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 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.     

FXR ligands protect against hepatocellular inflammation via SOCS3 induction

Because of the anti-inflammatory actions of farnesoid X receptor (FXR) agonists, FXR has received much attention as a potential therapeutic target. However, the molecular mechanisms of actions have not yet been elucidated. In the present study, we reported that in the animal model of LPS-induced liver injury, administration of the FXR natural ligand CDCA could attenuate hepatocyte inflammatory damage, reduce transaminase activities, suppress inflammation mediators (IL-6, TNF-α and ICAM-1) expression and inhibit STAT3 phosphorylation. These protective effects of FXR were accompanied by an increased expression of suppressor of cytokine signaling 3 (SOCS3), which is a negative feedback regulator of cytokine-STAT3 signaling. We then demonstrated that the beneficial effects of FXR agonist in STAT3 activation were weakened by small interfering RNA-mediated SOCS3 knockdown in hepacytes. Moreover we observed both natural ligand CDCA and synthetic ligand GW4064 could upregulate SOCS 3 expression by enhancing the promoter activity in hepatocytes. These results suggest modulation of SOCS3 expression may represent a novel mechanism through which FXR activation could selectively affect cytokine bioactivity in inflammation response. FXR ligands may be potentially therapeutic in the treatment of liver inflammatory diseases via SOCS3 induction.     

Pharmacophore-based discovery of FXR agonists. Part I: Model development and experimental validation

The farnesoid X receptor (FXR) is involved in glucose and lipid metabolism regulation, which makes it an attractive target for the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. In order to find novel FXR agonists, a structure-based pharmacophore model collection was developed and theoretically evaluated against virtual databases including the ChEMBL database. The most suitable models were used to screen the National Cancer Institute (NCI) database. Biological evaluation of virtual hits led to the discovery of a novel FXR agonist with a piperazine scaffold (compound 19) that shows comparable activity as the endogenous FXR agonist chenodeoxycholic acid (CDCA, compound 2).     

Inhibitory effects of bile acids and synthetic farnesoid X receptor agonists on rotavirus replication

Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r(2) = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.     

Triterpenes from Alisma orientalis act as farnesoid X receptor agonists

Alisma orientalis is a well-known traditional medicine exerting pharmacological effects including antidiabetes, antihepatitis, and antidiuretics, but the respective molecular mechanism is not completely clear. Farnesoid X receptor (FXR) is a member of nuclear receptor superfamily and viewed as one of the essential target proteins to develop antidiabetic treatments. In this study, the triterpenes, alisol M 23-acetate and alisol A 23-acetate, were isolated from A. orientalis and further evaluated for their activity against FXR. In the mammalian one-hybrid and transient transfection reporter assays, both triterpenes 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. These results highly indicated that alisol M 23-acetate and alisol A 23-acetate acted as FXR agonists so A. orientalis might exert therapeutic effect including antihyperglycemic effect through FXR pathway.     

Retinoid X receptor (RXR) agonist-induced antagonism of farnesoid X receptor (FXR) activity due to absence of coactivator recruitment and decreased DNA binding

The bile salt export pump (BSEP) plays an integral role in lipid homeostasis by regulating the canalicular excretion of bile acids. Induction of BSEP gene expression is mediated by the farnesoid X receptor (FXR), which binds as a heterodimer with the retinoid X receptor (RXR) to the FXR response element (FXRE) located upstream of the BSEP gene. RXR ligands mimic several partner ligands and show additive effects upon coadministration. Using real-time quantitative PCR and cotransfection reporter assays, we demonstrate that the RXR agonist LG100268 antagonizes induction of BSEP expression mediated by endogenous and synthetic FXR ligands, CDCA and GW4064, respectively. Moreover, this antagonism is a general feature of RXR agonists and is attributed to a decrease in binding of FXR/RXR heterodimers to the BSEP-FXRE coupled with the inability of RXR agonists to recruit coactivators to FXR/RXR. Our data suggest that FXR/RXR is a conditionally permissive heterodimer and is the first example of RXR ligand-mediated antagonism of FXR activity. Because FXR agonists lower triglyceride levels, our results suggest a novel role for RXR-mediated antagonism of FXR activity in the development of hypertriglyceridemia observed with RXR agonists in rodents and humans.     

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

A chemical,genetic, and structural analysis of the nuclear bile acid receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.     

Pyrazolidine-3,5-dione derivatives as potent non-steroidal agonists of farnesoid X receptor: virtual screening, synthesis, and biological evaluation

The identification of a novel pyrazolidine-3,5-dione based scaffold hit compound as Farnesoid X receptor (FXR) partial or full agonist has been accomplished by means of virtual screening techniques. A series of pyrazolidine-3,5-dione derivatives (1a-u and 7) was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activities against FXR. Most of them showed agonistic potencies and 10 of them (1a, 1b, 1d-f, 1j, 1n, 1t, 5b, and 7) exhibited lower EC(50) values than the reference drug CDCA. Molecular modeling studies for the representative compounds 1a, 1d, 1f, 1j, 1n, 1u, 5b, and 7 were also presented. The novel structural scaffold has provided a new direction for finding potent and selective FXR partial and full agonists (referred to as 'selective bile acid receptor modulators', SBARMs).     

Sesquiterpenoids from Atractylodes macrocephala act as farnesoid X receptor and progesterone receptor modulators

Two sesquiterpenoids, atractylenolide II and III, were isolated and identified from Atractylodes macrocephala (Asteraceae) to be subsequently evaluated for their activity against farnesoid X receptor (FXR) and progesterone receptor (PR) by transient transfection reporter assays. These sesquiterpenoids did not exert significant agonistic effect but antagonized the activity of chenodeoxycholic acid (CDCA), an endogenous FXR agonist, for FXR driven SHP promoter transactivation. Additionally, they transactivated CYP7A1 gene promoter activity by antagonizing FXR. Apart from acting as a FXR antagonist, atractylenolide III also showed agonistic activity against PR. All these results demonstrated that atractylenolide II and III are the active components of Atractylodes macrocephala to exert specific pharmacologic effects.     

Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. PPARalpha is also reported to influence bile acid formation and bile composition. Farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that mediates the effects of bile acids on gene expression and plays a major role in bile acid and possibly also in lipid metabolism. Thus, both PPARalpha and FXR appear to act on common metabolic pathways. To determine the existence of a molecular cross-talk between these two nuclear receptors, the regulation of PPARalpha expression by bile acids was investigated. Incubation of human hepatoma HepG2 cells with the natural FXR ligand chenodeoxycholic acid (CDCA) as well as with the nonsteroidal FXR agonist GW4064 resulted in a significant induction of PPARalpha mRNA levels. In addition, hPPARalpha gene expression was up-regulated by taurocholic acid in human primary hepatocytes. Cotransfection of FXR/retinoid X receptor in the presence of CDCA led to up to a 3-fold induction of human PPARalpha promoter activity in HepG2 cells. Mutation analysis identified a FXR response element in the human PPARalpha promoter (alpha-FXR response element (alphaFXRE)] that mediates bile acid regulation of this promoter. FXR bound the alphaFXRE site as demonstrated by gel shift analysis, and CDCA specifically increased the activity of a heterologous promoter driven by four copies of the alphaFXRE. In contrast, neither the murine PPARalpha promoter, in which the alphaFXRE is not conserved, nor a mouse alphaFXRE-driven heterologous reporter, were responsive to CDCA treatment. Moreover, PPARalpha expression was not regulated in taurocholic acid-fed mice. Finally, induction of hPPARalpha mRNA levels by CDCA resulted in an enhanced induction of the expression of the PPARalpha target gene carnitine palmitoyltransferase I by PPARalpha ligands. In concert, these results demonstrate that bile acids stimulate PPARalpha expression in a species-specific manner via a FXRE located within the human PPARalpha promoter. These results provide molecular evidence for a cross-talk between the FXR and PPARalpha pathways in humans.     

Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.     

Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay

Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR-CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.     

Pharmacological Activation of the Bile Acid Nuclear Farnesoid X Receptor Is Feasible in Patients with Quiescent Crohn's Colitis

Background

The bile acid-activated nuclear receptor Farnesoid X Receptor (FXR) is critical in maintaining intestinal barrier integrity and preventing bacterial overgrowth. Patients with Crohn''s colitis (CC) exhibit reduced ileal FXR target gene expression. FXR agonists have been shown to ameliorate inflammation in murine colitis models. We here explore the feasibility of pharmacological FXR activation in CC.

Methods

Nine patients with quiescent CC and 12 disease controls were treated with the FXR ligand chenodeoxycholic acid (CDCA; 15 mg/kg/day) for 8 days. Ileal FXR activation was assessed in the fasting state during 6 hrs after the first CDCA dose and on day 8, by quantification of serum levels of fibroblast growth factor (FGF) 19. Since FGF19 induces gallbladder (GB) refilling in murine models, we also determined concurrent GB volumes by ultrasound. On day 8 ileal and cecal biopsies were obtained and FXR target gene expression was determined.

Results

At baseline, FGF19 levels were not different between CC and disease controls. After the first CDCA dose, there were progressive increases of FGF19 levels and GB volumes during the next 6 hours in CC patients and disease controls (FGF19: 576 resp. 537% of basal; GB volumes: 190 resp. 178% of basal) without differences between both groups, and a further increase at day 8. In comparison with a separate untreated control group, CDCA affected FXR target gene expression in both CC and disease controls, without differences between both groups.

Conclusions

Pharmacological activation of FXR is feasible in patients with CC. These data provide a rationale to explore the anti-inflammatory properties of pharmacological activation of FXR in these patients.

Trial Registration

TrialRegister.nl NTR2009     

Farnesoid X receptor suppresses constitutive androstane receptor activity at the multidrug resistance protein-4 promoter

Multidrug resistance protein-4 (MRP4) is a member of the multidrug resistance associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive up-regulation in response to cholestatic injury or bile acid feeding. In this study we demonstrate that farnesoid X receptor (FXR) regulates MRP4 in vivo and in vitro. In vivo deletion of FXR induces MRP4 gene expression. In vitro treatment of HepG2 cells with FXR ligands, chenodeoxycholic acid (CDCA), cholic acid (CA) and the synthetic ligand GW-4064 suppresses basal mRNA level of the MRP4 gene as well as the co-treatment with CDCA and 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), an activator of constitutive androstane receptor (CAR). We found in the human MRP4 promoter a CAR responsive element (CARE) embedded within an FXR responsive element (FXRE). We cloned this region and found that FXR suppresses CAR activity in luciferase assay. Finally, we demonstrated that FXR competes with CAR for binding to this overlapping binding site. Our results support the view that FXR activation in obstructive cholestasis might worsen liver injury by hijacking a protective mechanism regulated by CAR and provides a new molecular explanation to the pathophysiology of cholestasis.