Alvaxanthone, a Thymidylate Synthase Inhibitor with Nematocidal and Tumoricidal Activities

Abstract

With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.

Keywords: cytotoxicity; inhibitor; nematocidal activity; thymidylate synthase; xanthones.

Figure 1

Viability of BJ, U-118 MG and SCC-15 cells after 48 h treatment with alvaxanthone or rheediaxanthone B, estimated by NR (A) and XTT (C) assays. Each bar represents the median of a population of 9 results, obtained in consequence of three independent experiments, each done in triplicate. The whiskers are the lower (25%) and upper (75%) quartile ranges. The results significantly different from control (in a view of the Kruskal–Wallis test) are marked * (p ≤ 0.05). (B) Cell morphology and neutral red accumulation following 48 h alvaxanthone or rheediaxanthone B treatment and 1 h incubation with neutral red. Red vesicles are lysosomes containing the dye.

Figure 2

Proliferation of cancer (U-118 MG and SCC-15) and normal (BJ) cells after 72 h incubation with 5–40 μM alvaxanthone or 1.25–40 μM rheediaxanthone B, determined with the CyQUANT GR Cell Proliferation Assay Kit. Median results are presented of three independent experiments, each performed in triplicate. The whiskers are lower (25%) and upper (75%) quartile ranges. The results significantly different from control (in view of the Kruskal–Wallis test) are indicated * (p ≤ 0.05). Images collected with Olympus IX-83 microscope with contrast phase (scale bar = 100 µm).

Figure 3

Influence of alvaxanthone or rheediaxanthone B on BJ, U-118 MG, and SCC-15 cells adhesion after 48-h treatment. The crystal violet assay was used. Results are medians of triplicate assays of three independent experiments expressed as a % of non-treated controls. The whiskers are lower (25%) and upper (75%) quartile ranges. * p ≤ 0.05, Kruskal–Wallis test (against non-treated control).

Figure 4

Changes of caspase-3/7 activities and intracellular ATP level in BJ, U-118 MG, and SCC-15 cells after 48-h incubation with 5–40 µM alvaxanthone or rheediaxanthone B. Median results are presented of three independent experiments, each performed in triplicate. The whiskers are lower (25%) and upper (75%) quartile ranges. The results significantly different from control (in view of the Kruskal–Wallis test) are indicated as * (p ≤ 0.05).

Figure 5

Predicted binding mode of alvaxanthone and rheediaxanthone against TS. (A,B) docking pose of alvaxanthone (A) and rheediaxanthone (B) against the catalytic site of TS from C. elegans, PDB ID 4IQQ (green lines). (C,D) docking pose of alvaxanthone (C) and rheediaxanthone (D) against the catalytic site of TS from homo sapiens PDB ID 5X5D (yellow lines). TS inhibitors are shown as cyan lines. dUMP is shown as sticks and colored as the protein. H-bonds are showed as black dashed lines. Only residues within 5 Å from the docked ligands are shown.