Accelerated denaturation of hemoglobin and the antimalarial action of chloroquine.

To study the antimalarial action of chloroquine, normal mouse erythrocytes were used as surrogates for erythrocytoid bodies. These bodies form in the endosomes of intraerythrocytic malaria parasites as they feed on their host and consist of erythrocyte cytoplasm enclosed in a vestige of the erythrocyte membrane. In suspensions of normal erythrocytes or lysates (equivalent to 5 microl of erythrocytes per ml in each case), hemoglobin underwent denaturation when it was incubated at 38 degrees C in 150 mM sodium acetate (pH 5). It is reasonable to assume that the same phenomenon occurs in acidic endosomes. Addition of 100 microM chloroquine to the incubation mixture caused the rate of hemoglobin denaturation to double to 40 nanomoles per hour per ml of packed erythrocytes. This effect required the presence of erythrocyte stroma and was inhibited by reducing the temperature to 24 degrees C or increasing the pH to 6. We propose that the primary antimalarial action of chloroquine is to bind to ferriprotoporphyrin IX (FP) and remove it from oxidized hemoglobin, thus producing toxic FP-chloroquine complexes and an excess of denatured globin. Furthermore, we suggest that these substances inhibit endosomal maturation and thereby cause hemoglobin accumulation in immature endosomes and masking of the lipids needed for FP dimerization. The term "masking" is used to signify that unsaturated lipids are present in parasitized erythrocytes but are specifically unavailable to promote FP dimerization.

Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs.

Two subclasses of quinoline antimalarial drugs are used clinically. Both act on the endolysosomal system of malaria parasites, but in different ways. Treatment with 4-aminoquinoline drugs, such as chloroquine, causes morphologic changes and hemoglobin accumulation in endocytic vesicles. Treatment with quinoline-4-methanol drugs, such as quinine and mefloquine, also causes morphologic changes, but does not cause hemoglobin accumulation. In addition, chloroquine causes undimerized ferriprotoporphyrin IX (ferric heme) to accumulate whereas quinine and mefloquine do not. On the contrary, treatment with quinine or mefloquine prevents and reverses chloroquine-induced accumulation of hemoglobin and undimerized ferriprotoporphyrin IX. This difference is of particular interest since there is convincing evidence that undimerized ferriprotoporphyrin IX in malaria parasites would interact with and serve as a target for chloroquine. According to the ferriprotoporphyrin IX interaction hypothesis, chloroquine would bind to undimerized ferriprotoporphyrin IX, delay its detoxification, cause it to accumulate, and allow it to exert its intrinsic biological toxicities. The ferriprotoporphyrin IX interaction hypothesis appears to explain the antimalarial action of chloroquine, but a drug target in addition to ferriprotoporphyrin IX is suggested by the antimalarial actions of quinine and mefloquine. This article summarizes current knowledge of the role of ferriprotoporphyrin IX in the antimalarial actions of quinoline drugs and evaluates the currently available evidence in support of phospholipids as a second target for quinine, mefloquine and, possibly, the chloroquine-ferriprotoporphyrin IX complex.

Chloroquine-induced masking of a lipid that promotes ferriprotoporphyrin IX dimerization in malaria.

Mice infected with the NYU-2 strain of Plasmodium berghei were used to study the effect of chloroquine on masking of a lipid that promotes ferriprotoporphyrin IX dimerization. More than 40% of this lipid was masked and unable to promote dimerization in membrane ghosts from erythrocytes of untreated, infected mice. Thus, preparations of membrane ghosts dimerized 57 +/- 6 nmol of ferriprotoporphyrin IX during a 2-h incubation, whereas the lipids extracted from these preparations dimerized 101 +/- 11 nmol of ferriprotoporphyrin IX (means +/- S.D. for four experiments). Exposure of membrane ghosts to sonication or cold significantly increased the extent of masking. In addition, chloroquine treatment of infected mice increased the extent of masking to approximately 90%. The lipid could be unmasked by extracting it into acetone or by aging erythrocyte membrane ghosts from untreated or chloroquine-treated, infected mice for 24 h at pH 7.4 and 25 degrees C. These findings indicate that masking and unmasking of a lipid is central to the regulation of ferriprotoporphyrin IX dimerization in malaria parasites. They also indicate that chloroquine impairs the function of this regulatory process.

Relationship of chloroquine-induced redistribution of a neutral aminopeptidase to hemoglobin accumulation in malaria parasites.

To study the relationship between neutral aminopeptidase activity and hemoglobin accumulation in malaria parasites, we treated mice infected with Plasmodium berghei NYU-2 with chloroquine intraperitoneally in doses ranging from 0.3 to 3 micromol per 25 g mouse. Preparations of infected erythrocytes (normalized to represent 1000 parasites per 1000 erythrocytes) hydrolyzed 1200 nmol of leucine-p-nitroanilide per minute per milliliter of packed erythrocytes, which was 10x more than that of uninfected preparations. The activity in infected preparations was distinguished by resistance to ferriprotoporphyrin IX and puromycin and susceptibility to inhibition by ethanol and Tris. Chloroquine treatment caused the activity in unwashed membrane ghosts of infected preparations to decrease by 50% despite an increase in total activity. Concomitantly, hemoglobin in washed membrane ghosts increased. Electron microscopy revealed that the hemoglobin was retained in endocytic vesicles. Chloroquine-induced redistribution of a neutral aminopeptidase may be the cause of hemoglobin accumulation in endocytic vesicles of malaria parasites.