In vitro activity: UNC0642 demonstrates high in vitro and cellular potency, low cell toxicity, and excellent selectivity. UNC0642 is competitive with the peptide substrate and non-competitive with the cofactor SAM. The Ki of UNC0642 is determined to be 3.7±1 nM. UNC0642 displays high in vitro potency for GLP (IC50< 2.5 nM), similar to G9a. UNC0642 is more than 300-fold selective for G9a and GLP over a broad range of kinases, GPCRs, transporters, and ion channels. UNC0642 exhibits high potency at reducing the H3K9me2 mark, low cell toxicity, and good separation of functional potency and cell toxicity in a number of cell lines. It reduces clonogenicity in PANC-1 cells, a pancreatic carcinoma cell line.

Kinase Assay: Selectivity of UNC0642 against a panel of 50 kinases was conducted using a standard off-chip mobility shift assay technology. The full list of the 50 kinases is included in Table S1. Selectivity of inhibitor 7 (UNC0642) against 44 GPCRs, ion channels, and transporters was performed in standard radioligand binding assays. The full list of the 44 targets is included in Table S2.

Cell Assay: Cells (MDA-MB-231, PC3, and U2OS) are treated with inhibitors (UNC0642) for 48 h. Cell viability assays are performed by incubating cells with 0.1 mg/mL of resazurin for 3 – 4 h. Resazurin reduction is monitored with 544 nm excitation, measuring fluorescence at 590 nm. In-cell western assay is performed as described previously.