1 Promega Corporation, R&D Department, Madison, WI, USA.
2 Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
SLAS Discov. 2018 Mar;23(3):242-254. doi: 10.1177/2472555217745657. Epub 2017 Dec 14.
The modification of a diverse array of substrates by Fe(II)/2-oxoglutarate-dependent dioxygenases is central to the modulation of distinct biological processes such as epigenetics, hypoxic signaling, and DNA/RNA repair. Of these, JumonjiC domain-containing histone lysine demethylases (JMJCs) and prolyl hydroxylases are potential drug targets due to their relevance to human diseases. Thus, assays to interrogate this enzyme superfamily are needed to identify selective and potent inhibitors as leads for drug development and that could also be useful research tools. Since succinate is a common product to all Fe(II)/2-oxoglutarate-dependent dioxygenase reactions, a method that detects succinate would be suitable to all members of this enzyme superfamily. We therefore developed a bioluminescent and homogenous succinate detection assay and validated its use with diverse sets of enzyme classes. We evaluated the substrate specificities of these enzymes, their apparent kinetic constants, and inhibition profiles and mode of action of reported and novel inhibitors. Our results indicate that succinate detection is a useful readout for the monitoring of enzymatic activities with distinct substrate entities, as well as for the discovery of novel inhibitors. By investigating a large number of Fe(II)/2-oxoglutarate-dependent enzymes, this method could have a significant impact on the field of dioxygenase research.
各种基质的修饰是通过 Fe(II)/2- 氧代戊二酸依赖性双加氧酶完成的，这对于调节不同的生物学过程至关重要，如表观遗传学、缺氧信号和 DNA/RNA 修复。在这些过程中，含有 JumonjiC 结构域的组蛋白赖氨酸去甲基酶 (JMJCs) 和脯氨酰羟化酶由于与人类疾病相关，是潜在的药物靶点。因此，需要进行这些酶超家族的检测分析，以鉴定选择性和有效的抑制剂作为药物开发的先导，同时也可以作为有用的研究工具。由于琥珀酸盐是所有 Fe(II)/2- 氧代戊二酸依赖性双加氧酶反应的常见产物，因此检测琥珀酸盐的方法适合该酶超家族的所有成员。因此，我们开发了一种生物发光和均相琥珀酸检测测定法，并通过不同的酶类对其进行了验证。我们评估了这些酶的底物特异性、表观动力学常数、抑制谱以及报道和新型抑制剂的作用模式。我们的结果表明，琥珀酸检测是监测具有不同底物实体的酶活性以及发现新型抑制剂的有用指标。通过研究大量的 Fe(II)/2- 氧代戊二酸依赖性酶，这种方法可能会对双加氧酶研究领域产生重大影响。
