Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development.

Abstract

Over the last century, malaria deaths have decreased by more than 85%. Nonetheless, there were 405 000 deaths in 2018, mostly resulting from Plasmodium falciparum infection. In the 21st century, much of the advance has arisen from the deployment of insecticide-treated bed nets and artemisinin combination therapy. However, over the past few decades parasites with a delayed artemisinin clearance phenotype have appeared in Southeast Asia, threatening further gains. The effort to find new drugs is thus urgent. A prominent process in blood stage malaria parasites, which we contend remains a viable drug target, is hemozoin formation. This crystalline material consisting of heme can be readily seen when parasites are viewed microscopically. The process of its formation in the parasite, however, is still not fully understood.In early work, we recognized hemozoin formation as a biomineralization process. We have subsequently investigated the kinetics of synthetic hemozoin (β-hematin) crystallization catalyzed at lipid-aqueous interfaces under biomimetic conditions. This led us to the use of neutral detergent-based high-throughput screening (HTS) for inhibitors of β-hematin formation. A good hit rate against malaria parasites was obtained. Simultaneously, we developed a pyridine-based assay which proved successful in measuring the concentrations of hematin not converted to β-hematin.The pyridine assay was adapted to determine the effects of chloroquine and other clinical antimalarials on hemozoin formation in the cell. This permitted the determination of the dose-dependent amounts of exchangeable heme and hemozoin in P. falciparum for the first time. These studies have shown that hemozoin inhibitors cause a dose-dependent increase in exchangeable heme, correlated with decreased parasite survival. Electron spectroscopic imaging (ESI) showed a relocation of heme iron into the parasite cytoplasm, while electron microscopy provided evidence of the disruption of hemozoin crystals. This cellular assay was subsequently extended to top-ranked hits from a wide range of scaffolds found by HTS. Intriguingly, the amounts of exchangeable heme at the parasite growth IC50 values of these scaffolds showed substantial variation. The amount of exchangeable heme was found to be correlated with the amount of inhibitor accumulated in the parasitized red blood cell. This suggests that heme-inhibitor complexes, rather than free heme, lead to parasite death. This was supported by ESI using a Br-containing compound which showed the colocalization of Fe and Br as well as by confocal Raman microscopy which confirmed the presence of a complex in the parasite. Current evidence indicates that inhibitors block hemozoin formation by surface adsorption. Indeed, we have successfully introduced molecular docking with hemozoin to find new inhibitors. It follows that the resulting increase in free heme leads to the formation of the parasiticidal heme-inhibitor complex. We have reported crystal structures of heme-drug complexes for several aryl methanol antimalarials in nonaqueous media. These form coordination complexes but most other inhibitors interact noncovalently, and the determination of their structures remains a major challenge.It is our view that key future developments will include improved assays to measure cellular heme levels, better in silico approaches for predicting β-hematin inhibition, and a concerted effort to determine the structure and properties of heme-inhibitor complexes.