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

The malarial parasite Plasmodium falciparum can only synthesize pyrimi dine nucleotides via the de novo pathway which is therefore a suitable target for development of antimalarial drugs. New assay procedures ha ve been developed using high-pressure liquid chromatography (HPLC) whi ch enable concurrent measurement of pyrimidine intermediates in malari a. Synchronized parasites growing in erythrocytes were pulse-labeled w ith [C-14] bicarbonate at 6-h intervals around the 48-h asexual life c ycle. Analysis of malarial extracts by HPLC showed that incorporation of [C-14]bicarbonate into pyrimidine nucleotides was maximal during th e transition from trophozoites to schizonts. The reaction, N-carbamyl- L-aspartate --> L-dihydroorotate (CA-asp --> DHO) catalyzed by malaria l dihydroorotase is inhibited by L-6-thiodihydroorotate (TDHO) in vitr o (K-i = 6.5 mu M), and TDHO, as the free acid or methyl ester, induce s a major accumulation of CA-asp in malaria. Atovaquone, a naphthoquin one, is a moderate inhibitor of dihydroorotate dehydrogenase in vitro (K-i = 27 mu M) but induces major accumulations of CA-asp and DHO. Pyr azofurin induces accumulation of orotate and orotidine in malaria, con sistent with inhibition of orotidine 5'-monophosphate (OMP) decarboxyl ase 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-Fluoro orotate also arrests the growth of P. falciparum with major accumulati ons of 5-fluorouridine mono-, di-, and triphosphates and the most sign ificant inhibition of de novo biosynthesis of pyrimidine nucleotides.