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.

GSK343 is a selective and SAM-competitive inhibitor of the histone lysine methyltransferase EZH2 with IC50 value of 4 nM [1].

EZH2 (enhancer of zestehomolog 2) is a catalytic subunit of the protein complex (polycomb repressive complex 2, PRC2). It catalyzes the methylation of H3K27 through transferring the methyl group of SAM to H3K27. The trimethylation of H3K27 subsequently results in the transcription suppression of target genes such as RUNX3, FOXC1 and BRCA1. The overexpression and mutation of EZH2 have been found in many sorts of tumors demonstrating that EZH2 is an attractive target for the treatment of cancers. As a potent EZH2 inhibitor, GSK343 inhibits the activity of the enzyme via competing with the cofactor SAM [1, 2].

GSK343 is a selective EZH2 inhibitor. It showed no significant inhibitory effects on other enzymes requiring SAM as cofactor, including DNMT, MLL, PRMT and SETMAR. GSK343 exerted a certain degree of effective on EZH1 with IC50 value of 240 nM since EZH1 was quite homologous to EZH2. In cultured HCC1806 breast cancer cells, treatment of GSK343 dose-dependently reduced H3K27me3 with IC50 value of 174 nM. Besides that, GSK343 was found to inhibit cell proliferation in some breast and prostate cancer cells. In LNCaP cells, GSK343 suppressed cell growth with IC50 value of 2.9 μM. In human EOC cells, GSK343 notably inhibited cell invasion and induced cell apoptosis. GSK343 was also found to induce LC3-II accumulation and autophagy in A549, MDA-MB-231 and HepG2 cell. It enhanced the antitumor activity of sorafenib which was a multikinase inhibitor and could decrease the expression of EZH2 in HepG2 cells [1, 3 and 4].

GSK343 is only used as a tool to investigate EZH2 in vitro because of its high clearance in the animal model [1].

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.
[2] Ferraro A, Boni T, Pintzas A.  EZH2 Regulates Cofilin Activity and Colon Cancer Cell Migration by Targeting ITGA2 Gene. PloS one, 2014, 9(12): e115276.
[3] Amatangelo M, Garipov A, Li H, et al.  Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition. Cell Cycle, 2013, 12(13): 2113-2119.
[4] Liu T P, Lo H L, Wei L S, et al.  S-Adenosyl-L-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells. Anti-cancer drugs, 2015, 26(2): 139.