Limonene exerts antileishmanial effects via mevalonate pathway in Leishmania amazonensis

Limonene, a monoterpene abundant in essential oils of citrus plants, exhibits well-documented activity against both promastigote and amastigote forms of Leishmania in vitro and in vivo. Leishmania (L.) amazonensis is one of the etiological agents of localized and diffuse cutaneous leishmaniasis in the Americas, clinical forms frequently associated with limited therapeutic efficacy and recurrent treatment failure. Here, we show that limonene inhibits the biosynthesis of key isoprenoids in L. amazonensis promastigotes, including ubiquinone, dolichol, and ergosterol. Mechanistically, limonene disrupts the mevalonate pathway, reducing mevalonate synthesis by 69 %. The consequent decrease in ubiquinone levels impairs mitochondrial redox homeostasis, leading to increased mitochondrial reactive oxygen species, with a marked rise in hydrogen peroxide production following limonene exposure. In contrast, superoxide levels decline and reach a significant reduction at the highest limonene concentration tested. These redox disturbances are accompanied by pronounced mitochondrial morphological alterations, including severe vacuolization and degranulation of cytosolic and nuclear contents. Together, these findings demonstrate that limonene exerts pleiotropic effects on parasite sterol and isoprenoid metabolism and induces mitochondrial dysfunction. Our data identify the mevalonate pathway as a critical molecular target underlying limonene's antileishmanial mechanism of action and highlight its potential as a scaffold for the development of new therapeutic strategies against cutaneous leishmaniasis caused by L. amazonensis.