Dados do Trabalho

Título

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242

Introdução

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug.

Objetivo(s)

In order to investigate SUB of Leishmania spp. further, we first determined the cellular location of the S8 domain-containing SUB in L.amazonensis, and then, to determine whether SUB could be a potential drug target, we use as an investigative pharmacological tool the evaluated the serine protease inhibitor, PF-429242 against promastigotes and amastigotes of L.amazonensis.

Material e Métodos

By electron microscopy and flow cytometry, we first show that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of the human was determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against dengue virus, arenaviruses, and hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro.

Resultados e Conclusão

PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07±0.20; 0.83±0.12;2.02±0.27 and 5.83±1.2 μM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 μM). We detected by flow cytometry that there is a greater expression of subtilisin in amastigote however, the PF-429242 had a low effect against this intracellular form with an IC50 of >100 μM for intracellular amastigotes for the LV78 strain as axenic amastigotes (94.12±2.8 μM). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

Palavras-chave

Leishmania, serine protease, subtilisin, PF-429242, cellular localization