Molecular targets of 5-fluoroorotate in the human malaria parasite, Plasmodium falciparum

Abstract

5-Fluoroorotate is known to have potent antimalarial activity against chloroquine-susceptible as well as chloroquine-resistant dones of Plasmodium fakiparum. It was hypothesized that this activity was mediated through synthesis of 5-fluoro-2'-deoxyuridylate, an inactivator of thymidylate synthase, or through incorporation of 5-fluoropyrimidine residues into nudeic acids. Treatment of P. fakciparum in culture with 100 nM 5-fluoroorotate resulted in rapid inactivation of malarial thymidylate synthase activity. A 50%o loss of thymidylate synthase activity as well as a 50%o decrease in parasite proliferation were seen with 5 nM 5-fluoroorotate. Dihydrofolate reductase activity, which resides on the same bifunctional protein as thymidylate synthase, was not affected by 5-fluoroorotate treatment. Incubation of malarial parasites with 3 to 10 FM radioactive 5-fluoroorotic acid for 48 h resulted in significant incorporation of radioactivity into the RNA fraction of P. falciparum; approximately 9%o of the uridine residues were substituted with 5-fluorouridine.However, compared with the 50%o inhibitory concentrations of 5-fluoroorotate, a 1,000-fold higher concentration of the pyrimidine analog was required to see significant modification of RNA molecules. Results of these studies are consistent with the hypothesis that thymidylate synthase is the primary target of 5-fluoroorotate in malarial parasites.The emergence of chloroquine-resistant variants of Plasmodiumfalciparum across the globe has intensified searches for new effective treatments against malaria (4,16,24,32,33,40,51). It has long been known that, unlike mammalian cells, malarial parasites are completely dependent on de novo pyrimidine biosynthesis (15,39,45). Orotic acid is the only preformed pyrimidine that is utilized by malarial parasites (19). This, in turn, has inspired evaluation of orotic acid analogs as potential antimalarial agents (18,27,(35)(36)(37)(38). 5-Fluoroorotate inhibited the in vitro proliferation of chloroquine-susceptible as well as chloroquine-resistant P. falciparum with a 50% inhibitory concentration of 6 nM (38). Mammalian cells in culture were not affected by this concentration of 5-fluoroorotate, particularly in the presence of uridine. The antimalarial activity of 5-fluoroorotate was also evaluated in mice infected with a lethal strain ofPlasmodium yoelii (18,36). A combination of 5-fluoroorotate and uridine could eliminate malarial parasites without obvious toxicity to mice.To fully understand the potent and selective antimalarial activity of 5-fluoroorotate, it was important to first identify the principal metabolic targets of this agent and then to determine their relative contributions to the selective cytotoxicity associated with this compound.Since the synthesis of the first 5-fluorinated pyrimidines, four decades of intense research have provided us with an enormous amount of information on the biochemical mechanisms underlying the antiproliferative activity of 5-fluoropyrimidines (21, 31). It appears that, depending on the...

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

“…3C), but fully killing parasites required 96 h (Fig. 3D), as measured by using clonal dilution methods (14). Given that a typical P. falciparum-infected mouse has ∼10 8 parasites, the same load used in the in vitro cidality assays, it appears that (+)-SJ733 acts at least fourfold faster in vivo than it does in vitro.…”

Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum

“…In light of recent evidence that the antimalarial activity of 5-FO probably involves inactivation of malarial thymidylate synthase (28), one would have expected resistance to occur through several potential mechanisms. However, in all cases examined, the only significant metabolic difference between 5-FO-susceptible and 5-FO-resistant parasites was in their ability to utilize exogenous radioactive orotic acid.…”

“…In the past it has been demonstrated that a combination of 5-fluoroorotate (5-FO) and uridine allowed for a 1,000-fold difference in the 50% inhibitory concentrations (IC50s) for malarial and mammalian cells in culture (27). The nanomolar concentrations of 5-FO that caused the inhibition of parasite proliferation also inactivated malarial thymidylate synthase activity and triggered cell death (28,32).…”

Antimalarial drug discovery: old and new approaches

“…Attempts are now underway to develop improved DHFR inhibitor antimalarials, including biguanides related to proguanil (Kinyanjui et al, 1999). In addition, inhibitors of other folate pathway (Rathod et al, 1992). Glycolysis is another cytosolic pathway of interest.…”