Redox Homeostasis within the Drug-Resistant Malarial Parasite Digestive Vacuole.

We have developed a cost-effective strategy for the complete synthesis of azetidinyl coumarin fluorophore derivatives that report changes in physiologic levels of glutathione (GSH), which includes a more cost- effective synthesis of the probe precursor hydroxyl derivative and its subsequent derivatization to promote subcellular localization. We functionalize coumarin derivatives with a cyano side chain similar to a previous strategy (Jiang X. et al., Nature Communications 2017, 8; 16087) and validate the 7-azetidinyl conformation as an explanation for enhanced GSH-dependent coumarin fluorescence. We couple the azetidinyl probe to different mass dextrans using either no linker or a 6C linker and also synthesize a morpholino derivative. We titrate the fluorescence of the different functionalized probes vs [GSH] in vitro. We load one dextran-conjugated probe within the digestive vacuole (DV) of live intraerythrocytic P. falciparum malarial parasites and also measure cytosolic localization of the morpholino probe. Using significantly improved single-cell photometry (SCP) methods, we show that the morpholino probe faithfully reports [GSH] from the live parasite cytosol, while the 70 kDa dextran-conjugated probe reports DV redox homeostasis for control chloroquine-sensitive (CQS) and artemisinin-sensitive (ARTS) transfectant parasites vs their genetically matched chloroquine-resistant (CQR)/artemisinin-sensitive (CQR/ARTS) and CQR artemisinin-resistant (CQR/ARTR) strains, respectively. We quantify rapid changes in DV redox homeostasis for these parasites ± drug pulses under live-cell perfusion conditions. The results are important for understanding the pharmacology of antimalarial drugs and the molecular mechanisms underlying CQR and ARTR phenomena.