In vitro evaluation of 4-phenyl-5-(4'-X-phenyl)-1,3,4-thiadiazolium-2-phenylaminide chlorides and 3[N-4'-X-phenyl]-1,2,3-oxadiazolium-5-olate derivatives on nitric oxide synthase and arginase activities of Leishmania amazonensis.

Leishmaniasis is a spectrum of infectious diseases caused by Leishmania protozoan parasites. The purpose of this study was to perform, in vitro, a comparative analysis of the activity amastigotes. Results showed excellent efficacy of all compounds against axenic amastigotes, compared to pentamidine isethionate, the reference drug used. The cytotoxic effect of these mesoionic compounds of six mesoionic compounds (three 1,3,4-thiadiazolium-2-aminide and three 1,2,3-oxadiazolium-5-olate class compounds) was evaluated in mouse peritoneal macrophages using MTT assay, low toxicity (≈ 10%) for these mammalian cells being observed. In an attempt to define a potential drug target, the activities of nitric oxide synthase (NOS) and arginase of the parasites treated with the mesoionic derivatives were evaluated. NOS was purified from a cell-free extract of infective promastigotes and axenic amastigotes and all derivatives tested were able to inhibit the enzyme as monitored by the decrease of NADPH consumption. Arginase activity from both stages of the parasite was measured using urea production and none of the compounds inhibited the enzyme activity of axenic amastigotes. However, the compounds without substituents (MI-H and SID-H) were able to inhibit arginase activity of these parasites.

In vitro sodium nitroprusside-mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes.

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.