3E) and artemisinin derivatives such as ART, ATS, and ATM (Fig. 3F) did not cause a major increase in the fluorescence ratio of recombinant hGrx1-roGFP2, even at add to favorites concentrations up to 100 ��M and after 24 h incubation. Short-term effects of antimalarial drugs and redox-active compounds on the cytosolic redox potential in Plasmodium Prior to investigating the effects of antimalarial drugs and redox-active compounds on hGrx1-roGFP2 in living cells, we (re)-assessed the IC50 values of the drugs in a standardized IC50 assay, which is based on a 72-hour incubation and incorporation of 3H-labeled hypoxanthine (please see details in Table 1 and the Materials and Methods section). Then, we first tested short-term incubations (up to 5 min) for which rather high drug concentrations were necessary in order to observe effects (for a summary please see Table 2).
Among the antimalarial drugs tested, only MB evoked an immediate increase in the fluorescence ratio 405/488 nm; this increase was observed in both parasite strains (3D7: Fig. 4A, B; Dd2: Fig. 4C, D) and was faster and higher in 3D7 than in Dd2. A direct interaction between MB and the probe (see above) definitely has to be considered when interpreting this result. Interestingly, PYO, a natural compound structurally related to MB, (Figs. 5A, B) and MNA also caused an increase in the fluorescence ratio 405/488 nm (Figs. 5C, D) and Dd2 (Figs. 5E, F) within 5 min. Effects were also observed for 1-chloro-2,4-dinitrobenzene (CDNB), a thioredoxin reductase inhibitor and an electrophilic xenobiotic compound that is detoxified by conjugation to GSH [33].
On the 3D7 strain, millimolar concentrations of CDNB caused a pronounced but transient increase in the fluorescence ratio (Figs. 5G, H). In contrast (and as expected), 1 mM of the glutathione biosynthesis inhibitor L-buthionine sulfoximine (BSO) did not immediately induce ratio changes (shown for Dd2hGrx1-roGFP2 in Fig. S6A). Artemisinin (Fig. S6B�CD) and quinoline-based (Fig. S7) antimalarials at 100 ��M did not change the fluorescence signal of the probe within 5 min, either. Figure 4 Immediate oxidation of the P. falciparum cytosol by methylene blue. Figure 5 hGrx1-roGFP2 visualizes cytosolic depletion of glutathione induced by pyocyanin, menadione, and CDNB. Table 2 Short-term effects of redox-active compounds and antimalarial drugs on the redox ratio of hGrx1-roGFP2 in living parasites.
Effects of antimalarial drugs on the glutathione redox potential of Plasmodium after 4 h incubation Brefeldin_A The clinically employed concentrations of antimalarials are usually in the nanomolar range and thus much lower than the 100 ��M employed above to see maximal effects in short-term incubations. Therefore we investigated whether hGrx1-roGFP2 can monitor changes in EGSH at pharmacologically meaningful drug concentrations.