thapsigargin and Malaria--Falciparum

Infection by Plasmodium falciparum is the leading cause of malaria in humans. The parasite contains a unique and essential plastid-like organelle called the apicoplast that, similar to the mitochondria and chloroplast, houses its own genome that must undergo replication and repair. The putative apicoplast replicative DNA polymerase, POM1, has no direct orthologs in mammals, making the P. falciparum POM1 an attractive antimalarial drug target. Here, we report on a fluorescent high-throughput DNA polymerase assay that relies on the ability of POM1 to perform strand-displacement synthesis through the stem of a DNA hairpin substrate, thereby separating a Cy3 dye from a quencher. Assay-validation experiments were performed using 384-well plates and resulted in a signal window of 7.90 and aZ' factor of 0.71. A pilot screen of a 2880-compound library identified 62 possible inhibitors that cause more than 50% inhibition of polymerase activity. The simplicity and statistical robustness of the assay suggest it is well suited for the screening of novel apicoplast polymerase inhibitors that may serve as lead compounds in antimalarial drug-discovery efforts.

Topics: Antimalarials; Apicoplasts; Chloroplasts; DNA; DNA-Directed DNA Polymerase; Drug Discovery; Exonucleases; Humans; Kinetics; Malaria, Falciparum; Mitochondria; Multienzyme Complexes; Nucleic Acid Synthesis Inhibitors; Peptide Library; Plasmodium falciparum; Protozoan Proteins; Spectrometry, Fluorescence

RBX11160 (OZ277) is a fully synthetic peroxidic antimalarial in clinical development. To study the possible mechanisms of action of RBX11160, we have examined its ability to inhibit PfATP6, a sarcoplasmic reticulum calcium ATPase and proposed target for semisynthetic peroxidic artemisinin derivatives. RBX11160 inhibits PfATP6 (apparent half-maximal inhibitory constant=7,700 nM) less potently than artemisinin (79 nM). Inhibition of PfATP6 is abrogated by desferrioxamine, an iron-chelating agent. Consistent with this finding, the killing of Plasmodium falciparum organisms by RBX11160 in vitro is antagonized by desferrioxamine. Artesunate and RBX11160 also act antagonistically against P. falciparum in vitro. A fluorescent derivative of RBX11160 localizes to the parasite cytosol in some parasites and to the food vacuole in other parasites. These data demonstrate that there are both similarities and differences between the antimalarial properties of RBX11160 and those of semisynthetic antimalarials such as artesunate and artemisinin.

Topics: Animals; Antimalarials; Artemisinins; Artesunate; Calcium-Transporting ATPases; Heterocyclic Compounds, 1-Ring; Malaria, Falciparum; Microscopy, Confocal; Peroxides; Plasmodium falciparum; Sesquiterpenes; Spiro Compounds

The regulation of intracellular Ca(2+) in the intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, was investigated using parasites 'isolated' from their host cells by saponin-permeabilisation of the erythrocyte membrane. The isolated parasites maintained tight control over their resting cytosolic Ca(2+) concentration which ranged from approximately 100 nM in the absence of extracellular Ca(2+) to approximately 700 nM in the presence of 1 mM extracellular Ca(2+). The parasite has two functionally discrete intracellular Ca(2+) stores. One is an 'endoplasmic reticulum (ER)-like' store, the other an 'acidic store'. The ER-like store was discharged by cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) of animal and plant cells, but not by thapsigargin (TG), a more specific inhibitor of SERCAs of animal cells. The acidic store was discharged by nigericin and by NH(4)(+). The amount of Ca(2+) in the ER-like store increased with increasing extracellular Ca(2+) concentration, whereas the amount of Ca(2+) in the acidic store did not. Ca(2+) released from the ER-like store by CPA was cleared from the parasite cytosol by uptake into the acidic store (over a range of extracellular Ca(2+) concentrations), consistent with the acidic store serving as a Ca(2+) reservoir within the intracellular parasite.

Topics: Animals; Calcium; Culture Media; Erythrocytes; Homeostasis; Hydrogen-Ion Concentration; Indoles; Malaria, Falciparum; Nigericin; Plasmodium falciparum; Quaternary Ammonium Compounds; Thapsigargin