Inhibition of dihydroorotate dehydrogenase by the immunosuppressive agent leflunomide

Leflunomide [HWA 486 or RS-34821, 5-methyl-N-(4-trifluoromethylphenyl)-4-isoxazole carboximide] is an immunosuppressive agent effective in the treatment of rheumatoid arthritis. In spite of its clinical potential, its mechanism of action has not been elucidated. Recent studies suggest that leflunomide may interfere with the metabolism of pyrimidine nucleotides. In our studies, the active metabolite of leflunomide, RS-61980 (A77 1726, 2-hydroxyethylidene-cyanoacetic acid-4-trifluoromethyl anilide), was cytostatic towards a human T-lymphoblastoma cell line (A3.01). The inhibition of growth could be overcome completely by uridine. The other nucleosides, cytidine, adenosine and guanosine, did not overcome the effect of the compound. Since uridine is a precursor for the salvage synthesis of UMP, we propose that RS-61980 may be inhibiting the de novo pathway of UMP synthesis. Using human cells, the six enzymes catalyzing de novo UMP biosynthesis were tested for their sensitivity towards RS-61980. Only one of the enzymes, dihydroortate dehydrogenase (DHODH, EC 1.3.3.1) was inhibited by RS-61980 with a Ki value of 2.7 +/- 0.7 microM. The other five enzymes were not affected. The inhibition exhibited mixed-type kinetics towards both substrates, dihydroorotic acid and coenzyme Q. These results suggest that the molecular target of leflunomide action is DHODH. The immunomodulating activity may be related to the inhibition of UMP synthesis in proliferating lymphocytes.