Interrogating the Druggability of the 2‑Oxoglutarate-Dependent Dioxygenase Target Class by Chemical Proteomics

The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class

Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 1. 3‑Amino-4-pyridine Carboxylate Derivatives

Optimization of KDM6B (JMJD3) HTS hit <b>12</b> led to the identification of 3-((furan-2-ylmethyl)­amino)­pyridine-4-carboxylic acid <b>34</b> and 3-(((3-methylthiophen-2-yl)­methyl)­amino)­pyridine-4-carboxylic acid <b>39</b> that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds <b>34</b> and <b>39</b> possess activity, IC₅₀ ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC₅₀ = 6–8 μM). Compounds <b>34</b> and <b>39</b> are also potent inhibitors of KDM5C (JARID1C) (RFMS IC₅₀ = 100–125 nM)