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

Subtilisin proteases are enzymes present in all organisms that play a critical role in the post-translational processing of proteins. In Leishmania major and L. donovani, subtilisin is essential for survival; however, there has been limited investigation into compounds targeting this enzyme for potential antileishmanial applications. In this study, we used electron microscopy and flow cytometry to demonstrate that subtilisin is widely distributed throughout the cytoplasm and membrane of the parasite in its promastigote form, with concentrated areas in the flagellar pocket.

Through in silico analysis, we examined the similarities between subtilisin from various Leishmania species and that of humans. We then assessed the interaction potential of the serine protease inhibitor PF-429242 against both life cycle forms of Leishmania using molecular docking. PF-429242 has been previously studied against dengue virus, arenaviruses, and hepatitis C virus, and it has shown antilipogenic effects in a mouse model, leading to hypolipidemia in human cells in vitro.

Our findings reveal that PF-429242 significantly inhibited the growth of L. amazonensis promastigotes across four strains, with IC50 values of 3.07 ± 0.20, 0.83 ± 0.12, 2.02 ± 0.27, and 5.83 ± 1.2 µM for the LTB0016, PH8, Josefa, and LV78 strains, respectively, while exhibiting low toxicity to host macrophages (CC50 = 170.30 µM). Flow cytometry analysis indicated higher levels of subtilisin expression in the amastigote form; however, PF-429242 demonstrated limited efficacy against this intracellular stage, with an IC50 of >100 µM for intracellular amastigotes and 94.12 ± 2.8 µM for axenic amastigotes of the LV78 strain.

In conclusion, while PF-429242 does not significantly impact the intracellular forms of Leishmania, it provides a valuable tool for exploring pharmacological and PF 429242 potential leishmanicidal targets.