Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum

Previous studies have shown that 100 nM 5-fluoroorotate (5-FO) is sufficient to block the in vitro proliferation of Plasmodium falciparum without causing toxicity to mammalian cells. In anticipation of potential drug resistance, a study was undertaken to identify P. falciparum cells that would proliferate in the presence of 5-FO. About 3 x 10⁶ UV-irradiated as well as nonirradiated parasites were subjected to a one-step selection with 100 nM 5-FO both in the absence and in the presence of preformed pyrimidines (uracil, uridine, thymine, and thymidine). The P. falciparum cells that emerged after 3 weeks were cloned, and the 90% inhibitory concentration of 5- FO for the cloned cells was found to be 100- to 400-fold greater than that for the parent cell line. Two clones that were further characterized retained resistance to 5-FO even after prolonged propagation in culture without drug pressure. Since the mutants were not cross-resistant to 5-fluorouracil or to dihydrofolate reductase inhibitors, it was unlikely that alteration of thymidylate synthase or overproduction of the bifunctional dihydrofolate reductase-thymidylate synthase was responsible for 5-FO resistance. Similarly, resistance was not due to the expression of a pyrimidine salvage pathway since the cells were not pyrimidine auxotrophs, they did not show increased utilization of pyrimidine nucleosides, and they did not show increased susceptibility to 5-fluoropyrimidine nucleosides. However, the mutant parasites did show as much as a 40-fold lower level of utilization of exogenous radioactive orotate, suggesting that the decreased susceptibility to 5-FO was due to decreased transport or decreased activation of this compound to fluorinated nucleotides. When the selection experiments were repeated, without mutagenesis, in the presence of 10^-7 M 5-FO with fewer than 10⁶ parasites or in the presence of more than 10^-7 M 5-FO with more than 10⁸ parasites, viable mutants could not be recovered from the cultures. The implications of these findings for the in vivo use of 5-FO for malaria chemotherapy are discussed.