A Photoswitchable Inhibitor of the Human Serotonin Transporter

Article abstract of DOI:10.1021/acschemneuro.9b00521

The human serotonin transporter (hSERT) terminates serotonergic signaling through reuptake of neurotransmitter into presynaptic neurons and is a target for many antidepressant drugs. We describe here the development of a photoswitchable hSERT inhibitor, termed azo-escitalopram, that can be reversibly switched between trans and cis configurations using light of different wavelengths. The dark-adapted trans isomer was found to be significantly less active than the cis isomer, formed upon irradiation.

Full text of DOI:10.1021/acschemneuro.9b00521

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Letter
A Photoswitchable Inhibitor of the Human Serotonin Transporter
Bichu Cheng, Johannes Morstein, Lucy Kate Ladefoged, Jannick
Bang Maesen, Birgit Schiøtt, Steffen Sinning, and Dirk Trauner
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00521 • Publication Date (Web): 10 Apr 2020
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A Photoswitchable Inhibitor of the Human Serotonin Transporter
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Bichu Cheng†,‡, , Johannes Morstein‡, Lucy Kate Ladefoged#, Jannick Bang Maesen§, Birgit Schiøtt#,
&
Steffen Sinning*,§, and Dirk Trauner*,‡
†School of Science, Harbin Institute of Technology (Shenzhen), Shenzhen 518055, China
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‡Department of Chemistry, New York University, 100 Washington Square East, New York, New York 10003, United States
§Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark
#Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark
&These authors contributed equally
serotonin reuptake inhibitors (SSRIs), including cital-
opram (2), are used clinically to treat anxiety and de-
pression (Fig. 1).6,7 Citalopram (2) was developed by
the pharmaceutical company Lundbeck and first mar-
keted in 1989 in Denmark. It binds with high affinity
and selectivity to SERT relative to other monoamine
transporters.8 Citalopram was originally used as a ra-
cemic mixture but Lundbeck later introduced the
more potent (S)-enantiomer as escitalopram (3).
ABSTRACT: The human serotonin transporter
(hSERT) terminates serotonergic signaling through
reuptake of neurotransmitter into presynaptic neu-
rons and is a target for many antidepressant drugs. We
describe here the development of a photoswitchable
hSERT inhibitor, termed azo-escitalopram, that can be
reversibly switched between trans and cis configura-
tions using light of different wavelengths. The dark-
adapted trans isomer, was found to be significantly
less active than the cis isomer, formed upon irradia-
tion.
Keywords: Photopharmacology, Serotonin, Trans-
porter, Photochromic Ligand, Neurotransmitter
Figure 1. Chemical structures of serotonin, citalopram, and
escitalopram.
Serotonin (1) or 5-hydroxytryptamine (5-HT) is a
monoamine neurotransmitter and plays a number of
important roles in physiology and neurophysiology.1,2
Most of the human body's serotonin is located in the
enterochromaffin cells in the gastrointestinal tract,
where it regulates intestinal movements. The remain-
der is synthesized from tryptophan in serotonergic
neurons of the central nervous system, and is released
into the synaptic cleft, where it activates serotonin re-
ceptors of the postsynaptic neurons. Serotonergic sig-
naling influences neurological processes including
sleep, mood, cognition, pain, hunger and aggression
behaviours.3 The serotonin transporter (SERT or 5-
HTT) is a member of the neurotransmitter sodium
symporter (NSS) family of transporters. It is responsi-
ble for the sodium- and chloride-dependent reuptake
of serotonin from the synaptic cleft back to the presyn-
aptic neuron, which terminates the serotonergic sig-
naling and simultaneously enables the recycling of
serotonin by the presynaptic neuron.4
Using in silico docking and experimental validation
with a combination of citalopram analogs and hSERT
mutants it was shown in 2010 that escitalopram binds
to the same site as 5-HT, the S1 site.9,10 Escitalopram
was proposed to reside in an orientation wherein the
protonated aliphatic tertiary amine forms a salt bridge
to the conserved Asp98 in subsite A, the fluorophenyl
group utilizes subsite B lined by Ala173, Asn177, and
Thr439, whereas the cyanophenyl group engages sub-
site C lined by Phe335 and Phe341. This orientation
was confirmed by X-ray crystallographic structure of
human SERT bound to the antidepressant escital-
opram in 2016.11,12 Escitalopram locks SERT in an out-
ward-open conformation by lodging in the central
binding site, directly blocking serotonin binding and
preventing the binding site from closing by protruding
the cyanophenyl moiety through the aromatic lid nor-
mally occluding the substrate.9–11 This implies that the
cyano group will be most forgiving to chemical modi-
fications because substituents would extend up
through the extracellular vestibule towards a second
allosteric site,13 which is also supported by structure-
Due to its importance in synaptic transmission and
serotonin homeostasis, SERT has been subject to in-
tense pharmacological studies, which gave rise to
many clinically approved antidepressants.5 Selective
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activity relationship studies on citalopram analogs,14
in a similar fashion to what we have shown for substi-
tutions on the 3-position of the tricyclic antidepres-
sant imipramine.15
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Scheme 1. Design and synthesis of azo-escitalopram (5).
In recent years, our group and others have developed a
range of synthetic azobenzene photoswitches, either as
freely diffusible photochromic ligands (PCLs), pho-
toswitchable tethered ligands (PTLs), or photoswitcha-
ble orthogonal remotely tethered ligands (PORTLs), that
can be used to optically control the function of many bi-
ological systems.16–20 These include ion channels21–23, G-
protein coupled receptors (GPCRs)24–26, enzymes27,28, li-
pids22,26,29, nuclear hormone receptors30, and recently
transporters31–33. We now describe the development of a
photoswitchable azobenzene derivative of escitalopram
that allows for the precise and reversible control of
hSERT activity with light.
design of an azobenzene analog which largely retains the
structure of the parent compound. This well-prece-
dented azologization approach35 yields a version of es-
citalopram that can undergo photoisomerization. We hy-
pothesized that the two photoisomers would exhibit
marked structural differences, resulting in different po-
tencies, which would enable the optical control of SERT
activity.
We termed the azostere of the escitalopram derivate 4
“azo-escitalopram” (5). The synthesis of 5 began from
commercially available escitalopram oxalate (6). Neu-
tralization of 6 afforded the free amine, which under-
went a rhodium-catalyzed nitrile hydration reaction to
afford the primary amide 7.36 An NBS mediated Hofmann
rearrangement and deprotection of the resulting carba-
mate 8 generated aniline 9.37 A Baeyer-Mills reaction of
aniline 9 with nitrosobenzene then afforded the desired
product 5, which was isolated in its hydrochloride salt
form (Scheme 1).
Extensive structure-activity studies at the 4- and 5-posi-
tions of the dihydroisobenzofuran moiety of escital-
opram were conducted by the Newman group.34 They
found that replacement of the cyano group with a bulkier
phenylvinyl (4) substituent retained most of the binding
affinity with SERT (racemic Ki = 9.32 nM, (S)-enantiomer,
Ki = 10.6 nM), and it showed higher binding affinity at
SERT compared to its saturated phenylethyl analogue
(racemic, Ki= 38.1 nM). Compound 4 is an azobenzene
isostere (“azostere”) which allows for straightforward
In its dark-adapted state, azo-escitalopram (5) exists in
its thermally stable trans configuration with high absorp-
tion at 340 nm. It could be isomerized to its cis-form with
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UV-A (λ = 365 nm) light. Isomerization from cis to trans
could be achieved with blue light (λ = 460 nm). Pho-
toisomerization could be repeated over many cycles
without obvious fatigue. In its cis form, 5 remained stable
in the dark (Scheme 2 B).
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the inhibitory potency of 5 is significantly increased
(P=0.0087, paired t-test) by photoswitching to the cis state
by irradiation with light at 365 nm.
A
B
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We found that the potency of escitalopram was, as expected,
unaffected by darkness or light at 365 nm whereas the po-
tency of 5 was low in the dark (trans-state) but increased
43-fold (p=0.0087, paired t-test) in the cis-state after illumi-
nation with 365 nm light. The potency of 5 in the cis-state
was found to be 18.9 nM as compared to 819 nM in the
trans-state. To confirmed that this increase in activity was
not due to rapid degradation to a more potent derivative,
we exposed 5 to glutathione (10 mM) for 1h in the presence
and absence of light. The azobenzene was found to be stable
under these conditions (Supporting Fig. 1)
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Scheme 2. Photoisomerization of azo-escitalopram (5) in
10% DMSO in PBS (50 µM). A) Absorption spectra of 5 in
the dark-adapted, UV-A adapted, and blue-adapted state. B)
Repeated cycles of trans-cis isomerization of 5 with 460 nm
and 365 nm light.
With ample supplies of azo-escitalopram (5) in hand, we
proceeded to test its action on the serotonin transporter.
We performed uptake assays on HEK293MSR cells ex-
pressing hSERT to study the potency of 5 in inhibition of
3H-5-HT uptake. A dilution series of 5 was prepared in
the dark, mixed with 3H-5-HT and then distributed to mi-
crowell plates where the compounds were either left in
the dark or illuminated with 365 nm light from a
handheld device for two seconds every minute in the
time leading up to the experiment. Cells were first prein-
cubated for 25 minutes with the inhibitor in either dark-
ness or under illumination with 365 nm for two seconds
every minute to achieve equilibrium. The uptake was in-
In order to show that the photoswitchable inhibitor could
be used to control the transport activity of hSERT in real
time, we investigated the ability of 5 to photoswitch during
an activity assay for the serotonin transporter (Figure 3).
We initially equilibrated the transporter with trans-5 at 460
nm for 20 minutes and then initiated uptake by addition of
3H-5-HT. The inhibitor was maintained in the trans configu-
ration for 8 minutes by brief illumination with light at 460
nm for two seconds every minute, after which the cells were
exposed to different illumination regimes either by contin-
ued illumination at 460 nm for two seconds every minute to
maintain the trans configuration or 5 was switched to the
cis configuration by illumination at 365 nm for two seconds
every minute for 22 minutes (figure 3A). We observed that
5-HT uptake was reduced significantly at 365 nm at three
subsequent time points (Figure 3A) but also by comparing
the normalized uptake rates in the linear phase (linear re-
gression for incubation time 8-24 minutes) of the uptake as-
say in four independent experiments (Figure 3B).
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itiated by addition of the inhibitor mixed with H-5-HT
and was allowed to proceed for 10 minutes in either
darkness or under illumination with 365 nm for two sec-
onds every minute. The uptake was terminated by aspi-
ration and washing.
Figure 2. Azo-escitalopram (5) is a potent, photoswitchable
inhibitor of the human serotonin transporter. (A)
HEK293MSR cells were transiently transfected with hSERT
in the pCDNA3 vector and exposed to the inhibitors either
in the dark or illuminated at 365 nm for two seconds every
minute prior to and during uptake of tritiated 5-HT. Shown
is a representative example of normalized transport activity
plotted as a function of drug concentration and fitted to a
sigmoidal dose-response curve. Error bars represent stand-
ard error mean. (B) Mean Ki from three independent exper-
iments show that the potency of the parent compound, es-
citalopram, is unaffected by irradiation at 365 nm whereas
Figure 3. The transport activity of hSERT can be controlled
in real time through different illumination regimes of azo-
escitalopram (5). (A) HEK293MSR cells were transiently
transfected with hSERT in the pCDNA3 vector and chal-
lenged with 200 nM 5 prior to and during uptake of tritiated
5-HT. Cells were briefly illuminated with 460 nm for two
seconds every minute during 20 minutes of pre-incubation
and for the first 8 minutes of the uptake assay. After 8
minutes of incubation with tritiated 5-HT half of the cells
were then subjected to brief illumination with 365 nm for
two seconds every minute (the shift in illumination regime
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is marked by an arrow). Shown is the global fit of normal-
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(54 of 75 poses). Overall, cis-5 exhibited most similarity
with the binding of the parent inhibitor, escitalopram
(Figure 4A), whereas trans-5 produced a range of clus-
ters, termed trans-C1–17 clusters, of which only two
were of a significant size (27 and 32 out of 100 poses)
(Supporting Table S1). For the trans-C1 cluster, the lig-
and is found more in the low-affinity allosteric S2 site ra-
ther than in S113,39,40, whereas the other major cluster
(trans-C12) resembles the binding mode of the parent in-
hibitor, escitalopram (Figure 4B), albeit with some nota-
ble perturbations. For example, the important salt bridge
between the protonated amine of the ligand and Asp98
is only found in less than one third of the trans-C12 poses
(10 of 32 poses), the cation-π interaction between the
protonated amine of the ligand and Tyr95 is found in
only approximately half of the trans-C12 poses (17 of 32
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ized data for four independent experiments where error
bars on individual point represent SEM. Uptake at individ-
ual time points at different illumination regimes (12-30
minutes) were compared using the two-stage linear step-up
procedure of Benjamini, Krieger and Yekutielia for paired t-
test (p<0.001: ***).38 (B) The uptake rates at 200 nM 5 was
obtained from the slopes of the linear phase of the uptake
assay at different illumination regimes (8-24 minutes in
(A)) by linear regression analysis and then normalized to
the uptake rates for cells challenged with 200 nM 5 illumi-
nated with 460 nm. Comparison of the normalized uptake
rates from four independent experiments exhibited highly
statistically significant reduction (p<0.001, t-test) in uptake
for hSERT exposed to cis-5 (365 nm) as opposed to trans-5
(460 nm).
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Figure 4. Induced fit docking of cis-5 (left) and trans-5 (right) demonstrates the molecular basis for hSERT selectivity for cis-
5. (A) Out of a total of 100 docking poses for cis-5 (light grey), 75 poses (cis-C2 cluster) assume a binding mode highly similar
to the high-affinity parent inhibitor, escitalopram (dark grey). (B) Out of a total of 100 docking poses for trans-5 (light grey),
only 32 poses (trans-C12 cluster) assume a binding mode somewhat similar to the high-affinity parent inhibitor, escitalopram
(dark grey), with notable disruption of the important salt bridge between the protonated amine of the inhibitor and the car-
boxylic acid side-chain of D98 as well as perturbations of the interactions between the inhibitor and the π-systems of Y95 and
Y176.
poses) and a favourable π-π interaction between the
fluorophenyl of the ligand and Tyr176 is found in less
than half of the trans-C12 poses (15 of 32 poses). Overall,
the significant similarity between cis-5 binding and es-
citalopram binding as well of the retainment of well-es-
tablished key protein-ligand interactions responsible for
the high affinity of escitalopram12 supports that cis-5
should exhibit superior potency for inhibition of hSERT
compared to trans-5 as the experimental data also shows
(Figure 2-3).
Since the trans form of 5 sterically resembles the potent
stilbene inhibitor 4, it was unexpected, albeit welcome,
that cis-5 was more potent than trans-5. In order to bet-
ter understand the cis-selectivity of 5, we performed in-
duced fit docking simulations (Figure 4). Docking of cis-
5 resulted in 100 poses which were clustered into 11
clusters, termed cis-C1–11 clusters (Supporting Table
S1). Only one large cluster was identified: This cis-C2
cluster held 75 poses and contained the best scoring
poses. Several elements in this cluster display the hall-
marks of high-affinity inhibitors of hSERT identified ear-
lier10–12, most notably a salt bridge between the proto-
nated amine of the ligand and Asp98 (55 of 75 poses),
cation-π interaction between the protonated amine of
the ligand and Tyr95 (73 of 75 poses) and a π-π interac-
tion between the fluorophenyl of the ligand and Tyr176
In conclusion, we have designed and synthesized azo-es-
citalopram (5) as the first photochromic inhibitor of
hSERT and the first photopharmacological tool for sero-
tonin biology at large. It can be reversibly switched be-
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tween trans (low-affinity) and cis (high-affinity) configu-
5 (20 µM in 1% DMSO/PBS) was treated with PBS or re-
duced glutathione (10 mM) with and without prior irra-
diation at 365 nm for 4 minutes. After 1 hour, the sam-
ples were irradiated at 460 nm for 4 minutes to reach
460 nm adapted photostationary states in all samples for
better direct comparison.
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rations using light of different colors. We find that the cis
configuration has nanomolar potency and is 43-fold
more potent as an inhibitor than the trans configuration.
Induced-fit docking calculations identify key protein-lig-
and interactions that are retained in cis-5 and perturbed
in trans-5. As such 5 could be a useful tool for the spatio-
temporal regulation of serotonin in neural networks
with the precision that light affords.
[3H]-5-HT uptake assay
The uptake measurements were performed as previ-
ously described.41 hSERT cDNA in the pcDNA3 vector
(Invitrogen) was used to transiently transfect monolayer
HEK-293 MSR cells (Invitrogen) in DMEM (BioWhitaker)
supplemented with 10% FCS (Gibco Life Technologies),
100ꢀU/ml penicillin, 100ꢀμg/mL streptomycin (BioWhit-
aker) and 6ꢀμg/mL of Geneticin (Invitrogen) at 95% hu-
midity and 5% CO2 at 37ꢀ°C. In brief, two days before the
uptake experiment, cells were detached from the culture
flask with trypsin/EDTA (BioWhitaker), transfected
with midiprep DNA-Lipofectamine 2000 (Life Technolo-
gies) or Ecotransfect (OZ Biosciences) complex and
seeded into white tissue culture treated 96-well micro-
titer plates (Nunc). Immediately before the uptake ex-
periment was initiated, medium was aspirated and cells
were washed once with PBSCM (regular PBS buffer sup-
plemented with calcium and magnesium: 137ꢀmM NaCl,
27ꢀmM KCl, 4.7ꢀmM Na2HPO4, 1.2ꢀmM KH2PO4, 0.1ꢀmM
CaCl2, 1ꢀmM MgCl2, pH 7.4). Cells were preincubated
with inhibitor for 20-25ꢀminutes. Uptake was initiated by
the addition of 40ꢀμL of a dilution of the [3H]-5-HT mixed
with inhibitor. Dilution series of inhibitors were done in
the dark. During preincubation and incubation the inhib-
itors were either left in the dark or exposed to irradiation
at 365 nm for 2 seconds every minute for the IC50 assay
or exposed to irradiation at 365 nm or 460 nm for 2 sec-
onds every minute for the realtime rescue of azo-escital-
opram potency. Uptake was terminated after 10ꢀminutes
(IC50 assay) or at variable time points for the assay for
realtime rescue of azo-escitalopram by aspiration and
washing with PBSCM. 50ꢀμL of Microscint 20 (Packard)
was dispensed into each well resulting in cell lysis and
release of accumulated radiolabelled substrate from the
adherent cells allowing direct quantitation on a Packard
Top-counter. Uptake data were fitted to sigmoidal dose-
response curves by nonlinear regression analysis using
the built-in tools in Prism8 (Graphpad).
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METHODS
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Unless stated otherwise, all reactions were performed in
oven-dried or heat gun dried glassware. Starting materi-
als, solvents and reagents were obtained from commer-
cial sources and used without further purification. Reac-
tions were magnetically stirred and monitored by ana-
lytical thin-layer chromatography (TLC). TLC plates
were visualized by exposure to ultraviolet light (UV, 254
nm). Flash column chromatography was performed em-
ploying silica gel (60 Å, 40-63 μm, Merck). Unless other-
wise noted, yields refer to chromatographic and spectro-
scopic (1H and 13C NMR) pure materials. Proton and
carbon nuclear magnetic resonance (1H, 13C NMR) spec-
tra were recorded on a Bruker Avance III HD 400 MHz
spectrometer with cryoprobe. Proton chemical shifts are
expressed in parts per million (δ scale) and are cali-
brated using the residual undeuterated solvent as an in-
ternal reference (CDCl3: δ 7.26). Data for 1H NMR spectra
are reported as follows: chemical shift (δ ppm) (multi-
plicity, coupling constant (Hz), integration). Multiplici-
ties are reported as follows: s = singlet, d = doublet, t =
triplet, q = quartet, hept = heptet, appt = apparent, m =
multiplet, br = broad or combinations thereof. Carbon
chemical shifts are expressed in parts per million (δ
scale) and are referenced to the carbon resonances of the
solvent (CDCl3: δ 77.0). Infrared (IR) spectra were rec-
orded on a Perkin Elmer Spectrum BX II (FTIR System)
and reported in frequency of absorption (cm–1). Mass
spectroscopy (MS) experiments were performed on a
Thermo Finnigan LTQ FT (ESI) instrument. Optical rota-
tions were measured at 20 °C with a Krüss P8000-T po-
larimeter at the sodium-D line (589 nm), and the concen-
trations (c) are reported in units of g/100mL with the in-
dicated solvent. UV/Vis spectra were recorded on a Var-
ian Cary 60 Scan UV/Vis spectrometer equipped with an
18-cell holder using disposable UV cuvettes (1.5 – 3.0 mL
chamber volume). Sample preparation and experiments
were performed under red light conditions in a dark
room. The solution was prepared from a 5 mM stock-so-
lution in DMSO and diluted to 50 μM in PBS + 9% DMSO
prior to the experiment. For illumination, a 12 x 5 mm
365 nm LED flashlight array (λmax = 373 ± 17 nm) and a
21 x 5 mm 460 nm LED flashlight array (λmax = 460 ± 10
nm) were used.
Computational modelling
Protein preparation: The SERT structure was obtained
from PDB entry 5I7111 and prepared using the Protein
Preparation Wizard42 available in Maestro (Schrödinger
Suite 2019, Schrödinger, LLC, New York, NY, 2019). This
structure was chosen as SERT was co-crystallized with
escitalopram in the central binding site. Residues with
missing atoms were modelled using Prime and the pro-
tonation states of titratable residues were assessed by
PROPKA,43 both available in Maestro. The resulting pro-
tein model included two structural sodium ions and one
structural water molecule coordinating one of the ions. A
Glutathione reduction assay
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cysteine bridge was modelled between Cys200 and
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J.M. thanks the German Academic Scholarship Founda-
tion for a fellowship and New York University for a Mac-
Cracken fellowship and a Margaret and Herman Sokol
fellowship. This work was supported by the National In-
stitutes of Health (Grant R01NS108151-01). We are
grateful to Simon Ravnkilde Sinning for designing and
producing custom 3D-printed plastic polymer masks en-
abling controlled illumination of desired wells in micro-
plates. We thank Grace Pan for assistance with the gluta-
thione assay.
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Cys209, Glu508 was modelled in the protonated state,
and His240 was modelled as the epsilon tautomer, while
all other residues were modelled in their default states.
Ligand preparation: The cis- and trans-azo-escitalopram
molecules were drawn based on escitalopram from the
PDB entry 5I71. The resulting bond orders and
atomtypes were manually checked. Each molecule was
first minimized in 5000 steps using a conjugant gradient
method and then submitted to a conformational search
using both the OPLS-2005 force field44 and MacroModel
available in Maestro. In the case of cis-azo-escitalopram,
both calculations were performed with a dihedral re-
straint on the N=N bond (0±50° using a force constant of
100 kcal/mol) in order to retain the cis-isomer. All other
settings were default. Induced fit docking: Both azo-es-
citalopram isomers were docked into SERT using the
standard IFD protocol45 available in Maestro. This proto-
cal utilizes a three step docking protocol to achieve flex-
ibility of both ligand and protein during docking. First, a
softened docking is performed using 50% vdW interac-
tion strength; secondly, the protein binding site
sidechains are optimized in the context of the initially
docked ligand; and thirdly, the ligand is re-docked into
the now optimized binding site. The 200 best poses were
allowed through the first docking step, while the 100
best poses were allowed through the final docking step.
The first docking was performed in standard precision,
while the last step was performed in extra precision. The
resulting docking poses were clustered based on their
conformation using the conformer cluster script availa-
ble in Maestro. The number of clusters to use was deter-
mined for each ligand based on the minimum of the cor-
responding Kelley potential.
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