All extracellular forms of Trypanosoma cruzi, the causative agent of Chagas disease, release extracellular vesicles (EVs) containing major surface molecules of the parasite. EV release depends on several mechanisms (internal and external). However, most of the environmental conditions affecting this phenomenon are still unknown. In this work, we evaluated EV release under different stress conditions and their ability to be internalized by the parasites. In addition, we investigated whether the release conditions would affect their immunomodulatory properties in preactivated bone marrow-derived macrophages (BMDM). Sodium azide and methyl-cyclo-β-dextrin (CDB) reduced EV release, indicating that this phenomenon relies on membrane organization. EV release was increased at low temperatures (4°C) and acidic conditions (pH 5.0). Under this pH, trypomastigotes differentiated into amastigotes. EVs are rapidly liberated and reabsorbed by the trypomastigotes in a concentration-dependent manner. Nitrosative stress caused by sodium nitrite in acid medium or S-nitrosoglutathione also stimulated the secretion of EVs. EVs released under all stress conditions also maintained their proinflammatory activity and increased the expression of iNOS, Arg 1, IL-12, and IL-23 genes in IFN-γ and LPS preactivated BMDM. In conclusion, our results suggest a budding mechanism of release, dependent on the membrane structure and parasite integrity. Stress conditions did not affect functional properties of EVs during interaction with host cells. EV release variations under stress conditions may be a physiological response against environmental changes.
This work reports a repurposing study of pyrazinoic acid (1) and methyl (2), ethyl (3) and 2-chloroethyl (4) ester derivatives with antimycobacterial activity, in assays against Trypanosoma cruzi. The compounds and benznidazole, the standard antitrypanosoma drug, were evaluated in concentrations ranging from 100 to 6.25 μg/mL. The results showed that compounds 2 and 3 (EC 50 = 182 and 447 μM) significantly reduced the infection rate of the parasite into the mammalian cells at 100 μg/mL (p < 0.05) in a similar way to benznidazole. In addition, all the compounds also significantly reduced the number of intracellular parasites (compound 1 at 50 μg/mL, and compounds 2-4 at 100 μg/mL, p < 0.05) in comparison to the control. Compounds 1 and 2 were more effective than benznidazole at 50 μg/mL (p < 0.001). Moreover, compounds 1-4 did not show significant cytotoxicity against THP-1, J774, and HeLa cells (>1000 μM), indicating that they possess considerable selectivity against the parasites. This report represents the first study of such compounds against T. cruzi, indicating the potential of pyrazinoates as antiparasitic agents. K E Y W O R D Santichagasic, antitrypanosoma agents, drug repositioning, pyrazinoates
Aims: We performed an initial in vitro study with a single molecule to evaluate the possibility to develop a larger series of hybrid compounds active in Chaga's disease. Hybridization is an important approach to confer to a single molecule the biological activity of two distinct molecules. We proposed thiosemicarbazone (TS9) hybridization with β-citronellol through carbamate linkage. Methodology: The cytotoxicity of the hybrid compound was evaluated against human THP-1 cells and all forms of Trypanosoma cruzi (T. cruzi). IC 50 value was determined against amastigotes and the selectivity index (SI) was estimated based on toxicity against THP-1 cells. Lipinski analysis was performed in order to estimate the hybrid drug-like properties. Results: The hybrid presented substantially less cytotoxicity against THP-1 cells than TS9 and biological similarities to both matrix moieties. The hybrid SI (3.9) was better than for TS9 (0.6) and
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