Identification of 5α, 6α-epoxycholesterol as a novel modulator of liver X receptor activity.

The liver X receptors (LXRα and LXRß) are members of the nuclear receptor superfamily that function as key transcriptional regulators of a number of biological processes, including cholesterol homeostasis, lipid metabolism, and keratinocyte differentiation. Natural ligands that activate LXRs include oxysterol derivatives such as 25-hydroxycholesterol, 27-hydroxycholesterol, 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Related oxysterols, such as 5α,6α-epoxycholesterol (5,6-EC) are present in a number of foods and have been shown to induce atherosclerosis in animal models. Intriguingly, these oxysterols have also been detected in atherosclerotic plaques. Using a variety of biochemical and cellular assays, we demonstrate that 5,6-EC is the first dietary modulator and an endogenous LXR ligand with cell and gene context-dependent antagonist, agonist, and inverse agonist activities. In a multiplexed LXR-cofactor peptide interaction assay, 5,6-EC induced the recruitment of a number of cofactor peptides onto both LXRα and LXRß and showed an EC(50) of approximately 2 µM in peptide recruitment. Furthermore, 5,6-EC bound to LXRα in a radiolabeled ligand displacement assay (EC(50) = 76 nM), thus demonstrating it to be one of the most potent natural LXRα ligands known to date. Analysis of endogenous gene expression in various cell-based systems indicated the potential of 5,6-EC to antagonize LXR-mediated gene expression. Furthermore, it also induced the expression of some LXR-responsive genes in keratinocytes. These results clearly demonstrate that 5,6-EC is an LXR modulator that may play a role in the development of lipid disorders, such as atherosclerosis, by antagonizing the agonistic action of endogenous LXR ligands.

Synthesis of 4-(3-biaryl)quinoline sulfones as potent liver X receptor agonists.

A series of 4-(3-biaryl)quinolines with sulfone substituents on the terminal aryl ring (8) was prepared as potential LXR agonists. High affinity LXRbeta ligands with generally modest binding selectivity over LXRalpha and excellent agonist potency in LXR functional assays were identified. Many compounds had LXRbeta binding IC(50) values <10 nM while the most potent had EC(50) values <1.0 nM in an ABCA1 mRNA induction assay in J774 mouse cells with efficacy comparable to T0901317. Sulfone 8a was further evaluated in LDL (-/-) mice and shown to reduce atherosclerotic lesion progression.

Identification of phenylsulfone-substituted quinoxaline (WYE-672) as a tissue selective liver X-receptor (LXR) agonist.

A series of phenyl sulfone substituted quinoxaline were prepared and the lead compound 13 (WYE-672) was shown to be a tissue selective LXR Agonist. Compound 13 demonstrated partial agonism for LXRbeta in kidney HEK-293 cells but did not activate Gal4 LXRbeta fusion proteins in huh-7 liver cells. Although 13 showed potent binding affinity to LXRbeta (IC(50) = 53 nM), it had little binding affinity for LXRalpha (IC(50) > 1.0 microM) and did not recruit any coactivator/corepressor peptides in the LXRalpha multiplex assay. However, compound 13 showed good agonism in THP-1 cells with respect to increasing ABCA1 gene expression and good potency on cholesterol efflux in THP-1 foam cells. In an eight-week lesion study in LDLR -/- mice, compound 13 showed reduction of aortic arch lesion progression and no plasma or hepatic triglyceride increase. These results suggest quinoxaline 13 may have an improved biological profile for potential use as a therapeutic agent.

Quinoline-3-carboxamide containing sulfones as liver X receptor (LXR) agonists with binding selectivity for LXRbeta and low blood-brain penetration.

A series of quinoline-3-carboxamide containing sulfones was prepared and found to have good binding affinity for LXRbeta and moderate binding selectivity over LXRalpha. The 8-Cl quinoline analog 33 with a high TPSA score, displayed 34-fold binding selectivity for LXRbeta over LXRalpha (LXRbeta IC(50)=16nM), good activity for inducing ABCA1 gene expression in a THP macrophage cell line, desired weak potency in the LXRalpha Gal4 functional assay, and low blood-brain barrier penetration in rat.

4-(3-Aryloxyaryl)quinoline sulfones are potent liver X receptor agonists.

A series of 4-(3-aryloxyaryl)quinolines with sulfone substituents on the terminal aryl ring (7) was prepared as LXR agonists. High affinity LXR ligands with excellent agonist potency and efficacy in functional assays of LXR activity were identified. In general, these sulfone agonists were equal to or superior to previously described alcohol and amide analogs in terms of affinity, functional potency, and microsomal stability. Many of the sulfones had LXRbeta binding IC(50) values <10nM while the most potent compounds in an ABCA1 mRNA induction assay in J774 mouse cells had EC(50) values <10nM and were as efficacious as T0901317.

3-(3-Aryloxyaryl)imidazo[1,2-a]pyridine sulfones as liver X receptor agonists.

Replacement of a quinoline with an imidazo[1,2-a]pyridine in a series of liver X receptor (LXR) agonists incorporating a [3-(sulfonyl)aryloxyphenyl] side chain provided high affinity LXR ligands 7. In functional assays of LXR activity, good agonist potency and efficacy were found for several analogs.

1-(3-Aryloxyaryl)benzimidazole sulfones are liver X receptor agonists.

A series of 1-(3-aryloxyaryl)benzimidazoles incorporating a sulfone substituent (6) was prepared. High affinity LXR ligands were identified (LXRbeta binding IC(50) values <10nM), some with excellent agonist potency and efficacy in a functional assay of LXR activity measuring ABCA1 mRNA increases in human macrophage THP1 cells. The compounds were typically stable in liver microsome preparations and had good oral exposure in mice.

4-(3-aryloxyaryl)quinoline alcohols are liver X receptor agonists.

A series of 4-(3-aryloxyaryl)quinolines with alcohol substituents on the terminal aryl ring was prepared as potential LXR agonists, in which an alcohol group replaced an amide in previously reported amide analogs. High affinity LXR ligands with excellent agonist potency and efficacy in a functional model of LXR activity were identified, demonstrating that alcohols can substitute for amides while retaining LXR activity. The most potent compound was 5b which had an IC(50)=3.3 nM for LXRbeta binding and EC(50)=12 nM (122% efficacy relative to T0901317) in an ABCA1 mRNA induction assay in J774 mouse cells.

Discovery and SAR of cinnolines/quinolines as liver X receptor (LXR) agonists with binding selectivity for LXRbeta.

A series of cinnolines/quinolines was prepared and it was found that 4-phenyl-cinnoline/quinolines with either a 2',3' or 2',5'-disubstituted benzyloxy moiety or the 1-Me-7-indole methoxy moiety on the meta position of the 4-phenyl ring showed good binding selectivity for LXRbeta over LXRalpha. The LXRbeta binding selective modulators displayed good activity for inducing ABCA1 gene expression in J774 macrophage cell line and poor efficacy in the LXRalpha Gal4 functional assay. 26, 37 and 41 were examined for their ability to induce SREBP-1c gene expression in Huh-7 liver cell line and they were weak partial agonists.

LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse.

Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.

Biarylether amide quinolines as liver X receptor agonists.

A series of 4-(amido-biarylether)-quinolines was prepared as potential LXR agonists. Appropriate substitution with amide groups provided high affinity LXR ligands, some with excellent potency and efficacy in functional assays of LXR activity. Novel amide 4g had a binding IC(50)=1.9 nM for LXRbeta and EC(50)=34 nM (96% efficacy relative to T0901317) in an ABCA1 gene expression assay in mouse J774 cells, demonstrating that 4-(biarylether)-quinolines with appropriate amide substitution are potent LXR agonists.

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 and implementation of transcriptional biomarkers of synthetic LXR agonists in peripheral blood cells.

BACKGROUND: LXRs (Liver X Receptor alpha and beta) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds. OBJECTIVE: Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators. METHODS: Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRalpha, LXRbeta, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated ex vivo with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623. RESULTS: A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRalpha and LXRbeta, and all cell types significantly increased ABCA1 and ABCG1 expression upon ex vivo LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription. CONCLUSION: Peripheral blood cells express LXRalpha and LXRbeta, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.

Carboxylic acid based quinolines as liver X receptor modulators that have LXRbeta receptor binding selectivity.

A series of potent and binding selective LXRbeta agonists was developed using the previously reported non-selective LXR ligand WAY-254011 as a structural template. With the aid of molecular modeling, it was found that 2,3-diMe-Ph, 2,5-diMe-Ph, and naphthalene substituted quinoline acetic acids (such as quinoline 33, 37, and 38) showed selectivity for LXRbeta over LXRalpha in binding assays.

Adipocyte fatty acid-binding protein (aP2), a newly identified LXR target gene, is induced by LXR agonists in human THP-1 cells.

The liver X receptors (LXRalpha and LXRbeta), ligand-activated transcription factors, belong to the superfamily of nuclear hormone receptors and have been shown to play a major role in atherosclerosis by modulating cholesterol and triglyceride metabolism. In this report, we describe a novel LXR target, the adipocyte fatty acid binding protein (aP2), which plays an important role in fatty acid metabolism, adipocyte differentiation and atherosclerosis. While LXR agonists induce aP2 mRNA expression in human monocytes (THP-1 cells) and macrophages in a time- and concentration-dependent manner, they have no effect on aP2 expression in human adipocytes. The increase in aP2 mRNA level was additive when THP-1 cells were treated with LXR and PPARgamma agonists. Also, an RXR agonist induced aP2 expression in these cells. While no additive effect was observed with LXR and RXR agonists, additive effects were observed with RXR and PPARgamma agonists. GW9662, a potent PPARgamma antagonist, inhibited PPARgamma-induced aP2 expression without affecting LXR-mediated aP2 expression indicating the induction is mediated directly through LXR activation. Analysis of human aP2 promoter revealed a potential LXR response element (LXRE). Gel shift data showed that the LXRalpha/RXRalpha heterodimer bound to the LXRE motif in aP2 promoter in vitro in a sequence-specific manner. Deletion and mutation analyses of the proximal aP2 promoter confirm that this is a functional LXRE. These data indicate for the first time that human macrophage aP2 promoter is a direct target for the regulation by LXR/RXR heterodimers.

Further modification on phenyl acetic acid based quinolines as liver X receptor modulators.

A series of phenyl acetic acid based quinolines was prepared as LXR modulators. An SAR study in which the C-3 and C-8 positions of the quinoline core were varied led to the identification of two potent LXR agonists 23 and 27. Both compounds displayed good binding affinity for LXRbeta and LXRalpha, and increased expression of ABCA1 in THP-1 cells. These two compounds also had desirable pharmacokinetic profiles in mice and displayed in vivo efficacy in a 12-week Apo E knockout mouse lesion model.

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.

Liver X receptor (LXR)-beta regulation in LXRalpha-deficient mice: implications for therapeutic targeting.

The nuclear receptors liver X receptor (LXR) LXRalpha and LXRbeta are differentially expressed ligand-activated transcription factors that induce genes controlling cholesterol homeostasis and lipogenesis. Synthetic ligands for both receptor subtypes activate ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol metabolism, increase reverse cholesterol transport, and provide atheroprotection in mice. However, these ligands may also increase hepatic triglyceride (TG) synthesis via a sterol response element binding protein 1c (SREBP-1c)-dependent mechanism through a process reportedly regulated by LXRalpha. We studied pan-LXRalpha/beta agonists in LXRalpha knockout mice to assess the contribution of LXRbeta to the regulation of selected target genes. In vitro dose-response studies with macrophages from LXRalpha-/- and beta-/- mice confirm an equivalent role for LXRalpha and LXRbeta in the regulation of ABCA1 and SREBP-1c gene expression. Cholesterol-efflux studies verify that LXRbeta can drive apoA1-dependent cholesterol mobilization from macrophages. The in vivo role of LXRbeta in liver was further evaluated by treating LXRalpha-/- mice with a pan-LXRalpha/beta agonist. High-density lipoprotein (HDL) cholesterol increased without significant changes in plasma TG or very low density lipoprotein. Analysis of hepatic gene expression consistently revealed less activation of ABCA1 and SREBP-1c genes in the liver of LXRalpha null animals than in treated wild-type controls. In addition, hepatic CYP7A1 and several genes involved in fatty acid/TG biosynthesis were not induced. In peripheral tissues from these LXRalpha-null mice, LXRbeta activation increases ABCA1 and SREBP-1c gene expression in a parallel manner. However, putative elevation of SREBP-1c activity in these tissues did not cause hypertriglyceridemia. In summary, selective LXRbeta activation is expected to stimulate ABCA1 gene expression in macrophages, contribute to favorable HDL increases, but circumvent hepatic LXRalpha-dominated lipogenesis.

Modulation of adipose tissue development by pharmacological inhibition of PAI-1.

OBJECTIVE: The effect of a novel small molecule plasminogen activator inhibitor (PAI-1) inhibitor on adipose tissue physiology was investigated. METHODS AND RESULTS: In human preadipocyte cultures, PAI-039 inhibited both basal and glucose-stimulated increases in active PAI-1 antigen, yet had no effect on PAI-1 mRNA, suggesting a direct inactivation of PAI-1. Differentiation of human preadipocytes to adipocytes was associated with leptin synthesis, which was significantly reduced in the presence of PAI-039, together with an atypical adipocyte morphology characterized by a reduction in the size and number of lipid containing vesicles. In a model of diet-induced obesity, pair-fed C57 Bl/6 mice administered PAI-039 in a high-fat diet exhibited a dose-dependent reduction in body weight, epididymal adipose tissue weight, adipocyte volume, and circulating plasma active PAI-1. Plasma glucose, triglycerides, and leptin were also significantly reduced in drug-treated mice, and concentrations of PAI-039 associated with these physiological effects were near the in vitro IC50 for the inhibition of PAI-1. CONCLUSIONS: Our results indicate that a small molecule inactivator of PAI-1 can neutralize glucose-stimulated increases in PAI-1 in human preadipocyte cultures, reduce adipocyte differentiation, and prevent the development of diet-induced obesity. These data suggest the pharmacological inhibition of PAI-1 could be beneficial in diseases associated with expansion of adipose tissue mass.

Gene-selective modulation by a synthetic oxysterol ligand of the liver X receptor.

Liver X receptors (LXRs) play key roles in the regulation of cholesterol homeostasis by limiting cholesterol accumulation in macrophages within arterial wall lesion sites by a mechanism that includes the upregulation of ATP binding cassette transporters. These atheroprotective properties distinguish LXRs as potential targets for pharmaceutical intervention in cardiovascular disease. Their associated activity for promoting lipogenesis and triglyceride accretion through the activation of sterol-response element binding protein 1c (SREBP-1c) expression, however, represents a potential proatherogenic liability. A newly characterized synthetic oxysterol, N,N-dimethyl-3beta-hydroxycholenamide (DMHCA), represents a gene-selective LXR modulator that mediates potent transcriptional activation of ABCA1 gene expression while exhibiting minimal effects on SREBP-1c both in vitro and in vivo in mice. DMHCA has the potential to stimulate cholesterol transport through the upregulation of LXR target genes, including ABCA1, in liver, small intestine, and peritoneal macrophages. Compared with known nonsteroidal LXR agonists, however, DMHCA exhibits only limited activity for increasing hepatic SREBP-1c mRNA and does not alter circulating plasma triglycerides. Cell-based studies also indicate that DMHCA enhances cholesterol efflux in macrophages and suggest a mechanism whereby this selective modulator can potentially inhibit cholesterol accumulation. DMHCA and related gene-selective ligands of LXR may have application to the study and treatment of atherosclerosis.