Multiple mechanisms of action of a triazole-derived salt against Leishmania amazonensis: Apoptosis-like death and autophagy

Abstract

Current chemotherapy for leishmaniasis faces significant limitations due to high toxicity, prolonged treatment regimens, and increasing parasite resistance, highlighting the urgent need for innovative treatment strategies. This study aimed to evaluate the in vitro activity of 1,2,3-triazole derivatives against promastigotes and amastigotes of Leishmania amazonensis, as well as their cytotoxicity in murine macrophages. Additionally, we investigated the mechanism of parasite death through different biochemical and cellular indicators of cell death parameters. Our results underscored the importance of the salt form, as the neutral form showed no inhibition of parasite growth. In contrast, the triazole-derived salt demonstrated 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 more active than miltefosine, the standard drug. Regarding the mode of action of the triazole-derived salt, 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 lipid body accumulation in the cytoplasm. Treatment with triazole-derived salt also produced several ultrastructural, biochemical, and cellular changes in the promastigote forms, such as the occurrence of apoptosis-like 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 interruption, in addition to DNA fragmentation. Despite MDC positive 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 parasite cells. Moreover, the association of the active compound with miltefosine showed an additive effect in treating L. amazonensis-infected macrophages. Altogether, these results highlight the therapeutic potential of the evaluated salt and support further studies to assess its in vivo efficacy in a murine model of cutaneous leishmaniasis.