Computational and Biological Evaluation of <i>N</i>-octadecyl-<i>N</i>′-propylsulfamide, a Selective PPARα Agonist Structurally Related to <i>N</i>-acylethanolamines

<div><p>To further understand the pharmacological properties of N-oleoylethanolamine (OEA), a naturally occurring lipid that activates peroxisome proliferator-activated receptor alpha (PPARα), we designed sulfamoyl analogs based on its structure. Among the compounds tested, N-octadecyl-N′-propylsulfamide (CC7) was selected for functional comparison with OEA. The performed studies include the following computational and biological approaches: 1) molecular docking analyses; 2) molecular biology studies with PPARα; 3) pharmacological studies on feeding behavior and visceral analgesia. For the docking studies, we compared OEA and CC7 data with crystallization data obtained with the reference PPARα agonist GW409544. OEA and CC7 interacted with the ligand-binding domain of PPARα in a similar manner to GW409544. Both compounds produced similar transcriptional activation by <i>in vitro</i> assays, including the GST pull-down assay and reporter gene analysis. In addition, CC7 and OEA induced the mRNA expression of CPT1a in HpeG2 cells through PPARα and the induction was avoided with PPARα-specific siRNA. <i>In vivo</i> studies in rats showed that OEA and CC7 had anorectic and antiobesity activity and induced both lipopenia and decreases in hepatic fat content. However, different effects were observed when measuring visceral pain; OEA produced visceral analgesia whereas CC7 showed no effects. These results suggest that OEA activity on the PPARα receptor (e.g., lipid metabolism and feeding behavior) may be dissociated from other actions at alternative targets (e.g., pain) because other non cannabimimetic ligands that interact with PPARα, such as CC7, do not reproduce the full spectrum of the pharmacological activity of OEA. These results provide new opportunities for the development of specific PPARα-activating drugs focused on sulfamide derivatives with a long alkyl chain for the treatment of metabolic dysfunction.</p></div

Docking of compounds in the ligand binding domain of PPARα.

<p>Docking energy (kcal/mol) and the percentage of the occurrence of hydrogen bonds (HB) to the hydroxyl group of Tyr 464 residue.</p

mRNA expression of CPT1a in HepG2 cells.

<p>(<b>A</b>) The CPT1a mRNA expression was determined by qRT-PCR after the incubation of cells with OEA (0.1, 1, 5 and 10 μM) for 48 h. (<b>B</b>) HepG2 cells were serum deprived for 16 h and transfected with PPARα-specific siRNA or control siRNA followed by incubation with 10 μM of OEA and CC7 for 48 h. Then, the CPT1a mRNA was determined by qRT-PCR. Expression was normalized to GAPDH expression. Experiments were repeated 3 times and results are presented as x-fold induction over vehicle treated cells. Columns represent the mean ± SEM, and these data were analyzed by Student’s t test. *P<0.05, **P<0.01 and ***P<0.01 vs vehicle or control siRNA.</p

Basal and ligand-induced activities of PPARα were determined using luciferase reporter gene assays.

<p>(<b>A</b>) MCF-7 cells were transiently transfected with a reporter construct containing the human CPT1 DR1-type PPRE (CPT1-PPRE) and the indicated expression vectors for PPARα, RXRα, SRC1 (co-activator) and NCoR (co-repressor). The columns represent the mean ± SEM of at least three experiments, and the data were analyzed by Student’s t test. **P<0.01 and ***P<0.001 compared to vehicle alone. (<b>B</b>) MCF-7 cells were treated for 16 h with 10 μM of OEA, CC7, CC12 or GW7647 in either presence or absence and of 100 nM of the HDAC inhibitor trichlorostatin A (TSA). Stimulation of luciferase activity was normalized to the basal activity of PPARα-RXRα in absence of ligands. The columns represent the mean ± SEM of at least three experiments, and these data were analyzed by Student’s t test. *P<0.05, **P<0.01 and ***P<0.001 compared to the absence of TSA and the presence of vehicle.</p

Computational docking representation of the binding modes of the optimal conformation/orientation in PPARα-LBD for GW7647, OEA, CC7 and CC12.

<p>(<b>A</b>) GW7647 pose #62, (<b>B</b>) OEA pose #45, (<b>C</b>) CC7 pose #36 and (<b>D</b>) CC12 pose #58 in ball and stick format colored by atom type. The co-crystallized conformation of GW405544 is shown in green, and the protein backbone is represented by ribbons (blue). The critical polar interactions (green line) and residues involved (Ser-280, Tyr-314, His-440 and Tyr-464) are shown. All agonist compounds formed a hydrogen bond with Tyr-464. The most ideal docking pose (#58) of CC12 only interacts with hydroxyl group of Ser-280.</p

Effect of IL-6 treatment on the expression of <i>Ppar-alpha</i> and its target gene <i>Cpt1</i> in the livers of WT mice fed a HFD.

<p>The qPCR analysis for the gene expression of <i>Ppar-alpha</i> and <i>Cpt1</i> <b>(A)</b> in the livers of mice fed STD, HFD or HFD+rIL-6 is depicted in the histogram. The expression of each gene was normalized using Biogazelle’s qBase^PLUS software with <i>Gapdh</i> and <i>Gus-beta</i> as reference genes. The columns represent CNRQ means ± SEM (n = 8 animals per group). (<b>B</b>) Western blot analysis of Cpt1 protein expression in liver of mice fed a STD, HFD or HFD+rIL-6 is shown in the upper panel. Representative blots of each protein from one sample out of eight per group are presented. The corresponding expression of actin is shown as a loading control per lane. The histogram below the blot depicts the levels of Cpt1 determined through densitometry corrected for actin. Comparisons between diet conditions were analyzed using one-way ANOVA. *<i>P</i><0.05, **<i>P</i><0.01 denote significant differences compared with the corresponding STD-fed group. ^#<i>P</i><0.05 denotes significant differences in expression between HFD-fed and HFD+rIL-6 groups.</p

Ligand-dependent interaction profiles between the PPARα and co-activators and between PPARα-RXRα heterodimers and co-activators on DNA.

<p>(<b>A</b>) GST pull-down assays performed with GST-SRC1_597–791 and [³⁵S]-labeled wild-type PPARα in the presence of vehicle (DMSO), OEA (1 μM), CC7 (1 μM), CC12 (1 μM) and GW7647 (1 μM). The percentage of precipitated PPARα proteins with respect to input was quantified. The columns represent the mean ± SEM of at least three experiments, and the data were analyzed by Student’s t test. **P<0.01 and ***P<0.001 compared to SRC1 interaction in presence of vehicle alone. (<b>B</b>) Ligand-dependent interaction profiles of PPARα-RXRα heterodimers with co-activators on DNA. Combined gel shift and super-shift experiments were performed with wild type PPARα-RXRα protein and [³²P]-labeled human CPT1-PPRE. PPARα-RXRα heterodimers were pre-incubated with solvent and different concentrations (1, 5 and 10 μM) of OEA, CC7 and CC12 followed by the addition of 2 μg of either GST alone (as a control) or GST-SRC1_597–791. Protein-DNA complexes were resolved from the free probe with 8% non-denaturing polyacrylamide gels. Representative experiments are shown.</p

Effect of IL-6 treatment on the expression of lipogenic enzymes in the livers of WT mice fed a HFD.

<p><b>(A)</b> The gene expression of <i>Acaca</i>, <i>Acacb</i>, <i>Fasn</i>, and <i>Scd1</i> in the livers of WT fed STD, HFD or HFD-treated chronically with rIL-6 (HFD+rIL-6) is depicted in the corresponding histogram. The gene expression was determined through qPCR analysis of the liver samples. The expression of each gene was normalized using Biogazelle’s qBase^PLUS software with <i>Gapdh</i> and <i>Gus-beta</i> as reference genes. The columns represent CNRQ means ± SEM (n = 8 animals per group). (<b>B</b>) Western blot analysis of the protein expression of Acc-alpha/beta, Fas, and Scd1 in liver samples from WT and mice fed a STD, HFD or HFD+rIL-6. Representative blots of each protein from two samples out of five per group are presented in the upper panel. The corresponding expression of adaptin is shown as a loading control per lane. The histograms below blots depict the levels of each Acc-alpha/beta, Fas and Scd1 protein determined through densitometry and corrected for adaptin. The values represent the means ± SEM (n = 8 samples per group). Comparisons between diet conditions for each gene/protein in A and B were analyzed using one-way ANOVA. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 denote significant differences compared with the corresponding STD-fed group.</p

Effect of chronic administration of IL-6 on the hepatic expression of <i>IL-6</i> and <i>Tnf-alpha</i> in the livers of WT mice fed a HFD.

<p>Endogenous expression of the genes <i>IL-6</i> and <i>Tnf-alpha</i> in liver samples from mice fed STD, HFD or HFD+rIL-6, determined through qPCR analysis. Normalization was performed by Biogazelle’s qBase^PLUS software with <i>Gapdh</i> and <i>Gus-beta</i> as reference genes. The columns represent CNRQ means ± SEM (n = 8 animals per group) and the significance of differences between groups was analyzed by one-way ANOVA. *<i>P</i><0.05 denotes significant differences compared with the corresponding STD-fed group.</p

Effect of AM251 on body weight in rats fed standard or obesogenic diets.

<p>Net body weight gain was evaluated in rats fed for 10 weeks on a STD, HCHD or HFD. Columns are means ± SEM (n = 16 animals per group). Data were analyzed by one-way ANOVA and Bonferroni’s <i>post-hoc</i> test. ***<i>p</i><0.001 denotes significant differences compared with the STD-fed group (<b>A</b>). Relative body weight loss was evaluated in STD, HCHD or HFD-fed animals after 14-day exposure to vehicle or AM251 (3 mg/kg, daily, i.p.). Columns are means ± SEM (n = 8 animals per group). Each pair of treatment groups was analyzed by Student’s <i>t</i> test. ***<i>p</i><0.001 denotes significant differences compared with the corresponding vehicle-treated group (<b>B</b>).</p