Henry M. Staines

Artemisinins: their growing importance in medicine

Artemisinins are derived from extracts of sweet wormwood (Artemisia annua) and are well established for the treatment of malaria, including highly drug-resistant strains. Their efficacy also extends to phylogenetically unrelated parasitic infections such as schistosomiasis. More recently, they have also shown potent and broad anticancer properties in cell lines and animal models. In this review, we discuss recent advances in defining the role of artemisinins in medicine, with particular focus on their controversial mechanisms of action. This safe and cheap drug class that saves lives at risk from malaria can also have important potential in oncology.

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum

A recent study on malaria‐infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host‐ or parasite‐derived and their molecular nature have not been resolved. Here we report a comparison of a malaria‐induced anion channel with an endogenous anion channel in Plasmodium falciparum‐infected human RBCs. Ion channel activity was measured using the whole‐cell, cell‐attached and excised inside‐out configurations of the patch‐clamp method. Parasitised RBCs were cultured in vitro, using co‐cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (∼15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I− > Br− > Cl−. In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (∼80 pS, outward conductance) was spontaneously active. The host membrane of malaria‐infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria‐infected RBCs were shown to have a low open‐state probability (Po) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole‐cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up‐regulated by the malaria parasite P. falciparum.

Increased permeability of the malaria-infected erythrocyte to organic cations.

The human malaria parasite, Plasmodium falciparum, induces in the plasma membrane of its host red blood cell new permeation pathways (NPP) that allow the influx of a variety of low molecular weight solutes. In this study we have demonstrated that the NPP confer upon the parasitised erythrocyte a substantial permeability to a range of monovalent organic (quaternary ammonium) cations, the largest having an estimated minimum cross-sectional diameter of 11-12 A. The rate of permeation of these cations showed a marked dependence on the nature of the anion present, increasing with the lyotropicity of the anion. There was no clear relationship between the permeation rate and either the size or the hydrophobicity of these solutes. However, the data were consistent with the rate of permeation being influenced by a combination of these two factors, with the pathways showing a marked preference for the relatively small and hydrophobic phenyltrimethylammonium ion over larger or less hydrophobic solutes. Large quaternary ammonium cations inhibited flux via the NPP, as did long-chain n-alkanols. For both classes of compound the inhibitory potency increased with the size and hydrophobicity of the solute. This study extends the range of solutes known to permeate the NPP of malaria-infected erythrocytes as well as providing some insight into the factors governing the rate of permeation.

Artemisinins and the biological basis for the PfATP6/SERCA hypothesis.

With the advent of artemisinin resistance, it is timely to revisit the biological basis for the controversial suggestion that this class of antimalarial exerts its activity by inhibiting a calcium ATPase (PfATP6) that is most similar to sarcoplasmic endoplasmic reticulum calcium ATPases (SERCAs). Herein, evidence is discussed that relates to this hypothesis as alternative suggestions for how artemisinins might act have been reviewed elsewhere.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum‐infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole‐cell configuration of the patch‐clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole‐cell recordings in malaria‐infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4‐fold and inward currents by approximately 2‐fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

Functional state of the plasma membrane Ca2+ pump in Plasmodium falciparum-infected human red blood cells.

1 The active Ca2+ transport properties of malaria‐infected, intact red blood cells are unknown. We report here the first direct measurements of Ca2+ pump activity in human red cells infected with Plasmodium falciparum, at the mature, late trophozoite stage. 2 Ca2+ pump activity was measured by the Co2+‐exposure method adapted for use in low‐K+ media, optimal for parasitised cells. This required a preliminary study in normal, uninfected red cells of the effects of cell volume, membrane potential and external Na+/K+ concentrations on Ca2+ pump performance. 3 Pump‐mediated Ca2+ extrusion in normal red cells was only slightly lower in low‐K+ media relative to high‐K+ media despite the large differences in membrane potential predicted by the Lew‐Bookchin red cell model. The effect was prevented by clotrimazole, an inhibitor of the Ca2+‐sensitive K+ (KCa) channel, suggesting that it was due to minor cell dehydration. 4 The Ca2+‐saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax) of parasitised red cells was marginally inhibited (2‐27 %) relative to that of both uninfected red cells from the malaria‐infected culture (cohorts), and uninfected red cells from the same donor kept under identical conditions (co‐culture). Thus, Ca2+ pump function is largely conserved in parasitised cells up to the mature, late trophozoite stage. 5 A high proportion of the ionophore‐induced Ca2+ load in parasitised red cells is taken up by cytoplasmic Ca2+ buffers within the parasite. Following pump‐mediated Ca2+ removal from the host, there remained a large residual Ca2+ pool within the parasite which slowly leaked to the host cell, from which it was pumped out.

Passive Ca2+ Transport and Ca2-dependent K+ transport in Plasmodium falciparum-infected red cells

Abstract. Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca2+ permeability. The magnitude of the increase is greater than that normally required to activate the Ca2+-dependent K+ channel (KCa channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the KCa channel and the Ca2+ permeability properties of pRBC. For pRBC suspended in media containing Ca2+, KCa channel activation was elicited by treatment with the Ca2+ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca2+ into pRBC in which the Ca2+ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (1013 cells · hr)−1), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca2+ influx increased to over 1 mmol (1013 cells · hr)−1, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca2+ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni2+. Possible roles for this pathway in pRBC are considered.

Malaria parasite Plasmodium gallinaceum up‐regulates host red blood cell channels

The properties of the malaria parasite‐induced permeability pathways in the host red blood cell have been a major area of interest particularly in the context of whether the pathways are host‐ or parasite‐derived. In the present study, the whole‐cell configuration of the patch‐clamp technique has been used to show that, compared with normal cells, chicken red blood cells infected by Plasmodium gallinaceum exhibited a 5–40‐fold larger membrane conductance, which could be further increased up to 100‐fold by raising intracellular Ca2+ levels. The increased conductance was not due to pathways with novel electrophysiological properties. Rather, the parasite increased the activity of endogenous 24 pS stretch‐activated non‐selective cationic (NSC) and 62 pS calcium‐activated NSC channels, and, in some cases, of endogenous 255 pS anionic channels.

Use of a Selective Inhibitor To Define the Chemotherapeutic Potential of the Plasmodial Hexose Transporter in Different Stages of the Parasite's Life Cycle

ABSTRACT During blood infection, malarial parasites use d-glucose as their main energy source. The Plasmodium falciparum hexose transporter (PfHT), which mediates the uptake of d-glucose into parasites, is essential for survival of asexual blood-stage parasites. Recently, genetic studies in the rodent malaria model, Plasmodium berghei, found that the orthologous hexose transporter (PbHT) is expressed throughout the parasites development within the mosquito vector, in addition to being essential during intraerythrocytic development. Here, using a d-glucose-derived specific inhibitor of plasmodial hexose transporters, compound 3361, we have investigated the importance of d-glucose uptake during liver and transmission stages of P. berghei. Initially, we confirmed the expression of PbHT during liver stage development, using a green fluorescent protein (GFP) tagging strategy. Compound 3361 inhibited liver-stage parasite development, with a 50% inhibitory concentration (IC50) of 11 μM. This process was insensitive to the external d-glucose concentration. In addition, compound 3361 inhibited ookinete development and microgametogenesis, with IC50s in the region of 250 μM (the latter in a d-glucose-sensitive manner). Consistent with our findings for the effect of compound 3361 on vector parasite stages, 1 mM compound 3361 demonstrated transmission blocking activity. These data indicate that novel chemotherapeutic interventions that target PfHT may be active against liver and, to a lesser extent, transmission stages, in addition to blood stages.

Exploiting the therapeutic potential of Plasmodium falciparum solute transporters.

Mammalian transport proteins are essential components of cellular function that have been very successfully exploited as drug targets. Over the past few years, a small but increasing number of Plasmodium transport proteins have been validated as being crucial for parasite survival. This is an essential early step towards identifying new targets for urgently needed antimalarial drugs. Presented here is an overview of our current understanding of the transport processes used by Plasmodium parasites, with an emphasis on their therapeutic potential. It demonstrates the largely untapped potential of targeting these important pathways (including P-type ATPases, ABC transporters and K+ channels) and highlights where these parasites might be most vulnerable to intervention.