2-oxoglutarate analogue-based biomolecular tools for exploring structure-activity relationships in non-heme iron enzymes.

Abstract

2-oxoglutarate (2OG)-dependent non-heme iron (NHFe) enzymes constitute a family of enzymes that use 2OG and oxygen to catalyze the hydroxylation of unactivated C(sp3)-H bonds. These enzymes are of biological importance and therapeutic interest due to their role in regulating various cellular processes. Herein, we have rationally designed two classes of 2OG analogues and have used them as tools to investigate the active site of a 2OG-dependent NHFe enzyme, prolyl hydroxylase domain 2 (PHD2). Using an activity assay in conjunction with steady-state kinetics, we identify a new class of aryl-conjugated 2OG analogues that exhibit 12-fold varied inhibition and compete with 2OG for the PHD2 active site. Immunoblot studies suggest that these analogues are biologically active and could target PHD2 intracellularly. Furthermore, computational modelling studies reveal that the analogues bind to the active site in a "flipped" conformation relative to 2OG, and functional group placement is responsible for their different inhibition capabilities. Our mutagenesis studies further validate this unique binding mode and suggest several interactions that are crucial for inhibition. Overall, these studies provide a toolkit of 2OG analogues to establish structure-activity relationships and identify interactions that could be useful for PHD2 inhibitor design.