Cellular Uptake of Chloroquine Is Dependent on Binding to Ferriprotoporphyrin IX and Is Independent of NHE Activity in Plasmodium falciparum

Abstract

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ–FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ–FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.

Two complementary fluorimetric assays for the determination of aminoquinoline binding and uptake by human erythrocytes in vitro

“…The amount of CQ entrapped in the membrane was approximately 10% of the total (0.1 pmol/10 6 RBC vs. 0.98 pmol/10 6 RBC). As expected, and in agreement with Bray and coworkers [7], when CQ and RBC were coincubated in the presence of FP (10 lM), the drug bound to RBC was more than twice that measured in the absence of FP (2.32 pmol/10 6 RBC) and CQ entrapped in the membrane increased from 10% to approximately 50% (1.3 pmol/10 6 RBC).…”

“…The uptake of the drug by RBC was quantified by counting the radioactivity that is associated with the cell pellet or that disappeared from the incubation medium after centrifugation [5][6][7]. To avoid the laborious synthesis and risky use of radiolabeled compounds, we developed two simple, low-cost, and reproducible methods based on the high fluorescence of the quinoline ring to evaluate binding/internalization of the quinolines to human RBC.…”

Chloroquine Resistance Modulated in Vitro by Expression Levels of the Plasmodium falciparum Chloroquine Resistance Transporter

“…Percent reductions, presented as mean Ϯ S.E., were determined by normalization of the data from each parasite line at the different loading amounts. DV extracts were prepared and analyzed by Western blotting as described previously (40,41).…”

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

“…CQR parasites are reported to have lower DV CQ concentrations [13], although the DV volume is usually not directly measured complicating the calculation of DV CQ concentration. CQ resistance may be conferred by reducing access of CQ to FPIX [14,15]. Alternatively, CQ resistance may involve an energy-dependent, efflux process [16,17].…”