Abstract
Dimethylated histone H3 lysine 9 (H3K9me2) is a crucial epigenetic mark associated with transcription repression. PHF8, also known as histone lysine demethylase 7B (KDM7B), harbours a plant homeodomain finger (PHD) and a Jumonji-C domain (JmjC), in which JmjC is able to remove dimethyl-lysine 9 on histone H3. Binding of PHD to H3K4me3 enhances the demethylation activity of H3K9me2 by JmjC, resembling a co-activator for transcription. PHF8 plays important roles in cell cycle progression, rDNA transcription and brain development. Mutations in PHF8 are associated with X-linked mental retardation since PHF8 interacts with other transcription factors, including ZNF711. Moreover, overexpression of PHF8 is found in prostate cancer and involved in carcinogenesis. In an aim to develop new inhibitors toward this important molecule, structure-based virtual docking approach was used to design the potential compounds and the site-moiety maps were established by using SiMMap. Here, the N-terminal region of PHF8 (residues 1-441) that consists of both PHD and JmjC domains was cloned into the pET-30a expression vector. The purified protein, his-PHF8 was confirmed to possess the H3K9me2 demethylation activity using immunoblotting assay with anti-H3K9me2 antibody. Kinetic parameters of demethylation reaction of PHF8 were determined based on a formaldehyde dehydrogenase (FDH)-coupled continuous fluorescent assay. Hits of forty candidate compounds were obtained by virtual docking, but only two of them — NSC 1A and NSC 2A were identified to have inhibitory effect toward PHF8, with the IC50 values of 50.1 ± 17.3 µM and 133.6 ± 27.4 µM, respectively. NSC 1A, with an inhibition constant (Ki) value of 26.2 μM, is a candidate lead compound for optimization and refinement of the drug design in the future. Based on the site-moiety map from SiMMap, the binding pose of NSC 1A to PHF8 possesses a unique hydrogen bonding anchor which was not observed from the poses of other candidate compounds. The interacting residues in this anchor are believed to play a crucial role in the demethylase activity.