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SUBTILISIN OF LEISHMANIA AMAZONENSIS AS POTENTIAL DRUGGABLE TARGET: SUBCELLULAR LOCALIZATION, IN VITRO LEISHMANICIDAL ACTIVITY AND MOLECULAR DOCKING OF PF-429242, A SUBTILISIN INHIBITOR
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Abstract
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. In this study, we first show, by electron microscopy and
flow cytometry, 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 humans were 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 the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved
to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro.
Here, 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 M against LTB0016, PH8,
Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 M).
We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form;
however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 M for
intracellular amastigotes, as well as against axenic amastigotes (94.12 2.8 M for the LV78 strain).
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.
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