Reporter Ligand NMR Screening Method for 2‑Oxoglutarate Oxygenase Inhibitors

Abstract

The human 2-oxoglutarate (2OG) dependent oxygenases belong to a family of structurally related enzymes that play important roles in many biological processes. We report that competition-based NMR methods, using 2OG as a reporter ligand, can be used for quantitative and site-specific screening of ligand binding to 2OG oxygenases. The method was demonstrated using hypoxia inducible factor hydroxylases and histone demethylases, and <i>K</i>_D values were determined for inhibitors that compete with 2OG at the metal center. This technique is also useful as a screening or validation tool for inhibitor discovery, as exemplified by work with protein-directed dynamic combinatorial chemistry