THE IMMUNOSUPPRESSIVE METABOLITE OF LEFLUNOMIDE IS A POTENT INHIBITOROF HUMAN DIHYDROOROTATE DEHYDROGENASE

The active metabolite of leflunomide, A771726, is a novel immunosuppre ssive compound that has been shown to be a powerful antiproliferative agent for mononuclear and T-cells. The molecular mechanism of action f or this compound has not been clearly established. In vitro cellular a nd enzymatic assays, however, demonstrate that leflunomide is an inhib itor of several protein tyrosine kinases, with IC50 values between 30 and 100 mu M. The in vivo properties of A771726 are reminiscent of ano ther immunosuppressive agent, brequinar sodium, which has been shown t o be a nanomolar inhibitor (K-i = 10-30 nM) of the enzyme dihydroorota te dehydrogenase (DHODase). On this;basis, we have investigated the ef fects of leflunomide and A771726 on the activity of purified recombina nt human DHODase. We find that A771726 is a potent inhibitor of DHODas e (K-i = 179 +/- 19 nM), while the parent compound, leflunomide, had n o inhibitory effect at concentrations as high as 1 mu M. Studies of th e dependence of inhibition on the concentrations of the substrates ubi quinone and dihydroorotate demonstrate that A771726 is a competitive i nhibitor of the ubiquinone binding site and is noncompetitive with res pect to dihydroorotate. The potency of A771726 as a DHODase inhibitor is thus 100-1000-fold greater than that reported for its inhibition of protein tyrosine kinases; These data suggest that an alternative expl anation for the immunosuppressive efficacy of A771726 may be the poten t inhibition of DHODase by this compound.