Uptake of a fluorescently tagged chloroquine analogue is reduced in CQ-resistant compared to CQ-sensitive Plasmodium falciparum parasites

BACKGROUND: Chloroquine (CQ) was the drug of choice for decades in the treatment of falciparum malaria until resistance emerged. CQ is suggested to accumulate in the parasite’s digestive vacuole (DV), where it unfolds its anti-malarial properties. Discrepancies of CQ accumulation in CQ-sensitive (CQS) and CQ-resistant (CQR) strains are thought to play a significant role in drug susceptibility. Analysis of CQ transport and intracellular localization using a fluorescently tagged CQ analogue could provide much needed information to distinguish susceptible from resistant parasite strains. The fluorescently tagged CQ analogue LynxTag-CQ™(GREEN) (CQ(GREEN)) is commercially available and was assessed for its suitability. METHODS: IC(50) values were determined for both CQ and CQ(GREEN) in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQ(GREEN) in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQ(GREEN) for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQ(GREEN) uptake rates were determined for the cytosol and DV in the presence and absence of verapamil. RESULTS: In CQS strains, twofold higher IC(50) values were determined for the CQ(GREEN) analogue compared to CQ. No significant differences in IC(50) values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQ(GREEN). Live cell imaging revealed that CQ(GREEN) fluorescence was mainly seen in the cytosol of most parasites, independent of the concentration used. Incubation periods of up to 7 h did not influence intracellular localization of CQ(GREEN). Nevertheless, CQ(GREEN) uptake rates in CQR strains were reduced by 50% compared to CQS strains. CONCLUSION: Although fluorescence of CQ(GREEN) was mainly seen in the cytosol of parasites, IC(50) assays showed comparable efficacy of CQ(GREEN) and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQ(GREEN) in CQR strains compared to CQS strains indicate parasite-specific responses to CQ(GREEN) exposure. The data contains valuable information when CQ(GREEN) is used as an analogue for CQ.