Discovery of SDS-347 as a specific peptide competitive inhibitor of G9a with promising anti-cancer potential

G9a is a histone H3K9 methyltransferase enzyme found highly upregulated in many cancers. H3 binds to the rigid I-SET domain and the cofactor, S-adenosyl methionine, binds to the flexible post-SET domain of G9a. Inhibition of G9a is known to inhibit the growth of cancer cell-lines. Recombinant G9a and H3 were used to develop radioisotope-based inhibitor screening assay. The identified inhibitor was evaluated for isoform selectivity. The mode of enzymatic inhibition was studied by enzymatic assays and bioinformatics. Anti-proliferative activity of the inhibitor was studied in cancer cell lines by utilizing MTT assay. The mechanism of cell death was studied by western blotting and microscopy. We developed a robust G9a inhibitor screening assay that led to the discovery of SDS-347 as a potent G9a inhibitor with IC50 of 3.06 μM. It was shown to reduce the levels of H3K9me2 in cell-based assay. The inhibitor was found to be peptide competitive and highly specific as it did not show any significant inhibition of other histone methyltransferases and DNA methyltransferase. Docking studies showed that SDS-347 could form direct bonding interaction with Asp1088 of the peptide-binding site. SDS-347 showed anti-proliferative effect against various cancer cell lines especially the K562 cells. Our data suggested that SDS-347 mediated antiproliferative action via ROS generation, induction of autophagy and apoptosis. Overall, the findings of the current study include development of a new G9a inhibitor screening assay and identification of SDS-347, as a novel, peptide competitive and highly specific G9a inhibitor with promising anticancer potential. [Display omitted] •We have developed a robust radioisotope based G9a histone methyltransferase inhibitor screening assay system.•We report the discovery of SDS-347 as a specific and peptide competitive G9a inhibitor.•Docking results have highlighted the key aminoacid residues of G9a involved in interaction with SDS-347.•SDS-347 was found to be cytotoxic agent against a panel of cancer cell lines.•SDS-347 mediated antiproliferative action via reactive oxygen species generation, induction of autophagy and apoptosis.