Estudo da atividade antileishmanial de um novo sal orgânico derivado de triazol em leishmania amazonensis

Abstract

Leishmaniasis is an infectious disease that is difficult to control and presents itself in three clinical forms: cutaneous, mucocutaneous and visceral leishmaniasis, caused by more than 20 species of parasites of the genus Leishmania spp., transmitted by the insect vector, the sand fly. Current chemotherapy for leishmaniasis faces significant limitations due to high toxicity, prolonged treatment regimens and increased parasite resistance, highlighting the urgent need for new treatment strategies. This study aimed to evaluate the in vitro activity of triazole derivatives against promastigotes and amastigotes of L. amazonensis, as well as its cytotoxicity in murine macrophages. In addition to investigate the mechanism of parasite killing. antipromastigote activity and cytotoxicity in peritoneal macrophages were determined by the colorimetric MTT method after 72 hours of treatment. The anti-amastigote effect was evaluated in macrophages infected with L. amazonensis after 72 hours of incubation, stained with Giemsa and counted by optical microscopy. The tests for analysis of the mechanism of action were performed using different biochemical and cellular indicators of cell death parameters, by fluorimetry and flow cytometry. Additionally, the ultrastructure and morphology of promastigotes treated with TZDS were analyzed by electron microscopy. The results found highlighted the importance of triazole salts, since the neutral form did not show inhibition of parasite growth. However, the triazole-derived salt demonstrated a promising selective index (SI = 34.28) and antileishmanial activity (IC50 = 0.13 μM and IC50 = 2.06 μM against promastigote and amastigote forms, respectively), proving to be more active than miltefosine, the standard drug. Regarding the TZDS mode of action, this compound induced significant mitochondrial alterations in the parasite, characterized by an increase in mitochondrial membrane potential (ΔΨm), elevated levels of total and mitochondrial Reactive Oxygen Species (ROS), and accumulation of lipid bodies in the cytoplasm. Treatment with triazole salt also produced several ultrastructural, biochemical, and cellular alterations in the promastigotes, consistent with apoptosislike cell death, including cell shrinkage and reduction in length, as well as exposure of phosphatidylserine in the outer leaflet of the plasma membrane and marked cell cycle arrest, in addition to DNA fragmentation. Despite the positive MDC (monodansylcadaverine) and the presence of membrane-bound vacuoles resembling autophagosomal structures observed by TEM analysis, autophagy is not a predominant process, with severe mitochondrial damage emerging as the primary event leading to parasite death. These findings demonstrate the promising antileishmanial potential of the triazole-derived salt, with its effect on multiple targets in L. amazonensis cells. Furthermore, the association of the active compound with miltefosine showed an additive effect in the treatment of macrophages infected by L. amazonensis. Overall, these results highlight the therapeutic potential of the evaluated salt and support further studies to evaluate its in vivo efficacy in a murine model of cutaneous leishmaniasis.