Bioorganic & Medicinal Chemistry Letters 24 (2014) 4181–4186

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Cryptochinones from Cryptocarya chinensis act as farnesoid X receptor agonists Hsiang-Ru Lin a,⇑, Tsung-Hsien Chou b, Din-Wen Huang c, Ih-Sheng Chen b,⇑ a

Department of Chemistry, College of Science, National Kaohsiung Normal University, No. 62, Shenjhong Rd., Yanchao District, Kaohsiung 82446, Taiwan, ROC School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan, ROC c Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC b

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Article history: Received 7 February 2014 Revised 11 July 2014 Accepted 16 July 2014 Available online 29 July 2014 Keywords: Cryptocarya chinensis Cryptochinones Farnesoid X receptor (FXR) Nuclear receptor

a b s t r a c t Cryptochinones A–D are tetrahydroflavanones isolated from the leaves of Cryptocarya chinensis, an evergreen tree whose extracts are believed to have a variety of health benefits. The origin of their possible bioactivity is unclear. The farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that has been widely targeted for developing treatments for chronic liver disease and for hyperglycemia. We studied whether cryptochinones A–D, which are structurally similar to known FXR ligands, may act at this target. Indeed, in mammalian one-hybrid and transient transfection reporter assays, cryptochinones A–D transactivated FXR to modulate promoter action including GAL4, SHP, CYP7A1, and PLTP promoters in dose-dependent manner, while they exhibited similar agonistic activity as chenodeoxycholic acid (CDCA), an endogenous FXR agonist. Through molecular modeling docking studies we evaluated their ability to bind to the FXR ligand binding pocket. Our results indicate that cryptochinones A–D can behave as FXR agonists. Ó 2014 Elsevier Ltd. All rights reserved.

Nuclear receptors (NRs) act as transcription factors, modulating transcription of target genes with many important roles, including the maintenance of cellular phenotypes, metabolism, and cell proliferation. They act by homodimerizing or heterodimerizing with other members of the nuclear receptor superfamily, which has over 30 known members.1 Most of these receptors exert their functions following ligand activation. Structurally they usually contain six functional domains (A–F) including the first transcription activation domain (AF-1, A/B), DNA binding domain (DBD, C), hinge domain (D), and the second transcription activation domain (AF2, E/F) also referred to as the ligand binding domain (LBD). NR dysfunction has been implicated in many diseases, including breast cancer, prostate cancer, and diabetes, thus NR modulation is a promising strategy for the development of specific therapeutic agents. The farnesoid X receptor (FXR, NR1H4), identified in 1995, is a member of the nuclear receptor superfamily.2 Endogenous ligands of FXR include bile acids, such as chenodeoxycholic acid (CDCA, 1, Fig. 1), cholic acid (CA), and deoxycholic acid (DCA).3 FXR forms a heterodimer with 9-cis retinoid acid-activated retinoid X receptor ⇑ Corresponding authors. Tel.: +886 7 7172930x7123; fax: +886 7 6051083 (H.-R.L.); tel.: +886 7 3121101x2191; fax: +886 7 3210683 (I.-S.C.). E-mail addresses: [email protected] (H.-R. Lin), [email protected]. tw (I.-S. Chen). http://dx.doi.org/10.1016/j.bmcl.2014.07.045 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

(RXR) to regulate the transcription of essential genes involved in bile acid and cholesterol metabolism, including small heterodimer partner (SHP), cholesterol 7a-hydroxylase (CYP7A1), and bile salt export pump (BSEP). As a result, FXR modulators offer promise for the treatments of diseases of cholesterol homeostasis and bile acid dysfunction.4–6 FXR is also associated with the progression of chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and primary biliary cirrhosis (PBC).7 For example, ligand-activated FXR was shown to directly transactivate the transcription of essential genes, including SHP, to inhibit the synthesis of bile acids from cholesterol, protecting the liver from toxicity associated with bile acid accumulation.8 These actions limit the overall size of the circulating bile pool while promoting choleresis and reducing hepatic exposure to bile acids. Clinical evidence of the promise of FXR agonists in therapy for cholestatic liver diseases has recently emerged, with the novel FXR agonist INT-747 (obeticholic acid, a CDCA derivative developed by Intercept Pharmaceuticals, Inc., USA), entering clinical trials for PBC and NASH. Apart from regulating lipid and bile acid metabolism, FXR was shown to modulate glucose metabolism and function in two important ways: (1) FXR activation induced a positive regulatory effect on glucose-induced insulin transcription; (2) FXR decreased blood glucose through downregulation of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-pase), critical enzymes in the hepatic gluconeogenesis

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Figure 2. Superimposition of 3D structures of 6-ethyl-chenodeoxycholic acid (scaffold in green color; oxygen atom in red color) and cryptochinone C (scaffold in purple color; oxygen atom in red color).

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Figure 1. Chemical structures of chenodeoxycholic acid (CDCA, 1), cryptochinone A (2), cryptochinone B (3), cryptochinone C (4), cryptochinone D (5), 6a-ethylchenodeoxycholic acid (6ECDCA, 6), ivermectin (7), and GW4064 (8).

pathway.9–11 Treating type 2 diabetes/NAFLD patients with INT-747 increased insulin sensitivity, reduced inflammation, and ameliorated fibrosis markers. In other studies, FXR agonists have ameliorated undesired symptoms resulting from diabetes progression, such as the improvement of erectile dysfunction and limiting nephropathy in diabetes patients.12,13 Based on these lines of evidences, herbal compounds acting as FXR agonists might be useful

as the treatments or supplements for the management of diabetes and chronic liver diseases. Cryptocarya chinensis (Hance) Hemsl. (Lauraceae) is a mediumsized evergreen tree distributed throughout southern China, Japan, and Taiwan.14 Extracts of C. chinensis are rich in natural products, including flavonoids, pyrones, lignans, pavines, aporphines, and benzylisoquinolines, reported to exhibit numerous biological effects, including antioxidant, immunological, antiarrhythmic, antitumor, and antituberculosis effects. In a previous study, several cryptochinone derivatives from C. chinensis curbed proliferation of MCF-7, NCI-H460, and SF-268 cell lines, though but the cryptochinones A–D were not cytotoxic.15,16 The ability of cryptochinones A–D to interact with FXR has not been demonstrated. Because we recognized structural similarities of these natural products to FXR ligands, we have evaluated whether they may act as FXR agonists. Cryptochinones A–D (2–5, Fig. 1) are tetrahydroflavanones and are significantly smaller in size than CDCAs (compounds 1 and 6, Fig. 1). 3D structural overlays of cryptochinone C and 6-ethylchenodeoxycholic acid (6ECDCA, compound 6, Fig. 1) shown in Figure 2 showed significant similarities in shape and positioning of functional groups. In particular, the 4-keto and 12-carbonyl groups of cryptochinone C superimposed very well with the 3- and 7-hydroxyl groups of 6ECDCA, which are known to be important for FXR agonism by the CDCAs. Given this structural similarity, we proposed that cryptochinones A–D might also behave as FXR agonists. Accordingly, we have used FXR-specific functional cell-based assays to assess their activity. We complemented these efforts with a molecular modeling study to define the basis for FXR agonism in this scaffold. The isolation of cryptochinones A–D was as mentioned previously.15 To assess FXR agonism of cryptochinones A–D we used a mammalian one-hybrid assay specific for FXR agonism as a primary screen, an assay protocol commonly used for identifying nuclear receptor ligands. The assay used an expression plasmid containing FXR LBD fused to GAL4 DNA binding domain.17,18 The transactivation activity of vehicle (DMSO, RLA = 1) served as the standard null reference, while 10 lM CDCA (RLA = 2.3; Sigma– Aldrich, St. Louis, MO, USA) was utilized as the positive control. Cryptochinones A–D had no effect upon the growth of HepG2 cells. As shown in Figure 3, however, cryptochinone A transactivated FXR LBD to stimulate GAL4 promoter by 2.1-fold of vehicle activity at 10 lM. At the same concentration, cryptochinones B–D (RLA about 1.6) exhibited slightly lower potency than cryptochinone A, which in turn was only slightly less active than CDCA. All of these cryptochinones exerted at least about 1.5-fold of

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vehicle activity at 1 lM but showed negligible activity at 100 nM. This result strongly indicates that cryptochinones A–D can function as FXR agonists. SHP is an orphan nuclear receptor, that is, it lacks an identified natural ligand. SHP acts as a coregulator of several nuclear receptors and other transcription factors, such as estrogen receptor, androgen receptor, and NF-kB. SHP plays a role in the regulation of hepatic bile acid synthesis, lipid metabolism, liver fibrosis, glucose metabolism, and insulin secretion.19 Human SHP gene transcription is known to be modulated by FXR, through its binding

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Figure 4. Transient transfection reporter assay in HepG2 cells. FXR agonistic effect was determined by the SHP promoter-driven luciferase activity. The relative luciferase activity (RLA) was quantified as normalized luciferase activity of tested compound/normalized luciferase activity of DMSO. The RLA data represented mean ± standard deviation for at least three individual determinations. CA: cryptochinone A, CB: cryptochinone B, CC: cryptochinone C, CD: cryptochinone D.

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Figure 3. Transient transfection reporter assay in HepG2 cells. FXR agonistic effect was determined by the GAL4 promoter-driven luciferase activity. The relative luciferase activity (RLA) was quantified as normalized luciferase activity of tested compound/normalized luciferase activity of DMSO. The RLA data represented mean ± standard deviation for at least three individual determinations. CA: cryptochinone A, CB: cryptochinone B, CC: cryptochinone C, CD: cryptochinone D.

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Figure 5. Transient transfection reporter assay in HepG2 cells. FXR agonistic effect was determined by the CYP7A1 promoter-driven luciferase activity. The relative luciferase activity (RLA) was quantified as normalized luciferase activity of tested compound/normalized luciferase activity of DMSO. The RLA data represented mean ± standard deviation for at least three individual determinations. CA: cryptochinone A, CB: cryptochinone B, CC: cryptochinone C, CD: cryptochinone D.

to specific sites 294 to 276 in the SHP gene promoter region, which facilitates SHP gene transcription. To further confirm whether cryptochinones A–D act as FXR agonists, the reporter plasmid containing human SHP gene promoter was employed in a transient transfection reporter assay. Null transactivation activity of vehicle (DMSO, RLA = 1) was used as reference negative control, and 10 lM CDCA (RLA = 1.92) was the positive control. The result in Figure 4 showed that cryptochinones C (RLA = 4.8) and D (RLA = 5.2) exhibited better activity than cryptochinones A and B at 10 lM. Both compounds showed effects about 3-fold of vehicle at 1 lM, with significantly enhanced SHP gene promoter transactivation even at the low dose of 100 nM. This result confirms the hypothesis that the cryptochinones act as FXR agonists to stimulate SHP transcription. Interestingly, all cryptochinones were more active than CDCA in this assay, contrary to the results in the previous assay system. Regulation of SHP promoter transactivation also involves peroxisome proliferator-activated receptor c (PPAR c), a NR that is also expressed in HepG2 cells.20 The outstanding SHP promoter transactivation effects exerted by cryptochinones A–D in this assay arise from their activity at other nuclear receptors, including PPAR c. In preliminary results (data not shown here) we indeed have found evidence for weak agonism of PPAR c by cryptochinones. More study is required to confirm this hypothesis. CYP7A1 is a microsomal cytochrome P450 isoenzyme, expressed primarily in the liver and plays a key role in the transformation of cholesterol to bile acid, catalyzing the first and ratedetermining step in the pathway. CYP7A1 modifies the steroid nucleus in several ways, including hydroxylation at 7a-position, epimerization of 3b-hydroxyl group, and saturation of the steroid nucleus. For example, CYP7A1 converts cholesterol to 7a-hydroxycholesterol, which is further modified by other P450 enzymes to generate the final bile acid. Activation of FXR results in the upregulation of the SHP gene, which in turn downregulates CYP7A1 gene transcription.21 Thus we also evaluated FXR agonism of cryptochinones A–D using a transient transfection reporter assay with reporter plasmid containing rat CYP7A1 promoter. Vehicle (DMSO, RLA = 1) again was used as a negative control, with 10 lM CDCA (RLA = 0.51) as the positive control. The results, shown in Figure 5,

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Figure 6. Transient transfection reporter assay in HepG2 cells. FXR agonistic effect was determined by the PLTP promoter-driven luciferase activity. The relative luciferase activity (RLA) was quantified as normalized luciferase activity of tested compound/normalized luciferase activity of DMSO. The RLA data represented mean ± standard deviation for at least three individual determinations. CA: cryptochinone A, CB: cryptochinone B, CC: cryptochinone C, CD: cryptochinone D.

demonstrate that cryptochinones A–D transrepressed rat CYP7A1 gene promoter in dose-dependent manner. Cryptochinones A (RLA = 0.68), B (RLA = 0.55), C (RLA = 0.69), and D (RLA = 0.57) showed transrepression effects about 60% of vehicle effect at 10 lM. Cryptochinones C and D showed negligible inhibition at 1 lM, but cryptochinones A and B showed weak activity at 1 lM. All of the cryptochinones were inactive at 100 nM in this assay. These natural products were also less effective FXR agonists than was CDCA in this assay. This positive result, however, further confirms that cryptochinones A–D act as modestly potent FXR agonists with respect to transrepression of CYP7A1 transcription. Phospholipid transfer protein (PLTP) is a member of the lipid transfer protein family and acts by transferring phospholipids from triglyceride rich particles, including very low density lipoprotein (VLDL) and chylomicrons, to high density lipoprotein (HDL) during

lipoprotein lipolysis.22 PLTP gene transcription was reported to be upregulated by FXR through the IR-1 element, a FXR response element, located within the 339/ 327 proximal promoter region of the human PLTP gene.23 By using a transient transfection reporter assay with reporter plasmid containing human PLTP promoter, we aimed to further evaluate the FXR agonistic effect of cryptochinones A–D. Results, shown in Figure 6, show that cryptochinones A–D stimulated PLTP gene transcription in dose-dependent manner. Vehicle (DMSO, RLA = 1) was again the standard reference, and 10 lM CDCA (RLA = 2) was again the positive control. Cryptochinones C (RLA = 2.1) and D (RLA = 1.95) exerted a similar effect as did CDCA at 10 lM, while cryptochinones A and B showed slightly lower transactivation activity. All these cryptochinones showed no obvious FXR agonism at 100 nM. The higher dose results, however, further support the proposal that the cryptochinones function as FXR agonists. To understand the physical basis for these properties, we used a computational study using the modeling program Discovery Studio 2.1 (Accelrys, San Diego, USA). We generated docking models to explore how cryptochinones A–D might interact with the FXR LBD to transactivate FXR. The crystal structure of rat FXR LBD complexed with 6ECDCA, disclosed in 2003, served as a starting point for the analysis.24 The FXR LBD contains 12 a-helices, like other nuclear receptors, forming a ligand binding pocket with volume of about 700 Å in which helices 4, 5, 8, and 9 are sandwiched between helices 1, 3, 7, and 10 at the top half of the pocket. Importantly, helices 3, 7, and 11 form a large cavity in the bottom half of the pocket to accommodate the FXR ligand. In the agonistic conformation, once a FXR agonist is bound to the pocket and located in the cavity, it further induces the movement of helix 12 (as shown in pink color in Fig. 7A) to close the entrance of pocket, exposing a coactivator-binding surface. 6ECDCA, a semisynthetic derivative of CDCA, contacts helices 3, 5, 7, 11, and 12 of the FXR LBD, making hydrophobic interactions. However, unlike estradiol, the A ring of 6ECDCA preferably orients toward helix 11 of the FXR LBD, while the 24-carboxylate moiety approaches helix 3 of the FXR LBD. Importantly, the crystal structure of 6ECDCA/rFXR LBD complex revealed three essential hydrogen bond interactions: (1) the 3a-hydroxyl group of 6ECDCA interacts with His 444 and Trp 466; (2) the 7a-hydroxyl group of 6ECDCA interacts with Tyr 366; (3) the 24-carboxylate group of 6ECDCA serves as a hydrogen bond acceptor, interacting with the

Figure 7. (A) Binding model of cryptochinone C in FXR ligand binding pocket. (B) Hydrophilic interactive binding mode of cryptochinone C in FXR ligand binding pocket. Oxygen atom in red color and hydrogen atom in white color. Green line indicates the hydrogen bond interaction.

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amino terminus of Arg 328. These three contacts were viewed as essential for agonizing FXR. SAR studies of CDCA derivatives confirmed the importance of the 3a/7a-hydroxyl group stereochemistry for FXR agonism.25 For example, ursodeoxycholic acid (UDCA) containing 3a/7b-hydroxyl groups, has no FXR transactivation activity, even though it contains the 24-carboxylate moiety. In recent years, new crystal structures of FXR LBD complexed with various agonists suggested that FXR agonists may lack one or even all three of these H-bond interactions. For example, the antiparasitic drug ivermectin (7, Fig. 1), which has a huge hydrophobic chemical skeleton, was demonstrated to serve as a FXR agonist. In the crystal structure of FXR LBD/ivermectin, hydrophobic interactions drive the stability of the complex.26 Interestingly, ivermectin lacks significant hydrophilic interactions with the FXR LBD, having none of the three appropriately-located H-bonding groups described above, although it contains numerous hydrophilic substituents. To evaluate possible binding modes of cryptochinones A–D, we used the ligandfit docking program and the crystal structure of human FXR LBD complexed with GW4064 (Protein Data Bank ID: 3DCT).27,28 Similar preferred binding modes emerged for all four cryptochinones, so only the binding mode of cryptochinone C is discussed here. As shown in Figure 7A, our docking model of cryptochinone C suggests that it can bind in the FXR ligand binding pocket by orienting the 12-carbonyl moiety toward helix 11 (as shown in yellow color in Fig. 7A and B) and by directing the 2-phenyl group toward helix 3 (as shown in blue color in Fig. 7A and B). In this arrangement, cryptochinone C mainly forms hydrophobic contacts with the FXR LBD, but also has two significant hydrogen bond interactions with the FXR ligand binding pocket. As shown in Figure 7B, the hydrogen bond interactions include (1) the 12-carbonyl group of cryptochinone C acting as the hydrogen bond acceptor to His 447; (2) the 4-keto group of cryptochinone C serving as a hydrogen bond acceptor with the hydroxyl group of Ser 332, whose side chain oxygen atom also interacts with the hydroxyl group of Tyr 369. Additionally, the 7-methoxy moiety of cryptochinone C resides within a hydrophobic zone formed by helices 3, 11, and 12, using residues Leu 451, Phe 461, Leu 465, and Trp 469. Importantly, cryptochinones A and B contain a 7-hydroxyl substituent that can make no significant hydrophilic contact in this pocket. The 7-methoxy substituent of cryptochinones C and D, on the other hand, contribute to weak hydrophobic contacts in this region. Although this docking model might explain how cryptochinones A–D interact with the FXR LBD, crystal structures of the FXR LBD complexed with these cryptochinones are needed to establish the nature of the interaction. GW4064 (8, Fig. 1), a novel FXR agonist developed by GlaxoSmithKline, binds to the human FXR LBD by hydrophobic interactions, through an interaction of its carboxylate group with Arg 331 on helix 3, and through binding of the isoxazole ring with groups on helix 11.28 Although the isoxazole ring of GW4064 is near His 447, there is no hydrogen bond apparent between His 447 and GW6064. However, in the rat FXR LBD/6ECDCA complex, the orientation and distance of the 3-hydroxyl and 24-carboxylate groups of 6ECDCA allow interaction with His 444 (His 447 of human FXR LBD) and Arg 328 (Arg 331 of human FXR LBD) respectively. In our human FXR LBD/ cryptochinone C docking model, cryptochinone C shows a different hydrophilic binding mode, with the 4-keto and 12-carbonyl groups simultaneously making hydrogen bond interactions with Ser 332 and His 447, as indicated by a suitable orientation and distance. Although cryptochinone C might locate in FXR LBD in the opposite orientation, by directing the 12carbonyl moiety toward helix 3 in a similar manner as GW4064, this binding mode only permits one of the two possible hydrogen bond interactions. As evidenced by the finding of similar FXR agonistic activity as CDCA, cryptochinone C more likely adopts the

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binding mode shown in Figure 7A. This docking model also correlates well with the 3D structural similarities mentioned previously. FXR is one of NRs targeted by pharmaceutical industry for developing treatments of diabetes and chronic liver diseases. Importantly, four relevant assays, together with molecular modeling studies suggesting a plausible binding mode, demonstrate that cryptochinones A–D function as FXR agonists. As such, these tetrahydroflavanones, or subsequent analogs, may show important therapeutic effects through selective modulation of the FXR pathway. Acknowledgments We thank Dr. Chiang J.Y. at College of Medicine, Northeastern Ohio University for kindly offering Dr. Lin H.R. the rat CYP7A1 reporter plasmid and Dr. Tu A.Y. at Department of Medicine, University of Washington for generously offering Dr. Lin H.R. the PLTP reporter plasmid.

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added into a well of 96-well plate. The b-galactosidase activity was measured as the luminescence strength detected by using a luminescence microplate reader. The normalized luciferase activity was calculated by the equation, normalized luciferase activity = luciferase activity/b-galactosidase activity. For the agonistic effect of test molecule, the normalized luciferase activity value was further converted to relative luciferase activity (RLA) by using vehicle (DMSO) as the standard whose activity was set to 1. Experiments were at least triplicated for each compound. Lin, H. R. Bioorg. Med. Chem. Lett. 2012, 22, 4787. Zhang, Y.; Hagedorn, C. H.; Wang, L. Biochim. Biophys. Acta 2011, 1812, 893. Kim, H. I.; Koh, Y. K.; Kim, T. H.; Kwon, S. K.; Im, S. S.; Choi, H. S.; Kim, K. S.; Ahn, Y. H. Biochem. Biophys. Res. Commun. 2007, 360, 301. Chiang, J. Y.; Kimmel, R.; Weinberger, C.; Stroup, D. J. Biol. Chem. 2000, 275, 10918. Huuskonen, J.; Olkkonen, V. M.; Jauhiainen, M.; Ehnholm, C. Atherosclerosis 2001, 155, 269. Urizar, N. L.; Dowhan, D. H.; Moore, D. D. J. Biol. Chem. 2000, 275, 39313. Mi, L. Z.; Deverakonda, S.; Harp, J. M.; Han, Q.; Pelliciari, R.; Wilson, T. M.; Khorasanizadeh, S.; Rastinejad, F. Mol. Cell 2003, 11, 1093.

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Cryptochinones from Cryptocarya chinensis act as farnesoid X receptor agonists.

Cryptochinones A-D are tetrahydroflavanones isolated from the leaves of Cryptocarya chinensis, an evergreen tree whose extracts are believed to have a...
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