Optical control of the nuclear bile acid receptor FXR with a photohormone

Article abstract of DOI:10.1039/c9sc02911g

Herein, we report a photoswitchable modulator for a nuclear hormone receptor that exerts its hormonal effects in a light-dependent fashion. The azobenzene AzoGW enables optical control of the farnesoid X receptor (FXR), a key regulator of hepatic bile aci

Full text of DOI:10.1039/c9sc02911g

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Optical control of the nuclear bile acid receptor
FXR with a photohormone†
Cite this: Chem. Sci., 2020, 11, 429
a
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have been paid for by the Royal Society
of Chemistry
Johannes Morstein,
Julie B. Trads,b Konstantin Hinnah,a Sabine Willems,c
David M. Barber,b Michael Trauner,d Daniel Merk
and Dirk Trauner
c
a
*
Herein, we report a photoswitchable modulator for a nuclear hormone receptor that exerts its hormonal
effects in a light-dependent fashion. The azobenzene AzoGW enables optical control of the farnesoid X
receptor (FXR), a key regulator of hepatic bile acid, lipid and glucose metabolism. AzoGW was derived
from the synthetic agonist GW4064 through an azologization strategy and is a metabolically stable,
highly selective photoswitchable FXR agonist in its dark-adapted form. Upon irradiation, the thermally
bistable ‘photohormone’ becomes significantly less active. Optical control of FXR was demonstrated in
a luminescence reporter gene assay and through light-dependent reversible transcription modulation of
FXR target genes (CYP7A1, Osta, Ostb) in liver cells.
Received 14th June 2019
Accepted 4th November 2019
DOI: 10.1039/c9sc02911g
rsc.li/chemical-science
Nuclear hormone receptors (NHRs) are ligand-activated tran-
In recent years, photopharmacology has become a broadly
scription factors which interact with DNA and regulate the applicable approach to manipulate and study biological
expression levels of their target genes.1,2 As such, they are systems.8–10 While ion channels, GPCRs, enzymes and many
involved in a broad spectrum of biological processes, ranging other biological targets have been successfully put under
from embryonic development, differentiation, homeostasis and reversible optical control with small molecule photoswitches,
metabolism to cell death (endocrine mode of control). A NHRs have not yet been systematically addressed. Recently, we
signicant portion (ca. 16%) of all small molecule drugs act on demonstrated that the azobenzene-containing retinoic acid
this biological target family, which consists of 48 different receptor a (RARa) agonist Azo80 11 allows for optical control of
receptors in humans.3 Endogenous ligands include steroid and RARa.12 We envisioned that photoswitchable NHR modulators,
thyroid hormones, retinoids, bile acids (BAs), fatty acids, and termed ‘photohormones’, could facilitate progress in the study
vitamin D.4
of NHRs. Herein, we describe the development of a photo-
The farnesoid X receptor (FXR) is activated by BAs as switchable FXR agonist, termed AzoGW. This photohormone
endogenous ligands and serves as a central regulator of allows for optical control of FXR as demonstrated through
mammalian metabolism, that controls genes regulating the a luminescent reporter gene assay and via transcriptional
levels of BAs, lipids, cholesterol, and glucose.5,6 It is involved in modulation in a liver cell model system.
various physiological and pathophysiological processes,
AzoGW was derived from the potent FXR agonist GW4064.
including liver protection and regeneration, inammation, and GW4064 is one of the most widely used FXR agonists with good
carcinogenesis. FXR acts on genes directly involved in BA selectivity over related NHRs.13 However, it has recently been
metabolism and exhibits a number of rapid non-genomic shown to exhibit some off-target effects on GPCRs. GW4064
effects, e.g. the control of glucose stimulated insulin secretion features a trans-stilbene moiety. Replacement of the stilbene
and glutamate transport.7
with an azobenzene (‘azologization’)12,14 furnished the photo-
switchable analog (Fig. 1A), which was synthesized in analogy to
its parent compound (Fig. 1B). Diazotization of m-amino-
benzoic acid and azo-coupling with phenol gave azobenzene 1.
A subsequent Fischer esterication afforded the corresponding
methyl ester 2. In parallel, 2,6-dichlorobenzaldoxime was
chlorinated to yield chloro-oxime 3. Treatment of 3 with 4-
methyl-3-oxopentanoate under basic conditions then gave iso-
xazole 4. DIBAL-H reduction yielded primary alcohol 5, which
aDepartment of Chemistry, New York University, New York, New York 10003, USA.
E-mail: dirktrauner@nyu.edu
bDepartment of Chemistry, Center for Integrated Protein Science, Ludwig Maximilians
University Munich, 81377 Munich, Germany
cInstitute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-
Strasse 9, 60438 Frankfurt, Germany
dHans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and was coupled with the azobenzene 2 under Mitsunobu condi-
Hepatology, Department of Internal Medicine III, Medical University of Vienna,
tions. Saponication of the resulting methyl ester then gave
AzoGW.
Waehringer Guertel 18-20, 1090 Vienna, Austria
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9sc02911g
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Fig. 1 Design and synthesis of the photoswitchable FXR agonist AzoGW. (A) Design of AzoGW from the potent FXR agonist GW4064 via
azologization. (B) Synthesis of AzoGW.
The photophysical properties of AzoGW were evaluated 365 nm (cis, gray) demonstrate wavelength-dependent switch-
using UV-Vis spectroscopy (Fig. 2A–D). The UV-Vis spectra of ing as expected for a ‘classical’ azobenzene (Fig. 2A). Photo-
AzoGW aer illumination with l ¼ 460 nm (trans, blue) and l ¼ switching could be repeated rapidly over multiple cycles
Fig. 2 Photophysical evaluation and molecular docking of AzoGW. (A) The UV-Vis spectra of AzoGW in the dark-adapted (trans, black), 365 nm
adapted (cis, gray) and 460 nm adapted (trans, blue) photostationary states. (B) Reversible cycling between photoisomers with alternating
illumination at the two distinct wavelengths 365 nm and 460 nm, indicating the fast kinetics of the isomerization. (C) Reversible cycling between
photoisomers with alternating illumination at varying wavelengths (between 340 nm and 420 nm). (D) AzoGW was repeatedly switched between
its cis- and trans-state for 50 cycles. (E) 1H-NMR-studies of AzoGW in the dark-adapted, 365 nm adapted and 460 nm adapted photostationary
states in DMSO-d6/D2O. (F) Molecular docking of trans-AzoGW (green) and cis-AzoGW (purple) to the FXR ligand binding site (PDB-ID: 3DCT15).
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Fig. 3 Pharmacological evaluation of AzoGW. (A) Dose responses of GW4064, trans-AzoGW and cis-AzoGW in a luminescent reporter cell line
after 22 h. Samples were run in duplicate and in two independent experiments. Error bars represent mean ꢀ SD (B) control and rescue
(reversibility) experiments using GW4064 (41 nM), AzoGW (367 nM), and DY 268 (2 mM). Samples were run at least in triplicate. Error bars
represent SEM ***p < 0.001, n.s., not significant, Student's t-test. (C) Selectivity panel for trans-AzoGW (3 mM) over several related nuclear
hormone receptors. Samples were run in duplicate and in four independent experiments. Error bars represent SEM. (D) Isothermal titration
calorimetry (ITC) experiment with recombinant FXR LBD and AzoGW (dark-adapted, 15 mM). KD ¼ 433 nm. (E) Bidirectional switching experiment
with AzoGW in luminescent reporter cell line using GW4064 (0.5 mM) and AzoGW (2 mM). To switch from dark to 370 nm cells were removed
from the dark after 6 hours, irradiated with 365 nm for 3 minutes and subsequently irradiated with the Cell DISCO (370 nm) system for 75 ms
every 15 s. To switch from 365 nm cells were removed from the Cell DISCO (370 nm) system after 6 hours, irradiated at 465 nm for 3 minutes and
incubated in the dark until 24 h.
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(Fig. 2B), the amount of active photoswitch could be titrated human FXR, where the ligand-binding dependent activation of
with different wavelengths of light (‘color-dosing’, Fig. 2C), and FXR is coupled to the expression of the reporter gene rey
no photobleaching, photodegradation, or fatigue was observed luciferase. We used varying concentrations of GW4064 and
aer numerous switching cycles (Fig. 2D). cis-AzoGW exhibits AzoGW (Fig. 3A). A Cell DISCO system was employed to achieve
a thermal relaxation half-life time of 52 h at room temperature switching of trans-AzoGW to cis-AzoGW (370 nm, 75 ms pulse
in physiological buffer (ESI Fig. S1†) and can thus be classied every 15 s).17 We found that trans-AzoGW (dark, EC50 ¼ 1.1 ꢁ
as a bistable photoswitch.
10ꢂ6 M) is approximately 100-fold more potent than the less
In some cases, the metabolic stability of azobenzenes is active cis-isomer of AzoGW (UV, EC50 ¼ 9.5 ꢁ 10ꢂ5 M). These
compromised by reductive cleavage by bacteria or glutathione results demonstrate that good optical control of FXR activation
(GSH) reduction.16 To assess the metabolic stability of AzoGW, we could be obtained with this tool at low micromolar concentra-
assayed its stability in the presence of the bacterial enzyme azor- tions. As a control experiment, we showed that light does not
eductase in E. coli (ESI Fig. S5†). We found that AzoGW exhibits affect transcription levels (Fig. 3B). Moreover, the FXR antago-
excellent stability whilst methyl red is degraded over time. Addi- nist DY 268 (2 mM) inhibits the effects of trans-AzoGW (367 nM)
tionally, we evaluated the stability of AzoGW in the presence of demonstrating target-specicity in our assay. A rescue experi-
GSH (ESI Fig. S6†). Although GSH enhances the thermal relaxation ment demonstrated reversibility of the reported effects. To this
of cis-AzoGW, we did not observe notable degradation, supporting end, AzoGW (367 nM) was added to cells as 365 nm-adapted cis-
that AzoGW is metabolically stable which makes it potentially AzoGW and aer 5 min the cells were illuminated with 460 nm
useful for applications in cultured cells or in vivo.
light for 2 min to reactivate AzoGW. Similar levels of tran-
We further investigated the binding mode of AzoGW by scription were observed compared to the experiment with dark-
molecular docking to a co-crystal of GW4064 and FXR (PDB-ID: adapted trans-AzoGW (Fig. 3B). In a hybrid reporter gene assay
3DCT15). The docking pose of trans-AzoGW matches the crys- for the nuclear hormone receptors PPARa/g/d, RXRa/b/g, LXRa/
tallized binding mode of GW4064 (ESI Fig. S7†). The cis-isomer b, RARa/b/g, FXR, VDR, and CAR, we veried that trans-AzoGW
of AzoGW deviates considerably from the binding pose of selectively activates FXR over other related receptors (Fig. 3C).
GW4064 and trans-AzoGW and the carboxylic acid group is less
Next, we analyzed the capacity of AzoGW for the optical
close to engaging residues (Fig. 2E). These results suggest control of FXR-dependent gene expression in untransfected
reduced potency of the cis-isomer in keeping with the predic- liver cells (HepG2). To this end, we analyzed expression levels of
tions of our chemoinformatic azologization study.12
CYP7A1 as well as Osta/b mRNA. CYP7A1 encodes a mono-
To explore if AzoGW allows for light-dependent activation of oxygenase that accounts for the rate-limiting step in bile acid
FXR, we carried out a luminescent reporter gene assay for biosynthesis. Osta and Ostb encode bile acids transporters
Fig. 4 (A) Role of FXR in physiology: Agonist induced activation of FXR, either by its endogenous ligands (BA) or synthetic ligands (GW4064,
AzoGW), leads to an up-or downregulation of its target genes. FXR indirectly suppresses CYP7A1 which catalyzes the rate determining step in BA
biosynthesis from cholesterol. Also, FXR promotes expression levels of the BA export pumps Osta and Ostb. (B–E) Effects of GW4064 and
AzoGW on FXR. Depicted are the mRNA expression levels of FXR, CYP7A1, Osta and Ostb, relative to the vehicle control (1% DMSO) after
incubation for 24 h in the dark (black) or under illumination with l ¼ 365 nm (gray) and l ¼ 460 nm (blue) respectively. Samples were run in three
independent experiments. Error bars represent SEM ***p < 0.001, **p < 0.01, *p < 0.1, n.s., not significant, Student's t-test.
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(Fig. 4A). As expected, treatment of HepG2 cells with GW4064 Herman Sokol fellowship. J. B. T. thanks the Danish National
resulted in a robust suppression of CYP7A1, whereas a strong Research Foundation Center for DNA Nanotechnology
upregulation of Osta and Ostb was observed (Fig. 4C–E). (DNRF81) and Aarhus University for nancial support. D. M. B.
Expression levels of FXR remained unaffected (Fig. 4B). We also thanks the European Commission for a Marie Skłodowska-
saw no differences in the light-excluded (dark) and irradiated Curie Intra-European Fellowship (PIEF-GA-2013-627990). We
samples (365 nm) with GW4064, conrming that light at the thank Dr Julia Ast, Dr David Hodson, and Dr Thierry Claudel for
intensities used in our experiments does not mediate changes experimental support in the initial stages of the project and
in FXR-dependent transcription levels.
helpful discussion. We thank Dr Christian Fischer, Christopher
Next, we investigated whether AzoGW could put FXR J. Arp, Grace Pan and Alan Liu for insightful discussion and
dependent gene expression under optical control. Indeed, proofreading of the manuscript.
incubation of HepG2 cells with different concentrations of
AzoGW showed light-dependent suppression of CYP7A1 and
upregulation of Osta and Ostb (Fig. 4C–E). As expected, trans-
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