Redox reactions of plasmodione with oxyhemoglobin or heme (Fe2+)

Summary 

Plasmodione (3-[4-(trifluoromethyl)benzyl]-menadione, PD) is active against malaria parasite Plasmodium falciparum at 10-8 M concentrations, and possess high selectivity index, 1:1000. Its antiplasmodial activity is manifested through multistep redox processes with the initial step(s) of formation of PDO, a 3-benzoylmenadione metabolite. Several current hypotheses about the mechanism(s) of its formation may not fully reflect the diversity of redox processes in the erythrocyte-parasite system. The aim of the visit is to determine and to discuss whether the 3-benzoyl PD metabolite, PDO, is formed during the reaction of PD with oxyhemoglobin (OxyHb) or heme (Fe2+). OxyHb can perform various oxidation and hydroxylation reactions of aromatic compounds. Although they are slow, they can be important because of the high concentration of OxyHb in erythrocytes. In this study, we will use the heavy 13C18-enriched-PD, characterized by a m/z of 18 units, in mixture (1:1) with PD to track and characterize the formation of its metabolites, e.g. PD-bzol and PDO by LC-MS. Further studies (possible contribution of flavoenzymes-electron transferases, studies with intact erythrocytes) will be performed in the applicant's laboratory. 

Goals of the STSM 

1 . Design of experimentation 

The high antiplasmodial activity of PD was discovered in the host laboratory [1]. Further studies were carried out to expand the range of PD analogs, to study their activity against different parasite strains, and to detail its activity mechanisms ([2], and refs. therein, Scheme 1). The latter direction partly involving the applicant's team, highlighted the possibility of formation of highly active PD benzoyl metabolite (PDO) by reactive oxygen species (ROS) during the redox cycling of PD radicals, formed after its reduction by P. falciparum flavoenzymes [2]. Several flavoenzymes-candidates are being studied in the host laboratory, such as 

P. falciparum ferredoxin-NADP+ reductase (PfFNR) and mitochondrial NADH dehydrogenase (PfNDH2), , using various 3-benz(o)ylmenadiones as potential substrates. Different approaches, including yeast genetics (yeast NDH1/NDH2 [3]), and fused-apicoplast Grx-roGFP-based imaging studies [2] were applied to demonstrate the implication of these flavoenzymes in PD bioactivation to some extent. The genetic validation of this mechanism in P. falciparum is currently underway in Strasbourg. In 2018, the host lab designed the synthesis of 13C18-enriched-PD [4] and successfully used it for demonstrating that PD generates two metabolites, PDO and PD-bzol, in a cycle fueled by PfFNR [2]. 

In order to elucidate the alternative pathways of PD transformation, we intend to investigate the possibility of PDO formation during PD reaction with OxyHb. OxyHb can perform various oxidation and hydroxylation reactions of aromatic compounds [5-7]. Although they are slow, they can be important because of the high concentration of OxyHb in erythrocytes. 

The goal of this STSM would be assessment of a possible alternative for PD bioactivation mechanism, i.e. the involvement of oxyHb or heme(Fe2+), although somewhat ignored mechanism of PDO formation, and, if successful, determination of its kinetic parameters. The possible involvement of free heme (Fe2+) will be envisionned in the benzylic oxidation, and its implication in ferroptosis induction through PDO formation, and parasite death. 

2. Discussions about European project Applications (Erasmus +) 

During the STSM trip, Prof. N. Čėnas will have discussions to set up a European consortium for future European applications (Erasmus +), including French teams (Host laboratory, team of Stefanie Blandin).