In vitro activity: BIX01294 is a selective inhibitor of G9a histone methyltransferase and does not affect SUV39H1(H320R) and PRMT1 within the tested concentration range. BIX-01294 specifically inhibited G9a (H3K9me2) and, to a lesser extent, the closely related GLP enzyme (primarily H3K9me3), with an IC50 of 1.7 μM for G9a and 38 μM for GLP. BIX-01294 inhibits G9a in an uncompetitive manner with SAM. BIX-01294 (4.1μM) reduces H3K9me2 Levels in Bulk Histone Preparations from wt ES cells, mouse embryonic fibroblasts and HeLa cells, but not in G9a deficient stem cells. BIX-01294 is a valuable inhibitor for the transient modulation of chromosomal H3K9me2. BIX-01294 Reduces H3K9me2 at Several G9a Target Genes including Bim1 and Serac1. BIX01294 could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1

Kinase Assay: Dissociation Enhanced Lanthanide Fluoro-Immuno Assay (DELFIA) was performed in white, opaque 384-well plates coated with Neutravidin. Test compounds were diluted to 12 μg/mL in 50mM Tris-HCl (pH 8.5) containing 4% DMSO and 10 μL was dispensed into the wells. Blank and control wells received only compound buffer. GST-G9a at 10 μg/mL and SAM at 40 μM were diluted in 50mM Tris HCl (pH 8.5)/10 mM DTT and added in a volume of 20 μL. Blank wells received Tris/DTT buffer only. The reactions were initiated by the addition of 800 nM H3(1-20)-cysbiotin substrate in 50 mM Tris (pH 8.5) in a volume of 10 μL, and incubated at room temperature for 60 mins. The plates were washed 3 times with 100 μL of Wash Buffer. Next, 50 μL of Fluoroimmunoassay (FI) Buffer containing 5 ng α-2X-di-meth H3-K9 and 5 ng goat anti-rabbit Eu chelate was added to all wells of the plate, and the plate was incubated for an additional 1 hr at room temperature. The plates were washed 3 times with 100 μL of Wash Buffer, and 50 μL of Enhancement Solution was added to each well. Time resolved fluorescence was measured after 45 mins on a Viewlux Microplate Imager imaging for 15 seconds.

Cell Assay: BIX-01294 (4.1 μM) reduced the H3K9me2 Levels in Bulk Histone Preparations from wild-type mouse ES cells, mouse embryonic fibroblasts and human HeLa cells, but not in G9a deficient mouse ES cells.

Mol Cell. 2007 Feb 9;25(3):473-81; J Biol Chem. 2010 May 28;285(22):16538-45.