The role of the haemoglobin degradation pathway in the uptake and activity of antimalarial drugs in Plasmodium falciparum

Here, we describe data indicating that CQ uptake and activity in Plasmodium falciparum is dependent on binding to ferriprotoporpohyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of haemoglobin and stop the generation of FPIX also inhibit the uptake and activity of CQ. We have developed an efficient method of isolating pure and intact parasite food vacuoles and vesicles, from which proteinase-rich lysates were extracted. Using these food vacuolar enzymes and human haemoglobin as a substrate, we developed a sensitive cell-free assay method that allowed us to generate CQ and AQ binding sites. This binding in the cell-free assay accounts for the extensive accumulation (2500-fold) in intact cells (R² = 0.98 p < 0.001), the first experimental evidence which unequivocally demonstrates that binding to FPIX drives the cellular uptake and activity of CQ into P. falciparum-infected erythrocytes. Additionally, by comparison of the cell-free binding profiles of AQ and CQ at similar inhibitor concentrations, we show that the uptake of AQ is driven by the same mechanism (R² = 0.99 p < 0.001). Inhibition of CQ uptake by EIPA occurs because of displacement of CQ from FPIX, suggesting that CQ uptake is NHE-independent. Furthermore, our data suggests that while CQ binds with haemozoin, the level of interaction we measured (4%) is insufficient to justify its role in the saturable uptake and activity of the drug. We provide evidence that proteasome function may be important in the uptake and activity of CQ, which is FPIX-dependent. Besides being potent antimalarials in their own right, the specific proteasome inhibitors employed had profoundly inhibited the uptake and activity of CQ. We report that the uptake and activity of the artemisinin derivatives (arteflene (AF), artemether (ART) and dihydroartemisinin (DHA)) is dependent on free iron rather than FPIX, from haemoglobin digestion. For the first time we show that the artemisinin derivatives, e.g. DHA accumulate extensively (5500-fold, ~ 2 times higher than CQ) into P. falciparum-infected erythrocytes. They all bind to haemozoin but with intriguing differences: DHA is hardly incorporated into haemozoin at all whilst ART binds considerably, suggesting that there may be differences in the mode of action of these drugs. However, the degree of interaction seen in all the compounds is not sufficient to account for their cellular accumulation into intact cells nor is it sufficient to account for the antimalarial activity. The drugs inhibit FPIX crystallisation into haemozoin in vitro but due to the limited interaction of these drugs with FPIX and the slope of the dose/response relationship, the process is unlikely to be effectively inhibited in vivo. This is underlined by the demonstration that removal of iron markedly antagonises the activity of artemisinin derivatives, whereas removal of FPIX is only slightly antagonistic or has no effect at all. We postulate therefore, based on the limited interaction of these drugs with FPIX, that the inhibition of FPIX crystallisation into haemozoin may be the result of the inhibitory effects of the drugs on a phase-transfer catalyst, present in the parasite lysate and that the antiamalarial activity depends on activation by free iron.