Miriam Krugliak

Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action

We propose here a new and detailed model for the antimalarial action of chloroquine (CQ), based on the its ability to inhibit degradation of heme by glutathione. Heme, which is toxic to the malaria parasite, is formed when the intraerythrocytic malaria parasite ingests and digests inside its food vacuole its host cell cytosol, which consists mainly of hemoglobin. The parasite protects itself against the toxicity of heme by polymerizing some of it to insoluble hemozoin (HZ). We show here that in Plasmodium falciparum at the trophozoite stage only ca. 30% of the heme is converted into hemozoin. We suggest that nonpolymerized heme exits the food vacuole and is subsequently degraded by glutathione, as has been shown before for uninfected erythrocytes. Marginal amounts of free heme could be detected in the membrane fraction of infected cells but nowhere else. It is well established that CQ and amodiaquine (AQ) accumulate in the parasites food vacuole and inhibit heme polymerization, thereby increasing its efflux out of the food vacuole. We found that these drugs competitively inhibit the degradation of heme by glutathione, thus allowing heme to accumulate in membranes. Incubation of intact infected cells with CQ and AQ results in a marked increase in membrane-associated heme in a dose- and time-dependent manner, and a relationship exists between membrane heme levels and the extent of parasite killing. Heme has been shown to disrupt the barrier properties of membranes and to upset ion homeostasis in CQ-treated malaria-infected cells. In agreement with the predictions of our model, increasing the cellular levels of glutathione leads to increased resistance to CQ, whereas decreasing them results in enhanced sensitivity to the drug. These results insinuate a novel mechanism of drug resistance.

Characterization of permeation pathways appearing in the host membrane of Plasmodium falciparum infected red blood cells.

The host cell membrane of Plasmodium falciparum infected cells becomes permeabilized at the trophozoite stage. A variety of otherwise impermeant substances such as carbohydrates, polyols, amino acids and anions easily gain access to the cytosol of infected cells. Using the isotonic-hemolysis method or uptake of labeled substances, we characterized the new permeation pathways as pores of approximately 0.7 nm equivalent radius. The pores bear a positively charged character which facilitates movement of small anions and excludes cations, so that the ionic composition and osmotic properties of infected cells are not drastically altered. Substances of a molecular size similar to that of disaccharides are fully excluded. Substances of limiting size might be accommodated in the pore, provided they bear a side group of hydrophobic character. The new permeation pathways may provide a vital route for acquisition or release of essential nutrients or catabolites.

Intraerythrocytic Plasmodium falciparum utilizes only a fraction of the amino acids derived from the digestion of host cell cytosol for the biosynthesis of its proteins

It is generally accepted that intraerythrocytic malaria parasites digest hemoglobin to supply the amino acids needed for the synthesis of their own proteins. This view has never been quantitatively tested. In this investigation we have measured the degradation of hemoglobin and the increase in parasite protein content as a function of parasite maturation in cultures of Plasmodium falciparum. Defined parasite stages were obtained either from tightly synchronized cultures or from asynchronous cultures after density-fractionation. We showed that both hemoglobin digestion and total parasite protein content increased with parasite maturation, from the early trophozoite stage onwards, although the total protein content of the parasite remained significantly lower than that of other eukaryotes. The parasite digested up to 65% of the host cells hemoglobin but utilized only up to about 16% of the amino acids derived from hemoglobin digestion. This large discrepancy is profoundly puzzling particularly in view of the need to detoxify the cell from the large quantities of ferriprotoporphyrin IX and iron released during hemoglobin digestion.

New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes

The permeability properties of the membrane of human erythrocytes infected with malaria parasites (Plasmodium falciparum) were studied by the method of osmotic hemolysis. At the trophozoite stage, the host membrane becomes permeable to substrates such as sorbitol and glucose. The new permeability pathway is insensitive to most inhibitors of the glucose carrier, but is highly susceptible to the membrane dipole modifier phloretin. It is blocked by disaccharides and oligosaccharides, both of which are impermeant to non-infected and infected cells. It has an enthalpy of activation of solute penetration of 10 +/- 1 kcal mol-1 (range of 5-37 degrees C). It appears that new permeability pathways with pore-like properties are induced in parasitized cells. The pore(s) admit(s) neutral and anionic substances of a discrete molecular volume, but exclude(s) cations. Apparently they play an essential role in parasite development.

The fate of ferriprotorphyrin IX in malaria infected erythrocytes in conjunction with the mode of action of antimalarial drugs

The intraerythrocytic malaria parasite digests considerable amounts of its host cell cytosol, which consists mostly of hemoglobin. In order to avert the toxicity of ferriprotorphyrin IX (FP) thus produced, it is generally accepted that FP is polymerized to the non-toxic hemozoin. Investigating the fate of FP in cultured Plasmodium falciparum -infected human red blood cells, revealed a straight correlation between amounts of digested hemoglobin and hemozoin, but the latter contained less FP than produced. The efficacy of FP polymerization is stage-dependent, increasing with parasite maturation. Different strains display dissimilar efficacy in hemozoin production. Unpolymerized FP possibly exits the food vacuole and is degraded by glutathione, thus accounting for the low levels of free FP found in infected cells. 4-aminoquinoline antimalarials demonstrably form complexes with FP and inhibit hemozoin production in vitro. Chloroquine, amodiaquine, quinine and mefloquine were found to inhibit hemozoin production in intact infected cells, but only the first two drugs caused a dose-dependent accumulation of FP in the membrane fraction of infected cells that correlated well with parasite killing, due to the permeabilization of membranes to ions. This differential effect is explained by the ability of chloroquine and amodiaquine to inhibit the degradation of membrane-associated FP by glutathione and the incapacity of quinine and mefloquine to do so. This discrepancy implies that the antimalarial mode of action of chloroquine and amodiaquine is different in its mechanistic details from that of quinine and mefloquine and is compatible with the diametric sensitivity of most strains to chloroquine and mefloquine and the disparate interaction of these drugs with enhancers of their antimalarial action.

Digestion of the host erythrocyte by malaria parasites is the primary target for quinolinecontaining antimalarials

Intraerythrocytic malaria parasites feed on their host cell cytosol. We show that human red blood cells infected with the malaria parasite Plasmodium falciparum, produce free amino acids the composition of which resembles that of globin, the most abundant red blood cell protein. The rate of amino acid production is almost equal to the rate of efflux of these acids from the infected cell. Production of amino acids increases with parasite age: the rates of production at the young ring and the mature trophozoite stages were 3.3 and 13.5 nmol/10(8) infected cells per min at 37 degrees, respectively, compared with 0.04 nmol/10(8) cells per min in uninfected cells. The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, inhibit amino acid production at the same concentrations at which they inhibit parasite growth, but have no effect on the endogenous parasite protein degradation. We suggest that parasite feeding on host cell cytosol is the primary target for the antimalarial action of these drugs. Chloroquine accumulation, the rate of amino acid production by infected cells and the inhibitory effect of the drug, were determined simultaneously at the different stages of parasite development. At all stages the rate of amino acid production and chloroquine accumulation were directly related and both were inversely related to the inhibitory efficiency of the drug. The lysosomotropic agents methylamine and NH4Cl at millimolar concentrations also inhibit amino acid production, suggesting that the process is pH dependent and localized in the vacuole. Host cytosol degradation and drug accumulation both take place in the parasite food vacuole. Our observations imply that the metabolically dependent acidification of this parasite organelle is involved in both processes.

Kinetics of inhibition of glutathione-mediated degradation of ferriprotoporphyrin IX by antimalarial drugs

We have shown previously that chloroquine and amodiaquine inhibit the glutathione-dependent degradation of ferriprotoporphyrin IX (FP). We have also demonstrated that treatment of human erythrocytes infected with Plasmodium falciparum with chloroquine or amodiaquine results in a dose- and time-dependent accumulation of FP in the membrane fraction of these cells in correlation with parasite killing. High levels of membrane FP are known to perturb the barrier properties of cellular membranes, and could thereby irreversibly disturb the ion homeostasis of the parasite and cause parasite death. We here report on the effect of various 4-aminoquinolines, as well as pyronaridine, halofantrine and some bis-quinolines, on glutathione-mediated destruction of FP in aqueous solution, when FP was bound non-specifically to a protein, and when it was dissolved in human erythrocyte ghost membranes. We showed that all drugs were capable of inhibiting FP degradation in solution. The inhibitory efficacy of some drugs declined when FP was bound non-specifically to protein. Quinine and mefloquine were unable to inhibit the degradation of membrane-associated FP, in line with their inability to increase membrane-associated FP levels in malaria-infected cells following drug treatment. The discrepancy between chloroquine and amodiaquine on the one hand, and quinine and mefloquine on the other, is discussed in terms of the particular location of drugs and FP in the phospholipid membrane, and may suggest differences in the mechanistic details of the antimalarial action of these drugs.

Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.

Antimalarial Activities of Dermaseptin S4 Derivatives

ABSTRACT The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC50) in the micromolar range. Among these, the substituted S4 analog K4K20-S4 was the most potent (IC50 = 0.2 μM), while its shorter version, K4-S4(1–13)a, retained a considerable potency (IC50 = 6 μM). Both K4K20-S4 and K4-S4(1–13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K4K20-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K4K20-S4, the most hemolytic dermaseptin. The same trend was observed with K4-S4(1–13)a. The d isomers of K4K20-S4 or K4-S4(1–13)a were as active as the l counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasites plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.

Alkalinization of the food vacuole of malaria parasites by quinoline drugs and alkylamines is not correlated with their antimalarial activity

Quinoline-containing antimalarial drugs accumulate inside the acid food vacuole of the parasite where they inhibit the digestion of ingested host cell cytosol, and consequently, parasite growth. In order to verify whether this inhibition is caused by drug-induced alkalinization of the food vacuole, we investigated the accumulation of acridine orange (AO) as a vacuolar pH probe in intact Plasmodium falciparum-infected human erythrocytes as affected by the drugs chloroquine (CQ), 7H-quinoleine (7HQ), quinine (Q) and mefloquine (MQ). It was established by various criteria that AO accumulates primarily in the acid compartment(s) of the parasite as a function of the pH difference between it and the extracellular medium. This pH gradient was dissipated by the drugs in the rank order MQ greater than CQ greater than Q greater than 7HQ. The kinetics of vacuolar alkalinization and the concentration ranges at which it was observed imply that the monoprotic drugs MQ and Q exerted their effect mostly by translocating protons across the vacuolar membrane, i.e. they could cross the membrane as a protonated species, while the diprotic drugs CQ and 7HQ raised the vacuolar pH mostly by proton trapping. Similarly, hydrophobic alkylamines raised the vacuolar pH by proton translocation, while their relatively more polar congeners and ammonia did so by proton titration. However, the alkalinizing effect of each drug was observed at a concentration which was 1-2 orders of magnitude larger than the IC50 of its antimalarial effect. These results mean that vacuolar alkalinization is not the primary effect of antiparasitic action of quinoline antimalarials.