Selective killing of the human malaria parasite Plasmodium falciparum by a benzylthiazolium dye

Abstract

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. The most virulent form of the disease is caused by Plasmodium falciparum which infects hundreds of millions of people and is responsible for the deaths of 1-2 million individuals each year. An essential part of the parasitic process is the remodeling of the red blood cell membrane and its protein constituents to permit a higher flux of nutrients and waste products into or away from the intracellular parasite. Much of this increased permeability is due to a single type of broad specificity channel variously called the new permeation pathway (NPP), the nutrient channel, and the Plasmodial surface anion channel (PSAC). This channel is permeable to a range of low molecular weight solutes both charged and uncharged, with a strong preference for anions. Drugs such as furosemide that are known to block anion-selective channels inhibit PSAC. In this study, we have investigated a dye known as benzothiocarboxypurine, BCP, which had been studied as a possible diagnostic aid given its selective uptake by P. falciparum infected red cells. We found that the dye enters parasitized red cells via the furosemide-inhibitable PSAC, forms a brightly fluorescent complex with parasite nucleic acids, and is selectively toxic to infected cells. Our study describes an antimalarial agent that exploits the altered permeability of Plasmodium-infected red cells as a means to killing the parasite and highlights a chemical reagent that may prove useful in high throughput screening of compounds for inhibitors of the channel.

Figure 1

Structure of PUR-1, also known as benzothiocarboxypurine (BCP), and two structural analogs, Pyr-1 (Pyrimidino-1) and Pyridino-1.

Figure 2

Chemical synthesis of PUR-1.

Figure 3

Selective staining of PRBC (and not normal red cells) by PUR-1, as visualized by confocal fluorescence microscopy.

Figure 4

Stage-dependent staining of synchronous PRBC by PUR-1.

Figure 5

Inhibition of PUR-1 staining of PRBC by the anion selective channel blocker, furosemide. Top left, normal red cells stained with PUR-1; top right, PRBC stained with PUR-1 (asynchronous parasitemia of ≈8%); bottom left, PRBC stained with PUR-1 in the presence of 10μM furosemide (1.51% staining); and PRBC stained with PUR-1 in the presence of 100μM furosemide (1.1% staining).

Figure 6

Inhibition of PUR-1 staining of PRBC by folic acid. Conditions were as described above for PUR-1 and furosemide combinations. Top left, normal red cells stained with PUR-1; top right, PRBC stained with PUR-1 (asynchronous parasitemia of 5.93% staining positive); bottom left, PRBC stained with PUR-1 in the presence of 500μM furosemide (1.53% staining); and PRBC stained with PUR-1 in the presence of 500μM folic acid (1.43% staining).