Discovery of New Liver X Receptor Agonists by Pharmacophore Modeling and Shape-Based Virtual Screening

Agonists of liver X receptors (LXR) α and β are important regulators of cholesterol metabolism, but agonism of the LXRα subtype appears to cause hepatic lipogenesis, suggesting LXRβ-selective activators are attractive new lipid lowering drugs. In this work, pharmacophore modeling and shape-based virtual screening were combined to predict new LXRβ-selective ligands. Out of the 10 predicted compounds, three displayed significant LXR activity. Two activated both LXR subtypes. The third compound activated LXRβ 1.8-fold over LXRα

Effect of capsaicin on vascular smooth muscle cell proliferation: raw data

<p>Cell proliferation was estimated by quantification of metabolic activity (Data_1A) and DNA synthesis (Data_1B) (both relative units). Cell death was estimated by quantification of the percentage of extracellular lactate dehydrogenase activity (Data_1C). Numbers in column headers denote capsaicin concentration (uM); Digitonin concentration was 100 ug/mL). Data correspond to Figure 1 in the associated article.</p

Rate of aerobic glycolysis in the presence of BA.

<p>MEF were treated with 10 µM BA or DMSO (0.1%) for 16 h before they were subjected to a glycolysis stress test as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115683#s2" target="_blank">Materials and Methods</a>”. In (A) the extracellular acidification rate (ECAR) is depicted upon exposing cells successively to glucose (10 mM), oligomycin A (2 µM) and deoxyglucose (DOG, 100 mM) (mean + SD; compiled raw data from three independent experiments with four technical replicates each). In (B) those data are analyzed in terms of basal (glycolytic ECAR after glucose addition), maximal (glycolytic ECAR after oligomycin) and spare (maximal minus basal activity) glycolytic activity (mean + SD,* p<0.05, t-test, DMSO vs. BA). Quantitation is based on the value at the final time point of each treatment condition. In (C) values of OCR and ECAR (of DMSO and BA-treated cells (16 h)) after addition of glucose were plotted against each other to visualize the shift from glucose oxidation to glycolysis. The observed shift upon addition of oligomycin A is added to the plot as a reference. (D) MEF were treated with DMSO (0.1%, D) and 10 µM BA for the indicated periods of time before total cell lysates were subjected to immunoblot analyses for pPDHE1 (Ser273) and total PDHE1 (molecular weight 43 kDa). Representative blots out of three experiments are shown. The graph below depicts compiled densitometric values of pPDHE/PDHE (n = 3; mean + SD; *p<0.05; t-test; DMSO vs BA at each time point). (E) MEF were treated with 10 µM BA or DMSO (0.1%) for 16 h before they were subjected to immunoblot analysis for GLUT1 (∼50 kDa), GLUT3 (∼55 kDa) and actin (42 kDa). Representative blots out of three independent experiments are shown. The graph below depicts compiled densitometric values of GLUT1/actin and GLUT3/actin, respectively (n = 3; mean + SD; *p<0.05; t-test; DMSO vs BA at each time point).</p

Mitochondrial function in the presence of BA.

<p>(A) MEF were treated with 10 µM BA or 0.1% DMSO for 16 h before they were subjected to flow cytometric analysis of the mitochondrial ROS production with the use of MitoSox Red and antimycin A (2 µM, 1 h) as positive control. (n = 3 (each in duplicate or triplicate); mean + SD,* p<0.05, t-test). (B) MEF were treated with DMSO or 10 µM BA for 16 h before total cell lysates were subjected to western blot analysis for UCP1, UCP2 (band at ∼25–35 kDa) and actin (42 kDa) as loading control. Representative blots of three independent experiments are depicted. The graph below depicts compiled densitometric values of UCP1/actin and UCP2/actin, respectively (n = 3; mean + SD; *p<0.05; t-test; DMSO vs BA). (C) MEF were treated with 10 µM BA or 0.1% DMSO for 16 h before they were subjected to flow cytometric analysis of the mitochondrial content by using MitoTracker Green. Valinomycin (200 nM, 16 h), a known disruptor of the mitochondrial membrane potential served as control for the potential-independent signal of MitoTracker Green. MEF were treated with 10 µM BA or DMSO (0.1%) for 16 h before they were subjected to a mitochondrial stress test as described under Materials. In (D) the mean oxygen consumption rate (OCR) of three independent experiments is depicted upon exposing cells successively to oligomycin (2 µM), FCCP (1.5 µM) and antimycin A + rotenone (A+R; 1+1 µM). In (E) those data are analyzed in terms of oxygen consumption rate under basal conditions, oxygen consumption for ATP synthesis, maximal respiratory rate, spare respiratory capacity and proton leak (mean + SD,* p<0.05, t-test, DMSO vs. BA).</p

The role of AMPK and LKB-1 for the BA-induced glycolytic switch.

<p>(A) MEF were treated with DMSO (0.1%) or BA (10 µM) for the indicated periods of time. Total cell lysates were subjected to immunoblot analysis for pAMPK (Thr172) and total AMPK (60 kDa) (left panel) or pAMPK (Thr172), pACC (Ser79) (∼245 kDa) and actin (42 kDa) (right panel). Representative blots out of three independent experiments are depicted. The graphs below depict compiled densitometric values of pAMPK/AMPK or pACC/actin, respectively (n = 3; mean + SD; *p<0.05; t-test; DMSO vs BA at each time point). WT and AMPK -/- MEF were treated with DMSO or BA (10 µM) for 16 h. Then the cellular glucose uptake rates (B) were assessed (n = 3 (in triplicate); mean + SD,* p<0.05, ANOVA, Dunnett's post-test vs DMSO ctrl), as well as the expression levels of GLUT1 (50 kDa), AMPK (60 kDa) and actin (42 kDa) by western blot analysis (C). One representative blot is depicted of three independent experiments. The graph below depicts compiled densitometric values of GLUT1/actin, respectively (n = 3; mean + SD; *p<0.05; t-test; DMSO vs BA). In (D) cells were subjected to determination of the extracellular acidification rate (ECAR) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115683#pone-0115683-g003" target="_blank">Fig. 3</a>. Representative results of three independent experiments (with three technical replicates each) are shown (mean + SD,* p<0.05, t-test). (E) WT and AMPK -/- MEF were treated with DMSO (0.1%) or BA (10 µM) for 16 h before they were subjected to a mitochondrial stress test and extracellular flux analysis. Compiled data of three independent experiments are depicted (mean + SD). (F) WT and LKB1-/- MEF were treated with BA (10 µM) for the indicated periods of time before total cell lysates were subjected to immunoblot analysis for pAMPK (Thr172), AMPK (60 kDa), LKB1(∼50 kDa) and actin (42 kDa). Representative blots out of three independent experiments are depicted. The graph below depicts compiled densitometric values of pAMPK/AMPK, respectively (n = 3; mean + SD; *p<0.05; ANOVA, Dunnett (vs 0 h treatment with BA).</p

Glucose-addiction in the presence of BA.

<p>MEF were treated with DMSO (0.1%, D) or BA (10 µM) in DMEM (containing 1% serum, glutamine (2 mM) and pyruvate (2 mM)) in the presence (25 mM) and absence of glucose (instead 25 mM mannitol as osmotic balance) for 48 h before ATP levels (A) and biomass (B) were assessed. Bar graphs depict compiled data of three experiments in quadruplicate. (mean + SD, * p<0.05, t-test DMSO vs BA).</p

Glycolytic Switch in Response to Betulinic Acid in Non-Cancer Cells

<div><p>The naturally occurring triterpenoid betulinic acid (BA) shows pronounced polypharmacology ranging from anti-inflammatory to anti-lipogenic activities. Recent evidence suggests that rather diverse cellular signaling events may be attributed to the same common upstream switch in cellular metabolism. In this study we therefore examined the metabolic changes induced by BA (10 µM) administration, with focus on cellular glucose metabolism. We demonstrate that BA elevates the rates of cellular glucose uptake and aerobic glycolysis in mouse embryonic fibroblasts with concomitant reduction of glucose oxidation. Without eliciting signs of obvious cell death BA leads to compromised mitochondrial function, increased expression of mitochondrial uncoupling proteins (UCP) 1 and 2, and liver kinase B1 (LKB1)-dependent activation AMP-activated protein kinase. AMPK activation accounts for the increased glucose uptake and glycolysis which in turn are indispensable for cell viability upon BA treatment. Overall, we show for the first time a significant impact of BA on cellular bioenergetics which may be a central mediator of the pleiotropic actions of BA.</p></div

Cytotoxicity of BA in MEF.

<p>MEF were treated with BA (10 µM and 30 µM) for 48 h before they were subjected to determination of membrane integrity (% LDH release) (A), potential of proapoptotic events (activation of caspase 3/7) (B), of ATP levels (C) and biomass (D). Bar graphs depict compilation of three independent experiments (expressed as fold of the DMSO mean value in B, C, and D), each in quadruplicate (mean + SD, * p<0.05, ANOVA, Dunnett's post test versus DMSO ctrl). Staurosporine (Stauro, 1 µM for 6 h), triton (1% for 1 h) or a combination of oligomycin A (OL; 2 µM) and DOG (10 mM; 5 h) served as positive controls in the assays.</p

NF-κB Inhibitors from <i>Eurycoma longifolia</i>

The roots of <i>Eurycoma longifolia</i> have been used in many countries of Southeast Asia to alleviate various diseases including malaria, dysentery, sexual insufficiency, and rheumatism. Although numerous studies have reported the pharmacological properties of <i>E. longifolia</i>, the mode of action of the anti-inflammatory activity has not been elucidated. Bioguided isolation of NF-κB inhibitors using an NF-κB-driven luciferase reporter gene assay led to the identification of a new quassinoid, eurycomalide C (<b>1</b>), together with 27 known compounds including 11 quassinoids (<b>2</b>–<b>12</b>), six alkaloids (<b>13</b>–<b>18</b>), two coumarins (<b>19</b>, <b>20</b>), a squalene derivative (<b>21</b>), a triterpenoid (<b>22</b>), and six phenolic compounds (<b>23</b>–<b>28</b>) from the extract of <i>E. longifolia.</i> Evaluation of the biological activity revealed that C₁₉-type and C₂₀-type quassinoids, β-carboline, and canthin-6-one alkaloids are potent NF-κB inhibitors, with IC₅₀ values in the low micromolar range, while C₁₈-type quassinoids, phenolic compounds, coumarins, the squalene derivative, and the triterpenoid turned out to be inactive when tested at a concentration of 30 μM. Eurycomalactone (<b>2</b>), 14,15β-dihydroklaieanone (<b>7</b>), and 13,21-dehydroeurycomanone (<b>10</b>) were identified as potent NF-κB inhibitors with IC₅₀ values of less than 1 μM

Eurycomalactone Inhibits Expression of Endothelial Adhesion Molecules at a Post-Transcriptional Level

The C-19 quassinoid eurycomalactone (<b>1</b>) has recently been shown to be a potent (IC₅₀ = 0.5 μM) NF-κB inhibitor in a luciferase reporter model. In this study, we show that <b>1</b> with similar potency inhibited the expression of the NF-κB-dependent target genes ICAM-1, VCAM-1, and E-selectin in TNFα-activated human endothelial cells (HUVECtert) by flow cytometry experiments. Surprisingly, <b>1</b> (2 μM) did not inhibit TNFα-induced IKKα/β or IκBα phosphorylation significantly. Also, the TNFα-induced degradation of IκBα remained unchanged in response to <b>1</b> (2 μM). In addition, pretreatment of HUVECtert with <b>1</b> (2 μM) had no statistically significant effect on TNFα-mediated nuclear translocation of the NF-κB subunit p65 (RelA). Quantitative RT-PCR revealed that <b>1</b> (0.5–5 μM) exhibited diverse effects on the TNFα-induced transcription of <i>ICAM-1</i>, <i>VCAM-1</i>, and <i>SELE</i> genes since the mRNA level either remained unchanged (ICAM-1, E-selectin, and VCAM-1 at 0.5 μM <b>1</b>), was reduced (VCAM-1 at 5 μM <b>1</b>), or even increased (E-selectin at 5 μM <b>1</b>). Finally, the time-dependent depletion of a short-lived protein (cyclin D1) as well as the measurement of <i>de novo</i> protein synthesis in the presence of <b>1</b> (2–5 μM) suggested that <b>1</b> might act as a protein synthesis inhibitor rather than an inhibitor of early NF-κB signaling