GSK J4 HCl

GSK J4 HCl is an ethyl ester derivative of the JMJD3 selective histone demethylase inhibitor GSK-J1 with an IC50 value greater than 50 μM in vitro. GSK J4 HCl is used to probe the consequences of demethylation of H3K27me3. In human primary macrophages, GSK-J4 inhibits the lipopolysaccharide-induced production of cytokines, including pro-inflammatory tumour necrosis factor (TNF). In addition, GSK-J4 prevents the lipopolysaccharide-induced loss of H3K27me3 associated with the TNF transcription start sites and blocked the recruitment of RNA polymerase II. ^[1]

GSK-J4 hydrochloride is a potent dual inhibitor of H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A. It inhibits LPS-induced TNF-α production in human primary macrophages. GSK-J4 hydrochloride is a cell permeable prodrug of GSK-J1.

• Histone Demethylase AlphaScreen

  Inhibition of histone demethylases is assessed using the histone demethylase AlphaScreen assay (Amplified Luminescence Proximity Homogenous Assay). This assay uses a biotinylated peptide substrate and relies on detection of the product methyl mark using a specific antibody coupled to protein-A acceptor beads and a Steptavidin donor bead to capture the peptide. In brief, recombinant demethylase enzymes are incubated in the presence of Fe2+ in the form of Ferrous Ammonium Sulphate (FAS), -ketoglutarate (KG) and biotinylated peptide substrate. L-Ascorbic Acid is included to provide a reducing environment and prevent oxidation of Fe2+. After incubation with peptide substrate the presence of the product is detected using AlphaScreen technology. The demethylase AlphaScreen assays are performed in 384-well plate format using white proxiplates. All steps are carried out in assay buffer (50 mM HEPES pH 7.5, 0.1% (w/v) BSA and 0.01 % (v/v) Tween-20). FAS is dissolved fresh each day in 20 mM HCl to a concentration of 400 mM and diluted to 1.0 mM in deionized water. All other components are dissolved fresh each day in deionized water. For IC50 determinations 5 μL of assay buffer containing demethylase enzyme is transferred to wells of a 384-well proxiplate. Titrations of compound (0.1 μL) are transferred to each well and the enzymes allowed to pre-incubate for 15 minutes with compound (final concentration of DMSO is 1%). The enzyme reaction is initiated by addition of 5 μL of a substrate mix consisting of α-KG, FAS, L-Ascorbic Acid and biotinylated peptide substrate and the reaction incubated for the indicated time at room temperature. The enzyme reaction is stopped after the indicated time by addinton of 5 μL of EDTA (7.5 mM final concentration in assay buffer). Streptavidin Donor beads (0.08 mg/ml) and Protein-A conjugated acceptor beads (0.08 mg/ml) are pre-incubated for 1 hour with an antibody to the product methyl mark and the presence of biotin-H3-product is detected by addition of 5 μL of the preincubated AlphaScreen beads (final concentrations of 0.02 mg/ml with respect to acceptor and donor beads). Detection is allowed to proceed for 1 hour at room temperature and the assay plates read in a BMG Labtech Pherastar FS plate reader. Data are normalized to the no enzyme control and the IC50 determined from the nonlinear regression curve fit using GraphPad Prism 5.

• Cell lines

  Mouse podocytes

• Concentrations

  5 μM

• Incubation Time

  48 h

• Method

  Cells were serum starved for 4 hours, followed by treatment with EPZ-6438 (10 μM) or GSK-J4 (5 μM) for 48 hours.

• Animal Models

  BALB/c mice

• Dosages

  10 mg/kg

• Administration

  i.p.