Liu Min, Choi Sungwoon, Cuny Gregory D, Ding Kai, Dobson Brittany C, Glicksman Marcie A, Auerbach Ken, Stein Ross L
Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, 65 Landsdowne Street, Fourth Floor, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2008 Aug 12;47(32):8367-77. doi: 10.1021/bi800732v. Epub 2008 Jul 18.
Cdk5/p25 is a member of the family of cyclin-dependent, Ser/Thr kinases and is thought to play a causal role in Alzheimer's disease (AD) due to its ability to phosphorylate the protein tau, and thus promote the latter's aggregation into intraneuronal tangles. Given this, we and others are seeking inhibitors of cdk5/p25 as possible disease-modifying therapeutics for AD. In this paper, we first report the kinetic mechanism for the cdk5/p25-catalyzed phosphorylation of tau and histone H-1-derived peptide (H1P). These studies served as a necessary kinetic backdrop for investigations of the mechanism of inhibition by prototype inhibitors N4-(6-aminopyrimidin-4-yl)-sulfanilamide (APS) and 1-(5-cyclobutyl-thiazol-2-yl)-3-isoquinolin-5-yl-urea (CTIU). We found that the cdk5/p25-catalyzed phosphorylation of tau follows a rapid equilibrium, random kinetic mechanism, as evidenced by initial velocity analysis indicating sequential addition of tau and ATP, and studies of the mechanism of inhibition by substrate analogue AMP, product ADP, and analogues of peptide substrate H1P. Identical mechanistic conclusions were drawn when H1P was the phosphoryl acceptor. Subsequent studies of inhibition by APS and CTIU revealed that both compounds can bind to all four steady-state forms of the enzyme, to form the complexes E:I, E:I:tau, E:I:ATP, and E:I:tau:ATP. These results contrast with reported claims that APS and CTIU are competitive inhibitors of the binding of ATP.
细胞周期蛋白依赖性丝氨酸/苏氨酸激酶Cdk5/p25是该家族的成员之一，因其能够磷酸化tau蛋白，进而促进其聚集成神经元内缠结，所以被认为在阿尔茨海默病（AD）中起致病作用。鉴于此，我们和其他研究人员正在寻找Cdk5/p25的抑制剂，将其作为可能用于AD的疾病修饰疗法。在本文中，我们首次报道了Cdk5/p25催化tau蛋白和组蛋白H-1衍生肽（H1P）磷酸化的动力学机制。这些研究为原型抑制剂N4-(6-氨基嘧啶-4-基)-磺胺（APS）和1-(5-环丁基噻唑-2-基)-3-异喹啉-5-基脲（CTIU）的抑制机制研究提供了必要的动力学背景。我们发现，Cdk5/p25催化的tau蛋白磷酸化遵循快速平衡随机动力学机制，这一结论得到了初始速度分析（表明tau蛋白和ATP依次添加）以及底物类似物AMP、产物ADP和肽底物H1P类似物抑制机制研究的证实。当H1P作为磷酰基受体时，得出了相同的机制结论。随后对APS和CTIU抑制作用的研究表明，这两种化合物都能与酶的所有四种稳态形式结合，形成E:I、E:I:tau、E:I:ATP和E:I:tau:ATP复合物。这些结果与报道称APS和CTIU是ATP结合的竞争性抑制剂的说法形成了对比。
