Unlike mammalian cells, malarial parasites are completely dependent on de novo pyrimidine metabolism. Even though these parasites do not use external uracil or uridine, orotic acid, an intermediate of pyrimidine biosynthesis, is successfully transported into the parasite and incorporated into parasite nucleic acids. On this basis, it was hypothesized that 5-fluoroorotate, a cytotoxic derivative of orotic acid, may be a potent and selective antimalarial agent. In vitro, 5-fluoroorotate caused 50% inhibition of the growth of Plasmodium falciparum at a concentration of 6.0 nM. In contrast, 5-fluorouracil, 5-fluorouridine, and 5-fluoro 2'-deoxyuridine were much less effective against malarial parasites. Chloroquine-susceptible and chloroquineresistant clones of P. falciparum were equally susceptible to 5-fluoroorotate. The toxicity of 5-fluoroorotate was evaluated on four human cell lines (HT-1080, IMR-90, HeLa S3, and HL-60) Two lines of evidence suggested that malarial parasites were unable to utilize exogenous preformed pyrimidines. First, early studies showed that radioactive carbon dioxide was incorporated into pyrimidine units of malarial nucleic acids while uracil, uridine, thymine, thymidine, cytidine, and deoxycytidine were not (3,13,35,39,40 species of Plasmodia were shown to carry enzymes for de novo pyrimidine biosynthesis but not for the salvage of preformed pyrimidines (10-12, 17, 19, 30, 32). In contrast, mammalian cells utilize preformed pyrimidine bases and nucleosides by salvage pathways (18,24,41).Any compound with schizonticidal activity has to be transported across the three membranes that separate the host plasma from the cytoplasm of the parasite. Gutteridge and Trigg demonstrated that radioactive orotic acid, an intermediate in de novo pyrimidine biosynthesis, was effectively transported and incorporated into Plasmodium nucleic acids (13). This led us to the hypothesis that an analog of orotic acid which inhibits de novo pyrimidine metabolism would inhibit the growth of malarial parasites.To test this hypothesis, we considered 5-fluoroorotate as a model antimalarial drug. The uptake and metabolism of 5-fluoro-pyrimidines have not been studied in malarial parasites. Nevertheless, we expected the fluorinated analog to be transported into the parasite as effectively as orotic acid because the substitution of a fluorine for a proton at the 5 position represents a minimum structural alteration. In mammalian cells, 5-fluoroorotate is metabolized to toxic 5-fluoropyrimidines with the aid of orotate phosphoribosyltransferase, orotidylate decarboxylase, and other enzymes of de novo pyrimidine metabolism (7,16,31). Since malarial parasites express enzymes of de novo pyrimidine biosynthesis, we inferred that P. falciparum, too, would have the capacity to activate 5-fluoroorotate to toxic metabolites.In this study, we examine the in vitro antimalarial properties of 5-fluoroorotate against the human pathogen P. falciparum in culture. We also report the effect of this compound on the proliferation of mam...
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