Allele‐Specific Inhibition of Histone Demethylases

Abstract

Histone demethylases play a critical role in mammalian gene expression by removing methyl groups from lysine residues in degree‐ and site‐specific manner. To specifically interrogate members and isoforms of this class of enzymes, we have developed demethylase variants with an expanded active site. The mutant enzymes are capable of performing lysine demethylation with wild‐type proficiency, but are sensitive to inhibition by cofactor‐competitive molecules embellished with a complementary steric “bump”. The selected inhibitors show more than 20‐fold selectivity over the wild‐type demethylase, thus overcoming issues typical to pharmacological and genetic approaches. The mutant–inhibitor pairs are shown to act on a physiologically relevant full‐length substrate. By engineering a conserved amino acid to achieve member‐specific perturbation, this study provides a general approach for studying histone demethylases in diverse cellular processes.

Advances and Opportunities in Epigenetic Chemical Biology

“…[254] Additionally, Wagner and co-workers have taken advantage of allele-specific chemical genetics as a means to studying pathways involving specific histone demethylases. [255] Using this technique, the authors mutated key residues in demethylase KDM4A to accommodate synthetically-accessible "bumped" 2ketoglutarate analogues as competitive inhibitors. The mutant KDM4A enzymes with an expanded active site maintained catalytic activity and were capable of removing two methyl groups, whereas the wild-type enzymes only remove a single methyl group.…”

“…Additionally, Wagner and co‐workers have taken advantage of allele‐specific chemical genetics as a means to studying pathways involving specific histone demethylases [255] . Using this technique, the authors mutated key residues in demethylase KDM4A to accommodate synthetically‐accessible “bumped” 2‐ketoglutarate analogues as competitive inhibitors.…”

Phosphine‐Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

“…Pharmacological inhibitors are commonly used for this purpose; while they work quickly and are usually reversible, available drugs can access only a limited portion of the proteome, typically can only deactivate protein function, and may have off‐target effects . Other techniques, including bump‐and‐hole approaches, chemically induced dimerization and dissociation, and controlled degradation have expanded conditional control over protein function. Here, we present an optimized and generally applicable approach that does not require small‐molecule ligand discovery, molecular engineering of the ligand, or genetically fused ligand‐binding domains, while enabling rapid OFF to ON switching of protein function with complete specificity.…”