Identification of the acidic compartment of Plasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine.

Abstract

Chloroquine (CQ), the most widely used antimalarial drug, is an acidotropic agent (De Duve, 1983) which accumulates to high levels in malaria-infected erythrocytes. A possible site of accumulation of the drug, the parasite's food vacuole, has been implicated in the mode of action of CQ. We have defined the various compartments of Plasmodium falciparumparasitized human erythrocytes in tenns of their pH and capacity to accumulate bases. The host cell and the parasite cytosols were differentially labeled in situ with pH-sensitive fluorescein, and the parasite food vacuole was revealed by tareting fluoresceinated dextran via endocytosis. The pH of the various compartments obtained from fluorescence excitation spectra were 6.9 for the cytosol of normal and infected erythrocytes and 5.2 for the parasite food vacuole. Determination of CQ and methylamine accumulation in infected erythrocytes, in conjunction with morphometric determination of the relative sizes of the various cellular compartments, provided an independent assessment of the vacuolar pH, yielding a value of 5.0-5.2. Perturbation of the proton gradient, either by lowering extracellular pH or by alkaliniation of the food vacuole with NH4CI or monensin, resulted in a concomitant and reversible decrease in accumulation of the probe. We conclude that drug accumulation in malariainfected erythrocytes can be fully accounted for by the steady-state proton gradients across the barriers delineating the various cellular compartments and the acidotropic properties of the drug.Key words: malaria/Plasmodium falciparum/intracellular pH/food vacuole/chloroquine/fluorescence Introducdton Chloroquine (CQ) is a widely used antimalarial drug. The development of resistance to this compound is one of the major reasons for the world wide resurgence of the disease during recent years (Peters, 1982). The mode of action of the drug has not been elucidated hitherto, let alone the mechanisms responsible for drug resistance. A major factor in the antimalarial action of the drug has been attributed to the ability of CQ-sensitive parasites to accumulate relatively high levels of the drug (Macomber et al., 1966). It has been proposed that this accumulation results from binding to a putative intraparasitic receptor, ferriprotoporphyrin IX (FP) with the consequent formation of a membrane lytic complex (Fitch, 1983). However, free FP has never been detected in malaria-infected red blood cells. Alternatively, by analogy with the lysosomotropic effects of weak bases on animal cells (Poole and Okhuma, 1981), it has been postulated that CQ accumulates to high levels in the food vacuoles of malaria parasites, causing their concomitant alkalinization (Homewood et al., 1972).In this study we defined the role of transmembrane proton gradients as the determining factor in CQ accumulation by Plasmodiumfalciparum-infected human erythrocytes (RBC). We have done this with the aid of fluorescent probes and labeling procedures which allowed us to gain access to various intracellular compartments of the in...

Inhibition of the peroxidative degradation of haem as the basis of action of chloroquine and other quinoline antimalarials

“…The location of this haem is not known, but as very little haem appears to escape the parasite (as judged by the total iron levels), much of the haem may be associated with membranes within the parasite. If even half of this amount of haem in located in the food vacuole, which represents less than 5 % of the total volume of the infected erythrocytes [30], a local haem concentration of a few millimolar could be reached. A concentration of haem in the millimolar range is sufficient to cause substantial membrane lysis and peroxidative damage, although the presence of specialized haem-binding proteins in the food vacuole environment may protect the food vacuole membrane from the lytic effects of the haem [31].…”

“…Chloroquine resistance is thought to arise, at least in part, from a decreased level of chloroquine uptake, although some studies suggest that an alteration in the drug target may also be involved [30,36,45]. Chloroquine resistance can be reversed in itro and in animal models by co-administration of compounds such as verapamil, chlorpromazine and desipramine [46][47][48].…”

Antimalarial drugs disrupt ion homeostasis in malarial parasites

“…Acidic compartments are organelles present in Plasmodium and several trypanosomatids (Docampo & Moreno 2001). For Plasmodium, it has been suggested that these organelles are the food vacuole in which hemoglobin is digested and antimalarial drugs such as chloroquine are accumulated (Yayon et al 1984). Chloroquine is though to act by preventing the monomeric hemin from forming the inert and insoluble polymer hemozoin which is derived from digestion of hemoglobin in the acidic vacuole (Slater & Ceram 1992).…”

Drug Resistance in Malaria-in a nutshell

“…The situation has presently worsened to such an extent that the genotype of malarial parasites, so obtained from the Central Americas where chloroquine was earlier believed to be effective, has also begun to exhibit features of resistance (Elbadry et al, 2013). It has been well-established that chloroquine, in its uncharged form, freely diffuses into the erythrocyte and subsequently into the digestive vacuole (DV).Inside the DV, chloroquine after undergoing protonation, is unable to penetrate the DV membrane back (Homewood et al, 1972;Yayon et al, 1984)). It then goes on to bind to the toxic hematin molecule and prevents the development of the non-toxic haemozoin crystal.…”