Plasmodium falciparum Ferredoxin-NADP⁺ Reductase-Catalyzed Redox Cycling of Plasmodione Generates Both Predicted Key Drug Metabolites: Implication for Antimalarial Drug Development

Abstract

Plasmodione (PD) is a potent antimalarial redox-active 3-benzyl-menadione acting at low nM range concentrations on different malaria parasite stages. Specific bioactivation of PD was proposed to occur via a cascade of redox reactions starting from one electron-reduction, then benzylic oxidation, leading to generation of several key metabolites including the benzylic alcohol (PD-bzol) and the 3-benzoylmenadione (PDO). In this study, we showed that benzylic oxidation of PD is closely related to the formation of a benzylic semiquinone radical, which can be produced under two conditions: UV-photoirradiation, or catalyzed by P. falciparum apicoplast ferredoxin-NADP + reductase (PfFNR) redox cycling in the presence of oxygen and the parent PD. Electrochemical properties of both PD metabolites were investigated in DMSO and in water. The single-electron reduction potential values of PD, PD-bzol, PDO and a series of 3-benzoylmenadiones were determined according to ascorbate oxidation kinetics. These compounds possess enhanced reactivity towards PfFNR as compared with model quinones. Optimal conditions were set up to obtain the best conversion of the starting PD to corresponding metabolites. UV-irradiation of PD in isopropanol under positive oxygen pressure led to an isolated yield of 31% PDO through the transient semiquinone species formed in a cascade of reactions. In the presence of PfFNR, PDO and PD-bzol could be observed during long lasting redox cycling of PD continuously fueled by NADPH regenerated by an enzymatic system. Finally, we observed and quantified the effect of PD on the production of oxidative stress in the apicoplast of transgenic 3D7 P. falciparum parasites by using the described genetically encoded glutathione redox sensor hGrx1-roGFP2 methodology. The observed fast ROS pulse released in the apicoplast is proposed to be mediated by PD redox cycling catalyzed by PfFNR.

Synthesis and Photochemical Properties of Fluorescent Metabolites Generated from Fluorinated Benzoylmenadiones in Living Cells

“…Benzylic oxidation indeed alters markedly the redox properties of the electroactive core ( i.e. , PDO is more prone to reduction than PD ). , For PDO , a third redox signal at −1.58 V (Figure S5 in the SI) is observed, which was attributed to one-electron redox process centered on the benzoyl carbonyl group Figure S7 in the SI).…”

“…, dihydroquinone), semiquinone radicals. , Following UV excitation, the 1,4-naphthoquinone scaffold is photoexcited and generates an excited triplet state that can be trapped by i PrOH ( i.e. , H atom donor) to lead to the semi-1,4-naphthoquinone radical. , In this way, UV irradiation of PD in i PrOH under oxygen pressure or an air atmosphere allowed the benzylic oxidation to PDO . The reactivity of F-PDO at 5 mM in an i PrOH/DCM ( v / v ) mixture was evaluated by irradiation at 350 nm for 24 h ( i.e.…”

“…Benzylic oxidation indeed alters markedly the redox properties of the electroactive core (i.e., PDO is more prone to reduction than PD). 17,29 For PDO, a third redox signal at −1.58 V (Figure S5 in the SI) is observed, which was attributed to one-electron redox process centered on the benzoyl carbonyl group. 23 PD-bzol differs with a much stronger effect on the one-electron reduced semiquinone due to an intramolecular hydrogen bond between the benzylic alcohol and the negatively charged semiquinone subunit (Figure S7 in the SI).…”

“…45,46 In this way, UV irradiation of PD in i PrOH under oxygen pressure or an air atmosphere allowed the benzylic oxidation to PDO. 17 The reactivity of F-PDO at 5 mM in an i PrOH/DCM (v/v) mixture was evaluated by irradiation at 350 nm for 24 h (i.e., a Rayonet photochemical reactor equipped with 16 UV lamps of 14 W and maintained at 16 °C) using high-resolution electrospray mass spectrometry (ESI-HRMS) monitoring.…”

Redox-Based Strategies against Infections by Eukaryotic Pathogens

“…Plasmodione (3-[4-(trifluoromethyl)benzyl]-menadione) is a new antimalarial drug. As a redox-active compound, it impairs the redox balance of parasites, mainly through the generation of benzhydrol and benzoyl metabolites leading to cell death [ 67 , 68 ]. Plasmodione and metabolites are actually substrates of mitochondrial respiratory chain flavoprotein NADH-dehydrogenases, leading to ROS production.…”