A Physico-Biochemical Study on Potential Redox-Cyclers as Antimalarial and Antischistosomal Drugs

Abstract

The role of redox enzymes in establishing a microenvironment for parasite development is well characterized. Mimicking human glucose-6-phosphate dehydrogenase and glutathione reductase (GR) deficiencies by redox-cycling compounds thus represents a challenge to the design of new preclinical antiparasitic drug candidates. Schistosomes and malarial parasites feed on hemoglobin. Heme, the toxic prosthetic group of the protein, is not digested and represents a challenge to the redox metabolism of the parasites. Here, we report on old and new redox-cycling compoundswhose antiparasitic activities are related to their interference with (met)hemoglobin degradation and hematin crystallization. Three key-assays allowed probing and differentiating the mechanisms of drug actions. Inhibition of β-hematin was first compared to the heme binding as a possible mode of action. All tested ligands interact with the hematin π-π dimer with K D similar to those measured for the major antiparasitic drugs. No correlation between a high affinity for hematin and the capacity to prevent β-hematin formation was however deduced. Inhibition of β-hematin formation is consequently not the result of a single process but results from redox processes following electron transfers from the drugs to iron(III)-containing targets. The third experiment highlighted that several redox-active compounds (in their reduced forms) are able to efficiently reduce methemoglobin to hemoglobin in a GR/NADPH-coupled assay. A correlation between methemoglobin reduction and inhibition of β-hematin was shown, demonstrating that both processes are closely related. The ability of our redox-cyclers to trigger methemoglobin reduction therefore constitutes a critical step to understand the mechanism of action of our drug candidates.

1,4-Naphthoquinones and Other NADPH-Dependent Glutathione Reductase- Catalyzed Redox Cyclers as Antimalarial Agents

“…It is likely that P. berghei can uptake GSH from the host, as opposed to P. falciparum [151]. However, of greater importance is the NADPH flux, which can be diverted to a futile cycle in the presence of redox-active agents by exploiting the GR reductive catalysis for drug bioactivation in infected red blood cells and/or drug combination in concert with metHb catabolism [22, 29, 43, 156]. …”

“…(1)). The molecular mechanisms by which inhibition of hemozoin formation occurs has been recently detailed and reviewed for different series of antimalarial drugs, including methylene blue [22], 1,4-naphthoquinones and xanthones [29]. In order to correlate the contribution of the redox-cycling capabilities of these molecules to the antiplasmodial activity, three key-assays were set up to allow probing and differentiating the mechanisms of drug actions with relevant heme species under quasi-physiological conditions.…”

The Antimalarial Activities of Methylene Blue and the 1,4-Naphthoquinone 3-[4-(Trifluoromethyl)Benzyl]-Menadione Are Not Due to Inhibition of the Mitochondrial Electron Transport Chain

“…In vitro, methylene blue and certain 1,4-naphthoquinones accept electrons from the flavoprotein glutathione reductase (GR), which is itself reduced by NADPH (22,25). After reduction, these compounds can transfer single electrons to key oxidants of P. falciparum, e.g., to the hemoglobin catabolites methemoglobin and Fe(III)-protoporphyrin IX (22,24,26). The generated Fe(II) species are toxic to the parasite since Fe(II)-containing oxyhemoglobin is a poor substrate of the hemoglobinase falcipain-2 (27), and Fe(II)-protoporphyrin IX was shown to inhibit the crystallization of ␤-hematin in vitro (28).…”

The Redox Cycler Plasmodione Is a Fast-Acting Antimalarial Lead Compound with Pronounced Activity against Sexual and Early Asexual Blood-Stage Parasites

“…Methylene blue, which is still the drug of choice in the treatment of methemoglobinemia, has recently regained interest for its antiplasmodial activities (66)(67)(68), especially as a companion drug in combination therapies (69,70) or as a potential transmission-blocking agent (17). Similar to benzylmenadiones, methylene blue seems to exert its antiplasmodial activity by causing a redox imbalance (9,71). However, the blue dye colors the body fluids, such as the urine, and the eyes of the patients, which is a limiting factor for drug development.…”

“…Studies on the mode of action have suggested that plasmodione, like other benzylmenadione derivatives, acts as a redox cycler, thereby disrupting the redox homeostasis of the parasitized erythrocyte (3,(8)(9)(10)(11). Interfering with the parasite's redox balance is a largely unexploited but highly effective strategy to restrict parasite development.…”