Fatty Acid Synthase as Interacting Anticancer Target of the Terpenoid Myrianthic Acid Disclosed by MS-Based Proteomics Approaches

Abstract

This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of Myrianthus arboreus and from Oenothera maritima Nutt. (Onagraceae), using MS-based chemical proteomic techniques. Application of drug affinity responsive target stability (DARTS) and targeted-limited proteolysis coupled to mass spectrometry (t-LiP-MS) led to the identification of the enzyme fatty acid synthase (FAS) as an interesting macromolecular counterpart of myrianthic acid. This result, confirmed by comparison with the natural ursolic acid, was thoroughly investigated and validated in silico by molecular docking, which gave a precise picture of the interactions in the MA/FAS complex. Moreover, biological assays showcased the inhibitory activity of myrianthic acid against the FAS enzyme, most likely related to its antiproliferative activity towards tumor cells. Given the significance of FAS in specific pathologies, especially cancer, the myrianthic acid structural moieties could serve as a promising reference point to start the potential development of innovative approaches in therapy.

Keywords: DARTS; drug discovery; fatty acid synthase; functional proteomics; molecular docking; preclinical investigations; t-LiP.

Figure 1

Chemical structure of Myrianthic acid (MA).

Figure 2

(A) Western blotting analysis carried out on the DARTS samples reveals FAS protection upon MA interaction. GAPDH was used as a loading normalizer. Western blotting densitometric analysis for FAS (B) was performed through ImageJ. Undigested proteins (i.e., Lysate sample) were rated as 100%.

Figure 3

Predicted binding mode according to the first hypothesis of ursolic acid (panel A, colored by atom type: C green, O red, polar H white) and MA (panel B, colored by atom type: C light blue, O red, polar H white) in the MAT domain binding site (PDB: 5MY0, key residues are reported as sticks and colored by atom type: C grey, O red, N blue, S yellow, polar H light grey). The hydroxyl groups interacting with the catalytic triad are highlighted in blue for ursolic acid (A) and green for MA (B). H-bonds are represented by cyan dotted lines.

Figure 4

Predicted binding mode according to the second hypothesis of ursolic acid (panel A, colored by atom type: C cyan, O red, polar H white) and MA (panel B, colored by atom type: C green, O red, polar H white) in the MAT domain binding site (PDB: 5MY0, key residues are reported as sticks and colored by atom type: C grey, O red, N blue, S yellow, polar H light grey). The carboxylic groups interacting with the catalytic triad are highlighted in blue for ursolic acid (A) and green for MA (B). H-bonds are represented by cyan dotted lines.

Figure 5

Cell survival index, evaluated by the MTT assay and live/dead cell ratio, for MDA-MB-231 and MCF-7 BC cell lines following 48 and 72 h of incubation with the indicated concentrations (0–50 µM) of myrianthic acid (MA), ursolic acid (UA), and Orlistat, as indicated in the legend. Data are expressed as a percentage of untreated control cells and are reported as the mean of five independent experiments ± SEM (n = 30). The cell survival index was calculated as described in the experimental section and plotted in line graphs against the different concentrations of the tested molecules. * p ˂ 0.05 vs. control cells; ** p < 0.01 vs. control cells; *** p < 0.001 vs. control cells.

Figure 6

Cell survival index, evaluated by the MTT assay and live/dead cell ratio, for MCF-10A and HDFa following 48 and 72 h of incubation with the indicated concentrations (0–50 µM) of myrianthic acid (MA), ursolic acid (UA), and Orlistat, as indicated in the legend. Data are expressed as a percentage of untreated control cells and are reported as the mean of five independent experiments ± SEM (n = 30). The cell survival index was calculated as described in the experimental section and plotted in line-graphs against the different concentrations of the tested molecules. * p ˂ 0.05 vs. control cells.

Figure 7

Bar graphs encompassing representative data from the FAS activity assay in tumor cell lysates obtained from the indicated cell lines by monitoring the oxidation of NADPH, as described in the experimental section. In the absence of molecules under consideration, 100% was attributed to FAS activity. Results are expressed as percentage (%) of total FAS activity in presence of 10 µM of MA, UA, and Orlistat, respectively, and are reported as mean of three independent experiments ± SEM (n = 15). *** p < 0.001 vs. untreated controls.

Figure 8

Bar graphs relative to the FAS activity assay in the indicated BC cells protein extracts by monitoring the oxidation of NADPH, as described in the experimental section. Cells were treated in vitro for 48 h with 10 µM of MA, UA, and Orlistat, as indicated in the legend, and then appropriately lysed to obtain cellular extracts. FAS activity in lysates from untreated control cells had 100% attributed to it. Results are expressed as percentage of FAS activity and are reported as mean of three independent experiments ± SEM (n = 15). * p ˂ 0.05 vs. control cells; ** p < 0.01 vs. control cells; *** p < 0.001 vs. control cells.

Figure 9

Western blot analysis showing the effects of 10 µM concentrations of myrianthic acid (MA), ursolic acid (UA), and Orlistat following 48 h of incubations in MDA-MB-231 cells on the expression of FAS. The shown blots are representative of three independent experiments and are cropped from different parts of the same gel (provided in Supplementary Materials), as explicit by using clear delineation with dividing lines and white space.