Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease. The parasite needs to enter in the host cell to grow and expand infection. Trypomastigotes are classicaly infective forms of T. cruzi, and they are able to invade host cell. After the invasion of the mammalian cell, parasites start amastigogenesis process, in which trypomastigotes forms differentiate into amastigotes that proliferate.Those steps of intracelular invasion and amastigogenesis are essential to parasite. Currently, two drugs are used to treatment: benznidazol and nifurtimox. Both drugs show side adverses effects that causes treatment interruption by the patient, due to of the low efficacy in chronic phase of Chagas diseases, making important the development of new drugs. Basically, two aproaches are applied in drugs development: phenotype and targed-based. In this work, we used two strategies in the targed-based approach. The first was to study the parasite biology relationship with host cell. In this context, we search ROS relation, especifically H2O2, in the intracelular infection. Once ROS are produced in the phagolysosome and in this compartment has signaling to amastigogenesis, we investigated the H2O2 influence in this process.We observed that H2O2 increased 30% of differentiation after two hours of induction by low pH. However, among antioxidant molecules tested, only PEG-catalase, enzyme that neutralizes directly H2O2 inhibited amastigogeneses improvement ROS-induced. We observed that H2O2 increased a serine/threonine phosphatase activity, as well as PP2A inhibitor okadaic acid (OKA) reverted H2O2 effect upon amastigogenesis. Corroborating ROS involvement, observed that differentiaton induced by acid pH produced H2O2 after one hour, being reduced later. When we evaluated the superoxide dismutase gene expression of T. cruzi, we observed that mitochondrial isoform (Fe-SODA) were up regulated after 60 minutes and 90 minutes differentiation induction. Furthermore, we detected a reduction in the oxygen consumption and maximal eletron tranference system capacity, as well as loss in the mitochondrial membrane potential (ΔΨm) after 60 minutes and 120 minutes of amastigogenese induction. In consideration of these results, wherein mitochondrial dysfunction is followed by loss of ΔΨm, H2O2 production observed can be from mitochondrial by mitochondrial permeability transition pore (mPTP). Indeed, mPTP inhibitor cyclosporin and mitochondrial antioxidant mitoTEMPO reduced H2O2 generation induced by low pH. Together, this results showed that H2O2 act as signaling molecule to differentiation. The second strategy were trypomastigote surface screening by phage display library. After biopanning, were identified two high affinity clones, N3 and N1. The peptide express in N3 clone were synthetized and in vitro bound assays showed peptide specificity to infective forms of T. cruzi. Furthermore, peptide were active inhibiting Vero cells invasion. Thus, N3 peptide bound in a surface molecule of T. cruzi that is important to cell invasion.