title: Mechanism of ascaridole activation in Leishmania.
abstract: Endoperoxides (EP) are an emerging class of drugs which have potential in antiparasitic therapy, but also in other fields. For malaria therapy the EP artemisinin (Art) and its derivatives are successfully used. We have shown in the past that the EP ascaridole (Asc) is useful for the treatment of cutaneous leishmaniasis in a mouse model. Biomimetic experiments suggested that these drugs need activation in the respective target pathogens to exert their function. In spite of this idea, direct activation of EP to radicals inside cells has never been demonstrated. Therefore, this study was initiated to explore the activation of Asc in biomimetic systems and inside Leishmania in comparison to Art. Using electron paramagnetic resonance spectroscopy (EPR) in combination with spin-trapping we identified the secondary alkyl radical intermediates arising from reduction by Fe2+in cell-free systems. Combined GC/NMR analysis confirmed the loss of isopropyl residues from Asc during this process as intermediates. This activation of Asc was stimulated by low molecular Fe2+complexes or alternatively by hemin in conjunction with thiol reductants, such as cysteine (Cys). In Leishmania tarentolae promastigotes (LtP) as model for pathogenic forms of Leishmania carbon-centered radicals were identified in the presence of Asc by EPR spin-trapping. Both Asc and Art inhibited the viability in LtP with IC50values in the low micromolar range while IC50values for J774 macrophages were considerably higher. A similar structure without EP bridge (1,4-cineole) resulted in no detectable radicals and possessed much less cytotoxicity in LtP and no selectivity for LtP compared to J774 cells. The Asc-derived radical formation in LtP was inhibited by the iron chelator deferoxamine (DFO), and stimulated by Cys (a suitable reductant for hemin). The IC50values for LtP viability in the presence of Asc or Art were increased significantly by the spin trap DMPO, while Cys and DFO increased only IC50values for Art. In a heme association assay Asc demonstrated a lower binding affinity to heme than Art. ICP-OES measurements revealed that in LtP the total iron concentrations were twice as high as values in J774 macrophages. Since low molecular iron was important in Asc activation we studied the influence of Asc on the labile iron pool (LIP) in LtP. Low temperature EPR experiments demonstrated that Asc shifts the redox balance of iron in the LIP to its oxidized state. These data demonstrate that univalent cleavage of Asc/Art in LtP is an essential part of their pharmacological mechanism. The structure of the EP determines whether activation by low molecular iron or heme is favored and the availability of these intracellular activators modulates their cytotoxicity. These findings may be helpful for synthesis of new Asc derivatives and understanding the action of EP in other cell types.
authors.affiliation: Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Geroldinger
authors.affiliation: Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Tonner
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Tulln, Austria.
authors.forename: Hubert
authors.initials: H
authors.lastname: Hettegger
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Tulln, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Bacher
authors.affiliation: Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria; Parasitology Department, Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba.
authors.forename: Lianet
authors.initials: L
authors.lastname: Monzote
authors.affiliation: Department of Environmental Geosciences, University of Vienna, Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Walter
authors.affiliation: Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Katrin
authors.initials: K
authors.lastname: Staniek
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Tulln, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rosenau
authors.affiliation: Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria. Electronic address: Lars.Gille@vetmeduni.ac.at.
authors.forename: Lars
authors.initials: L
authors.lastname: Gille
mesh_terms: D000818:Animals; D002460:Cell Line; D002849:Chromatography, Gas; D000081005:Cyclohexane Monoterpenes; D004578:Electron Spin Resonance Spectroscopy; D007891:Leishmania / Q000187:drug effects* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D039821:Monoterpenes / Q000494:pharmacology*; D010545:Peroxides / Q000494:pharmacology*
chemical_list: D000081005:Cyclohexane Monoterpenes; D039821:Monoterpenes; D010545:Peroxides; C043372:ascaridole
keywords: Artemisinin (Pubchem CID: 68827); Ascaridole; Ascaridole (Pubchem CID: 10545); Electron spin resonance; Endoperoxides; Iron; Leishmania