In vitro activity: JIB-04 induces transcriptional changes in a cancer-selective manner, including the downregulation of proliferative genes and the upregulation of the anti-proliferative/pro-apoptotic genes. JIB-04 blocks growth of lung and prostate cancer lines with IC50 as low as 10 nM, while produces less anti-proliferative activities on HBECs and PrSCs/PrECs.

Kinase Assay: Active JMJD2E aa 1-350 is purified from E.coli and used in vitro in the presence of α-ketoglutarate, 5-10 μM iron, ascorbic acid and a histone peptide substrate in a coupled reaction with formaldehyde dehydrogenase supplemented with NAD+ to quantify NADH production or using Epigentek kit P-3081. For histone demethylation reactions quantified by Western analysis, a His-tagged hJMJ2D aa 1-350 expression construct, the kind gift of Drs. Y. Shi and J. Whetstine, is expressed and purified from E.coli following the Qiagen Ni-NTA agarose manual instructions and the protocol of Whestine et al 54. Briefly, protein is eluted in 50 mM TrisHCl pH 7.8 + 0.3 M NaCl + 10% Glycerol + 200 mM immidazol, and dialyzed against 20 mM TrisHCl pH 7.4, 0.15 M NaCl, 0.2 mM PMSF, 0.5 mM DTT, 8% glycerol. Enzyme is aliquoted, flash frozen and stored at -80°C. For activity assays by Western blot, ~1.5 μg of enzyme are combined with 0.3 μg H3K9me3 substrate (Active Motif #31213) in 10 μM (NH4)2Fe(SO4)2, 1 mM α-ketoglutarate, and 2 mM sodium L-ascorbate in 50 mM Hepes pH 7.9 in the presence of vehicle or drug and incubated for 30 min-2 hrs at 37°C. SDS loading buffer is added to the reactions, and after boiling, samples are run on NuPage 4-12% Bis-Tris gels, transferred to nitrocellulose and blotted using Upstate #07-523 to detect H3K9me3. For the detection of H3 total signal, we uses Active Motif #39763 (primary) and IRDye 680 conjugated donkey anti-rabbit IgG (secondary, LI-COR # 926-32223) and imaged blots in an Odyssey Infrared Imaging system kindly made available by Dr. M. Cobb. For in vitro IC50 determinations and competition studies, typically 100-200 ng of purified protein are incubated with vehicle, JIB-04 or analogs, as indicated in figure legends and activity measured by ELISA (Epigentek kit P-3081 for H3K9me3 demethylation, P-3083 for H3K4me3 demethylation, and P-3085 for H3K27me3 demethylation) in reactions containing 50mM Hepes pH 7.5, 0.01% Tween 20, 120nM (NH4)2Fe(SO4)2, 1 mM α-ketoglutarate, 2 mM sodium L-ascorbate and 50ng peptide substrate. Final enzyme concentrations in the reactions were as follows: 206 nM JMJD2A, 12 nM JMJD2B, 60 nM JMJD2C, 90 nM JMJD2D E.coli, 30 nM JMJD2D Sf9, 30 nM JMJD2E, 30 nM Jarid1a, 35 nM JMJD3. Background readings are given by heat inactivated enzymes, 0.5-1 mM 2,4 PDCA or reactions with no 2-OG. hJMJD2A (aa1-350) purified in E.coli is the kind gift of Dr. Jose Rizo-Rey and is assayed at 400 ng/reaction due to its intrinsic low activity. GraphPad Prism software is used for IC50 calculations and curve fitting. E.coli JMJD2D purified by us and Sf9 JMJD2D from BPS gave undistinguishable results. Note that for substrate competition assays, in order to remain in the linear range of the assay and not saturate binding capacity of the ELISA plate, reactions with> 0.75μM H3K9me3 containes unbiotinylated substrate or are diluted at the detection step and signals adjusted per dilution factor. For the direct quantification of H3K9me3 demethylase activity in cell lysates, treated cells (plated at 2 million/10cm dish) or tumor homogenates in PBS were sonicated (3x 4 sec) and equal amounts of protein are incubated with a histone H3K9me3 substrate in a reaction buffer containing cofactors for 2h at 37°C before specific immune-detection of the H3K9me2 product using Epigentek kit P-3081 reagents. 500 ng of E.coli purified PHD2 protein in 40mM Tris pH 7.4, 100mM NaCl, 20% glycerol, 5mM β-mercapto-ethanol, 10mM maltose are used to obtain activity in the linear range. Biotinylated peptides derived from the HIF-1 ODD (Biotin-Acp-DLDLEALAPYIPADDDFQL or Biotin-Acp-DLDLEALAP(OH)YIPADDDFQL as a hydroxylated control) are immobilized on Neutravidin-coated 96-well plates. Enzyme is incubated in the coated wells in reaction buffer (20 mM Tris-Cl pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 2 mM DTT, 0.12 μM ferrous sulfate, 0.5 mM 2-oxoglutarate and 1 mM ascorbate) for 45 min at room temperature in the presence of the indicated drugs. The competitive analog of α-ketoglutarate, DMOG, is used as a positive control for inhibition. Peptide hydroxylation is detected using a polyclonal rabbit antibody raised against a hydroxylated HIF peptide epitope, (rabbit anti-hydroxyproline 4817, made in house), followed by addition of a goat anti-rabbit HRP-conjugated secondary antibody. Luminescence is measured in an EnVision plate reader. The activity of LSD1 recombinant protein is measured using Epigentek kit P-3075 according to the manufacturer’s protocol with the proprietary inhibitor.

Cell Assay: For cell viability assays, cells are plated at 1500-3000 cells/well in 96 well plates and treated the next day with increasing doses of compound over 4 days and their viability assessed by standard MTS assays using Promega’s Cell Titer or Cell Titer-Glo reagents according to the manufacturer’s protocols. Absorbance at 490 nm and 650 nm or luminescence is measured by a Spectra Max or a FlouroStar Omega plate reader. Data are normalized to the untreated controls (100% viability). Each cell line is tested in 2-5 independent assays, each containing 4-8 replicates. IC50 values are calculated using DIVISA, a high-throughput software, developed in hous, for storing and analyzing drug sensitivity assays. Dose-response curves are plotted using a non-linear regression model and IC50s are determined from the fitted curves. The average IC50 derived from 2-5 independent assays, each containing 4-8 replicates is reported.

Nat Commun. 2013;4:2035.