Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects on P. falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S. cerevisiae growth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. An hnm1⌬ yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound. Based on these data for S. cerevisiae, labeling studies in P. falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies in P. falciparum and in vitro studies with recombinant P. falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity.Plasmodium falciparum, the causative agent of the most severe form of human malaria, is responsible for over 2 million deaths annually (49). The emergence of parasites resistant to the most commonly used antimalarials, such as chloroquine, mefloquine, and pyrimethamine, has hampered efforts to combat this disease, emphasizing the need to develop new compounds for malaria treatment and prophylaxis.The rapid multiplication of P. falciparum in human erythrocytes requires active synthesis of new membranes. Therefore, developing drugs that target membrane biogenesis is an attractive strategy to fight malaria. The finding that quaternary ammonium choline analogs inhibit the synthesis of new membranes and block the growth of the parasite has stimulated efforts to develop this class of compounds for antimalarial chemotherapy (4-6, 11, 12). With a combinatorial chemistry approach to obtain compounds with greater specificity and potency against malaria, more than 420 choline analogs have been synthesized, and their structures were optimized with quantitative structural-activity criteria (11,12,44,45). These compounds displayed a very close correlation between inhibition of parasite growth in vitro and specific inhibition of parasite membrane biogenesis (1,47,48).
