Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites.

A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.

In vitro and in vivo antiplasmodial activities of risedronate and its interference with protein prenylation in Plasmodium falciparum.

The increasing resistance of malarial parasites to almost all available drugs calls for the identification of new compounds and the detection of novel targets. Here, we establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages of Plasmodium falciparum (50% inhibitory concentration [IC50] of 20.3±1.0 µM). We also suggest a mechanism of action for risedronate against the intraerythrocytic stage of P. falciparum and show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate. Our in vivo experiments further demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasite Plasmodium berghei in mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.