Discussion

Histone lysine methylation is an important regulator of transcriptional activation and repression and aberrant histone methylation patterns have been associated with oncogenesis. The KDM4 family of histone lysine demethylases, which contain the Jumonji catalytic domain use Fe(II) and α-ketoglutarate (2OG) as cofactors to demethylate the trimethylated and dimethylated residues at lysines 9 and 36 of histone 3. Overexpression of KDM4A has been observed in prostate cancer, triple-negative breast tumours and lung cancer, so inhibition of KDM4A may be beneficial for the treatment of these cancer types. In order to design KDM4 subfamily specific inhibitors a screening campaign using the AlphaScreen™ technology was carried out to identify novel ligand-efficient hit matter, followed by KDM4A protein-ligand crystallography to inform a structure based drug design approach.
Structure-based inhibitor design was hindered by compounds, shown to have inhibitory activity in the AlphaScreen™ assay, not yielding protein-ligand structures. Therefore, a KDM4A thermal shift assay was employed alongside LogD7.4 determination to assess whether either could be used a predictor of protein ligand crystallography success. Data on 71 compounds is presented showing that the majority of compounds with a ΔTm ≥ 1 and a negative LogD7.4 generated KDM4A ligand bound structures.
Based on the data obtained we demonstrated the use of the thermal shift assay as a predictor of successful binding in a crystallographic soaking experiment, which we are exploiting to design more potent and selective KDM4 inhibitors.