Xu X, Williams J W, Gong H, Finnegan A, Chong A S
Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA.
Biochem Pharmacol. 1996 Aug 23;52(4):527-34. doi: 10.1016/0006-2952(96)00303-6.
Previous studies have demonstrated that the active metabolite of leflunomide, A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], is capable of inhibiting the activities of tyrosine kinases and dihydroorotate dehydrogenase (DHO-DHase). In the present study, we define the relative contribution of these activities to the ability of A77 1726 to inhibit proliferation of the murine leukemia cell line LSTRA. A77 1726 inhibited LSTRA cell growth and proliferation (IC50 = 10-30 microM); this inhibition, however, could be reversed by the addition of exogenous uridine, suggesting that the anti-proliferative activity of A77 1726 may be due to inhibition of de novo pyrimidine nucleotide synthesis. Quantitation of nucleotide levels revealed that A77 1726, at an IC50 of about 10 microM, selectively inhibited pyrimidine nucleotide but not purine nucleotide synthesis. In vitro enzyme assays confirmed that A77 1726 directly inhibited the activity of DHO-DHase, the fourth enzyme in the de novo pathway of pyrimidine nucleotide synthesis (IC50 = 220 nM). LSTRA cells overexpress p56lck and have elevated levels of tyrosine phosphorylated intracellular proteins. A77 1726 reduced the intracellular levels of tyrosine phosphorylated proteins with relatively high IC50 values ranging from 50 to 100 microM. A77 1726 also inhibited p56lck activity in LSTRA membrane preparation and immunoprecipitates; the IC50 values for inhibition of immunoprecipitated p56lck autophosphorylation and exogenous substrate histone 2B were 80 and 40 microM, respectively. The anti-tyrosine phosphorylation activity of A77 1726 was not affected by uridine. These studies therefore demonstrate the two activities of A77 1726: inhibition of pyrimidine nucleotide synthesis and interference with tyrosine phosphorylation.
先前的研究表明，来氟米特的活性代谢产物A77 1726 [N-(4-三氟甲基苯基)-2-氰基-3-羟基巴豆酰胺]能够抑制酪氨酸激酶和二氢乳清酸脱氢酶（DHO-DHase）的活性。在本研究中，我们确定了这些活性对A77 1726抑制小鼠白血病细胞系LSTRA增殖能力的相对贡献。A77 1726抑制LSTRA细胞生长和增殖（IC50 = 10 - 30微摩尔）；然而，这种抑制作用可通过添加外源性尿苷而逆转，这表明A77 1726的抗增殖活性可能是由于抑制了嘧啶核苷酸的从头合成。核苷酸水平的定量分析显示，A77 1726在约10微摩尔的IC50浓度下，选择性地抑制嘧啶核苷酸合成而非嘌呤核苷酸合成。体外酶活性测定证实，A77 1726直接抑制嘧啶核苷酸从头合成途径中的第四个酶DHO-DHase的活性（IC50 = 220纳摩尔）。LSTRA细胞过度表达p56lck，细胞内酪氨酸磷酸化蛋白水平升高。A77 1726以相对较高的IC50值（范围为50至100微摩尔）降低细胞内酪氨酸磷酸化蛋白的水平。A77 1726还抑制LSTRA细胞膜制剂和免疫沉淀物中的p56lck活性；抑制免疫沉淀的p56lck自磷酸化和外源性底物组蛋白2B的IC50值分别为80和40微摩尔。A77 1726的抗酪氨酸磷酸化活性不受尿苷影响。因此，这些研究证明了A77 1726的两种活性：抑制嘧啶核苷酸合成和干扰酪氨酸磷酸化。
