A Photoswitchable Agonist for the Histamine H3 Receptor, a Prototypic Family A G-Protein-Coupled Receptor

Article abstract of DOI:10.1002/anie.201813110

Spatiotemporal control over biochemical signaling processes involving G protein-coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H₃ receptor (hH₃R). Upon illumination, key compound 65 decreases its affinity for the hH₃R by 8.5-fold and its potency in hH₃R-mediated G_i protein activation by over 20-fold, with the trans and cis isomer both acting as full agonist. In real-time two-electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light-induced modulation of hH₃R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.

Full text of DOI:10.1002/anie.201813110

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: A Photoswitchable Agonist for the Histamine H3 Receptor, a
prototypic family A G protein-coupled receptor
Authors: Niels J. Hauwert, Tamara A.M. Mocking, Daniel Da Costa
Pereira, Ken Lion, Yara Huppelschoten, Henry F. Vischer,
Iwan J.P. De Esch, Maikel Wijtmans, and Rob Leurs
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813110
Angew. Chem. 10.1002/ange.201813110
Link to VoR: http://dx.doi.org/10.1002/anie.201813110
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Angewandte Chemie International Edition
10.1002/anie.201813110
COMMUNICATION
A Photoswitchable Agonist for the Histamine H Receptor,
3
a prototypic family A G protein-coupled receptor
+
+
Niels J. Hauwert , Tamara A.M. Mocking , Daniel Da Costa Pereira, Ken Lion, Yara Huppelschoten,
Henry F. Vischer, Iwan J.P. De Esch, Maikel Wijtmans*, Rob Leurs*
[
7]
[8]
[9]
[10]
[11]
Abstract
3
CXCR3 , CB1 , H R , mGlu5 and GLP1 . Yet, almost all these
examples include at least one but more frequently two
antagonistic/partial agonist isomeric forms. In contrast, freely
Spatiotemporal control over biochemical signaling processes
involving G protein-coupled receptors (GPCRs) is highly desired for
dissecting their complex intracellular signaling. We developed a set
diffusible affinity and potency photoswitches in which both isomers
[1a]
act as full agonists are scarce
even though such compounds
would be very useful for photopharmacology approaches and
complementary to agonist-to-antagonist switches.
of sixteen photoswitchable ligands for the human histamine H
receptor (hH R). Upon illumination key compound 65 decreases its
affinity for the hH R 8.5-fold and its potency in hH R-mediated G
3
3
3
3
i
protein activation over 20-fold, with the trans and cis isomer both
acting as full agonist. Furthermore, in real-time two-electrode voltage
clamp experiments in Xenopus oocytes, 65 shows rapid light-
3
The histamine H R receptor is an intensively studied GPCR that is
known to play an important role in sleep disorders and cognition-
related diseases such as Alzheimer’s and Parkinson’s disease. The
®
induced modulation of hH
3
R activity. Ligand 65 shows good binding
first H
3
R antagonist pitolisant (Wakix ) has been approved by the
[
12]
selectivity amongst the histamine receptor subfamily and has good
photolytic stability. In all, 65 (VUF15000) is the first photoswitchable
GPCR agonist confirmed to be modulated through its affinity and
potency upon photoswitching while maintaining its intrinsic activity,
rendering it a new chemical biology tool for spatiotemporal control of
GPCR activation.
European Medicines Agency for the treatment of narcolepsy.
[
9]
Recently, we published a toolbox of photoswitchable antagonists
that competitively inhibit histamine-induced activity. In the
current work, we aimed to develop high-potency
photoswitchable agonists that can simplify spatiotemporal studies of
the signaling network of the R. We disclose unique
photoswitchable H R agonists that can be optically converted to
3
H R
3
H R
H
3
3
isomers differing in their affinity and potency.
Introduction
In recent years, photopharmacology has been gaining momentum as
[
1]
a strategy to optically control biochemical processes. The use of
light as an external trigger to change ligand shape and consequently
its pharmacological properties allows the probing of biological
[
2]
systems with great spatiotemporal resolution. The azobenzene
[
1a]
moiety is often used in photoswitchable ligands due to its limited
size, high photostability and tunability of the peak absorption
Figure 1. General design and concept of photoswitchable H R full agonists.
3
Main Text
[
3]
wavelength λmax
. Its thermodynamically stable trans isomer typically
involves a flat elongated structure, whereas its photoinduced cis
configuration has a bent geometry with considerably shorter end-to-
The scaffold design was inspired by hH R full agonist VUF5980
3
[
4]
[13]
end distance. Whereas photopharmacology is well established in
the field of enzyme and ion channel modulation, it is an upcoming
previously published by our lab (Figure 1). To date, virtually every
published hH R full agonist contains
3
a 4-substituted imidazole
[
1a]
technology for G protein-coupled receptors (GPCRs).
constitute one of largest families of transmembrane proteins, their
dysfunction is associated with plethora of diseases and
consequently GPCRs are one of the most successful classes of drug
GPCRs
moiety combined with a basic or neutral side chain, as is the case for
VUF5980. We left the imidazole portion of the molecule unchanged,
and considered the diphenylacetylene moiety to be an attractive
a
[
3]
candidate for an “azologization” strategy.
Introducing the
[
5]
targets. Recently, various GPCRs have been successfully targeted
azobenzene at this position furthermore allows for great flexibility in
the diversification of the scaffold. Based on the steep SAR observed
[6]
,
using photopharmacology approaches, including µ-opioid
[
13]
with VUF5980 it was postulated that small changes would have
significant impact on the affinity and potency for hH R. Therefore,
3
[
a]
N.J. Hauwert MSc.*, T.A.M. Mocking MSc.*, D. Da Costa Pereira, Z.
Lion MSc., Y. Huppelschoten MSc., Dr. H.F.Vischer, Prof. Dr.
I.J.P.de Esch, Dr. M. Wijtmans, Prof. Dr. R. Leurs
primarily the azobenzene was decorated with small substituents (i.e.
methyl and fluorine groups) on both phenyl rings.
*
Authors contributed equally
Division of Medicinal Chemistry
Amsterdam Institute for Molecules Medicines and Systems (AIMMS)
Vrije Universiteit Amsterdam
De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands
E-mail: M.Wijtmans@vu.nl, R.Leurs@vu.nl
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
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COMMUNICATION
o
room temperature (20 C, Table
1
). The observed thermal
relaxation half-lives were
impractically long for direct
quantification,
extrapolations
therefore
high
of
temperature thermal relaxation
were used to quantify half-lives
o
[14]
at 20 C (Table 1, Figure S1).
Compound 69 shows the fastest
thermal relaxation in 50 mM Tris-
HCl pH 7.4 buffer, having a half-
life of 26.6 days, while 72 shows
the slowest relaxation with
half-life of 147 days.
a
Based
on
its
favorable
Scheme 1. General synthetic scheme for photoswitchable H
experimental procedures
3
R agonists. See supporting information for detailed
pharmacological profile (vide
infra) and synthetic tractability,
To
imidazole-4-carbaldehyde
protected using
synthesize
the
ligands,
2
was
N,N-
dimethylsulfamoylchloride (DMS-
Cl, Scheme 1) to afford 3, which
was reduced to 4. Alcohol 4 was
converted to chloride 5 using an in
situ mesylation. A diverse set of
anilines 6-15 was oxidized to the
corresponding
nitrosobenzenes
16-25 using Oxone™. After work-
up, they were directly used in a
Mills reaction with 3-amino-
phenylboronic acid pinacol esters
2
6-32 to yield azobenzene-pinacol
esters 33-48. Cross coupling with
chloride yielded 49-64 in
generally good yields. Acidic
deprotection yielded final
5
compounds 65-80 which were
used for biological evaluation.
Compounds 65-80 all have
λ
max
values for the -* transition of the
trans isomer between 313 and 330
nm (Table 1). The observed limited
variation is due to the absence of
strong electron-donating or
withdrawing substituents. Similarly,
max values for the n-* transition of
the cis isomer differed marginally,
-
λ
1
ranging between 417 and 430 nm. Figure 2: (A) Representative part of H NMR spectra of 10 mM 65 in DMSO-d
6
illuminated at 360 ± 20 nm displayed at
various time points (seconds). The presented peak belongs to the hydrogen atoms explicitly drawn in the structure
shown above the spectrum. Full spectra are available in Figure S4. (B) Representative part of LC–MS chromatograms
Upon continuous illumination with
3
60 ± 20 nm, the values for the belonging to the illuminated NMR sample in Figure 2A. Full chromatograms are available in Figure S5. (C) UV–Vis
spectra of 25 μM of 65 in 50 mM Tris–HCl pH 7.4 buffer + 1% DMSO-d
illuminated for 300 s using 360 ± 20 nm light. PSS trans represents subsequent illumination for 300 s using 434 ± 9 nm.
D) Repeated isomerization of 25 μM of 65 in 50 mM Tris–HCl pH 7.4 buffer + 1% DMSO-d analyzed at 320 nm. PSS
7.5% cis, except for 69 which has cis was obtained by using illuminations for 40 s at 360 ± 20 nm. PSS trans was obtained by using illuminations for 40 s
at 434 ± 9 nm. (E) Absorbance at 320 nm of 25 μM of 65 in 50 mM Tris–HCl pH 7.4 buffer + 1% DMSO-d . UV–Vis
spectra were obtained with 1 s intervals under alternating illumination with 360 ± 20 nm and 434 ± 9 nm perpendicular to
6
. PSS cis represents a sample which has been
photostationary states (PSS) of 65-
8
9
8
0
ranged between 92.3 and
(
6
6
2.6% cis. Compounds 65-80 all
showed slow thermal relaxation at the light source of the UV–Vis spectrometer.
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COMMUNICATION
compound 65 was subjected to in-depth photochemical
observed.
1
characterization using H-NMR and LC-MS analysis during
illumination with 360 ± 20 nm. The well-resolved signal of the
1
The long thermal relaxation half-lives allowed for detailed
pharmacological evaluation using hH R competition binding as well
benzylic CH
2
group provided a clear handle for quantification in H-
3
NMR analysis (Figure 2A). An overestimation of isomerization
percentage is observed in LCMS analysis at 254 nm compared to
as functional experiments. For this, the compound solutions were
1
either illuminated using 360 ± 20 nm to reach a PSS cis or kept in
H-NMR analysis (Figure 2B, Table S1), which can be explained by
the dark to ensure >99% trans isomer. Then, the affinity of both
the differences in extinction coefficients for the trans and cis isomer
at 254 nm (Figure 2C, S2, S3). Compound 65 shows excellent
resistance to photobleaching upon >1000 times isomerization
3
isomers for the hH
3
R was assessed in competition binding with [ H]-
α
3
N -methylhistamine (NAMH). All compounds display hH R binding
affinity, which decreases upon illumination reaching up to a 21-fold
(
Figure 2D). The dynamic isomerization was studied using UV-Vis
affinity difference in the case of 76. In terms of absolute affinity, 65
spectroscopy under alternating illumination (Figure 2E). At 25 µM of
displays the highest affinities for the hH
3 i
R with a pK value of 8.42 ±
6
65 in 50 mM Tris-HCl pH 7.4 buffer + 1% DMSO-d , a half-life of 4.2
0
.04 for its trans isomer and a pK of 7.49 ± 0.05 for its cis isomer,
i
±
0.16 s for 360 ± 20 nm and 5.7 ± 0.19 s for 434 ± 9 nm was
resulting in an 8.5-fold shift upon illumination (Figure 3A, Table 1).
Fluorine-substituted analogues 67 and 80 perform similarly to 65 in
competition binding displaying only a marginally lower affinity (Table
1
R
1
). Notably, para-methyl substitution on the
position (78)
decreases the binding affinity and abrogates the photoisomerization
induced affinity shift compared to 65 (Table 1). Reduction of the size
of the para-substituents to either chlorine (71) or fluorine (68)
gradually rescues hH
3
R affinity and reestablishes the shift in affinity
to 6- and 15-fold, respectively. Methyl addition to either the ortho
1
(
76) or meta (77) position of R still results in decent binding affinities
and high (21-fold) to good (8.5-fold) affinity shifts upon illumination.
2
Addition of substituents at the R position results in a clear affinity
cliff, with fluorine substitutions (79 and 80) still being allowed, but
addition of a methyl substituent (72-75) highly decreased the binding
affinity of the cis isomer. Moreover, for the trans isomers, 4-Me (73)
and 6-Me (75) substitution is still tolerated yet showing a log-unit
decrease in hH
3
R affinity compared to 65, while 2-Me (72) and 5-Me
R affinity and consequently reduce or
(
74) groups highly reduce hH
3
even abolish (72) the affinity shift (Table 1).
Based on the observed affinities and photo-induced affinity shifts,
the efficacy to stimulate hH
evaluated for ligands 65 and 76 in a [ S]-GTPS binding assay.
Highest affinity ligand 65 (pK trans = 8.42 ± 0.04) also displays the
highest potency (pEC50 trans = 7.60 ± 0.13) to induce G activation,
which upon photoisomerization decreases 20-fold (pEC50 at PSS cis:
.30 ± 0.13) with both isomers being full agonists with both having
intrinsic activities of α = 1.0 ± 0.03, as compared to histamine
Figure 3B). Since the observed shift in hH R affinity was 8.5-fold,
the larger (20-fold) shift in functional potency indicates that for 65 the
3 i
R-mediated G protein activation was
35
i
i
6
(
3
[
15]
efficacy (propensity to activate a GPCR ) is also affected upon
trans-cis isomerization. Interestingly, large photo-induced
a
decrease in potency of 23-fold was also obtained for 76 (pEC50
trans: 6.78 ± 0.11, PSS cis: 5.41 ± 0.11, α = 1.00 ± 0.0). This shift in
potency of 76 is completely explained by the observed change of its
affinity (vide supra).
3
Figure 3. Representative curves of 65 (A) in competition binding with [ H]-NAMH Compound 65 (VUF15000) was selected as tool compound for
3
5
or (B) in G
i
protein activation as measured by [ S]-GTPS accumulation on
R. Black lines refer to a
further analysis as it has good synthetic tractability and because its
HEK293T cell homogenates transiently expressing hH
3
sample containing >99% trans, while magenta lines refer to a sample illuminated to superior potency is a clear advantage for pharmacological studies.
PSS with 360 ± 20 nm prior to the assay. (C) Schematic drawing of the TEVC
setup used for dynamic hH R and GIRK current activation in Xenopus laevis
3
oocytes expressing hH R and GIRK. (D) Representative part of a GIRK-mediated
3
current trace during continuous perfusion with 1 μM 65 under illumination of the histamine receptor subtypes
oocyte with alternating 360 ± 20 and 434 ± 9 nm wavelength as measured by
TEVC. Error bars shown are mean ± SD.
As the imidazole-based pharmacophore/scaffold used in the design
of these photoswitchable ligands is prone to interact with other
[
13]
,
65 was tested for its subtype
R over hH
R (Table S2), while a 31-fold selectivity is observed over its
selectivity. Binding of 65 is >300-fold selective for hH
and hH
3
1
R
2
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Angewandte Chemie International Edition
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COMMUNICATION
closest homologue hH
nM (trans) to low µM (PSS cis) binding affinities for both mouse and
rat H R with a 4-fold and 8-fold shift in binding affinity upon
photoisomerization, respectively (Table S2).
4
R (Table S2). Interestingly, 65 displays high
3
Real-time photomodulation of hH
using two-electrode voltage clamp (TEVC) on Xenopus laevis
oocytes expressing both hH R and G protein-coupled inwardly-
rectifying potassium (GIRK)-channels (Figure 3C). In this expression
system, histamine application results in hH R-mediated GIRK
activation, which is insensitive to optical modulation. As expected
3
R activity by 65 was measured
3
3
[
9]
35
based on our data with the [ S]-GTPS binding assay, trans-65
elicits an agonistic response in this system, which could be reduced
by switching to the less active cis isomer upon illumination with 360
±
20 nm. Retrieval of the agonistic response could be provoked by
either actively switching the cis isomer back into its trans isomer by
illuminating with 434 ± 9 nm or by stopping illumination, due to
continuous perfusion of the trans isomer (Figure 3D). Dynamic
photoswitching of 65 could be performed repeatedly, illustrating that
the use of two specific wavelengths allows optical control of the
hH
3
R
activation mediated by 65. Furthermore, photoswitchable
R activation and deactivation kinetics,
agonist 65 shows rapid hH
3
aiding in its use in in vivo experimentation.
Conclusion
3
We have synthesized and characterized 16 photoswitchable hH R
agonists that change their affinity and potency upon illumination,
indicating a successful azologization strategy. All possess long
thermal relaxation half-lives at room temperature making them useful
for a variety of pharmacological studies. Compound 65 (VUF15000)
was selected as key compound on the basis of synthetic tractability
and highest absolute hH
displays a high potency and 20-fold potency shift while maintaining
full intrinsic activity in G protein activation making it especially
3
R affinity. Moreover, upon illumination 65
i
attractive as a tool compound. With a 20-fold shift in potency, 65 is
one of the best photoswitchable GPCR agonists reported so far.
Electrophysiology experiments showed the dynamic optical
modulation of hH
3
R activation induced by 65 in real time, setting the
stage for further unraveling of the downstream signaling of hH
3
R
with great spatiotemporal precision. Recently, photopharmacology
approaches with freely diffusible GPCR ligands have for the first time
been used successfully in vivo to modulate tadpole and zebrafish
[
10, 16]
behavior
and to elucidate the role of the metabotropic glutamate
receptor 4 in the nervous system using a mouse model of chronic
[17]
pain.
3
In view of the widespread distribution of the H R in the
central and peripheral nervous system, photopharmacology
approaches with tools such as 65 offer new means (complimentary
[
18]
to optogenetic approaches ) to investigate the spatial and temporal
details of H R modulation of important processes like arousal,
cognition and neuropathic pain.
3
[12a-e]
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Angewandte Chemie International Edition
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COMMUNICATION
3
Table 1. Structure-affinity relationship of photoswitchable azobenzene-derived H R agonists.
[b]
[b]
[c]
1/2
[d]
Compound
number
1
2
pK
i
trans ±
SEM
pK at PSS
cis ± SEM
i
pK
SEM
i
shift ±

max trans
(nm)

max cis
(nm)
t
PSS ±
R
R
[a]
(days)
SEM
[a]
1
8.74 ± 0.10
-
-
-
-
-
6
6
6
6
6
7
7
5
6
7
8
9
0
1
H
H
H
8.42 ± 0.04
8.28 ± 0.08
8.35 ± 0.09
7.69 ± 0.08
8.00 ± 0.02
7.86 ± 0.03
6.76 ± 0.07
5.57 ± 0.09
6.90 ± 0.06
5.75 ± 0.03
7.15 ± 0.03
7.72 ± 0.03
7.39 ± 0.08
5.72 ± 0.14
7.81 ± 0.07
8.39 ± 0.06
7.49 ± 0.05
7.09 ± 0.03
7.42 ± 0.05
6.51 ± 0.08
7.26 ± 0.09
6.85 ± 0.04
5.98 ± 0.07
5.45 ± 0.03
5.77 ± 0.13
5.13 ± 0.17
5.94 ± 0.06
6.40 ± 0.04
6.46 ± 0.06
5.71 ± 0.06
6.54 ± 0.06
7.36 ± 0.03
-0.93 ± 0.06
-1.19 ± 0.04
-0.93 ± 0.04
-1.18 ± 0.09
-0.74 ± 0.10
-1.02 ± 0.03
-0.78 ± 0.10
-0.12 ± 0.10
-1.13 ± 0.08
-0.62 ± 0.19
-1.21 ± 0.04
-1.32 ± 0.05
-0.94 ± 0.09
-0.01 ± 0.16
-1.27 ± 0.02
-1.03 ± 0.03
320
323
320
322
313
324
326
323
327
322
324
326
323
330
324
322
427
425
425
426
417
420
428
428
430
427
426
426
428
429
425
427
106
128
96.1 ± 1.9
95.7 ± 0.27
94.1 ± 1.3
95.9 ± 1.6
82.6 ± 1.9
95.3 ± 0.22
97.5 ± 0.48
92.3 ± 4.9
96.3 ± 1.1
94.5 ± 1.4
95.8 ± 0.92
96.5 ± 1.6
95.4 ± 0.39
94.0 ± 4.4
96.6 ± 0.51
94.6 ± 1.3
2-F
3-F
4-F
2,6-F
2-Cl
4-Cl
H
H
101
H
95.9
26.6
96.1
29.7
147
H
H
H
2-Me
4-Me
5-Me
6-Me
H
7
2
3
7
H
42.9
122
74
75
76
77
78
79
80
H
H
125
2-Me
3-Me
4-Me
H
35.6
77.0
34.1
91.7
84.6
H
H
4-F
6-F
H
[
13]
[
a] Adapted from Wijtmans et al. [b] Determined at 25 μM in 50 mM Tris-HCl pH 7.4 buffer + 1% DMSO-d6. [c] Thermal relaxation half-life times as
[
1
4
]
o
determined according to the method of Priimagi et al. in 50 mM Tris-HCl pH 7.4 buffer + 1% DMSO-d
6
extrapolating to 20
C
.
Arrhenius plots are
available in Figure S1. [d] Photostationary state area percentages after illumination with 360 ± 20 nm at 1 mM in DMSO-d
LC−MS analysis at 254 nm. All pharmacology experiments were at least performed as triplicate.
6
and as determined by
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COMMUNICATION
Acknowledgements
up the photochemistry equipment and Fons Lefeber (Leiden
University) for NMR assistance. Kristoffer Sahlholm (Karolinska
institute) is kindly acknowledged for providing the pcDNA3.1-Kir3.1
and -Kir3.4 plasmids.
We acknowledge The Netherlands Organisation for Scientific
Research (NWO) for financial support (TOPPUNT, “7 ways to 7TMR
modulation (7-to-7)”, 718.014.002). All authors participate in the
European Cooperation in Science and Technology Action CM1207
Keywords: Photopharmacology • VUF15000 • H R • Agonism •
3
[GPCR-Ligand Interactions, Structures, and Transmembrane
Dynamic modulation
Signaling: A European Research Network (GLISTEN)]. We thank
Hans Custers for HRMS analyses, Andrea van de Stolpe for setting
[
10] S. Pittolo, X. Gomez-Santacana, K. Eckelt,
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Angewandte Chemie International Edition
10.1002/anie.201813110
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Shedding light on G protein-
coupled receptor activation:
VUF15000 is a photoswitchable
+
Niels J. Hauwert , Tamara A.M.
+
Mocking , Daniel Da Costa Pereira, Ken
Lion, Yara Huppelschoten, Henry F.
Vischer, Iwan J.P. De Esch, Maikel
Wijtmans*, Rob Leurs*
histamine H
full G protein activation in both its
trans and cis isomer. Moreover, it
shows dynamic optical H receptor
3
receptor agonist showing
i
Page No. – Page No.
3
modulation in electrophysiology
experiments. VUF15000 can serve as
a valuable photochromic tool
A Photoswitchable Agonist for the
3
Histamine H Receptor, a prototypic
compound for unraveling the H
3
family A G protein-coupled receptor
receptor signaling cascade with
spatiotemporal precision.
This article is protected by copyright. All rights reserved.