RESUMO
Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science 307: 1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.
Assuntos
Inibidores da Fusão de HIV/farmacologia , Inibidores da Fusão de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Receptores CCR5/metabolismo , Internalização do Vírus/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores da Fusão de HIV/química , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologiaRESUMO
Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.
Assuntos
Cumestrol/farmacologia , Fitoestrógenos/farmacologia , Receptores de Esteroides/antagonistas & inibidores , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Linhagem Celular , Células Cultivadas , Receptor Constitutivo de Androstano , Cumestrol/química , Cumestrol/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Coativador 1 de Receptor Nuclear , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fitoestrógenos/química , Fitoestrógenos/metabolismo , Receptor de Pregnano X , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we describe the cloning and functional characterization of two novel vitamin D receptor (VDR) paralogs from the freshwater teleost medaka (Oryzias latipes). VDR sequences were identified through mining of the medaka genome database in which gene organization and structure was determined. Two distinct VDR genes were identified in the medaka genome and mapped to defined loci. Each VDR sequence exhibits unique intronic organization and dissimilar 5' untranslated regions, suggesting they are not isoforms of the same gene locus. Phylogenetic comparison with additional teleosts and mammalian VDR sequences illustrate that two distinct clusters are formed separating aquatic and terrestrial species. Nested within the teleost cluster are two separate clades for VDRalpha and VDRbeta. The topology of teleost VDR sequences is consistent with the notion of paralogous genes arising from a whole genome duplication event prior to teleost radiation. Functional characterization was conducted through the development of VDR expression vectors including Gal4 chimeras containing the yeast Gal4 DNA binding domain fused to the medaka VDR ligand binding domain and full-length protein. The common VDR ligand 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D(3)] resulted in significant transactivation activity with both the Gal4 and full-length constructs of medaka (m) VDRbeta. Comparatively, transactivation of mVDRalpha with 1alpha,25(OH)(2)D(3) was highly attenuated, suggesting a functional divergence between these two nuclear receptor paralogs. We additionally demonstrate through coactivator studies that mVDRalpha is still functional; however, it exhibits a different sensitivity to 1alpha,25(OH)(2)D(3), compared with VDRbeta. These results suggest that in mVDRalpha and VDRbeta have undergone a functional divergence through a process of sub- and/or neofunctionalization of VDR nuclear receptor gene pairs.
Assuntos
Evolução Molecular , Peixes/genética , Receptores de Calcitriol/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Cricetinae , Cricetulus , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Oryzias/genética , Filogenia , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transativadores/metabolismoRESUMO
Defining complete sets of gene family members from diverse species provides the foundation for comparative studies. Using a bioinformatic approach, we have defined the entire nuclear receptor complement within the first available complete sequence of a non-human vertebrate (the teleost fish Fugu rubripes). In contrast to the human set (48 total nuclear receptors), we found 68 nuclear receptors in the Fugu genome. All 68 Fugu receptors had a clear human homolog, thus defining no new nuclear receptor subgroups. A reciprocal analysis showed that each human receptor had one or more Fugu orthologs, excepting CAR (NR1I3) and LXRbeta (NR1H2). These 68 receptors add striking diversity to the known nuclear receptor superfamily and provide important comparators to human nuclear receptors. We have compared several pharmacologically relevant human nuclear receptors (FXR, LXRalpha/beta, CAR, PXR, VDR and PPARalpha/gamma/delta) to their Fugu orthologs. This comparison included expression analysis across five Fugu tissue types. All of the Fugu receptors that were analyzed by PCR in this study were expressed, indicating that the majority of the additional Fugu receptors are likely to be functional.
Assuntos
Receptores Citoplasmáticos e Nucleares/genética , Takifugu/genética , Sequência de Aminoácidos , Animais , Receptor Constitutivo de Androstano , Feminino , Expressão Gênica , Variação Genética , Genoma , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
The nuclear receptors pregnane X receptor (PXR, NR1I2) and constitutive active receptor (CAR, NR1I3) have both been proposed to function as xenosensors, but the details of their respective physiological roles are still being elucidated. We have contrasted these two receptors in a variety of experiments including gene expression assays, cell-based ligand profiling assays, and crystallographic/structural modeling analyses. These data highlight key differences between PXR and CAR.
Assuntos
Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Receptor Constitutivo de Androstano , Expressão Gênica , Humanos , Ligantes , Modelos Moleculares , Receptor de Pregnano XRESUMO
The NR1I subfamily of nuclear receptors contains a phylogenetically diverse array of receptors related to the mammalian pregnane X receptor (PXR) (NR1I2) and constitutive androstane receptor (CAR) (NR1I3). We have carried out an extensive comparative analysis of this subgroup with representatives from fish, birds, amphibians, and mammals. Four novel receptors were isolated from fish, dog, pig, and monkey for this study and combined with a previously reported set of related receptors including human PXR, rabbit PXR, mouse PXR, chicken CXR, frog benzoate X receptors (BXRalpha, BXRbeta), and human and mouse CAR. A broad range of xenobiotics, steroids, and bile acids were tested for their ability to activate the ligand binding domain of each receptor. Three distinct groups of receptors were identified based on their pharmacological profiles: 1) the PXRs were activated by a broad range of xenobiotics and, along with the mammalian PXRs, included the chicken and fish receptors; 2) the CARs were less promiscuous, had high basal activities, and were generally repressed rather than activated by those compounds that modulated their activity; and 3) the BXRs were selectively activated by a subset of benzoate analogs and are likely to be specialized receptors for this chemical class of ligands. The PXRs are differentiated from the other NR1I receptors by a stretch of amino acids between helices 1 and 3, which we designate the H1-3 insert. This insert was present in the mammalian, chicken, and fish PXRs but absent in the CARs and BXRs. Modeling studies suggest that the H1-3 insert contributes to the promiscuity of the PXRs by facilitating the unwinding of helices-6 and -7, thereby expanding the ligand binding pocket.
Assuntos
Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Ácidos e Sais Biliares/farmacologia , Sítios de Ligação , Galinhas , Clonagem Molecular , Receptor Constitutivo de Androstano , Cães , Evolução Molecular , Haplorrinos , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Receptor de Pregnano X , Estrutura Secundária de Proteína , Coelhos , Ratos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/química , Receptores de Esteroides/genética , Alinhamento de Sequência , Esteroides/farmacologia , Suínos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Xenobióticos/farmacologia , Xenopus laevis , Peixe-ZebraRESUMO
The nuclear receptor (NR) superfamily is a large group of related, pharmacologically important receptors, comprising the targets for over 10% of commonly prescribed drugs. Cross-genome analysis of NR sequence, structure, and biological function, provides an important source of information on the function of human NRs and thus plays a role in NR drug discovery. For example, research on the pregnane X receptor (PXR; NR1I2), constitutive androstane receptor (CAR; NR1I3), hepatocyte nuclear factor 4 (HNF4; NR2A1), and farnesoid X receptor (FXR) illustrate how the study of nonhuman orthologs has provided new insights into NR biology and has increased our understanding of human NRs and orphan NR function. Understanding differences between humans and pharmacological model species may provide useful tools for the development of new NR-binding drugs.
Assuntos
Genoma Humano , Receptores Citoplasmáticos e Nucleares/genética , Tecnologia Farmacêutica/métodos , Sequência de Aminoácidos/genética , Animais , Receptor Constitutivo de Androstano , Humanos , Dados de Sequência Molecular , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Many of the commonly observed reproductive toxicities associated with therapeutic compounds can be traced to a disruption of the steroidogenic pathway. We sought to develop an in vitro assay that would predict reproductive toxicity and be high throughput in nature. H295R cells, previously validated as having an intact and functional steroidogenic pathway, were treated with 83 known-positive and 79 known-negative proprietary and public-domain compounds. The assay measured the expression of the key enzymes STAR, 3ßHSD2, CYP17A1, CYP11B2, CYP19A1, CYP21A2, and CYP11A1 and the hormones DHEA, progesterone, testosterone, and cortisol. We found that a Random Forest model yielded a receiver operating characteristic area under the curve (ROC AUC) of 0.845, with sensitivity of 0.724 and specificity of 0.758 for predicting in vivo reproductive toxicity with this in vitro assay system.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Modelos Biológicos , 3-Hidroxiesteroide Desidrogenases/metabolismo , Linhagem Celular Tumoral , Colforsina/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Desidroepiandrosterona/metabolismo , Humanos , Hidrocortisona/metabolismo , Imidazóis/toxicidade , Modelos Estatísticos , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Testosterona/metabolismoRESUMO
Glucocorticoid receptor (GR) agonists have been used for more than half a century as the most effective treatment of acute and chronic inflammatory conditions despite serious side effects that accompany their extended use that include glucose intolerance, muscle wasting, skin thinning, and osteoporosis. As a starting point for the identification of GR ligands with an improved therapeutic index, we wished to discover selective nonsteroidal GR agonists and antagonists with simplified structure compared to known GR ligands to serve as starting points for the optimization of dissociated GR modulators. To do so, we selected multiple chemical series by structure guided docking studies and evaluated GR agonist activity. From these efforts we identified 5-arylindazole compounds that showed moderate binding to the glucocorticoid receptor (GR) with clear opportunities for further development. Structure guided optimization was used to design arrays that led to potent GR agonists and antagonists. Several in vitro and in vivo experiments were utilized to demonstrate that GR agonist 23a (GSK9027) had a profile similar to that of a classical steroidal GR agonist.
Assuntos
Desenho de Fármacos , Indazóis/química , Indazóis/farmacologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Indazóis/síntese química , Indazóis/farmacocinética , Masculino , Camundongos , Modelos Moleculares , NF-kappa B/metabolismo , Conformação Proteica , Ratos , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Especificidade por SubstratoRESUMO
The nuclear receptor constitutive androstane receptor (CAR) (NR1I3) regulates hepatic genes involved in xenobiotic detoxification as well as genes involved in energy homeostasis. We provide data that extend the role of CAR to regulation of serum triglyceride levels under conditions of metabolic/nutritional stress. The typically high serum triglyceride levels of ob/ob mice were completely normalized when crossed onto a Car(-/-) (mice deficient for the Car gene) genetic background. Moreover, increases in serum triglycerides observed after a high-fat diet (HFD) regime were not observed in Car(-/-) animals. Conversely, pharmacological induction of CAR activity using the selective mouse CAR agonist TCPOBOP during HFD feeding resulted in a CAR-dependent increase in serum triglyceride levels. A major regulator of hepatic fatty oxidation is the nuclear receptor PPARalpha (NR1C1). The expression of peroxisome proliferator-activated receptor alpha (PPARalpha) target genes was inversely related to the activity of CAR. Consistent with these observations, Car(-/-) animals exhibited increased hepatic fatty acid oxidation. Treatment of mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) significantly decreased expression of PPARalpha mRNA as well as Cyp4a14, CPT1alpha, and cytosolic Acyl-CoA thioesterase (CTE) in the liver. These data have implications in disease therapy such as for diabetes and nonalcoholic steatohepatitis (NASH).
Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Triglicerídeos/sangue , Animais , Sequência de Bases , Receptor Constitutivo de Androstano , Primers do DNA/genética , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Análise de Sequência com Séries de Oligonucleotídeos , PPAR alfa/genética , PPAR alfa/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Estresse Fisiológico , Fatores de Transcrição/agonistas , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
The orphan nuclear receptor CAR (NR1I3) has been characterized as a central component in the coordinate response to xenobiotic and endobiotic stress. In this study, we demonstrate that CAR plays a pivotal function in energy homeostasis and establish an unanticipated metabolic role for this nuclear receptor. Wild-type mice treated with the synthetic CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) exhibited decreased serum concentration of the thyroid hormone (TH) thyroxine (T(4)). However, treatment of Car(-/-) mice with TCPOBOP failed to elicit these changes. To examine whether CAR played a role in the regulation of TH levels under physiological conditions, wild-type and Car(-/-) mice were fasted for 24 h, a process known to alter TH metabolism in mammals. As expected, the serum triiodothyronine and T(4) concentrations decreased in wild-type mice. However, triiodothyronine and T(4) levels in fasted Car(-/-) mice remained significantly higher than those in fasted wild-type animals. Concomitant with the changes in serum TH levels, both CAR agonist treatment and fasting induced the expression of CAR target genes (notably, Cyp2b10, Ugt1a1, Sultn, Sult1a1, and Sult2a1) in a receptor-dependent manner. Importantly, the Ugt1a1, Sultn, Sult1a1, and Sult2a1 genes encode enzymes that are capable of metabolizing TH. An attenuated reduction in TH levels during fasting, as observed in Car(-/-) mice, would be predicted to increase weight loss during caloric restriction. Indeed, when Car(-/-) animals were placed on a 40% caloric restriction diet for 12 weeks, Car(-/-) animals lost over twice as much weight as their wild-type littermates. Thus, CAR participates in the molecular mechanisms contributing to homeostatic resistance to weight loss. These data imply that CAR represents a novel therapeutic target to uncouple metabolic rate from food intake and has implications in obesity and its associated disorders.
Assuntos
Receptores Citoplasmáticos e Nucleares/fisiologia , Hormônios Tireóideos/metabolismo , Fatores de Transcrição/fisiologia , Animais , Northern Blotting , Restrição Calórica , Receptor Constitutivo de Androstano , DNA Complementar/metabolismo , Regulação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Químicos , Obesidade , Piridinas/farmacologia , Tiroxina/sangue , Fatores de Tempo , Tri-Iodotironina/sangueRESUMO
Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.
Assuntos
Antineoplásicos/farmacologia , Hidrocarboneto de Aril Hidroxilases/biossíntese , Oxirredutases N-Desmetilantes/biossíntese , Paclitaxel/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Animais , Antineoplásicos/metabolismo , Citocromo P-450 CYP3A , Indução Enzimática , Técnicas In Vitro , Proteínas de Membrana , Camundongos , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Paclitaxel/metabolismo , Receptor de Pregnano X , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genéticaRESUMO
The nuclear pregnane X receptor (PXR) and constitutive androstane receptor (CAR) play central roles in protecting the body against environmental chemicals (xenobiotics). PXR and CAR are activated by a wide range of xenobiotics and regulate cytochrome P450 and other genes whose products are involved in the detoxification of these chemicals. In this report, we have used receptor-selective agonists together with receptor-null mice to identify PXR and CAR target genes in the liver and small intestine. Our results demonstrate that PXR and CAR regulate overlapping but distinct sets of genes involved in all phases of xenobiotic metabolism, including oxidative metabolism, conjugation, and transport. Among the murine genes regulated by PXR were those encoding PXR and CAR. We provide evidence that PXR regulates a similar program of genes involved in xenobiotic metabolism in human liver. Among the genes regulated by PXR in primary human hepatocytes were the aryl hydrocarbon receptor and its target genes CYP1A1 and CYP1A2. These findings underscore the importance of these two nuclear receptors in defending the body against a broad array of potentially harmful xenobiotics.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Hepatócitos/metabolismo , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Fatores de Transcrição/fisiologia , Xenobióticos/metabolismo , Animais , Células Cultivadas , Receptor Constitutivo de Androstano , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , Humanos , Inativação Metabólica/fisiologia , Intestino Delgado/metabolismo , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Oxirredução , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/deficiência , Receptores de Esteroides/genética , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
The orphan nuclear constitutive androstane receptor (CAR) is proposed to play a central role in the response to xenochemical stress. Identification of CAR target genes in humans has been limited by the lack of a selective CAR agonist. We report the identification of 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel human CAR agonist with the following characteristics: (a) potent activity in an in vitro fluorescence-based CAR activation assay; (b) selectivity for CAR over other nuclear receptors, including the xenobiotic pregnane X receptor (PXR); (c) the ability to induce human CAR nuclear translocation; and (d) the ability to induce the prototypical CAR target gene CYP2B6 in primary human hepatocytes. Using primary cultures of human hepatocytes, the effects of CITCO on gene expression were compared with those of the PXR ligand rifampicin. The relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.
Assuntos
Oximas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/farmacologia , Fatores de Transcrição/agonistas , Células Cultivadas , Receptor Constitutivo de Androstano , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Receptores Citoplasmáticos e Nucleares/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/fisiologiaRESUMO
The nuclear xenobiotic receptor PXR is activated by a wide variety of clinically used drugs and serves as a master regulator of drug metabolism and excretion gene expression in mammals. St. John's wort is used widely in Europe and the United States to treat depression. This unregulated herbal remedy leads to dangerous drug-drug interactions, however, in patients taking oral contraceptives, antivirals, or immunosuppressants. Such interactions are caused by the activation of the human PXR by hyperforin, the psychoactive agent in St. John's wort. In this study, we show that hyperforin induces the expression of numerous drug metabolism and excretion genes in primary human hepatocytes. We present the 2.1 A crystal structure of hyperforin in complex with the ligand binding domain of human PXR. Hyperforin induces conformational changes in PXR's ligand binding pocket relative to structures of human PXR elucidated previously and increases the size of the pocket by 250 A(3). We find that the mutation of individual aromatic residues within the ligand binding cavity changes PXR's response to particular ligands. Taken together, these results demonstrate that PXR employs structural flexibility to expand the chemical space it samples and that the mutation of specific residues within the ligand binding pocket of PXR tunes the receptor's response to ligands.