Lipid-mediated haemozoin (Hz) formation was investigated using experimental and computational techniques. Glycerolipids (predominantly diacylglycerols), not phospholipids, were found entrapped by Hz crystals isolated from Plasmodium falciparum and consequently are proposed to nucleate Hz crystals in vivo. The initial stages of Hz formation were investigated using molecular dynamics simulations which involved modelling ferrihaem species pertinent to Hz formation (π-π and μ-propionato dimers) at lipid-water interfaces comprising a representative glycerolipid 1,2-dioleoyl-sn-glycerol (DOG) or phospholipid 1,2dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Simulations showed that π-π dimers partition into both lipids and remain near the hydrated lipid-water interface. Dimer aggregation, which was only observed in DOG, is proposed to be crucial for nucleating Hz. Indeed, β-haematin (synthetic Hz) production was found experimentally to be greater in DOG than DOPC (65 vs. 24%). Density functional theory calculations indicate that iron coordination by the carboxylate group of the propionate side chain, not the propionic acid group, is feasible in the aggregate. This can only occur, however, if ferrihaem is in a zwitterionic form with an axial water ligand, one propionic acid and one propionate side chain. UV-visible spectroscopy indicates that the presence of solvent water molecules is crucial for maintaining this species over a tautomeric form consisting of two propionic acid groups and an axial hydroxide ligand. Anhydrous conditions promote the latter tautomer which is unlikely to form Hz. A mechanism describing the initial stages of Hz formation is proposed based on these findings, which supports proposals that Hz crystal growth occurs in the lipid subphase.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZPresistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.

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The biomineral haemozoin, or its synthetic analogue β-haematin (βH), has been the focus of several target-based screens for activity against Plasmodium falciparum parasites. Together with the known βH crystal structure, the availability of this screening data makes the target amenable to both structure-based and ligand-based virtual screening. In this study, molecular docking and machine learning techniques, including Bayesian and support vector machine classifiers, were used in sequence to screen the in silico ChemDiv 300k Representative Compounds library for inhibitors of βH with retained activity against P. falciparum. We commercially obtained and tested a prioritised set of inhibitors and identified the coumarin and iminodipyridinopyrimidine chemotypes as potent in vitro inhibitors of βH and whole cell parasite growth.