Davis J P, Cain G A, Pitts W J, Magolda R L, Copeland R A
Inflammatory Diseases Research and Chemical and Physical Sciences, DuPont Merck Research Laboratories, Wilmington, Delaware 19880-0400, USA.
Biochemistry. 1996 Jan 30;35(4):1270-3. doi: 10.1021/bi952168g.
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.
来氟米特的活性代谢产物A771726是一种新型免疫抑制化合物，已被证明是单核细胞和T细胞的强力抗增殖剂。该化合物的分子作用机制尚未明确。然而，体外细胞和酶分析表明，来氟米特是几种蛋白酪氨酸激酶的抑制剂，IC50值在30至100微摩尔之间。A771726的体内特性让人联想到另一种免疫抑制剂布喹那钠，后者已被证明是二氢乳清酸脱氢酶（DHODase）的纳摩尔级抑制剂（Ki = 10 - 30 nM）。在此基础上，我们研究了来氟米特和A771726对纯化的重组人DHODase活性的影响。我们发现A771726是DHODase的强效抑制剂（Ki = 179 ± 19 nM），而母体化合物来氟米特在高达1微摩尔的浓度下没有抑制作用。对抑制作用依赖于底物泛醌和二氢乳清酸浓度的研究表明，A771726是泛醌结合位点的竞争性抑制剂，对二氢乳清酸是非竞争性的。因此，A771726作为DHODase抑制剂的效力比其对蛋白酪氨酸激酶抑制作用的报道效力高100 - 1000倍。这些数据表明，A771726免疫抑制疗效的另一种解释可能是该化合物对DHODase的强效抑制作用。
