Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, Texas 78712, United States.
J Am Chem Soc. 2011 Feb 9;133(5):1553-62. doi: 10.1021/ja109207m. Epub 2011 Jan 11.
In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK(a) ca. 8.8), and 1 does not inactivate papain (Cys pK(a) ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.
为了开发新型的二甲基精氨酸二甲氨基水解酶（DDAH）共价修饰物，用于生物应用，我们测试了一组使用高通量筛选鉴定的“片段”大小的抑制剂，以评估其时间依赖性抑制作用。一类结构的失活剂，4-卤代吡啶，显示出时间和浓度依赖性的 DDAH 失活，其中一个例子 4-溴-2-甲基吡啶（1）的失活机制被详细描述。卤代吡啶的中性形式与过量的谷胱甘肽反应性不强。然而，1 很容易与 DDAH 的活性位点半胱氨酸 249 发生选择性、共价和不可逆的反应，同时失去其卤化物。该活性位点半胱氨酸不是特别反应性（pK(a)约 8.8），并且 1 不会使木瓜蛋白酶失活（Cys pK(a)约≤4），这表明，与许多试剂不同，半胱氨酸亲核性不是选择性的主要因素。相反，第二个部分（天冬氨酸 66）结合并稳定更具反应性的吡啶翁形式的失活剂，对于容易发生反应是必需的。这种约束赋予了这些失活剂独特的选择性特征。据我们所知，卤代吡啶以前没有被报道为蛋白质修饰剂，因此它们代表了一种新型的静止亲和标签的首例。
