Computational and biological evaluation of N-octadecyl-N'-propylsulfamide, a selective PPARα agonist structurally related to N-acylethanolamines

Abstract

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 in vitro 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. In vivo 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.

Figure 1. OEA induces ligand-dependent PPARα complex formation on DNA.

The electrophoretic mobility shift assay (EMSA) was performed with hepatic nuclear extracts of rats treated with OEA and/or vehicle and [³²P]-labeled rat acyl-CoA oxidase (ACOX)-response element (PPRE). For super-shift experiments, 1 μL of anti-PPARα antibody was added. Protein-DNA complexes (RXRα-PPARα and RXRα-PPARα-Ab) were separated from free probe on 8% non-denaturing polyacrylamide gels. Representative gels are shown. The first lane of each gel represents the probe alone as a negative control; all of the other lanes contained nuclear extract. The percentage of protein complexed with DNA was quantified after 6 determinations. The data are represented as the mean ± SEM and were analyzed by Student’s t test. **P<0.01 and ***P<0.001 compared to the vehicle group.

Figure 2. Computational docking representation of the binding modes of the optimal conformation/orientation in PPARα-LBD for GW7647, OEA, CC7 and CC12.

(A) GW7647 pose #62, (B) OEA pose #45, (C) CC7 pose #36 and (D) 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.

Figure 3. Ligand-dependent interaction profiles between the PPARα and co-activators and between PPARα-RXRα heterodimers and co-activators on DNA.

(A) 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) 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.

Figure 4. Basal and ligand-induced activities of PPARα were determined using luciferase reporter gene assays.

(A) 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) 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.

Figure 5. mRNA expression of CPT1a in HepG2 cells.

(A) 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) 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.

Figure 6. Effects of acute and subchronic treatment with OEA and CC7 (5 mg/kg, i.p.) on lipid metabolism and feeding behavior in Wistar rats.

(A) Effects on food intake in rats deprived of food for 24 h after acute treatment with OEA and CC7 (N = 7). The data are represented as the mean ± SEM and were analyzed by two-way ANOVA and post hoc Bonferroni’s tests. #P<0.05, *P<0.05 and **P<0.01 compared to vehicle-treated rats. (B) Body weight gain after a 7-day treatment with OEA and CC7 in free-feeding rats (N = 8). The data are represented as the mean ± SEM and were analyzed by two-way ANOVA and post hoc Bonferroni’s tests. *P<0.05, **P<0.01, ***P<0.001 compared to vehicle-treated rats. (C) Cholesterol and triglyceride content in the plasma after a 7-day treatment with OEA and CC7 in free-feeding rats (N = 8). The bars represent the mean ± SEM. Data were analyzed by Student’s t test. **P<0.01 compared to vehicle-treated rats. (D) Total fat content in the liver after a 7-day treatment with OEA and CC7 in free-feeding rats (N = 8). The bars represent the mean ± SEM. Data were analyzed by Student’s t test. *P<0.05 compared to vehicle-treated rats.

Figure 7. Effects of OEA and CC7 (5 mg/kg, i.p.) on abdominal constrictions (writhing) caused by i.p. injection of 0.6% acetic acid in CD1 mice.

Drugs were administered 15± SEM. Data were analyzed by Student’s t test. **P<0.01 compared to vehicle- and acetic acid-treated mice.