Discovery of novel liver X receptor inverse agonists as lipogenesis inhibitors.

Based on the co-crystal structures of LXRß and its agonists (spiro [pyrrolidine-3,3'-oxindole] derivatives) discovered by us previously, we designed and synthesized a compound library to explore the agonistic activities. The library was screened with luciferase reporter assays, interestingly, it resulted in the discovery of 10 LXR inverse agonists besides 5 LXR agonists. To clarify the mechanism of the actions, we conducted molecular dynamics (MD) simulations on the LXR and inverse agonists complexes, and revealed that H3, H11 and H12 configurations are the key to turn on agonism or inverse agonism status for LXR. Binding tightly with H3, pushing H11 out and destabilizing H12 could form a bigger hydrophobic groove to accommodate NCOR1 to turn on LXR inverse agonism. The inverse agonist 10rr was further studied, and found as a lipogenesis inhibitor through down-regulating LXR target genes SREBP-1c, ACC, FAS and SCD-1, and demonstrated lipid-lowering effects in 3T3-L1 cells, HepG2 cells and mice with Triton WR-1339-induced hyperlipidemia. Therefore, we have proved that LXR inverse agonists can be promising agents for hyperlipidemia treatment.

LXRα Regulates ChREBPα Transactivity in a Target Gene-Specific Manner through an Agonist-Modulated LBD-LID Interaction.

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.

Peptide Amphiphile Supramolecular Nanostructures as a Targeted Therapy for Atherosclerosis.

The rising prevalence of cardiovascular disease worldwide necessitates novel therapeutic approaches to manage atherosclerosis. Intravenously administered nanostructures are a promising noninvasive approach to deliver therapeutics that reduce plaque burden. The drug liver X receptor agonist GW3965 (LXR) can reduce atherosclerosis by promoting cholesterol efflux from plaque but causes liver toxicity when administered systemically at effective doses, thus preventing its clinical use. The ability of peptide amphiphile nanofibers containing apolipoprotein A1-derived targeting peptide 4F to serve as nanocarriers for LXR delivery (ApoA1-LXR PA) in vivo is investigated here. These nanostructures are found to successfully target atherosclerotic lesions in a mouse model within 24 h of injection. After 8 weeks of intravenous administration, the nanostructures significantly reduce plaque burden in both male and female mice to a similar extent as LXR alone in comparison to saline-treated controls. Furthermore, they do not cause increased liver toxicity in comparison to LXR treatments, which may be related to more controlled release by the nanostructure. These findings demonstrate the potential of supramolecular nanostructures as safe, effective drug nanocarriers to manage atherosclerosis.

Molecular characterization and homology modeling of liver X receptor in Asian seabass, Lates calcarifer: predicted functions in reproduction and lipid metabolism.

Liver X receptor (LXR) is a ligand-activated transcription factor that plays vital roles in maintaining cholesterol and lipid homeostasis. Much work has been done on mammalian LXRs, but the role of LXR in fish remains unclear. In the present study, LXR gene was identified from adult Asian seabass, Lates calcarifer, and its predicted protein structure was docked with several cholesterol derivatives at the binding site. The LXR cDNA consisted of 1495 bp encoding a putative LXR protein of 494 amino acids. The Asian seabass LXR retained many important structural features found in LXRs of other fishes and mammals, such as putative signal peptide, activation function-1 (AF-1) domain, DNA-binding domain (DBD), ligand-binding domain (LBD), activation function-2 (AF-2) domain, and eight conserved cysteine residues. The deduced amino acid sequence of LXR shared significant identity with those of other species ranging from 65.7 to 95.8%. The homology modeling and in silico molecular docking demonstrated that Asian seabass LXR could interact with cholesterol derivatives at amino acid residues Phe274 and Ile312. Real-time PCR further revealed that LXR transcripts are ubiquitously expressed in all tissues examined, with the highest levels detected in the gonad followed by the liver. Given the well-known importance of cholesterol-mediated signaling in these tissues, Asian seabass LXR may reasonably be involved in reproduction and lipid metabolism.

Effects of antiepileptic drugs on lipogenic gene regulation and hyperlipidemia risk in Taiwan: a nationwide population-based cohort study and supporting in vitro studies.

To characterize the association between epilepsy, use of antiepileptic drugs (AEDs), and the risk of hyperlipidemia, we conducted a nationwide population-based cohort study with data obtained from the National Health Insurance Research Database of Taiwan. The effects of AEDs on lipogenic gene expression were also examined in vitro. We identified 3617 cases involving patients, whose epilepsy was newly diagnosed between 2000 and 2011, and selected a comparison cohort comprising 14,468 patients without epilepsy. The Cox proportional hazards model was used to evaluate the association between epilepsy, AED use, and hyperlipidemia. The incidence rate of hyperlipidemia was higher in the epilepsy cohort than in the comparison cohort, with an adjusted hazard ratio (aHR) of 1.21 [95% confidence interval (CI): 1.06-1.38] after adjusting for comorbidities and medications. Epilepsy patients not taking AEDs had a higher risk of hyperlipidemia (aHR 1.65; 95% CI 1.35-2.03). Among AEDs, only valproate treatment showed a higher risk of hyperlipidemia (aHR 1.53; 95% CI 1.01-2.33), although the dose-dependent effect did not reach statistical significance. In vitro studies with two hepatic cell lines showed that valproate may exert its effects by activating the liver X receptor alpha (LXRα) signaling pathway, inducing the expression of lipogenesis-related genes and increasing cellular lipid contents. In silico calculations concluded that valproate can bind stably with the ligand-binding domain of LXRα. Thus, valproate-induced hepatic lipogenic gene expression may occur through LXRα activation. Predicting the 'off-target' effects of valproate may prove valuable in developing antiepileptic agents with fewer adverse reactions. Monitoring blood lipid levels throughout the course of treatment is recommended.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20LWKPGAQDASSQAQGGSSCILRE42. However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT.

A de novo substructure generation algorithm for identifying the privileged chemical fragments of liver X receptorß agonists.

Liver X receptorß (LXRß) is a promising therapeutic target for lipid disorders, atherosclerosis, chronic inflammation, autoimmunity, cancer and neurodegenerative diseases. Druggable LXRß agonists have been explored over the past decades. However, the pocket of LXRß ligand-binding domain (LBD) is too large to predict LXRß agonists with novel scaffolds based on either receptor or agonist structures. In this paper, we report a de novo algorithm which drives privileged LXRß agonist fragments by starting with individual chemical bonds (de novo) from every molecule in a LXRß agonist library, growing the bonds into substructures based on the agonist structures with isomorphic and homomorphic restrictions, and electing the privileged fragments from the substructures with a popularity threshold and background chemical and biological knowledge. Using these privileged fragments as queries, we were able to figure out the rules to reconstruct LXRß agonist molecules from the fragments. The privileged fragments were validated by building regularized logistic regression (RLR) and supporting vector machine (SVM) models as descriptors to predict a LXRß agonist activities.

1α-Hydroxy derivatives of 7-dehydrocholesterol are selective liver X receptor modulators.

The nuclear receptors liver X receptor (LXR) α and LXRß are involved in the regulation of lipid metabolism, inflammation, immunity, cellular proliferation, and apoptosis. Oxysterols are endogenous LXR ligands, and also interact with other nuclear and membrane receptors. We previously reported that a phytosterol derivative with a 1α-hydroxy group acts as a potent LXR agonist with intestine-selective action and that 25-hydroxy and 26/27-hydroxy metabolites of 7-dehydrocholesterol (7-DHC) exhibit partial LXR agonism. In this study, we report that 1α-hydroxy derivatives of 7-DHC, 1α-OH-7-DHC and 1,25-(OH)2-7-DHC, act as LXR modulators. Luciferase reporter gene assays showed that 1α-OH-7-DHC activates LXRα and LXRß and that 1,25-(OH)2-7-DHC activates both LXRs and vitamin D receptor. Examination of cofactor peptide association showed that the 1α-hydroxy derivatives, specifically 1,25-(OH)2-7-DHC, induce association of coactivator/corepressor peptide in a different manner from the agonist T0901317. Docking modeling and alanine mutational analysis of LXRα demonstrated that 1,25-(OH)2-7-DHC interacts with LXRα residues in a manner distinct from potent agonists, such as T0901317 and 24(S),25-epoxycholesterol. 1α-OH-7-DHC and 1,25-(OH)2-7-DHC induced expression of LXR target genes in a cell type- and gene-selective manner. 1,25-(OH)2-7-DHC effectively suppressed lipopolysaccharide-stimulated proinflammatory gene expression in an LXR-dependent manner. Therefore, 1α-hydroxy derivatives, such as 1,25-(OH)2-7-DHC, are unique LXR modulators with selective agonistic activity and potent transrepression function. These oxysterols have potential as LXR-targeted therapeutics for inflammatory disease.

Role of the liver X receptors in skin physiology: Putative pharmacological targets in human diseases.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily that have been shown to regulate various physiological functions such as lipid metabolism and cholesterol homeostasis. Concordant reports have elicited the possibility to target them to cure many human diseases including arteriosclerosis, cancer, arthritis, and diabetes. The high relevance of modulating LXR activities to treat numerous skin diseases, mainly those with exacerbated inflammation processes, contrasts with the lack of approved therapeutic use. This review makes an assessment to sum up the findings regarding the physiological roles of LXRs in skin and help progress towards the therapeutic and safe management of their activities. It focuses on the possible pharmacological targeting of LXRs to cure or prevent selected skin diseases.

Identification of a Novel Liver X Receptor Agonist that Regulates the Expression of Key Cholesterol Homeostasis Genes with Distinct Pharmacological Characteristics.

Activation of liver X receptor (LXR) is associated with cholesterol metabolism and anti-inflammatory processes, which makes it beneficial to antiatherosclerosis therapy. Nevertheless, existing agonists that target LXR, for example TO901317, are related to unwanted side effects. In the present study, using a screening method we identified IMB-808, which displayed potent dual LXRα/ß agonistic activity. In vitro, IMB-808 effectively increased the expressing quantity of genes related to reverse cholesterol transport process as well as those associated with cholesterol metabolism pathway in multiple cell lines. Additionally, IMB-808 remarkably promoted cholesterol efflux from RAW264.7 as well as THP-1 macrophage cells and reduced cellular lipid accumulation accordingly. Interestingly, compared with TO901317, IMB-808 almost did not increase the expressing quantity of genes related to lipogenesis in HepG2 cells, which indicated that IMB-808 could exhibit fewer internal lipogenic side effects with a characteristic of selective LXR agonist. Furthermore, in comparison with the full LXR agonist TO901317, IMB-808 recruits coregulators differently and possesses a distinct predictive binding pattern for the LXR ligand-binding domain. In summary, our study demonstrated that IMB-808 could act as an innovative partial LXR agonist that avoids common lipogenic side effects, providing insight for the design of novel LXR modulators. Our data indicate that this compound might be used as a promising therapeutic agent for the prospective treatment of atherosclerosis in the future.