A photochromic agonist of AMPA receptors

Article abstract of DOI:10.1002/anie.201109265

Light switch: A photochromic agonist ATA-3 (see scheme) of AMPA receptors, arguably the most important class of ionotropic glutamate receptors, is shown to be subtype selective, activates the AMPA receptor GluA2 in the dark, and is quickly turned off when

Full text of DOI:10.1002/anie.201109265

Angewandte
Chemie
DOI: 10.1002/anie.201109265
Optochemical Genetics
A Photochromic Agonist of AMPA Receptors**
Philipp Stawski, Martin Sumser, and Dirk Trauner*
Optochemical genetics uses small photoswitchable molecules
to control neuronal activity. These can be attached covalently
or noncovalently to their target proteins, which can be ligand
or voltage-gated ion channels as well as G-protein coupled
receptors. The integration of the resulting hybrid photo-
receptors into excitable cells allows for the control of neural
networks with the temporal and spatial precision that only
light provides.[1]
Among different targets for optochemical genetics, iono-
tropic glutamate receptors (iGluRs) stand out owing to their
central role in synaptic transmission. These ion channels are
gated by glutamate (1) and fall into three major classes, each
of which is marked by a distinct pharmacology (Figure 1b).[2]
The so-called AMPA receptors (GluAs), named after the
selective agonist 2-amino-3-(5-methyl-3-hydroxyisoxazol-4-
yl)propanoic acid (2), can be considered the workhorses of
glutamatergic synapses. They are responsible for the major
part of the excitatory neurotransmission in the mammalian
central nervous system and are mostly positioned at the
center of the postsynaptic density. Kainate receptors (GluKs),
named after kainic acid (3), in contrast, are located primarily
on the periphery of the synaptic cleft and play more of
a supporting and modulatory role. NMDA-receptors, named
after their selective response to the agonist N-methyl-d-
aspartate (4), are both voltage- and ligand-sensitive ion
channels and function as coincidence detectors of postsynap-
tic depolarization and synaptic glutamate release.[3]
Our group has a longstanding interest in converting
iGluRs into photoreceptors for optochemical applications. So
far, our efforts have been focused on kainate receptors owing
to the well-defined architecture of their clamshell-like ligand-
binding domain (LBD) and their readily interpretable
pharmacology (Figure 1a). Our efforts have yielded
LiGluR, a light-gated ionotropic glutamate receptor, wherein
a derivative of glutamate was covalently tethered to the
surface of the LBD through a photoswitchable linker
(“photoswitched tethered ligand”, PTL).[4] Shortly thereafter,
we introduced a photochromic ligand (PCL) for kainate
receptors that functions as a “reversibly caged glutamate”
(Figure 1c,d).[5] This molecule, termed 4-GluAzo (5), is an
Figure 1. a) General structure of a glutamate receptor (derived from
protein data bank (PDB): 3KG2) with a close-up of an LBD (PDB:
2P2A). b) The universal agonist and three subtype-selective agonists of
iGluRs. c) Schematic mode of action of a photochromic agonist. d) 4-
GluAzo (5), a PCL acting on kainate receptors.
azobenzene derivative of glutamate that changes its affinity
and efficacy to GluK1 and GluK2 upon photoisomerization.
As such, it can be used to reversibly control neuronal activity
with different wavelengths of light.
[*] Dipl.-Chem. P. Stawski, Dr. M. Sumser, Prof. Dr. D. Trauner
Department of Chemistry and Center for Integrated Protein Science,
Ludwig-Maximilians-Universitꢀt
The rational design of suitable photoswitches for AMPA,
kainate and NMDA receptors has been greatly facilitated by
the availability of numerous X-ray crystal structures.[6] The
clamshell-like ligand-binding domains of these receptors have
been crystallized in conjunction with a variety of agonists,
such as AMPA (2) and domoic acid, antagonists, such as
DNQX, as well as modulators, such as cyclothiazide. Very
recently, the crystal structure of a full tetrameric GluA2
receptor assembly has been disclosed.[7] This seminal work
provided insights into the overall architecture and symmetry
Butenandtstrasse 5–13 (F4.086), 81377 Munich (Germany)
E-mail: dirk.trauner@lmu.de
Homepage: http://www.cup.uni-muenchen.de/oc/trauner/
[**] P.S. is grateful to the Fonds der Chemischen Industrie for a Kekulꢁ
Fellowship. We thank S. Strych, D. T. Hog, D. Woodmansee, and V.
Erdmann for their helpful discussions. This work was funded by
SFB 870 and the ERC (grant no. 268795).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201109265.
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of AMPA receptors in particular and ionotropic glutamate
receptors in general.
kainate receptors (Figure 2). This structure suggested to us
that extending the benzene to an azobenzene would allow the
molecule to reach the solvent-exposed surface while still
permitting the ligand binding domain (LBD) to close
sufficiently for receptor activation. It could also be deduced
that this elongation would require a meta-substitution with
respect to the first benzene ring of the azobenzene unit.
To test our hypothesis that ATAs would function as
photochromic agonists, we synthesized several variants (7a–
d; Figure 2). These molecules have different substitution
patterns and photophysical properties owing to the presence
or absence of substituents in the 4’-position of the azoben-
zene. The dimethylamino substituent in 7c, for instance, shifts
the absorption spectrum of the trans-isomer toward the red
(lmax = 456 nm), which allows the use of longer wavelengths
that are better tolerated by cells during prolonged exposure.
The synthesis of ATA-3 (7c) was modeled after the
published route to BnTetAMPA (6),[8] but relied on an
improved protecting-group strategy (Schemes 1 and 2). It
Although the clamshell-like LBDs of AMPA and kainate
receptors share a similar overall architecture, there are subtle
differences that apparently prevent 4R-substituted glutamate
derivatives, such as 4-GluAzo (5), from acting as agonists.
One of the reasons for this inhibition may be that the LBD of
AMPA receptors is closed more tightly around the ligand in
the activated form. Thus, side chains attached to a glutamate
molecule cannot be as easily accommodated as in the case of
GluKs.
Accordingly, glutamate derivatives with a photoswitchable
side chain, such as 4-GluAzo (5) were deemed unsuitable for
the light-dependent stimulation of AMPA receptors and we
decided to radically change our molecular design. We now
report a new class of molecules termed ATAs (azobenzene
tetrazolyl AMPAs, 7a–d, Figure 2). These compounds are
photochromic derivatives of AMPA itself, which selectively
target GluA receptors expressed in HEK cells and neurons.
One of them (7c) effectively triggers neuronal firing in the
dark and quickly inactivates when irradiated with blue–green
light. As such, it could be a useful tool for the study of neural
circuitry controlled by AMPA receptors and a potential
therapeutic tool for the restoration of vision with artificial
photoswitches.
Scheme 1. Synthesis of racemic key intermediate 13.
commenced with the known hydroxy isoxazole 8,[9] which was
protected as an allyl ether to afford methyl ester 9.[10] This
intermediate was then converted into nitrile 10 in a two-step
procedure.[11] Lithiation of 10, followed by conjugate addition
of the obtained organolithium compound to dehydroalanine
11, yielded racemic AMPA derivative 12. This step required
careful optimization but could be carried out reliably and on
large scale at very low temperatures. A 1,3-dipolar cyclo-
addition of hydrazoic acid to nitrile 12 then furnished
tetrazole 13, which served as a common intermediate in the
synthesis of all the ATAs (7a–d).
Figure 2. The structure of BnTetAMPA (6) and its complex with the
GluA2 LBD (PDB: 2P2A) and the structures of ATA-1 (7a) and ATA-2–4
(7b–d) shown in their respective trans-form. The green circle indicates
the previous “exit tunnel”, whereas the orange circle shows the
approximate location of the newly identified “exit tunnel”.
Mitsunobu coupling of tetrazole 13 with azobenzene 14[13]
gave the N2-alkylated tetrazole 15 as the major isomer
(Scheme 2). The undesired, N1-alkylated regioisomer could
be separated by HPLC after removal of the allyl group under
mild conditions,[12] which yielded hydroxy isoxazole 16.
Subsequent global deprotection using trifluoroacetic acid
(TFA) gave the free amino acid 7c after reversed phase
chromatography. The synthesis of 7b and 7d as well as the
para-substituted control compound 7a was carried out
analogously.[13]
The design of 7a–d is based on a recently published X-ray
structure of the GluA2 LBD in conjunction with the potent
and highly selective agonist BnTetAMPA (6).[8] This molecule
is a derivative of AMPA, wherein the methyl substituent on
the isoxazole ring is replaced with a tetrazole benzylated in
the N2-position. As seen in the crystal structure, the benzyl
substituent occupies a cleft in the receptor that is different
from the “exit tunnel” that we previously exploited in the
As anticipated, ATA-3 (7c) and ATA-4 (7d) showed
significant red-shifted absorption spectra compared to ATA-
2
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Scheme 2. Synthesis of the PCL ATA-3 (7c). DEAD=diethyl azodicar-
Figure 3. Light-induced current recorded from GluA2-expressing cells
boxylate.
a) Action spectrum of ATA-3 (7c), b) action spectrum recorded with
intermittent darkness from a different cell (c=50 mm).
1 (7a) and ATA-2 (7b).[13] In contrast to its congeners, ATA-3
(7c) could not be actively switched from its cis- to its trans-
state in physiological buffer solution because of the complete
overlap of the p–p* and n–p* absorption bands. However,
thermal relaxation was found to be sufficiently fast under
these conditions.
With 7a–d in hand, we assessed their biological activity in
mouse cortical slices and HEK293T cells using whole-cell
patch-clamp electrophysiology (Figures 3 and 4). Whereas 7a
was found to be completely inactive in both preparations and
7b gave inconclusive results, compounds 7c and 7d proved to
be effective trans-agonists of GluA2 transiently expressed in
HEK293T cells. This result confirmed that the meta-substitu-
tion at the azobenzene moiety is a necessary structural
requirement.
likely show considerably higher activity, as has been observed
for other AMPA-derivatives.[14]
We evaluated the ability of 7c to control action potentials
in excitable cells. Action potentials were recorded from layer
2/3 neurons in mouse cortical slices. When 7c was applied at
50 mm concentration, trains of action potentials could be
generated reliably upon switching from 480 nm light to
darkness (Figure 4). Owing to the moderate intensity of the
Since 7c showed the highest activity and best kinetics we
decided to focus our further investigations on this PCL. Its
action spectrum at GluA2 recorded in HEK293T cells is
shown in Figure 3. As designed, 7c elicited the strongest
inward current in its dark-adapted trans-state. Irradiation
with varying wavelengths of light then produced photosta-
tionary states that gave less or no current at all (Figure 3a). In
addition, the kinetics of ATA-3 (7c) currents in the dark and
at different wavelengths of light were determined (Fig-
ure 3b). We found that the fastest abrogation of current was
achieved by switching from darkness to l = 480 nm light
[toff = (47.2 Æ 7.7) ms].[13] When 7c was tested against GluK2,
no response (neither agonistic nor antagonistic) was
observed.[13] This result demonstrates the selectivity of the
ATA chemotype for AMPA over kainate receptors.
Figure 4. Reversible generation of action potentials by 7c in mouse
cortical neurons (resting potential À65 mV).
light used (20 mWmÀ1 m2)[13] recordings could be performed
over prolonged periods without any apparent photodamage.
The activity of 7c in neural networks was further probed
using known glutamate receptor pharmacology. GYKI-
52466,[15] an AMPA-selective antagonist, blocked the effect
of the PTL as did the AMPA/kainate antagonist CNQX.
Additional experiments with the selective NMDA antagonist
AP5[16] failed to prevent action potential firing.[13] This result
As a final characterization step in non-excitable cells, we
determined the efficacy of 7c at GluA2 expressed in
HEK293T cells. The compound acts as a partial agonist with
an EC50 of 24 mm in the dark and is virtually inactive under
480 nm illumination.[13] However, enantiopure material will
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demonstrates that 7c can discriminate between all three
subtypes of iGluRs and selectively acts on AMPA receptors.
In summary, we have developed a photochromic agonist
of AMPA receptors that stimulates neurons in the dark but
can be rapidly turned off when irradiated with blue–green
light at moderate intensities. ATA-3 (7c) uses a novel “exit
tunnel” that should allow for the design of new, highly
AMPA-selective ligands, which are not necessarily photo-
chromic. Given the prominence of AMPA receptors in
synaptic transmission, this molecule could be used as an
effective tool to optically control neuronal activity in a wide
variety of networks. Its response to light matches the logic of
OFF-bipolar cells, and by extension OFF-retinal ganglion
cells, which increase firing in the darkness and show
decreased activity when light reaches their receptive field.
As such, 7c could be a useful component of our ongoing
efforts to restore vision with small photochromic molecules.
Received: December 31, 2012
Published online: && &&, &&&&
Keywords: bioorganic chemistry · diazo compounds ·
.
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optochemical genetics · photopharmacology · receptors
[1] T. Fehrentz, M. Schçnberger, D. Trauner, Angew. Chem. 2011,
123, 12362 – 12390; Angew. Chem. Int. Ed. 2011, 50, 12156 –
12182.
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Chemie
Communications
Optochemical Genetics
P. Stawski, M. Sumser,
D. Trauner*
&&&& — &&&&
A Photochromic Agonist of AMPA
Receptors
Light switch: A photochromic agonist
ATA-3 (see scheme) of AMPA receptors,
arguably the most important class of
ionotropic glutamate receptors, is shown
to be subtype selective, activates the
AMPA receptor GluA2 in the dark, and is
quickly turned off when irradiated with
blue–green light. It can be used to
effectively control neuronal activity in the
mammalian brain.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!