A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs

Abstract

Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P. falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P. falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by PfGR in CQ-resistant versus CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.

The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost

“…It is therefore possible that the passage of other molecules through the NPC indirectly influences the action of these drugs (e.g., by changing drug activation or accumulation in the FV). For instance, glucose depletion influences FV pH (Saliba et al, 2003), which affects drug accumulation of some 4-AQs (Jida et al, 2017;Yayon et al, 1984). However, MFQ, which may have a different mode and site of action than other 4-AQs (Cowman et al, 1994;Wong et al, 2017), was still dependent on the NPCs in our assay , arguing against an indirect effect based on mode of action.…”

The Nitrobenzoxadiazole Derivative NBDHEX Behaves as Plasmodium falciparum Gametocyte Selective Inhibitor with Malaria Parasite Transmission Blocking Activity

“…The comparative analysis of the above cysteine modifications showed that these were detectable only in gametocyte-infected RBC treated with 1 and not with 4, suggesting that the complete reduction of the 7-nitro group of 1 to its 7-amino counterpart likely takes place only after the previous adduct formation step(s). Recent observations that NBD derivatives are substrates of NADPH-dependent P. falciparum oxidoreductases led to propose a role for these molecules as "redox cyclers", i.e., compounds depleting the cell cytosol reducing power [41]. This may lead to speculate that gametocyte enzymes, yet to be identified, are likely responsible for the nitroreduction process that seems to be involved in the generation/stabilization of the adducts between 1 and cysteine residues.…”

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“…According to Jida et al, (2016) the accumulation of Fluo-CQ, a pH-sensitive fluorescent chloroquine analog in the DV did not occur in the glucose-free medium. This was due to the lack of energy input to acidify the DV, which is necessary for the accumulation and trapping of Fluo-CQ in the DV.…”

“…This was due to the lack of energy input to acidify the DV, which is necessary for the accumulation and trapping of Fluo-CQ in the DV. The acidification of DV was observed through the strong fluorescence signal of Fluo-CQ 23 .…”