No abstract

Mast cells play a central role in the early clearance of the intestinal parasite Giardia lamblia. In a previous study, we reported that G. lamblia live trophozoites or trophozoite-derived total soluble extract induced direct activation (IgE-independent) of mast cells and release of IL-6 and TNF-α. To identify the Giardia molecules and the mast cell receptors involved in this activation, trophozoite-derived total soluble proteins separated into three fractions (F1-F3) were evaluated for its ability to activate mast cells in vitro. F2 activated mast cells in a greater extent than F1 and F3. Furthermore, F2 induced the release of IL-6 and TNF-α by mast cells. TLR2 and TLR4 expression increased slightly after mast cell stimulation with either F2 or total soluble extract; however, these receptors were not involved in F2 or total soluble extract-induced proinflammatory cytokine production. Proteins present in F2 as unique and high-intensity bands identified by liquid chromatography coupled with tandem mass spectrometry, include molecules with important biological activities such as enolase and arginine deiminase (ADI). Recombinant ADI and enolase were tested for their ability to activate mast cells, but only ADI induced a significant release of IL-6 and TNF-α. ADI product, citrulline but not ammonium, also induced mast cell release of TNF-α. Interestingly, recombinant ADI still stimulated the secretion of TNF-α by mast cells in a arginine-free medium, although in a lower extend that in the presence of arginine, indicating that either ADI itself can stimulate mast cells or through its metabolic product, citrulline.

Leishmanicidal drugs have many side effects, and drug resistance to all of them has been documented. Therefore, the development of new drugs and the identification of novel therapeutic targets are urgently needed. Leishmania mexicana trypanothione reductase (LmTR), a NADPH-dependent flavoprotein oxidoreductase important to thiol metabolism, is essential for parasite viability. Its absence in the mammalian host makes this enzyme an attractive target for the development of new anti-Leishmania drugs. Herein, a tridimensional model of LmTR was constructed and the molecular docking of 20 molecules from a ZINC database was performed. Five compounds (ZINC04684558, ZINC09642432, ZINC12151998, ZINC14970552, and ZINC11841871) were selected (docking scores −10.27 kcal/mol to −5.29 kcal/mol and structurally different) and evaluated against recombinant LmTR (rLmTR) and L. mexicana promastigote. Additionally, molecular dynamics simulation of LmTR-selected compound complexes was achieved. The five selected compounds inhibited rLmTR activity in the range of 32.9% to 40.1%. The binding of selected compounds to LmTR involving different hydrogen bonds with distinct residues of the molecule monomers A and B is described. Compound ZINC12151998 (docking score −10.27 kcal/mol) inhibited 32.9% the enzyme activity (100 µM) and showed the highest leishmanicidal activity (IC50 = 58 µM) of all the selected compounds. It was more active than glucantime, and although its half-maximal cytotoxicity concentration (CC50 = 53 µM) was higher than that of the other four compounds, it was less cytotoxic than amphotericin B. Therefore, compound ZINC12151998 provides a promising starting point for a hit-to-lead process in our search for new anti-Leishmania drugs that are more potent and less cytotoxic.