Targeting phosphoglycerate kinases by tatridin A, a natural sesquiterpenoid endowed with anti-cancer activity, using a proteomic platform

Abstract

Tatridin A (TatA) is a germacrane sesquiterpenoid containing one E-double bond and one Z-double bond in its 10-membered ring, which is fused to a 3-methylene-dihydrofuran-2-one moiety. Tatridin A bioactivity has been poorly investigated despite its interesting chemical structure. Here, a functional proteomic platform was adapted to disclose its most reliable targets in leukemia monocytic cells, and phosphoglycerate kinases were recognized as the most affine enzymes. Through a combination of limited proteolysis and molecular docking, it has been discovered that tatridin A interacts with the active domains of phosphoglycerate kinase 1, altering its hinge region, and it can be accountable for tatridin A inhibition potency on enzyme activity. A more detailed tatridin A biological profile showed that it is also fully active against gastric cancer cells, downregulating the mRNA levels of chemokine receptor 4 and β-catenin and inhibiting the invasiveness of living KATO III cells as a direct consequence of phosphoglycerate kinase 1 antagonism.

Keywords: CXCR4; PGK1; cancer dissemination; functional proteomics; gastric cancer; sesquiterpenes.

FIGURE 1

(A) Chemical structure of Tatridin A (TatA). (B) Coomassie-stained gel showing the protection of proteins to protease upon TatA interaction (red arrows indicate the cutting sites). (C) Proteome Discoverer-retrieved protection ratio of the four proteins shared among all DARTS experiments at three TatA concentrations. (D) Immunoblotting analysis of one DARTS experiment revealing PGK1 and PGK2 protected in a TatA concentration-dependent fashion, together with the densitometric analysis. GAPDH is resistant to subtilisin under these experimental conditions and is used as the loading control.

FIGURE 2

(A) Selected TatA peptide reported with its parent and daughter m/z value used in the MRM approach, its length, and the calculated fold change (FC and the associated p-value) as the ratio of the peptide area due to TatA protection. (B) Best predicted docking pose of TatA on PGK1. TatA is reported in light blue sticks, the PGK1 site for 3PG and ATP is reported in red (in particular, amino acids K215, G372, G373, D374, T375, G395, and G396), the hinge helix is reported in blue, and the amino acids involved in the interaction are reported in the 3D representation. (C) TatA is bound to PGK2.

FIGURE 3

TatA is able to inhibit PGK1 activity on (A) the recombinant enzyme, (B) THP-1 cell lysate, and (C) KATO III cell lysate. The graphs were prepared using Prism software.

FIGURE 4

Quantitative real-time PCR (qRT-PCR) analysis of PGK1 (A), CXCR4 (B), and β-catenin (C) in KATO III cells after 24 h treatment with 100 μM TatA. HPRT1 was used as the housekeeping gene. Experiments were carried out in triplicate. Data are expressed as mean ± s.d. (t-test; **p-value ≤0.01, ***p-value ≤0.001).

FIGURE 5

(A) Representative microscopic images of the bottom surface of Transwell filters stained with crystal violet (magnification ×10) showing KATO III cells treated with or without 100 μM TatA for 24 h. (B) Quantification of invasive cells. The data are shown as the mean number of cells per eight visual fields (magnification ×10) of three replicate wells ± s.d. (t-test; **p-value ≤0.01).