Triggers and Targets of Ascaridole Action in Leishmania

Leishmaniasis, a disease caused by protozoal parasites, is endemic in 98 countries and represents a serious health problem. The incidence of leishmaniasis is on the rise because of the increases in the disease vectors due to global warming. Presently, chemotherapy remains the mainstay for controlling leishmaniasis. Ascaridole (Asc) is a chemically defined endoperoxide compound that is biosynthesized in specific plants. Essential oils from plants that contain Asc have been successfully tested in the experimental treatment of cutaneous leishmaniasis in a mouse model. In pharmacological terms, Asc is a pro-drug that requires activation. However, the mechanistic determinants of Asc activation and action in Leishmania are not clear. We propose to study the antileishmanial mechanism of action of Asc at the molecular, subcellular and cellular levels in the model organism L. tarentolae in comparison to mammalian macrophage/monocyte cell lines. The work will elucidate the specific triggers of Asc activation in these cells and the preferred targets that cause cytotoxicity in Leishmania and might contribute to selectivity. Specifically, the role of heme iron and the low molecular labile iron pool in Asc activation will be studied with electron spin resonance (ESR) spectroscopy. Mechanism studies will be supported by the synthesis of Asc- related endoperoxides and the use of non-endoperoxide Asc analogues to enable structure-activity relationship studies. The role of selective toxicity will be addressed by comparative studies with monocyte/macrophage cell lines as well as with mammalian detoxification enzymes. Findings of the mechanism studies relevant for treatment will be validated in a mouse model of cutaneous leishmaniasis. Based on the details of the mechanism of action of Asc, the synergistic role of chemotherapeutic metal-chelating ligands in their action against Leishmania will be identified. The increased knowledge of the mechanistic details will help us to understand the conditions for the effective application of Asc-related endoperoxides as antileishmanial drugs.

Triggers and Targets of Ascaridole Action in Leishmania Leishmaniasis is an infective disease of humans and other mammals caused by unicellular parasites (Leishmania). Depending on the Leishmania species, this disease can even lead to death. Up to 1 million new cases and about 70,000 deaths occur every year. The insidious property of Leishmania is that they hide inside macrophages from the immune system, which also complicates pharmacological treatment. The essential oil of the plant Chenopodium ambrosioides was shown to possess antileishmanial properties in model experiments. A major ingredient of this oil is the endoperoxide ascaridole. This project was set up to explore the antileishmanial mechanism of ascaridole and related endoperoxides. Basic research using sophisticated radical detection and other bioanalytical methods in cell culture models of Leishmania and host macrophages was carried out. Experimental data revealed that for the antileishmanial effect of the endoperoxide ascaridole its activation by iron compounds is necessary leading to carbon-centered radical intermediates in Leishmania. Therefore, the properties of the intracellular iron in Leishmania and host macrophages was studied. Leishmania contain about twice as high total iron concentrations as host macrophages. Moreover, the contribution of the labile iron pool (redox-active iron that can react with peroxides) is much higher in Leishmania than in host macrophages. This in part explains the higher sensitivity of Leishmania than host macrophages for ascaridole and other endoperoxides. Mitochondria and oxidation-prone cellular components (thiol groups) were identified as targets of ascaridole-derived radicals compromising the viability of Leishmania. Ascaridole confirmed its activity against intracellular parasites in a cell model of mouse macrophages containing intracellular Leishmania. To understand the role of the molecular structure of ascaridole and especially of the endoperoxide group, we studied another natural endoperoxide artemisinin, which is used in malaria therapy, and synthetic endoperoxides derived from anthracene. This comparison revealed that most of these compounds possess antileishmanial activity. However, the structure beyond the endoperoxide group determines the actual activation partners for forming radicals, the penetration of the compounds into cells and, therefore, the selectivity of these compounds for Leishmania versus host macrophages. In addition, for artemisinin the intracellular degradation of heme (iron derived from digestion of red blood cells in macrophages) in Leishmania was identified as important activator. Beyond the cellular models, selected endoperoxides were studied in a mouse model of cutaneous leishmaniasis triggered by infection with Leishmania amazonensis. Several endoperoxides including ascaridole and artemisinin were able to reduce the parasite burden and the lesion size in these infections. These mechanistic findings and first in vivo results suggest that natural and synthetic endoperoxides could be promising candidates for antileishmanial drugs and predict possible intracellular reaction partners. These results provide the basis for the further development of endoperoxide-derived antileishmanial drugs.