CYTOTOXIC EFFECTS OF INHIBITORS OF DE-NOVO PYRIMIDINE BIOSYNTHESIS UPON PLASMODIUM-FALCIPARUM

The malarial parasite Plasmodium falciparum can only synthesize pyrimidine nucleotides via the de novo pathway which is therefore a suitable target for development of antimalarial drugs. New assay procedures have been developed using high-pressure liquid chromatography (HPLC) which enable concurrent measurement of pyrimidine intermediates in malaria. Synchronized parasites growing in erythrocytes were pulse-labeled with [C-14] bicarbonate at 6-h intervals around the 48-h asexual life cycle. Analysis of malarial extracts by HPLC showed that incorporation of [C-14]bicarbonate into pyrimidine nucleotides was maximal during the transition from trophozoites to schizonts. The reaction, N-carbamyl-L-aspartate --> L-dihydroorotate (CA-asp --> DHO) catalyzed by malarial dihydroorotase is inhibited by L-6-thiodihydroorotate (TDHO) in vitro (K-i = 6.5 mu M), and TDHO, as the free acid or methyl ester, induces a major accumulation of CA-asp in malaria. Atovaquone, a naphthoquinone, is a moderate inhibitor of dihydroorotate dehydrogenase in vitro (K-i = 27 mu M) but induces major accumulations of CA-asp and DHO. Pyrazofurin induces accumulation of orotate and orotidine in malaria, consistent with inhibition of orotidine 5'-monophosphate (OMP) decarboxylase with subsequent dephosphorylation of the OMP accumulated. Although TDHO, atovaquone, and pyrazofurin arrest the growth of P. falciparum, only moderate decreases in UTP, CTP, and dTTP were observed. 5-Fluoroorotate also arrests the growth of P. falciparum with major accumulations of 5-fluorouridine mono-, di-, and triphosphates and the most significant inhibition of de novo biosynthesis of pyrimidine nucleotides.