Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors.
Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors.
复制标题
DOI：
10.1038/ja.2013.106
复制
发表时间：
2014-01
期刊：
影响因子：
--
通讯作者：
Burkart MD
中科院分区：
文献类型：
--
作者：
Kosa NM;Foley TL;Burkart MD
磷酸泛酰巯基乙胺基转移酶（E.C. 2.7.8.-）激活生物合成途径，在细菌中合成初级和次级代谢产物。这些酶的抑制剂具有作为抗生素化合物的潜力，其通过独特的作用模式发挥作用并具有临床实用性。在这里，我们报告了一个直接和连续的测定这类酶的基础上监测偏振的荧光磷酸泛酰巯基乙胺类似物，因为它是从低分子量辅酶A底物转移到更高分子量的蛋白质受体。我们证明了这种方法的实用程序的磷酸泛酰巯基乙胺基转移酶SFP，从这个类的典型代表的生化表征。我们还建立了这种技术的可移植性，以适应其他同系物的测定功能与人类磷酸泛酰巯基乙胺基转移酶，一个目标，其中微孔板检测方法目前还不存在。这些目标的比较提供了一个基础，以预测候选抑制剂的治疗指数，并提供了一个有价值的表征酶的活性。
Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity.