Initial studies on mechanism of action and cell death of activeN-oxide-containing heterocycles inTrypanosoma cruziepimastigotesin vitro

Abstract

SUMMARYChagas disease, endemic in 21 countries across Latin America, kills more people in the region each year than any other parasite-borne disease. Therapeutic options have problems ranging from toxicity, poor efficacy, drug resistance and high cost. Thus, cheaper and less toxic treatments are necessary. From our in-house chemical library of agents againstTrypanosoma cruzithe most relevantN-oxide-containing heterocycles were selected for mode of action and type of death studies. Also included in these studies were two active nitrofuranes. Epimastigotes ofT. cruziwere used as the biological model in this study. The metabolic profile was studied by1H NMR in association with the MTT assay. Excreted catabolites data, using1H NMR spectroscopy, showed that most of the studiedN-oxides were capable of decreasing both the release of succinate and acetate shedding, the compounds therefore possibly acting on mitochondria. Only quinoxalines and the nitrofuraneNf1showed significant mitochondrial dehydrogenase inhibitions, but with different dose–time profiles. In the particular case of quinoxalineQx2the glucose uptake study revealed that the integrity of some pathways into the glycosome could be affected. Optic, fluorescence (TUNEL and propidium iodide) and transmission electron microscopy (TEM) were employed for type of death studies. These studies were complemented with1H NMR to visualize mobile lipids. At low concentrations none of the selected compounds showed a positive TUNEL assay. However, both quinoxalines, one furoxan and one benzofuroxan showed a necrotic effect at high concentrations. Curiously, one furoxan,Fx1, one benzofuroxan,Bfx1, and one nitrofurane,Nf1, caused a particular phenotype, with a big cytoplasmatic vacuole being observed while the parasite was still alive. Studies of TEM and employing a protease inhibitor (3-methyladenine) suggested an autophagic phenotype forBfx1andNf1and a ‘BigEye’ phenotype forFx1.