g., are activated via reductive cleavage of their peroxide bond by intracellular iron (Fe2+) or heme �Cgenerating, Y-27632 CAS carbon-centered free radicals [9], [10] with the potential to alkylate vital cellular components including the tripeptide glutathione (��-Glu-Cys-Gly, GSH). Methylene blue (MB), quinoline [11], and artemisinin-based [6] antimalarial drugs have been shown to accumulate in the food vacuole and inhibit the formation of hemozoin [12]. Non-crystallized toxic heme exits the food vacuole and is degraded in the cytosol with GSH as a cofactor. This process is inhibited by chloroquine (CQ) and amodiaquine (AQ) [13], leading to an intensive discussion about the role of oxidative stress in the mechanism of action of 4-aminoquinolines [14], [15], [16].

Furthermore, MB is known to be a redox cycler and a substrate and inhibitor of glutathione reductase (PfGR) in the cytosol [17], [18], [19], and the mutant P. falciparum chloroquine resistance transporter (PfCRT) may also be involved in the transport of glutathione [20]. Hence, redox metabolism seems to play an important role in antimalarial drug action and resistance and deserves to be studied in more detail. The P. falciparum glutathione redox system comprises the electron donor NADPH, a highly active PfGR located in the cytosol and the apicoplast, and reduced/oxidized glutathione (GSH/GSSG) [21], [22], [23]. Glutathione levels have been shown to be regulated via glutathione biosynthesis, glutathione efflux, and reduction via GR. De novo, GSH is sequentially synthesized in the cytosol by ��-glutamylcysteine synthetase (��-GCS) and glutathione synthase.

The GSH/GSSG redox couple is the major redox buffer in the cytosol [21], [22] and functions as an indicator of the cellular redox status and oxidative stress [24]. In malaria parasites, GSH is a key player in the detoxification of reactive oxygen (ROS) and nitrogen species (RNS), which are produced by antimalarial drugs, hemoglobin digestion, and the host’s immune system [25], [26]. The midpoint redox potential of the GSH:GSSG redox couple (E0′GSH) at pH 7.0, physiologic ionic strength and 25��C is ?240 mV [24]. Importantly, changes in EGSH appear to correlate with the biological status of cells including proliferation (?240 mV), differentiation (?200 mV), and apoptosis (?170 mV) [24]. Using global values (2.39 mM GSH; 8.4 ��M GSSG [27]), the EGSH for the Plasmodium trophozoite cytosol at pH 7.2 and 37��C has so far only been roughly estimated [22]. Conventionally, GSSG and GSH are measured via reverse-phase high-performance liquid chromatography or enzymatically via glutathione reductase-dependent reactions and the 5,5��-dithiobis(2-nitrobenzoic acid) Anacetrapib (also known as DTNB or Ellman’s reagent) recycling assay [28].