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Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.

Original publication




Journal article


Eur J Med Chem

Publication Date





316 - 337


Histone demethylases, KDM inhibitors, KDM4 subfamily, KDM5 subfamily, Pyridopyrimidinones, Cell Line, Tumor, Crystallography, X-Ray, Drug Screening Assays, Antitumor, Enzyme Inhibitors, Humans, Hydrophobic and Hydrophilic Interactions, Jumonji Domain-Containing Histone Demethylases, Molecular Structure, Protein Binding, Pyridines, Pyrimidinones, Structure-Activity Relationship