Ursolic acid-rich extract presents trypanocidal action in vitro but worsens mice under experimental acute Chagas disease.

Chagas disease is a neglected tropical disease with only two drugs available for treatment and the plant Cecropia pachystachya has several compounds with antimicrobial and anti-inflammatory activities. This study aimed to evaluate a supercritical extract from C. pachystachya leaves in vitro and in vivo against Trypanosoma cruzi. A supercritical CO2 extraction was used to obtain the extract (CPE). Cytotoxicity and immunostimulation ability were evaluated in macrophages, and the in vitro trypanocidal activity was evaluated against epimastigotes and trypomastigotes forms. In vivo tests were done by infecting BALB/c mice with blood trypomastigotes forms and treating animals orally with CPE for 10 days. The parasitemia, survival rate, weight, cytokines and nitric oxide dosage were evaluated. CPE demonstrated an effect on the epi and trypomastigotes forms of the parasite (IC50 17.90 ± 1.2 µg/mL; LC50 26.73 ± 1.2 µg/mL) and no changes in macrophages viability, resulting in a selectivity index similar to the reference drug. CPE-treated animals had a worsening compared to non-treated, demonstrated by higher parasitemia and lower survival rate. This result was attributed to the anti-inflammatory effect of CPE, demonstrated by the higher IL-10 and IL-4 values observed in the treated mice compared to the control ones. CPE demonstrated a trypanocidal effect in vitro and a worsening in the in vivo infection due to its anti-inflammatory activity.

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic ß-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity.

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic ß-cells and is important for insulin exocytosis. To better understand IP7 regulation in ß-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic ß-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated.