Methylembelin affects the invasion and proliferation of Trypanosoma cruzi.

BARRERA P; SPINA RM; FERESIN G; TAPIA A; LOZANO E; SOSA MA

Buenos Aires

Congreso; Drug Discovery for Neglected Diseases International Congress 2018. 4th Scientific Meeting of ResNet NPND; 2018

Instituto de la Química y Metabolismo del Fármaco.Consejo Nacional de Investigaciones Científicas y Técnicas Facultad de Farmacia y Bioquímica ? Universidad de Buenos Aires

Abstract. Trypanosoma cruzi is a monoflagellate parasite that causes Chagas´ disease, one of the major health issue in Latin America. At present the chemotherapy against T. cruzi is unsatisfactory as it provokes severe toxicity and resistant strains. Thus, investigation efforts have been directed to the identification of new drug leads. Recent reports, showed that the semisynthetic benzoquinone Methylemebelin (ME) reduces the number of amastigotes in infected cells, being the effect higher than the reference drug Benznidazole (BZN). In this work, the effect of ME related to the invasiveness of T. cruzi in the host cells, intracellular differentiation, and it release from cells was evaluated. Invasion assays: trypomastigotes Dm28c strain were treated with 16µM and 8µM ME for24 h, and then added to growing Vero cells (DMEM). After 24 h, cells were stained with Giemsa to estimate the percentage of infection. In other assay the benzoquinone (16µM) was added during the infection. Negative and positive controls were included (without ME and with BZN). To study the effects of ME on intracellular amastigote-to-trypomastigote differentiation, and its subsequent release to the extracellular medium, Vero cells (6 days postinfection) were treated with 16µM ME for 24 h, and then, the intracellular amastigotes were immunostained with anti ?Ssp-4 mAb 2C2 and observed with Olympus Confocal Microscope FV1000-EVA using Alexa 488- conjugated anti IgG. Nucleus and kinetoplast were stained with Hoechst. Samples were observed with Olympus Confocal Microscope FV1000-EVA (Olympus). Images were processed with Image J software: first cellular nucleus signal was eliminated by the use of algorithm and then fluorescence emitted by amastigotes (Alexa 488) was compare with that emitted by all stages of T. cruzi (Hoechst). This relationship was compared between controls (untreated infected cells) and treated cells. Results showed that ME reduce the parasite invasiveness to Vero cells at higher extent than BZN, although no apparent effect was observed on intracellular differentiation neither on the release of trypomastigotes to extracellular medium by treatment with the benzoquinone. Overall these results suggest that ME could act on invasion and/or amastigote replication. New efforts to elucidate the molecular target of this compound are needed.