Steve Baldwin, Marie Parker, Ralph Hyde

Malaria, resulting from infection by protozoa of the genus Plasmodium, causes more than 300 million clinical cases per annum and leads to more than 1.5 million deaths world-wide in tropical and subtropical areas. Unfortunately, resistance to existing antimalarial drugs, including those most recently introduced such as artemisinin (Fig. 1), is rapidly spreading. Therefore the identification of new drug targets is an important goal. One such target might be purine salvage, because the intraerythrocytic stages of Plasmodium (Fig. 3) lack the enzymes required for de novo purine nucleotide biosynthesis and so are absolutely dependent upon host purines. Many mammalian cells similarly utilise salvage pathways, and take up the necessary purines via nucleoside transport proteins in the plasma membrane. In the hope of exploiting differences in the substrate and inhibitor specificity of human and parasite transporters for therapeutic purposes, a parallel program of research into the structure and function of mammalian and protozoan purine transporters is therefore underway in our laboratory.

Mammalian cells possess both active, sodium-linked nucleoside transporters (concentrative nucleoside transporters, CNTs) and passive, equilibrative nucleoside transporters. The latter are the most widespread and can be further subdivided into two subclasses on the basis of their sensitivity to the transport inhibitor nitrobenzylthioinosine (NBMPR). Transporters of the es (equilibrative sensitive) subclass are inhibited by nanomolar concentrations of NBMPR (K_i 0.1-10 nM). In contrast, transporters of the ei (equilibrative insensitive) subclass are relatively insensitive to NBMPR even at micromolar concentrations. In general, transporters of the es-type are also potently inhibited by the coronary vasodilators dipyridamole, dilazep and lidoflazine analogues such as draflazine, while ei-type transporters are less sensitive to these inhibitors. Our laboratory cloned the first examples of es- and ei-type transporters, hENT1 and hENT2 respectively, from human placenta in 1997, in collaboration with Jim Young and Carol Cass at the University of Alberta in Edmonton, Canada. Both hENT1 and hENT2 transport a broad range of purine and pyrimidine nucleosides, while hENT2 is also capable of transporting nucleobases. These proteins turned out to be members of a novel family that we have designated the equilibrative nucleoside transporter (ENT) family. Very recently, we have cloned a third member of the family, hENT3, but its substrate selectivity remains to be determined. All three transporters are predicted to possess 11 transmembrane (TM) segments, and we now have direct experimental evidence for much of the topology shown in Fig. 2.

Such evidence has included the results of glycosylation scanning mutagenesis experiments, and accessibility of cysteine residues to membrane-impermeable reagents. For example, with our Canadian collaborators we have recently shown that cysteine 140 in TM4 of the rat ei-type transporter rENT2, is responsible for inhibition of transport by extracellular p-chloromercuribenzene sulphonate, and so must be exofacial (Fig. 2). Interestingly, substrate protects rENT2 against inhibition, implying that TM4 contributes to the substrate translocation channel of the transporter. Through examination of the properties of chimeric transporters we had previously established that the TM3-6 region contains residues responsible for interactions of the ENTs with NBMPR and coronary vasodilators (Fig. 2).

In an attempt to identify the transporter(s) responsible for purine uptake by the causative agent of malaria, Plasmodium falciparum, we searched the unfinished sequence data arising from the Malaria Genome Sequencing Project for homologues of mammalian active and passive nucleoside transporters. This approach led to identification of fragments encoding a putative ENT homologue, and thence to isolation of a complete coding sequence for this protein, which we termed PfENT1. The 422-residue protein is predicted to adopt an 11-TM topology similar to that of its human counterparts and is expressed during the intraerythrocytic stage of the Plasmodium life cycle.

Expression of PfENT1 in Xenopus oocytes showed that it was indeed a transporter, catalysing the saturable uptake of a wide range of both nucleosides and nucleobases. Interestingly, PfENT1 was found to differ profoundly from its mammalian counterparts in several respects. For example, it was able efficiently to transport 3´-deoxynucleoside analogues such as the antiviral drug AZT, which are poor substrates for human ENTs. Similarly, it was not inhibited by characteristic inhibitors of the mammalian transporters such as NBMPR and coronary vasodilators. These findings suggest that it will be possible to develop inhibitors specific for the parasite transporter for use in anti-malarial chemotherapy. The dependence of Plasmodium on purines from the host, and our observation that PfENT1 appears to represent the sole route for uptake of purines by the parasite, suggest that the transporter itself may be a worthwhile chemotherapeutic target. Alternatively, its unique substrate specificity might be exploited to deliver cytotoxic purine analogues selectively into the parasite cytosol while sparing human cells.

Glenn McConkey; University of Leeds
Jim Young, Carol Cass; University of Alberta, Edmonton, Canada

Parker, M.D., Hyde, R.J., Yao, S.Y.M., McRobert, L., Cass, C.E., Young, J.D., McConkey, G.A. & Baldwin, S.A. (2000) Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for antimalarial chemotherapy. Biochem. J. 349, 67-75.

Yao, S.Y.,M., Sundaram, M., Chomey, E.G., Cass, C.E., Baldwin, S.A. and Young, J.D. (2001) Identification of Cys¹⁴⁰ in helix 4 as an exofacial cysteine residue within the substrate translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2. Biochem. J. 353, 387-393.

We acknowledge the support of BBSRC, MRC and The Wellcome Trust