Optical control of the nuclear bile acid receptor FXR with a photohormone† †Electronic supplementary information (ESI) available. See 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.

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.
Nuclear hormone receptors (NHRs) are ligand-activated transcription factors which interact with DNA and regulate the expression levels of their target genes. 1,2 As such, they are involved in a broad spectrum of biological processes, ranging from embryonic development, differentiation, homeostasis and metabolism to cell death (endocrine mode of control). A signicant portion (ca. 16%) of all small molecule drugs act on this biological target family, which consists of 48 different receptors in humans. 3 Endogenous ligands include steroid and thyroid hormones, retinoids, bile acids (BAs), fatty acids, and vitamin D. 4 The farnesoid X receptor (FXR) is activated by BAs as endogenous ligands and serves as a central regulator of mammalian metabolism, that controls genes regulating the levels of BAs, lipids, cholesterol, and glucose. 5,6 It is involved in various physiological and pathophysiological processes, including liver protection and regeneration, inammation, and carcinogenesis. FXR acts on genes directly involved in BA metabolism and exhibits a number of rapid non-genomic effects, e.g. the control of glucose stimulated insulin secretion and glutamate transport. 7 In recent years, photopharmacology has become a broadly applicable approach to manipulate and study biological systems. [8][9][10] While ion channels, GPCRs, enzymes and many other biological targets have been successfully put under reversible optical control with small molecule photoswitches, NHRs have not yet been systematically addressed. Recently, we demonstrated that the azobenzene-containing retinoic acid receptor a (RARa) agonist Azo80 11 allows for optical control of RARa. 12 We envisioned that photoswitchable NHR modulators, termed 'photohormones', could facilitate progress in the study of NHRs. Herein, we describe the development of a photoswitchable FXR agonist, termed AzoGW. This photohormone allows for optical control of FXR as demonstrated through a luminescent reporter gene assay and via transcriptional modulation in a liver cell model system.
AzoGW was derived from the potent FXR agonist GW4064. GW4064 is one of the most widely used FXR agonists with good selectivity over related NHRs. 13 However, it has recently been shown to exhibit some off-target effects on GPCRs. GW4064 features a trans-stilbene moiety. Replacement of the stilbene with an azobenzene ('azologization') 12,14 furnished the photoswitchable analog (Fig. 1A), which was synthesized in analogy to its parent compound (Fig. 1B). Diazotization of m-aminobenzoic 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 4methyl-3-oxopentanoate under basic conditions then gave isoxazole 4. DIBAL-H reduction yielded primary alcohol 5, which was coupled with the azobenzene 2 under Mitsunobu conditions. Saponication of the resulting methyl ester then gave AzoGW.
The photophysical properties of AzoGW were evaluated using UV-Vis spectroscopy ( Fig. 2A-D). The UV-Vis spectra of AzoGW aer illumination with l ¼ 460 nm (trans, blue) and l ¼ 365 nm (cis, gray) demonstrate wavelength-dependent switching as expected for a 'classical' azobenzene ( Fig. 2A). Photoswitching could be repeated rapidly over multiple cycles    (Fig. 2B), the amount of active photoswitch could be titrated with different wavelengths of light ('color-dosing', Fig. 2C), and no photobleaching, photodegradation, or fatigue was observed aer numerous switching cycles (Fig. 2D). cis-AzoGW exhibits a thermal relaxation half-life time of 52 h at room temperature in physiological buffer (ESI Fig. S1 †) and can thus be classied as a bistable photoswitch.
In some cases, the metabolic stability of azobenzenes is compromised by reductive cleavage by bacteria or glutathione (GSH) reduction. 16 To assess the metabolic stability of AzoGW, we assayed its stability in the presence of the bacterial enzyme azoreductase in E. coli (ESI Fig. S5 †). We found that AzoGW exhibits excellent stability whilst methyl red is degraded over time. Additionally, we evaluated the stability of AzoGW in the presence of GSH (ESI Fig. S6 †). Although GSH enhances the thermal relaxation of cis-AzoGW, we did not observe notable degradation, supporting that AzoGW is metabolically stable which makes it potentially useful for applications in cultured cells or in vivo.
We further investigated the binding mode of AzoGW by molecular docking to a co-crystal of GW4064 and FXR (PDB-ID: 3DCT 15 ). The docking pose of trans-AzoGW matches the crystallized binding mode of GW4064 (ESI Fig. S7 †). The cis-isomer of AzoGW deviates considerably from the binding pose of GW4064 and trans-AzoGW and the carboxylic acid group is less close to engaging residues (Fig. 2E). These results suggest reduced potency of the cis-isomer in keeping with the predictions of our chemoinformatic azologization study. 12 To explore if AzoGW allows for light-dependent activation of FXR, we carried out a luminescent reporter gene assay for human FXR, where the ligand-binding dependent activation of FXR is coupled to the expression of the reporter gene rey luciferase. We used varying concentrations of GW4064 and AzoGW (Fig. 3A). A Cell DISCO system was employed to achieve switching of trans-AzoGW to cis-AzoGW (370 nm, 75 ms pulse every 15 s). 17 We found that trans-AzoGW (dark, EC 50 ¼ 1.1 Â 10 À6 M) is approximately 100-fold more potent than the less active cis-isomer of AzoGW (UV, EC 50 ¼ 9.5 Â 10 À5 M). These results demonstrate that good optical control of FXR activation could be obtained with this tool at low micromolar concentrations. As a control experiment, we showed that light does not affect transcription levels (Fig. 3B). Moreover, the FXR antagonist DY 268 (2 mM) inhibits the effects of trans-AzoGW (367 nM) demonstrating target-specicity in our assay. A rescue experiment demonstrated reversibility of the reported effects. To this end, AzoGW (367 nM) was added to cells as 365 nm-adapted cis-AzoGW and aer 5 min the cells were illuminated with 460 nm light for 2 min to reactivate AzoGW. Similar levels of transcription were observed compared to the experiment with darkadapted trans-AzoGW (Fig. 3B). In a hybrid reporter gene assay for the nuclear hormone receptors PPARa/g/d, RXRa/b/g, LXRa/ b, RARa/b/g, FXR, VDR, and CAR, we veried that trans-AzoGW selectively activates FXR over other related receptors (Fig. 3C).
Next, we analyzed the capacity of AzoGW for the optical control of FXR-dependent gene expression in untransfected liver cells (HepG2). To this end, we analyzed expression levels of CYP7A1 as well as Osta/b mRNA. CYP7A1 encodes a monooxygenase that accounts for the rate-limiting step in bile acid 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. (Fig. 4A). As expected, treatment of HepG2 cells with GW4064 resulted in a robust suppression of CYP7A1, whereas a strong upregulation of Osta and Ostb was observed (Fig. 4C-E). Expression levels of FXR remained unaffected (Fig. 4B). We also saw no differences in the light-excluded (dark) and irradiated samples (365 nm) with GW4064, conrming that light at the intensities used in our experiments does not mediate changes in FXR-dependent transcription levels.
Next, we investigated whether AzoGW could put FXR dependent gene expression under optical control. Indeed, 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-AzoGW is a more potent agonist of FXR, leading to a stronger modulation of FXR target genes. Relative abundance of FXR did not change signicantly. This supports that the observed expression level modulations exclusively originate in differences in the effect of trans-AzoGW and cis-AzoGW on FXR. A rescue experiment was conducted to assess the reversibility of the inhibitory and promoting effect of AzoGW (Fig. 4, blue bars) concluding that illumination with blue light (l ¼ 460 nm) signicantly restores the activity of trans-AzoGW (referred to as the dark-adapted state).
In summary, we report the development of a photohormone for the nuclear hormone receptor FXR. AzoGW exhibits excellent photo-and metabolic stability and allows for photocontrol of FXR. Most importantly, FXR-dependent transcription was brought under optical control in an untransfected liver cell line, demonstrating the potential of this tool in cellular systems. Our study underscored that optogenetic and photopharmacological technologies can be applied to nuclear receptors, which include thyroid hormone receptors (TRs), peroxisome proliferatoractivated receptors (PPARs), and estrogen receptors (ERs). In addition, to azologization strategies, many NHRs could potentially be addressed through the design of photoswitchable amphiphilic (lipid-like) agonists. [18][19][20] Photohormones could enable precise genomic regulation in complex signaling networks and rapid control of non-genomic NHR effects. As such, they could lead to new insights into NHR physiology and nd therapeutic applications using endoscopic/endoluminal light delivery. 21,22 Conflicts of interest M. T. served as a speaker and/or consultant for Albireo, Boehringer Ingelheim, BiomX, Falk, Gilead, Intercept, Novartis, Phenex, Regulus and Shire, and received travel support from Falk, Gilead, and Intercept, as well as grants/research support from Albireo, Cymabay, Falk, Gilead and Intercept. He is also co-inventor of patents on the medical use of 24-norursodeoxycholic acid.