Cytotoxic effects of dihydroorotase inhibitors upon human CCRF-CEM leukemia.

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.

Chromatographic analysis of purine precursors in mouse L1210 leukemia.

A number of antagonists of nucleotide metabolism with anti-cancer activity affect the de novo purine pathway. To determine the biochemical mechanisms of cytotoxicity of these drugs, assay procedures have been developed for measurement of the levels of intermediates proximal to IMP in the pathway for de novo purine biosynthesis in mouse L1210 leukemia cells. Purine precursors have been synthesized in vitro from [14C]glycine using enzymes from chicken liver. These 14C-labeled intermediates have been used as marker compounds to define retention times for metabolites of leukemia cells separated by HPLC and the chromatographic mobilities of these intermediates after two-dimensional thin-layer chromatography. These new chromatographic procedures have been used in combination to determine the steady-state concentrations for purine precursors in mouse L1210 leukemia cells in the exponential phase of growth: N-formylglycineamide ribotide (16 microM); N-formylglycineamidine ribotide (4.7 microM); 5-aminoimidazole ribotide (4.0 microM); 4-carboxy-5-aminoimidazole ribotide (0.46 microM); N-succino-5-aminoimidazole-4-carboxamide ribotide (11 microM); 5-aminoimidazole-4-carboxamide ribotide (16 microM); 5-formamidoimidazole-4-carboxamide ribotide (2.7 microM); and IMP (57 microM). The metabolic effects of tiazofurin (25 microM) upon mouse L1210 leukemia cells growing in culture define a "metabolic crossover point" at the reaction catalyzed by IMP dehydrogenase (EC 1.1.1.205) which confirms previous reports of inhibition of this enzyme.

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase.

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)