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 regulates organic solute transporters alpha and beta in the adrenal gland, kidney, and intestine

Expression of the farnesoid X receptor (FXR; NR1H4) is limited to the liver, intestine, kidney, and adrenal gland. However, the role of FXR in the latter two organs is unknown. In the current study, we performed microarray analysis using RNA from H295R cells infected with constitutively active FXR. Several putative FXR target genes were identified, including the organic solute transporters alpha and beta (OSTalpha and OSTbeta). Electromobility shift assays and promoter-reporter studies identified functional farnesoid X receptor response elements (FXREs) in the promoters of both human genes. These FXREs are conserved in both mouse genes. Treatment of wild-type mice with 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR agonist, induced OSTalpha and OSTbeta mRNAs in the intestine and kidney. Both mRNAs were also induced when wild-type, but not FXR-deficient (FXR-/-), adrenals were cultured in the presence of GW4064. OSTalpha and OSTbeta mRNA levels were also induced in the adrenals and kidneys of wild-type, but not FXR-/-, mice after the increase of plasma bile acids in response to the hepatotoxin alpha-naphthylisothiocyanate. Finally, overexpression of human OSTalpha and OSTbeta facilitated the uptake of conjugated chenodeoxycholate and the activation of FXR target genes. These results demonstrate that OSTalpha and OSTbeta are novel FXR target genes that are expressed in the adrenal gland, kidney, and intestine.     

Anthranilic acid derivatives as novel ligands for farnesoid X receptor (FXR)

Nuclear farnesoid X receptor (FXR) has important physiological roles in various metabolic pathways including bile acid, cholesterol and glucose homeostasis. The clinical use of known synthetic non-steroidal FXR ligands is restricted due to toxicity or poor bioavailability. Here we report the development, synthesis, in vitro activity and structure–activity relationship (SAR) of anthranilic acid derivatives as novel FXR ligands. Starting from a virtual screening hit we optimized the scaffold to a series of potent partial FXR agonists with appealing drug-like properties. The most potent derivative exhibited an EC₅₀ value of 1.5 ± 0.2 μM and 37 ± 2% maximum relative FXR activation. We investigated its SAR regarding polar interactions with the receptor by generating derivatives and computational docking.     

DAX1 suppresses FXR transactivity as a novel co-repressor

Bile acid receptor FXR (farnesoid X receptor) is a key regulator of hepatic bile acid, glucose and lipid homeostasis through regulation of numerous genes involved in the process of bile acid, triglyceride and glucose metabolism. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains and acts primarily as a co-repressor of many nuclear receptors. Here, we demonstrated that DAX1 is co-localized with FXR in the nucleus and acted as a negative regulator of FXR through a physical interaction with FXR. Our study showed that over-expression of DAX1 down-regulated the expression of FXR target genes, whereas knockdown of DAX1 led to their up-regulation. Furthermore, three LXXLL motifs in the N-terminus of DAX1 were required for the full repression of FXR transactivation. In addition, our study characterized that DAX1 suppresses FXR transactivation via competing with co-activators such as SRC-1 and PGC-1α. In conclusion, DAX1 acts as a co-repressor to negatively modulate FXR transactivity.     

Transient impairment of the adaptive response to fasting in FXR-deficient mice

The farnesoid X receptor (FXR) has been suggested to play a role in gluconeogenesis. To determine whether FXR modulates the response to fasting in vivo, FXR-deficient (FXR^−/−) and wild-type mice were submitted to fasting for 48 h. Our results demonstrate that FXR modulates the kinetics of alterations of glucose homeostasis during fasting, with FXR^−/− mice displaying an early, accelerated hypoglycaemia response. Basal hepatic glucose production rate was lower in FXR^−/− mice, together with a decrease in hepatic glycogen content. Moreover, hepatic PEPCK gene expression was transiently lower in FXR^−/−mice after 6 h of fasting and was decreased in FXR^−/−hepatocytes. FXR therefore plays an unexpected role in the control of fuel availability upon fasting.     

Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.     

5Alpha-bile alcohols function as farnesoid X receptor antagonists

The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A ring to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A ring, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5beta-configuration in FXR activation. The results showed that the 5beta-(A/B cis) bile alcohols 5beta-cyprinol and bufol are potent FXR agonists, whereas their 5alpha-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A-ring orientation of bile salts in agonist/antagonist function.     

Structural optimization and in vitro profiling of N-phenylbenzamide-based farnesoid X receptor antagonists

Activation of the nuclear farnesoid X receptor (FXR) which acts as cellular bile acid sensor has been validated as therapeutic strategy to counter liver disorders such as non-alcoholic steatohepatitis by the clinical efficacy of obeticholic acid. FXR antagonism, in contrast, is less well studied and potent small molecule FXR antagonists are rare. Here we report the systematic optimization of a novel class of FXR antagonists towards low nanomolar potency. The most optimized compound antagonizes baseline and agonist induced FXR activity in a full length FXR reporter gene assay and represses intrinsic expression of FXR regulated genes in hepatoma cells. With this activity and a favorable toxicity-, stability- and selectivity-profile it appears suitable to further study FXR antagonism in vitro and in vivo.     

核受体FXR调控SHP的机制及FXR-SHP轴在肝脏中作用的研究进展

法尼醇X受体(Farnesoid X Receptor,FXR)属于代谢性核受体,是需配体激活的转录因子,在肝脏胆汁酸、脂质代谢过程,肝脏炎症和肿瘤的发展过程中起着重要的调节作用。小异二聚体伴侣受体(Small Heterodimer Partner,SHP)是核受体超家族中的一个特殊成员,在特异的组织中作为转录调节的共抑制因子,抑制其他多种转录因子的活性,在众多代谢通路中起到了负性调节作用。近年来研究发现,核受体FXR通过对SHP的调控来实现其在肝脏的多种功能。本文着重对FXR调节SHP的机制及FXR-SHP轴在肝脏中作用进行综述。     

Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.     

Combinational effects of farnesoid X receptor antagonist and statin on plasma lipid levels and low-density lipoprotein clearance in guinea pigs

Aims

We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model.

Main methods

Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment.

Key findings

Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10 mg/kg) or atorvastatin (10 mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%.

Significance

Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options.     

Conformationally constrained farnesoid X receptor (FXR) agonists: heteroaryl replacements of the naphthalene

To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.     

激活FXR抑制结肠癌细胞浸润转移及MMP-7的表达

目的:探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞浸润转移的机制。方法:在体外培养人结肠癌细胞HT-29,应用GW4064作用于结肠癌细胞,以四唑氮蓝还原法(MTT)检测细胞活性的变化。用transwell小室研究结肠癌细胞的迁移及浸润。用RT-PCR检测FXRm RNA及MMP-7mRNA表达的变化,用western blot检测FXR及MMP-7蛋白表达的变化。结果:MTT结果显示GW4064作用于人结直肠HT-29细胞的生长抑制率呈浓度依赖性;transwell小室结果显示GW4064抑制结肠癌细胞的浸润转移,与对照组相比,差异具有统计学意义(P0.05),RT-PCR及Western blot显示GW4064促进FXR m RNA及蛋白表达,抑制MMP-7mRNA及蛋白的表达,与对照组相比差异有统计学意义(P0.05)。结论:GW4064抑制结肠癌细胞的生长及转移,上调HT-29细胞FXR m RNA及蛋白的表达,下调HT-29细胞MMP-7 m RNA及蛋白的表达。FXR被激活后抑制结肠癌细胞转移,MMP-7可能是其作用通路之一。     

FXR与糖尿病相关性研究进展

国际糖尿病联盟(IDF)最新数据表明目前中国糖尿病患者在剧增。糖尿病是以胰岛素分泌相对或和绝对不足导致的慢性高血糖为特征的代谢性疾病,法尼醇X受体(farnesoid X receptor,FXR,NR1H4)是能被胆汁酸激活的转录因子,FXR能对胆汁酸的代谢进行调节,胆汁酸代谢与糖尿病相关,胆汁酸代谢在β细胞的功能是通过FXR介导的,本文回顾国内外有关法尼醇X受体通过抑制肝糖原异生、增加肝糖原储存、影响胰岛素信号、增加胰岛素的分泌和增强胰岛素的敏感性等机制发挥调节血糖平衡作用的研究,意在探索FXR与糖尿病的相关性,为糖尿病的发病机制提供新的理论依据。