Assessing histone demethylase inhibitors in cells : lessons learned

摘要: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and tight associations with diseases including cancer mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, efforts ongoing develop potent, selective cell-active ‘probe’ molecules this target class. Robust cellular assays assess the specific engagement cells well selectivity a prerequisite development high-quality inhibitors. Here we describe use high-content immunofluorescence assay method demonstrating cells. A panel Jumonji C subfamily KDMs was developed encompass all major branches JmjC phylogenetic tree. These compare compound activity against wild-type proteins catalytically inactive version KDM, which residues involved active-site iron coordination mutated inactivate enzyme activity. mutants critical assessing effect revealing indirect effects on histone methylation status. reported make ectopically expressed demethylases, demonstrate profile several recently identified classes structurally matched controls. generated data correlate results endogenous inhibition confirm observed biochemical isolated enzymes. We find that both permeability competition 2-oxoglutarate affect translation inhibition. High-content-based been established eight members 2-oxoglutarate-dependent oxygenases covering JmjC-KDM usage full-length, mutant protein, structure-matched control compounds, allowed detection nonspecific causing changes result toxicity. offer demethylase family-wide tool cell on-target efficacy. In addition, presented may inform further studies cell-based