t0901317 and 24-25-epoxycholesterol

Hepatitis C virus (HCV) is a major human pathogen that causes many serious diseases, including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Treatments for this virus are inadequate, and improved antiviral therapies are necessary. Although the precise mechanisms regulating HCV entry into hepatic cells are still unknown, the low-density lipoprotein receptor (LDLR) has been shown to be essential for entry of infectious HCV particles. Liver X receptors (LXR) were recently reported to control LDLR expression through the regulation of the expression of the Idol (inducible degrader of the LDLR) protein, which could trigger the ubiquitination and degradation of LDLR. In this study, we analyzed the antiviral effect of Idol in vitro. The results demonstrated that Huh7.5.1 cells that exogenously expressed Idol were resistant to HCV infection. Next, the treatment of HCV-infected Huh7.5.1 cells with either synthetic LXR agonists (GW3965 or T0901317) or the natural LXR ligand 24(S),25-epoxycholesterol inhibited HCV infection in a dose-dependent manner. Furthermore, a combination of LXR agonists and HCV RNA replication inhibitors exerted additive effects against HCV, as revealed by isobologram analysis. In conclusion, our data indicate that molecules such as LXR agonists, which could stimulate the expression of Idol, represent a new class of potential anti-HCV compounds, and these compounds could be developed for therapeutic use against HCV infection, either as a monotherapy, or in combination with other anti-HCV drugs.

Topics: Antiviral Agents; Benzoates; Benzylamines; Cell Line; Cholesterol; Dose-Response Relationship, Drug; Hepacivirus; Hepatocytes; Humans; Hydrocarbons, Fluorinated; Liver X Receptors; Orphan Nuclear Receptors; Sulfonamides; Ubiquitin-Protein Ligases; Virus Internalization

Liver X receptors alpha and beta (LXRalpha, LXRbeta) are key regulators of cholesterol homeostasis. The effects of LXR ligands on endothelial cells are largely unknown. While oxysterol LXR agonists can increase the endothelial-leukocyte interaction, synthetic LXR agonists are anti-atherogenic and anti-inflammatory. Mechanistic differences may underlie such findings.. LXRalpha and LXRbeta were found to be expressed in human endothelial cells. While synthetic LXR agonists could blunt the LPS-induced up-regulation of adhesion molecules (ICAM-1, VCAM-1, E-Selectin), 22-hydroxycholesterol and 24,25-epoxycholesterol enhanced such response. Microarray profiling further showed that the endothelial gene expression fingerprints of 22-hydroxycholesterol and T0901317 largely differed and unexpectedly shared only a restricted number of genes. Indeed, 22-hydroxycholesterol down-regulated eNOS and up-regulated a vast cohort of inflammatory mediators such as adhesion molecules, cytokines, enzymes and transcription factors. Other LXR-activating oxysterols such as 24,25-epoxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol could also stimulate the endothelial expression of inflammatory markers, although significant differences were observed. These effects persisted in LXR-silenced cells, confirming the mechanistic dissociation of oxysterol and LXR pathways. Furthermore, the oxysterol-induced expression of inflammatory markers was not secondary to cell apoptosis and may relate to oxidative stress.. LXR-activating oxysterols comprehensively activate the expression of endothelial inflammation markers independently from LXRs. At proper dosage, synthetic LXR agonists are safe on endothelial cells and may even transrepress inflammatory reactions.

Topics: Anticholesteremic Agents; Benzoates; Benzylamines; Cells, Cultured; Chemokines; Cholesterol; E-Selectin; Endothelial Cells; Gene Expression Profiling; Gene Expression Regulation; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Inflammation Mediators; Intercellular Adhesion Molecule-1; Lipopolysaccharides; Liver X Receptors; Orphan Nuclear Receptors; RNA Interference; RNA, Messenger; Signal Transduction; Sulfonamides; Transcription Factors; Transfection; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Recent evidence suggests that the liver X receptor (LXR) is a potential anticancer target in prostate carcinoma. There is little characterization, however, of which of the two LXR isoforms, LXRalpha or LXRbeta, regulates the LXR-responsive genes ATP-binding cassette subfamily members A1 (ABCA1) and G1 (ABCG1) in transformed prostatic epithelial cells. In this study, small interfering RNA (siRNA) was used to determine whether LXRalpha or LXRbeta is involved in regulating ABCA1 and ABCG1 mRNA expression in LNCaP and PC-3 cells. Treatment of both cell lines with the synthetic LXR ligand T0901317 and oxysterols: 25-hydroxycholesterol (25HC) and 24(S), 25-epoxycholesterol (24,25EC), resulted in more than a 10-fold increase of ABCA1 and ABCG1 mRNA expression. Transfection of LNCaP cells with siRNA against either LXRbeta or LXRalpha failed to inhibit T0901317 and 25HC-mediated increase of ABCA1 mRNA. siRNA silencing of LXRbeta did, however, inhibit ABCA1 mRNA expression in 24,25EC-treated LNCaP cells. In contrast, LXRbeta siRNA inhibited T0901317, 25HC, and 24,25EC induction of ABCA1 mRNA in PC-3 cells and ABCG1 mRNA in both LNCaP and PC-3 cells. Additional experiments revealed that T0901317 and 25HC induction of ABCA1 mRNA expression was significantly inhibited by the p38 stress kinase antagonist SB202190 and PKA inhibitor H89. Our study is the first to show that LXRbeta, but not LXRalpha, is the major regulatory isoform of ABCG1 mRNA expression in LNCaP and PC-3 cells. Our study also reveals that ABCA1 gene expression is differentially regulated by synthetic and natural LXR ligands, possibly involving kinase mediated signal transduction.

Topics: ATP Binding Cassette Transporter 1; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; Cholesterol; DNA-Binding Proteins; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Ligands; Liver X Receptors; Male; Neoplasms, Hormone-Dependent; Orphan Nuclear Receptors; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Tumor Cells, Cultured

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.

Topics: Androstenes; Animals; Benzoates; Benzylamines; Carbazoles; Cell Line, Tumor; Cholesterol; Ciona intestinalis; DNA-Binding Proteins; Evolution, Molecular; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Liver X Receptors; Mice; Molecular Structure; Orphan Nuclear Receptors; Phylogeny; Receptors, Cytoplasmic and Nuclear; Structure-Activity Relationship; Sulfonamides; Xenopus laevis; Zebrafish

The liver X receptor (LXR) agonists, 24(S),25-epoxycholesterol and T0901317, were previously shown to be capable of inducing CYP3A expression in primary cultured rodent hepatocytes through activation of the pregnane X receptor (PXR). In this study, the abilities of these two LXR agonists to regulate CYP3A4 and CYP2B6 mRNA expression in primary cultures of human hepatocytes were evaluated. Treatment with 10 or 30 microM of the endogenous oxysterol, 24(S),25-epoxycholesterol, had no effect on CYP3A4 mRNA content in five preparations of primary cultured human hepatocytes, while 30 microM 24(S),25-epoxycholesterol treatment increased CYP2B6 mRNA content by approximately two-fold. By comparison, treatment with the synthetic LXR agonist, T0901317, potently increased CYP3A4 and CYP2B6 mRNA levels in the human hepatocyte cultures, producing multi-fold increases at 10nM. Using a HepG2-based transactivation assay, T0901317 activated human PXR with an EC(50) approximately 20nM, which was more than 10-fold lower than that of the potent PXR ligand, SR-12813, while treatment with 24(S),25-epoxycholesterol failed to induce reporter expression in this assay. Therefore, while 24(S),25-epoxycholesterol-mediated PXR activation and CYP3A induction does not appear to be conserved from rodent to human, T0901317 is among the most potent known activators of human PXR.

Topics: Adult; Aged, 80 and over; Aryl Hydrocarbon Hydroxylases; Cell Line; Cells, Cultured; Cholesterol; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; DNA-Binding Proteins; Female; Gene Expression Regulation, Enzymologic; Hepatocytes; Humans; Hydrocarbons, Fluorinated; Infant; Liver X Receptors; Male; Middle Aged; Orphan Nuclear Receptors; Oxidoreductases, N-Demethylating; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; RNA, Messenger; Sulfonamides; Transfection

A novel series of hexafluorocarbinols were discovered as potent activators of the liver X receptor-alpha using a fluorescence polarization assay. Structure-activity relationship study led to the identification of compounds that are more potent agonists than the endogenous ligand, 24(S), 25-epoxycholesterol, with similar efficacy. Several compounds, including T0901317, were shown to have desirable pharmacokinetic profiles suitable for in vivo studies.

Topics: Administration, Oral; Animals; Cholesterol; DNA-Binding Proteins; Fluorescence Polarization; Hydrocarbons, Fluorinated; Ligands; Liver X Receptors; Male; Molecular Structure; Orphan Nuclear Receptors; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Structure-Activity Relationship; Sulfonamides

The x-ray crystal structures of the human liver X receptor beta ligand binding domain complexed to sterol and nonsterol agonists revealed a perpendicular histidinetryptophan switch that holds the receptor in its active conformation. Hydrogen bonding interactions with the ligand act to position the His-435 imidazole ring against the Trp-457 indole ring, allowing an electrostatic interaction that holds the AF2 helix in the active position. The neutral oxysterol 24(S),25-epoxycholesterol accepts a hydrogen bond from His-435 that positions the imidazole ring of the histidine above the pyrrole ring of the tryptophan. In contrast, the acidic T0901317 hydroxyl group makes a shorter hydrogen bond with His-435 that pulls the imidazole over the electron-rich benzene ring of the tryptophan, possibly strengthening the electrostatic interaction. Point mutagenesis of Trp-457 supports the observation that the ligand-histidine-tryptophan coupling is different between the two ligands. The lipophilic liver X receptor ligand-binding pocket is larger than the corresponding steroid hormone receptors, which allows T0901317 to adopt two distinct conformations. These results provide a molecular basis for liver X receptor activation by a wide range of endogenous neutral and acidic ligands.

Topics: Anticholesteremic Agents; Binding Sites; Cholesterol; Crystallography, X-Ray; DNA-Binding Proteins; Histidine; Humans; Hydrocarbons, Fluorinated; Hydrogen Bonding; In Vitro Techniques; Ligands; Liver; Liver X Receptors; Models, Molecular; Mutagenesis, Site-Directed; Orphan Nuclear Receptors; Protein Conformation; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Sulfonamides; Tryptophan

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.

Topics: Animals; Anticholesteremic Agents; ATP-Binding Cassette Transporters; Biological Transport; Cell Line; Cell Nucleus; Cholesterol; DNA-Binding Proteins; Dose-Response Relationship, Drug; Ergosterol; Genes, Reporter; Glutathione Transferase; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Intestinal Mucosa; Ligands; Liver; Liver X Receptors; Mice; Models, Chemical; Orphan Nuclear Receptors; Phytosterols; Protein Binding; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Time Factors; Transfection