Previous studies have demonstrated that the active metabolite of
leflunomide,
A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], is capable of inhibiting the activities of
tyrosine kinases and
dihydroorotate dehydrogenase (DHO-DHase). In the present study, we define the relative contribution of these activities to the ability of
A77 1726 to inhibit proliferation of the murine
leukemia cell line LSTRA.
A77 1726 inhibited LSTRA cell growth and proliferation (IC50 = 10-30 microM); this inhibition, however, could be reversed by the addition of exogenous
uridine, suggesting that the anti-proliferative activity of
A77 1726 may be due to inhibition of de novo
pyrimidine nucleotide synthesis. Quantitation of
nucleotide levels revealed that
A77 1726, at an IC50 of about 10 microM, selectively inhibited
pyrimidine nucleotide but not
purine nucleotide synthesis. In vitro
enzyme assays confirmed that
A77 1726 directly inhibited the activity of DHO-DHase, the fourth
enzyme in the de novo pathway of
pyrimidine nucleotide synthesis (IC50 = 220 nM). LSTRA cells overexpress p56lck and have elevated levels of
tyrosine phosphorylated intracellular
proteins.
A77 1726 reduced the intracellular levels of
tyrosine phosphorylated
proteins with relatively high IC50 values ranging from 50 to 100 microM.
A77 1726 also inhibited p56lck activity in LSTRA membrane preparation and immunoprecipitates; the IC50 values for inhibition of immunoprecipitated p56lck autophosphorylation and exogenous substrate
histone 2B were 80 and 40 microM, respectively. The anti-
tyrosine phosphorylation activity of
A77 1726 was not affected by
uridine. These studies therefore demonstrate the two activities of
A77 1726: inhibition of
pyrimidine nucleotide synthesis and interference with
tyrosine phosphorylation.