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Biochem Pharmacol. 2019 Nov 28:113737. doi: 10.1016/j.bcp.2019.113737. [Epub ahead of print]

Activation of Artemisinin and Heme Degradation in Leishmania tarentolae Promastigotes: A Possible Link.

Author information

1
Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria.
2
Department of Environmental Geosciences, University of Vienna, Vienna, Austria.
3
Department of Pharmacology, Institute of Post Graduate Medical Education & Research, Kolkata, India.
4
Institute of Pharmacy and Food, Havana University, Havana, Cuba.
5
Parasitology Department, Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba.
6
Institute of Animal Nutrition and Functional Plant Compounds, Department of Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
7
Institute for Medical Biochemistry, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria.
8
Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria. Electronic address: Lars.Gille@vetmeduni.ac.at.

Abstract

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

KEYWORDS:

Leishmania; artemisinin; endoperoxide; heme; radicals

PMID:
31786259
DOI:
10.1016/j.bcp.2019.113737

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