Dihydroorotase and dihydroorotate dehydrogenase, two enzymes of the pyrimidine 
biosynthetic pathway, were purified from Plasmodium berghei to apparent 
homogeneity. Orotate and a series of 5-substituted derivatives were found to 
inhibit competitively the purified enzymes from the malaria parasite. The order 
of effectiveness as inhibitors on pyrimidine ring cleavage reaction for 
dihydroorotase was 5-fluoro orotate greater than 5-amino orotate, 5-methyl 
orotate greater than orotate greater than 5-bromo orotate greater than 5-iodo 
orotate with Ki values of 65, 142, 166, 860, 2200 and greater than 3500 microM, 
respectively. 5-Fluoro orotate and orotate were the most effective inhibitors 
for dihydroorotate dehydrogenase. In vitro, 5-fluoro orotate and 5-amino orotate 
caused 50% inhibition of the growth of P. falciparum at concentrations of 10 nM 
and 1 microM, respectively. In mice infected with P. berghei, these two orotate 
analogs at a dose of 25 mg/kg body weight eliminated parasitemia after a 4-day 
treatment, an effect comparable to that of the same dose of chloroquine. The 
infected mice treated with 5-fluoro orotate at a lower dose of 2.5 mg/kg had a 
95% reduction in parasitemia. The effects of the more potent compounds tested in 
combination with inhibitors of other enzymes of this pathway on P. falciparum in 
vitro and P. berghei in vivo are currently under investigation. These results 
suggest that the pyrimidine biosynthetic pathway in the malarial parasite may be 
a target for the design of antimalarial drugs.