Azurin Induces Apoptosis in Leishmania Promastigotes, Showing Potent Anti-Leishmanial Activity In Vitro

Leishmaniasis, a neglected tropical protozoan disease, affects millions globally, yet current therapies are plagued by numerous shortcomings, including toxicity, drug resistance, and limited efficacy. There is an urgent need for novel drugs and targets to combat this debilitating condition. Azurin, a bacterial protein, has garnered significant attention due to its broad biological activities, including anticancer, antiviral, and antiparasitic properties. Its recent success against malaria, another protozoan disease, positions it as a promising candidate for exploring its therapeutic potential against Leishmania parasites, addressing a critical gap in current treatment strategies.

Researchers conducted an integrated in silico and in vitro investigation to assess azurin's anti-leishmanial potential. In silico, essential Leishmania enzyme structures were docked with the P18 and P28 regions of azurin (chain A). For in vitro studies, Leishmania promastigotes were treated with azurin. Apoptosis induction was evaluated via AO/EB staining, reactive oxygen species (ROS) production using DCFDA assay, and intracellular calcium levels with Fluo-4-AM assay. The study also assessed azurin's effect on promastigote numbers within macrophages, comparing treated groups to a non-treated control to determine its efficacy.

In silico analysis revealed that Leishmania enzymes C-S14D-3L4D and E-NMT-2WUU exhibited the best interactions with azurin peptides during post-docking and simulation studies, suggesting specific molecular targets. In vitro experiments confirmed azurin's therapeutic activity, demonstrating that azurin treatment induced apoptosis in promastigotes. This apoptotic effect was mediated by significant production of reactive oxygen species (ROS) and activation of ER stress signaling pathways. Furthermore, an increase in intracellular Ca+2 levels was observed, contributing to the pro-apoptotic cascade. The study determined an IC50 value for promastigotes at 20.72 ± 2.492 µg/mL, indicating potent activity at relatively low concentrations. Importantly, azurin treatment drastically reduced the number of promastigotes within macrophages compared to the non-treated control group, highlighting its ability to target intracellular parasites. This multi-pronged action underscores azurin's potential as an effective anti-leishmanial agent.

Azurin treatment achieved an IC50 of 20.72 ± 2.492 µg/mL against Leishmania promastigotes, demonstrating potent anti-parasitic activity at lower concentrations.