Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magnetooptical detection of hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying hemozoin production over the intraerythrocytic cycle, we reveal that hemozoin formation is already initiated by ~ 6-12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6-10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.

Plasmodium falciparum parasites undergo multiple genome duplication events during their development. Within the intraerythrocytic stages, parasites encounter an oxidative environment and DNA synthesis necessarily proceeds under these circumstances. In addition to these conditions, the extreme AT bias of the P. falciparum genome poses further constraints for DNA synthesis. Taken together, these circumstances may allow appearance of damaged bases in the Plasmodium DNA . Here, we focus on uracil that may arise in DNA either via oxidative deamination or thymine‐replacing incorporation. We determine the level of uracil at the ring, trophozoite, and schizont intraerythrocytic stages and evaluate the base‐excision repair potential of P. falciparum to deal with uracil‐ DNA repair. We find approximately 7–10 uracil per million bases in the different parasite stages. This level is considerably higher than found in other wild‐type organisms from bacteria to mammalian species. Based on a systematic assessment of P. falciparum genome and transcriptome databases, we conclude that uracil‐ DNA repair relies on one single uracil‐ DNA glycosylase and proceeds through the long‐patch base‐excision repair route. Although potentially efficient, the repair route still leaves considerable level of uracils in parasite DNA , which may contribute to mutation rates in P. falciparum .

Synthesis of acidic new crown ethers containing a diarylphosphinic acid unit has been accomplished. The aromatic rings of the crown ethers were substituted with tertbutyl and nitro groups. Nitro substitution of the crown ethers was investigated. pKa determination of the new proton-ionizable crown ethers has been performed, showing the effect of the substituents of the aromatic rings on the acidity. An anomaly was discovered in the pKa values and an explanation was given based on quantum mechanical calculations and molecular dynamics simulations.

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

Understanding and characterizing the molecular background of the maintenance of genomic integrity might be a major factor in comprehending the exceptional ability of the malaria parasite, Plasmodium falciparum to adapt at a fast pace to antimalarials. A balanced nucleotide pool is an essential factor for high-fidelity replication. The lack of detailed studies on deoxynucleotide-triphosphate (dNTP) pools in various intraerythrocytic stages of Plasmodium falciparum motivated our present study. Here, we focused on the building blocks of DNA and utilized an EvaGreen-based dNTP incorporation assay to successfully measure the temporal dynamics of dNTPs in every intraerythrocytic stage and in drug-treated trophozoites. Our findings show that the ratio of dNTPs in the ring-stage parasites significantly differs from the more mature trophozoite and schizont stages. We were also able to detect dGTP levels that have never been shown before and found it to be the least abundant dNTP in all stages. Treatment with WR99210, a TS-DHFR inhibitor drug, affected not only dTTP, but also dGTP levels, despite its presumed selective action on pyrimidine biosynthesis. Results from our studies might assist in a better understanding of genome integrity mechanisms and may potentially lead to novel drug related aspects involving purine and pyrimidine metabolic targets.

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