Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell‐permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure–activity relationships, leading to the development of a small molecule with around 75‐fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.

Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.

Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective,c ell-permeable inhibitors or suitable tool compounds for these enzymes.W ed escribe the discovery of an ew class of inhibitor that is highly potent towardst he histone lysine demethylases KDM2A/7A. Amodular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of asmall molecule with around 75fold selectivity towards KDM2A/7A versus other KDMs,a s well as cellular activity at low micromolar concentrations.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.