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 [14C]
bicarbonate at 6-h intervals around the 48-h asexual life cycle. Analysis of malarial extracts by HPLC showed tht incorporation of [14C]
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 (Ki = 6.5 microM), 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 (Ki = 27 microM) 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.