Bioluminescent High-Throughput Succinate Detection Method for Monitoring the Activity of JMJC Histone Demethylases and Fe(II)/2-Oxoglutarate-Dependent Dioxygenases:

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...