Artemisinin and its derivatives (ART) are the first-line treatment in malaria. ART clears parasitaemia and resolves symptoms very rapidly (1). ART monotherapy is however associated with high rates of recrudescence (2-3) requiring the combination of ART with an antimalarial of a different chemical class (ART combination therapy or ACT). Despite ACT's outstanding activity, recrudescences of Plasmodium falciparum infections have been reported (4-6).The mechanisms leading to recrudescence are not known. ART-mediated recrudescence is associated with the accumulation of dormant ring stage parasites and disruption of the parasite's cell cycle. The objective of this proposal is to elucidae the molecular mechanism of ART-induced dormancy. ART is a sesquiterpene trioxane lactone containing an endoperoxide bridge that is a source of free radicals. The reductive cleavage of the bridge is essential for ART anti-malarial activity. We propose that the oxidative stress elicited by ART activates an eIF2? (eukaryotic initiation factor 2?) kinase in some of the parasites, leading to a global inhibition of protein synthesis and the dormant state. We document a high level of eIF2? phosphorylation in ART-treated, dormant ring stages of P. berghei parasites, and show that dormancy is significantly extended by Salubrinal (Sal). Sal is a specific inhibitor of eIF2?-P phosphatase (7-8). We propose to identify the eIF2? kinase that is activated by the ART-induced oxidative stress. There are three eIF2? kinases in the Plasmodium genome: eIK1, eIK2 and PK4(9). To find which one responds to ART stress we will analyze the effect of ART on the kinase knockouts of P. berghei. We expect that the relevant mutants will not generate dormant rings. We have already obtained clones of eIK2 (-) parasites (8) and will test them for recrudescence. The PbeIK2 (-) blood stages will be injected in mice and the animals treated with ART. After the parasites are cleared we will check for recrudescence of the infection. We will generate eIK1 knockout clones and test them as above. HoweverPK4 (-) mutants cannot be generated because PK4 is essential for the development of erythrocytic stages (10-11). Evidence for a possible role of PK4 in ART-mediated dormancy will be obtained by analysis of PK4 transcription prior to and following treatment with sub-lethal doses of ART. We will search databases containing small chemicals for inhibitors of the "dormancy kinase". The identification of the kinase should facilitate future development of drugs that prevent dormancy and overcome ART treatment failures.
The most efficient drug to treat malaria infection is ART, but in many patients the infection recurrs. This is because a small number of ring stages enter a dormancy state and can re-enter the life cycle after days. We propose to elucidate the molecular mechanism of dormancy.