GSK-J1 An inhibitor for human H3K27me3 and H3K4me3/2/1 demethylases

Histone Demethylase AlphaScreen. Inhibition of histone demethylases was assessed using the histone demethylase AlphaScreen assay (Amplified Luminescence Proximity Homogenous Assay) (Kawamura et al. 2010). The demethylase AlphaScreen assays were performed in 384-well plate format using white proxiplates (Perkin Elmer). All reagents were from Sigma Aldrich unless otherwise stated and were of the highest purity. Bovine serum albumin (BSA) used in the AlphaScreen assays was fatty acid and globulin free (Sigma A7030). Hydroxy Ethyl Piperazine Ethane Sulfonic acid (HEPES) buffer was from Apollo Scientific. All steps were carried out in assay buffer (50 mM HEPES pH 7.5, 0.1% (w/v) BSA and 0.01 % (v/v) Tween- 20). FAS was 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 were dissolved fresh each day in deionized water. For IC50 determinations 5 µl of assay buffer containing demethylase enzyme was transferred to wells of a 384-well proxiplate. Final assay concentrations of enzymes were: JMJD3 (1 nM), JMJD2E (2 nM), JMJD2C (5 nM), JMJD1A (0.2 nM), JARID1B (0.5 nM), JARID1C (0.5 nM). Titrations of compound (0.1 µl) were transferred to each well and the enzymes pre-incubated for 15 minutes with compound (final concentration of DMSO was 1%). The enzyme reaction was initiated by addition of 5 µl of a substrate mix consisting of aKG (see table for final assay concentration), FAS (see table for final assay concentration), L-Ascorbic Acid (100 µM) and biotinylated peptide substrate (see table for details and final assay concentrations). Enzyme reactions were allowed to proceed for the following times: JMJD3 (5 min), JMJD2E (20 min), JMJD2C (25 min), JMJD1A (5 min), JARID1B (15 min), JARID1C (15 min). The enzyme reaction was stopped after the indicated time by addition of 5 ml of EDTA (7.5 mM final assay concentration). Streptavidin Donor beads (0.08 mg/ml) and Protein-A conjugated acceptor beads (0.08 µg/ml) were pre-incubated for 1 hour with an antibody to the product methyl mark (see table for details) and the presence of the product methyl mark was detected by addition of 5 ml of the pre-incubated alphascreen beads (final concentrations of 0.02 mg/ml with respect to acceptor and donor beads). Detection was allowed to proceed for 1 hour at room temperature and the assay plates read in a BMG Labtech Pherastar FS plate reader. Data were normalized to the no enzyme control and the IC50 determined from the nonlinear regression curve fit using GraphPad Prism 5.

Profiling across Fe2+ / 2-Oxoglutarate Oxygenases. The binding of inhibitors was profiled across a range of Fe2+ / KG dependent oxygenases by using melting temperature (Tm) shift analysis (Niesen et al. 2007) to assess changes in the thermal stability upon binding of inhibitor. Protein unfolding was monitored in the presence of the fluorophore SYPRO Orange (Invitrogen) that has a strong fluorescent signal when bound to hydrophobic patches as a protein unfolds. The Tm shift assay was performed in 96-well plate format using white thin walled 0.2 ml PCR plates (Appleton Woods). Sypro Orange dye (Invitrogen) was diluted 1:1000 in 50 µM HEPES pH7.4, 150 µM NaCl, 50 µM NiCl2 and enzyme added to a concentration of 1 µM. For Tm shift assays 19 µl of enzyme was transferred to each well of a 96-well PCR plate and compound (1.0 µl) added to a final concentration of 20 µM (final DMSO concentration was 0.5%). Thermal unfolding of protein was monitored over 71 cycles in a Stratagene Mx3005p real time PCR machine by increasing the temperature by 3°C every minute. Increase in fluorescence was monitored at 492 nM excitation and 610 nM emission. Fluorescence intensity values were plotted as a function of temperature in GraphPad Prism 5 and Tm values were calculated by fitting the data to the Boltzmann equation.

For further experimental details, please see the supplementary information.