In vitro biochemical assays against histone methyltransferases

Activity against EZH2 was assessed using 5 member PRC2 complex (Flag-EZH2, EED, SUZ12, AEBP2 and RbAp48). The assay protocol may be summarized as follows: 10 mM stocks of compounds were prepared from solid in 100% DMSO. An 11 point serial dilution master plate was prepared in 384 well format (1:3 dilution, columns 6 and 18 were equal volume DMSO controls) and dispensed to assay ready plates using acoustic dispensing technology to create a 100 nL stamp of compound and DMSO controls. The assay additions consisted of equal volume additions of 10 nM EZH2 and the substrate solution (5 ?g/mL HeLa nucleosomes and 0.25 ?M [3H]-SAM) dispensed into assay plates using a multi-drop combi dispense. Reaction plates were incubated for 1 hr and quenched with an equal volume addition of 0.5 mg/mL PS-PEI Imaging Beads (RPNQ0098) containing 0.1 mM unlabeled SAM. The plates were sealed, dark adapted for 30 mins, and a 5-min endpoint luminescence image was acquired using a Viewlux imager. Plate statistics such as Z’ and signal to background as well as dose response curves were analyzed using Activity BaseXE. The in vitro biochemical activity of EZH1 was assessed as part of a 5 member PRC2 complex using a 384 well SPA assay identical to EZH2. Buffer components, reagent dispensing, compound plate preparation, quench conditions and data analysis were identical for EZH1 and EZH2 with final assay concentrations of 20 nM EZH1, 5 μM/mL HeLa nucleosomes and 0.25 μM [3H]-SAM. Further data analysis, pIC50 pivots and visualizations were enabled by TIBCO Spotfire. Compounds were profiled at Reaction Biology Corp. (Malvern, PA) to assess inhibition in their panel of histone methyltransferase assays. Methyltransferase activity was assessed using HotSpot technology, a miniaturized radioisotope-based filter binding assay. Inhibitors were dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations up to 100 μM with a final DMSO concentration of 2%. Buffer containing the methyltrasferase at the listed concentration and its preferred substrate as shown in the accompanying table was preincubated with compound for 10 mins. Reactions were initiated by the addition of 1 μM S-adenosyl-L-[methyl-3H]methionine (SAM), allowed to incubate for 60 mins at 30℃ followed by transfer to P81 filter-paper and PBS wash before detection.

Cell lines

Breast cancer cell lines (HCC1806, Sk-Br-3 and ZR-75-1) and prostate cancer cell lines (DU145, PC3 and LNCaP)

Preparation method

The solubility of this compound in DMSO is limited. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

~ 50 μM; 6 days

Applications

In HCC1806 breast cancer cells, GSK343 inhibited trimethylation of H3K27 (H3K27me3) with the IC50 value of 174 nM. In breast cancer cells and prostate cancer cells, GSK343 potently inhibited cell proliferation. In addition, the prostate cancer cell line LNCaP was the most sensitive to GSK343, with the IC50 value of 2.9 μM.

References:

[1]. Verma S K, Tian X, LaFrance L V, et al.Identification of potent, selective, cell-active inhibitors of the histone lysine methyltransferase EZH2. ACS medicinal chemistry letters, 2012, 3(12): 1091-1096.