
O
NH
O
NH
e,f
Pbf
9
H2N
Pbf
14a m = 1, n = 7 (28%)
14b m = 6, n = 2 (26%)
21 m= 1, n = 7 (21%)
H2N
N
Ns
N
N
N
H
10a m = 1, n = 7 (10%) 12a m = 1, n = 7 (21%)
10b 12b m = 6, n = 2 (8%)
17 m = 1, n = 7 (11%) 19 m = 1, n = 7 (28%)
N
N
m
N
H
N
m
n
n
H
H
H
NHBoc
Ns
NHBoc
m = 6, n = 2 (8%)
10
11
12
13
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CONHTrt
7
8
N
N
N
O
O
NH
N
N
O
O
NH
NH2
N
H
N
H
N
g
R
Pbf h,i
R
N
H
N
n
N
N
N
H
N
N
n
N
N
Me2N
Et2N
m
m
H
NHBoc
H
H
H
H
O
Ns
O
NH2
CONHTrt
CONH2
15a m = 1, n = 7 (25%)
15b m = 6, n = 2 (21%)
22 m =1, n = 7 (14%)
13a m = 1, n = 7 (27%)
13b m = 6, n = 2 (10%)
20 m = 1, n = 7 (20%)
11a
m = 1, n = 7 (25%)
11b m = 6, n = 2 (15%)
18
9
10-15
m = 1, n = 7 (17%)
O
O
NH
O
O
NH
N NH2
h,i
g
Pbf
7
R
N
N
N
N
N
R
N
N
n
N
m
H
17-22
n
m
H
H
NHBoc
H
H
H
H
Ns
NH2
16
Scheme 2. Solid-phase synthesis of photochromic compounds 10–15 (a–b) and 17–22. Reagents and conditions: a) BAL PS
resin, NaBH(OAc)3, DMF/AcOH (9:1); b) Boc-Arg(Pbf)-OH, HATU, DIPEA, DCM/DMF (9:1); c) N-Teoc 9-aminononan-1-ol or N-
Teoc 4-aminobutan-1-ol, Bu3P, ADDP, DCM/THF (1:1); d) TBAF, THF, 55°C; e) Fmoc-Asn(Trt)-OH, HATU, DIPEA, DCM/DMF
(9:1); f) 20% piperidine, DMF; g) carboxy azobenzene, HATU, DIPEA, DCM/DMF (9:1); h) DBU, ß-mercaptoethanol, DMF; i)
TFA/DCM/TIPS/H2O (75:20:2.5:2.5). ADDP = 1,1’- azodicarbonyldipiperidine, Boc = tert-butyl carbamate, DBU = 1,8-
diazabicyclo[5.4.0]undecane, DIPEA = N,N-Diisopropylethylamine, DMF = N,N-dimethylformamide, Fmoc = fluorenylme-
thyloxycarbonyl, HATU = N-((dimethylamino)-1H-1,2,3-triazolo[4,5,b]pyridin-1-ylmethylene)-N-methylmethanaminium hex-
afluorophosphate N-oxide, Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Teoc = 2-(trimethylsilyl)ethyloxycarbonyl,
TFA = trifluoroacetic acid, THF = tetrahydrofuran, TIPS = triisopropylsilane, Trt = trityl.
**Statistical difference between inhibition by dark adapted
trans state and illumination adapted cis state of the photo-
chromic antagonists (P<0.001, students t-test)
Compounds carrying an unsubstituted azobenzene
moiety (10a–11a, 10b–11b and 17–18) were initially
screened in the dark to investigate whether this azoben-
zene scaffold could be accommodated in the ion channel
vestibule (Figure S2, Supporting Information). This
showed to be the case for the AMPAR targeting com-
pounds 10b–11b and the NMDAR targeting compound 18,
which showed inhibitions around 70–80% in the dark-
adapted trans form. The substituted analogues were
screened both in the dark (black bars) and under illumi-
nation with blue light (470 nm, grey bars, Figure 1). En-
couragingly, compounds 12b–15b all showed more than
70% inhibition of the AMPAR in the dark-adapted trans
form, while in comparison the NMDAR targeting com-
pounds (12a–15a and 19–22) showed significantly lower
inhibition in the 20–50% range.
Interestingly, illumination of compounds 12b–15b with
blue light (470 nm) led to a reduction in inhibition, espe-
cially for compound 14b, which showed a remarkable 30%
drop in inhibition upon illumination (Figure 1, grey bars).
This difference was even more pronounced when inhibi-
tion was measured at a 10 nM concentration of the photo-
chromic compound, where 14b had approximately three
times higher inhibition in the dark than when illuminated
(dark state inhibition: 60% ± 5%, n=3; Illuminated state
Figure 1. Screening of compounds. Percentage inhibi-
tion by 100 nM photochromic ligand at (A) NMDAR subtype
GluN1/2A and (B) AMPAR subtype GluA1, in the dark (black
bars) and under illumination with blue light (470 nm, grey
bars). Bar graphs are showing mean ± SEM (standard error of
the mean) for n = 3-16 oocytes at a membrane potential of
−80 mV. *Statistical difference between inhibition by dark
adapted trans state and illumination adapted cis state of the
photochromic antagonists (P<0.05, students t-test).
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inhibition: 20% ± 5%, n=3) (Figure 2B). At the same con-
centration of 14b less than 5% inhibition was observed at
the NMDAR, both in the dark and under illumination,
showing that 14b had retained selectivity toward the
AMPAR (Figure 2A-B).
Page 4 of 8
NMDAR as well to investigate subfamily selectivity, and
14b showed significantly lower potencies (IC50 > 1 µM),
thus displaying high selectivity toward the AMPAR sub-
family. This study includes GluA1 and GluN1/2A as repre-
sentative subtypes of the AMPAR and NMDAR subfamilies,
respectively. On that note, polyamines, such as spermine,
can act as potentiators by binding to a low affinity binding
site on the amino terminal domain (ATD) of the GluN1/2B
NMDAR subtype.20 Thus, the polyamine tail of 14b could
potentially interact with the ATD of this particular subtype,
consequently leading to an overall loss of inhibition and
increased antagonistic selectivity towards the AMPAR sub-
family. This selectivity is further substantiated by the
inherent specificity for the Ca2+-permeable subtype of
AMPARs that polyamine toxins demonstrate.
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Table 1. Potency of 14b at NMDAR and AMPARs subtypes.
[d]
Receptor
Illumination IC50 (µM)[a]
nH
NMDAR[b]
Dark
1.1 [0.7–1.9]
-0.8
-0.9
-0.5
-1.0
470 nm
Dark
1.2 [0.8–1.9]
AMPAR[c]
0.008 [0.0043–0.015]
0.061 [0.049–0.077]
470 nm
[a] IC50 values at NMDAR subtype GluN1/2A and AMPAR
subtype GluA1 recombinantly expressed in oocytes and a
membrane potential of −80 mV. Numbers in brackets denote
the 95% confidence interval for IC50. [b] Inhibition of the
Figure 2. Pharmacological characterization of photo-
chromic antagonist 14b. A: Representative electrophysio-
logical trace showing shift in inhibition upon illumination
with blue light (470 nm, highlighted in grey). B: Screening of
percentage inhibition by 10 nM photochromic ligand at the
NMDAR subtype GluN1/2A and the AMPAR subtype GluA1 in
the dark (black bars) and under illumination with blue light
(470 nm, grey bars). Bargraphs are showing mean ± SEM
(standard error of the mean) for n = 3-10 oocytes held at a
membrane potential of −80 mV. C and D: Determination of
IC50 values at NMDAR subtype GluN1/2A and AMPAR sub-
type GluA1. Data points represent the mean ± SEM (standard
error of the mean) for n = 3-10 oocytes held at a membrane
potential of -80 mV. **Statistical difference between inhibi-
tion by dark adapted trans state and illumination adapted cis
state of 14b (P<0.001, students t-test).
current elicited by 100 μM L-Glu and 100 μM Gly by simulta-
neous co-application of the antagonist to oocytes injected
with a 1:10 ratio of cRNA coding for GluN1 and GluN2A subu-
nits, respectively. [c] Inhibition of the current elicited by 300
μM L-Glu by simultaneous co-application of the antagonist to
oocytes injected with cRNA coding for GluA1. [d] nH indicates
the Hill slope of the curves.
CONCLUSION
In summary, we have designed and synthesized a se-
ries of photochromic analogues of ArgTX-93 (1) and Ar-
gTX-48 (2) by incorporating photoswitchable azobenzene
moieties in place of their headgroups. Through a combi-
nation of different azobenzene moeitites and modifica-
tion of the polyamine toxin scaffold, we have tuned the
spectroscopic properties toward red-shifted absorption,
while increasing the difference between the conforma-
tional space mapped by the cis and trans isomers. Phar-
macological characterization of the developed com-
pounds have resulted in the discovery of a photochromic
antagonist, 14b, that acts as a highly potent and selective
dark-adapted trans antagonist of Ca2+-permeable AM-
PARs. This photochromic antagonist, 14b, binds outside
the orthosteric binding site, thus we expect that 14b can
be used to add an additional input signal to Ca2+-
permeable AMPA receptors, that does not perturb with
binding of the endogenous agonist. To this end, given the
importance of AMPARs in neuronal activity, we envision
that 14b could be a highly useful tool for studying the role
of this receptor in excitatory neuronal communication.
Full dose-response curves were generated for 14b at
the AMPAR and NMDAR subtypes to determine the IC50
values, both in the dark and under illumination (Figure
2C-D). The dark-adapted trans isomer of 14b showed to
be a highly potent inhibitor of the AMPAR, having an IC50
value as low as 8 nM [4.3–15], thus being more potent
than the parent polyamine toxin, 2 (Table 1).14 Upon illu-
mination, 14b showed significantly lower potency, inhibit-
ing with an IC50 value of 61 nM [49–77]. Thus, 14b acts as a
trans antagonist, where the inhibition can be reduced by
8-fold upon stimulation with blue light. Moreover, the
difference in potency between the cis and trans isomers is
most likely even bigger, as illumination cannot convert
14b fully into the cis configuration, thus a part of the
observed inhibition is caused by the 10–20% still found as
the trans isomer. IC50 values were determined on the
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ng/nL of GluA1; 0.5 ng/nL of GluN1 and GluN2A in a 1:10 mix-
EXPERIMENTAL SECTION
ture) and incubated in 1-3 days in Barth’s medium (in mM: 88
NaCl, 1 KCI, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 2.4 NaHCO3,
10 HEPES; pH 7.4) with gentamicin (0.10 mg/mL).and used for
recordings 1 to 3 days post-injection.
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Experimental Chemistry.
General procedures.
Unless otherwise stated, starting materials were obtained from
commercial suppliers and were used without further purifica-
tion. The BAL resin used was 2-(3,5-dimethoxy-4-
formylphenoxy)ethyl (DFPE) polystyrene with a loading of 0.87
mmol/g, which was purchased from Novabiochem. N-(3-
Aminopropyl)-2-nitrobenzenesulfonamide,21 N-(8-aminooctyl)-
Voltage-clamp recordings from Xenopus oocytes. Two-
electrode voltage-clamp (TEVC) recordings were made 1–3 days
post-injection. Oocytes were placed in a 500 µL recording cham-
ber with a single perfusion line delivering 5 mL/min perfusion.
Voltage and current electrodes were filled with 3 M KCl. Record-
ings were made using a Warner OC725B two-electrode voltage
clamp (Warner Instruments, Hamden, CT) configured as rec-
ommended by the manufacturer. Oocytes were perfused with a
solution comprised of (mM): 115 NaCl, 2 KCl, 5 HEPES and 1.8
BaCl2, pH 7.6. All experiments were performed at room tempera-
ture (23 °C). Data acquisition and voltage control was accom-
plished using a CED 1401plus analog-digital converter (Cam-
bridge Electronic Design, Cambridge, UK) interfaced with a PC
running WinWCP software (available from Strathclyde Electro-
physiology Software, University of Strathclyde, Glasgow, UK).
2-nitrobenzenesulfonamide,15
2-(trimethylsilyl)ethyl
(4-
hydroxybutyl)carbamate,22 (E)-4-(phenyldiazenyl)benzoic acid23
and (E)-3- (phenyldiazenyl)benzoic acid23 were prepared accord-
ing to literature procedures. Tetrahydrofuran (THF), N,N-
dimethylformamide (DMF) and dichloromethane (DCM) were
dried, degassed and scrubbed using a Glass Contour Solvent
Purification System (SPS) immediately before use.
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Preparative HPLC was performed on a Agilent 1100 system using
a C18 reverse phase column (Zorbax 300 SB-C18, 21.2 × 250 mm)
with a linear gradient of the binary solvent system of wa-
ter/acetonitrile/formic acid (A: 95/5/0.1% and B: 5/95/0.1%) with
a flow rate of 20 mL/min and UV detection at 215 nm and 254
1
nm. H and 13C NMR spectra were recorded on an Avance 400
Screen of photochromic antagonists and determination
of IC50. A screen of the photochromic antagonists was carried
out by measuring the maximal current resulting from bath ap-
plication of a saturating concentration of agonist to the oocyte
expressing either the NMDAR subtype GluN1/2A or AMPAR
subtype GluA1, held at a membrane potential of -80 mV (300 µM
spectrometer (at 400 or 100 MHz, respectively), using CD3OD,
CDCl3 or DMSO-d6 solvents. Chemical shifts are reported in ppm
(δ) using residual solvent as an internal standard; CDCl3: 7.26
(1H), 77.16 (13C) ppm; CD3OD: 3.31, 49.00 ppm; DMSO-d6: 2.50,
39.52 ppm. Coupling constants (J) are given in Hz. The purity of
the compounds was determined by LC-MS and all compounds
are >95% pure. LC-MS analysis was performed on an Agilent
6410 Triple Quadrupole Mass Spectrometer instrument using
electron spray coupled to an Agilent 1200 HPLC system (ESI-
LC/MS) with autosampler and diode-array detector using a
gradient (0–100% B over 3 min, then 100% B for 3 min) of the
binary solvent system of water/acetonitrile/TFA (A: 95/5/0.1%
and B: 5/95/0.1%) with a flow rate of 1 mL/min. During ESI-
LC/MS analysis evaporative light scattering (ELS) traces were
obtained with a Sedere Sedex 85 Light Scattering Detector,
which was used for estimation of the purity of the final products.
Analyses at intermediate stages of the synthesis were performed
by cleaving samples from the resin as follows: a 1 mL screwcap
vial was charged with a 1 mg sample of the resin and 0.4 mL
TFA/DCM/EDT (75:20:5). The vial was agitated periodically over
1 h and filtered using a syringe filter and disposable syringe. The
crude product was analyzed by LC-MS. High resolution mass
spectra (HRMS) were obtained using a Micromass Q-Tof II
instrument. Absorption spectra were recorded on a Thermo
Scientific UV-Vis spectrophotometer. (See supporting infor-
mation)
L-glutamate for GluA1; 100 µM L-glutamate and 100 µM glycine
for GluN1/2A). Subsequently varying concentrations of antago-
nist was applied in the presence of agonist. The resulting current
was normalized to the response produced by the agonist alone.
Measurements in the dark were carried out in a dark room.
Measurements at 470 nm were conducted by addition of photo-
chromic antagonist in the dark, followed by illumination using a
handheld flashlight emitting light with intensity of 470 nm.
Dose-response measurements of polyamine inhibition of the
receptors were performed by measuring agonist-evoked currents
in step-wise increasing concentrations of polyamine toxin. For
determination of IC50, data were pooled among individual exper-
iments at 3 to 10 individual oocytes and the composite dose-
response data were fitted using GraphPad Prism software
(GraphPad Inc, San Diego, CA, USA) by the equation:
1
ꢀꢁꢂꢃꢄꢅꢂꢁ =
1+10((Logꢆꢇ
ꢊꢋ)∗ꢌ
)
ꢈꢉ
ꢍ
Response is the agonist-evoked current response measured at
a given inhibitor concentration normalized to the agonist-
evoked current response in absence of inhibitor, IC50 is the
concentration of inhibitor that produces a half-maximal
inhibition, X is the logarithm of the concentration of the
inhibitor concentration, and nH is the Hill slope.
Experimental biology
Expression of glutamate receptors in Xenopus oocytes.
The cDNA encoding rat GluA1 (flip isoform), rat GluN1-1a and rat
GluN2A subunits inserted in the vectors pGEM-HE and pCIneo
were used for preparation of capped cRNA transcripts. cDNA
was linearized using the NheI or NotI restriction enzymes (New
England Biolabs, Ipswich, MA) and cRNA transcription was
performed using the Amplicap-max T7 high Yield Message Mak-
er mRNA-capping kit (Ambion, Austin, TX, USA) The quality
and quantity of the synthesized cRNA was assessed by gel elec-
trophoresis and spectroscopy. Stage V and VI oocytes were sur-
gically removed from the ovaries of Xenopus laevis frogs and
prepared as described in Poulsen et al.24 under license 2014-15-
0201-00031 from the Danish Veterinary and Food Administra-
tion. Oocytes were injected with 13-46 nL of diluted cRNA (1
ASSOCIATED CONTENT
Supporting Information. Preparation of building blocks
1
and H and 13C NMR spectra of final compounds, as well as
Figures S1 and S2. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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Page 6 of 8
photochromic agonist of ionotropic glutamate receptors. J. Am.
*Phone: +45 3533 6114.
Chem. Soc. 2006, 129 (2), 260-261; (b) Stawski, P.; Sumser, M.;
Trauner, D., A photochromic agonist of AMPA receptors. Angew.
Chem. Int. Ed. 2012, 51 (23), 5748-5751.
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2
3
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8
E-mail: kristian.stromgaard@sund.ku.dk.
Author contribution
9.
Barber, D. M.; Liu, S. A.; Gottschling, K.; Sumser, M.;
†NGN and MHP contributed equally
Hollmann, M.; Trauner, D., Optical control of AMPA receptors using
a photoswitchable quinoxaline-2,3-dione antagonist. Chem. Sci. 2017,
8 (1), 611-615.
Notes
10.
Manev, H.; Favaron, M.; Guidotti, A.; Costa, E., Delayed
The authors declare no competing financial interest.
increase of Ca2+ influx elicited by glutamate: role in neuronal death.
Mol. Pharmacol. 1989, 36 (1), 106-112.
ACKNOWLEDGMENT
9
11.
(a) Strømgaard, K.; Mellor, I., AMPA receptor ligands:
Synthetic and pharmacological studies of polyamines and polyamine
toxins. Med. Res. Rev. 2004, 24 (5), 589-620; (b) Olsen, C. A.;
Kristensen, A. S.; Strømgaard, K., Small molecules from spiders used
as chemical probes. Angew. Chem. Int. Ed. 2011, 50 (48), 11296-11311.
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We are grateful for support from the GluTarget (University
of Copenhagen) for a Ph.D. fellowship to N.G.N and M.H.P.
ABBREVIATIONS
12.
Strømgaard, K.; Jensen, L. S.; Vogensen, S. B., Polyamine
toxins: development of selective ligands for ionotropic receptors.
Toxicon 2005, 45 (3), 249-254.
ADDP, 1,1-(azodicarbonyl)dipiperidine; AMPA, α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid; ArgTX, argio-
toxin; BAL, backbone amide linker; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane;
DIPEA, diisopropylamine; DMF, N,N-dimethylformamide;
13.
Blaschke, M.; Keller, B. U.; Rivosecchi, R.; Hollmann, M.;
Heinemann, S.; Konnerth, A., A single amino acid determines the
subunit-specific spider toxin block of α-amino-3-hydroxy-5-
methylisoxazole-4-propionate/kainate receptor channels. Proc. Natl.
Acad. Sci. USA 1993, 90 (14), 6528-6532.
HATU,
tetramethyluronium hexafluorophosphate; NMDA, N-
methyl- -aspartate; Ns, 2-nitrobenzenesulfonamide; Pbf,
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
14.
Poulsen, M. H.; Lucas, S.; Bach, T. B.; Barslund, A. F.;
D
Wenzler, C.; Jensen, C. B.; Kristensen, A. S.; Strømgaard, K.,
Structure-activity relationship studies of argiotoxins: selective and
potent inhibitors of ionotropic glutamate receptors. J. Med. Chem.
2013, 56 (3), 1171-1181.
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; TBAF,
tetra-n-butylammonium fluoride; TFA, trifluoroacetic acid;
Teoc, 2-(trimethylsilyl)ethyloxycarbonyl; TIPS, triiso-
propylsilane.
15.
Nørager, N. G.; Jensen, C. B.; Rathje, M.; Andersen, J.;
Madsen, K. L.; Kristensen, A. S.; Strømgaard, K., Development of
potent fluorescent polyamine toxins and application in labeling of
ionotropic glutamate receptors in hippocampal neurons. ACS Chem.
Biol. 2013, 8 (9), 2033-2041.
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