N-Acylthiadiazolines, a new class of liver X receptor agonists with selectivity for LXRbeta

We have identified a novel liver X receptor (LXR) agonist (2) that activates the LXRbeta subtype with selectivity over LXRalpha. LXRbeta selectivity was confirmed using macrophages derived from LXR mutant mice. Despite its selectivity and modest potency, the compound can induce APO-AI-dependent cholesterol efflux from macrophages with full efficacy. Our results indicate that it is possible to achieve significant LXRbeta selectivity in a small molecule while maintaining functional LXR activity.     

Combination of virtual screening and high throughput gene profiling for identification of novel liver X receptor modulators

We conducted virtual docking studies using GLIDE with modified LXRbeta ligand-binding domain (LBD) on internal compound collection followed by the gene profiling with ArrayPlate mRNA assay. A total of 69 compounds were found to upregulate LXRalpha and certain LXR regulated genes from 1308 compounds selected by virtual screen (hit rate: 5.3%). Compound 4 was shown to significantly induce the expression of LXR target genes such as ABCA1, ABCG1, APOE, SCD-1, and SREBP-1c in THP-1 differentiated macrophages. In vitro binding assay confirmed that 4 binds to both LXRalpha and LXRbeta directly and recruits coactivator peptide SRC-1. It functions as a full LXR agonist in stimulating cholesterol efflux in THP-1 differentiated macrophages and induces lipogenesis in HepG2 cells. This study demonstrates that the combination of virtual screen and high throughput gene profiling is an efficient approach for rapid identification of novel LXR modulators.     

Design, structure activity relationships and X-Ray co-crystallography of non-steroidal LXR agonists

The Liver X Receptor (LXR) alpha and beta isoforms are members of the type II nuclear receptor family which function as a heterodimer with the Retinoid X Receptor (RXR). Upon agonist binding, the formation of the LXR/RXR heterodimer takes place and ultimately the regulation of a number of genes begins. The LXR isoforms share 77% sequence homology, with LXRalpha having highest expression in liver, intestine, adipose tissue, and macrophages and LXRbeta being ubiquitously expressed. The aim of this article is to review the reported medicinal chemistry strategies towards the optimisation of novel non-steroidal chemotypes as LXR agonists. An analysis of the structural features important for LXR ligand binding will be given, utilising both structural activity relationship data obtained from LXR assays as well as X-ray co-crystallographic data obtained with LXR ligands and the LXR ligand binding domain (LBD). The X-ray co-crystallographic data analysis will detail the key structural interactions required for LXR binding/agonist activity and reveal the differences observed between chemotype classes. It has been postulated that a LXRbeta selective compound may have a beneficial outcome on the lipid profile for a ligand by dissociating the favourable and unfavourable effects of LXR agonists. Whilst there have been a few examples of compounds showing a modest level of LXRalpha selectivity, obtaining a potent LXRbeta selective compound has been more challenging. Analysis of the SAR and X-ray co-crystallographic data suggests that the rational design of a LXRbeta selective compound will not be trivial.     

Selective activation of liver X receptors by acanthoic acid-related diterpenes

Terpenoids constitute a large family of natural steroids that are widely distributed in plants and insects. We investigated the effects of a series of diterpenes structurally related to acanthoic acid in macrophage functions. We found that diterpenes with different substitutions at the C4 position in ring A are potent activators of liver X receptors (LXRalpha and LXRbeta) in both macrophage cell lines from human and mouse origin and primary murine macrophages. Activation of LXR by these diterpenes was evaluated in transient transfection assays and gene expression analysis of known LXR-target genes, including the cholesterol transporters ABCA1 and ABCG1, the sterol regulatory element-binding protein 1c, and the apoptosis inhibitor of macrophages (Spalpha). Moreover, active diterpenes greatly stimulated cholesterol efflux from macrophages. It is interesting that these diterpenes antagonize inflammatory gene expression mainly through LXR-dependent mechanisms, indicating that these compounds can activate both LXR activation and repression functions. Stimulation of macrophages with acanthoic acid diterpenes induced LXR-target gene expression and cholesterol efflux to similar levels observed with synthetic agonists 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)-amino]propyloxy]phenylacetic acid hydrochloride (GW3965) and N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide [T1317 (T0901317)]. These effects observed in gene expression were deficient in macrophages lacking both LXR isoforms (LXRalpha,beta(-/-)). These results show the ability of certain acanthoic acid diterpenes to activate efficiently both LXRs and suggest that these compounds can exert beneficial effects from a cardiovascular standpoint through LXR-dependent mechanisms.     

Novel roles of liver X receptors exposed by gene expression profiling in liver and adipose tissue

Liver X receptor (LXR) alpha and LXRbeta are nuclear oxysterol receptors whose biological function has so far been elucidated only with respect to cholesterol and lipid metabolism. To expose novel biological roles for LXRs, we performed genome-wide gene expression profiling studies in liver and white and brown adipose tissue from wild-type (LXRalpha(+/+)beta(+/+)) and knockout mice (LXRalpha(-/-)beta(-/-)) treated with a synthetic LXR agonist. By an adapted statistical analysis, we detected 319 genes significantly regulated by LXR agonist treatment in wild-type but not in knockout mice, fulfilling most stringent criteria with an overall confidence of 94%. Down-regulation of essential enzymes of gluconeogenesis in liver could point to possible beneficial effects of LXR agonists in diabetes mellitus. LXR agonist treatment also altered expression of genes involved in steroid hormone synthesis and growth hormone receptor signaling, emphasizing a potential impact on endocrine function. Notably, LXR agonist treatment up-regulated CYP4A10 and CYP4A14 together with cytochrome P450 reductase, indicating a possible enhancement of microsomal lipid peroxidation. In conclusion, these gene expression profiling data identify novel areas of regulation by LXRs and provide a highly valuable basis for further research on the biological functions of these nuclear receptors and the pharmacological characteristics of their ligands.     

Liver x receptors: potential novel targets in cardiovascular diseases

The Liver X Receptors, LXRalpha and LXRbeta are members of the nuclear hormone receptor superfamily which have recently been implicated as novel pharmacological targets for the treatment of cardiovascular diseases. The identification of natural and synthetic ligands for LXRs and the generation of LXR-deficient mice have been crucial for our understanding of the function of these receptors and for the identification of LXR-regulated target genes, particularly with respect to the role of LXRs in regulating cholesterol homeostasis. Synthetic LXRalpha/beta agonists induce cholesterol efflux and reverse cholesterol transport, improve glucose metabolism, inhibit macrophage-derived inflammation, and suppress the proliferation of vascular smooth muscle cells. By regulating the expression of multiple genes involved in these pathways, LXR agonists prevent the development and progression of atherosclerosis and inhibit neointima formation following angioplasty of the arterial wall. In this review, we will summarize the important roles of LXR in metabolism and vascular biology and discuss its implications as potential molecular drug target for the treatment of cardiovascular diseases.     

The effect of T0901317 on ATP-binding cassette transporter A1 and Niemann-Pick type C1 in apoE-/- mice

Although a range of studies indicated Liver X receptor (LXR) activation inhibited the development of atherosclerosis in animal models, the mechanism of this effect for LXR agonists has not been fully understood. A recent study has suggested LXR activators increased the amount of free cholesterol in the plasma membrane of human macrophages by inducing Niemann-Pick type C1 (NPC1) gene expression. Therefore, we hypothesize that LXRs may also promote NPC1 expression in vivo. Here we investigated the effect of a synthetic LXR agonist T0901317 on ATP-binding cassette transporter A1 (ABCA1) and NPC1 in apolipoprotein E knockout (apoE-/-) mice. Male apoE-/- mice were randomized into four groups: baseline group (n = 10), vehicle group (n = 14), prevention group (n = 14), and treatment group (n = 14). En face analysis and Oil red O staining were used to examine the aortic atherosclerotic lesions. Macrophage content of aortic root atherosclerotic lesions and cholesterol efflux form peritoneal macrophages were measured. Gene and protein expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting, respectively. T0901317 treatment reduced aortic atherosclerotic lesion area by 64.2% in prevention group (P < 0.001) and 58.3% in treatment group (P < 0.001) and resulted in a reduction in macrophage content. Plasma triglyceride, total cholesterol, high-density lipoprotein cholesterol, and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 also promoted ABCA1 and NPC1 gene and protein levels in the aorta, liver, and small intestine of apoE-/- mice and significantly increased cholesterol efflux from peritoneal macrophages. T0901317 upregulates ABCA1 and NPC1. This study gives us a new insight into the mechanism for antiatherogenic effect of LXR synthetic agonists.     

Liver X receptors as potential therapeutic targets for multiple diseases

Liver X receptor alpha (LXRalpha) and liver X receptor beta(LXRbeta are oxysterol receptors that regulate multiple target genes involved in cholesterol homeostasis. Recent studies also suggest that the pair of receptors may also be involved in glucose metabolism, inflammation and Alzheimer's disease by regulating critical molecules involved in these pathophysiological processes. Although the prototypic LXR agonists induce liver triglyceride accumulation by regulating the hepatic lipogenesis pathway, it is hoped that a subtype-specific agonist or selective modulators would provide the desired cardioprotection and other benefits without the undesirable concomitant induction of lipogenesis. This review intends to summarize the most recent progress in the field and provide an assessment of LXRs as potential therapeutic targets.     

Indazole-based liver X receptor (LXR) modulators with maintained atherosclerotic lesion reduction activity but diminished stimulation of hepatic triglyceride synthesis

A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly weaker with respect to potency and efficacy on LXRalpha than on LXRbeta. Lead compounds in this series 12 (WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13 showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.     

Discovery of phenyl acetic acid substituted quinolines as novel liver X receptor agonists for the treatment of atherosclerosis

A structure-based approach was used to optimize our new class of quinoline LXR modulators leading to phenyl acetic acid substituted quinolines 15 and 16. Both compounds displayed good binding affinity for LXRbeta and LXRalpha and were potent activators in LBD transactivation assays. The compounds also increased expression of ABCA1 and stimulated cholesterol efflux in THP-1 cells. Quinoline 16 showed good oral bioavailability and in vivo efficacy in a LDLr knockout mouse model for lesions.     

Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.     

LXR agonists for the treatment of atherosclerosis

Liver X receptor (LXR) alpha/beta nuclear receptors are intracellular sterol sensors that regulate expression of genes controlling cholesterol absorption, excretion, catabolism and cellular efflux in target organs, including small intestine, liver and macrophages. Through co-ordination of the expression of target genes in multiple tissues, LXR agonists increase the flux of cholesterol from the periphery to the liver, where it is metabolized and excreted into the bile. Synthetic dual LXR alpha/beta agonists decrease atherosclerosis in mice, however, upregulation of lipogenic target genes and triglyceride elevation in rodents reveals a narrow therapeutic window. LXR subtype-selective agonists or LXR modulators may dissociate the anti-atherosclerotic and lipogenic effects of current dual LXR agonists.     

The selective Alzheimer's disease indicator-1 gene (Seladin-1/DHCR24) is a liver X receptor target gene

The nuclear hormone receptors liver X receptor alpha (LXRalpha) and LXRbeta function as physiological receptors for oxidized cholesterol metabolites (oxysterols) and regulate several aspects of cholesterol and lipid metabolism. Seladin-1 was originally identified as a gene whose expression was down-regulated in regions of the brain associated with Alzheimer's disease. Seladin-1 has been demonstrated to be neuroprotective and was later characterized as 3beta-hydroxysterol-Delta24 reductase (DHCR24), a key enzyme in the cholesterologenic pathway. Seladin-1 has also been shown to regulate lipid raft formation. In a whole genome screen for direct LXRalpha target genes, we identified an LXRalpha occupancy site within the second intron of the Seladin-1/DHCR24 gene. We characterized a novel LXR response element within the second intron of this gene that is able to confer LXR-specific ligand responsiveness to reporter gene in both HepG2 and human embryonic kidney 293 cells. Furthermore, we found that Seladin-1/DHCR24 gene expression is significantly decreased in skin isolated from LXRbeta-null mice. Our data suggest that Seladin-1/DHCR24 is an LXR target gene and that LXR may regulate lipid raft formation.     

Atorvastatin inhibits ABCA1 expression and cholesterol efflux in THP-1 macrophages by an LXR-dependent pathway

The effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux remains controversial. In an effort to clarify this issue, ABCA1 expression and apolipoprotein AI (apoAI)-mediated cholesterol efflux after atorvastatin treatment were investigated in THP-1 macrophages. Atorvastatin from 2 microM to 40 microM dose-dependently inhibited ABCA1 expression in human monocyte-derived macrophages and phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 monocytes. ApoAI-mediated cholesterol efflux was reduced in PMA-stimulated THP-1 cells treated with atorvastatin, this effect was abolished with acetylated low-density lipoprotein (LDL) pretreatment. Atorvastatin treatment also dose-dependently reduced liver X receptor alpha (LXRalpha) expression and Rho activation. Rho activation by farnysylpyophosphate (FPP) and lysophosphatidic acid (LPA) did not salvage, but further depressed, the cholesterol efflux and ABCA1 expression in the presence of atorvastatin. Without atorvastatin, Rho activation by mevalonate, FPP, and LPA diminished apoAI-mediated cholesterol efflux, and Rho activation by GTPgammaS also decreased ABCA1 messenger ribonucleic acid (mRNA) by 16%. Furthermore, Rho inhibition by C3 exoenzyme increased ABCA1 mRNA by 48% despite a 17% decrease in apoAI-mediated cholesterol efflux. LXRalpha agonists (T01901317 and 22(R)-hydroxycholesterol) prevented any reductions in cholesterol efflux or ABCA1 expression associated with atorvastatin treatment. Furthermore, Western blot analysis demonstrated the reciprocal inhibition of Rho and LXRalpha. In conclusion, atorvastatin decreases ABCA1 expression in noncholesterol-loaded macrophages in an LXRalpha- but not Rho-dependent pathway; this effect can be compromised after acetylated LDL cholesterol loading.     

PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI

PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/³H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20 mg/kg/day) did not change ³H-tracer plasma appearance, but surprisingly decreased fecal ³H-free sterol excretion by 43% (P < 0.01) over 48 h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with ³H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived ³H-CE uptake by adipose tissue (P < 0.005) with concomitant 22% decrease in HDL derived ³H-CE uptake by the liver (P < 0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived ³H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.     

Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines

A potent, selective, orally active LXR agonist was identified from focused libraries of tertiary amines. GW3965 (12) recruits the steroid receptor coactivator 1 to human LXRalpha in a cell-free ligand-sensing assay with an EC(50) of 125 nM and profiles as a full agonist on hLXRalpha and hLXRbeta in cell-based reporter gene assays with EC(50)'s of 190 and 30 nM, respectively. After oral dosing at 10 mg/kg to C57BL/6 mice, 12 increased expression of the reverse cholesterol transporter ABCA1 in the small intestine and peripheral macrophages and increased the plasma concentrations of HDL cholesterol by 30%. 12 will be a valuable chemical tool to investigate the role of LXR in the regulation of reverse cholesterol transport and lipid metabolism.