6-L-Thiodihydroorotate (
TDHO) and
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (
HDDP) are potent inhibitors of mammalian
dihydroorotase in vitro (R. I. Christopherson, K. J. Schmalzl, E. Szabados, R. J. Goodridge, M. C. Harsanyi, M. E. Sant, E. M. Algar, J. E. Anderson, A. Armstrong, S. C. Sharma, W. A. Bubb, and S. D. Lyons, Biochemistry, 28: 463-470, 1989). Using human CCRF-CEM
leukemia cells growing in culture,
TDHO and
HDDP as the free
acids have 50% inhibitory concentration (IC50) values of 32 microM and greater than 1000 microM, respectively, whereas for
TDHO methyl
ester, the IC50 value is 25 microM, and for
HDDP dimethyl
ester, the IC50 value is 21 microM. These IC50 values were not affected by addition of
dihydroorotate,
uridine, or
deoxycytidine to the culture medium.
TDHO methyl
ester (25 microM) had only slight inhibitory effects upon the
dihydroorotase reaction of de novo
pyrimidine biosynthesis in growing
leukemia cells, cells arrested in G2 + M phases of the cell cycle. At 250 microM
TDHO methyl
ester, analysis of
cell extracts by high-performance liquid chromatography showed that after 4 h carbamyl
aspartate had accumulated from undetectable levels to 760 microM, whereas
UTP decreased from 580 to 110 microM and
CTP from 350 to 86 microM, indicating inhibition of
dihydroorotase in growing
leukemia cells.
IMP accumulated from 63 to 350 microM, total guanylates increased while adenylates decreased, and the adenylate energy charge decreased from 0.91 to 0.69 after 4 h. The cellular concentration of 5-phosphoribosyl 1-pyrophosphate increased from 180 to 290 microM due to sparing from
pyrimidine nucleotide biosynthesis resulting in complementary stimulation of the de novo
purine pathway.
HDDP dimethyl
ester at concentrations of up to 250 microM had no discernable effect upon
pyrimidine or
purine nucleotide biosynthesis. At 25 microM
HDDP-dimethyl
ester, cells arrested in G2 + M phases initially, with accumulation of cells in G1/G0 at later times. These data suggest that the primary mechanisms of growth inhibition for
TDHO and
HDDP involve inhibition of cell cycle progression from late G2 or M phase to G1 phase and that blockade of the
pyrimidine pathway by
TDHO is a secondary effect found at higher concentrations.