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.

依赖2-氧戊二酸的双加氧酶（2-oxoglutarate-dependent dioxygenase）靶点类别包含约60种酶，其中多个亚家族与人类疾病密切相关，例如脯氨酰羟化酶（prolyl hydroxylases）以及Jumonji型赖氨酸去甲基化酶（Jumonji-type lysine demethylases）。当前的药物研发策略大多基于靶向铁/2-氧戊二酸辅因子结合位点的小分子抑制剂。本研究开发了一种基于偶联于磁珠的非选择性活性位点配体组合的化学蛋白质组学方法，可从人类细胞中亲和捕获约40种不同的双加氧酶。采用游离配体竞争结合模式对磁珠结合的酶进行基于质谱的定量分析，能够全面测定辅底物2-氧戊二酸以及2-羟基戊二酸（2-hydroxyglutarate）等肿瘤代谢物（oncometabolites）的结合亲和力。本研究还对一系列具有代表性的类药抑制剂化合物开展了活性谱表征。研究结果表明，内源性辅因子介导的细胞内竞争以及活性位点的高度同源性，为该靶点类别的药物研发带来了显著挑战。