RESUMO
Farnesoid X receptor (FXR) is a nuclear receptor that regulates the synthesis and enterohepatic circulation of bile acids (BAs). It also regulates lipid and carbohydrate metabolism, making FXR ligands potential therapeutic agents for systemic and/or hepatic metabolic disorders. We previously synthesized a series of FXR antagonists and showed that oral administration of FLG249 reduced the expression of several FXR target genes in the mouse ileum. Here, we investigated the effects of FLG249 on lipid metabolism in mice fed a high-fat diet (HFD). When FLG249 was administered for 4 weeks to HFD-induced obese mice, it altered the expression of genes related to BA metabolism, ceramide synthesis and fatty acid ß-oxidation, improving lipid metabolism in the liver and ileum without decreasing body weight. These findings suggest that FLG249 has the potential to be a low toxicity pharmaceutical compound and likely acts as a nonsteroidal FXR antagonist to improve lipid metabolism disorders.
Assuntos
Colesterol , Dieta Hiperlipídica , Fígado , Camundongos Endogâmicos C57BL , Obesidade , Receptores Citoplasmáticos e Nucleares , Triglicerídeos , Animais , Dieta Hiperlipídica/efeitos adversos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Masculino , Fígado/metabolismo , Fígado/efeitos dos fármacos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/sangue , Colesterol/sangue , Triglicerídeos/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácidos e Sais Biliares/metabolismo , Camundongos , Camundongos Obesos , Íleo/metabolismo , Íleo/efeitos dos fármacosRESUMO
Farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)γ are nuclear receptor 1 superfamily of transcription factors. FXR and PPARγ agonists have been individually investigated in clinical trial of anti-diabetic agents in the patients with nonalcoholic fatty liver disease (NAFLD). Regarding recent agonist development, the partial agonists for FXR and PPARγ are drawing attention from the standpoint of avoiding overactive responses caused by full agonists. In this article, we report that 18 with a benzimidazole scaffold possesses FXR/PPARγ dual partial agonistic activity. In addition, 18 shares the ability to reduce cyclin-dependent kinase 5-mediated phosphorylation of PPARγ-Ser273 and the metabolic stability in mouse liver microsome assay. To date, there are no published reports on FXR/PPARγ dual partial agonists with biological profiles similar to 18. Thus, the analog would be a feasible candidate as an unprecedented approach to NAFLD associated with type 2 diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 2 , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , PPAR gama/agonistas , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fatores de Transcrição , Hipoglicemiantes/farmacologiaRESUMO
BACKGROUND AND AIMS: Chronic HBV infection is a major health problem worldwide. Currently, the first-line treatment for HBV is nucleos(t)ide analogs or interferons; however, efficient therapeutic approaches that enable cure are lacking. Therefore, anti-HBV agents with mechanisms distinct from those of current drugs are needed. Sodium taurocholate cotransporting polypeptide (NTCP) was previously identified as an HBV receptor that is inhibited by several compounds. Farnesoid X receptor (FXR) activation also inhibits NTCP function. APPROACH AND RESULTS: In this study, we investigated the inhibitory effect of bile acid (BA) derivatives-namely obeticholic acid (OCA), 6α-ethyl-24-nor-5ß-cholane-3α,7α,23-triol-23 sulfate sodium salt (INT-767; a dual agonist of FXR and Takeda G protein-coupled receptor [TGR5]), and 6α-ethyl-23(S)-methyl-cholic acid (INT-777; a TGR5 agonist)-3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064; a FXR agonist), cyclosporin A, and irbesartan. OCA and INT-777 suppressed HBV infection in HepG2-human NTCP-C4 cells. Interestingly, INT-767 showed potent inhibition by attaching to HBV particles rather than binding to NTCP. As an entry inhibitor, INT-767 was stronger than various natural BAs. Furthermore, in chimeric mice with humanized liver, INT-767 markedly delayed the initial rise of HBsAg, HBeAg, and HBV DNA and reduced covalently closed circular DNA. The strong inhibitory effect of INT-767 may be due to the cumulative effect of its ability to inhibit the entry of HBV and to stimulate FXR downstream signaling, which affects the postentry step. CONCLUSIONS: Our results suggest that BA derivatives, particularly INT-767, are prospective candidate anti-HBV agents. Clarifying the underlying mechanisms of BA derivatives would facilitate the development of anti-HBV agents.
Assuntos
Antivirais/farmacologia , Hepatite B Crônica/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Acoplados a Proteínas G/agonistas , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Ácidos e Sais Biliares/farmacologia , Ácidos e Sais Biliares/uso terapêutico , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Ácido Quenodesoxicólico/uso terapêutico , Ácidos Cólicos/farmacologia , Ácidos Cólicos/uso terapêutico , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/virologia , Humanos , Masculino , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Estudos Prospectivos , Receptores Citoplasmáticos e Nucleares/metabolismo , Simportadores/metabolismo , Quimeras de TransplanteRESUMO
Secondary bile acids (SBAs) with high hydrophobicity are abundant in the colonic lumen. However, both aggravating and protective roles of SBAs have been proposed in the pathogenesis of inflammatory bowel diseases (IBDs). We observed that oral administration of hyodeoxycholic acid (HDCA), a hydrophilic bile acid, prevented the development of dextran sulfate sodium (DSS)-induced colitis in mice. We then examined the individual effects of DSS and HDCA as well as their combined effects on fecal bile acid profile in mice. HDCA treatment increased the levels of most of fecal bile acids, whereas DSS treatment had limited effects on the levels of fecal bile acids. The combined treatment with DSS and HDCA synergistically increased the levels of fecal chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) in feces, which are potent activators of the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5). The overall hydrophobicity of fecal bile acids was not modified by any treatments. Our data suggest that the preventive effect of HDCA on DSS-induced colitis in mice is due to the synergism between DSS and HDCA in increasing the levels of the fecal bile acids with potencies to activate FXR and TGR5.
Assuntos
Colite , Animais , Ácidos e Sais Biliares , Ácido Quenodesoxicólico/efeitos adversos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/prevenção & controle , Ácido Desoxicólico/efeitos adversos , Sulfato de Dextrana , Camundongos , Receptores Acoplados a Proteínas GRESUMO
As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. With respect to the bone metabolism, FXR positively regulates bone metabolism through both bone formation and resorption of the bone remodeling pathways. Some of FXR agonists possessing isoxazole moiety are undergoing clinical trials for the treatment of non-alcoholic steatohepatitis. To date, therefore, the activation of FXR leads to considerable interest in FXR as potential therapeutic targets. We have identified a series of nonsteroidal FXR agonists bearing N1-methyl benzimidazole and isoxazole moieties that are bridged with aromatic derivatives. They showed affinity to FXR, but also weak affinity toward the vitamin D receptor (VDR) that involves regulation of calcium and phosphate homeostasis and is activated by bile acids. The deployment of FXR agonists without activity against VDR as off-target is therefore crucial in the development of FXR ligands. Our efforts focusing on increasing the agonist properties towards FXR led to the discovery of 19, which activates FXR at and below nanomolar levels (EC50 = 26.5 ± 10.5 nM TR-FRET and 0.8 ± 0.2 nM luciferase, respectively) and functions as a FXR agonist: the affinity toward FXR over eight nuclear receptors, including VDR [IC50 (VDR) / EC50 (FXR) > 5000] and TGR5, effects FXR target genes, and activates bone morphogenetic protein-2-induced differentiation of mouse bone marrow-derived mesenchymal stem cell-like ST2 cells into osteoblast.
Assuntos
Benzimidazóis/farmacologia , Receptores de Calcitriol/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/agonistas , Benzimidazóis/síntese química , Benzimidazóis/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Receptores de Calcitriol/metabolismo , Relação Estrutura-AtividadeRESUMO
Antagonizing transcriptional activity of farnesoid X receptor (FXR) in the intestine has been reported as an effective means for the treatment of nonalcoholic fatty liver disease, type 2 diabetes and obesity. We describe herein that the building blocks necessary to maintain the antagonism of our chemotype were investigated in order to modulate in vivo pharmacokinetic behavior and the tissue distribution without blunting the activity against FXR. A comprehensive understanding of the structure-activity relationship led to analog 30, which is superior to 12 in terms of its pharmacokinetic profiles by oral administration and its tissue distribution toward target tissues (liver and ileum) in rats while preserving the in vitro activity of 12 against FXR. Thus, 30 should be a candidate compound to investigate the effects of inhibiting FXR activity while simultaneously improving the outcome of nonalcoholic fatty liver disease, type 2 diabetes and obesity.
Assuntos
Benzimidazóis/farmacocinética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Administração Intravenosa , Administração Oral , Animais , Fármacos Antiobesidade/administração & dosagem , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacocinética , Benzimidazóis/administração & dosagem , Benzimidazóis/síntese química , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacocinética , Íleo/metabolismo , Fígado/metabolismo , Masculino , Estrutura Molecular , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
We describe here a novel chemotype with substituted benzimidazole scaffold for nonsteroidal farnesoid X receptor (FXR) antagonists starting from the identification of a screening hit, BB-4. Structure diversity in four regions A-D of BB-4 or 1 is discussed. In particular, regions A and C had an effect on an antagonism against FXR as demonstrated by the derivatives represented by 7 and 15, respectively. Thus, compound 19 arising from the combination of regions A and C underscored an important fact on antagonism against FXR, also showing the reduced small heterodimer partner and the increased cholesterol 7α-hydroxylase expression levels.
Assuntos
Benzimidazóis/farmacologia , Descoberta de Drogas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Benzimidazóis/química , Linhagem Celular Tumoral , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Espectroscopia de Prótons por Ressonância Magnética , RNA Mensageiro/genética , Relação Estrutura-AtividadeRESUMO
The aim of this study is to examine the ability of the bile acid analogues obtained by chemical modification of ursodeoxycholic acid (UDCA) for TGR5 activation. Eleven UDCA analogues including 3- or 7-methylated UDCAs and amino acid conjugates were investigated as to their ability to activate TGR5 by means of the luciferase assay. It was noteworthy that 7α-methylated UDCA, namely 3α,7ß-dihydroxy-7α-methyl-5ß-cholanoic acid, had a significantly high affinity for and ability to activate TGR5 as compared to UDCA. Additionally, FXR activation ability of 7α-methylated UDCA was low relative to that of UDCA. However, other modification of UDCA, such as the introduction of methyl group at its C-3 position and oxidation or epimerization of hydroxyl group in the C-3 position, could not elicit such remarkable effect. The present findings would provide a useful strategy for the development of TGR5-selective agonist.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Ácido Ursodesoxicólico/análogos & derivados , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-Atividade , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/farmacologiaRESUMO
We describe the discovery of analog 15 (FLG249), which is an orally active and nonsteroidal farnesoid X receptor (FXR) antagonist in mice with unique profiles, such as a propensity for ileum distribution and the significant control in the expression level of three FXR target genes in mouse ileum. Key design features incorporated in 15 were the introduction of metabolically stable groups in potent and metabolically labile antagonist 9. Our pursuit ultimately identified FXR antagonist 15, which has enabled its assessment in a drug discovery program.
RESUMO
TGR5 is a G protein-coupled receptor that is activated by bile acids, resulting in an increase in cAMP levels and the subsequent modulation of energy expenditure in brown adipose tissue and muscle. Therefore, the development of a TGR5-specific agonist could lead to the prevention and treatment of various metabolic disorders related to obesity. In the present study, we evaluated the ability of bile alcohols, which are structurally and physiologically similar to bile acids and are produced as the end products of cholesterol catabolism in evolutionarily primitive vertebrates, to act as TGR5 agonists. In a cell-based reporter assay and a cAMP production assay performed in vitro, most bile alcohols with a side chain containing hydroxyl group(s) were highly efficacious agonists for TGR5 comparable to its most potent ligand in the naturally occurring bile acid, lithocholic acid. However, the abilities of the bile alcohols to activate TGR5 varied with the position and number of the hydroxyl substituent in the side chain. Additionally, the conformation of the steroidal nucleus of bile alcohols is also important for its activity as a TGR5 agonist. Thus, we have provided new insights into the structure-activity relationships of bile alcohols as TGR5 agonists.
Assuntos
Membrana Celular/metabolismo , Colestanóis/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Colestanóis/química , Colestanóis/farmacologia , Humanos , Hidróxidos/química , Ligantes , Conformação Molecular , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The biliary bile salts of the medaka, the Japanese rice fish (Oryzias latipes) were isolated and identified. Only bile acids were present, and all were N-acylamidated with taurine. Three bile acids, constituting 98% of total bile acids, were isolated by chromatography and their structure inferred from their properties compared to those of synthetic standards when analyzed by liquid chromatographytandem mass spectrometry. The dominant bile acid was the 25R-epimer (82%) of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-oic acid. The 25S-epimer was also present (11%), as was cholic acid (5%). Complete (1)H and (13)C NMR signal assignments of the C-25 epimers were made by using a combination of several 1D- and 2D-NMR techniques. The (1)H and (13)C NMR chemical shifts and spectral patterns of the hydrogen and carbon atoms, being close to the asymmetric centered at C-25, provided confirmatory evidence in that they distinguished the two epimeric diastereomers. The medaka is the first fish species identified as having C(27) biliary bile acids as dominant among its major bile salts.
Assuntos
Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Bile/química , Colestanóis/química , Colestanóis/metabolismo , Oryzias/fisiologia , Animais , Estrutura Molecular , Oryzias/genética , FilogeniaRESUMO
BACKGROUND: Some patients with cholestasis of unknown cause may have inborn errors of bile acid metabolism (IEBAM) thus causing abnormalities of bile acid biosynthesis. Although seven types of bile acid synthetic defects have thus far been reported for this disorder, no detailed information on its incidence and so on in Japan is yet available. In order to elucidate the current status of IEBAM in Japan, in July 1996 a diagnostic determination system was established for high-risk screening for IEBAM. METHODS: Urinary bile acids were analyzed on gas chromatography-mass spectrometry (GC-MS) and quantitative analysis was done using selected ion monitoring (SIM). RESULTS AND CONCLUSIONS: In a total of 576 samples analyzed over the 10 year period prior to June 2005, 159 patients were found with cholestasis of unknown etiology. Of these patients, 10 (6.3%) were found to have IEBAM by this system, while 91 (61.1%) had cholestasis without a definitive diagnosis. This diagnostic determination system with GC-MS of urinary bile acids is therefore considered useful for detecting IEBAM.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/etiologia , Erros Inatos do Metabolismo de Esteroides/diagnóstico , Adolescente , Adulto , Ácidos e Sais Biliares/urina , Criança , Colestase/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lactente , Recém-Nascido , Síndrome de Smith-Lemli-Opitz/diagnóstico , Adulto Jovem , Síndrome de Zellweger/diagnósticoRESUMO
TGR5, a metabotropic receptor that is G-protein-coupled to the induction of adenylate cyclase, has been recognized as the molecular link connecting bile acids to the control of energy and glucose homeostasis. With the aim of disclosing novel selective modulators of this receptor and at the same time clarifying the molecular basis of TGR5 activation, we report herein the biological screening of a collection of natural occurring bile acids, bile acid derivatives, and some steroid hormones, which has resulted in the discovery of new potent and selective TGR5 ligands. Biological results of the tested collection of compounds were used to extend the structure-activity relationships of TGR5 agonists and to develop a binary classification model of TGR5 activity. This model in particular could unveil some hidden properties shared by the molecular shape of bile acids and steroid hormones that are relevant to TGR5 activation and may hence be used to address the design of novel selective and potent TGR5 agonists.
Assuntos
Ácidos e Sais Biliares/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Ácidos e Sais Biliares/química , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Hormônios/química , Hormônios/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure-activity relationship (SAR) exploration of nonacidic FXR antagonist 6 focusing on two regions in the structure and biological evaluation of nonacidic 10 with the characteristic N-acylated piperidine group obtained from SAR studies. As the robust affinity to FXR is feasible with our nonacidic analogue, 10 is among the most promising candidates for in vivo testing.
RESUMO
Cerebrotendinous xanthomatosis (CTX) is a rare, autosomal recessive, inborn disruption in bile acid synthesis characterized by severe systemic xanthomas, cataracts and neurological injuries occurring before adolescence without elevation of the serum cholesterol or triglyceride levels. CTX is caused by a deficiency of the mitochondrial enzyme sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. We herein report a 50-year-old Japanese woman with late-onset CTX who had no relevant symptoms before the development of bilateral Achilles tendon xanthomas in middle age. A genetic analysis revealed a compound heterozygous mutation in the CYP27A1 gene with a previously known missense mutation (NM_000784.3:c.1421 G>A) and a novel frame shift mutation of NM_000784.3:c.1342_1343insCACC.
Assuntos
Colestanotriol 26-Mono-Oxigenase/genética , Mutação/genética , Xantomatose Cerebrotendinosa/diagnóstico , Tendão do Calcâneo , Feminino , Mutação da Fase de Leitura , Humanos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , XantomatoseRESUMO
The farnesoid X receptor (FXR) is a major nuclear receptor of bile acids; its activation suppresses sterol regulatory element-binding protein 1c (SREBP1c)-mediated lipogenesis and decreases the lipid contents in the liver. There are many reports showing that the administration of ursodeoxycholic acid (UDCA) suppresses lipogenesis and reduces the lipid contents in the liver of experimental animals. Since UDCA is not recognized as an FXR agonist, these effects of UDCA cannot be readily explained by its direct activation of FXR. We observed that the dietary administration of UDCA in mice decreased the expression levels of SREBP1c and its target lipogenic genes. Alpha- and ß-muricholic acids (MCA) and cholic acid (CA) were the major bile acids in the mouse liver but their contents decreased upon UDCA administration. The hepatic contents of chenodeoxycholic acid and deoxycholic acid (DCA) were relatively low but were not changed by UDCA. UDCA did not show FXR agonistic or antagonistic potency in in vitro FXR transactivation assay. Taking these together, we deduced that the above-mentioned change in hepatic bile acid composition induced upon UDCA administration might cause the relative increase in the FXR activity in the liver, mainly by the reduction in the content of ß-MCA, a farnesoid X receptor antagonist, which suggests a mechanism by which UDCA suppresses lipogenesis and decreases the lipid contents in the mouse liver.
Assuntos
Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Ácido Ursodesoxicólico/administração & dosagem , Animais , Linhagem Celular , Ácido Quenodesoxicólico/metabolismo , Ácidos Cólicos , Ácido Desoxicólico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Ácido Ursodesoxicólico/farmacologiaRESUMO
The bile acid intermediate 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxisomal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.
Assuntos
Trifosfato de Adenosina/metabolismo , Colestanóis/metabolismo , Ácidos Cólicos/metabolismo , Fígado/metabolismo , Peroxissomos/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Transporte Biológico Ativo , Membrana Celular/metabolismo , Ácidos Cólicos/química , Endopeptidase K/farmacologia , Inibidores Enzimáticos/farmacologia , Immunoblotting , Masculino , Nucleotídeos/farmacologia , Oxirredução , Ratos , Ratos Wistar , Fatores de TempoRESUMO
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, 3alpha,7alpha-dihydroxy-5beta-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.
Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/farmacologia , Técnicas Biossensoriais/métodos , Linhagem Celular , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/farmacologia , Chlorocebus aethiops , Proteínas de Ligação a DNA/química , Transferência Ressonante de Energia de Fluorescência , Histona Acetiltransferases , Humanos , Cinética , Ligantes , Luciferases/metabolismo , Dados de Sequência Molecular , Coativador 1 de Receptor Nuclear , Fragmentos de Peptídeos/química , Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição/químicaRESUMO
A key intermediate in the biosynthetic pathway by which C(24) bile acids are formed from cholesterol has long been considered to be varanic acid, (24ξ,25ξ)-3α,7α,12α-24-tetrahydroxy-5ß-cholestan-27-oic acid. The (24R,25R)-epimer of this tetrahydroxy bile acid, in the form of its taurine N-acyl amidate, was thought to be the major biliary bile acid in lizards of the family Varanidae. We report here that a major biliary bile acid of three lizard species - the Komodo dragon (Varanus komodoensis), Gray's monitor (Varanus olivaceus), and the Gila monster (Heloderma suspectum) - is a novel epimer of varanic acid. The epimer was shown to be (24R,25S)-3α,7α,12α,24-tetrahydroxy-5ß-cholestan-27-oic acid (present in bile as its taurine conjugate). The structure was established by mass spectroscopy and by (1)H and (13)C nuclear magnetic spectroscopy, as well as by synthesis of the compound.
Assuntos
Ácidos e Sais Biliares/química , Sistema Biliar/metabolismo , Colestanóis/química , Lagartos , Animais , Ácidos e Sais Biliares/isolamento & purificação , Ácidos e Sais Biliares/metabolismo , Colestanóis/isolamento & purificação , Colestanóis/metabolismo , EstereoisomerismoRESUMO
Three C(27) bile acids were found to be major biliary bile acids in the capuchinbird (Perissocephalus tricolor) and bare-throated bellbird (Procnias nudicollis), both members of the Cotingidae family of the order Passeriformes. The individual bile acids were isolated by preparative RP-HPLC, and their structures were established by RP-HPLC, LC/ESI-MS/MS and NMR as well as by a comparison of their chromatographic properties with those of authentic reference standards of their 12α-hydroxy derivatives. The most abundant bile acid present in the capuchinbird bile was the taurine conjugate of C(27) (24R,25R)-3α,7α,24-trihydroxy-5ß-cholestan-27-oic acid, a diastereomer not previously identified as a natural bile acid. The four diastereomers of taurine-conjugated (24ξ,25ξ)-3α,7α,24-trihydroxy-5ß-cholestan-27-oic acid could be distinguished by NMR and were resolved by RP-HPLC. The RRT of the diastereomers (with taurocholic acid as 1.0) were found to be increased in the following order: (24R,25R)<(24S,25R)<(24S,25S)<(24R,25S). Two epimers (25R and 25S) of C(27) 3α,7α-dihydroxy-5ß-cholestan-27-oic acid were also present (as the taurine conjugates) in both bird species. Epimers of the two compounds could be distinguished by their NMR spectra and resolved by RP-HPLC with the (25S)-epimer eluting before the (25R)-epimer. Characterization of the taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5ß-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5ß-cholestan-27-oic acid should facilitate their detection in peroxisomal disease and inborn errors of bile acid biosynthesis.