Targeting the Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery: Technologies for In Vitro β-Hematin Formation Assay
Babu L. Tekwani and Larry A. Walker
Pages 63-79 (17)
Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. The malaria parasite digests hemoglobin within the digestive vacuole through a sequential metabolic process involving multiple proteases. Massive degradation of hemoglobin generates large amount of toxic heme. Malaria parasite, however, has evolved a distinct mechanism for detoxification of heme through its conversion into an insoluble crystalline pigment, known as hemozoin. Hemozoin is identical to β-hematin, which is constituted of cyclic heme dimers arranged in an ordered crystalline structure through intermolecular hydrogen bonding. The exact mechanism of biogenesis of hemozoin in malaria is still obscure and is the subject of intense debate. Hemozoin synthesis is an indispensable process for the parasite and is the target for action of several known antimalarials. The pathway has therefore attracted significant interest for new antimalarial drug discovery research. Formation of β-hematin may be achieved in vitro under specific chemical and physiochemical conditions through a biocrystallization process. Based on these methods several experimental approaches have been described for the assay of formation of β-hematin in vitro and screening of compounds as inhibitors of hemozoin synthesis. These assays are primarily based on differential solubility and spectral characteristics of monomeric heme and β-hematin. Different factors viz., the malaria parasite lysate, lipids extracts, preformed β-hematin, malarial histidine rich protein II and some unsaturated lipids have been employed for promoting β-hematin formation in these assays. The assays based on spectrophotometric quantification of β-hematin or incorporation of 14C-heme yield reproducible results and have been applied to high throughput screening. Several novel antimalarial pharmacophores have been discovered through these assays.
malaria, plasmodium falciparum, hemozoin, beta-hematin, heme, antimalarials, quinolines
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA.