Table 1.
THDP-17 was administered orally after 12 h fasted mice.
Figure 1.
Inhibitory effect on glutaminase activity of several compounds tested in this study.
Data are shown in log scale (inhibitor concentration), being 100% activity measured without inhibitor. There was not significant differences among compounds tested (Student t test; p>0.05). THDP-17 was chosen on toxic effect base.
Figure 2.
Molecular structure of N-(3-methyl 3-butenyl)-N′ phenyl thiourea (THDP17).
Figure 3.
THDP17 inhibition kinetics. A lineal double reciprocal plot (1/v against 1/[Gln]) in absence (0) and presence (10 µM) of the inhibitor is shown.
Vmax and KM values for PAG were 0.67 mmol min−1, and 19.33 mM, respectively. In the presence of THDP-17 both parameters were modified: Vmax-ap = 0.38 mmol min−1, and KM-ap = 13.62 mM, indicating an uncompetitive inhibition. All experiments were performed in triplicates; p>0.05.
Figure 4.
In vivo inhibitory effect on glutaminase activity at different concentrations of THDP17 and DON (6-Diazo-5-oxo-L-norleucine) was tested in Caco-2 cell cultures.
PAG inhibition by THDP-17 in cell cultures is effective at concentration higher than 5 µM, showing an inhibition of 18±2.1% and 46±3.4% at 20 and 100 µM, respectively (* p<0.05).
Table 2.
THDP-17 and/or DMSO were then administered orally to animals after free access to food and water overnight, and mice were sacrificed at 24 h and 72 h randomly.
Figure 5.
Metformin inhibits glutaminase activity in a dose dependent manner: from 17.5% at 10 mM up to 68% at 100 mM (A) (* p<0.05).
This effect, analyzed by Dixon plot, showed competitive inhibition kinetics, with a Ki of 14.28 mM (B). In Caco2 cells, metformin 20 mM showed 24% inhibition of glutaminase activity at 72 hours compared to control cultures (p<0.05) (glutamate production was decreased from 26.85±0.74 µM to 19.9±2.05 µM; p<0.05) (C).