THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy

<div><p>Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing V_max and K_m values of 384.62 µmol min⁻¹, 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.</p></div

<i>In vivo</i> 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.

<p>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).</p

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).

<p>This effect, analyzed by Dixon plot, showed competitive inhibition kinetics, with a K_i 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).</p

THDP-17 was administered orally after 12 h fasted mice.

<p>Animals were divided in 4 groups and deaths after treatment were recorded.</p><p>THDP-17 was administered orally after 12 h fasted mice.</p

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.

<p>*Animal without water.</p><p>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.</p