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.     

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.     

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.     

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.     

Structure-guided design of N-phenyl tertiary amines as transrepression-selective liver X receptor modulators with anti-inflammatory activity

A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.     

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 and atherosclerosis

Cardiovascular disease, the primary cause of death and illness in the industrialized world, is typically due to complications of atherosclerosis, a multifactorial disease of the arterial intima. The liver X receptors (LXRs), LXRalpha and LXRbeta, are intracellular receptors that appear to play an important role in protection against atherosclerosis; however, LXR activation also leads to a dramatic increase in liver and serum triglycerides. This presents a challenge to developing drugs via these targets. This article discusses the role of LXRs in atherosclerosis and lipid regulation and the possibility of designing LXR ligands that may be anti-atherogenic without side effects.     

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.     

尼古丁对巨噬细胞肝X受体α表达及胆固醇外流的影响

目的通过研究尼古丁对巨噬细胞的肝X受体α(LXRα)及其下游的一些目的基因表达和胆固醇外流的影响,探讨尼古丁对LXR信号系统的作用。方法分离人外周血单核细胞,并转化为巨噬细胞。在尼古丁的作用下,观察巨噬细胞的aopA-Ⅰ介导的胆固醇外流的变化和LXR以及其下游一些目的基因mRNA表达。结果尼古丁明显影响巨噬细胞中一些涉及胆固醇代谢及炎症反应的基因表达,同时降低aopA-Ⅰ介导的胆固醇外流。结论巨噬细胞在尼古丁的作用下,由aopA-Ⅰ介导的胆固醇外流降低,这种效应与尼古丁下调LXRα及其下游的影响胆固醇代谢的目的基因有关,同时,也促进一些炎症因子基因的表达。提示尼古丁在动脉粥样硬化中的作用与其影响巨噬细胞LXR信号途径有关,从而影响泡沫细胞的形成。     

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.     

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.     

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.