Possible primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi

LAURELLA LAURA; LOZANO, ESTEBAN; LOMBARDO ELISA; PUENTE VANESA, ; SPINA RENATA; SOSA MIGUEL; DEMARIA A; SULSEN VALERIA

CABA

Congreso; Drug Discovery for Neglected Diseases International Congress 2018; 2018

Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model [1]. Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets.The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin [2]; the thiol interaction was determined spectrophotometrically through its reaction with 5,5?-dithiobis-2-nitrobenzoate in the presence of glutathione [3]; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry [2]. The superoxide dismutase activity was spectrophotometrically tested by measuring the inhibition of superoxide-driven NADH oxidation [4]. Trypanothione reductase activity was determined following NADPH oxidation [4]; cell viability and mitochondrial membrane potential were assessed by means of propidium iodide plus rhodamine 123 staining; sterols were qualitatively and quantitatively determined by TLC [2]; ultrastructural changes were analyzed by transmission electron microscopy. Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected, as indicated by propidium iodide labeling. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Structural changes observed in the parasites under transmission electron microscopy showed that deoxymikanolide could induce a delay in the cell cycle of the parasites, an intense vacuolization, and the appearance of autophagosome-like structures triggered by mitochondrial dysfunction. Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.