A Photoswitchable Dualsteric Ligand Controlling Receptor Efficacy

Article abstract of DOI:10.1002/anie.201701524

The investigation of the mode and time course of the activation of G-protein-coupled receptors (GPCRs), in particular muscarinic acetylcholine (mACh or M) receptors, is still in its infancy despite the tremendous therapeutic relevance of M receptors and GPCRs in general. We herein made use of a dualsteric ligand that can concomitantly interact with the orthosteric, that is, the neurotransmitter, binding site and an allosteric one. We synthetically incorporated a photoswitchable (photochromic) azobenzene moiety. We characterized the photophysical properties of this ligand called BQCAAI and investigated its applicability as a pharmacological tool compound with a set of FRET techniques at the M₁ receptor. BQCAAI proved to be an unprecedented molecular tool; it is the first photoswitchable dualsteric ligand, and its activity can be regulated by light. We also applied BQCCAI to investigate the time course of several receptor activation processes.

Full text of DOI:10.1002/anie.201701524

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International Edition: DOI: 10.1002/anie.201701524
German Edition: DOI: 10.1002/ange.201701524
Photopharmacology
A Photoswitchable Dualsteric Ligand Controlling Receptor Efficacy
Luca Agnetta+, Michael Kauk+, Maria Consuelo Alonso Canizal, Regina Messerer,
Ulrike Holzgrabe,* Carsten Hoffmann,* and Michael Decker*
Abstract: The investigation of the mode and time course of the
activation of G-protein-coupled receptors (GPCRs), in partic-
ular muscarinic acetylcholine (mACh or M) receptors, is still in
its infancy despite the tremendous therapeutic relevance of
M receptors and GPCRs in general. We herein made use of
a dualsteric ligand that can concomitantly interact with the
orthosteric, that is, the neurotransmitter, binding site and an
allosteric one. We synthetically incorporated a photoswitchable
(photochromic) azobenzene moiety. We characterized the
photophysical properties of this ligand called BQCAAI and
investigated its applicability as a pharmacological tool com-
pound with a set of FRET techniques at the M1 receptor.
BQCAAI proved to be an unprecedented molecular tool; it is
the first photoswitchable dualsteric ligand, and its activity can
be regulated by light. We also applied BQCCAI to investigate
the time course of several receptor activation processes.
types.[3] M1 receptors are abundantly expressed in the cortex,
hippocampus, and striatum and play a key role in learning and
memory processes. Agonists have been suggested for the
treatment of cognitive impairment such as that associated
with schizophrenia and Alzheimerꢀs disease (AD).[4] Unfortu-
nately, the tremendous efforts to develop therapeutically
applicable selective M1 ligands have not been successful
owing to the high sequence homology of the orthosteric
binding sites, that is, the binding sites of ACh. Owing to the
plethora of very diverse physiological roles associated with
the five mAChR subtypes, the lack of selectivity led to
numerous side effects for the experimental therapeutics. As
numerous details of the molecular basis of receptor function
remain to be elucidated, suitable molecular tools are neces-
sary.
The less conserved allosteric binding sites of the M re-
ceptors, including M1, led to intensive efforts to develop
selective allosteric modulators, that is, compounds that affect
the binding of an orthosteric ligand or the endogenous
neurotransmitter ACh either positively, neutrally, or nega-
tively (Figure 1).[5] Such ligands are able to overcome the
problem of subtype selectivity for the M1 subtype by making
use of cooperativity in the selective binding and activation of
the receptor.[6]
T
he activity of numerous nervous processes in the human
body, such as smooth-muscle contraction, the cardiac rate and
force, and glandular secretion, is regulated by the peripheral
nervous system, and its parasympathic nerves are regulated
by muscarinic acetylcholine receptors (mAChRs) through the
metabotropic action of acetylcholine (ACh).[1] Hence, these
receptors have been identified and utilized as therapeutic
targets for the treatment of a broad spectrum of diseases.[2]
mAChRs belong to the class A family of G-protein-
coupled receptors (GPCRs) and comprise five distinct sub-
To take this concept one step further, dualsteric (or
bitopic) ligands are developed herein that covalently connect
a high-affinity/low-selectivity orthosteric moiety to highly
selective allosteric building blocks for concomitant interac-
[*] L. Agnetta,[+] R. Messerer, Prof. Dr. U. Holzgrabe, Prof. Dr. M. Decker
Pharmaceutical and Medicinal Chemistry
Institute of Pharmacy and Food Chemistry
Julius Maximilian University of Wꢀrzburg
Am Hubland, 97074 Wꢀrzburg (Germany)
E-mail: ulrike.holzgrabe@uni-wuerzburg.de
michael.decker@uni-wuerzburg.de
M. Kauk,[+] M. C. A. Canizal, Prof. Dr. C. Hoffmann
Department of Pharmacology and Toxicology
Julius Maximilian University of Wꢀrzburg
Versbacher Strasse 9, 97078 Wꢀrzburg (Germany)
E-mail: c.hoffmann@toxi.uni-wuerzburg.de
M. Kauk,[+] M. C. A. Canizal, Prof. Dr. C. Hoffmann
Rudolf Virchow Center for Experimental Biomedicine
Julius Maximilian University of Wꢀrzburg
Josef Schneider Strasse 2, 97080 Wꢀrzburg (Germany)
Prof. Dr. C. Hoffmann
Current address: Institute for Molecular Cell Biology
CMB—Center for Molecular Biomedicine
University Hospital Jena, Friedrich Schiller University Jena
Hans-Knçll-Strasse 2, 07745 Jena (Germany)
[+] These authors contributed equally to this work.
Figure 1. Structures of the non-selective M1 agonist iperoxo, the
positive allosteric modulator (PAM) benzyl quinolone carboxylic acid
(BQCA), and a representative dualsteric M1 ligand consisting of the
two building blocks.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201701524.
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tion with both binding sites. Recently, our groups have
reported several sets of dualsteric M1 and M2 agonists for
which dynamic ligand binding is assumed, leading to partial
agonism for the M1 receptor (Figure 1)[7] as well as for the M2
receptor.[8]
Based on these findings, dualsteric ligands hold great
potential both as future therapeutics and as pharmacological
tools owing to their unprecedented selectivity at M receptor
subtypes, their specificity for signaling pathways (“biased
signaling”), their capability for partial agonism, and their
potential for studying the process of receptor activation at the
molecular level.[6,9] We have now made use of a photophar-
macological approach by incorporating a photoswitchable (or
photochromic) azobenzene as a linker into the dualsteric
ligands described to specifically design dualsteric ligands for
investigating spatiotemporal receptor activation processes
with high precision.
The rapidly expanding field of photopharmacology aims
to introduce light sensitivity into experimental therapeutics or
drugs to control and/or investigate biological processes.[10] In
this regard, molecular photoswitches that reversibly change
their structure and physicochemical properties upon irradi-
ation with light play an important role. The light-induced
isomerization of the azobenzene photoswitch from the trans
form to the thermodynamically less stable cis form is
associated with significant changes in geometry and polar-
ity.[11] When an azobenzene unit is incorporated into a bioac-
tive compound, this change can be translated into an
alteration in the biological activity towards the respective
target.[12]
Figure 2. Structure of the photoswitchable dualsteric M1 ligand
BQCAAI in the trans and cis form.
group of the quaternary iperoxo ammonium salt has been
replaced by an azobenzene moiety.
Following the design strategy for the dualsteric iperoxo/
BQCA-type hybrids, we connected the superagonist iperoxo
to a positive allosteric modulator with an N-benzyl quinolone
carboxylic acid type structure. To directly investigate the
effect of the spacer on the intrinsic activity of these hybrids at
the M1 receptor, we replaced the aliphatic carbon chain
(polymethylene linker) with an azobenzene linker. Introduc-
ing the photoswitch into this part of the molecule should
significantly change the relative position of the two pharma-
cophores, namely from a linear to a rectangular arrangement
(Figure 2), and thus lead to a different binding mode.
Fluorescence detection and fluorescence resonance
energy transfer (FRET) techniques are now well-established
in pharmacological research for characterizing various pro-
cesses,[13] such as receptor activation,[14] G-protein activa-
tion,[15] and arrestin signaling,[16] or further downstream
detection of calcium, diacylglycerol (DAG), or cyclic
AMP.[17] The advantage of such approaches compared to
other methods is that they can be performed in living cells
under almost physiological conditions.
Herein, we report the synthesis, characterization, and
pharmacological testing of a benzyl quinolone carboxylic
acid–azobenzene–iperoxo (BQCAAI) hybrid compound
(Figure 2 and Scheme 1), which represents the first photo-
switchable dualsteric ligand described to date. For compar-
ison, we also synthesized a derivative with a benzene-
containing alkyne linker (18; see the Supporting Information,
Scheme S3) and photoiperoxo (Scheme S1), a photochromic
iperoxo derivative, in which a hydrogen atom of a methyl
The synthesis of BQCAAI began with the construction of
the quinolone skeleton through a microwave-assisted Gould–
Jacobs reaction.[18] Benzylation and subsequent hydrolysis
gave acid 4, which reacted with 4-aminobenzylamine to form
amide 5. To introduce the azobenzene moiety into the target
molecule, a Mills reaction was performed. The hydroxy
function of the azobenzene compound was replaced by
a bromine atom in an Appel reaction to give compound 6.
Finally, iperoxo, which had been synthesized using a conver-
gent synthetic pathway,[19] was connected to the azobenzene
group in a microwave-assisted reaction to afford the photo-
switchable dualsteric ligand BQCAAI (Scheme 1).
As a prerequisite for the light-dependent control of the
intrinsic activity (efficacy) at the human M1 (hM1) receptor,
the photoswitchable compound needs to effectively respond
to light. To this end, the structural change between the two
photoisomers should be fast and significant, and a high degree
of photoconversion (the trans/cis ratios should differ signifi-
cantly) is necessary. Furthermore, the stability towards
thermal isomerization as well as the intended pharmacolog-
ical/biological applications have to be taken into account.
First, BQCAAI was characterized by UV/Vis spectroscopy,
which revealed clear photoswitchability (photochromic
behavior) and the typical absorption bands of azobenzenes.
The absorption maxima at around 325 nm and 430 nm are due
to the p–p* and n–p* transitions, respectively, which allows
for distinct photoswitching between the trans and cis forms.
This process is reversible as switching can be repeated over
many cycles without loss of photochromic behavior (Figures 3
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and can be regarded as an antag-
onist. This was confirmed by
studying the calcium release
upon ligand binding (Figure S2c)
with a calcium- and DAG-sensi-
tive fluorescent probe. Whereas
iperoxo induced a rapid calcium
response, no calcium release was
observed for compound 12, even
more than 250 s after application
(Figure S2d). When compound 12
was first applied for 20 s followed
by addition of iperoxo, an imme-
diate calcium response was detect-
able for iperoxo (Figure S2e),
clearly indicating that compound
12 does not exhibit agonism
although binding to the receptor
has been indirectly shown (Fig-
ure S2b).
The same setting was used for
investigations with BQCAAI,
which showed that both isomers
interact with the M1 receptor (Fig-
ure 4a). A receptor response of
25% for the trans form and a sig-
nificantly reduced signal of 14%
for the cis form were detected.
Interestingly, the activation is
Scheme 1. Synthesis of the benzyl quinolone carboxylic acid–azobenzene–iperoxo hybrid BQCAAI.
and 4). Second, the photostationary distribution of BQCAAI
in the dark was determined by HPLC analysis to be 80% in
favor of the trans form (20% of the cis form). Upon
irradiation with UV light (l = 365 nm), the ratio changed to
52% in favor of the thermodynamically less stable cis isomer
(Figure 4). Finally, BQCAAI shows excellent thermal stabil-
ity. When the compound is kept in the dark, the photosta-
tionary state is stable for several hours after UV light
irradiation (Figure S1).
For pharmacological characterization of BQCAAI and its
individual building blocks, a range of different fluorescence or
FRET methods were applied. The orthosteric building block
iperoxo, a synthetic agonist for all mAChR subtypes,[20] was
used as a reference for all experiments and showed full
agonism (EC50 = 0.57 mm). In contrast, the azobenzene-modi-
fied iperoxo derivative (photoiperoxo) 12 was unable to
induce conformational changes at the M1 receptor, both in the
trans and in the cis form (Figure S2a), which is in agreement
with previous findings for the M1 receptor, namely that
increasing the linker length of iperoxo derivatives leads to
antagonism at this receptor.[21] To evaluate the affinity of
compound 12 to the receptor, a competition experiment was
performed (Figure S2b). Iperoxo (10 mm solution) induced
a 70% receptor response, and compound 12, when applied
alone, did not induce any conformational changes. When
iperoxo and compound 12 were applied together, a signal
reduction of on average 50% was observed, indicating that
both ligands compete for binding at the receptor. From this
experimental setting (see Ref. [21a]), we conclude that
photoiperoxo must have a distinct affinity for the receptor
slower than with iperoxo. Comparable results were obtained
for derivative 18 (RM405) with a linear alkynylbenzene linker
(Figure 4 f),[21a] but the process was significantly slower than
for related dualsteric ligands bearing a polymethylene linker.
This finding suggests that the receptor activation kinetics of
a dualsteric ligand and the structure of the linker moiety are
closely related.
To gain further insight into the characteristic properties of
trans- and cis-BQCAAI with respect to receptor agonism,
G-protein activation was investigated using G-protein FRET
sensors. As shown in Figure 4b, both isomers were able to
induce a G-protein response. With regard to iperoxo, the
trans isomer induced a more pronounced signal than the
cis isomer.
However, both of these FRET sensors are excited at
436 nm, a wavelength that induces isomerization of the
azobenzene moiety favoring the trans isomer. Given the
rapid isomerization from the cis to the trans isomer (Fig-
ure 3b), we wanted to investigate if our detection system
interferes with the true efficacy of the two ligand config-
urations owing to the slower kinetics of the G protein
response compared to receptor activation. Repeating the
experiment with a lower data sampling frequency (1 Hz,
taking 1 data point every second, instead of 10 Hz, with taking
data points every 100 ms) reduces the light exposure by 90%
and should hence influence the cis-to-trans isomerization
(Figure 4c). When the light exposure was reduced, Gq
activation by the trans isomer was not affected, whereas the
signal of the cis isomer was significantly reduced by more than
30%.
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Figure 3. Photochemical characterization. a) Absorption spectra of BQCAAI, show distinct photochromic behavior. b) The photoswitching process
can be repeated over many cycles without noticeable photofatigue. c) HPLC chromatogram of BQCAAI showing the photostationary states in the
dark (red) and after irradiation with UV light (blue).
Subsequently, we used a dual Ca2+/DAG fluorescent
probe with 488 nm and 562 nm excitation wavelengths,[21a]
which can be used simultaneously or separately, with the
benefit that one excitation signal lies within the absorption
spectrum of BQCAAI and the other one does not (Fig-
ure 3a). Monitoring calcium only without DAG for BQCAAI
shows a fast signal for the trans isomer (Figure 4d, red trace)
whereas the cis isomer does not induce a signal over more
than 200 s (Figure 4e, red). Exciting both fluorescent probes
simultaneously results in the same observation for the trans
isomer (Figure 4d, green) but in an opposite result for the cis
isomer (Figure S3), which induces both a calcium and a DAG
response, but with a rather long time delay of 45 s. This
behavior is likely due to an induced switching process by the
light used for detection, which would allow for a certain
proportion of the cis-BQCAAI to switch back to trans-
BQCAAI. This assumption is strengthened by the absence of
a calcium signal evoked by cis-BQCAAI when only the
calcium signal is monitored without DAG detection (Fig-
ure 4e).
based on receptor conformational changes has an influence
itself because of the current need for an excitation wavelength
of 436 nm. The use of a dual Ca2+/DAG sensor helped to
overcome this problem. The receptor subtype selectivity of
these ligands and the possible application of BQCAAI to
investigate the receptor activation of other M subtypes are
currently unclear and are subject to ongoing research.
Acknowledgements
L.A. and M.K. were supported by the International Doctoral
Program “Receptor Dynamics: Emerging Paradigms for
Novel Drugs” funded within the framework of the Elite
Network of Bavaria. M.C.A.C. was supported by the Marie
Curie Initial Training Network (ITN) “WntsApp” (Grant
608180). We thank Montana Molecular and in particular Dr.
Anne Marie Quinn for technical support and help with the
use of the dual sensor.
Taken together, we have clear evidence that BQCAAI is
not only the first reported photoswitchable dualsteric ligand
for GPCRs, but also the first “dimmable” one, in the sense
that cis-BQCAAI acts as an antagonist while trans-BQCAAI
is an agonist. We have shown that a direct detection system
Conflict of interest
The authors declare no conflict of interest.
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Figure 4. Characterization of the dualsteric BQCAAI hybrid compound at the receptor level and its effect on downstream signaling. a) A
representative single-cell recording of HEK293 cells stably expressing the M1-IN3-CFP receptor sensor. A solution of 100 mm iperoxo was used as
the reference. b) Representative FRET trace of HEK293 cells expressing a Gq sensor. c) Schematic comparison of the Gq activation of both
isomers at different irradiation frequencies. d) trans-BQCAAI induced a detectable Ca2+ and DAG response that is significantly lower than the
response to iperoxo. e) cis-BQCAAI was not able to induce a Ca2+ response over more than 200 s. f) The trans-BQCAAI isomer showed reduced
activation kinetics at the receptor level compared to a dualsteric ligand with a polymethylene linker. Replacing the polymethylene linker with
a benzene-containing linker resulted in comparable activation kinetics.
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Keywords: acetylcholine receptors · azobenzenes ·
dualsteric ligands · G-protein-coupled receptors ·
photopharmacology
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Manuscript received: February 11, 2017
Revised: March 30, 2017
Final Article published: && &&, &&&&
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Communications
Photopharmacology
A dualsteric acetylcholine receptor ligand
that interacts with two binding sites of the
receptor and contains a photoswitchable
azobenzene unit was synthesized. FRET
studies reveal that the activity of the
ligand can be reduced with light and that
it represents an unprecedented molecular
tool to investigate the time course of
receptor activation processes.
L. Agnetta, M. Kauk, M. C. A. Canizal,
R. Messerer, U. Holzgrabe,*
C. Hoffmann,*
M. Decker*
&&&& — &&&&
A Photoswitchable Dualsteric Ligand
Controlling Receptor Efficacy
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