Division of Medicinal Chemistry, University of Texas, Austin, Texas 78712, USA.
J Am Chem Soc. 2009 Oct 28;131(42):15096-7. doi: 10.1021/ja906432e.
Asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA) is an endogenously produced inhibitor of human nitric oxide synthase and an emerging biomarker for cardiovascular disease. Concentrations of ADMA are controlled by two isoforms of its catabolic enzyme dimethylarginine dimethylaminohydrolase (DDAH), the dysregulation of which has been studied as a mediating factor for endothelial dysfunction. A two-part, click-chemistry mediated activity-based probe, N-but-3-ynyl-2-chloroacetamidine, is shown to label myc-tagged DDAH-1 expressed in HEK 293T cells, but not an inactive mutant or inhibited enzyme. A two-color Western blotting technique is used to determine the in vivo IC(50) value for a reversible inhibitor of DDAH-1, N(5)-(1-iminopropyl)-l-ornithine, indicating this compound's bioavailability and its competition for binding to the active site. This probe provides a novel tool for the analysis of DDAH-1 activity in normal and pathophysiological states and should allow more meaningful studies of the etiology of endothelial dysfunction.
不对称 N(omega),N(omega)- 二甲基精氨酸 (ADMA) 是一种内源性的人一氧化氮合酶抑制剂，也是心血管疾病的新兴生物标志物。ADMA 的浓度受其代谢酶二甲基精氨酸二甲氨基水解酶 (DDAH) 的两种同工型控制，其失调已被研究为内皮功能障碍的介导因素。一种两部分的、点击化学介导的活性探针 N-丁-3-炔基-2-氯乙酰胺，被证明可以标记在 HEK 293T 细胞中表达的 myc 标记的 DDAH-1，但不能标记无活性的突变体或抑制的酶。使用双色 Western blot 技术来确定 DDAH-1 的可逆抑制剂 N(5)-(1-亚氨基丙基)-l-鸟氨酸的体内 IC(50) 值，表明该化合物的生物利用度及其与活性位点的竞争。该探针为正常和病理生理状态下 DDAH-1 活性的分析提供了一种新工具，应该允许对内皮功能障碍的病因进行更有意义的研究。
