In vitro activity: In MCF7, 22RV1, and IMR90 cells, UNC0631 potently reduces H3K9me2 levels, and shows excellent separation of functional potency versus cell toxicity. UNC0631 thus can be used as a tool for the biomedical research community to further investigate the biology of G9a and its role in chromatin remodeling.

Kinase Assay: This assay utilizes SAHH to hydrolyze the methyltransfer product SAH to homocysteine and adenosine in the presence of adenosine deaminase which converts adenosine to inosine. The homocysteine concentration is then determined through conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore, ThioGlo. For IC50 determinations, assay mixtures are prepared in 25 mM potassium phosphate buffer pH 7.5, 1 mM EDTA, 2 mM MgCl2, 0.01% Triton X-100 with 5 μM SAHH, 0.3 U/mL of adenosine deaminase, 25 μM SAM, and 15 μM ThioGlo. G9a, GLP, SETD7, SETD8, PRMT3 and SUV39H2 are assayed at 25 nM, 100 nM, 200 nM, 250 nM, 500 nM and 100 nM, respectively. Inhibitors are added at concentrations ranging from 4 nM to 16 μM. After 2 min incubation, reactions are initiated by addition of the histone peptides: 10 μM H3(1–25) for G9a, 20 μM H3(1–25) for GLP, 100 μM H3(1–25) for SETD7, 500 μM H4(1–24) for SETD8, 10 μM H4(1–24) for PRMT3 and 200 μM H3K9Me1 (1–15) for SUV39H2. The methylation reaction is followed by monitoring the increase in fluorescence using Biotek Synergy2 plate reader with 360/40 nm excitation filter and 528/20 nm emission filter for 20 min in 384 well-plate format. Activity values are corrected by subtracting background caused by the peptide or the protein. IC50 values are calculated using Sigmaplot. Standard deviations are calculated from two independent experiments.

Cell Assay: MDA-MB-231, PC3, HCT116 cells are cultured in RPMI with 10% FBS, 22RV1 cells in alphaMEM and 10% FBS, MCF7 and IMR90 cells in DMEM with 10% FBS. Cells are treated with inhibitors for 48 h. The media is removed and replaced with DMEM 10% FBS without phenol red supplemented with 1mg/mL of MTT and incubated for 1–2 h. Live cells reduce yellow MTT to purple formazan. The resulting formazanis solubilized in acidified isopropanol and 1% Triton. Formazan signal absorbance is measured at 570 nm and corrected for the 650 nm background. IC50s are calculated using GraphPad Prizm statistical package with sigmoidal variable slope dose response curve fit.

J Med Chem. 2011 Sep 8;54(17):6139-50.