Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.     

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.     

Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR

Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism.     

Identification of PLTP as an LXR target gene and apoE as an FXR target gene reveals overlapping targets for the two nuclear receptors

Affymetrix microarray data and Northern blot assays demonstrated that phospholipid transfer protein (PLTP) was induced 6-fold when either murine or human macrophages were incubated in the presence of ligands for the liver X receptor (LXR) and the retinoid X receptor. Two functional LXR response elements (LXREs) were identified and characterized in the proximal promoter of the human PLTP gene. One LXRE corresponds to a traditional direct repeat separated by 4 bp. However, the second LXRE is novel in that it corresponds to an inverted repeat separated by 1 bp, and is identical to the farnesoid X receptor response element. These studies demonstrate that PLTP is a direct target for activated LXR and farnesoid X receptor (FXR). In addition, apolipoprotein E (apoE), a known LXR target gene in macrophages, was shown to be activated in liver cells by FXR ligands. Taken together, the current data suggest that a small number of genes that currently include PLTP, apoE, and apoC-II, are induced in macrophages by activated LXR and in liver by activated FXR.     

Essential roles of bile acids and their nuclear receptors,FXR and PXR,in the cholestatic liver disease

Bile acids (BAs) are steroid acids found predominantly in the bile of mammals and other vertebrates. Though BAs have been known as digestive juice, recent studies have revealed that BAs act as signaling molecules to control metabolism and inflammation. Today, BAs are considered as potential therapeutic molecules for treatment of complex metabolic liver disease. However, the detergent properties of BAs lead to hepatic injury and intrahepatic cholestasis when BAs are accumulated in the liver with impaired bile flow into gall bladder. Cholestasis is a pathological condition of hepatic retention of cytotoxic bile acids. To date, hydrophilic ursodeoxycholic acid has been currently used to treat cholestasis, but the efficacy of UDCA for cholestasis is still limited. Given that BAs are endogenous ligands of several nuclear receptors, including Farnesoid X receptor and Pregnane X receptor, novel synthetic ligands for those nuclear receptors are promising for the treatment of cholestatic liver diseases.     

A chemical,genetic, and structural analysis of the nuclear bile acid receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.     

A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay

Aims: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small‐cell variant (SCV) by real‐time PCR (qPCR) in clinical samples. Methods and Results: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. Conclusions: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large‐cell and small‐cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. Significance and impact of study: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.     

A controlled pore glass bead assay for the measurement of cytoplasmic and nuclear glucocorticoid receptors

An assay for the quantitation of cytoplasmic and nuclear glucocorticoid receptors in lymphoid tissue has been developed using controlled pore glass (CPG) beads. Soluble receptor--3H-steroid complex (cytosol or nuclear extract) is adsorbed quantitatively within the crevasses of porous glass beads. Excess labeled steroid as well as most non-specifically bound steroid is easily washed away, leaving the hormone-receptor complex retained by the beads. Bound 3H-steroid is eluted with ethanol and measured for radioactivity. This procedure which is simple, rapid, and highly reproducible is carried out using frozen samples (stable for many months) containing as few as 1 X 10(7) cells. A comparison of the CPG assay to dextran coated charcoal and a whole cell assay demonstrates that CPG and dextran coated charcoal give equivalent measurements of cytosolic receptor concentration, while the CPG and whole cell assays provide equivalent values for total receptor content.     

Diphenylmethane skeleton as a multi-template for nuclear receptor ligands: preparation of FXR and PPAR ligands

Novel, potent farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor alpha (PPARalpha) agonists were obtained by using a diphenylmethane skeleton as a substitute for a steroid skeleton.     

Nitrification and denitrification in soil: A comparison of enzyme assay,incubation and enumeration methods

Summary Three methods for assaying nitrification and denitrification were compared in agricultural field plots in the Southeastern USA. Nitrification was measured using the chlorate inhibition enzyme assay, by measuring (NO ₃ ⁻ −N) production in controlled incubations and by most probable number (MPN) determinations. Denitrification was measured by the phase I enzyme assay, by incubation of soil cores and by MPN determinations. The methods were compared in terms of their representation of seasonal patterns and treatment differences. The enzyme assays were most effective for showing treatment differences because they measure maximum potential enzyme biomass activity which is an integrated product of treatment effects. The incubation methods required minimal alteration ofin situ soil conditions but were confounded by other biological processes and by high spatial and temporal variabiltiy. MPN determinations were time consuming and were least effective for illustrating treatment differences and seasonal patterns.     

A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.     

The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes

Bile acids are synthesized from cholesterol in the liver and are excreted into bile via the hepatocyte canalicular bile salt export pump. After their passage into the intestine, bile acids are reabsorbed in the ileum by sodium-dependent uptake across the apical membrane of enterocytes. At the basolateral domain of ileal enterocytes, bile acids are extruded into portal blood by the heterodimeric organic solute transporter OSTalpha/OSTbeta. Although the transport function of OSTalpha/OSTbeta has been characterized, little is known about the regulation of its expression. We show here that human OSTalpha/OSTbeta expression is induced by bile acids through ligand-dependent transactivation of both OST genes by the nuclear bile acid receptor/farnesoid X receptor (FXR). FXR agonists induced endogenous mRNA levels of OSTalpha and OSTbeta in cultured cells, an effect that was not discernible upon inhibition of FXR expression by small interfering RNAs. Furthermore, OST mRNAs were induced in human ileal biopsies exposed to the bile acid chenodeoxycholic acid. Reporter constructs containing OSTalpha or OSTbeta promoters were transactivated by FXR in the presence of its ligand. Two functional FXR binding motifs were identified in the OSTalpha gene and one in the OSTbeta gene. Targeted mutation of these elements led to reduced inducibility of both OST promoters by FXR. In conclusion, the genes encoding the human OSTalpha/OSTbeta complex are induced by bile acids and FXR. By coordinated control of OSTalpha/OSTbeta expression, bile acids may adjust the rate of their own efflux from enterocytes in response to changes in intracellular bile acid levels.     

A nuclear binding assay for measurement of biologically active androgen receptors in animal tissues and human prostate cancer

A nuclear binding (NB) assay has been developed for the measurement in intact viable cells of biologically active (functional) estrogen and progesterone receptors, i.e. those capable of binding to nuclear acceptor sites [Spelsberg et al., Endocrinology 121: 631 (1987)]. This paper describes the application of this assay to analyses of androgen receptors in the guinea pig seminal vesicle and in human prostatic carcinoma. Cells from fresh animal seminal vesicles or human prostate carcinoma are isolated using collagenase and are incubated with [3H]R1881 for 1 h at 22 degrees C, after which nuclei are isolated at 4 degrees C and assayed for DNA and radioactivity. This NB assay demonstrates a saturable, temperature dependent, steroid and tissue specific nuclear binding of [3H]R1881 for the guinea pig-seminal vesicle system. The nuclear binding is of high affinity and low capacity. The NB assay reveals several important aspects of the androgen and estrogen receptors in target tissues: (1) the nuclear acceptor sites for androgen receptor (AR) are steroid receptor specific; (2) there are different concentrations of the androgen and estrogen receptors between the epithelium and the fibromuscular components of the guinea pig seminal vesicle; and finally (3) some biopsies of human prostate cancer appear to contain biologically inactive AR. This assay may be useful in the analyses of functional receptors in biopsies of human cancer cells.