In parts of southeast Asia, malaria parasites are showing resistance to the active ingredient in artemisinin-based antimalarial drugs. Delineation of a cell-signalling pathway might help to explain this phenomenon. See Letter p.683
Imagine an unfinished jigsaw puzzle. There are two separate parts, which are clearly from the same picture, but the pieces linking the two fragments elude you. This is the situation facing the malaria research community. The first part of the puzzle is the fact that, in regions of southeast Asia, Plasmodium falciparum malaria parasites in the ring stage (an early blood-borne stage of the parasite's life cycle) are showing varying degrees of resistance to dihydroartemisinin1,2, the active component of the artemisinin class of antimalarial drugs3. The second is the culprit in this resistance, mutations in a protein called P. falciparum Kelch13 (PfKelch13), about which little is known4, so that a mechanistic understanding of how resistance is conferred is missing. In this issue, Mbengue et al.5 (page 683) provide some of the missing pieces to connect these two parts of the puzzle.
Drug resistance can arise in various ways: a biological target can change or mutate so as to reduce a drug's ability to bind to it; the drug can become physically separated from the target, such that it can no longer exert its effect; or the biological target can increase to high enough levels to overcome the presence of the drug. Mbengue and colleagues show that dihydroartemisinin (DHA) is a potent inhibitor of a P. falciparum enzyme called phosphatidylinositol 3-kinase (PfPI3K), which phosphorylates an important phospholipid, phosphatidylinositol (PI), to produce phosphatidylinositol 3-phosphate (PI3P) in ring-stage parasites6. However, mutations in PfPI3K do not correlate with resistance to DHA, indicating that this is not a main cause of resistance. The authors provide no evidence that the malaria parasite separates itself from DHA by destroying it or expelling it from its site of action, nor that the parasite hides in a protective state until DHA has dissipated7. This leaves the hypothesis that an increase in PfPI3K levels might confer resistance — but how is this linked to PfKelch13?
Clues about the function of PfKelch13 come from the roles of similar proteins in mammalian cells. The human equivalent of PfKelch13 binds to protein targets to promote molecular 'tagging', in which ubiquitin molecules are added to the substrate (a process called polyubiquitination). These ubiquitin-tagged protein complexes can trigger other biochemical processes, but they are also recognized and broken down by a cellular structure called the proteasome. Mbengue and co-workers reasoned that PfKelch13 might function in a similar manner in malarial ring-stage parasites, with PfPI3K as its target (Fig. 1). They hypothesized that mutations in PfKelch13 inhibit its association with PfPI3K. With less PfKelch13–PfPI3K binding, there would be less PfPI3K polyubiquitination, resulting in less degradation and higher cellular levels of PfPI3K. Indeed, the authors confirmed that increased levels of PfPI3K and PI3P were associated with certain PfKelch13 mutations. They had connected the two jigsaw pieces.
This work contains some surprises. First, it seems that this mechanism of action is specific to ring-stage parasites, implying that other reported biological consequences of DHA affect the parasite at other stages of its life cycle. By contrast, it is commonly assumed that a drug that kills parasites in different stages of its life cycle is driven by a single biological target, rather than different targets affecting different stages.
Second, the authors demonstrated that DHA inhibits PfPI3K more potently than it does related human kinases, and that this inhibition is reversible. DHA has a weak oxygen–oxygen (peroxide) bond, and the researchers provide data to suggest that this bond, along with other molecular features of DHA, is necessary for reversible PfPI3K inhibition. This is interesting, because all other mechanisms by which DHA kills parasites involve breaking the peroxide bond8,9.
The third surprise is that DHA is different in structure from most types of molecule that inhibit kinases, although Mbengue et al. use modelling to provide some possible explanations for this discrepancy. It will be fascinating to carry out in-depth studies of interactions between DHA and PfPI3K.
Finally, PfKelch13 mutations correlate with only a slight increase in levels of PfPI3K in resistant parasites, but the extent of ring-stage resistance to DHA seems to be of a high magnitude, indicating that small alterations in PfPI3K levels can have a large effect on resistance. This suggests that resistance could be overcome by increasing the dose of artemisinin in drug combinations, to inhibit PfPI3K more potently — and such a study10 has been independently conducted.
Many questions remain unanswered, the most important of which is why parasites survive when levels of PfPI3K and PI3P increase. Furthermore, exactly how DHA inhibits PfPI3K remains to be seen. How do PfPI3K and PfKelch13 bind together, and why do mutations hinder this binding? Finally, why does polyubiquitination increase following PfPI3K–PfKelch13 binding, and does tagging lead to intracellular signalling events or solely to destruction of the complex by the proteasome?
Will this knowledge of the ring-stage target of DHA help us to design better drugs, as the authors claim? Yes and no. Because resistance is due to factors other than PfPI3K mutation, new PfPI3K inhibitors will not only need to be very potent, but also, crucially, must show potency against the key ring-stage-resistant parasites. We know that mutations in PfKelch13 can lead to less PfKelch13–PfPI3K binding, and that this leads to resistance. Only time and a much greater understanding of the underlying biology will tell us whether the parasite might overcome PfPI3K inhibitors through mutations that further reduce binding between PfKelch13 and PfPI3K. From a drug-development perspective, given that there are other antimalarial drugs11,12 in clinical development that have a weak peroxide bond but that differ from DHA, it will be intriguing to see whether they show inhibition of PfPI3K and resistance against the ring stage similar to those demonstrated by DHA13.
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