Nuclear Receptor Modulators Research Articles

Carotenoid metabolism in mammals, including man: formation, occurrence, and function of apocarotenoids: Thematic Review Series: Fat-Soluble Vitamins: Vitamin A

Vitamin A was recognized as an essential nutrient 100 years ago. In the 1930s, it became clear that dietary β-carotene was cleaved at its central double to yield vitamin A (retinal or β-apo-15'-carotenal). Thus a great deal of research has focused on the central cleavage of provitamin A carotenoids to form vitamin A (retinoids). The mechanisms of formation and the physiological role(s) of noncentral (eccentric) cleavage of both provitamin A carotenoids and nonprovitamin A carotenoids has been less clear. It is becoming apparent that the apocarotenoids exert unique biological activities themselves. These compounds are found in the diet and thus may be absorbed in the intestine, or they may form from enzymatic or nonenzymatic cleavage of the parent carotenoids. The mechanism of action of apocarotenoids in mammals is not fully worked out. However, as detailed in this review, they have profound effects on gene expression and work, at least in part, through the modulation of ligand-activated nuclear receptors. Understanding the interactions of apocarotenoids with other lipid-binding proteins, chaperones, and metabolizing enzymes will undoubtedly increase our understanding of the biological roles of these carotenoid metabolites.

Effect of Diet and Nutrients on Molecular Mechanism of Gene Expression Mediated by Nuclear Receptor and Epigenetic Modulation

Major research progress in the last few decades has elucidated the complex nexus between nutrition and health. Diet and lifestyle influence epigenetic changes that are heritable. However, a statement of reservation is needed here, viz. that it is often difficult to distinguish between epigenetic changes that are inherited from one generation to the next and true mutations, for instance in mitochondrial DNA. The last topic is a big one in its own right and will not be further dis- cussed in this article. Epigenetic changes induced by dietary nutrients ultimately culminate in changes of the expression of genes through transcription and translation. The interaction between dietary nutrients and nuclear receptors triggers the signaling pathway, leading to modulation of epigenetic change and gene expression. Knowledge about nuclear receptors is important for explaining dietary modulation of transcription via recruitment of large protein complexes. These proteins are capable of causing modification of chromosomal components, can influence chromatin proteins, and affect the binding of proteins to particular parts of the DNA molecules controlling the expression of individual genes. Chromatin complexes between DNA and proteins can be destabilized by recruitment of transcriptional coactivators by histone acetylation. How- ever, in the presence of hormone antagonists or in the absence of relevant ligands, recruitment of other cellular core pro- teins may stabilize chromatin by their influence on histone deacetylases, thus antagonizing the effect of enzymes causing histone acetylation. This article reviews the current knowledge on nutritional modulation of bioactive molecules by epige- netic changes, if they can regulate genetic expressions. The molecular mechanism of action of various dietary nutrients on gene expression mediated by nuclear receptors is also discussed.

Effect Of Diet and Nutrients on Molecular Mechanism of Gene Expression Mediated By Nuclear Receptor and Epigenetic Modulation

Effect Of Diet and Nutrients on Molecular Mechanism of Gene Expression Mediated By Nuclear Receptor and Epigenetic Modulation

Nuclear receptors and their selective pharmacologic modulators.

Nuclear receptors are ligand-activated transcription factors and include the receptors for steroid hormones, lipophilic vitamins, sterols, and bile acids. These receptors serve as targets for development of myriad drugs that target a range of disorders. Classically defined ligands that bind to the ligand-binding domain of nuclear receptors, whether they are endogenous or synthetic, either activate receptor activity (agonists) or block activation (antagonists) and due to the ability to alter activity of the receptors are often termed receptor "modulators." The complex pharmacology of nuclear receptors has provided a class of ligands distinct from these simple modulators where ligands display agonist/partial agonist/antagonist function in a tissue or gene selective manner. This class of ligands is defined as selective modulators. Here, we review the development and pharmacology of a range of selective nuclear receptor modulators.

Interlink between Nuclear Receptors, Posttranslational Modifications and the Biological Clock in Health and Diseases

The day and night cycle influences the body function in health and disease through the modulation of the biological clock in via the suprachiasmatic nucleus (SCN) which in turn acts as the master clock in aligning the downstream clocks in the peripheral organs. The peripheral clocks can also be entrained independently by different factors such as external nutritional, hormonal and chemical cues that in many cases come from ligand modulation of nuclear receptors and kinase activators. Disruption of the molecular clock is associated with many diseases including cancer, immune diseases and aging. In addition, administration of toxic drugs such as anti-cancer can disturb the clock. These examples lead to changes in the sleep pattern of the patient and consequently can accelerate the manifestation of disease. This review details the function of the molecular clock in health and disease and how nuclear receptors and posttranslational modifications interact with the clock to regulate its function. In addition this review describes how chronotherapy can be applied in the clinical setting to treat cancer patients

The therapeutic potential of RORγ modulators in the treatment of human disease.

Nuclear receptors (NR) are ligand-regulated transcription factors that bind DNA in proximity to their target genes and exert their effects as a result of binding by small molecule ligands such as sterols, lipids, fatty acids, retinoids, and steroid hormones. The retinoic acid receptor-related orphan receptors or RORs (NR1F1–NR1F3) are nuclear receptors that regulate multiple cellular processes, including metabolism, cellular differentiation, and apoptosis, in a range of tissues and organs. These receptors bind as monomers to ROR response elements commonly called ROREs present in promoter regions of target genes and tether chromatin remodeling enzymes, facilitating recruitment of transcription machinery. Several recent reports have highlighted the potential role for RORs in human disease, and more importantly, studies have demonstrated that these receptors can be modulated by exogenous synthetic ligands, paving the way for development of novel therapeutics. Here we review the current status of synthetic ligand development as well as the structural aspects governing modulation of ROR signaling pathways as they relate to metabolic diseases and autoimmune disorders.

Small Molecule Modulation of Nuclear Receptor Conformational Dynamics: Implications for Function and Drug Discovery

Nuclear receptors are targets for a wide range of ligands, both natural and synthetic, that regulate their activity and provide a means to pharmacologically modulate the receptor. Recent emphasis in the nuclear receptor field has focused on selective nuclear receptor modulators, which can display graded transcriptional responses and tissue selective pharmacological responses that deviate from the prototypical agonist or antagonist. Understanding the molecular mechanism of action of these selective modulators will provide significant insight toward the development of the next generation of modulators. Although most nuclear receptor structural studies have primarily focused on obtaining ligand-receptor cocrystal structures, recent studies implicate an important role for protein dynamics in the mechanism of action of nuclear receptor ligands. Here we review nuclear receptor studies reporting how ligands modulate the conformational dynamics of the nuclear receptor ligand-binding domain (LBD). A particular emphasis is placed on protein NMR and hydrogen/deuterium exchange (HDX) techniques and how they provide complementary information that, when combined with crystallography, provide detailed insight into the function of nuclear receptors.

What lies beneath: natural products from marine organisms as nuclear receptor modulators

The marine environment has long been known to be species-rich and to be a source of molecules with interesting and beneficial biochemical and clinical properties. However, despite some notable successes the potential of the 'marine pipeline' has yet to be fully realized. Recent studies involving members of the nuclear receptor superfamily illustrate the chemical richness of molecules from marine species and helps to re-emphasize the translational possibilities of natural products in drug discovery. In this issue of the Biochemical Journal Wang et al. describe the identification and characterization of such a compound, an agonist for the retinoic acid receptor isolated from the sponges Luffariella sp. and Fascaplysinopsis.

Marine natural products as modulators of nuclear receptors

Nuclear receptors (NRs) represent one of the most important drug targets in terms of potential therapeutic application, playing a role in every aspect of development, physiology and disease in humans. In recent years many natural products were found to act as ligands for NRs. As consequence of our long standing interest in the chemistry of bioactive marine products, we found that some new natural products were endowed with modulatory activity towards FXR, PXR and PPARγ receptors. The chemistry and the biological activity of these modulators will be discussed.

Constitutive expression and phenobarbital modulation of drug metabolizing enzymes and related nuclear receptors in cattle liver and extra-hepatic tissues

In humans and rodents, phenobarbital (PB) induces hepatic and extra-hepatic drug metabolizing enzymes (DMEs) through the activation of specific nuclear receptors (NRs). In contrast, few data about PB transcriptional effects in veterinary species are available.The constitutive expression and modulation of PB-responsive NR and DME genes, following an oral PB challenge, were investigated in cattle liver and extra-hepatic tissues (duodenum, kidney, lung, testis, adrenal and muscle).Likewise to humans and rodents, target genes were expressed to a lower extent compared to the liver with few exceptions.Phenobarbital significantly affected hepatic CYP2B22, 2C31, 2C87, 3A and UDP-glucuronosyltransferase 1A1-like, glutathione S-transferase A1-like and sulfotransferase 1A1-like (SULT1A1-like) mRNAs and apoprotein amounts; in extra-hepatic tissues, only duodenum showed a significant down-regulation of SULT1A1-like gene and apoprotein. Nuclear receptor mRNAs were never affected by PB.Presented data are the first evidence about the constitutive expression of foremost DME and NR genes in cattle extra-hepatic tissues, and the data obtained following a PB challenge are suggestive of species-differences in drug metabolism; altogether, these information are of value for the extrapolation of pharmacotoxicological data among species, the characterization of drug–drug interactions as well as the animal and consumer’s risk caused by harmful residues formation.

Noncanonical Mechanisms to Regulate Nuclear Receptor Signaling

Nuclear receptor (NR)-targeted therapies comprise a large class of clinically employed drugs. A number of drugs currently being used against this protein class were designed as structural analogs of the endogenous ligand of these receptors. In recent years, there has been significant interest in developing newer strategies to target NRs, especially those that rely on mechanistic pathways of NR function. Prominent among these are noncanonical means of targeting NRs, which include selective NR modulation, NR coactivator interaction inhibition, inhibition of NR DNA binding, modulation of NR cellular localization, modulation of NR ligand biosynthesis and downregulation of NR levels in target tissues. This article reviews each of these promising emerging strategies for NR drug development and highlights some of most significant successes achieved in using them.

Detection of bioavailable peroxisome proliferator-activated receptor gamma modulators by a cell-based luciferase reporter system

In vitro cell-based reporter assays are a useful tool for the discovery and characterization of nuclear receptor modulators. However, the properties of a given molecule can differ when tested in vitro and in vivo as a result of the molecule’s bioavailability. In this work, we describe a methodology that allows the detection of the PPARγ (peroxisome proliferator-activated receptor gamma) agonist bezafibrate in rat serum by an in vitro cell-based reporter assay. This methodology could be adapted to the detection and characterization of bioavailable PPARγ or other nuclear receptor modulators in serum, extending the possibilities of the classical in vitro assays.

Zearalenone exposure modulates the expression of ABC transporters and nuclear receptors in pregnant rats and fetal liver

The mycotoxin zearalenone (ZEN) is produced by a variety of Fusarium fungi and contaminates numerous cereals, fruits and vegetables. Interacting with the estrogen receptors, ZEN and reduced metabolites zearalenols cause hormonal effects in animals. Few data are available on the effects of repeated exposure to ZEN, particularly during pregnancy. The aim of our work was to assess the impact of this toxin on the expression of ABC transporters and nuclear receptors in fetal liver and pregnant rats that were exposed daily (gestation day 7–20) to 1mg/kg ZEN. Significant variations were observed, depending on the tissue type, the tissue origin (maternal or fetal), and the time of analysis after the last exposure to ZEN (4h or 24h). The modulations of expression were independent of the magnitude of tissue impregnation by ZEN and its metabolites. The maternal uterus was the most sensitive tissue: Abcb1a, Abcb1b and Abcg2 mRNA and protein expressions were induced at both times, while Abcc1, Abcc3 and Esr1 mRNA and protein expressions were inhibited then induced 4h and 24h after exposure, respectively. In the fetal liver, Abcb1a and Esr1 protein expression was inhibited at both times, while mRNA expression was induced 24h after the last exposure to ZEN. These results suggested that ZEN exposure could impact maternal and fetal exposure to ABC transporters substrates, and influence fetus development through nuclear receptor modulation.

RXR antagonism induces G0/G1 cell cycle arrest and ameliorates obesity by up‐regulating the p53–p21$^{\rm{Cip1}}$ pathway in adipocytes

The peroxisome proliferator activated receptor-γ (PPARγ) agonist, pioglitazone (PIO), exerts anti-diabetic properties associated with increased fat mass, whereas the retinoid X receptor (RXR) antagonist HX531 demonstrates anti-obesity and anti-diabetic effects with reduced body weight and fat pad mass. The cell cycle abnormality in adipocytes has not been well-investigated in obesity or during treatment with modulators of nuclear receptors. We therefore investigated cell size and cell cycle distributions of adipocytes in vivo and examined the expression of cell cycle regulators in cultured human visceral preadipocytes. The cell size distribution and cell cycle analyses of in vivo adipocytes derived from OLETF rats demonstrated that HX531 brought about G0/G1 cell cycle arrest associated with the inhibition of cellular hypertrophy, which resulted in the reduction of fat pad mass. In contrast, PIO promoted proliferation activities associated with the increase in M + late M:G0 + G1 ratio and the appearance of both small and hypertrophied adipocytes. In cultured human visceral preadipocytes HX531 up-regulated cell cycle regulators, p53, p21(Cip1), cyclin D1, Fbxw7 and Skp2, which are known contributors towards G0 /G1 cell cycle arrest. The knockdown of p53 with a shRNA lentivirus reversed the HX531-induced up-regulation of p21(Cip1), which is one of the major p53-effector molecules. We conclude that HX531 exerts anti-obesity and anti-diabetes properties by up-regulating the p53-p21(Cip1) pathway, resulting in G0/G1 cell cycle arrest and the inhibition of cellular hypertrophy of adipocytes.

Expression and induction by dexamethasone of ABC transporters and nuclear receptors in a human T-lymphocyte cell line

The efficacy of drugs acting within lymphocytes, like antiretroviral drugs in the treatment of HIV infection, depends on their intracellular concentrations modulated by efflux proteins like ABCB1 (P-glycoprotein). In lymphocytes, two glucocorticoids, prednisone and prednisolone, have been shown to induce ABCB1 activity. Yet, no data exist regarding dexamethasone (DEX). We report the modulation of ABC transporters and nuclear receptors’ expression by DEX in a commonly used model of human lymphocytes. CCRF-CEM cells were exposed to DEX (100 nM, 2 μM) for 24 to 72 hours. ABCB1 activity was measured using DiOC6 efflux in flow cytometry. Gene expression levels were quantified by qRT-PCR. ABCB1 activity and mRNA expression increased with DEX concentrations and incubation times. DEX (1 μM, 24 h) increased significantly ABCB1 and GR mRNA expression levels by around 8- and 3·5-fold, respectively (P<10−6). ABCB1 induction by DEX in CCRF-CEM cells suggests a potential risk of interaction in lymphocytes when associating DEX to ABCB1 substrates in antiretroviral multitherapies in vivo.

Quantitative NMR-Derived Interproton Distances Combined with Quantum Mechanical Calculations of 13C Chemical Shifts in the Stereochemical Determination of Conicasterol F, a Nuclear Receptor Ligand from Theonella swinhoei

Here we report the first application of combined accurate ROE-distance analysis with DFT calculations of NMR chemical shifts to achieve the relative configuration assignment of a marine natural product, conicasterol F, a new polyhydroxylated steroid isolated from the marine sponge Theonella swinhoei. We demonstrate the substantial advantages of this combined approach as a tool for structural studies of natural products, providing a powerful alternative to, or information to underpin, total synthesis when more classical NMR data analysis fails to provide unequivocal results. In this paper, we also describe the isolation and structure elucidation of conicasterol F and its 24-ethyl derivative, theonellasterol I, and their pharmacological evaluation as human nuclear receptor modulators.

Structure Activity Relationships of Nuclear Receptor, GPCR and Kinase Modulators Revealed with Differential HDX

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) has emerged as a powerful technology for analysis of protein conformational dynamics and ligand interactions. The regulation of transcriptional output by nuclear receptors (NRs) is driven by alterations in the conformational ensemble of the receptor upon ligand binding and previously we have shown that HDX can be used to determine a novel mechanism of ligand activation of PPARγ, detailed analysis of binding modes of ligands within the ligand binding pocket of two ER isoforms, and how ERα ligands can be classified and correlated to their pharmacology based on receptor HDX signatures. More recently, we have applied HDX to probe the conformational dynamics of intact full length nuclear receptor complexes upon interaction with DNA and coactivator proteins. These studies have demonstrated that DNA binding alters conformational dynamics of the nuclear receptor heterodimer in regions remote of the DBD. These alterations in conformational selection appear to be important for coactivator binding to the heterodimer suggesting that DNA acts as an allosteric ligand. In addition to work on NRs, the lab recently demonstrated the use of HDX for probing ligand interaction with G protein coupled receptor (GPCRs) and kinases. We have extended these studies to probe differential receptor perturbation upon interacting with functionally selective ligands, and with kinases probed both ligand and co-regulatory protein interactions. Results from these studies will be presented.

Identical Gene Regulation Patterns of T3 and Selective Thyroid Hormone Receptor Modulator GC-1

Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TRβ subtype vs. TRα, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T(3), and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TRβ, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T(3), at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TRβ to directly induce this gene but this gene-selective activity is not related to unusual T(3)-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T(3) and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.

NUCLEAR RECEPTORS

NUCLEAR RECEPTORS

Abstract A28: The link between the metabolic effects of nuclear receptor (NR) modulators and anticarcinogenic properties on leukemia cancer cells can be revealed through studying the Warburg effect

Abstract The Warburg effect is the observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by aerobic oxidation of pyruvate in mitochondria. Genetic studies in humans and rodents support the notion that metabolic enzymes controlling glucose metabolism can directly contribute to carcinogenesis. Current knowledge of nuclear receptor (NR) signaling networks indicates that NRs control a wide variety of metabolic processes by regulating the expression of genes encoding key enzymes, transporters, and other proteins involved in metabolic homeostasis. Thus, a better understanding of the mechanistic links between cellular metabolism control by NRs and cancer cells' growth control may ultimately lead to better treatments for human cancer. We created a library of 134 NR modulators including compounds targeting the estrogen receptor (ER), androgen receptor (AR), farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR), retinoic acid receptor (RAR), retinoid X receptor (RXR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), liver X receptor (LXR), thyroid receptor (TR), and pregnane X receptor (PXR). We used high-throughput assays to measure cell viability, apoptosis, and cell proliferation of leukemic cell lines after treatment with this library. Afterwards, compounds which highly induced apoptosis and inhibited cell proliferation were picked for further investigation of their mechanism of action. We linked the effect of those compounds on apoptosis to their effect on lactate levels. In addition, we tested the effect of these compounds on the activity of pyruvate dehydrogenase complex (PDC) which catalyzes the irreversible oxidative decarboxylation of pyruvate into acetyl-CoA an important step in commitment of glucose into full oxidation rather than glycolysis, PDH dipstick assay was used for this purpose. Of the 134 nuclear receptors modulators that we have tested, nine compounds induced significant apoptosis in Jurkat cells. These compounds mainly targeted ER and RAR. On the other hand, only six compounds from this list significantly reduced the lactate levels in Jurkat cells. Finally, the compounds modulating the RAR pathway were the only compounds that showed significant effects on PDC activity. This work may open a door to further explore the RAR modulators as strong potential anticarcinogenic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A28.