Natural products as modulators of retinoic acid receptor-related orphan receptors (RORs).

Covering: 1994 to 2020 Retinoic acid receptor-related orphan receptors (RORs) belong to a subfamily of the nuclear receptor superfamily and possess prominent roles in circadian rhythm, metabolism, inflammation, and cancer. They have been subject of research for over two decades and represent attractive but challenging drug targets. Natural products were among the first identified ligands of RORs and continue to be of interest to this day. This review focuses on ligands and indirect modulators of RORs from natural sources and explores their roles in a therapeutic context.

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations.

BACKGROUND: The human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line. RESULTS: We developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time. CONCLUSIONS: The Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.

Characterization of a Structural Leoligin Analog as Farnesoid X Receptor Agonist and Modulator of Cholesterol Transport.

The ligand-activated farnesoid X receptor is an emerging therapeutic target for the development of drugs against metabolic syndrome-related diseases. In this context, selective bile acid receptor modulators represent a novel concept for drug development. Selective bile acid receptor modulators act in a target gene- or tissue-specific way and are therefore considered less likely to elicit unwanted side effects. Based on leoligin, a lignan-type secondary plant metabolite from the alpine plant Leontopodium nivale ssp. alpinum, 168 synthesized structural analogs were screened in a farnesoid X receptor in silico pharmacophore-model. Fifty-six virtual hits were generated. These hits were tested in a cell-based farnesoid X receptor transactivation assay and yielded 7 farnesoid X receptor-activating compounds. The most active one being LT-141A, with an EC50 of 6 µM and an Emax of 4.1-fold. This analog did not activate the G protein-coupled bile acid receptor, TGR5, and the metabolic nuclear receptors retinoid X receptor α, liver X receptors α/ß, and peroxisome proliferator-activated receptors ß/γ. Investigation of different farnesoid X receptor target genes characterized LT-141A as selective bile acid receptor modulators. Functional studies revealed that LT-141A increased cholesterol efflux from THP-1-derived macrophages via enhanced ATP-binding cassette transporter 1 expression. Moreover, cholesterol uptake in differentiated Caco-2 cells was significantly decreased upon LT-141A treatment. In conclusion, the leoligin analog LT-141A selectively activates the nuclear receptor farnesoid X receptor and has an influence on cholesterol transport in 2 model systems.

Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages.

Leoligin is a natural lignan found in Edelweiss (Leontopodium nivale ssp. alpinum). The aim of this study was to examine its influence on cholesterol efflux and to address the underlying mechanism of action. Leoligin increases apo A1- as well as 1% human plasma-mediated cholesterol efflux in THP-1 macrophages without affecting cell viability as determined by resazurin conversion. Western blot analysis revealed that the protein levels of the cholesterol efflux transporters ABCA1 and ABCG1 were upregulated, whereas the SR-B1 protein level remained unchanged upon treatment with leoligin (10 µM, 24 h). Quantitative reverse transcription PCR further uncovered that leoligin also increased ABCA1 and ABCG1 mRNA levels without affecting the half-life of the two mRNAs in the presence of actinomycin D, a transcription inhibitor. Proteome analysis revealed the modulation of protein expression fingerprint in the presence of leoligin. Taken together, these results suggest that leoligin induces cholesterol efflux in THP-1-derived macrophages by upregulating ABCA1 and ABCG1 expression. This novel activity suggests leoligin as a promising candidate for further studies addressing a possible preventive or therapeutic application in the context of atherosclerosis.

Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages.

Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease.

Honokiol: a non-adipogenic PPARγ agonist from nature.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators. METHODS: We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists. RESULTS: The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain. CONCLUSION: We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo. GENERAL SIGNIFICANCE: This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.

Impact of trans-resveratrol-sulfates and -glucuronides on endothelial nitric oxide synthase activity, nitric oxide release and intracellular reactive oxygen species.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides) and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings.

Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

The Takeda G protein-coupled receptor 5 (TGR5) is activated endogenously by primary and secondary bile acids. This receptor is considered a candidate target for addressing inflammatory and metabolic disorders. We have targeted TGR5 with structure-based methods for ligand finding using the recently solved experimental structures, as well as structures obtained from molecular dynamics simulations. Through addressing the orthosteric as well as a putative allosteric site, we identified agonists and positive allosteric modulators. While the predicted binding locations were not in line with their efficacy, our work contributes activating small-molecule ligands that we have thoroughly characterized in vitro.

Characterization of Constituents with Potential Anti-Inflammatory Activity in Chinese Lonicera Species by UHPLC-HRMS Based Metabolite Profiling.

This study centered on detecting potentially anti-inflammatory active constituents in ethanolic extracts of Chinese Lonicera species by taking an UHPLC-HRMS-based metabolite profiling approach. Extracts from eight different Lonicera species were subjected to both UHPLC-HRMS analysis and to pharmacological testing in three different cellular inflammation-related assays. Compounds exhibiting high correlations in orthogonal projections to latent structures discriminant analysis (OPLS-DA) of pharmacological and MS data served as potentially activity-related candidates. Of these candidates, 65 were tentatively or unambiguously annotated. 7-Hydroxy-5,3',4',5'-tetramethoxyflavone and three bioflavonoids, as well as three C32- and one C34-acetylated polyhydroxy fatty acid, were isolated from Lonicera hypoglauca leaves for the first time, and their structures were fully or partially elucidated. Of the potentially active candidate compounds, 15 were subsequently subjected to pharmacological testing. Their activities could be experimentally verified in part, emphasizing the relevance of Lonicera species as a source of anti-inflammatory active constituents. However, some compounds also impaired the cell viability. Overall, the approach was found useful to narrow down the number of potentially bioactive constituents in the complex extracts investigated. In the future, the application of more refined concepts, such as extract prefractionation combined with bio-chemometrics, may help to further enhance the reliability of candidate selection.

Lignan derivatives from Krameria lappacea roots inhibit acute inflammation in vivo and pro-inflammatory mediators in vitro.

The roots of Krameria lappacea are used traditionally against oropharyngeal inflammation. So far, the astringent and antimicrobial properties of its proanthocyanidin constituents are considered to account for the anti-inflammatory effect. The aim of the present study was to characterize pharmacologically a lipophilic extract of K. lappacea roots and several isolated lignan derivatives (1-11) in terms of their putative anti-inflammatory activity. The dichloromethane extract (ID50 77 µg/cm²) as well compounds 1-11 (ID50 0.31-0.60 µmol/cm²) exhibited topical antiedematous properties comparable to those of indomethacin (ID50 0.29 µmol/cm²) in a mouse ear in vivo model. Two of the most potent compounds, 2-(2-hydroxy-4-methoxyphenyl)-5-(3-hydroxypropyl)benzofuran (5) and (+)-conocarpan (7), were studied regarding their time-dependent edema development and leukocyte infiltration up to 48 h after croton oil-induced dermatitis induction, and they showed activity profiles similar to that of hydrocortisone. In vitro studies of the isolated lignan derivatives demonstrated the inhibition of NF-κB, cyclooxygenase-1 and -2, 5-lipoxygenase, and microsomal prostaglandin E2 synthase-1 as well as antioxidant properties, as mechanisms possibly contributing to the observed in vivo effects. The present findings not only support the ethnopharmacological use of K. lappacea roots but also reveal that the isolated lignan derivatives contribute strongly to the anti-inflammatory activity of this herbal drug.

Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists are used for the treatment of type 2 diabetes and metabolic syndrome. However, the currently used PPAR gamma agonists display serious side effects, which has led to a great interest in the discovery of novel ligands with favorable properties. The aim of our study was to identify new PPARgamma agonists by a PPAR gamma pharmacophore-based virtual screening of 3D natural product libraries. This in silico approach led to the identification of several neolignans predicted to bind the receptor ligand binding domain (LBD). To confirm this prediction, the neolignans dieugenol, tetrahydrodieugenol, and magnolol were isolated from the respective natural source or synthesized and subsequently tested for PPAR gamma receptor binding. The neolignans bound to the PPAR gamma LBD with EC(50) values in the nanomolar range, exhibiting a binding pattern highly similar to the clinically used agonist pioglitazone. In intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPAR gamma-mediated, but not human PPAR alpha- or -beta/delta-mediated luciferase reporter expression, with a pattern suggesting partial PPAR gamma agonism. The coactivator recruitment study also demonstrated partial agonism of the tested neolignans. Dieugenol, tetrahydrodieugenol, and magnolol but not the structurally related eugenol induced 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPAR gamma expression. In conclusion, we identified neolignans as novel ligands for PPAR gamma, which exhibited interesting activation profiles, recommending them as potential pharmaceutical leads or dietary supplements.

Impact of natural products on the cholesterol transporter ABCA1.

ETHNOPHARMACOLOGICAL RELEVANCE: In different countries and areas of the world, traditional medicine has been and is still used for the treatment of various disorders, including chest pain or liver complaints, of which we now know that they can be linked with altered lipid and cholesterol homeostasis. As ATP-binding cassette transporter A1 (ABCA1) plays an essential role in cholesterol metabolism, its modulation may be one of the molecular mechanisms responsible for the experienced benefit of traditional recipes. Intense research activity has been dedicated to the identification of natural products from traditional medicine that regulate ABCA1 expression. AIMS OF THE REVIEW: This review surveys natural products, originating from ethnopharmacologically used plants, fungi or marine sources, which influence ABCA1 expression, providing a reference for future study. MATERIALS AND METHODS: Information on regulation of ABCA1 expression by natural compounds from traditional medicine was extracted from ancient and modern books, materia medica, and electronic databases (PubMed, Google Scholar, Science Direct, and ResearchGate). RESULTS: More than 60 natural compounds from traditional medicine, especially traditional Chinese medicine (TCM), are reported to regulate ABCA1 expression in different in vitro and in vivo models (such as cholesterol efflux and atherosclerotic animal models). These active compounds belong to the classes of polyketides, terpenoids, phenylpropanoids, tannins, alkaloids, steroids, amino acids and others. Several compounds appear very promising in vivo, which need to be further investigated in animal models of diseases related to ABCA1 or in clinical studies. CONCLUSION: Natural products from traditional medicine constitute a large promising pool for compounds that regulate ABCA1 expression, and thus may prevent/treat diseases related to cholesterol metabolism, like atherosclerosis or Alzheimer's disease. In many cases, the molecular mechanisms of these natural products remain to be investigated.

Soraphen A enhances macrophage cholesterol efflux via indirect LXR activation and ABCA1 upregulation.

Increased cholesterol efflux from macrophage foam cells in the subendothelial space confers protection against atherosclerosis. Soraphen A, a myxobacterial macrolactone, is an inhibitor of acetyl coenzyme A carboxylases (ACC), which control fatty acid synthesis and oxidation. To assess a potential direct link between macrophage cholesterol efflux and ACC inhibition, we examined [3H]-cholesterol efflux from human THP-1-derived foam cells in the presence of soraphen A. We dissected underlying molecular events by western blot analyses, RT-qPCR, reporter gene and coactivator recruitment assays as well as relative quantification of free and total cholesterol. Soraphen A increased cholesterol efflux from macrophage foam cells via upregulation of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1). Soraphen A enhanced transcription of ABCA1 in an LXR-dependent manner, however, without direct binding to the ligand-binding domain of this nuclear receptor. Soraphen A elevated the cellular level of free cholesterol, and failed to activate LXR upon exogenous supplementation with fatty acids or inhibition of cholesterol synthesis. Thus, impeded conversion from acetyl- to malonyl-CoA by soraphen A may lead to more unesterified cholesterol and thus potential LXR agonists. The present study reveals ACC inhibition as a previously unrecognized mechanism to regulate macrophage cholesterol efflux via indirect LXR activation and ABCA1 upregulation.

Natural products as modulators of the nuclear receptors and metabolic sensors LXR, FXR and RXR.

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets. This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.

In Silico Workflow for the Discovery of Natural Products Activating the G Protein-Coupled Bile Acid Receptor 1.

The G protein-coupled bile acid receptor (GPBAR1) has been recognized as a promising new target for the treatment of diverse diseases, including obesity, type 2 diabetes, fatty liver disease and atherosclerosis. The identification of novel and potent GPBAR1 agonists is highly relevant, as these diseases are on the rise and pharmacological unmet therapeutic needs are pervasive. Therefore, the aim of this study was to develop a proficient workflow for the in silico prediction of GPBAR1 activating compounds, primarily from natural sources. A protocol was set up, starting with a comprehensive collection of structural information of known ligands. This information was used to generate ligand-based pharmacophore models in LigandScout 4.08 Advanced. After theoretical validation, the two most promising models, namely BAMS22 and TTM8, were employed as queries for the virtual screening of natural product and synthetic small molecule databases. Virtual hits were progressed to shape matching experiments and physicochemical clustering. Out of 33 diverse virtual hits subjected to experimental testing using a reporter gene-based assay, two natural products, farnesiferol B (27) and microlobidene (28), were confirmed as GPBAR1 activators reaching more than 50% receptor activation at 20 µM with EC50s of 13.53 µM and 13.88 µM, respectively. This activity is comparable to that of the endogenous ligand lithocholic acid (1). Seven further virtual hits showed activity reaching at least 15% receptor activation either at 5 or 20 µM, including new scaffolds from natural and synthetic origin.

6-Dihydroparadol, a Ginger Constituent, Enhances Cholesterol Efflux from THP-1-Derived Macrophages.

SCOPE: Ginger is reported to be used for the prevention and treatment of cardiovascular diseases (CVD). Cholesterol efflux from macrophage foam cells is an important process in reverse cholesterol transport, whose increase may help to prevent or treat CVD. In this study, we investigated the effects of 6-dihydroparadol from ginger on macrophage cholesterol efflux. METHODS AND RESULTS: We show that 6-dihydroparadol concentration-dependently enhances both apolipoprotein A1- and human plasma-mediated cholesterol efflux from cholesterol-loaded THP-1-derived macrophages using macrophage cholesterol efflux assay. 6-Dihydroparadol increases protein levels of both ATP-binding cassette transporters A1 and G1 (ATP-binding cassette transporter A1 [ABCA1] and ATP-binding cassette transporter G1 [ABCG1]) according to Western blot analysis. The ABCA1 inhibitor probucol completely abolishes 6-dihydroparadol-enhanced cholesterol efflux. Furthermore, increased ABCA1 protein levels in the presence of 6-dihydroparadol were associated with both increased ABCA1 mRNA levels and increased ABCA1 protein stability. Enhanced ABCG1 protein levels were only associated with increased protein stability. Increased ABCA1 protein stability appeared to be the result of a reduced proteasomal degradation of the transporter in the presence of 6-dihydroparadol. CONCLUSION: We identified 6-dihydroparadol from ginger as a novel promoter of cholesterol efflux from macrophages that increases both ABCA1 and ABCG1 protein abundance. This newly identified bioactivity might contribute to the antiatherogenic effects of ginger.

Novel interactomics approach identifies ABCA1 as direct target of evodiamine, which increases macrophage cholesterol efflux.

Evodiamine, a bioactive alkaloid from the fruits of the traditional Chinese medicine Evodia rutaecarpa (Juss.) Benth. (Fructus Evodiae, Wuzhuyu), recently gained attention as a dietary supplement for weight loss and optimization of lipid metabolism. In light of its use by patients and consumers, there is an urgent need to elucidate the molecular targets affected by this natural product. Using a novel interactomics approach, the Nematic Protein Organisation Technique (NPOT), we report the identification of ATP-binding cassette transporter A1 (ABCA1), a key membrane transporter contributing to cholesterol efflux (ChE), as a direct binding target of evodiamine. The binding of evodiamine to ABCA1 is confirmed by surface plasmon resonance (SPR) experiments. Examining the functional consequences of ABCA1 binding reveals that evodiamine treatment results in increased ABCA1 stability, elevated cellular ABCA1 protein levels, and ultimately increased ChE from THP-1-derived human macrophages. The protein levels of other relevant cholesterol transporters, ABCG1 and SR-B1, remain unaffected in the presence of evodiamine, and the ABCA1 mRNA level is also not altered.

Erythrodiol, an Olive Oil Constituent, Increases the Half-Life of ABCA1 and Enhances Cholesterol Efflux from THP-1-Derived Macrophages.

Cholesterol efflux (ChE) from macrophages is an initial step of reverse cholesterol transport (RCT). The ATP-binding cassette transporter A1 (ABCA1) is a key transporter for ChE and its increased expression is regarded to attenuate atherosclerosis. Thus, the identification and characterization of molecules raising ABCA1 and thereby stimulating ChE is of pharmacological relevance. In this study, we tested dietary compounds from olive oil for their capacity of enhancing cellular ABCA1 protein level. We identified erythrodiol (Olean-12-ene-3ß,28-diol) as an ABCA1 stabilizer and revealed its positive influence on ChE in THP-1-derived human macrophages. Among the nine tested compounds from olive oil, erythrodiol was the sole compound raising ABCA1 protein level (at 10 µM). None of the tested compounds impaired viability of THP-1 macrophages from 5 to 20 µM as determined by resazurin conversion. Western blot analyses of key membrane transporters contributing to ChE showed that the protein level of ABCG1 and scavenger receptor class B member 1 (SR-B1) remain unaffected by erythrodiol. Besides, erythrodiol (10 µM) did not influence the mRNA level of ABCA1, ABCG1, and SR-B1, as determined by quantitative reverse transcription PCR, but significantly inhibited the degradation of ABCA1 as evident by an increased half-life of the protein in the presence of cycloheximide, an inhibitor of de novo protein synthesis. Therefore, erythrodiol promotes ChE from THP-1-derived human macrophages by stabilizing the ABCA1 protein. This bioactivity makes erythrodiol a good candidate to be further explored for therapeutic or preventive application in the context of atherosclerosis.

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

BACKGROUND: Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. METHODS AND RESULTS: Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 µmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. CONCLUSIONS: Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease.

Linked magnolol dimer as a selective PPARγ agonist - Structure-based rational design, synthesis, and bioactivity evaluation.

The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and its hetero-dimerization partner retinoid X receptor α (RXRα) are considered as drug targets in the treatment of diseases like the metabolic syndrome and diabetes mellitus type 2. Effort has been made to develop new agonists for PPARγ to obtain ligands with more favorable properties than currently used drugs. Magnolol was previously described as dual agonist of PPARγ and RXRα. Here we show the structure-based rational design of a linked magnolol dimer within the ligand binding domain of PPARγ and its synthesis. Furthermore, we evaluated its binding properties and functionality as a PPARγ agonist in vitro with the purified PPARγ ligand binding domain (LBD) and in a cell-based nuclear receptor transactivation model in HEK293 cells. We determined the synthesized magnolol dimer to bind with much higher affinity to the purified PPARγ ligand binding domain than magnolol (K i values of 5.03 and 64.42 nM, respectively). Regarding their potency to transactivate a PPARγ-dependent luciferase gene both compounds were equally effective. This is likely due to the PPARγ specificity of the newly designed magnolol dimer and lack of RXRα-driven transactivation activity by this dimeric compound.