GSK-J1 is a highly potent H3K27 histone demethylase inhibitor with IC50 of 28 nM and 53 nM in cell-free assays for JMJD3 (KDM6B) and UTX (KDM6A), respectively, >10-fold selectivity over other tested demethylases.

JMJD3 (KDM6B):28 nM, UTX (KDM6A):53 nM

在HEK-293细胞中，GSK-J1抑制暂时转染的JMJD3和UTX的活性。GSK-J1还通过增加总核H3K27me3水平来抑制人初级巨噬细胞产生的TNF-α。[1] 在MC3T3-E1细胞中，GSK-J1抑制Runx2和Osterix表达以及ALP活性，并增加了全球H3K27me3的水平。[2]

Purified JmjD3 (1 μM) and UTX (3 μM) is incubated with 10 μM peptide [BiotinKAPRKQLATKAARK(me3 )SAPATGG] in 50 mM HEPES pH 7.5, 150 mM KCl, 50 μM (NH4)2SO4·FeSO4·Water, 1 mM 2-oxoglutarate, and 2 mM ascorbate (JmjD3, 3 minutes at 25°C; UTX, 20 minutes at 25°C) with various concentration of the inhibitor (0, 0.005, 0.01, 0.02, 0.05, 0.1 μM). 10 mM EDTA is added to stop the reaction. The reaction is desalted by zip tip and spotted on a MALDI plate with α-cyano-4-hydroxycinnamic acid MALDI matrix. Samples are analysed on a MALDI-TOF R system.