Using cell fractionation and measurement of Fe(III)heme-pyridine, the antimalarial chloroquine (CQ) has been shown to cause a dose-dependent decrease in hemozoin and concomitant increase in toxic “free” heme in cultured Plasmodium falciparum that is directly correlated with parasite survival. Transmission electron microscopy techniques have further shown that heme is redistributed from the parasite digestive vacuole to the cytoplasm and that CQ disrupts hemozoin crystal growth, resulting in mosaic boundaries in the crystals formed in the parasite. Extension of the cell fractionation study to other drugs has shown that artesunate, amodiaquine, lumefantrine, mefloquine and quinine, all clinically important antimalarials, also inhibit hemozoin formation in the parasite cell, while the antifolate pyrimethamine and its combination with sulfadoxine do not. This study finally provides direct evidence in support of the hemozoin inhibition hypothesis for the mechanism of action of CQ and shows that other quinoline and related antimalarials inhibit cellular hemozoin formation.

Inhibition of hemozoin biocrystallization is considered the main mechanism of action of 4-aminoquinoline antimalarials including chloroquine (CQ) but cannot fully explain the activity of ferroquine (FQ) which has been related to redox properties and intramolecular hydrogen bonding. Analogues of FQ, methylferroquine (Me-FQ), ruthenoquine (RQ), and methylruthenoquine (Me-RQ), were prepared. Combination of physicochemical and molecular modeling methods showed that FQ and RQ favor intramolecular hydrogen bonding between the 4-aminoquinoline NH group and the terminal amino group in the absence of water, suggesting that this structure may enhance its passage through the membrane. This was further supported by the use of Me-FQ and Me-RQ where the intramolecular hydrogen bond cannot be formed. Docking studies suggest that FQ can interact specifically with the {0,0,1} and {1,0,0} faces of hemozoin, blocking crystal growth. With respect to the structure-activity relationship, the antimalarial activity on 15 different P. falciparum strains showed that the activity of FQ and RQ were correlated with each other but not with CQ, confirming lack of cross resistance. Conversely, Me-FQ and Me-RQ showed significant cross-resistance with CQ. Mutations or copy number of pfcrt, pfmrp, pfmdr1, pfmdr2, or pfnhe-1 did not exhibit significant correlations with the IC(50) of FQ or RQ. We next showed that FQ and Me-FQ were able to generate hydroxyl radicals, whereas RQ and me-RQ did not. Ultrastructural studies revealed that FQ and Me-FQ but not RQ or Me-RQ break down the parasite digestive vacuole membrane, which could be related to the ability of the former to generate hydroxyl radicals.

Several blood-feeding organisms, including the malaria parasite detoxify haem released from host haemoglobin by conversion to the insoluble crystalline ferriprotoporphyrin IX dimer known as haemozoin. To date the mechanism of haemozoin formation has remained unknown, although lipids or proteins have been suggested to catalyse its formation. We have found that beta-haematin (synthetic haemozoin) forms rapidly under physiologically realistic conditions near octanol/water, pentanol/water and lipid/water interfaces. Molecular dynamics simulations show that a precursor of the haemozoin dimer forms spontaneously in the absence of the competing hydrogen bonds of water, demonstrating that this substance probably self-assembles near a lipid/water interface in vivo.

No abstract

Formation of beta-hematin in acidic acetate solution has been investigated using quantitative infrared spectroscopy, X-ray diffraction, and scanning and transmission electron microscopy. The process occurs via rapid precipitation of amorphous (or possibly nanocrystalline) hematin, followed by slow conversion to crystalline beta-hematin. Definitive evidence that the reaction occurs during incubation in acetate medium, rather than during the drying stage, is provided by X-ray diffraction and infrared spectroscopy of the wet material. The reaction follows a sigmoidal function indicative of a process of nucleation and growth and was modeled using the Avrami equation. Reaction rates and the dimensionality of growth (as indicated by the value of the Avrami constant) are strongly influenced by stirring rate. The reaction follows Arrhenius behavior, and there is a strong dependence of both the rate constant and the Avrami constant on acetate concentration. Acetate may act as a phase transfer catalyst, solubilizing hematin and facilitating its redeposition as beta-hematin. The pH dependence of the process indicates that only the monoprotonated species of hematin is active in forming beta-hematin. The formation of beta-hematin closely parallels many mineralization processes, and this suggests that hemozoin formation may be a unique biomineralization process. Inferences are drawn with respect to the formation of hemozoin in vivo.