The nuclear receptor superfamily and drug discovery.
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Drug Design↗
Biomedical subjects
John T Moore
Publications and source records attributed to John T Moore.
Metabolites of progesterone and the pregnane X receptor: a novel pathway regulating uterine contractility in pregnancy?
OBJECTIVE: The purpose of this study was to determine the role of 5beta-dihydroprogesterone (5beta-DHP), acting through the nuclear receptor pregnane X receptor (PXR), in regulating uterine contractility. STUDY DESIGN: Uterine contractility was studied in tissues from women, rats, and mice. Messenger RNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR), and protein was measured using enzyme assays, immunofluorescence microscopy, and Western analyses. RESULTS: Human and rat uterine tissues contain mRNA and protein for 5beta-reductase and for PXR. Acute in vitro treatment with 5beta-DHP causes rapid uterine relaxation that is not mediated by PXR. Chronic in vivo administration of 5beta-DHP to mice with intact PXR, but not in mice with disrupted PXR, causes an increased effect of 1400W, a specific inhibitor of inducible nitric oxide synthase (iNOS). This suggests that 5beta-DHP increased iNOS-modulated uterine tone, as occurs during pregnancy. CONCLUSION: These data support the hypothesis that metabolites of progesterone may act chronically through a PXR-mediated mechanism to regulate uterine contractility.
Analysis of Variance↗
A structural basis for constitutive activity in the human CAR/RXRalpha heterodimer.
The X-ray crystal structure of the human constitutive androstane receptor (CAR, NR1I3)/retinoid X receptor alpha (RXRalpha, NR2B1) heterodimer sheds light on the mechanism of ligand-independent activation of transcription by nuclear receptors. CAR contains a single-turn Helix X that restricts the conformational freedom of the C-terminal AF2 helix, favoring the active state of the receptor. Helix X and AF2 sit atop four amino acids that shield the CAR ligand binding pocket. A fatty acid ligand was identified in the RXRalpha binding pocket. The endogenous RXRalpha ligand, combined with stabilizing interactions from the heterodimer interface, served to hold RXRalpha in an active conformation. The structure suggests that upon translocation, CAR/RXRalpha heterodimers are preorganized in an active conformation in cells such that they can regulate transcription of target genes. Insights into the molecular basis of CAR constitutive activity can be exploited in the design of inverse agonists as drugs for treatment of obesity.
Binding Sites↗
The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction.
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.
Animals↗
Liver X receptors are regulators of adipocyte gene expression but not differentiation: identification of apoD as a direct target.
The liver X receptors alpha and beta (LXRalpha and LXRbeta) have been shown to play important roles in lipid homeostasis in liver and macrophages, however, their function in adipose tissue is not well defined. Both LXRs are highly expressed in fat, and the expression of LXRalpha increases during adipogenesis. Furthermore, LXRalpha expression is induced by peroxisome proliferator-activated receptor gamma (PPARgamma), the master regulator of fat cell differentiation. Here we investigate the role of LXRs in adipocyte differentiation and gene expression and their potential crosstalk with the PPARgamma pathway. We demonstrate that LXR agonists have no significant effect on the differentiation of 3T3-F442A or 3T3-L1 preadipocytes in vitro and do not alter the expression of differentiation-linked PPARgamma target genes in vivo. Moreover, retroviral expression of LXRalpha in NIH-3T3 cells does not alter the adipogenic potential of these cells and neither augments nor inhibits the action of PPARgamma. However, transcriptional profiling studies reveal that LXRs are important regulators of adipocyte gene expression. We identify the multifunction lipid carrier protein apolipoprotein D and the lipogenic protein Spot 14 as LXR responsive genes both in vitro and in vivo. Thus, although LXRs do not influence adipocyte differentiation per se, these receptors are likely to play an important role in the modulation of lipid metabolism in adipocytes.
3T3 Cells↗
CAR: detailing new models.
Functional analysis has broadened our understanding of the physiological roles of the two related nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3). Initial research focused on the role of these two receptors in xenobiotic detoxification and, more recently, additional functional roles for CAR have been identified. Specifically, CAR activity has been shown to ameliorate the effects of hyperbilirubinemia, caloric restriction and toxic bile acids. Thus, the physiological role of CAR has broadened to include responses to metabolic and nutritional stress. These data highlight potential new opportunities in targeting CAR for drug discovery.
Animals↗
The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily.
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.
Amino Acid Sequence↗
Estrogen receptor beta protein in human breast cancer: correlation with clinical tumor parameters.
The recent discovery of a second estrogen receptor (ER), designated ERbeta, raises pressing questions about its role in estrogen regulation of human breast cancer cells and its significance for the prediction of recurrence and treatment responses in clinical breast cancer. Most of what we know about ERbeta expression comes from studies examining a limited number of samples at the RNA level. We have now generated a monoclonal antibody useful for the detection of ERbeta at the protein level in archival, formalin-fixed breast tumors and have examined its expression using immunohistochemistry in a pilot series of 242 breast cancer patients. Coexpression of ERbeta and ERalpha was found in the majority of the tumors, with 76% of the tumors expressing ERbeta as determined by immunohistochemistry. ERalpha, but not ERbeta, was strongly associated with progesterone receptor expression, suggesting that ERalpha is the predominant regulator of this estrogen-induced gene in breast tumors. Although ERalpha expression was positively correlated with low tumor grade, diploidy, and low S-phase fraction, all biological parameters of a good prognostic profile, ERbeta trended toward an association only with aneuploidy; no association with tumor grade or S-phase fraction was seen for ERbeta. We found that ERbeta expression does cause false positive readings for ERalpha. These results suggest that ERbeta expression is not a surrogate for ERalpha in clinical breast tumors and, as such, could be a useful biomarker in its own right.
Antibodies, Monoclonal↗
Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes.
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.
Cells, Cultured↗
Functional and structural comparison of PXR and CAR.
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.
Animals↗
Beyond the human genome: examples of nuclear receptor analysis in model organisms and potential for drug discovery.
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.
Amino Acid Sequence↗
A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.
A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.
Crystallography, X-Ray↗
Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition.
Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.
Amino Acid Motifs↗
Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARalpha.
Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR, which in turn recruit histone deacetylases to the chromatin. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-alpha ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn alpha-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of the co-repressor motif is further reinforced by the antagonist, which blocks the AF-2 helix from adopting the active position. Biochemical analyses and structure-based mutagenesis indicate that this mode of co-repressor binding is highly conserved across nuclear receptors.
Amino Acid Motifs↗
Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids.
The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.
Amino Acid Sequence↗
Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification.
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.
Animals↗
Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors.
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.
Amino Acid Sequence↗
Genomics versus orphan nuclear receptors--a half-time report.
Following the successful cloning of the orphan nuclear receptors during the 1990s we entered the 21st century with knowledge of the full complement of human nuclear receptors. Many of these proteins are ligand-activated transcription factors that act as the cognate receptors for steroid, retinoid, and thyroid hormones. In addition to these well characterized endocrine hormone receptors, there are a large number of orphan receptors of which less is known about the nature and function of their ligands. The task of deciphering the physiological function of these orphan receptors has been aided by a new generation of genomic technologies. Through application of chemical, structural, and functional genomics, several orphan nuclear receptors have emerged as pharmaceutical drug targets for the treatment of important human diseases. The significant progress that has been made in the functional analysis of more than half of the nuclear receptor gene family provides an opportunity to review the impact of genomics in this endeavor.
Genomics↗