2‐Oxoglutarate Analog‐Based Biomolecular Tools for Exploring Structure–Activity Relationships in Nonheme Iron Enzymes

2-oxoglutarate (2OG)-dependent nonheme iron (NHFe) enzymes constitute a family of enzymes that use 2OG and oxygen to hydroxylate unactivated C(sp³)-H bonds. These enzymes are biologically important and therapeutically relevant due to their role in key cellular processes. However, selective targeting remains challenging due to high structural conservation in their active sites. Herein, two classes of 2OG analogs are rationally designed and used 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, a new class of aryl-conjugated 2OG analogs is identified that exhibits 12-fold varied inhibition and competes with 2OG for the PHD2 active site. Immunoblot studies suggest that these analogs are biologically active and can target PHD2 intracellularly. Furthermore, computational modeling studies reveal that the analogs bind to the active site in a "flipped" conformation relative to 2OG, and functional group placement is responsible for their different inhibition capabilities. 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 analogs to establish structure-activity relationships and identify interactions that can be useful for PHD2 inhibitor design.