Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide
Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide
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DOI：
10.1042/bj3360299
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发表时间：
1998-12-01
影响因子：
4.1
通讯作者：
Ruuth, E
中科院分区：
文献类型：
--
作者：
Bruneau, JM;Yea, CM;Ruuth, E
来氟米特目前处于治疗类风湿性关节炎的第三阶段临床试验。在这项研究中，我们致力于研究来氟米特的活性代谢物A77 1726在人类起源的细胞和组织中的作用机制。人与放射性标记的A77 1726的高亲和力结合蛋白是从溶解的U937膜上通过纯化过程跟踪结合活性而纯化的，鉴定为线粒体酶二氢酸脱氢酶(DHO-DH)。人和鼠的酶在SDS/PAGE(43 KDa)上显示相同的pi和分子量值，这与以前的报道形成了鲜明的对比，表明人酶的分子质量为50 kDa。A77 1726及其类似物HR325对U937和人脾膜制剂的DHO-DH活性有抑制作用。HR325对植物血凝素刺激的人外周血单核细胞具有抗增殖作用，其浓度与引起DHO蓄积和尿苷耗竭的浓度相同。加入外源性尿苷后，细胞的抗增殖作用部分消失。这与我们最近的证明是一致的，即A77 1726对脂多糖刺激的小鼠脾细胞的体外抗增殖作用是由DHO-DH抑制介导的[Williamson，Yea，Robson，Curnock，Gadher，Hambleton，Woodward，Bruneau；Hambleton，Moss等人，(1995)J.Biol。化学。22467-22472]。因此，A77 1726及其类似物对DHO-DH的抑制是化合物在体外对人类细胞产生抗增殖作用的原因，也可能是其在体内产生部分作用的原因。
Leflunomide is currently in phase-III clinical trials for the treatment of rheumatoid arthritis. In this study, we have focused our efforts on the study of the mechanism of action of the active metabolite of leflunomide, A77 1726, in cells and tissue of human origin. The human high-affinity binding protein for radiolabelled A77 1726 was purified from solubilized U937 membranes by following the binding activity through the purification process and was characterized as the mitochondrial enzyme dihydro-oro-tate dehydrogenase (DHO-DH). The human and murine enzyme displayed identical pi and molecular mass values on SDS/PAGE (43 kDa), which contrasts notably with previous reports suggesting a molecular mass of 50 kDa for the human enzyme. DHO-DH activity was inhibited by A77 1726 and its analogue HR325 with similar potency in U937 and human spleen membrane preparations, HR325 was found to be anti-proliferative for phytohaemagglutinin-stimulated human peripheral blood mononuclear cells, at the same concentrations that caused accumulation of DHO and depletion of uridine. Supplementation of the cultures with exogenous uridine led to partial abrogation of the anti-proliferative effect. This is in line with our recent demonstration that the anti-proliferative effect bz vitro of A77 1726 on lipopolysaccharide-stimulaled mouse spleen cells was mediated by DHO-DH inhibition [Williamson, Yea, Robson, Curnock, Gadher, Hambleton, Woodward, Bruneau; Hambleton, Moss et al., (1995) J. Biol. Chem. 270, 22467-22472]. Thus, DHO-DH inhibition by A77 1726 and its analogues is responsible for the anti-proliferative effects in vitro of the compounds on human cells and is likely to be responsible for some of its effects in vivo.