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Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described. This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin. The interaction between atovaquone and proguanil was synergistic against atovaquonesensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure. While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested. The relevance of in vitro interactions to in vivo treatment is discussed.Combination drug regimens for the treatment of cancer, AIDS, and tuberculosis often achieve a therapeutic efficacy greater than that achieved with monotherapy. Other benefits may include decreased toxicity, the delay or prevention of drug resistance development, and the favorable effects of synergistic drug interactions. Antimalarial drug resistance is becoming a major public health disaster in many areas of the tropical world (31). In the search for effective combination regimens, Malarone (the combination of atovaquone and proguanil), LapDap (the combination of chlorproguanil and dapsone), and Coartem (the combination of artemether and lumefantrine) have recently become available or are in development. Other combination therapies involving artemisinin derivatives such as artesunate-mefloquine have many benefits and are thought to be very effective against drug resistance (31). It is hoped that combination chemotherapy will delay the onset of resistance to new agents and reduce the effects of resistance to existing agents (24,30).In vitro interactions between antiplasmodials, as represented in isobolograms, provide an essential background for clinical studies. However, they do not necessarily determine the efficacy of a combination in the host, since this also depends on pharmacokinetic characteristics. Synergism, indifference (addition), and antagonism are the expected outcomes of drug-drug interactions. The antimalarial Malarone (GlaxoSmithKline) is a recently introduced combination regimen for the treatment (18, 19) and prophylaxis (14, 23) of falciparum malaria. It has been previously reported that the synergistic interaction between the components of Malarone, atovaquone (ATV) and proguanil (PG), is reduced to an indifferent interaction in ATV-resistant Plasmodium yoelii parasites (25). However, this interaction has been demonstrated to remain synergistic in ATV-resistant isolate C2B of P. falciparum derived from a clinical trial of ATV alone, although the parasites showed a 95-fold decrease in sensitivity to ATV compared to that of the pretreatment isolate (2). Despite recent reports of Malarone treatment failures (7,8), no studies have investigated the in vitro interaction between these two drugs against isolates fr...

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