Biochemical characterisation of putrescine and spermidine uptake as a potential therapeutic target against the human malaria parasite, Plasmodium falciparum

Abstract

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy.