Isopentyl caffeate targets Gp63 (Leishmanolysin) in Leishmania amazonensis

Objectives

Several drugs are commonly used in the management of Leishmaniasis, but their toxicity, cost, and efficacy depend on the form of the disease.The development of new therapeutics remains important to expand treatment options and address existing limitations. Previously, we reported that isopentyl caffeate (ICaf) exhibited potent activity against promastigotes and amastigotes of Leishmania amazonensis without notable toxicity in a murine model. This study investigated the effects of ICaf on the metallopeptidase gp63 (leishmanolysin), a key virulence factor in Leishmania that supports parasite survival by modulating host immune responses, degrading host proteins, and facilitating macrophage invasion.

Methods and Results

Molecular docking analysis revealed that ICaf interacted with key amino acids within the active site of gp63, achieving a docking score of -5.5 kcal/mol. The gp63-ICaf interaction was stabilised through a combination of hydrogen bonding, hydrophobic interactions, metal-acceptor interactions and van der Waals forces. Experimental data supported these in silico findings, with ICaf showing a typical dose-dependent inhibition of gp63 proteolytic activity, resulting in an IC₅₀ of 1.51 µM and a K_i of 9.89 µM. The formation of a stable gp63-ICaf complex was demonstrated by gel electrophoresis assay. Furthermore, pretreatment of promastigotes with ICaf reduced cell-associated gp63 proteolytic activity, as evidenced by decreased hydrolysis of the fluorogenic peptide substrate Z-Phe-Arg-AMC and gelatin incorporated into electrophoresis gel. Flow cytometry and confocal microscopy, both using anti-gp63 antibodies, further confirmed a decrease in gp63 expression on the parasite surface.

Conclusions

These findings suggest that ICaf is a promising strategy against leishmaniasis, offering a potential new approach to disease treatment.