Dados do Trabalho

Título

Effects of serine protease inhibitor PF-429242 on Leishmania infantum

Introdução

Visceral leishmaniasis (VL) is fatal if untreated and its control is based on chemotherapy, however the drugs available are toxic and parasite resistance has been reported. Therefore, the search for new antileishmanial compounds is urgent. Proteases are virulence factors in Leishmania and consequently emerge as potential new drug targets. PF-429242 is an inhibitor of subtilisin, an important protease found in Leishmania. However, studies regarding the effect of PF-429242 on Leishmania are scarce. Previously, we determined the effect of PF-429242 on Leishmania infantum, etiological agent of VL in the Americas. PF-429242 was active on both promastigotes (IC50 = 2.78 µM) and amastigotes (IC50 = 14.07 µM).

Objetivo (s)

In this context, we evaluated the mechanism involved in the death of L. infantum after treatment with PF-429242.

Material e Métodos

Leishmania infantum promastigotes in log phase growth or macrophages infected with L. infantum amastigotes were treated with PF-429242, at concentrations of 1x and 2x the IC50 value, for 72h. After this period, the cells were washed with PBS and stained with different probes or processed for transmission electron microscopy (TEM).

Resultados e Conclusão

TEM and staining of L. infantum promastigotes with MitoTrackerRed, rhodamine 123 and MitoSOX, revealed that the mitochondria was a potential target of PF-429242. Additionally, PF-429242 caused an accumulation of neutral lipids in promastigotes, which was demonstrated by Nile Red staining and TEM. In addition, PF-429242 also induced oxidative stress, what was seen by H2DCFDA staining. Furthermore the formation of autophagic vacuoles in L. infantum promastigotes was observed by MDC staining and TEM. However, the killing induced by PF-429242 in L. infantum promastigotes appeared to be unrelated to apoptosis and/or necrosis as there was no phosphatidylserine externalization, DNA fragmentation or alterations in the permeability of the parasite plasma membrane, as assessed by annexin V-FITC, TUNEL and propidium iodide staining, respectively. On L. infantum intracellular amastigotes, TEM analysis indicated that mitochondrial damage and autophagy could also be involved in the death of these forms after treatment with PF-429242. This work highlights effects PF-429242 against L. infantum and confirms the importance of proteases as drug targets in these parasites.

Palavras-chave

Visceral leishmaniasis, Leishmania infantum, Leishmaniasis chemotherapy, Protease, Subtilisin, PF-429242, Mitochondria, Autophagy.

Agradecimentos

FAPERJ, CAPES and CNPq.