Category: Assay Development and Screening

1212-C - Bioluminescent High-Throughput Succinate Detection Method for Monitoring the Activity of JMJC Histone Demethylases and Fe(II)/2-Oxoglutarate-Dependent Dioxygenases

Tuesday, February 6, 2018
2:00 PM - 3:00 PM

The structurally conserved Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily is composed of approximately 60 enzymes that catalyze the hydroxylation of a diverse array of substrates. These enzymes are central to the modulation of distinct biological processes such as epigenetics, hypoxic signaling, and DNA/RNA repair. In addition to JumonjiC-containing histone demethylases (JMJCs), this superfamily of dioxygenases include the ten-eleven-translocation (TET) DNA dioxygenases, mRNA demethylases such as FTO, ALKBH5 that impacts mRNA translation and transport, and the proyl- and asparaginyl-hydroxylases EGLN1-3 and the hypoxia inducing factor 1 alpha inhibitor FIH. Due to their relevance to human cell health and disease, members of the Fe(II)/2-oxoglutarate-dependent dioxygenases are attractive drug target candidates for treatment of a number of pathological conditions including obesity and type II diabetes, cancer, inflammatory diseases and ischemic processes. 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 were able to detect enzymatic activities from several members of the Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily, demonstrating that succinate detection can be used as a universal method regardless of substrate requirements. We also demonstrated that it could be used to determine substrate requirements/preferences as in the case of JumonjiC histone demethylases, as well as to determine the apparent kinetic values of the substrates used in the demethylase/hydroxylase reactions. Furthermore, its robustness and resistance to compound interference against the LOPAC 1280 library exemplified the utility of this assay for high-throughput screening applications. Primary hits such as hispidin were identified in compound screening against JMJD2C and were validated in reconfirmation and counterscreen experiments, query searches using PubChem Database, and were also tested on another JMJ family member, JMJD2E demethylase. To our knowledge, this is the first report that hispidin is a JumonjiC histone demethylase inhibitor. Finally, this method was shown to be useful for inhibitor mode of action studies as well as profiling experiments. 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.

Hicham Zegzouti

Senior Research Scientist
Promega Corporation
Madison, WI

Hicham Zegzouti is a Senior Research Scientist in the Assay Design Division of Research & Development at Promega Corporation. He is interested in the molecular and biochemical processes that regulate cellular signaling and cell-cell communications. His group develops Assay technologies to interrogate diverse enzyme activities and cellular pathways such as kinases, glycosyltransferases, epigenetic enzymes and other drug targets.