The Immunosuppressive Metabolite of Leflunomide Is a Potent Inhibitor of Human Dihydroorotate Dehydrogenase

Abstract

The active metabolite of leflunomide. A771726, is a novel immunosuppressive compound that has been shown to be a powerful antiproliferative agent for mononuclear and T-cells. The molecular mechanism of action for this compound has not been clearly established. In vitro cellular and enzymatic assays, however, demonstrate that leflunomide is an inhibitor of several protein tyrosine kinases, with IC50 values between 30 and 100 microM. The in vivo properties of A771726 are reminiscent of another immunosuppressive agent, brequinar sodium, which has been shown to be a nonomolar inhibitor (Ki = 10-30 nM) of the enzyme dihydroorotate dehydrogenase (DHODase). On the basis, we have investigated the effects of leflunomide and A771726 on the activity of purified recombinant human DHODase. We find that A771726 is a potent inhibitor of DHODase (Ki = 179 +/- 19 nM), while the parent compound, leflunomide, had no inhibitory effect at concentrations as high as 1 microM. Studies of the dependence of inhibition on the concentrations of the substrates ubiquinone and dihydroorotate demonstrate that A771726 is a competitive inhibitor of the ubiquinone binding site and is noncompetitive with respect to dihydroorotate. The potency of A771726 as a DHODase inhibitor is thus 100-100-fold greater than that reported for its inhibition of protein tyrosine kinases. These data suggest that an alternative explanation for the immunosuppressive efficacy of A771726 may be the potent inhibition of DHODase by this compound.

Modulation of inflammation and metalloproteinase expression in synovial tissue by leflunomide and methotrexate in patients with active rheumatoid arthritis: Findings in a prospective, randomized, double-blind, parallel-design clinical trial in thirty-nine patients at two centers

“…It has been suggested that the effects of methotrexate on the production of tissue-degrading factors are related to adenosine receptor stimulation (52) rather than to the effects on folate metabolism (53). The primary mode of action of leflunomide is thought to be inhibition of pyrimidine biosynthesis (21,(53)(54)(55)(56)(57), but other mechanisms are involved as well (22,24,58,59). Direct cell-cell contact between activated T cells and monocytes might be involved in MMP-1 induction (60).…”

“…Leflunomide is a prodrug that is rapidly converted in the cell into the active metabolite A77 1726. Leflunomide exerts its effects by various mechanisms; the proposed primary mode of action in RA is inhibition of the enzyme dihydroorotate dehydrogenase (19)(20)(21). Leflunomide also interferes with the phosphorylation of tyrosine kinases, resulting in effects on signal transduction pathways (22)(23)(24).…”

The immunomodulatory drug Leflunomide inhibits cell cycle progression of B-CLL cells

“…Leflunomide specifically inhibits the activity of dihydro-orotate dehydrogenase (DHODH) in a non-competitive manner. 1 DHODH is the rate-limiting enzyme in the de novo biosynthesis of pyrimidines. While its antiproliferative effect on lymphocytes and mononuclear cells is completely reversible by exogenous uridine, blockade of DHODH is considered to be the main mode of action.…”

The efficacy and safety of leflunomide in patients with active rheumatoid arthritis: A five‐year followup study

“…The primary mode of action is thought to be selective inhibition of de novo pyrimidine synthesis by blocking the rate-limiting enzyme dihydroorotate dehydrogenase (4). Activated CD4ϩ T cells proliferate rapidly during the progression of RA, a process involving de novo pyrimidine synthesis (5).…”