JIB-04 199596-05-9

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Molecular Weight:

308.76 JIB-04 is a pan-selective Jumonji Histone Demethylase Inhibitor with IC50 of 230, 340, 855, 445, 435, 1100, and 290 nM for JARID1A, JMJD2E, JMJD3, JMJD2A, JMJD2B, JMJD2C, and JMJD2D, respectively.


Biological 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.


In
two separate xenograft mouse models (H358 or A549), JIB-04 diminishes
tumor growth, lowers Jumonji histone demethylase activity in tumors, and
prolongs cancer survival.


Jumonji demethylase/prolyl hydroxylase/LSD1 activity assays


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.


Method






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.






Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Baboon	Dog	Monkey	Rabbit	Guinea pig	Rat	Hamster	Mouse
Weight (kg)	12	10	3	1.8	0.4	0.15	0.08	0.02
Body Surface Area (m2)	0.6	0.5	0.24	0.15	0.05	0.025	0.02	0.007
Km factor	20	20	12	12	8	6	5	3

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×	mouse Km(3)	= 11.2 mg/kg
	rat Km(6)

Chemical Information

Molecular Weight (MW)	308.76
Formula	C17H13ClN4
CAS No.	199596-05-9

Storage	3 years -20℃Powder
	6 months-80℃in solvent (DMSO, water, etc.)
Synonyms	NSC 693627

Solubility (25°C) *	In vitro	DMSO	25 mg/mL heating (80.96 mM)
		Water	<1 mg/mL (
		Ethanol	<1 mg/mL (
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Name	5-Chloro-2-[(E)-2-[phenyl(pyridin-2-yl)methylidene]hydrazin-1-yl]pyridine

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Product Categories : Epigenetics > Histone Demethylase Inhibitor