Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E17). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar “intrinsic” reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.
With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite Plasmodium falciparum, we examined the single-electron reduction by P. falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) of a series of nitrofurans and nitrobenzenes (n = 23), and their ability to inhibit P. falciparum glutathione reductase (PfGR). The reactivity of nitroaromatics in PfFNR-catalyzed reactions increased with their single-electron reduction midpoint potential (E17). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards PfGR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against P. falciparum strain FcB1 increased with their E17 values, octanol/water distribution coefficients at pH 7.0 (log D), and their activity as PfGR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of PfGR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.
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.
Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.
Aerobic cytotoxicity of 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ), a bioreductively activated hypoxia-specific anticancer agent, is responsible for TPZ side effects in chemotherapy. In order to clarify its mechanisms, we examined the aerobic cytotoxicity of TPZ and its main metabolites, 3-amino-1,2,4-benzotriazine-1-oxide and 3-amino-1,2,4-benzotriazine in murine hepatoma MH22a cells, and their reduction by NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP+ reductase (FNR). Analogous studies of several quinones and nitroaromatic compounds with similar values of single-electron reduction midpoint potentials (E17) were carried out. In single-electron reduction by P-450R and FNR, the reactivity of TPZ and its monoxide was similar to that of quinones and nitroaromatics, and increased with an increase in their E17. The cytotoxicity of TPZ and its metabolites possessed a prooxidant character, because it was partly prevented by an antioxidant N,N’-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. Importantly, the cytotoxicity of TPZ and, possibly, its 1-N-oxide, was much higher than that of quinones and nitroaromatics with similar values of E17 and redox cycling activities. A possible additional factor in the aerobic cytotoxicity of TPZ is its reductive activation in oxygen-poor cell nuclei, leading to the formation of DNA-damaging species similar to those forming under hypoxia.
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